Evolutionary Dynamics of Mammalian Karyotypes: Reprint of:'Cytogenetic and Genome Research 2012, Vol. 137, No. 2-4&#039

Evolutionary Dynamics of Mammalian Karyotypes Editors Roscoe Stanyon, Florence Alexander Graphodatsky, Novosibirsk 48...

Author: R. Stanyon | A. Graphodatsky

24 downloads 513 Views 6MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

Evolutionary Dynamics of Mammalian Karyotypes

Editors

Roscoe Stanyon, Florence Alexander Graphodatsky, Novosibirsk

48 figures, 28 in color, and 30 tables, 2012

Basel Freiburg Paris London New York New Delhi Bangkok Beijing Tokyo Kuala Lumpur Singapore Sydney •

•

•

•

•

•

•

•

•

•

•

S. Karger Medical and Scientific Publishers Basel Freiburg Paris London New York New Delhi Bangkok Beijing Tokyo Kuala Lumpur Singapore Sydney •

•

•

•

•

•

•

•

•

•

•

Disclaimer The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the journal is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.

Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center (see ‘General Information’). © Copyright 2012 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel

Vol. 137, No. 2–4, 2012

Contents

83 A Short Introduction to Cytogenetic Studies in Mammals with Reference

to the Present Volume Graphodatsky, A. (Novosibirsk); Ferguson-Smith, M.A. (Cambridge); Stanyon, R. (Florence) 97 Genome Size Evolution: Sizing Mammalian Genomes Redi, C.A. (Pavia); Capanna, E. (Roma) 113 The Evolution of Marsupial and Monotreme Chromosomes Deakin, J.E. (Canberra, A.C.T.); Graves, J.A.M. (Canberra, A.C.T./Melbourne, Vic.); Rens, W. (Cambridge) 130 Chromosome Evolution in Xenarthra: New Insights from an Ancient Group Svartman, M. (Belo Horizonte) 144 The Chromosomes of Afrotheria and Their Bearing on Mammalian

Genome Evolution Svartman, M. (Belo Horizonte); Stanyon, R. (Florence) 154 Chromosome Evolution in Eulipotyphla Biltueva, L.; Vorobieva, N. (Novosibirsk) 165 Chromosome Evolution in Bats as Revealed by FISH: The Ongoing Search

for the Ancestral Chiropteran Karyotype Volleth, M. (Magdeburg); Eick, G. (Eugene, Oreg.) 174 Comparative Chromosome Painting in Carnivora and Pholidota Perelman, P.L.; Beklemisheva, V.R.; Yudkin, D.V. (Novosibirsk); Petrina, T.N.; Rozhnov, V.V. (Moscow); Nie, W. (Kunming); Graphodatsky, A.S. (Novosibirsk) 194 Comparative Molecular Cytogenetics in Cetartiodactyla Rubes, J.; Musilova, P.; Kopecna, O.; Kubickova, S.; Cernohorska, H. (Brno); Kulemsina, A.I. (Novosibirsk) 208 Chromosome Evolution in Perissodactyla Trifonov, V.A. (Novosibirsk); Musilova, P. (Brno); Kulemsina, A.I. (Novosibirsk) 218 Low, Complex and Probably Reticulated Chromosome Evolution of

Sciuromorpha (Rodentia) and Lagomorpha Richard, F. (Paris/Versailles); Dutrillaux, B. (Paris)

© 2012 S. Karger AG, Basel Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Access to full text and tables of contents, including tentative ones for forthcoming issues: www.karger.com/cgr_issues

233 Non-Sciuromorph Rodent Karyotypes in Evolution Romanenko, S.A. (Novosibirsk); Volobouev, V. (Paris) 246 Molecular Cytogenetic Studies in Strepsirrhine Primates, Dermoptera and

Scandentia Nie, W. (Kunming) 259 Chromosome Evolution in New World Monkeys (Platyrrhini) de Oliveira, E.H.C. (Ananindeua/Belém); Neusser, M.; Müller, S. (Munich) 273 Evolutionary Molecular Cytogenetics of Catarrhine Primates: Past, Present

and Future Stanyon, R. (Florence); Rocchi, M. (Bari); Bigoni, F. (Florence); Archidiacono, N. (Bari)

285 Author Index Vol. 137, No. 2–4, 2012 286 Author Index Vol. 137, 2012 after 286 Contents Vol. 137, 2012

82

Contents

Cytogenet Genome Res 2012;137:83–96 DOI: 10.1159/000341502

Published online: July 26, 2012

A Short Introduction to Cytogenetic Studies in Mammals with Reference to the Present Volume A. Graphodatsky a M.A. Ferguson-Smith b R. Stanyon c a

Institute of Molecular and Cellular Biology, Siberian Division of the Russian Academy of Sciences, Novosibirsk , Russia; b Department of Veterinary Medicine, University of Cambridge, Cambridge, UK; c Department of Evolutionary Biology, University of Florence, Florence, Italy

Key Words Chromosome painting ⴢ Eutherian ancestral karyotype ⴢ Genome evolution ⴢ Phylogenomics

Abstract Genome diversity has long been studied from the comparative cytogenetic perspective. Early workers documented differences between species in diploid chromosome number and fundamental number. Banding methods allowed more detailed descriptions of between-species rearrangements and classes of differentially staining chromosome material. The infusion of molecular methods into cytogenetics provided a third revolution, which is still not exhausted. Chromosome painting has provided a global view of the translocation history of mammalian genome evolution, well summarized in the contributions to this special volume. More recently, FISH of cloned DNA has provided details on defining breakpoint and intrachromosomal marker order, which have helped to document inversions and centromere repositioning. The most recent trend in comparative molecular cytogenetics is to integrate sequencing information in order to formulate and test reconstructions of ancestral genomes and phylogenomic hypotheses derived from comparative cytogenetics. The integration of comparative cytogenetics and sequencing promises to provide an understanding of

© 2012 S. Karger AG, Basel 1424–8581/12/1374–0083$38.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/cgr

what drives chromosome rearrangements and genome evolution in general. We believe that the contributions in this volume, in no small way, point the way to the next phase in cytogenetic studies. Copyright © 2012 S. Karger AG, Basel

Cytogenetic analysis of mammalian karyotypes has gone through a number of phases strictly related to technical advances. Hsu, in his entertaining book entitled ‘Human and Mammalian Cytogenetics: an Historical Perspective’, referred to the earliest phases as the ‘dark ages’ and the scientists who pursued chromosome studies were quaintly deemed ‘knights’ [Hsu, 1979]. Then came the ‘hypotonic miracle’ of the 1950s, which culminated in deciphering the correct human diploid chromosome number. It is a curious fact that diploid numbers of various mammals, including relatives of man, such as the chimpanzees and macaques, were already correctly described by this time. Yet hypotonic treatment along with tissue culture and the discovery of mitogens led to an explosion in papers on the diploid number and chromosome morphology of literally hundreds of mammals. More than 400 of these karyotypes were published in ‘An Atlas of Mammalian Chromosomes’ edited by Hsu along with Benirschke as a multi-volume set [Hsu and BeRoscoe Stanyon Department of Evolutionary Biology University of Florence IT–50125 Florence (Italy) roscoe.stanyon @ unifi.it

nirschke, 1967–1991]. On the basis of classical cytogenetics it was easily shown that different species had different number and form of chromosomes, i.e. they had different karyotypes. Karyotypes could vary greatly even between related species, and hypotheses about ancestral genomes were framed in terms of the diploid number (2n) and the number of chromosome arms (fundamental number, FN). We now know that mammalian chromosome numbers range from 2n = 6 in the Indian muntjac to 2n = 102 in the viscacha rat. Just when classical studies of chromosomes seemed somewhat in a stall, then of course came chromosome banding, beginning with Q- and going on in short order to G-, R- and C-banding and many others, fluorescent and non-fluorescent. The immediate utility of banding was matching homologous chromosomes of the same cell and describing in more detail translocations and inversions in human clinical cases. In no time at all these technical advances were applied to animal species and in particular the mammals. Again an explosion of reports was forthcoming. Literally, hundreds of species were and are still being described at this level. Workers began looking for between-species homologies. These were most obvious between closely related species such as humans and their primate relatives, the great apes and Old World monkeys, with some notable failures such as the lesser apes. Even though it was clear that homology was proposed essentially on the basis of morphological similarity, bolder individuals looked even further afield and matched human chromosome banding with that of other mammals such as cats [O’Brien and Nash, 1982] and rabbits [Dutrillaux et al., 1980]. From these banding era comparisons it was rightly concluded that large blocks of mammalian genomes were conserved over millions of years. A curious fact almost ignored was that, even though the conclusions were right, about 50% of the homologies based on banding proposed between human/cat or human/rabbit was shown to be wrong in the chromosome painting era. About the same percentages were found in early banding homologies between macaques and green monkeys [Stock and Hsu, 1973]. We must conclude that establishing chromosomal homology between distantly related species or species characterized by rapid chromosomal evolution remained speculative until the advent of molecular cytogenetics and especially chromosome painting in the late 1980s and early 1990s [Wienberg et al., 1990]. Of course, by that time in situ hybridization was nothing new. It had begun even before the advent of banding with the publication of Gall and Pardue in 1969 [Gall and 84

Cytogenet Genome Res 2012;137:83–96

Pardue, 1969]. These early techniques have much in common with those of today. The chromosomal DNA on the slide and the probe both needed to be denatured. However, most molecular cytogeneticists now have little appreciation of how time-consuming and difficult these techniques were. The probe had to be radioactively labeled and after hybridization the slides were covered with a photograph emulsion. After sometimes weeks of exposure, the photograph emulsion was developed. Except for the most highly repetitive parts of the genome, the autoradiographs were difficult to analyze. The radioactive in situ hybridization procedure made the normal staining, microphotography, film development, printing and manually cutting and assembling karyotypes seem like a breeze (all procedures notably shortened today with the advent of digital imaging). Modern molecular cytogenetics was really born only with the discovery of nonradioactive methods [Langer et al., 1981] and probes, which initially came out of the human genome project. These initial hybridization techniques relied on biotin labeling and detection with avidin coupled with a fluorescent reporter molecule (fluorescence in situ hybridization, FISH).

A History of Chromosome Painting

Early hybridization of whole chromosome-specific probes (whole chromosome paints, WCP) used plasmid libraries and nick translation. This was sometimes termed CISS (chromosomal in situ suppression) hybridization [Cremer et al., 1988], due to the use of Cot-1 DNA to block the repetitive fraction of the genome from participating in the in situ hybridization. Later the easier, more visual ‘chromosome painting’ [Pinkel et al., 1988] became the exclusively used term. Soon after, chromosome painting began to establish its utility for both medical analysis and comparative research. Wienberg et al. [1990] were the first to show that chromosome painting could be used to compare genomes. Studies soon followed which mapped the chromosomal homology of entire genomes [Jauch et al., 1992; Wienberg et al., 1992]. The chromosome painting procedure based on plasmid libraries was still expensive and time-consuming, and, more importantly, it was also limited by the amount of probe DNA available which was rapidly consumed by the nick translations. Then Carter et al. [1992] and Telenius et al. [1992] introduced the use of a degenerate oligonucleotide-primed PCR (DOP-PCR) for amplifying chromosome sorts. The Graphodatsky /Ferguson-Smith /Stanyon

application of DOP-PCR methods using a random or universal primer made chromosome paints from flowsorted chromosomes available almost without limit. The 6MW primer reported in these papers became the standard primer for the PCR of chromosome sorts. However, alternative random primers are possible such as the FS primer for mouse chromosomes [Rabbitts et al., 1995] and many others. Note that these whole chromosome paints should not be referred to as libraries, a frequent error even today. Another advantage was that it soon became clear that the quality of these paints was much improved and had vast untapped potential to chart genomes even between distantly related species [Scherthan et al., 1994]. A number of modifications were necessary for long-range phylogenetic painting. Among these was an increase in the amount of probe used and an increase in hybridization times, from several days to a week. Pretreatment of the metaphase chromosomes was also important both to improve target DNA disposability and better banding. As is well known, chromosome paints rapidly became indispensable for a wide range of clinical and comparative research [Ferguson-Smith, 1997]. Part of this popularity was due to the fact that not only the paints were better, but also were cheaper and became more widely available. Chromosome painting was rapidly shown to be superior to chromosome banding for uncovering chromosome rearrangements in clinical cytogenetics and in reconstructing ancestral associations in comparative research. The results were much more reliable because homology is established not on the basis of morphological similarities, but on the basis of DNA content. The laboratory of Ferguson-Smith became the center of an important cytogenetic industry: the comparison and mapping of between-species chromosomal homology no longer at the morphological level, but at the DNA level. It is difficult to find a comparative cytogeneticist working today who in some way, directly or indirectly, is not connected to what took place in the Cambridge laboratory on Tennis Court Road. Flow-sorting and PCR introduced other additional possibilities to comparative cytogenetics. Chromosome paints could be produced from virtually any species for which an adequately dividing cell culture could be established. The possibility to flow-sort and establish chromosome paint sets from many species made reciprocal painting [Wienberg and Stanyon, 1997] and even multidirectional painting possible. Such reciprocal and multidirectional chromosome painting allows a much more precise inference of breakpoints involved in chromosome

rearrangements. Literally hundreds of species were flowsorted at Cambridge, many of which were distributed to researchers over the world through the Cambridge Resource Centre for Comparative Genomics which was funded by the Wellcome Trust [Ferguson-Smith et al., 2005]. Later a flow-sorting lab was established by Wienberg and Stanyon at the National Cancer Institute, Frederick (USA), and a second, more limited source of chromosome paints then became available.

Cytogenetic Studies in Mammals


Spectral Karyotyping, M-FISH, Chromosome Bar Coding Additional specialized applications based on chromosome sorting and painting were also developed. The most spectacular of these is the staining of each of the 24 human chromosomes in different colors at the same time using whole chromosome painting probes. One method is known as spectral karyotyping, SKY [Schrock et al., 1996], and another is M-FISH [Speicher et al., 1996]. Although the results are fairly comparable, the methods are diverse. They require different combinations of filters and imaging systems. Another method is chromosome bar coding, which ‘bands’ chromosomes [Müller and Wienberg, 2001]. All these techniques resort to pools of chromosome paints. Although the resulting images are striking, all have had little impact in comparative molecular cytogenetics. Apparently the rapidly decreasing hybridization efficiencies in the long-range phylogenetic FISH make these methods unproductive except in the most closely related species. Therefore, we will not dwell further on these methods. Microdissection and FISH with Cloned DNA Chromosome paints are the largest probes used in molecular cytogenetics. Chromosome paints are very good at documenting interchromosomal rearrangements (translocations), but rarely provide information on intrachromosomal rearrangements (e.g. inversions). These limitations could be overcome by physically isolating a slice of a chromosome by microdissection [Guan et al., 1992] or by cloning DNA in various vectors such as YACs and BACs [Boyle et al., 1992; Kirsch et al., 2000]. Microdissection probes can be pooled for multicolor banding hybridizations [Liehr et al., 2006], which have had some limited application in both primates and rodents. BACFISH has become somewhat more popular for establishing marker order along chromosomes and investigating intrachromosomal rearrangements. One surprising results was the discovery of evolutionary new centromeres [Rocchi et al., 2012] in a number of diverse mammalian 85

orders. The use of BAC-FISH to determine marker order and centromere repositioning is also reviewed in this volume in the article by Stanyon et al.

Search for Ancestral Genomes

The reconstruction of the history of life on our planet is one of the fundamental goals of evolutionary biology. Darwin first showed that evolution operated through natural selection and descent from common ancestors. Darwin’s theory that all forms of life on this planet are connected by descent from common ancestors is now an undisputed fact. This fact means that all forms of life on our planet are related and all species have common ancestors. Since the days of classical staining, cytogeneticists have been interested in establishing the content of ancestral karyotypes. In discussion of ancestral karyotypes in the classical staining era, cytogeneticists often fell into one camp or another. Many students of chromosomes thought that the ancestral mammalian karyotypes had a high diploid number and that evolution predominantly proceeded by chromosomal fusions. Others hypothesized that fissions were the main evolutionary mechanism and proposed low ancestral diploid numbers while others took the middle ground, suggesting that both mechanisms were important. An intriguing summary of these classically stained chromosome data is found in the paper by Matthey [1972] just at the beginning of the next great phase of cytogenetics, chromosome banding. Matthey, who among other things invented the term ‘fundamental number’, held the view that chromosome number could go up or down and therefore proposed that the ancestral placental mammalian karyotype should be close to the median of the 1,063 mammalian species he took into consideration. He decided on 48 chromosomes, surprisingly in line with modern chromosome painting hypotheses. In the same paper he discussed the karyotypes of marsupials and presented 2 hypotheses: an ancestral diploid number of 14 or 22. In the end he settled on 22 as the most probable ancestral diploid number for marsupials. The fascinating story of how molecular cytogenetics has finally allowed us to know that the ancestral number is more likely 14 is told in the contribution of Deakin et al. in this volume (see their figure 2). These authors also discuss why the ancestral chromosome number of monotremes, 2n = 46, is much higher than the 2n = 14 ancestral karyotype of marsupials and why the low diploid number is specific for 86


the marsupials, not for all non-placental mammals. They then go on to derive many of the karyotypes in these species. Ancestral karyotypes were also proposed for various points on the mammalian tree on the basis of banded chromosomes. Foremost among these were those of Dutrillaux and coworkers who proposed ancestral karyotypes for primates [Dutrillaux, 1979, 1988; Dutrillaux and Couturier, 1981] and carnivores [Dutrillaux and Couturier, 1983]. For example, they suggested an ancestral diploid number of 2n = 54 for the New World monkeys, the same number as discussed by de Oliviera et al. in this issue, but with some differences in content. Ancestral karyotypes, based on painting data and occasionally BAC-FISH and supported by gene sequencing data, are presented in a number of the reviews in this special issue. Perelman et al. thoroughly discuss the various hypotheses for ancestral karyotypes in the carnivores and Rubes reviews the progress in determining the ancestral karyotype of the Cetartiodactyla. Trifonov et al., also in this issue, discuss the utility of gene mapping data in reconstructing the ancestral karyotype of the Perissodactyla and higher-order branching in placental mammals. In their comparison of cytogenetic data for Sciuromorpha and Lagomorpha, Richards and Dutrillaux hypothesize that uncertainties in determining the content of ancestral genomes can inform us about the tempo and mode of speciation in these taxa. Their conclusions would be interesting to test in a broad array of mammals. The review of Nie in this volume also discusses the content of ancestral genomes and the various landmark rearrangements identified as characteristic for each different lineage, that potentially unite certain lineages within Strepsirrhine primates, Dermoptera and Scandentia. Finally, a catarrhine ancestral karyotype is also presented by Stanyon et al., which includes the probable marker order and centromere positions. Tenets of Ancestral Genome Reconstruction Establishing the ancestral eutherian karyotype has been one of the aims of interspecies chromosome painting studies. As discussed above, molecular cytogenetics allows great confidence in mapping chromosome homology between species. We can determine how many chromosome rearrangements are needed to transform the genome of one species into another. It is another but related task fundamental to phylogenetic reconstruction, to determine what chromosomes were present in ancestral species. Cladistic analysis of evolutionary relationships Graphodatsky /Ferguson-Smith /Stanyon

depends on the polarity of chromosome rearrangements. Two species can be very similar to each other only because they have both retained conserved, ancestral chromosomes. Other species which have very different karyotypes can at the same time be linked by common derived rearrangements. Hypotheses on the content of ancestral genomes aid in recognizing conserved, primitive characters versus derived rearrangements. Some basic tenets of constructing ancestral genomes from chromosome painting data were summarized by Wienberg and Stanyon [1995, 1997, 1998]. A chromosomal synteny may often be disrupted independently by chromosomal rearrangements, but it is highly unlikely that the same syntenic group was brought together independently in different lineages, especially in the same order. Parsimony analysis is then commonly used to determine ancestral chromosomal syntenies (the largest linkage group known) and associations. Commonality, combined with the inclusion of appropriate ‘outgroups’, helps determine if a chromosome is syntenic and what associations were present in an ancestral genome. An outgroup is one or more species from the next phylogenetic branch out from the group under discussion. For instance, the best outgroup for great apes and humans would be the Old World monkeys. When a chromosomal synteny is found intact between various species, this condition is likely to be ancestral (symplesiomorphic). For instance, a chromosomal synteny found in widely divergent mammalian orders would suggest that it was present in the ancestor of all mammals. In the early stages of reconstructing the ancestral genome of living placental mammals, it became clear that all chromosomes homologous to human chromosome 3 and human chromosome 21 were syntenic in the ancestor [Müller et al., 2000]. This syntenic chromosome association, written as 3/21, was later disrupted in the evolutionary line leading to humans. Today it is not found in any Old World monkeys, apes or humans, but it is found in New World monkeys, prosimians and is universal in almost all other mammals, including marsupials, and even in chickens. Therefore, the fission of 3/21 would qualify as a phylogenetic landmark linking Catarrhine primates. Most authors appeal to the concept of parsimony and consider that likely ancestral chromosomes are commonly present in species of a number of divergent eutherian orders [Svartman et al., 2004]. The Ancestral Karyotype of Placental Mammals Over the last few years various hypotheses were advanced on the content of the ancestral karyotype of all

living placental mammals. The diploid number in these hypotheses ranges from 44 to 50 chromosomes [Svartman et al., 2004]. Wienberg and Stanyon [1997] were the first to formulate hypotheses about the probable content of the mammalian ancestral karyotype based on chromosome painting data, but it was Chowdhary et al. [1998] who published one of the first complete reconstructions. They proposed a diploid number of 48, with chromosomes 4, 7, 8, 10, 16p present as single chromosomes, and that the association 12/22a with an additional chromosome 22b was also likely part of the eutherian ancestral karyotype. However, with the inclusion of improved chromosome painting data from further taxa, it was deemed likely that chromosomes 2pq, 2q, 3/21, 5, 6, 4/8p, 7p, 7q/16p, 8q, 9, 10p, 10q, 11, 12q/22a, 12p/22b, 13, 14/15, 16q/19q, 17, 18, 20, X and Y were syntenic in the ancestral eutherian karyotype [Murphy et al., 2001; Richard et al., 2003]. An unsettled question was the possibility that 10/12q/22a might be present as an ancestral placental mammal association and there were some doubts whether chromosome 1 might be a single syntenic unit in the ancestor. Then, Murphy et al. [2003] demonstrated through comparative gene mapping data and reciprocal chromosome painting with homologs to human (HSA) chromosome 1 in 7 species from 6 eutherian orders of mammals that the segments previously considered equivalents of HSA1p and 1q in these species were not identical. In the meantime, a single HSA1 homolog was also found in 3 highly divergent eutherian orders. These data strongly indicated that a single large chromosome, corresponding to HSA1, was present in the ancestral eutherian karyotype [Murphy et al., 2003]. The contribution of Svartman and Stanyon in this issue discusses the 2 hypotheses in regard to the association 1/19. Due to its presence in Afrotheria, the 1/19 association was considered a possible candidate for inclusion in the ancestor of all placental mammals [Yang et al., 2003]. The alternative hypothesis was that the 1/19 association was instead a derived trait linking Afrotheria [Frönicke et al., 2003; Svartman et al., 2004; Liu et al., 2011]. As discussed by Stanyon and Svartman in this issue, the 1/19 and the 5/21 associations are both derived in Afrotheria. Again in this volume, Svartman suggests that the xenarthran ancestral karyotype has conserved better and is closest to the ancestral karyotype of all placental mammals. It can be noted that the 2 supposed xenarthran synapomorphic associations HSA2/8 and 7/10 [Yang et al., 2006; Liu et al., 2011] were also detected in the marsupial and chicken genomes [Graphodatsky et al., 2011]. Therefore, Xenarthra may be a better candidate for, or at least represent more



87

closely, the basal placental mammal. Clearly, this possibility deserves further study, both with further chromosome painting and sequencing. Questions also arose whether there were conflicting results from other levels of genome analysis [Froenicke et al., 2006]. In a paper in Science, Murphy et al. [2005], while confirming many of the conclusions from molecular cytogenetics about the boreoeutherian ancestral genome, proposed 5 additional syntenic associations that had apparently gone undetected by chromosome painting. However, later more refined methods and additional data [Ma et al., 2006] gave no support to these additional associations [Rocchi et al., 2006].

Chromosome Homology and Evolution in Mammals, Birds and Reptiles: Hypothesis on the Role of Non-Coding DNA

(Section contributed by M.A.F.-S.) While animal karyotypes display a great diversity in number and morphology of chromosomes, their genomes are remarkably conserved. This conservation is resolved by chromosome painting into small numbers of large chromosome blocks. Rearrangement of these blocks into different combinations explains much of the diversity in karyotypes. Evolutionary relationships of representative species of all mammalian orders can be determined from studying the pattern of associations of these syntenic blocks [Ferguson-Smith and Trifonov, 2007]. For example, the association of blocks from HSA3p and 21 can be found in the karyotypes of all mammals except the apes and Old World monkeys, as are blocks from HSA14 and 15. Other associations are signatures that characterize 1 group of species, for example the HSA5/21 association in Afrotheria. The chromosome-specific DNA used to delineate these homologies is prepared from flow-sorted chromosomes, amplified and labeled by PCR. Unfortunately, the success of chromosome painting in determining chromosome homologies does not extend to all vertebrates. It is unsuccessful between placental mammals and marsupials or monotremes, or birds, or reptiles, although it works well enough within each of these groups to postulate ancestral karyotypes for each. Except for part of the X chromosome [Glas et al., 1999], homologies cannot be demonstrated by painting between human and marsupials, although cross-species painting is effective in determining chromosome homology between all extant marsupials [Rens et al., 1999, 2001, 2003]. 88


The chromosomes of egg-laying monotremes, which are the most basal clade of mammals and have a unique multiple X and Y sex chromosome system [Rens et al., 2004, 2007], also cannot be painted by human paint probes, although painting between platypus and echidna has been very informative [Rens et al., 2007]. Where cross-species painting fails to show homologous regions in more distantly related taxa such as chicken and platypus, recourse has been made to the more time-consuming gene mapping comparisons [Rens et al., 2007]. Marsupials and monotremes diverged from the mammalian lineage much earlier than placental mammals, approximately 148 and 166 million years ago (Mya), respectively [Bininda-Emonds et al., 2007]. This suggests that successful painting might be limited to taxa arising from a common ancestor no earlier than about 100 Mya. Thus cross-species painting would not be expected to work between mammals and birds. Painting chicken chromosome 4q to HSA4 is the only apparent exception to this general rule [Chowdhary and Raudsepp, 2000], although gene mapping and sequencing confirms that there are blocks of conserved homology between their chromosomes. The chicken karyotype has served as the model for comparative chromosome studies in birds. It has 9 pairs of easily recognized macrochromosomes, plus the Z and W sex chromosomes, and 29 pairs of microchromosomes, many of which sort together in the flow karyotype and are difficult to characterize. Some microchromosomes can be isolated from single chromosome sorts or by microdissection and others rely on BAC mapping for identification [Griffin et al., 1999; Masabanda et al., 2004]. Macrochromosome probes have been used almost exclusively in comparative painting studies in over 50 species from at least 10 different avian orders. The main findings are extensive chromosome conservation in birds with a comparatively small number of interchromosomal rearrangements in those species with diploid numbers from 78 to 92. Raptors tend to have smaller chromosome numbers, from 48 to 66, resulting from fusions of ancestral macro- and microchromosomes. The stone curlew (Burhinus oedicnemus, 2n = 42) has one of the smallest chromosome numbers with multiple fusions of chicken microchromosomes [Nie et al., 2009]. The chicken karyotype is thought to be close to the ancestral avian karyotype. Chromosome paints have been made recently in the Cambridge laboratory from several reptiles, including lizards (Scincus scincus, Hemidactylus turcicus, Hemidactylus platyurus, Anolis carolinensis and Gekko japonicus), Graphodatsky /Ferguson-Smith /Stanyon

turtle (Trachemys scripta elegans) and crocodile (Crocodylus niloticus). Cross-species painting between these species has been very informative, although the hybridization efficiency has been less than in birds or mammals: this is understandable in view of the longer divergence times. The results are consistent with greater genome conservation and even fewer evolutionary rearrangements than in birds [Giovannotti et al., 2009; Trifonov et al., 2011]. In view of the failure of chromosome painting to demonstrate homologies between taxa that have diverged more than 100 Mya, recent results on successful painting between birds and reptiles were unexpected. The chicken Z probe was found to hybridize without apparent rearrangement to an autosome in all the above reptilian species despite over 250 million years divergence [Pokorna et al., 2010]. This observation prompted experiments with chicken macrochromosome probes onto reptilian chromosomes. Extensive homology was discovered between chicken, turtle and crocodile chromosomes [Kasai et al., 2012b] and between chicken and squamate species [Pokorna et al., 2012]. Thus, painting between chicken and turtle showed that chicken macrochromosomes 1–3 and 11 were each represented by a single turtle chromosome, and the arms of chicken 4, 6 and 8 were homologous to turtle 5 and 7p, 7q, and 8q, respectively. However, painting between chicken and crocodile revealed fission of 2 ancestral avian chromosomes with rearrangement by centric fusion into 4 crocodile chromosomes, contributing to the reduced chromosome number characteristic of the order. High chromosome conservation is thus a feature of birds and reptiles, and the results already suggest that the presence of ancestral avian chromosomes extends into at least the early Jurassic era. The ability of cross-species chromosome painting to demonstrate chromosome homology between some vertebrate groups and not others is of some interest. The usual explanation is that chromosome painting involves hybridization of protein-coding sequences, and presumably also of regulatory sequences, to their complementary sequences on the chromosome. Painting signals tend to be more extensive the closer the relationship between 2 species. Its success must depend on the level of conservation, and thus the length of time elapsed since the divergence of the 2 species from a common ancestor. Repetitive sequences can be excluded because they are usually not specific to one particular chromosome and are suppressed during the painting procedure. Although vertebrate protein-coding sequences tend to be highly conserved, many human cloned genes do not hybridize easily to their ho-

mologues by FISH mapping, even in closely related species. For success the orthologue of each gene must be found and used. Also, protein-coding sequences represent less than 2% of genomes and seem an unlikely explanation for the depth and extent of hybridization signals observed across homologous regions. An alternative hypothesis is that painting does not depend on gene sequence but on an as yet undefined functional class of chromosome-specific non-coding DNA (CSNCD) in greater abundance than protein-coding sequences. CSNCD could have evolved to ensure chromosome synapsis in meiosis and to reduce illegitimate recombination between non-homologues. Different levels of conservation of CSNCD can account, for example, for the success of painting between chicken and reptiles and its lack of success between human and marsupials. In other words, CSNCD in marsupials is expected to have diverged substantially from humans, as cross-species painting is ineffective between them, whereas the chicken CSNCD is shared to a considerable extent with reptiles. New chromosomes resulting from rearrangement during evolution will still maintain CSNCD, albeit in a different combination. A recent study on the proportion of the genome that is functional suggests that 6.5–10% of the human genome is under functional constraint, and similar proportions are found for rat and mouse genomes [Meader et al., 2010]. These authors demonstrate in many pairs of species that the amount of functional DNA shared between 2 species decreases as the divergence between them increases. So far, the analysis has not extended to chromosome-specific DNA where one predicts that the proportion under functional constraint could be much higher. Other factors could contribute to the painting results between chicken and reptiles. Compared to humans, chicken, turtles and crocodiles have much smaller genome sizes (between 1.0 and 2.0 Gb) [Kasai et al., 2012a] and perhaps have not accumulated those DNAs that tend to diverge from the more conserved DNA classes in animals with larger genomes. Also, there is evidence that chicken and reptile genomes share a higher percentage of long conserved non-coding sequences with one another than with mammals [Janes et al., 2011]. Nonetheless, the CSNCD hypothesis remains the most plausible mechanism to explain the observations obtained by cross-species chromosome painting in vertebrates. The hypothesis may be testable by comparative sequencing of chromosome-specific DNA when suitable tools are developed to interrogate genome databases, so that shared CSNCD can be distinguished from diverged CSNCD.



89

Fig. 1. Examples of homology between human chromosome 5, various mammalian species and the chicken. Placental mammals were compared on the basis of chromosome painting, while species marked by an asterisk were compared on the basis of sequence alignments (http://www.ensembl.org/) without Zoo-FISH data.

The Importance of Marsupials, Monotremes and other Vertebrates for Understanding the Evolution of the Placental Genome

In this issue, Redi and Capanna present a fascinating contribution on the relationships between genome size, taxonomic divisions and molecular mechanisms. These are very basic facts of genome structure and composition, which support the higher taxonomic divisions because they appear to vary among the superordinal clades. They also raise the good possibility, in line with the above speculations of M.A.F.-S., that new classes of DNA apparently raised the genome size of placentals, which inhibit good signals from placental paints on marsupials and monotremes. As can well be appreciated from a causal glace at this volume, genome-wide comparative chromosome maps between humans and representative species of all extant eutherian orders have been established (fig. 1). However, the lack of good results using placental painting probes on marsupials and other vertebrates is not just an interesting theoretical question; it points out a critical fact. We do not have appropriate outgroups necessary to fulfill a fundamental requirement of cladistic analysis. As ably discussed above and in this volume by Deakin et al., there is outside of some hybridization data from the X chromosome a total lack of comparative chromosome painting data between eutherian and other mammals: monotremes and marsupials as well as other more distantly re90


lated vertebrates such as birds and reptiles. The only alternative at this moment is to compare the chromosome painting results to sequence assemblies (fig. 2). Wang et al. [2011] and Deakin et al. (this volume) discuss how it is possible to make a virtual map of the chromosome homologies between marsupials and placentals. Other researchers have demonstrated how the genome sequence data can be used to electronically paint chromosomes. Initial results are quite informative [Kemkemer et al., 2006]. These comparisons show that at the time of the ancestral placental, mammalian associations (4/8, 10/12/22, 7/16, 16/19) are present in other vertebrates, lending good support to the hypothesis that they were also present in the ancestral placental genome. A 1/19 association is indeed also present in Monodelphis domestica, but this association is 1p36/19q13 while that in the Afrotheria is 1p/19p as discussed in the contribution by Svartman and Stanyon in this issue (see their figures 1 and 2). We decided it might be worthwhile to compare the associations present in some non-placentals for which genome assembly data are available: the gray short-tailed opossum (M. domestica, MDO), the tammar wallaby (Macropus eugenii, MEU), the platypus (Ornithorhynchus anatinus, OAN), the chicken (Gallus gallus, GGA) and the green anole lizard (Anolis carolinensis, ACA). We are well aware that there are limitations based on various methods utilized to determine homology with human chromosomes and the diverse levels of coverage of the genome asGraphodatsky /Ferguson-Smith /Stanyon

Fig. 2. Genome-wide chromosomal correspondence among mammalian and avian species with the human ideogram as the reference. ECA = Horse (Equus caballus), GGA = chicken (Gallus gallus), HSA = human, MDO = opossum (Monodelphis domestica), OAN = platypus (Ornithorhynchus anatinus), and OCU = rabbit (Oryctolagus cuniculus). (All data from USC genome browser.)

semblies, which makes them difficult to thoroughly compare. Further, results can vary according to the analysis methods selected. We emphasize that this is a preliminary analysis valuable for speculation and pointing to aspects of genome architecture that might merit further attention. There are various sources which can be utilized for the human/opossum homology from the original genome as-

sembly publication [Mikkelsen et al., 2007] to that of Wang et al. [2011]. We also assembled an electronic painting of the opossum/human homologies (online suppl. fig. 1; for all online supplementary material, see www. karger.com/doi/10.1159/000341502) based on a manual inspection of coding genes (USC genome browser, http:// genome.ucsc.edu/cgi-bin/hgGateway). Marilyn Renfree



91

Fig. 3. Electronic chromosome painting idiogram of the chicken

(GGA) showing the color coded homology with human chromosomes. The idiogram was produced by a manual examination of coding gene homology with the human genome. Chromosome segments between coding genes homologous to the same human chromosome were considered to be homologous to that human

was kind enough to send M.A.F.-S. a human/tammar wallaby homology map, which was used for comparison. We also made an electronic painting karyotype of the chicken (fig. 3) for comparison to the other genomes based on a manual inspection of coding genes (USC genome browser). Comparisons with the lizard genome [Alfoldi et al., 2011] were made by taking the association data from the scaffolds of the genome assembly (USC genome browser). For all these comparisons we considered only major segmental associations which could possibly 92


chromosome. Segmental homology !3 kb was ignored as it was considered to be under the resolution of chromosomes paints. The homologous regions to anole lizard (ACA) and turkey (MGA) were taken from a manual examination of scaffolds. (All data from USC genome browser.)

be seen at the cytogenetic level. We then listed associations which were found in at least 2 species (online suppl. table 1). Among these vertebrates the highest affinity, as expected, is between tammar wallaby and opossum: there were 37 common associations: 1/6, 1/11, 1/17, 1/19, 2/3, 2/8, 2/11, 2/13, 2/15, 2/16, 2/20, 3/7, 3/9, 3/X, 4/8, 4/11, 5/14, 5/17, 5/18, 5/20, 6/8, 6/17, 6/18, 7/9, 7/10, 8/18, 8/19, 9/16, 9/17, 9/20, 10/15, 11/13, 12/22, 14/15, 15/X, 16/19, 21/X. It is interesting to note that the tammar wallaby, opossum Graphodatsky /Ferguson-Smith /Stanyon

and platypus share 10 associations: 2/3, 2/13, 2/16, 2/20, 3/9, 5/18, 6/17, 7/10, 7/16, 8/18. Therefore, the opossum and tammar wallaby share 27 associations not found in the platypus. In the same manner there are a few associations held by MDO and other vertebrates but not MEU: 2/5, 5/9, 10/12, and conversely there are also associations held by MEU with another vertebrate but not with MDO: 2/21, 7/12. It is informative to examine the associations held in common between non-placental mammals and other vertebrates. The 49 associations held in common between at least one mammal and the chicken or green anole lizard are compared in online supplement table 1. Of these 49 associations only 7 were apparently conserved in the ancestral eutherian karyotype. Certainly, not all these associations are homologous and some may be due to convergence. However, the implications seem inescapable. It has long been appreciated that the platypus genome has a notable similarity with birds and reptiles [Rens et al., 2007; O’Brien, 2008; Veyrunes et al., 2008; Warren et al., 2008; Alfoldi et al., 2011]. We show here that this similarity extends to the common syntenic associations between platypus, birds and lizard. Perhaps surprisingly, there is also by far a greater similarity in association structure between opossum/tammar wallaby with birds and lizards than with placental mammals. Therefore, it does not seem to be an exaggeration to suggest that placentals have had a virtual genomic revolution at least at the association level. If the speculations of M.A.F.-S. presented earlier hold out, then there was a parallel revolution in the non-coding part of the genome. Perhaps we can further speculate that we have something entirely new in the chromosome makeup of placentals. In this regard, previously workers, to address the ancestral genome structure of eutherians, were apparently more interested in looking at the associations, which were held in common between marsupials and placentals. We suggest that it is equally important, and perhaps even more informative, to look at the ancestral vertebrate associations that are conserved in marsupials and monotremes but lost in placentals. The loss of the majority of ancient vertebrate associations helps us better appreciate the magnitude of what happened at the origin of placentals. On the basis of the association data we find no support for the suggestion of Ruiz-Herrera et al. [2012] that the placental ancestral karyotype is ancestral for all mammalian species. Further, we have to note that we were not able to utilize their homology maps of some of these species because of an unfortunate, inconsistent use of color-coding to human chromosome homology. Cytogenetic Studies in Mammals

Future Directions in Molecular Cytogenetics

Although we now have, as this volume amply illustrates, a substantial, broad view of chromosome evolution in mammals, there are clearly many glaring gaps, in particular the insufficiency of taxa sampling. Phylogenomic analysis is better the more taxonomically rich are the assemblages available for analysis. Even in the best-studied mammalian orders, primates and carnivores, only a minority of species have been studied. Many contributors to the present special issue remark on how it would be beneficial to study additional species. This lack is particularly clear as pointed out by authors of papers in this special issue such as Volleth and Eick for bats and Romanenko and Volobouev for rodents. For both these enormous orders we have only just begun to scratch the surface of the chromosome diversity present. Additional species would also be helpful all over the mammalian tree and even in smaller clades such as Afrotheria and Xenarthra. We are sure that cytogeneticists will continue to fill in the missing species, taking full advantage of the progress that has been made up to this point. Indeed, several contributions to this review issue actually contributed new original data. We can mention that, among others, Biltueva and Vorobieva added chromosome painting data on Iberian shrew and Altai mole, while Perelman et al. report on chromosome painting of Genetta pardina. Clearly the most widely utilized molecular cytogenetic method is chromosome painting. Very few species, most of them primates, have been studied with higher resolution methods such as BAC-FISH. Studies to define marker order (intrachromosomal rearrangements) and to describe inversions etc. by defining breakpoints via BAC-FISH need to be applied to a wider range of species. There is much work to be done at this level. Nonetheless, one of the strengths of chromosome painting versus sequencing analyses is that the economy of cytogenetic analysis has allowed a larger, more appropriate sampling of taxa [Froenicke et al., 2006; Rocchi et al., 2006]. Instead, up to now, sequencing efforts have been devoted mostly to species of economic interest or to those that are of particular significance for understanding human evolution such as the higher primates. However, clearly sequencing efforts are rapidly widening their scope and, as costs drop, the number of vertebrate species that will be sequenced will be increasing exponentially. Consequently, cytogenetics will be called upon to aid sequencing efforts and can take advantage of the detailed information coming from these studies.


93

Integration of Cytogenetics and Genomic Sequencing

The integration of cytogenetic and sequencing information will be vital to helping resolve long-standing issues in cytogenetics, many of which are discussed in the contributions to this issue. Among these is resolving problems in reconstructing ancestral karyotypes at various points of the mammalian tree. Volleth and Eick discuss how Robertsonian translocations have obscured the karyotype composition of the chiropteran ancestor. The problem of weighing cytogenetic traits in phylogenomic analysis remains a point of discussion without clear resolution. For instance, the dangers of confusing convergence with homology exist even in the chromosome painting era. It is helpful to know the gene content especially at junctures of segments. A number of authors in this special issue attempt to use and integrate other types of biomolecular data, in part to eliminate the homoplasy that can plague cytogenetic reconstructions. Perelman et al. in carnivores compared results of reciprocal human-dog painting data and syntenic blocks order identified in Ensemble and in Evolutionary Highway. Trifonov et al. used genome sequencing data to test the homology of associations in Perissodactyla. Nie followed a similar procedure to refute the proposal of Picone et al. [2011] that Dermoptera and Lagomorpha formed a monophyletic clade supported by 3 synapomorphies, i.e. HSA4/18, 7/19 and 1/10, by showing that these associations are due to convergence. As these authors showed, convergent association and homologous associations can sometimes be distinguished by reference to sequencing data since the contiguous segments should have the same gene content and order. Other cases of homoplasy such as those due to lineage sorting and reticulate evolution will not be ruled out by this method. So a combination of cytogenetic and sequence analysis could provide precious information on speciation processes.

Rates of Chromosome Evolution

Early painting comparisons of humans and other primates showed that there is no molecular clock for chromosome evolution. In other words, evolutionary rates in chromosome rearrangements vary greatly from one evolutionary line to another. In this special issue a number of contributions present examples of contrasting rates of chromosome evolution. Perelman et al. discuss both the high chromosome conservation in felids and the rapid chromosome evolution in canids and skunks. They also 94


provide new data on another carnivore species with highly rearranged karyotype: the pardine genet. Rubes et al. discuss the conserved karyotypes of whales versus the highly rearranged ones of pigs. Trifonov et al. show how varied the evolutionary rates are for Perissodactyla, with extremely fast rates of chromosome evolution and centromere formation in equids. Richard and Dutrillaux reported on the 2 groups Sciuromorpha and Lagomorpha, normally known for their low number of rearrangements, but even here there are exceptions. De Oliviera et al. detail the remarkable example of chromosome variability in New World monkeys marked by vastly different rates of chromosome evolution. They contrast and compare various hypotheses of phylogenomics based on chromosome painting with reconstructions coming from other biomolecular comparisons. There seems to be little room for the old concept that mammals have a default chromosome rearrangement rate with just a few exceptions. There are just too many exceptions and just too wide a range to accept that there is a default rate. However, the reasons for the differences have eluded our understanding. As discussed in the contribution by Stanyon et al., numerous hypotheses have been proposed including social organization, demographic parameters, meiotic drive, recombination suppression, as well as chromosome architecture such as segmental duplications and repeats. In addition to their well-known and extremely rapid chromosome evolution, Romanenko and Volobouev detail the special genomic features of rodents. Certainly one of the most remarkable features of rodents is not only their outstanding variability in chromosome numbers and/or chromosome morphology, but also their heterogeneous heterochromatin, B and sex chromosomes. Such variability is present in other mammals, but perhaps not so dramatically. It is probable that a comprehensive understanding of the forces responsible for varying rates of chromosome evolution and special characteristics of the mammalian genome will be at least in part found at the sequencing level in the coming years. A closer integration between cytogenetics and sequencing efforts will certainly be necessary if cytogeneticists want to maintain their relevance in evolutionary studies. One encouraging trend seen in the papers of this volume is the willingness of researchers to integrate and compare the cytogenetic data with phylogenomic information that comes from a wide array of biomolecular studies. We believe that the contributions here in no small way point the way to the next phase in cytogenetic studies. Graphodatsky /Ferguson-Smith /Stanyon

Acknowledgements This study was funded in part by programs MCB, RAS and SB RAS Programs and by research grants of the Russian Fund for Basic Research to A.G., PRIN (Programmi di Ricerca di Interesse

Nazionale) to R.S., and a Royal Society Grant to M.A.F.-S. R.S. thanks F. Garofalo and P. D’Addabbo for assistance in the electronic chromosome painting of opossum and chicken karyotypes.

References Alfoldi J, Di Palma F, Grabherr M, Williams C, Kong L, et al: The genome of the green anole lizard and a comparative analysis with birds and mammals. Nature 477:587–591 (2011). Bininda-Emonds OR, Cardillo M, Jones KE, MacPhee RD, Beck RM, et al: The delayed rise of present-day mammals. Nature 446: 507–512 (2007). Boyle AL, Feltquite DM, Dracopoli NC, Housman DE, Ward DC: Rapid physical mapping of cloned DNA on banded mouse chromosomes by fluorescence in situ hybridization. Genomics 12:106–115 (1992). Carter NP, Ferguson-Smith MA, Perryman MT, Telenius H, Pelmear AH, et al: Reverse chromosome painting: a method for the rapid analysis of aberrant chromosomes in clinical cytogenetics. J Med Genet 29: 299–307 (1992). Chowdhary BP, Raudsepp T: HSA4 and GGA4: remarkable conservation despite 300-Myr divergence. Genomics 64:102–105 (2000). Chowdhary BP, Raudsepp T, Fronicke L, Scherthan H: Emerging patterns of comparative genome organization in some mammalian species as revealed by Zoo-FISH. Genome Res 8:577–589 (1998). Cremer T, Tesin D, Hopman AH, Manuelidis L: Rapid interphase and metaphase assessment of specific chromosomal changes in neuroectodermal tumor cells by in situ hybridization with chemically modified DNA probes. Exp Cell Res 176:199–220 (1988). Dutrillaux B: Chromosomal evolution in primates: tentative phylogeny from Microcebus murinus (Prosimian) to man. Hum Genet 48: 251–314 (1979). Dutrillaux B: New interpretation of the presumed common ancestral karyotype of platyrrhine monkeys. Folia Primatol 50:226–229 (1988). Dutrillaux B, Couturier J: The ancestral karyotype of platyrrhine monkeys. Cytogenet Cell Genet 30:232–242 (1981). Dutrillaux B, Couturier J: The ancestral karyotype of Carnivora: comparison with that of platyrrhine monkeys. Cytogenet Cell Genet 35:200–208 (1983). Dutrillaux B, Viegas-Pequignot E, Couturier J: Great homology of chromosome banding of the rabbit (Oryctolagus cuniculus) and primates, including man [author’s transl.]. Ann Genet 23:22–25 (1980). Ferguson-Smith MA: Genetic analysis by chromosome sorting and painting: phylogenetic and diagnostic applications. Eur J Hum Genet 5:253–265 (1997).


Ferguson-Smith MA, Trifonov V: Mammalian karyotype evolution. Nat Rev Genet 8: 950– 962 (2007). Ferguson-Smith MA, Yang F, Rens W, O’Brien PC: The impact of chromosome sorting and painting on the comparative analysis of primate genomes. Cytogenet Genome Res 108: 112–121 (2005). Froenicke L, Caldes MG, Graphodatsky A, Müller S, Lyons LA, et al: Are molecular cytogenetics and bioinformatics suggesting diverging models of ancestral mammalian genomes? Genome Res 16:306–310 (2006). Frönicke L, Wienberg J, Stone G, Adams L, Stanyon R: Towards the delineation of the ancestral eutherian genome organization: comparative genome maps of human and the African elephant (Loxodonta africana) generated by chromosome painting. Proc Biol Sci 270: 1331–1340 (2003). Gall JG, Pardue ML: Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci USA 63: 378–383 (1969). Giovannotti M, Caputo V, O’Brien PC, Lovell FL, Trifonov V, et al: Skinks (Reptilia: Scincidae) have highly conserved karyotypes as revealed by chromosome painting. Cytogenet Genome Res 127:224–231 (2009). Glas R, De Leo AA, Delbridge ML, Reid K, Ferguson-Smith MA, et al: Chromosome painting in marsupials: genome conservation in the kangaroo family. Chromosome Res 7: 167–176 (1999). Graphodatsky AS, Trifonov VA, Stanyon R: The genome diversity and karyotype evolution of mammals. Mol Cytogenet 4:22 (2011). Griffin DK, Haberman F, Masabanda J, O’Brien P, Bagga M, et al: Micro- and macrochromosome paints generated by flow cytometry and microdissection: tools for mapping the chicken genome. Cytogenet Cell Genet 87: 278–281 (1999). Guan XY, Meltzer PS, Cao J, Trent JM: Rapid generation of region-specific genomic clones by chromosome microdissection – isolation of DNA from a region frequently deleted in malignant-melanoma. Genomics 14:680– 684 (1992). Hsu TC: Human and Mammalian Cytogenetics: an Historical Perspective (Springer Verlag, New York 1979). Hsu TC, Benirschke K: An Atlas of Mammalian Chromosomes (Springer Verlag, Berlin 1967–1991).

Janes DE, Chapus C, Gondo Y, Clayton DF, Sinha S, et al: Reptiles and mammals have differentially retained long conserved noncoding sequences from the amniote ancestor. Genome Biol Evol 3:102–113 (2011). Jauch A, Wienberg J, Stanyon R, Arnold N, Tofanelli S, et al: Reconstruction of genomic rearrangements in great apes and gibbons by chromosome painting. Proc Natl Acad Sci USA 89:8611–8615 (1992). Kasai F, O’Brien PC, Ferguson-Smith MA: Reassessment of genome size in turtle and crocodile based on chromosome measurement by flow karyotyping: Close similarity to chicken. Biol Lett (2012a), Epub ahead of print. Kasai F, O’Brien PC, Martin S, Ferguson-Smith MA: Extensive homology of chicken macrochromosomes in the karyotypes of Trachemys scripta elegans and Crocodylus niloticus revealed by chromosome painting despite long divergence times. Cytogenet Genome Res (2012b), Epub ahead of print. Kemkemer C, Kohn M, Kehrer-Sawatzki H, Minich P, Hogel J, et al: Reconstruction of the ancestral ferungulate karyotype by electronic chromosome painting (E-painting). Chromosome Res 14: 899–907 (2006). Kirsch IR, Green ED, Yonescu R, Strausberg R, Carter N, et al: A systematic, high-resolution linkage of the cytogenetic and physical maps of the human genome. Nat Genet 24:339–340 (2000). Langer PR, Waldrop AA, Ward DC: Enzymatic synthesis of biotin-labeled polynucleotides: Novel nucleic acid affinity probes. Proc Natl Acad Sci USA 78:6633–6637 (1981). Liehr T, Starke H, Heller A, Kosyakova N, Mrasek K, et al: Multicolor fluorescence in situ hybridization (FISH) applied to FISHbanding. Cytogenet Genome Res 114: 240– 244 (2006). Liu Y, Ye J, Fu B, Ng BL, Wang J, et al: Molecular cytogenetic characterization of the genome organization of the 6-banded armadillo (Euphractus sexcinctus). Cytogenet Genome Res 132:31–40 (2011). Ma J, Zhang L, Suh BB, Raney BJ, Burhans RC, et al: Reconstructing contiguous regions of an ancestral genome. Genome Res 16: 1557– 1565 (2006). Masabanda JS, Burt DW, O’Brien PC, Vignal A, Fillon V, et al: Molecular cytogenetic definition of the chicken genome: the first complete avian karyotype. Genetics 166: 1367– 1373 (2004).


95

Matthey R: The chromosome formulae of eutherian mammals, in Chiarelli B, Cappanna E (eds): Cytotaxonomy and Vertebrate Evolution, pp 531–553 (Academic Press, London 1972). Meader S, Ponting CP, Lunter G: Massive turnover of functional sequence in human and other mammalian genomes. Genome Res 20: 1335–1343 (2010). Mikkelsen TS, Wakefield MJ, Aken B, Amemiya CT, Chang JL, et al: Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences. Nature 447: 167–177 (2007). Müller S, Wienberg J: ‘Bar-coding’ primate chromosomes: molecular cytogenetic screening for the ancestral hominoid karyotype. Hum Genet 109:85–94 (2001). Müller S, Stanyon R, Finelli P, Archidiacono N, Wienberg J: Molecular cytogenetic dissection of human chromosomes 3 and 21 evolution. Proc Natl Acad Sci USA 97: 206–211 (2000). Murphy WJ, Stanyon R, O’Brien SJ: Evolution of mammalian genome organization inferred from comparative gene mapping. Genome Biol 2: 1–5 (2001). Murphy WJ, Frönicke L, O’Brien SJ, Stanyon R: The origin of human chromosome 1 and its homologs in placental mammals. Genome Res 13:1880–1888 (2003). Murphy WJ, Larkin DM, Everts-van der Wind A, Bourque G, Tesler G, et al: Dynamics of mammalian chromosome evolution inferred from multispecies comparative maps. Science 309:613–617 (2005). Nie W, O’Brien PC, Ng BL, Fu B, Volobouev V, et al: Avian comparative genomics: reciprocal chromosome painting between domestic chicken (Gallus gallus) and the stone curlew (Burhinus oedicnemus, Charadriiformes) – an atypical species with low diploid number. Chromosome Res 17: 99–113 (2009). O’Brien SJ: The platypus genome unraveled. Cell 133:953–955 (2008). O’Brien SJ, Nash WG: Genetic mapping in mammals: chromosome map of domestic cat. Science 216:257–265 (1982). Picone B, Masters J, Silvestro D, Sineo L, Del Pero M: A phylogenetic analysis of human syntenies revealed by chromosome painting in euarchontoglires orders. J Mamm Evol 18: 131–146 (2011). Pinkel D, Landegent J, Collins C, Fuscoe J, Segraves R, et al: Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc Natl Acad Sci USA 85:9138–9142 (1988). Pokorna M, Giovannotti M, Kratochvil L, Kasai F, Trifonov VA, et al: Strong conservation of the bird Z chromosome in reptilian genomes is revealed by comparative painting despite 275 million years divergence. Chromosoma 120:455–468 (2010). Pokorna M, Giovannotti M, Kratochvil L, Caputo V, Olmo E, et al: Conservation of chro-

96

mosomes syntenic with avian autosomes in squamate reptiles revealed by comparative chromosome painting. Chromosoma (2012), Epub ahead of print. Rabbitts P, Impey H, Heppell-Parton A, Langford C, Tease C, et al: Chromosome specific paints from a high resolution flow karyotype of the mouse. Nat Genet 9:369–375 (1995). Rens W, O’Brien PC, Yang F, Graves JA, Ferguson-Smith MA: Karyotype relationships between four distantly related marsupials revealed by reciprocal chromosome painting. Chromosome Res 7:461–474 (1999). Rens W, O’Brien PC, Yang F, Solanky N, Perelman P, et al: Karyotype relationships between distantly related marsupials from South America and Australia. Chromosome Res 9:301–308 (2001). Rens W, O’Brien PC, Fairclough H, Harman L, Graves JA, Ferguson-Smith MA: Reversal and convergence in marsupial chromosome evolution. Cytogenet Genome Res 102: 282– 290 (2003). Rens W, Grutzner F, O’Brien P C, Fairclough H, Graves JA, Ferguson-Smith MA: Resolution and evolution of the duck-billed platypus karyotype with an X1Y1X 2Y2X 3Y3X4Y4 X 5Y5 male sex chromosome constitution. Proc Natl Acad Sci USA 101:16257–16261 (2004). Rens W, O’Brien PC, Grutzner F, Clarke O, Graphodatskaya D, et al: The multiple sex chromosomes of platypus and echidna are not completely identical and several share homology with the avian Z. Genome Biol 8:R243 (2007). Richard F, Lombard M, Dutrillaux B: Reconstruction of the ancestral karyotype of eutherian mammals. Chromosome Res 11: 605–618 (2003). Rocchi M, Archidiacono N, Stanyon R: Ancestral genomes reconstruction: an integrated, multi-disciplinary approach is needed. Genome Res 16:1441–1444 (2006). Rocchi M, Archidiacono N, Schempp W, Capozzi O, Stanyon R: Centromere repositioning in mammals. Heredity 108:59–67 (2012). Ruiz-Herrera A, Farre M, Robinson TJ: Molecular cytogenetic and genomic insights into chromosomal evolution. Heredity 108:28–36 (2012). Scherthan H, Cremer T, Arnason U, Weier HU, Lima-de-Faria A, Fronicke L: Comparative chromosome painting discloses homologous segments in distantly related mammals. Nat Genet 6:342–347 (1994). Schrock E, du Manoir S, Veldman T, Schoell B, Wienberg J, et al: Multicolor spectral karyotyping of human chromosomes. Science 273: 494–497 (1996). Speicher MR, Gwyn Ballard S, Ward DC: Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet 12:368–375 (1996). Stock AD, Hsu TC: Evolutionary conservatism in arrangement of genetic material. A comparative analysis of chromosome banding between the rhesus macaque (2n equals 42,


84 arms) and the African green monkey (2n equals 60, 120 arms). Chromosoma 43: 211– 224 (1973). Svartman M, Stone G, Page JE, Stanyon R: A chromosome painting test of the basal eutherian karyotype. Chromosome Res 12: 45–53 (2004). Telenius H, Pelmear AH, Tunnacliffe A, Carter NP, Behmel A, et al: Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes Chromosomes Cancer 4: 257–263 (1992). Trifonov VA, Giovannotti M, O’Brien PC, Wallduck M, Lovell F, et al: Chromosomal evolution in Gekkonidae. I. Chromosome painting between Gekko and Hemidactylus species reveals phylogenetic relationships within the group. Chromosome Res 19:843– 855 (2011). Veyrunes F, Waters PD, Miethke P, Rens W, McMillan D, et al: Bird-like sex chromosomes of platypus imply recent origin of mammal sex chromosomes. Genome Res 18: 965–973 (2008). Wang C, Deakin JE, Rens W, Zenger KR, Belov K, et al: A first-generation integrated tammar wallaby map and its use in creating a tammar wallaby first-generation virtual genome map. BMC Genomics 12: 422 (2011). Warren WC, Hillier LW, Marshall Graves JA, Birney E, Ponting CP, et al: Genome analysis of the platypus reveals unique signatures of evolution. Nature 453:175–183 (2008). Wienberg J, Stanyon R: Chromosome painting in mammals as an approach to comparative genomics. Curr Opin Genet Dev 5: 792–797 (1995). Wienberg J, Stanyon R: Comparative painting of mammalian chromosomes. Curr Opin Genet Dev 7:784–791 (1997). Wienberg J, Stanyon R: Comparative chromosome painting of primate genomes. Ilar J 39: 77–91 (1998). Wienberg J, Jauch A, Stanyon R, Cremer T: Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization. Genomics 8:347–350 (1990). Wienberg J, Stanyon R, Jauch A, Cremer T: Homologies in human and Macaca fuscata chromosomes revealed by in situ suppression hybridization with human chromosome specific DNA libraries. Chromosoma 101: 265–270 (1992). Yang F, Alkalaeva EZ, Perelman PL, Pardini AT, Harrison WR, et al: Reciprocal chromosome painting among human, aardvark, and elephant (superorder Afrotheria) reveals the likely eutherian ancestral karyotype. Proc Natl Acad Sci USA 100:1062–1066 (2003). Yang F, Graphodatsky AS, Li T, Fu B, Dobigny G, et al: Comparative genome maps of the pangolin, hedgehog, sloth, anteater and human revealed by cross-species chromosome painting: further insight into the ancestral karyotype and genome evolution of eutherian mammals. Chromosome Res 14: 283– 296 (2006).

Graphodatsky /Ferguson-Smith /Stanyon

Published online: May 23, 2012


Genome Size Evolution: Sizing Mammalian Genomes C.A. Redi a E. Capanna b a b

Fondazione IRCCS Policlinico San Matteo, Dipartimento di Biologia e Biotecnologie ‘Lazzaro Spallanzani’, Pavia, e Dipartimento di Biologia e Biotecnologie ‘Charles Darwin’, Università di Roma ‘La Sapienza’, Roma, Italia

Key Words Evolution ⴢ Genome ⴢ Genome size ⴢ Mammals ⴢ Mobile elements ⴢ Transposons

Abstract The study of genome size (GS) and its variation is so fascinating to the scientific community because it constitutes the link between the present-day analytical and molecular studies of the genome and the old trunk of the holistic and synthetic view of the genome. The GS of several taxa vary over a broad range and do not correlate with the complexity of the organisms (the C-value paradox). However, the biology of transposable elements has let us reach a satisfactory view of the molecular mechanisms that give rise to GS variation and novelties, providing a less perplexing view of the significance of the GS (C-enigma). The knowledge of the composition and structure of a genome is a pre-requisite for trying to understand the evolution of the main genome signature: its size. The radiation of mammals provides an approximately 180-million-year test case for theories of how GS evolves. It has been found from data-mining GS databases that GS is a useful cyto-taxonomical instrument at the level of orders/superorders, providing genomic signatures characterizing Monotremata, Marsupialia, Afrotheria, Xenarthra,



Laurasiatheria, and Euarchontoglires. A hypothetical ancestral mammalian-like GS of 2.9–3.7 pg has been suggested. This value appears compatible with the average values calculated for the high systematic levels of the extant Monotremata (⬃2.97 pg) and Marsupialia (⬃4.07 pg), suggesting invasion of mobile DNA elements concurrently with the separation of the older clades of Afrotheria (⬃5.5 pg) and Xenarthra (⬃4.5 pg) with larger GS, leaving the Euarchontoglires (⬃3.4 pg) and Laurasiatheria (⬃2.8 pg) genomes with fewer transposable elements. However, the paucity of GS data (546 mammalian species sized from 5,488 living species) for species, genera, and families calls for caution. Considering that mammalian species may be vanished even before they are known, GS data are sorely needed to phenotype the effects brought about by their variation and to validate any hypotheses on GS evolution in mammals. Copyright © 2012 S. Karger AG, Basel

For more than 50 years, we have been collecting data on genome size (GS) and have discovered that the genomes of eukaryotic organisms vary over 200,000-fold in size. Over this period, we have built up an impressive dataset now available for data-mining, raising new opportunities in genome studies. Sequencing, comparative and Carlo Alberto Redi Dipartimento di Biologia e Biotecnologie ‘Lazzaro Spallanzani’ Via A. Ferrata, 9 IT–27100 Pavia (Italy) Tel. +39 0382 986 306, E-Mail carloalberto.redi @ unipv.it

engineering genomes studies, to mention only a few, will all benefit immensely from precise knowledge of the GS. More generally, new avenues are opening up to expand our understanding of the biological meaning of the GS. The diversity of the possible applications derived from this knowledge should lead to cutting-edge solutions covering important unmet needs for the benefit of human health and animal reproduction.

Historical Perspective

It is impossible to avoid the beginning of a paper devoted to the evolution of mammalian GS without the canonical sentence: ‘The GS varies dramatically in any plant and zoological taxa’. This is the first firm conclusion one can reach from looking at GS variations at any hierarchical level. It is also impossible not to anticipate that we still know little about the fundamental principles governing this variability, how it arises, and how it becomes established. We acknowledge that GS evolution is a much more complex phenomenon than people tend to imagine when relating a GS value to the phenotype of a certain animal. Many renowned colleagues have studied GS evolution in detail, sometimes reaching different and contrasting views; however, thanks to their work we currently conceive GS evolution as due to the action of a plethora of factors acting on the composition and organization of a genome. In other words, the size of a genome should be looked at as the result of the interplay of genetic (i.e. intrinsic) and environmental (i.e. extrinsic) factors. These factors probably act together in a complex network of relationships in which each is connected simultaneously to several other factors by feedback and forward and backward effects which ultimately lead to the shaping of a GS.

Terminology and Picograms

The terminology we now use has emerged from the confused misuse of terms up to a few years ago. The word genome was coined in 1920 by Hans Winkler while he was studying parthenogenesis in animals and plants. He defined ‘Genom’ as the haploid set of chromosomes. Today, the term is used in 2 distinct ways to indicate either the total number of genes or the whole amount of nuclear DNA. Considering both Winkler’s definition and the fact that in almost all organisms only a small fraction of DNA comprises coding genes, the definition of the genome as 98


the total amount of DNA present in mature germ cells seems plausible. The term ‘genome size’ was first used by Hinegardner [1976] in the title of his paper ‘Evolution of Genome Size’ in which, according to Greilhuber et al. [2005], ‘it was probably intended to denote the mass or quantity of DNA in a non-replicated haploid genome (e.g. in fish sperm nuclei)’. When speaking of GS, the historically based consensus on the meaning of GS should be clear: the total amount of a mature germ cell’s DNA. The ‘C’ terminology, introduced by Hewson Swift in 1950 in an unfortunately ambiguous manner (since he used C to refer to class, category, content, or constant in his first paper on animal nuclei written for his PhD dissertation), has been resolved thanks to a personal communication by H. Swift himself to M.D. Bennett [Greilhuber et al., 2005]: the term ‘C-value’ was intended to mean ‘constant’ (i.e. the amount of DNA that was characteristic of a particular genotype) and it was defined as ‘DNA content of the unreplicated haploid chromosome complement’ [Bennett and Leitch, 2005]. That genomes vary in size was appreciated by the pioneers of microphotometry and prompted the formulation of the C-value paradox [Thomas, 1971; Cavalier-Smith, 1985]. The most popular idea is that GS is not related to either morphological complexity or to gene numbers even though van Nimwegen [2003] suggested scaling laws in the functional content of genomes in relation to the design of organisms. The identification of the contribution of repetitive sequences to GS variation began to provide a partial resolution of the C-value paradox. Thus, in 2003, the scientific community agreed to prefer the use of the term ‘C-value enigma’ [Gregory, 2001] during a discussion at the Royal Botanic Gardens in Kew (UK), the site of the historical Kew chromosome meetings. In fact, the C-value enigma conceptually better encapsulates all the relevant questions related to GS variations. These questions include: what is the type of noncoding DNA that gives different genomes different sizes? What is the origin and the intra- and intergenomic transfer of this noncoding DNA? What, if any, are the functions (at any hierarchical organization level) that these DNA sequences provide? Early cytogeneticists quantified genomes by analyzing the partition in chromosomes that were counted in cytological preparations. The development of microphotometry allowed DNA measurements throughout the cell cycle so that Swift [1950] showed that Feulgen DNA in corn nuclei was distributed in 1C, 2C, 4C, 8C, and 16C levels. He observed the 1C DNA content only at the end of meiosis. Thus, to refer to the whole chromosome complement with a chromosome number, irrespective of the degree of Redi /Capanna

generative polyploidy, and to eliminate ambiguities in reporting DNA contents, Bennett’s student, Johann Greilhuber, proposed an extended GS terminology [Greilhuber et al., 2005, 2007; Greilhuber and Doležel, 2009] including the term ‘holoploid genome’ and the full term ‘holoploid genome size’ to indicate its size, with the Cvalue being its abbreviation. Meanwhile, other studies [Hardie et al., 2002; Doležel et al., 2003; Greilhuber and Doležel, 2009; Doležel and Greilhuber, 2010] determined that the correct relationship between the number of base pairs and the DNA mass in picograms could be derived from the formula: number of base pairs = mass in pg ! 0.978 ! 109. In other words, 1 pg of DNA = 978 Mb.

data from the whole genomes of over 1,000 organisms (http://www.ncbi.nlm.nih.gov/genome). (k) The UCSC Genome Browser website contains the reference sequences and working draft assemblies for a large collection of genomes (http://genome.ucsc.edu/). (l) VEGA, the Vertebrate Genome Annotation browser, focuses on human, mouse, and zebrafish (http://www. sanger.ac.uk/resources/databases/vega/).

Sizing Mammalian Genomes

Several web sites host open access databases on, or provide links to, animal (including mammals) genome resources: (a) The Animal Genome Size Database offers C-values for about 1,300 invertebrates and 2,500 vertebrates (www. genomesize.com). (b) The DBA Mammalian Genome Size Database has 237 data sets (www.unipv.it/webbio/dbagsdb.htm). (c) The DOE Joint Genome Institute – Consortium of U.S. Department of Energy researchers develops and exploits new technologies as a means for discovering and characterizing the basic principles and relationships underlying living systems (http://www.jgi.doe.gov/index. html). (d) DOGS, the Database of Genome Sizes, covers 301 organisms (http://www.cbs.dtu.dk/databases/DOGS/). (e) EBI, the European Bioinformatics Institute, gives access to completed genomes and genome shotgun sequences (www.ebi.ac.uk/genomes). (f) The Ensembl project produces genome databases for vertebrates and other eukaryotic species (http://www. ensembl.org/index.html). (g) GOLD, the Genomes Online Database, represents a web resource for genome projects worldwide (www. genomesonline.org). (h) JGI, the Joint Genome Institute, offers a genome portal to download sequences (www.jgi.doe.gov). (i) KEGG, the Kyoto Encyclopedia of Genes and Genomes, provides (among other things) a database about genome projects with 243 entries (www.genome.ad.jp/ kegg). (j) NCBI, the National Center for Biotechnology Information, provides (among other things) sequence and map

Although ‘Evolution of Genome Size in Mammals’ had a nice ring about it as a title, ‘Sizing Mammalian Genomes’ has been chosen as more appropriate for what we are dealing with, since we still have a great need to measure GS in order to draw conclusions on its evolution. Knowledge about the extent of GS conservation and divergence will help unravel the genetic and evolutionary mechanisms that shape genomes and may reveal the processes that have conditioned the great success of mammalian evolution. However, extreme caution should be exercised when inferring evolutionary pathways from GS, trying to find an association between GS variation and features shared by mammals at any level of hierarchical organization. This caution is imposed by the simple fact that of the 5,488 species of mammals currently alive [Schipper et al., 2008; IUCN list http://www.iucnredlist. org/], detailed surveys of animal GS databases (see above) found the GS for just 546 species (January 31st, 2012). Moreover, GS data from the past can be counted on the fingers of one hand and have been roughly calculated from the cell volume measured in fossil bones. In other words, we have no data regarding the composition of ancient genomes (with just a very few exceptions, see below). This paucity of GS data from both extant and extinct species is one cause of the severe difficulty that we have in learning about the evolution of GS. In fact, we can try to infer and hypothesize trends in changing composition, organization, and sizes of genomes only on the basis of studies of living species and a handful of investigations on ancient DNA. The molecular analysis of those mammals whose genome has already been sequenced is being used to address the C-enigma, approaching a clarification that now seems less far away: the differential temporal (heterochronical) and spatial (heterotopical) expression of the 20,000– 30,000 genes shared from the lower invertebrates to the mammals can provide a framework for hypotheses forwarded to understand the formation of a nematode or a

Mammalian Genome Sizes


Genome Size Databases and Data-Mining

99

whale from these same coding genes. The remaining mass of DNA (the formerly named junk DNA) constitutes the part of the genome able to finely tune a specific phenotype to a specific ecological niche, making use of the common set of coding genes. Multiple copies of genes, pseudogenes, introns, duplications (of several magnitude), different quantities of ribosomal RNA genes and repeated DNA sequences with varying degrees of repetition (clustered or dispersed) all contribute to GS variation [Vinogradov, 2000]. As mentioned above, the intrinsic difficultly in determining the way(s) a GS evolves remains the fact that we must reason almost exclusively on the basis of GS values from extant mammals: we can only perform horizontal analyses (from extant GS) and not vertical ones (from extinct GS). To make a comparison, it is like studying the speciation process based only on existing, living species. We have no data on the GS of those species that precede im lauf der zeit the present day range of GS that we are dealing with. To try to get a picture of GS evolution, we need to study the mode and tempo of GS variation [Oliver et al., 2007]. We can hope that in the near future some useful data may be provided from sequencing projects of more ancient DNA, such as those that led to the sequencing of the Neanderthal [Green et al., 2010] and mammoth [Miller et al., 2008] genomes. At present, we have only a handful of ancient DNA data. On the other hand, a certain degree of encouragement can be derived from some very useful data regarding all those mammalian genomes that have been fully sequenced or are presently being sequenced. The mammalian genomes already sequenced and their phylogenomic distribution can be found at several websites. For example, GOLD (Genomes Online Database, http:// www.genomesonline.org/cgi-bin/GOLD/phylogenetic_ distribution.cgi) gives a clear picture of what has been done and what is presently underway (a total of 269 genomes, 144 of which are from primates, 34 from rodentia, etc.). These data are very valuable since they allow deconstruction and comparison of several genomes pertaining to different orders and families. Having said that, the reader will immediately understand that the reason we decided that we must entitle this section ‘Sizing Mammalian Genomes’ was to stress the urgent need to measure the GS of the greatest possible number of living mammals with the maximum accuracy, especially those currently being sequenced or about to be sequenced. The main project in this regard is probably one of the ‘Center-Initiated Projects’ of The Genome Institute at Washington University (http://genome.wustl.edu/ 100


collaborate/) being carried out and supervised by Kerstin Lindblad-Toh and Eric Lander of the Broad Institute (MIT and Harvard, USA). The aim of the project is ‘sequencing and analysis of ⬃200 additional mammals to identify functional constraint in the human genome at roughly single base resolution’. In addition to other mammals, it includes 2 primates, 9 non-primate Euarchontoglires (various rodents including chinchilla, hamster, beaver, and porcupine), 10 Laurasiatheria (including a number of bats, hippopotamus, rhinoceros, and seal), 4 Afrotheria (including aardvark and manatee), and 2 Xenarthra (including anteater). So far, as already mentioned, we have measured the GS of only 546 species. Identifying a relationship between GS size and phenotype diversity would probably have a tremendous impact on a broad range of fields of biomedical sciences, contributing to the re-foundation of present day medicine as evolutionary medicine [Nesse et al., 2010], a process that cannot be worked out irrespective of a detailed dissection of the composition of the genome, assigning each DNA component a functional role. This goes beyond being merely academic and theoretical, albeit fascinating, knowledge, since it will also provide practical benefits for human health, contribute to the concrete needs of biotechnology entrepreneurs to know the GS of certain species in order to refine their possible genome sequencing projects (which means saving money and time), and help international programs aimed at nature conservation [Vinogradov, 2004a]. It is worth noting that this is a perspective that takes into account the neverending debate over symmorphosis [Weibel et al., 1991] and allows diseases to be seen as distortions of the properly, evolutionary-selected structure/function relationships. Indeed, shedding light on the GS of the maximum possible number of mammalian species will have a tremendous impact. It will expand the profitable applications that can be driven by comparative genomics to identify the functional elements in the human genome, a powerful tool leading to cutting-edge solutions to important unmet clinical needs.

Mammalian Phylogenomics

The main divisions of existing mammals are the placental mammals (Eutheria), the marsupials (Metatheria), and the monotremes (Prototheria, such as the platypus). Great help in understanding the GS evolution of these mammals is being provided by the ever more detailed mammalian evolution tree [Murphy et al., 2001, 2004; Redi /Capanna

Graphodatsky et al., 2011; Helgen, 2011; Meredith et al., 2011] and sequencing genome projects (already completed or still running) for many mammals, including those for the mammoth [Miller et al., 2008] and the Neanderthal [Green et al., 2010]. There are sometimes apparent conflicts between different techniques (e.g. cytogenetic and bioinformatic techniques) used to reconstruct the ancestral genome [Froenicke et al., 2006], although these are likely to be resolved thanks to both an increased number of sized and sequenced genomes and the future integration of different data sets. Ohno, Tinbergen, Mayr Analysis of GS shows that 2 rounds of total genome doubling occurred in early vertebrate evolution, paving the way from fishes to mammals and leading to Susumo Ohno’s concept of evolution by gene duplications [Ohno, 1970, 1999]. However, at the same time, Ohno [1969] told us that, as far as regards GS variation, mammalian evolution ‘is not very interesting’, since the so very fascinating diversification of mammalian species occurred in spite of the lack of a ‘great’ GS variation accounting for this diversification. Interestingly, Mammalia is the class of animals with the least variable GS, since this varies by only 2 or 3 orders of magnitude [Bachmann, 1972; Manfredi Romanini, 1973; Pellicciari et al., 1982]. However, in order to continue to give credit to Ohno’s statement, we must agree that the 2 or 3 orders of magnitude variation is not great which would imply that we speculatively expect that GS variation should be of 4 (?) – 5 (?) – 6 (?!) orders of magnitude to be able to account, in our eyes, for mammalian diversification. In addition, the knowledge we have accumulated on the regulation of gene expression by transcription, post-transcription, translation, and post-translation mechanisms (noteworthy in the microRNA world) could theoretically account well for mammalian diversification [Taft and Mattick, 2004; Prud’homme et al., 2007; Slotkin and Martienssen, 2007] in the presence of only small variations in GS, thereby rendering it theoretically unnecessary to invoke a great GS variation. These arguments are of relevance since if a 2- or 3-fold variation is conceived as a great variation, then it means that the GS is not under strict control by natural selection; on the other hand, if we consider it a small variation, then we must envisage that the mammalian GS is under strict control by natural selection to justify its small variation. The risk is entering a tangled jam of biological concepts if we do not reason on higher numbers of GS (see above). Thus, we still need to size genomes if we want to know what mechanisms account for mamMammalian Genome Sizes

malian GS variation (whether small or big, it does not matter) and whether these mechanisms are causative or correlated to the diversification of species (the phenotypes of organisms) we observe in mammals. The ultimate understanding of the relevance of GS should become possible through detailed molecular dissections and functional analyses of the many different types of DNA sequences that build up a genome. This has recently been done across 33 mammalian genomes by Romiguier and co-workers [2010] who related the GC-content dynamics with species life-history traits and cytology (body mass and GS), finding significant effects consistent with the GC-biased gene conversion model. In doing this, since the Darwinian world leaves chemical signatures on GS and without going back to Aristotle’s material and final causes, we must frame our conceptual efforts within an updated version of the 4 questions Tinbergen asked back in 1963 (What is the mechanism? How did the mechanism develop? How has it given a selective advantage? What is its phylogeny?) in the light of Ernst Mayr’s [1974, 1993] distinction between proximate (how mechanisms work) and ultimate (evolutionary) explanations reconsidered and reconstructed [Ariew, 2003]. Surviving the Cretaceous The anatomical transition from the Mesozoic theriomorph reptiles to the several paraphyletic lineages of allotherian mammals, proliferating between the late Triassic until the end of the Mesozoic era [Luo and Wible, 2005; Luo, 2007; Price et al., 2007], is quite well recorded: (i) about 100–85 myr ago, there was the initial radiation of modern eutherian lineages, and (ii) the radiation of eutherian mammals in the early Tertiary age (65–60 myr ago) was not primarily of existing placental lineages but rather of now extinct groups; it is possible to trace the primary radiation of placental mammals back into the mid-Cretaceous, stressing that the most intriguing aspect of mammalian phylogeny is the inference that more than 40 lineages of living mammals survived from the Cretaceous to the present [Bininda-Emonds et al., 2007; Penny and Phillips, 2007] without any suppression by dinosaurs [Wilson et al., 2012]. The crucial macro-evolutionary events in the evolution of existing placental mammals occurred either before or after the Cretaceous-Tertiary boundary: the former events shaped the divergence between orders, the latter tailored the radiation of families [Meredith et al., 2011]. The phylogenetic split of contemporary Mesozoic mammals in the early-middle Jurassic was a critical transition accompanied by major ecomorphological diversification (notably scansorial adCytogenet Genome Res 2012;137:97–112

101

Fig. 1. The mean genome size (GS; pg of DNA 8 ␴) is a useful cytotaxonomical tool at the level of orders and superorders, providing genomic signatures characterizing Monotremata, Marsupialia, Afrotheria, Xenarthra, Laurasiatheria and Euarchontoglires. The figure has been redrawn from a publication by Redi et al. [2007].

aptation which made it possible for therians to exploit nocturnal and arboreal niches) which took multiple directions [Venditti et al., 2011] leading to the appearance of more of the derived mammalian clades. Highlighting the acquisition of morphological diversification, Venditti et al. [2011] reached the conclusion that ‘diversification seems to arise whenever, wherever, and at whatever rate it is advantageous’. In the post-Cretaceous-Tertiary boundary there was a widespread contraction in overall GS which occurred in multiple lineages of mammals, as suggested by the very limited numbers of very young, long terminal repeats [Rho et al., 2009]. Genomic Signatures The analysis of the available GS makes it possible to relate the size of genomes and their DNA compositions to the life-history traits that characterize the orders and superorders that had diverged by 75 myr ago (fig. 1). At the level of families and genera, the GS values show a scattered distribution pattern so that no relationships can be picked up; however, at the level of orders and superorders it seems that the GS is a useful cytotaxonomic tool, rep102


resenting an excellent genetic signature. The existence of survivors of that Mesozoic flourishing of new mammalian species, the Monotremata (GS ⬃2.97 pg), helps to clarify the phyletic relationships of the extant mammals with those of the extinct Allotheria [Luo et al., 2011]. Another paleogeographic circumstance makes mammals a very interesting group on which to perform GS molecular analyses: the evolutionary expansion of Metatheria and notably of Eutheria coincided with the supercontinent Pangaea crashing into the northern, Laurasia, and the southern, Gondwana, hemispheres. This fact led to evolutionary processes affecting old mammalian clades in isolated contexts. Recent molecular data have split the placental orders into 4 phylogenetic clades, namely Afrotheria, Xenarthra, Laurasiatheria, and Euarchontoglires; Afrotheria and Xenarthra occupy basal positions followed by the Boreoeutheria which embraces Euarchontoglires and Laurasiatheria [Madsen et al., 2001; Murphy et al., 2001; Delsuc et al., 2002; Murphy et al., 2004; Delsuc et al.,2005; Svartman et al., 2006]. This process, as it has been recognized in recent years, left genetic signatures specifically characterizing the 4 clades, the constitution Redi /Capanna

of retroposons [Kriegs et al., 2006] and the GS [Redi et al., 2005, 2007] (see fig. 1). The analysis of the presence/absence of retroposon insertions (i.e. the analysis of multiple long interspersed and long terminal repeat elements), which is virtually homoplasy-free, provides highly significant evidence for the monophyletic clades. A list of important rare genomic changes useful in placental mammal systematics is provided in table 1 of ‘Molecules Consolidate the Placental Mammal Tree’ by Springer et al. [2004], notably that of the AfroSINE [Nikaido et al., 2003] whose distribution supports the monophyly of Afrotheria. Afrotheria (the first of the 4 superorders of placental mammals to diverge from the rest) is the deepest node of eutherians; thus these data are open to data-mining for the reconstruction of the ancestral mammalian genome. The presently known GS attribute the largest genomes among all eutherian mammals to the few (7) Afrotheria whose genome has been sized [Redi et al., 2007], thus suggesting a shift toward smaller genomes during the transition of basal clades to the Boreoeutheria that conquered the Northern hemisphere. This suggestion requires extensive exploration of Afrotheria and Xenarthra genomes in the light of the possible opposite suggestion that the ancestral mammalian genome was in the range of 2.9–3.7 pg [Organ et al., 2011] (see below).

genomic scrap yard from which evolution may serve itself. Since the discovery of noncoding DNA and its impact on GS variation, the word ‘paradox’ has been replaced by ‘enigma’ in an attempt to identify the topic, more appropriately, as a ‘perplexing subject’ made up of several independent components [Gregory, 2005]. Although little is known about the evolutionary dynamics of the specific components that lead to GS variation, it is now generally agreed that the C-value enigma can be largely explained by the differential amplification and proliferation among organisms of the repetitive fraction of the genome, notably by retrotransposable elements.

The C-value of eukaryotic nuclei varies ⬃200,000fold, but there is only about a 20-fold variation in the number of protein-coding genes. Hence, most of the Cvalue variation is ascribed to the repetitive fraction, redundancy in genomes likely being the key to understanding GS evolution [Redi et al., 2001]. In the majority of mammals, protein-coding exons contribute merely 2% to the genome. The rest is composed of almost equal portions of repetitive DNA and unique sequences of mainly unknown function. The ubiquitous repetitive elements, cytologically detectable or not, account for varying Cvalues even among closely related taxa [Manfredi Romanini, 1973, 1985]. Despite earlier negative attributes, repetitive DNA is nowadays not regarded as useless. Indeed, these sequences provide an efficient mechanism for genomic shuffling. Several hypotheses have been proposed for possible intracellular molecular function(s) of redundant noncoding DNA (e.g. skeletal, buffering) grounding the ecophysiological cytogenetics topic [Vinogradov, 1997; Kozlowski et al., 2003]. Makalowski [2000] bridged the difference of opinion with his concept of a

Mobile Elements The retrotransposons are considered the most important factor of genomic inflation in both plants and animals because of their propensity to increase their copy number during transposition, a well documented phenomenon in grasses in which long terminal repeat (LTR)retrotransposons constitute more than half of the genome of some species [Kumar and Bennetzen, 1999; Schulman and Kalendar, 2005]. For example, in the span of just a few million years, the maize genome doubled in size due to transposable element (TE) activity, and at present about 70% of the maize nuclear genome is composed of LTR-retrotransposons [SanMiguel and Bennetzen, 1998]. Another striking example is the differential lineage-specific amplification of TEs which are responsible for the increase in Gossypium GS from 880–2,460 Mb [Hawkins et al., 2006]. TEs can be separated into 2 major classes, DNA transposons and retrotransposons, and are scattered throughout the genomes of many plants and animals where they can form a large proportion of the genome. The differential accumulation or deletion of TEs is the most common source of GS variation, and it is increasingly seen as the major originator of genetic changes which may or may not offer populations the chance to adapt and to change thus opening the scenario for possible evolution of the species. It has been established for many organisms that TEs are the main components of complex genomes and that transposition can be regarded as the predominant force driving their structural changes, besides polyploidy [Bennetzen et al., 2005; Vitte and Panaud, 2005]. With this evolutionary scenario in mind, the challenge is to trace the variation of GS accompanying mammalian phylogeny and the contribution of TEs to the GS variation. Considering that an essential fraction of 20,000–30,000 coding genes is necessary to ‘build’ a mammal, we have to search for a possible adaptive mean-



Mode and Tempo of Genome Size Variation

103

ing of variation of the GS (i.e. of the variation of GS due to the noncoding fraction of the genome). Interest in the role played by TEs increased further as drafts of the human genome became available in 2001 [Lander et al., 2001], revealing that around 45% of the DNA consists of TEs. But even then, their role in gene regulation was not fully recognized. TEs were frequently considered junk DNA evolving as retroviruses; nowadays, they are considered driving forces of genome evolution and thus of GS evolution and are acknowledged as a main component of most genomes: McClintock’s ideas that they can control gene activity are fully accepted, and there is general consensus within the scientific community that transposons are an important creative force [Wagner and Lynch, 2010] and have roles in the evolution of mammalian gene regulation [Feschotte, 2008]. Efforts are now being made to identify all the possible functional elements, as in the ENCODE (Encyclopedia of DNA Elements, http:// genome.ucsc.edu/ENCODE/) project. In addition, TEs are prone to horizontal transfer which results in invasion of new genomes in which they can increase their copy number. Thus, understanding TE biology or, in other words, discovering the mechanisms by which TEs move around, accumulate in and are lost from the genomes is the key to understanding the evolution of GS. TEs moving around and transferring horizontally from one genome to another can disrupt genes, induce genomic rearrangements, influence gene expression, reshape genetic circuitries, and mobilize various types of non-autonomous sequences. Mobile elements provide genetic material to generate new regulatory functions [Prud’homme et al., 2007; Slotkin and Martienssen, 2007] which can be advantageous to the host (exaptation). Making use of 29 mammal genomes, an international team lead by Kerstin Lindblad-Toh [Lindblad-Toh et al., 2011] detected approximately 280,000 mobile element exaptations which occurred during the ⬃90 myr of the eutherian radiation from marsupials. They estimated that 100–200 additional eutherian mammals should provide an increased resolution to enable single-nucleotide resolution analysis. Interestingly, the vast majority of these exaptations are present within the Laurasiatheria and Euarchontoglires branches which have smaller genomes than Afrotheria and Xenarthra (see below). Short and Long Interspersed Elements Retrotransposable elements are major constituents of genomes, and comparative mammalian genomics has revealed the existence of a large number of non-proteincoding genomic regions. The current model of eukary104


otic genome evolution in relation to the activity of TEs postulates that GS should result from 2 balanced forces: an increase as a result of retrotransposition and a decrease caused by recombinations and deletions [Petrov, 2002a]. GS variability can largely be accounted for by these latter processes. For example, in the human GS (3.5 pg), of which nearly 45% are TEs [Lander et al., 2001], the 2 major classes of retroposons are the non-LTR short interspersed elements (SINEs) and long interspersed elements (LINEs) which account for 12% and 17%, respectively, of the genome [Cordaux and Batzer, 2009]. SINEs emerged in evolution many times: at least 23 primary SINE families independently appeared in the evolution of placental mammals, and so far 51 mammalian SINE families have been described [Kramerov and Vassetzky, 2011]. The mammalian tree constructed by Kramerov and Vassetzky [2011] using the de novo emergence of SINEs in placental mammals fully overlaps the mammalian time tree produced by Hedges and co-workers [2006]. Some of these loci have been derived from a newly defined family of ancient SINEs called AmnSINE1 (Amniota SINE1), because they were found to be present in mammals as well as in birds [Nishihara et al., 2006; Hirakawa et al., 2009]. The long interspersed nucleotide element LINE-1 is an autonomous retroelement that makes up about 17% of the human genome. LINE-1 can jump to chromosomes with broken DNA strands and then slip into it and repair the damage [Morrish et al., 2002; 2007]. LINE-1 and other non-LTR retrotransposon elements (such as Alu and SVA) have proliferated over the past 80 million years of primate evolution, now accounting for something like 30% of the human genome: L1 and Alu elements alone have contributed ⬃750 Mb to the human genome [Lander et al., 2001]. Several other comparisons of fully sequenced genomes highlight the role played by TEs in mammalian genome evolution. It appears quite clear that TEs constitute from at least 30% to more than half of the sequences of mammalian genomes [Lander et al., 2001; Han et al., 2007; Mikkelsen et al., 2007; Pontius et al., 2007]. Recently published genomes of 3 early diverged mammals, the platypus (Ornithorhynchus anatinus) [O’Brien, 2008; Warren et al., 2008], the short-tailed opossum (Monodelphis domestica) [Mikkelsen et al., 2007; Samollow, 2008], and the tammar wallaby (Macropus eugenii) [Renfree et al., 2011] allow us to get a scent of the mode/tempo of the TE relationship in a well time-frameworked specific example of the evolution process. The platypus genome is composed of a large proportion (⬃50%) of interspersed repeats [O’Brien, 2008; Warren et al., 2008] mostly represented Redi /Capanna

by the still active LINE-2 and its non-autonomous companion, the mammalian-wide interspersed repeat (MIR). The platypus genome contains fewer non-protein-coding RNAs than do other mammalian species with a unique class of over 40,000 noncoding RNAs known as snoRTE. Probably, its genome was never invaded by TEs because it lacks the L1-mediated retrotransposition mechanism of the therians [Warren et al., 2008]. In other words, the bona fide small platypus genome is not reduced in size from a bigger common ancestral mammalian genome of ⬃170 myr ago but probably never expanded as a consequence of the extensive retrotransposition of non-protein-coding RNAs that occurred in the metatherian and eutherian lineages starting ⬃150 myr ago. Thus, the platypus GS supports the paraphyletic condition of this animal with respect to the present-day marsupials and placentals. The opossum genome contains a higher proportion of TEs (⬃52%), compared with the ⬃44% and ⬃38% in human and mouse genomes, respectively [Gentles et al., 2007]. Furthermore, compared with the genomes of other mammals, the opossum genome is significantly enriched in non-LTR elements from the L1, CR1, and RTE families. The total proportion of repetitive sequence in the tammar was found to be 52.8%. This is similar to the repeat content of the opossum genome (52.2%). The proportions of LINEs and SINEs in the opossum and tammar were also similar; however, the overall content of LTR elements was significantly below that observed for any other mammal (only 3.91%) with the exception of the platypus (about 0.47%). The small size of the tammar genome (averaged at 2.9 Gb since 3 approaches produced quite different values) could be questioned since the GS of all other Macropus species is considerably larger than that of humans, as is that of all other marsupials [Garagna and Formenti, 1981]; it could, however, be explained by the greatly reduced number and size of centromeres. Genome Size Evolution in Mammals After the separation of Gondwana into South America and Africa-Australia (⬃80 myr ago), the South American and Australian marsupials followed independent evolutionary pathways [Luo et al., 2003]. The paleobiogeographic patterns of retroposon insertion suggest a single marsupial migration from South America to Australia assigning the South American opossums (Didelphimorphia) the position of the first branch of the marsupial tree [Nilsson et al., 2010]. Both groups have retained a genome larger than that of the modal mammalian values [Garagna and Formeni, 1981]; it is therefore tempting to spec-

ulate a parallelism with the bigger GS of the Southern hemisphere placental clades (Afrotheria and Xenarthra) versus those of the Northern hemisphere clades [Redi et al., 2005, 2007]. Apart from the debate [Petrov, 2002a; Vinogradov, 2004b] concerning the biological meaning of the GS, we already suggested a possible functional interpretation for the larger genomes in the Southern hemisphere clades, especially Afrotheria. There are numerous data (both from small taxonomic groups and across a whole class) indicating that the body mass-corrected metabolic rate of homeotherms decreases with latitude, i.e. there is a positive association [Lovegrove, 2000]. The general reason is straightforward: there is no need for additional body warming, which is necessary at higher latitudes (but there are also other causes in separate cases, e.g. the shortage of water). At the same time, there is a negative correlation between body mass-corrected metabolic rate and GS [Vinogradov, 1995]. Taken together, these 2 phenomena could provide a functional explanation for the GS signatures of superorders. Thus, the molecular genetic signatures provided by the GS of marsupials, Afrotheria and Xenarthra suggest against a shortfuse ‘burst’ model to explain modern-style mammalian evolution. In other words, they seem to indicate that the mass extinction at the end of the Cretaceous had little discernible effect on mammalian evolution. Alternatively, they provide additional molecular evidence supporting the molecular phylogenetic hypothesis that the present-day superorders were established long before. It has been suggested that organisms may have a ‘oneway ticket to genomic obesity’ [Bennetzen and Kellogg, 1997] because of several other mechanisms responsible for expansion of GS [Dufresne and Jeffery, 2011]. These include variation in intron size, expansion of tandemly repetitive DNA sequences, segmental duplication, accumulation of pseudogenes, transfer of organellar DNA to the nucleus and, of course, polyploidy. However, just one controversial case of tetraploidy has been reported in mammals, that of the South American red vizcacha rat Tympanoctomys barrerae (Rodentia, Octodontidae). Gallardo and co-workers [1999; 2006] described this as a case of allotetraploidy, while Svartman and co-workers [2005], on the basis of classical and molecular cytogenetic evidence, discarded polyploidy and suggested amplification and dispersion of repetitive sequences as the best explanation for the large GS of this mammal. It is possible that differences in GS are not only the outcome of an organism’s tolerance for accrual of non-genic DNA, but also a consequence of its efficiency at removing non-essential DNA [Petrov et al., 2000; Petrov, 2002b]. Many organisms



105

with a small genome are striking for their relatively small proportion of non-genic DNA. Evidence of a deletional bias among organisms with smaller versus larger genomes led to the ‘mutational equilibrium model’ of DNA loss [Petrov, 2002a]. Other suggested mechanisms of DNA loss include unequal intrastrand homologous recombination between 2 tandem repeats in the same orientation, such as the LTRs of retrotransposable elements, illegitimate recombination, and double-stranded break repair. The dynamic interactions between mobile elements and the host genome have been presented by several researchers [Hedges and Batzer, 2005; Kriegs et al., 2006; Belancio et al., 2008; Venner et al., 2009; Agren and Wright, 2011; Levin and Moran, 2011, to mention a few] showing how transposons are able to shape genome evolution. An interesting close examination of the mechanisms that underlie the appearance and disappearance of microsatellite DNA sequences in a genome (the human) was carried out by Kelkar and co-workers [2011]. A fascinating mathematical modeling of GS evolution was conducted by Li and Zhang [2010] who argued convincingly that at the macroevolutionary level the Cambrian explosion was triggered by intrinsic properties of the primordial genome (something that has to be seen within the Tinbergen-Mayr conceptual framework), the ‘intrinsic reason of genome evolution determined the Cambrian explosion’ as they wrote. The authors generated an empirical formula to calculate GS evolution (taking into account both the coding/noncoding fractions of a genome and the protein lengths) from which they claim ‘transition of biological complexity essentially corresponds to a critical turning point in genome size evolution’. Based on this, they invented a ‘C-value clock’ [see also Sharov, 2006] representing the trend in GS evolution (not the raw GS of a species) able to predict the time of the Cambrian explosion which has ‘a profound relationship’ with the C-value enigma [Li and Zhang, 2010]. Interestingly, using paleogenomic data, Organ and co-workers [2011] calculated a mammalian-like GS for the ancestral amniote (average haploid GS of 2.9–3.7 pg), suggesting that GS had ‘no directional change within the synapsid lineage leading to mammals’. The hypothetical ancestral GS of 2.9–3.7 pg appears compatible with the average values for the high systematic levels of the Monotremata (⬃2.97 pg) and the Marsupialia (⬃4.07 pg), suggesting TE invasion concurrently with the separation of the older clades of Afrotheria (⬃5.5 pg) and Xenarthra (⬃4.5 pg) with larger GS, leaving the Euarchontoglires (⬃3.4 pg) and Laurasiatheria (⬃2.8 pg) relatively free of TEs.

106


However, there is clearly a need for both better homogeneity in GS evaluation and high-coverage sequencing [Milinkovitch et al., 2010] before firm conclusions on GS evolution can be reached, since GS estimates are frequently discordant (the need for the DNA stoichiometry) and genome sequencing is often done at low coverage. Thus, despite a large body of data, it is clear that there are still great difficulties in differentiating artifacts from true changes in modes and tempo of genome evolution [Cavalier-Smith, 2005; Oliver et al., 2007].

Extinct (Neanderthal, Mammoths) versus Extant Placental Genome Size

Thanks to the recent acquisition of the genome sequences of the chimpanzee, rhesus macaque, and orangutan, it has been possible to reconstruct the dynamics and the timing of the non-LTR retrotransposition-driven genomic expansions in the human lineage. It seems that the scale and complexity of the past and current amplification rates of TEs have not been uniform over time: (i) DNA transposons, which constitute about 3% of the human genome, were active during early primate evolution until ⬃37 myr ago [Pace and Feschotte, 2007] but not in the macaque genome [Han, 2007]; (ii) the amplification of L1 families occurred over the past ⬃70 myr, with the most prolific amplification of L1 subfamilies occurring 12–40 myr ago [Khan et al., 2006]; (iii) the majority of Alu elements were inserted ⬃40 myr ago, coincidentally with a burst of amplification with approximately one new Alu insertion in every birth [Shen et al., 1991]. An obvious consequence of the accumulation of TE copy numbers is the contribution of these elements to GS variation [Liu et al., 2003]. As the human genome has accumulated ⬃2,000 L1, ⬃7,000 Alu, and ⬃1,000 SVA copies over the past ⬃6 myr of human evolution [Chimpanzee Sequencing and Analysis Consortium, 2005], a change in GS of 18 Mb can be calculated with an increase of ⬃0.01 pg of DNA in the same ⬃6 myr mainly due to the Alu sequences. This is extremely interesting when comparing the chimpanzee and human genomes with the slowly evolving orangutan genome (as shown by a lower rate of gene family turnover, less segmental duplication, and fewer rearrangements) in which there are only quiescent Alu repeats [Locke et al., 2011]. This fact could explain why the orangutan genome is not larger than that of other primates, as could be expected on the basis of the orangutan’s extremely low energy usage (for a eutherian mammal) [Pontzer et al., 2010] and small sized communities. Redi /Capanna

Hominids Thus, it seems reasonable to assume that hominid genome evolution (and, more generally, mammalian genome evolution) was probably related, among other factors, to the effect of TEs being able to create a variety of genomic novelties including exon shuffling, the formation of new genes and promoters, RNA-editing-mediated exonization, etc., on the genomic functioning leading to the rewiring of gene networks. All of these TE-related phenomena probably contributed to genetic innovation much more than the GS variation due to amplification per se. In support of the view that retrotransposons had a crucial role in accelerating the evolution of mammalian genomes is the fact that the sequences of these retrotransposons can undergo large-scale modification through the antiviral mechanism of DNA editing [Carmi et al., 2011]. The recent acquisition of the draft genome sequence of the sister group of all present-day humans, the Neanderthal [Green et al., 2010], supports this view. Many of the 111 Neanderthal-specific segmental duplications (average size 22,321 bp and total length 1,862 kb) show increased copy number in humans without showing any significant changes in GS. On the other hand, Somel and co-workers [2011] have identified miR-92a, miR-454, and miR-320b as possible regulators of humanspecific neural development thus supporting the idea that microRNAs developmentally regulate the expression of a few key genes that may have been a major driving force of the rapid evolution of the human brain (particularly of the prefrontal cortex) compared to the chimpanzee brain. Developmental remodeling in the human brain could possibly have been due to de novo LINE-1 retrotransposition events, since LINE-1 elements are active in the human brain [Beck et al., 2010] and randomly add extra ‘jumping’ copies to the neuron genome [Coufal et al., 2009] thus altering the genetic landscape of the human brain [Baillie et al., 2011] by post-transcriptional control of neuronal differentiation and contributing to neural plasticity. This last phenomenon could have been the major driving force triggering the development of hominid lineages rather than the raw GS variation between humans and primates. Mammoth The study of ancient DNA allows fruitful comparisons for highlighting the contribution of several DNA classes to GS variation and evolution since the phylogenetic relationships between the extinct and the existing species of the same genus are usually well known. Fortunately, DNA has very recently been successfully extracted from Mammalian Genome Sizes

bones of other extinct animals and then sequenced. These valuable studies recovered GS data from the past, notably the data for the woolly mammoth (Mammuthus primigenius) already extinct by about 10,000 years ago. Mammoths belong to Afrotheria, a group of mammals exhibiting extreme morphological diversity and with large genomes [Redi et al., 2007]. Recent paleogenomic studies in which genetic information was successfully recovered from both the mitochondrial and nuclear genomes of this extinct species [Miller et al., 2008] showed that the mammoth genome contains a larger proportion of interspersed repetitive repeats than any other mammalian genome reported so far. The proliferation of the retrotransposon families LINE/RTE (accounting for 12% of the genome) and the 2 dominant SINE types, the Afrotherian lineage active AFROSINE and the relative newcomer to the proboscidian lineage AFROLA (accounting for 1.93 and 4.24% of the genome, respectively) are probably the main causes of the enlarged GS [Zhao and Schuster, 2009] by building up an unusually high proportion of interspersed repeats. Genomic Ecology Signal transduction networks convey information about extracellular and intracellular environments to the nucleus, while coordinated relocation of large DNA sections is feasible thanks to natural genetic engineering systems [Shapiro, 2005]. Repetitive DNA elements appear as appropriate candidates for being the physical basis that couples the nucleus and its environment. A recent example of such inside-outside cross-talk is the finding that human Alu element retrotransposition can be induced by exposure to the topoisomerase II inhibitor etoposide which is mediated in trans by endogenous LINEs [Hagan et al., 2003]. The fact that retrotransposition can be induced by genotoxic stress supports a speculative scenario in which abundant TEs could account for GS variations triggered by environmental signals. This led to speculations that genotoxic stresses could have occurred in a scenario of genomic ecology with great geographical global climatic events such as those that occurred during the Triassic period when the climate changed from hot and dry with arid deserts to warm and humid with lush rainforests. It can be speculated that concomitantly to these global environmental changes ALU, LINE-1, and SVA mobile elements (still active in the human genome) [Mills et al., 2007] could have undergone a burst of amplification in the prehominid lineage genome.


107

How Many Mammals Are There on Earth: Future Genome Size in Mammals

A large group of more than 1,700 worldwide experts stated in 2008 that the number of species of living mammals was 5,487, stressing the fact that species are still being discovered at a surprisingly high rate [Schipper et al., 2008]. As of January 31st 2012, the International Union for the Conservation of Nature and Natural Resources, IUCN listed 5,488 species. Unfortunately, more than 25% of all land mammals are at risk of extinction, and a large number of marine mammals are strongly threatened [Mora et al., 2011] because of accidental mortality and pollution. It is likely that some species may be vanished even before they are discovered and described. Considering that we are reasoning on GS variation and evolution based on data from 546 species, it is immediately apparent that we lack almost 90% of the potential values useful for working out a detailed picture. As said above, it is therefore necessary to use extreme caution when making claims on this topic. On the other hand, 546 it is an almost sufficient number to obtain reasonable GS dynamics at the level of orders and superorders even though the orders with the largest number of species, Chiroptera (⬃1,000 species) and Rodentia (⬃3,200 species), account for the vast majority of mammals. Clearly, the ‘Genome 10K Community of Scientists’ [2009] project to get a whole-genome sequence for 10,000 vertebrate species is highly welcome! As also said before, we have certainly made progress from the pioneering days of the first quantitative measurements of DNA when, on the basis of the few data then collected, we could only forward a concept based on a ‘paradox’ for the apparent lack of a direct linear relationship between the GS and the complexity of an organism’s features. We had already understood that selective pressures were sometimes operating and shaping the evolution of mammalian genomes, while in other cases this was not apparently the case. This fact still leaves us today unable to give an unambiguous solution, since we are not yet in the position to say whether the GS variations we measure are always causally responsible for differences between groups or are just the expression of dynamic processes of DNA metabolism. As highlighted above, most patterns of GS variations are more easily explained as consequences of the dynamic nature of the genome composition. Thus, extreme caution must be exercised in order do not fallaciously mistake correlation for causation. All of the empirical knowledge we have accumulated in nearly 50 years and the consequent theoretical 108


argumentation point to complex networks of elements shaping a genome’s composition, organization, and size in an Evo-Devo perspective [Prud’homme et al., 2007] able to produce phenotypic novelties which become exposed to Darwinian selection. De Omnibus Rebus et Quibusdam Aliis The study of the evolutionary meaning of GS is fascinating and has given rise to ceaseless debates as well as to a vast body of literature supporting or refuting the governance of phenotypic features by GS. Avise and Ayala [2009] quite recently stated that ‘most scientists agree that evolution provides the unifying framework for interpreting biological phenomena that otherwise can often seem unrelated and perhaps unintelligible’. In the quest for evolutionary explanations for GS, particular mention and gratitude must be given to all those colleagues who measured GS and to the few who made excellent syntheses of these data, notably Dmitri Petrov [Petrov, 2001, 2002a, b; Knight et al., 2005; Oliver et al., 2007] and Alexander Vinogradov [Vinogradov, 1995, 1997, 1998, 1999, 2000, 2004a, b; 2005; Vinogradov and Anatskaya, 2006]. The study of genome composition, organization, and size and of their phenotypic correlates (no matter whether nucleotypic, nucleoskeletal, or whatsoever other causative or coevolutionary relationship) constitutes an integrated field of analysis bringing together cytology, cytogenetics, physiology, and ecology [‘ecophysiological cytogenetics’, Vinogradov, 1997; Kozlowski et al, 2003]. The aim is to dissect the mammalian genome in order to identify those sequences responsible for the GS effects brought about on life history parameters apparently independently of the information encoded by genes. Clarification of the ‘essential’ mammalian genome should be a large step towards understanding key problems in biology: the challenge to explain structure-function relationships (symmorphosis: ‘the quantitative match of design and function parameters’) [Weibel et al., 1991] could become more tractable and ultimately be solved thanks to the integration of cell biology and ecology. The reader can gain profitable insights from 2 milestone syntheses on the genome as a life-history train in mammals: Martin and Palumbi [1993] and Bromham [2011]. Having looked at GS variation and some evolutionary trends in GS, one cannot avoid considering the effects brought about by GS on the physiology of organisms, particular traits of the life-history cycle and, more generally, phenotypic correlates. Some of what has been discovered is summarized below as succinctly as possible and without discussion. Redi /Capanna

Nuclear and Cellular Volumes The GS correlates positively with nuclear and cellular volumes [Cavalier-Smith, 1985; Gregory, 2000]. Thus, small genomes appear well adapted to the metabolic requirements for flight [Hughes and Hughes, 1995]. In fact, the mammals with an active capacity for flight, bats, are those with the smallest genome [Capanna and Manfredi Romanini, 1971]. This correlation also holds true for birds which, among vertebrates, are those, like bats, with the highest metabolic rate and also the smallest genome. Furthermore, larger genomes are found in running species of birds than in birds that fly [Tiersch and Wachtel, 1991; Vinogradov, 2004b]. Bats have a small GS thanks to a minimum of repetitive DNA elements [van den Bussche et al., 1995]. This paucity of repetitive DNA elements is also present in the tetraodontoid fish Fugu rubripes which has an extremely small genome (0.41 pg) with some 10% of repetitive DNA sequences, but it possesses nearly the same number of genes as humans (http://genome.jgi-psf. org/Takru4/Takru4.info.html). These findings suggest that even the smallest genomes (bats, puffer fish, flying birds) need a minimum of repetitive DNA to correctly express genes responsible for complex functions (the ‘eurygenic’ system of gene regulation envisaged by Zuckerkandl and Hennig [1995] and Vinogradov [2004a]): this is a characteristic that should facilitate the search for and identification of regulatory sequences in noncoding DNA, a hot topic of present day biomedicine (see ENCODE). Metabolic Rates Vinogradov’s [1995; 1997; 1998] studies on the nucleotypic effect in homeotherms, with the finding of a clear negative GS correlation with body mass-corrected basal metabolic rate, shows that a sizeable part of the genome affects evolutionary adjustment of the metabolic rate. Mammals originated as the last group in the vertebrates thanks to their small body size, nocturnal behavior, homeothermy, and viviparity. All of these features are energy-demanding, and the clear relationship between GS and metabolic rate [Vinogradov, 1995] underlies the great interest that the study of GS has in the understanding of mammalian evolution. One particularly interesting study case is the relationship between GS and the risk of extinction which was wonderfully presented by Vinogradov [2004a], who showed that larger genomes increased the risk of extinction in reptiles and birds while in mammals, luckily, no effect was observed. This was a nicely examined case of the dual role played by GS itself: the maladaptive effects of a large genome can be counteracted by beMammalian Genome Sizes

nevolent adaptive effects which are revealed in ecological niches with low energy supply in which lower basal metabolic rates are advantageous [Vinogradov, 2004a]. Developmental Time (Rate) Back in 1988, John and Miklos [1988] stated that GS should not be correlated with developmental time, stressing the extreme variability in developmental time versus the small GS variability in mammals. In an analysis carried out by Gregory [2002] this was shown to be the case, with the single exceptions of rodents whose developmental rate (the inverse of time) is negatively correlated with GS. Population Size Lynch and Conery [2003] suggested that the small population size (Ne) of many eukaryotic species facilitates the fixation of mutations that increase GS. Increasing GS can then evolve as a non-adaptive consequence of a small Ne. This idea contrasts with the more generally accepted views on neutral and adaptive meanings of GS evolution [see Petrov, 2002a; Bennet and Leitch, 2005]. In an ongoing debate, Lynch’s group [Rho et al., 2009; Lynch, 2011; Lynch et al., 2011] reasserted that the population size expansions that occurred in the post Cretaceous-Tertiary boundary were the driving cause of increased selection against proliferation of mobile elements, something that could justify the widespread reductions in GS that occurred in multiple lineages of mammals during the same period. However, the recent, detailed analysis carried out by Whitney and Garland [2010] shows that neither GS nor any of its components (e.g. transposon numbers, transposons as a fraction of the genome, intron numbers, intron size) are related to Ne.

Acknowledgements This paper is dedicated to the memory of Maria Gabriella Manfredi Romani who pioneered the concept of GS which is still today creating research avenues. We gratefully acknowledge Silvia Garagna and Maurizio Zuccotti for their critical reading of this paper. We thank the following organizations for continuously supporting us: University of Pavia, the Region of Lombardy, Fondazione Alma Mater Ticinensis, and Fondazione IRCCS Policlinico San Matteo (Pavia), ‘Bando Giovani Ricercatori 2009’ (to CAR).


109

References Agren JA, Wright SI: Co-evolution between transposable elements and their hosts: a major factor in genome size evolution? Chrom Res 19:777–786 (2011). Ariew A: Ernst Mayr’s ‘ultimate/proximate’ distinction reconsidered and reconstructed. Biol Philos 18: 553–565 (2003). Avise JC, Ayala FJ: In the light of evolution III: two centuries of Darwin. Proc Natl Acad Sci USA 106:9933–9938 (2009). Baillie JK, Barnett MW, Upton KR, Gerhardt DJ, Richmond TA, et al: Somatic retrotransposition alters the genetic landscape of the human brain. Nature 479:534–537 (2011). Bachmann K: Genome size in mammals. Chromosoma 37:85–93 (1972). Beck CR, Collier P, Macfarlane C, Malig M, Kidd JM, et al: LINE-1 retrotransposition activity in human genomes. Cell 141: 1159–1170 (2010). Belancio VP, Hedges DJ, Deininger P: Mammalian non-LTR retrotransposons: for better or worse, in sickness and in health. Genome Res 18:343–358 (2008). Bennett MD, Leitch IJ: Genome size evolution in plants, in Gregory TR (ed): The Evolution of the Genome, pp 89–162 (Elsevier, San Diego 2005). Bennetzen JL, Kellogg EA: Do plants have a one way ticket to genomic obesity? The Plant Cell 9:1509–1514 (1997). Bennetzen JL, Ma J, Devos KM: Mechanisms of recent genome size variation in flowering plants. Ann Bot 95:127–132 (2005). Bininda-Emonds OR, Cardillo M, Jones KE, MacPhee RD, Beck RMD, et al: The delayed rise of present-day mammals. Nature 446: 507–512 (2007). Bromham L: The genome as a life-history character: why rate of molecular evolution varies between mammal species. Phil Trans R Soc B 366:2503–2513 (2011). Capanna E, Manfredi Romanini MG: Nuclear DNA content and morphology of the karyotype in certain Paleartic microchiroptera. Caryologia 24: 471–482 (1971). Carmi S, Church GM, Levanon EY: Large-scale DNA editing of retrotransposons accelerates mammalian genome evolution. Nat Commun 2:519 doi:10.1038/ncomms1525 (2011). Cavalier-Smith T: Introduction: the evolutionary significance of genome size, in CavalierSmith T (ed): The Evolution of Genome Size, pp 1–36 (John Wiley & Sons Inc, Chichester, New York 1985). Cavalier-Smith T: Economy, speed and size matter: evolutionary forces driving nuclear genome miniaturization and expansion. Ann Bot 95: 147–175 (2005). Chimpanzee Sequencing and Analysis Consortium. Initial sequence of the chimpanzee genome and comparison with the human genome. Nature 437:69–87 (2005).

110

Cordaux R, Batzer M: The impact of retrotransposons on human genome evolution. Nature Rev Genet 10:691–703 (2009). Coufal NG, Garcia-Perez JL, Peng GE, Yeo GW, Mu Y, et al: L1 retrotransposition in human neural progenitor cells. Nature 460: 1127– 1131 (2009). Delsuc F, Scally M, Madsen O, Stanhope MJ, De Jong WW, et al: Molecular phylogeny of living xenarthrans and the impact of character and taxon sampling on the placental tree rooting. Mol Biol Evol 19:1656–1671 (2002). Delsuc F, Brinkmann H, Philippe H: Phylogenomics and the reconstruction of the tree of life. Nat Rev Genet 6:361–375 (2005). Doležel J, Greilhuber J: Nuclear genome size: are we getting closer? Cytometry A 77A:635– 642 (2010). Doležel J, Bartoš J, Voglmayr H, Greilhuber J: Nuclear DNA content and genome size of trout and human. Cytometry A 51:127–128 (2003). Dufresne F, Jeffery N: A guided tour of large genome size in animals: what we know and where we are heading. Chromosome Res 19: 925–938 (2011). Feschotte C: Transposable elements and the evolution of regulatory networks. Nat Rev Genet 9:397–405 (2008). Froenicke L, Caldés MG, Graphodatsky A, Müller S, Lyons LA, et al: Are molecular cytogenetics and bioinformatics suggesting diverging models of ancestral mammalian genomes? Genome Res 16:306–310 (2006). Gallardo MH, Bickham JW, Honeycutt RL, Ojeda RA, Köhler N: Discovery of tetraploidy in a mammal. Nature 401:341 (1999). Gallardo MH, González CA, Cebrián I: Molecular cytogenetics and allotetraploidy in the red vizcacha rat, Tympanoctomys barrerae (Rodentia, Octodontidae). Genomics 88: 214–221 (2006). Garagna S, Formenti D: DNA content variability in several species of Australian and South American marsupials. Boll Zool 48:255–261 (1981). Genome 10K Community of Scientists. Genome 10K: A proposal to obtain whole-genome sequence for 10,000 vertebrate species. J Hered 100:659–674 (2009). Gentles AJ, Wakefield MJ, Kohany O, Gu W, Batzer MA, et al: Evolutionary dynamics of transposable elements in the short-tailed opossum Monodelphis domestica. Genome Res 17:992–1004 (2007). Graphodatsky AS, Trifonov VA, Stanyon R: The genome diversity and karyotype evolution of mammals. Mol Cytogenet 4:22 (2011). Green RE, Krause J, Briggs AW, Maricic T, Stenzel U, et al: A draft sequence of the Neandertal genome. Science 328:710–722 (2010). Gregory TR: Nucleotypic effects without nuclei: genome size and erythrocyte size in mammals. Genome 43:895–901 (2000).


Gregory TR: Coincidence, coevolution, or causation? DNA content, cell size, and the C-value enigma. Biol Rev Camb Philos Soc 76:65–101 (2001). Gregory TR: Genome size and developmental parameters in the homeothermic vertebrates. Genome 45:833–838 (2002). Gregory TR: Genome size evolution in animals, in Gregory TR (ed): The Evolution of the Genome, pp 3–87 (Elsevier, San Diego 2005). Greilhuber J, Doležel J: 2C or not 2C: a closer look at cell nuclei and their DNA content. Chromosoma 118:391–400 (2009). Greilhuber J, Doležel J, Lysák M, Bennett MD: The origin, evolution, and proposed stabilization of the terms ‘genome size’ and ‘C-value’ to describe nuclear DNA contents. Ann Bot 95:255–260 (2005). Greilhuber J, Temsch EM, Loureiro JCM: Nuclear DNA content measurement in Doležel J, Greilhuber J, Suda J (eds): Flow Cytometry with Plant Cells, pp 67–101 (Wiley, Weinheim 2007). Hagan CR, Sheffield RF, Rudin CM: Human Alu element retrotransposition induced by genotoxic stress. Nat Genet 35:219–220 (2003). Han K, Konkel MK, Xing J, Wang H, Lee J, et al: Mobile DNA in old world monkeys: a glimpse through the Rhesus macaque genome. Science 316:238–240 (2007). Hardie DC, Gregory TR, Hebert PD: From pixels to picograms: a beginners’ guide to genome quantification by Feulgen image analysis densitometry. J Histochem Cytochem 50: 735–749 (2002). Hawkins JS, Kim HR, Nason JD, Wing RA, Wendel JF: Differential lineage-specific amplification of transposable elements is responsible for genome size variation in Gossypium. Genome Res 16:1252–1261 (2006). Hedges DJ, Batzer MA: From the margins of the genome: mobile elements shape primate evolution. Bioessays 27:785–794 (2005). Hedges SB, Dudley J, Kumar S: TimeTree: a public knowledge-base of divergence times among organisms. Bioinformatics 22: 2971– 2972 (2006). Helgen KM: Evolution. The mammal family tree. Science 334:458–459 (2011). Hinegardner R: Evolution of genome size, in Ayala FJ (ed): Molecular Evolution, pp 179–199 (Sinauer Associates Inc, Sunderland 1976). Hirakawa M, Nishihara H, Kanehisa M, Okada N: Characterization and evolutionary landscape of AmnSINE1 in Amniota genomes. Gene 441:100–110 (2009). Hughes AL, Hughes MK: Small genomes for better flyers. Nature 377:391 (1995). John B, Miklos G: The Eukaryote Genome in Development and Evolution (Allen and Unwin, London 1988). Kelkar YD, Eckert KA, Chiaromonte F, Makova KD: A matter of life or death: how microsatellites emerge in and vanish from the human genome. Genome Res 21:2038–2048 (2011).

Redi /Capanna

Khan H, Smit A, Boissinot S: Molecular evolution and tempo of amplification of human LINE-1 retrotransposons since the origin of primates. Genome Res 16: 78–87 (2006). Knight CA, Molinari NA, Petrov DA: The Large genome constraint hypothesis: evolution, ecology and phenotype. Ann Bot 95:177–190 (2005). Kozlowski J, Konarzewski M, Gawelczyk AT: Cell size as a link between noncoding DNA and metabolic rate scaling. Proc Natl Acad Sci USA 100:14080–14085 (2003). Kramerov DA, Vassetzky NS: Origin and evolution of SINEs in eukaryotic genomes. Heredity 107:487–495 (2011). Kriegs JO, Churakov G, Kiefmann M, Jordan U, Brosius J, Schmitz J: Retroposed elements as archives for the evolutionary history of placental mammals. PLoS Biol 4:e91 (2006). Kumar A, Bennetzen JL: Plant retrotransposons. Annu Rev Genet 33:479–532 (1999). Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, et al: Initial sequencing and analysis of the human genome. Nature 409:860– 921 (2001). Levin HL, Moran JV: Dynamic interactions between transposable elements and their hosts. Nat Rev Genet 12:615–627 (2011). Li DJ, Zhang S: The Cambrian explosion triggered by critical turning point in genome size evolution. Biochem Biophysic Res Commun 392:240–245 (2010). Lindblad-Toh K, Garber M, Zuk O, Lin MF, Parker BJ, et al: A high-resolution map of human evolutionary constraint using 29 mammals. Nature 478:476–482 (2011). Liu G, NISC Comparative Sequencing Program, Zhao S, Bailey JA, Sahinalp SC, et al: Analysis of primate genomic variation reveals a repeat-driven expansion of the human genome. Genome Res 13:358–368 (2003). Locke DP, Hillier LW, Warren WC, Worley KC, Nazareth LV, et al: Comparative and demographic analysis of orang-utan genomes. Nature 469:529–533 (2011). Lovegrove BG: The zoogeography of mammalian basal metabolic rate. Am Nat 156:201–219 (2000). Luo ZX: Transformation and diversification in early mammal evolution. Nature 450: 1011– 1019 (2007). Luo ZX, Wible JR: A Late Jurassic digging mammal and early mammalian diversification. Science 308:103–107 (2005). Luo ZX, Ji Q, Wible JR, Yuan CX: An Early Cretaceous tribosphenic mammal and metatherian evolution. Science 302: 1934–1940 (2003). Luo ZX, Yuan CX, Meng QJ, Ji Q: A Jurassic eutherian mammal and the divergence of marsupials and placentals. Nature 476: 442–445 (2011). Lynch M: Statistical inference on the mechanisms of genome evolution. PLoS Genet 7:e1001389 (2011). Lynch M, Conery JS: The origins of genome complexity. Science 302:1401–1404 (2003).


Lynch M, Bobay LM, Catania F, Gout JF, Rho M: The repatterning of eukaryotic genomes by random genetic drift. Annu Rev Genomics Hum Genet 12:347–366 (2011). Madsen O, Scally M, Douady CJ, Kao DJ, DeBry RW, et al: Parallel adaptive radiations in two major clades of placental mammals. Nature 409:610–614 (2001). Makalowski W: Genomic scrap yard: how genomes utilize all that junk. Gene 259: 61–67 (2000). Manfredi Romanini MG: The nuclear DNA content and the evolution of vertebrates, in Chiarelli B, Capanna E (eds): Cytotaxonomy and Vertebrate Evolution, pp 39–81 (Academic Press, London-New York 1973). Manfredi Romanini MG: The nuclear content of desoxyribonucleic acid and some problems of mammalian phylogenesis. Mammalia 49: 369–385 (1985). Martin AP, Palumbi SR: Body size, metabolic rate, generation time, and the molecular clock. Proc Natl Acad Sci USA 90:4087–4091 (1993). Mayr E: Teleological and teleonomic: a new analysis. Boston Stud Philos Sci 14:91–117 (1974). Mayr E: Proximate and ultimate causation. Biol Philos 8:95–98 (1993). Meredith RW, Janečka JE, Gatesy J, Ryder OA, Fisher CA, et al: Impacts of the Cretaceous terrestrial revolution and KPg extinction on mammal diversification. Science 334: 521– 524 (2011). Mikkelsen TS, Wakefield MJ, Aken B, Amemiya CT, Chang JL, et al: Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences. Nature 447: 167–177 (2007). Milinkovitch MC, Helaers R, Depiereux E, Tzika AC, Gabaldón T: 2x genomes – depth does matter. Genome Biol 11:R16 (2010). Miller W, Drautz DI, Ratan A, Pusey B, Qi J, et al: Sequencing the nuclear genome of the extinct woolly mammoth. Nature 456:387–390 (2008). Mills RE, Bennett EA, Iskow RC, Devine SE: Which transposable elements are active in the human genome? Trends Genet 23: 183– 191 (2007). Mora C, Tittensor DP, Adl S, Simpson AG, Worm B: How many species are there on earth and in the ocean? PLoS Biol 9:e1001127 (2011). Morrish TA, Gilbert N, Myers JS, Vincent BJ, Stamato TD, et al: DNA repair mediated by endonuclease-independent LINE-1 retrotransposition. Nat Genet 31: 159–165 (2002). Morrish TA, Garcia-Perez JL, Stamato TD, Taccioli GE, Sekiguchi J, Moran JV: Endonuclease-independent LINE-1 retrotransposition at mammalian telomeres. Nature 446: 208– 212 (2007). Murphy WJ, Elzirik E, Johnson WE, Zhang YP, Ryder OA, O’Brien SJ: Molecular phylogenetics and the origins of placental mammals. Nature 409:614–618 (2001).

Murphy WJ, Pevzner PA, O’Brien SJ: Mammalian phylogenomics comes of age. Trends Genet 20:631–639 (2004). Nesse RM, Bergstrom CT, Ellison PT, Flier JS, Gluckman P, et al: Making evolutionary biology a basic science for medicine. Proc Natl Acad Sci USA 107:1800–1807 (2010). Nikaido M, Nishihara H, Hukumoto Y, Okada N: Ancient SINEs from African endemic mammals. Mol Biol Evol 20:522–527 (2003). Nilsson MA, Churakov G, Sommer M, Tran NV, Zemann A, et al: Tracking marsupial evolution using archaic genomic retroposon insertions. PloS Biol 8:e1000436 (2010). Nishihara H, Smit AF, Okada N: Functional noncoding sequences derived from SINEs in the mammalian genome. Genome Res 16: 864–874 (2006). O’Brien SJ: The platypus genome unraveled. Cell 133:953–955 (2008). Ohno S: The mammalian genome in evolution and conservation of the original X-linkage group, n Benirschke K (ed): Comparative Mammalian Cytogenetics, pp 18–29 (Springer-Verlag, Berlin-Heidelberg-New York 1969). Ohno S: Evolution by gene duplication. (Springer-Verlag, Berlin-Heidelberg-New York 1970). Ohno S: Gene duplication and the uniqueness of vertebrate genomes circa 1970–1999. Cell Dev Biol 10: 517–522 (1999). Oliver MJ, Petrov DA, Ackerly D, Falkowski P, Schofield OM: The mode and tempo of genome size evolution in eukaryotes. Genome Res 17:594–601 (2007). Organ CL, Canoville A, Reisz RR, Laurin M: Paleogenomic data suggest mammal-like genome size in the ancestral amniote and derived large genome size in amphibians. J Evol Biol 24: 372–380 (2011). Pace JK, Feschotte C: The evolutionary history of human DNA transposons: evidence for intense activity in the primate lineage. Genome Res 17:422–432 (2007). Pellicciari C, Formenti D, Redi CA, Manfredi Romanini MG: DNA content variability in Primates. J Hum Evol 11:131–141 (1982). Penny D, Phillips MJ: Mass survivals. Nature 446:501–502 (2007). Petrov DA: Evolution of genome size: new approaches to an old problem. Trends Genet 17: 23–28 (2001). Petrov DA: Mutational equilibrium model of genome size evolution. Theor Popul Biol 61: 531–544 (2002a). Petrov DA: DNA loss and evolution of genome size in Drosophila. Genetica 115: 81–91 (2002b). Petrov DA, Sangster TA, Johnston JS, Hartl DL, Shaw KL: Evidence for DNA loss as a determinant of genome size. Science 287: 1060– 1062 (2000). Pontius JU, Mullikin JC, Smith DR; Agencourt Sequencing Team, Lindblad-Toh K, et al: Initial sequence and comparative analysis of the cat genome. Genome Res 17:1675–1689 (2007).


111

Pontzer H, Raichlen DA, Shumaker RW, Ocobock C, Wich SA: Metabolic adaptation for lowenergy throughput in orangutans. Proc Natl Acad Sci USA 107:14048–14052 (2010). Price SA, Vos RA, Gittleman JL, Purvis A: The delayed rise of present-day mammals. Nature 446:507–512 (2007). Prud’homme B, Gompel N, Carroll SB: Emerging principles of regulatory evolution. Proc Natl Acad Sci USA 104:8605–8612 (2007). Redi CA, Garagna S, Zacharias H, Zuccotti M, Capanna E: The other chromatin. Chromosoma 110:136–147 (2001). Redi CA, Zacharias H, Merani S, Oliveira Miranda M, Aguilera M, et al: Genome sizes in Afrotheria, Xenarthra, Euarchontoglires, and Laurasiatheria. J Hered 96: 485–493 (2005). Redi CA, Garagna S, Zuccotti M, Capanna E: Genome size: a novel genomic signature in support of Afrotheria. J Mol Evol 64: 484– 487 (2007). Renfree MB, Papenfuss AT, Deakin JE, Lindsay J, Heider T, et al: Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development. Genome Biol 12: 414 (2011). Rho M, Zhou M, Gao X, Kim S, Tang H, Lynch M: Independent mammalian genome contractions following the KT boundary. Genome Biol Evol 1: 2–12 (2009). Romiguier J, Ranwez V, Douzery EJ, Galtier N: Contrasting GC-content dynamics across 33 mammalian genomes: relationship with lifehistory traits and chromosome sizes. Genome Res 20:1001–1009 (2010). Samollow PB: The opossum genome: insights and opportunities from an alternative mammal. Genome Res 18:1199–1215 (2008). SanMiguel P, Bennetzen JL: Evidence that a recent increase in maize genome size was caused by the massive amplification of intergene retrotransposons. Ann Bot 82: 37–44 (1998). Schipper J, Chanson JS, Chiozza F, Cox NA, Hoffmann M, et al: The status of the worlds land and marine mammals: diversity, threat, and knowledge. Science 322:225–230 (2008). Schulman AH, Kalendar R: A movable feast: diverse retrotransposons and their contribution to Barley (Hordeum vulgare) genome dynamics. Cytogenet Genome Res 110: 598– 605 (2005).

112

Shapiro JA: A 21st century view of evolution: genome system architecture, repetitive DNA, and natural genetic engineering. Gene 345: 91–100 (2005). Sharov AA: Genome increase as a clock for the origin and evolution of life. Biol Direct 1: 17 (2006). Shen MR, Batzer MA, Deininger PL: Evolution of the master Alu gene(s). J Mol Evol 33:311– 320 (1991). Slotkin RK, Martienssen R: Transposable elements and the epigenetic regulation of the genome. Nat Rev Genet 8:272–285 (2007). Somel M, Liu X, Tang L, Yan Z, Hu H, et al: MicroRNA-driven developmental remodeling in the brain distinguishes humans from other primates. PLoS Biol 9:e1001214 (2011). Springer MS, Stanhope MJ, Madsen O, de Jong WW: Molecules consolidate the placental mammal tree. Trends Ecol Evol 19: 430–438 (2004). Svartman MT, Stone G, Stanyon R: Molecular cytogenetics discards polyploidy in mammals. Genomics 85: 425–430 (2005). Svartman MT, Stone G, Stanyon R: The ancestral eutherian karyotype is present in Xenarthra. PLoS Genet 2:e109 (2006). Swift HH: The constancy of deoxyribose nucleic acid in plant nuclei. Proc Natl Acad Sci USA 36:643–654 (1950). Taft RJ, Mattick JS: Increasing biological complexity is positively correlated with the relative genome-wide expansion of non-protein-coding DNA sequences (2004). Available at http://www.arxiv.org/abs/q-bio.GN/ 0401020. Thomas CA: The genetic organization of chromosomes. Ann Rev Genet 5:237–256 (1971). Tiersch TR, Wachtel SS: On the evolution of genome size in birds. J Hered 82: 363–368 (1991). Tinbergen N: On aims and methods of ethology. Z Tierpsychol 20:410–433 (1963). van den Bussche RA, Longmire JL, Baker RJ: How bats achieve a small C-value: frequency of repetitive DNA in Macrotus. Mam Gen 6: 521–525 (1995). van Nimwegen E: Scaling laws in the functional content of genomes. Trends Genet 19: 479– 484 (2003). Venditti C, Meade A, Pagel M: Multiple routes to mammalian diversity. Nature 479: 393–396 (2011). Venner S, Feschotte C, Bièmont C: Dynamics of transposable elements: towards a community ecology of the genome. Trends Genet 25: 317–323 (2009). Vinogradov AE: Nucleotypic effect in homeotherms: body-mass-corrected basal metabolic rates of mammals is related to genome size. Evolution 49:1249–1259 (1995).


Vinogradov AE: Nucleotypic effect in homeotherms: body-mass independent resting metabolic rate of passerine birds is related to genome size. Evolution 51:220–225 (1997). Vinogradov AE: Buffering: a possible passivehomeostasis role for redundant DNA. J Theor Biol 193:197–199 (1998). Vinogradov AE: Intron–genome size relationship on a large evolutionary scale. J Mol Evol 49:376–384 (1999). Vinogradov AE: Larger genomes for molluskan land pioneers. Genome 43:211–212 (2000). Vinogradov AE: Genome size and extinction risk in vertebrates. Proc R Soc Lond B 271: 1701–1705 (2004a). Vinogradov AE: Testing genome complexity. Science 304:389–390 (2004b). Vinogradov AE: Genome size and chromatin condensation in vertebrates. Chromosoma 113:362–369 (2005). Vinogradov AE, Anatskaya OV: Genome size and metabolic intensity in tetrapods: a tale of two lines. Proc R Soc Lond B 273: 27–32 (2006). Vitte C, Panaud O: LTR retrotransposons and flowering plant genome size: emergence of the increase/decrease model. Cytogenet Genome Res 110:91–107 (2005). Wagner GP, Lynch VJ: Evolutionary novelties. Curr Biol 20:R48–R52 (2010). Warren WC, Hillier LW, Marshall Graves JA, Birney E, Ponting CP, et al: Genome analysis of the platypus reveals unique signatures of evolution. Nature 453:175–184 (2008). Weibel ER, Taylor CR, Hoppeler H: The concept of symmorphosis: a testable hypothesis of structure-function relationship. Proc Natl Acad Sci USA 88:10357–10361 (1991). Whitney KD, Garland T: Did genetic drift drive increases in genome complexity? PLoS Genet 6: e1001080 (2010). Wilson GP, Evans AR, Corfe IJ, Smits PD, Fortelius M, Jernvall J: Adaptive radiation of multituberculate mammals before the extinction of dinosaurs. Nature 483:457–460 (2012). Winkler H: Verbreitung und Ursache der Parthenogenesis im Pflanzen- und Tierreiche. (Gustav Fischer Verlag, Jena 1920). Zhao F, Qi J, Schuster SC: Tracking the past: interspersed repeats in an extinct Afrotherian mammal, Mammuthus primigenius. Genome Res 19:1384–1392 (2009). Zuckerkandl E, Hennig W: Tracking heterochromatin. Chromosoma 104:75–83 (1995).

Redi /Capanna



The Evolution of Marsupial and Monotreme Chromosomes J.E. Deakin a J.A.M. Graves a, b W. Rens c a

Evolution, Ecology and Genetics, Research School of Biology, The Australian National University, Canberra, A.C.T., and b La Trobe Institute of Molecular Science, La Trobe University, Melbourne, Vic., Australia; c Department of Veterinary Medicine, University of Cambridge, Cambridge, UK

Key Words Ancestral karyotypes ⴢ Chromosome painting ⴢ Gene mapping ⴢ Genome evolution ⴢ Sex chromosomes

cently sequenced marsupial and monotreme genomes with cytogenetic analysis, which has provided a greater understanding of the events that have shaped not only marsupial and monotreme genomes, but the genomes of all mammals. Copyright © 2012 S. Karger AG, Basel

Abstract Marsupial and monotreme mammals fill an important gap in vertebrate phylogeny between reptile-mammal divergence 310 million years ago (mya) and the eutherian (placental) mammal radiation 105 mya. They possess many unique features including their distinctive chromosomes, which in marsupials are typically very large and well conserved between species. In contrast, monotreme genomes are divided into several large chromosomes and many smaller chromosomes, with a complicated sex chromosome system that forms a translocation chain in male meiosis. The application of molecular cytogenetic techniques has greatly advanced our understanding of the evolution of marsupial chromosomes and allowed the reconstruction of the ancestral marsupial karyotype. Chromosome painting and gene mapping have played a vital role in piecing together the puzzle of monotreme karyotypes, particularly their complicated sex chromosome system. Here, we discuss the significant insight into karyotype evolution afforded by the combination of re-



Deep divisions in class Mammalia provide an opportunity to look back in time and reconstruct the events shaping their genomes. Mammals are divided into 3 major lineages that diverged from a common ancestor between 161 and 217 million years ago (mya) (fig. 1) [Phillips et al., 2009]. The egg-laying monotremes (subclass Prototheria) are the earliest offshoot of the class Mammalia and are represented by 1 extant species of platypus and 4 species of echidna. Marsupial (infraclass Metatheria) and eutherian mammals combined make up the subclass Theria. Marsupials and eutherians diverged 143– 178 mya [Phillips et al., 2009; Luo et al., 2011]. This deep divergence of eutherian, marsupial and monotreme lineages is particularly valuable for gaining insight into the evolution of mammalian genomes and the reconstruction of ancestral chromosomes.

Janine E. Deakin Evolution, Ecology and Genetics, Research School of Biology The Australian National University Canberra, ACT 0200 (Australia) Tel. +61 261 254 902, E-Mail janine.deakin @ anu.edu.au

Theria

RI AN S EU TH E

il De v

Wa ll

ab y

MARSUPIALS Op os su m

Ec hid na

Pla typ us

RE PT I LE S

MONOTREMES

malian chromosome evolution and the reconstruction of the plesiomorphic karyotype of all mammals. Here, we describe the current knowledge of marsupial and monotreme chromosomes and the insight gained into chromosome evolution within these lineages.

0

Chromosome Evolution in Marsupials

100

200

300 MYA

Fig. 1. The phylogenetic relationship of key marsupial and mono-

treme species to other vertebrate groups.

One of the major differences between these 3 mammalian lineages is their mode of reproduction. Marsupials give birth to altricial young, and evolution has resulted in more emphasis on a sophisticated lactation system than in prolonged in utero development as observed in eutherian species. It is important to stress that, despite laying eggs, monotremes possess features that are distinctively mammalian; they are fur-bearing animals and, even in the absence of nipples, feed their young milk that is produced by glands and oozes onto the skin of the mother’s abdomen to be licked by their young. The unique features of marsupials and monotremes have intrigued biologists since they were first described. Among those features are their chromosomes, which differ markedly between these 2 groups. Marsupials are renowned for their low diploid numbers and characteristically large and well-conserved chromosomes, whereas monotremes have high diploid numbers, chromosome size differences reminiscent of reptiles and complicated sex chromosome systems. Great insight into the evolution of mammalian chromosomes can be gained from including representative marsupials and monotremes in comparative genomic studies. Although many studies have reconstructed the ancestral eutherian karyotypes from comparisons of different eutherian species, an understanding of the homologies and chromosome rearrangements between all 3 mammalian lineages is required to accurately elucidate the rearrangements that have occurred throughout mam114


Marsupials are a diverse group of mammals consisting of over 300 species, divided between the Americas and Australasia. The American (Ameridelphia) and Australian (Australiadelphia) species diverged from a common ancestor about 80 mya and it is generally acknowledged that the ameridelphian family Didelphidae were the first offshoot of the marsupial lineage, diverging from the lineage that gave rise to the American families, then to the Australidelphia species around 80 mya [Beck, 2008; Meredith et al., 2008]. One feature of marsupial karyotypes that has fascinated cytogeneticists is their remarkable conservation. This is quite unlike eutherian mammals, in which extensive chromosome rearrangement has occurred between species. Marsupial genomes are typically packaged into relatively few, very large chromosomes, which have facilitated cytogenetic studies. Basic karyotype studies reporting chromosome number, size and morphology for many species in each marsupial family showed that marsupial chromosomes had changed little since divergence from a common ancestor [Hayman and Martin, 1969] and certainly much less than the chromosomes of their eutherian counterparts. This finding was later supported by G-banding studies [Rofe and Hayman, 1985]. The advent of molecular cytogenetic techniques has enabled us to more thoroughly investigate the level of karyotypic conservation and delve much deeper into the analysis of chromosome evolution in this group of mammals. The limited range of diploid numbers was a telling sign of the limited amount of karyotypic variation amongst marsupials. Diploid numbers were bimodal in marsupials, with 2n = 14 and 2n = 22 complements common both in Australidelphia and Ameridelphia [Sharman, 1974; Hayman, 1990]. This led to 2 alternative hypotheses for the ancestral marsupial chromosome number [Sharman, 1973; Hayman and Martin, 1974; Reig et al., 1977; Rofe and Hayman, 1985]. Rofe and Hayman [1985] proposed a 2n = 14 ancestral marsupial karyotype, a chromosome complement common among several Australian families and observed to have changed little between American and Australian species, with fissions Deakin /Graves /Rens

giving rise to higher diploid numbers that are seen in many families [Hayman and Martin, 1974]. The alternative hypothesis proposed that the 2n = 22 complement is ancestral and that lower diploid numbers are the result of fusion events [Sharman, 1973; Svartman and ViannaMorgante, 1998]. The use of a well-supported phylogenetic tree provided further evidence for a 2n = 14 ancestor [Westerman et al., 2010], but the 2 hypotheses could not be distinguished without reference to an outgroup. Two of the most speciose groups of marsupials have very different rates of karyotypic evolution. The family Dasyuridae is known for its extreme level of karyotypic conservation, with 40 of the 68 known dasyurid species examined to date possessing the conserved 2n = 14 karyotype [Hayman and Martin 1974; Young et al., 1982; Rofe and Hayman, 1985]. In contrast, the family Macropodidae (kangaroos, wallabies) has been subject to major chromosome rearrangements, with diploid numbers ranging from a species with the lowest marsupial diploid number of 2n = 10, 11 to 2n = 24, all of which can be derived from a 2n = 22 macropodid ancestor by simple (mostly Robertsonian) fusions and fissions [Rofe, 1978; Hayman, 1990]. For this reason, their chromosomes have been comprehensively studied, first by G-banding and more recently by new molecular cytogenetic techniques which provide better resolution of the chromosome restructuring events that have occurred to result in the karyotypes of extant macropod species. Chromosome painting has afforded an overview of large-scale rearrangements, and physical gene mapping has been used to resolve breakpoints and identify smaller inversions. Reconstruction of the Ancestral Marsupial Karyotype from Cross-Species Chromosome Painting Cross-species chromosome painting on different marsupial species has proven exceptionally valuable for determining which chromosome regions are genetically homologous between closely and distantly related marsupials in order to determine how chromosomes have evolved in marsupials and to reconstruct the ancestral marsupial karyotype. The first such study used chromosome paints derived from 3 Australian species of the order Diprotodontia: the tammar wallaby (Macropus eugenii, 2n = 16), the brushtail possum (Trichosurus vulpecula, 2n = 22) and the long nosed potoroo (Potorous tridactylus, 2n = 12,XX female: 2n = 13,XY1Y2 male). These were compared in mostly reciprocal painting experiments to the fat tailed dunnart (Sminthopsis crassicaudata, 2n = 14) of the order Dasyuromorphia, a group with extremely conserved 2n = 14 karyotypes [Rens et al., 1999]. These Marsupial and Monotreme Chromosomes

experiments detected 15 conserved chromosome segments, and the arrangement of these segments in the 2n = 14 ancestral karyotype was reconstructed from Gbanding studies. The 2n = 14 karyotype of S. crassicaudata was thought to have remained very similar to that of the ancestral marsupial, separated by only 6 inversions. To further test the hypothesis of the 2n = 14 ancestral marsupial karyotype, detailed chromosome painting was extended to a diprotodontid species, (the southern hairynosed wombat, Lasiorhinus latifrons) and a dasyurid (the striped-face dunnart, S. macroura), both of which have 2n = 14 karyotypes. Chromosome paints derived from the tammar wallaby, also a member of the order Diprotodontia, were used to determine the chromosome homologies between these species. This study provided confirmation of previous G-banding studies that the 2n = 14 karyotype present in different orders of Australian marsupials are virtually identical. Rearrangements detected in 2n = 14 species were the result of inversions. The wombat essentially retains the predicted ancestral arrangement, with the exception of 2 inversions on chromosome 1 [De Leo et al., 1999]. Several additional inversions have taken place on S. macroura chromosomes, a finding similar to that observed for the closely related S. crassicaudata [Rens et al., 1999]. Thus, even distantly related Australian marsupials share a conserved 2n = 14 karyotype, from which other karyotypes could easily be derived. To determine whether this was also true for American species, and marsupials in general, Rens et al. [2001] used probes derived from the Brazilian gray short-tailed opossum (Monodelphis domestica) to paint onto chromosomes of 3 Australian species used in previous studies (S. crassicaudata, M. eugenii, T. vulpecula) and vice versa. All but one of the conserved segments identified in reciprocal painting experiments on Australian species [Rens et al., 1999] were also conserved in M. domestica. Indeed, 4 autosomes were homologous between M. domestica (1, 2, 5, and 8) and S. crassicaudata (2, 4, 6, and 5). Only 2 rearrangements differentiate the chromosomes of these species; M. domestica chromosomes 3 and 6 are homologous to S. crassicaudata chromosome 1, and chromosomes 4 and 7 are homologous to S. crassicaudata 3. Tellingly, this same rearrangement was observed by G-banding between a 2n = 14 American species Marmosops incanus (gray slender opossum) and M. domestica [Svartman and Vianna-Morgante, 1999], suggesting that the 2n = 14 American and Australian marsupial chromosome complements were homologous, as had been proposed from early G-banding studies of 2n = 14 karyotypes [Rofe and Hayman, 1985]. Cytogenet Genome Res 2012;137:113–129

115

Fig. 2. Arrangement of 19 conserved chromosome segments identified by cross-species chromosome painting [Rens et al., 2003] in species from different marsupial lineages. The arrangement of conserved segments has been inferred from G-banding for Caluromys lanatus and Didelphis marsupialis and hybridization of tammar wallaby paints for Dorcopsis and Lasiorhinus latifrons. For species in which the distribution of ITS signals has been determined, their location is indicated to the left of chromosomes. Red bars indicate ITS resulting from a centric fusion, whereas blue bars indicate ITS resulting from an inversion.

A better resolution of conserved segments was achieved by using chromosome paints derived from Aepyprymnus rufuscens (rufous bettong), the marsupial with the highest reported diploid chromosome number of 2n = 32. These paints detected 19 conserved chromosome segments when hybridized to 1 American (M.domestica) and 4 Australian species (S. crassicuadata, T. vulpecula, M. eugenii, and P. tridactylus) [Rens et al., 2003]. When these data were combined with G-banding data on many more species, it was possible to determine the chromosome rearrangements that have occurred during marsupial evo116


lution and reconstruct the marsupial ancestral karyotype (fig. 2) [Rens and Ferguson-Smith, 2010]. Examination of the conserved segment organisation across 2n = 14 ameridelphian and austradelphian species showed that the 2n = 14 karyotypes observed are virtually identical, apart from a few intrachromosomal rearrangements [Rens and Ferguson Smith, 2010]. However, the arrangement of these 19 conserved segments differed markedly in 2n = 22 American and Australian species. Therefore, 2n = 22 karyotypes in the 2 superorders [Sharman, 1973] are not the same, so they do not represent an ancestral arrangement. However, this does not rule out the possibility that the marsupial ancestor had a 2n = 22 complement that underwent fusion events early in marsupial evolution to form the common 2n = 14 karyotype, from which unrelated 2n = 22 karyotypes later evolved. The observation of interstitial telomere signals (ITS) in the chromosomes of 2n = 14 American species supported the origin of the large chromosomes by fusion of smaller chromosomes [Svartman and Vianna-Morgante, 1998, discussed further below], although it did not reveal when this occurred. To further test the alternative hypotheses of the ancestral marsupial karyotype, Westerman et al. [2010] used a well-resolved marsupial phylogenetic tree along with cytogenetic information to show that the conserved 2n = 14 karyotype appears almost unchanged in several American and Australian lineages, so that the most likely chromosome arrangement in the marsupial ancestor was 2n = 14, and higher diploid numbers are derived from fission events. However, this dichotomy cannot be resolved by any technique with out reference to an outgroup. Comparative Gene Mapping and Ancestral Karyotype Reconstruction Chromosome painting has provided an excellent foundation for comparing the homology of marsupial chromosomes and reconstruction of a plesiomorphic karyotype, but to resolve the debate over the ancestral chromosome complement, comparisons to outgroups need to be made. With the exception of the X chromosome [Glas et al., 1999b], marsupial chromosome paints do not hybridize to eutherian chromosomes. Fortunately, the sequencing of several marsupial genomes and the availability of bacterial artificial chromosomes (BAC) clones for these species have enabled gene maps to be constructed from orthologous genes in divergent species such as chicken and human. The opossum genome was deeply sequenced [Mikkelsen et al., 2007], and 97% of this sequence was anDeakin /Graves /Rens

chored by mapping the ends of sequence scaffolds to opossum metaphase chromosomes using fluorescence in situ hybridization (FISH) [Duke et al., 2007]. The Tasmanian devil (Sarcophilus harrissi) has been the subject of 2 independent sequencing efforts, both of which employed next-generation sequencing technology [Miller et al., 2011; Murchison et al., 2012]. One of these projects sequenced flow-sorted devil chromosomes to assign sequences to chromosomes, but sequence arrangement on devil chromosomes was not determined [Murchison et al., 2012]. The tammar wallaby genome has only been lightly sequenced, and more than 300,000 sequence scaffolds remain unanchored [Renfree et al., 2011]. By extrapolating data from chromosome painting and the anchored opossum genome assembly, we can predict the genic content of chromosomes of other marsupials. Access to sequence data and BAC libraries for the tammar wallaby and devil has enabled cytogenetic gene maps to be efficiently generated, providing finer resolution of the homology and rearrangements between marsupial chromosomes as well as permitting comparisons to outgroups such as chicken and human to be made. Such comparisons are critical for a definitive reconstruction of the ancestral marsupial karyotype. A map of tammar wallaby chromosome 5 was the first dense cytogenetic gene map constructed for any marsupial and a trial for an efficient approach to assigning genome sequence to chromosomes. A block of genes conserved between opossum chromosome 7 and the human X chromosome was identified, and 52 of the 58 genes occurring in this block were mapped to the short arm of tammar wallaby chromosome 5 [Deakin et al., 2008]. Rather than mapping every gene within an opossum-human conserved block, only genes at either end of all other opossum-human evolutionary conserved blocks were mapped, and the genic content of these blocks inferred from the opossum genome assembly. This resulted in a cytogenetic map of 141 genes across 15 opossum-human conserved blocks and the virtual assignment of 2,320 protein-coding genes [Deakin et al., 2008]. Three inversions that had not been detected by chromosome painting were revealed by comparison of the tammar wallaby map with the opossum genome assembly. Chromosome painting determined that tammar wallaby chromosome 5, consisting of conserved segments C11 and C12, was homologous to most of opossum chromosome 4 and a small region on chromosome 7, respectively [Rens et al., 2003]. Gene mapping more accurately determined the boundaries of these conserved segments. In the tammar wallaby, C12 did not encompass the entire

short arm and proximal region of the long arm of chromosome 5, but was instead restricted to the pericentric region, and part of C11 was located on the distal region of the short arm [Deakin et al., 2008]. The location of C12 on opossum chromosome 7 was inferred from known chromosome homologies rather than direct hybridization, due to difficulties experienced in hybridizing the C12 paint to opossum chromosomes [Rens et al., 2003]. Comparative mapping data has allowed this segment to be accurately assigned to the pericentric region of chromosome 7. The same mapping strategy was used to assign blocks conserved between opossum and human to all tammar wallaby autosomes [Mohammadi et al., 2009; Deakin, 2010], and a virtual map of all chromosomes has been constructed [Wang et al., 2011]. A similar approach has been used to generate a map of the devil genome, where 105 genes corresponding to the ends of opossum-wallaby conserved blocks were mapped to devil chromosomes [Deakin et al., 2012]. Comparisons of gene arrangements between devil, tammar wallaby and opossum revealed substantial chromosome rearrangement between these species, making it necessary for more genes to be mapped before a virtual map of the genome can be constructed. Of particular interest is the gene order on chromosome 1 of the Tasmanian devil, since it is this chromosome that has been extensively rearranged in the transmissible Devil Facial Tumour Disease [Deakin et al., 2012]. Confusingly, devil chromosome 1 has been mislabelled as chromosome 2 in some of the literature on this interesting tumour [e.g. Pearse and Swift, 2006], contradicting the long-established standard dasyurid karyotype [Martin, 1967]. The normal devil chromosome 1, like the ancestral chromosome 1 from which it evolved, consists of conserved segments C1 to C6, but these blocks are highly rearranged in devil. The most conserved chromosome corresponded to segments C11 and C12 on devil chromosome 3. Acceptance of a 2n = 14 ancestral marsupial karyotype predicts that segments C10, C11 and C12 would have formed chromosome 3, as we observe in the devil. The alternative hypothesis of a 2n = 22 ancestor distributes these conserved segments across 2 chromosomes [Svartman and Vianna-Morgante, 1998]. We observe that genes spanning parts of all 3 segments in a representative outgroup species, the chicken (Gallus gallus), were all found together on chicken chromosome 1. This suggests that they were part of the same chromosome in the ancestor of all mammals, which was subsequently subject to various fission and fusion events in some lineages but re-

Marsupial and Monotreme Chromosomes


117

mains as a single chromosomes in 2n = 14 species. This refutes the hypothesis that the marsupial ancestor had a 2n = 22 karyotype [Deakin et al., 2008]. Thus all evidence, from extensive G-banding studies to cross-species chromosome painting, phylogenetics and gene mapping, support the original hypothesis of Hayman and Martin [1974] for an marsupial ancestor with a 2n = 14 chromosome complement, which has been retained in 6 of the 7 marsupial orders. The highly conserved karyotype among species of the family Dasyuridae, including the devil, means that by anchoring genome sequence to chromosomes in this species [Deakin et al., 2012], we have essentially determined the gene order on all dasyurid chromosomes. It is important to note that the devil genome sequence assembly, which assigned sequence to chromosomes by sequencing of flow-sorted chromosomes [Murchison et al., 2012], perpetuates the mislabelling of devil chromosomes 1 and 2 noted above. Of course, there could be small-scale rearrangements that have escaped detection by G-banding that could differentiate members of this family, but most genes would be predicted to be present in the same order. Once further gene mapping is completed for this species, enabling the generation of a virtual map of the genome, it will be possible to predict the gene order in the ancestral 2n = 14 plesiomorphic karyotype, taking into account the inversions differentiating the standard dasyurid karyotype from that of the marsupial ancestor. Interpreting Interstitial Telomeric Sequences in Marsupial Chromosome Evolution Svartman and Vianna-Morgante [1998] explored the alternative hypothesis of a 2n = 22 marsupial ancestor. They argued that the 2n = 14 complement is derived from fusion of chromosomes from a 2n = 22 ancestor, using the presence of ITS in 2n = 14 and 2n = 18 species, and the absence of ITS in 2n = 22 species as evidence of such fusion events. They claim, for example, that ITS signals detected at the pericentric region of M. domestica chromosome 1 is evidence of a fusion between 2 and 10 of a putative 2n = 22 plesiomorphic karyotype. However, several other studies detecting ITS in American and Australian marsupial species demonstrated the co-localisation of these ITS with constitutive heterochromatin, suggesting that these ITS represent a component of satellite DNA and did not necessarily imply a past fusion event [Pagnozzi et al., 2000, 2002; Metcalfe et al., 2007]. However, Svartman [2009] claimed that, at least for M. incanus, the ITS signals are not within the boundaries of the pericentric heterochromatin, leaving it open for debate as to 118


whether or not ITS can be used as evidence of past fusion events in marsupials. Mapping ITS onto marsupial chromosome homology maps (fig. 2) makes clear that many ITS do not correspond to past fusion events. For instance, ITS detected in the pericentric region of 2 Sminthopsis species are unlikely to be evidence of fusion of chromosomes derived from a 2n = 22 complement. ITS signals are detected on chromosomes 1, 2, 3, and 6 in these species. If the marsupial ancestor was 2n = 22, chromosome 1 in these species would be the result of a fusion between a chromosome containing conserved segments C1, C2 and C3 with a chromosome consisting of segments C4–C6. However, chromosome painting indicates that this chromosome has been subject to at least 2 inversions [De Leo et al., 1999]. Thus, telomeric remnants of a fusion event would not be expected at the centromere on this chromosome in Sminthopsis species. Similarly, ITS on chromosome 6 are not evidence of a past fusion event, as this chromosome corresponds to a single chromosome in the predicted 2n = 22 ancestral karyotype [Metcalfe et al., 2007]. Furthermore, ITS were detected at pericentric locations on all chromosomes of tammar wallaby and the slender mouse opossum (Marmosops parvidens) [Pagnozzi et al., 2002], which were certainly not all formed by a fusion event. Indeed, tammar wallaby chromosome 4 consisting of conserved segments C4, C5 and C6, which form a single chromosome in the predicted ancestral marsupial 2n = 22 karyotype, has a strong ITS signal [Metcalfe et al., 2007]. ITS signals, therefore, do not provide conclusive evidence of past fusion events. Rather, some ITS on chromosomes not formed by centric fusions actually appear to correspond to remnants of telomeres from inversion events. This accounts for the ITS signals in 2n = 14 Sminthopsis species and the tammar wallaby chromosomes 4 and 5. Likewise, the ITS on M. domestica chromosome 1 are more likely to be the remnants of an inversion of the short arm containing segments C7 and C8 than a centric fusion. Karyotypic Evolution in Macropodiformes Although marsupials are renowned for karyotypic conservation, some groups have been particularly prone to karyotypic change. This is exemplified by members of the suborder Macropodiformes, which consists of 3 extant families (Macropodidae, Potoroidae, Hypsiprymnodontidae). The Macropodiformes have an ancestral 2n = 22 complement, which differs from the 2n = 22 karyotype of ameridelphian species. This was initially proposed from G-banding studies [Rofe, 1978] and has Deakin /Graves /Rens

since been verified using cross-species chromosome painting on members of the Macropodidae and Potoroidae families [Glas et al., 1999a; O’Neill et al., 1999; Rens et al., 1999, 2003]. This macropodiformes ancestral chromosome karyotype can be derived from the 2n = 14 ancestral marsupial complement by 5 fissions, 1 fusion and several inversions and centric shifts (fig. 3) [Rofe, 1978; Hayman, 1990; Eldridge and Close, 1993; Rens et al., 1999]. The macropodid ancestral 2n = 22 karyotype is observed to have been retained in some species in all 3 Macropodiformes families. Examples of various macropodiform karyotypes are shown in figure 4. The family Macropodidae (kangaroos and wallabies) have experienced extensive karyotypic change since their divergence approximately 23 mya. As a result, their chromosome evolution has been intensively studied by Gbanding [Rofe, 1978; Eldridge et al., 1989, 1990, 1991, 1992a, b; Eldridge and Pearson, 1997; Alsop et al., 2005], chromosome painting [Toder et al., 1997; Glas et al., 1999a; O’Neill et al., 1999] and FISH detection of ITS [Metcalfe et al., 1997, 1998, 2002, 2004, 2007]. It is clear that the 18 autosomal conserved segments identified by Rens et al. [2003] have undergone considerable reshuffling in this family. Even species within the same genus possessing the same diploid number and morphologically similar chromosomes do not have the same arrangement of the 18 conserved segments, and it appears they have undergone independent Robertsonian fusions of different ancestral chromosomes. This is illustrated by 2 species belonging the genus Macropus with 2n = 16 karyotypes, the tammar wallaby and the gray kangaroo (M. giganteus). Three of their chromosomes have been formed by the fusion of different conserved segments. For example, M. eugenii chromosome 1 was formed by the fusion of ancestral macropodiform (AnMac) chromosome 1 (C8, C9) to AnMac chromosome 10 (C1), whereas AnMac chromosome 1 fused to AnMac chromosome 8 (C2,C3) to form M. giganteus chromosome 1 (fig. 4). The swamp wallaby (Macropus/Wallabia bicolor) possesses one of the most derived marsupial karyotypes, having only 4 very large autosomes and an XX female, XY1Y2 male sex chromosome system that resulted from an X-autosome fusion (see section on sex chromosomes). Chromosome painting demonstrated that chromosome 1 in this species has been formed by a centric fusion of AnMac chromosomes 6 and 10 and the tandem fusion of AnMac chromosome 4 and 10 [Toder et al., 1997]. The distribution of telomeric sequences on M. bicolor chromosome 1 indicate that in addition to these fusion events, an inversion event has probably taken place involving Marsupial and Monotreme Chromosomes

Fig. 3. Derivation of the Macropodiformes ancestral 2n = 22

karyotype from the plesiomorphic 2n = 14 marsupial chromosome complement. Chromosomes are colour-coded as for the 19 conserved chromosome segments in figure 2. The sites of fusions, inversions and centric shifts are indicated.

conserved segment C4, as there are 3 sets of ITS rather than the expected 2 [Metcalfe et al., 1998] (fig. 4). A centric fusion has given rise to M. bicolor chromosome 2 and the remaining 2 autosomes are equivalent to tammar wallaby chromosomes 3 and 5. Convergence and Reversal in Marsupial Karotypic Evolution Macropodid karyotypes also afford an opportunity to assess the extent of convergence and reversal in marsupial karyotypic evolution. Comparisons of the arrangement of 18 autosomal conserved chromosome segments across macropodids reveal several examples of convergence (independent origin of the same segment associations in different species) and reversal (a secondarily derived arrangement resembling that of the ancestor). Six AnMac chromosomes (1, 5, 6, 8, 9, and 10) have been frequently involved in chromosome restructuring throughout macropodid evolution (fig. 4) [Hayman, 1990; Eldridge and Close, 1993; Eldridge and Johnston, 1993; Bulazel et al., 2007]. The fusion of AnMac chromosome 9 Cytogenet Genome Res 2012;137:113–129

119

Fig. 4. Arrangement of 19 conserved chromosome segments in Macropodiformes. The phylogeny is based on that determined by Westerman et al. [2010]. Chromosomes are colour-coded as for the 19 conserved chromosome segments in figure 2. The location of ITS in species tested to date are indicated to the left of chromosomes. Red bars indicate ITS resulting from a centric fusion, whereas blue bars indicate ITS resulting from an inversion.

(C18) to AnMac chromosome 8 (C2 and C3) occurred independently in P. tridactylus and Dendrologous matschiei (Matschiei’s tree kangaroo). The fusion of AnMac chromosomes 3 and 6 in these species provide an example of reversal, as these chromosomes have reverted to the 2n = 14 ancestral association of segments C10, C11 and C12 on a single chromosome [Rens et al., 2003]. Comparisons made more widely across marsupials reveal examples of convergence. The fission of segments C8 /C9 and C13/14 in the ameridelphid species Didelphis marsupials, as well as in the australidelphid species T. vulpecula, are examples of convergence, as they occurred independently in these species [Rens et al., 2003]. Chromosome painting and gene mapping indicate that the fis120


sion of segments C10, C11 and C12 arose from different breakpoints in American and Australian species (fig. 2), providing another example of convergence [Rens et al., 2003; Deakin et al., 2008]. Marsupial Sex Chromosomes The X chromosome in placental mammals is unusually well conserved, perhaps because translocations and rearrangements interrupt an X inactivation mechanism that is coordinately controlled over the whole X chromosome. However, in contrast to the generally high conservation of marsupial autosomes, marsupial X chromosomes vary significantly in morphology, size and banding pattern, even between species with the conserved Deakin /Graves /Rens

2n = 14 karyotype [Rofe and Hayman, 1985]. Moreover, translocations or fusions between autosomes and the sex chromosomes are widespread amongst marsupials [Toder et al., 1997]. For instance, hybridization of tammar wallaby chromosome paints on the swamp wallaby (M. bicolor, 2n = 10,XX female, 2n = 11,XY1Y2 male) chromosomes revealed that the short arm of the swamp wallaby X chromosome was homologous to the tammar wallaby X, whereas the long arm shared homology with tammar wallaby chromosomes 2 and 7 (fig. 4), as does Y2, representing the autosome to which the X was fused. The tammar wallaby Y chromosome hybridized predominantly to Y1, corresponding to the original Y chromosome. The tammar wallaby Xp paint, corresponding to a region shared between the tammar wallaby X and Y chromosomes was also detected on both X and Y2. P. tridactylus also has an X chromosome formed by the fusion of segments C5 and C6 to C19, resulting in 2n = 12,XX females and 2n = 13,XY1Y2 males (fig. 4). The X chromosome of all macropodids includes the nucleolus organizer (NOR) and nucleolus-associated heterochromatin. This region lies on chromosome 5p in the ancestral 2n = 14 karyotype, but was translocated to the X and Y in an ancestral macropodid; the NOR has since degenerated on the Y in all but one species and has amplified and elaborated on the X in others. The X chromosome in most marsupials is represented by a single conserved chromosome segment (C19) [Rens et al., 2003]. Gene mapping and chromosome painting of the tammar wallaby X chromosome onto human chromosomes demonstrated homology between the wallaby X and two-thirds of the human X chromosome [Glas, 1999b]. The remainder of the human X chromosome maps to chromosome 5 in tammar wallaby and chromosome 3 in dasyurids, and to chromosomes 4 and 7 in the opossum (M. domestica) and also forms a separate evolutionary block in chicken. Thus, the marsupial X represents the original therian X chromosome to which an autosomal region was added early in the eutherian lineage. The X chromosome of eutherian mammals is highly conserved in gene content, and usually in gene order, even among the most distantly related species [Delgado et al., 2009]. This has been ascribed to the dosage compensation mechanism that is coordinated over the whole X [Ohno, 1967] by the XIST gene that transcribes a key non-coding RNA involved in X chromosome inactivation in eutherians. Surprisingly, comparisons of the order of genes on the X chromosomes of tammar wallaby, devil and opossum revealed extensive rearrangement [Deakin et al., 2008, 2012], which would account for the varia-

tion observed in G-banding patterns. This may relate to differences in the control of X chromosome inactivation between marsupial and placental mammals, particularly the absence of XIST in marsupials. Genes that flank the XIST locus in eutherians map to opposite ends of the X chromosome in 2 opossum species, M. domestica [Davidow et al., 2007; Hore et al., 2007; Shevchenko et al., 2007] and Didelphis virginiana [Shevchenko et al., 2007], and tammar wallaby [Deakin et al., 2008]. This correlates with the locus-specific and partial X inactivation observed for genes on the marsupial X chromosome [Deakin et al., 2009; Al Nadaf et al., 2010]. Perhaps the acquisition of XIST and the evolution of a more tightly controlled inactivation mechanism than the partial inactivation observed in marsupials [Deakin et al., 2009; Al Nadaf et al., 2010] selected against rearrangements that might disrupt a whole X inactivation mechanism. The basic marsupial Y chromosome is very small, in some species no more than 12 Mb [Toder et al., 2000 1997]. It represents a degraded relic of the original therian proto-Y chromosome, which was originally homologous to the original proto-X chromosome. To date, 12 genes have been mapped on the tammar wallaby Y chromosome, 11 of which have copies on the X chromosome from which they originated [Sankovic et al., 2006; Murtagh et al., 2012]. Orthologues of 10 of the tammar wallaby Y-borne gene sequences have been identified in the devil testis transcriptome [Murtagh et al., 2012]. This low tally of genes on the marsupial Y chromosome, nevertheless, far exceeds the 4 genes retained on the human Y from the original proto-XY, implying that the marsupial Y has been subject to a slower rate of degradation than the eutherian Y. Sex determination (or at least testis determination) in marsupials is thought to be via a male-dominant gene because XXY animals have testes and XO animals lack them. However, some sexual dimorphisms are evidently a function of the dosage of X chromosomes (or the presence of a paternal X) since XXY animals possess a pouch and mammary glands, and XO animals have a scrotum. Marsupials were crucial in the identification of the mammal sex determining gene; the first candidate ZFY was first eliminated by the finding that this gene is autosomal in marsupials [Sinclair et al., 1988]. This led to the discovery, in humans and mice, of the sex determining gene SRY, which is ubiquitous in eutherian mammals and has an orthologue on the tiny marsupial Y; however, marsupials also have a Y-borne copy of the human sex reversing gene ATRY, which presented a rival candidate.



121

Conclusions and Future Directions for Marsupial Chromosome Evolution The application of molecular cytogenetics to understanding the evolution of marsupial chromosomes has greatly advanced this field of research. The ancestral marsupial karyotype has been confidently reconstructed. There remain some important gaps, which can be filled in by a more thorough investigation of chromosome homologies and rearrangements in different taxa. For example, chromosome painting is yet to be performed on any members of the family Peramelidae (bandicoots), which predominantly have a 2n = 14 karyotype, and Petauroidea species with 2n = 22. Moreover, chromosome painting on at least one representative 2n = 14 and 2n = 22 American species will prove useful, as the conserved segment composition of chromosomes for all except M. domestica has been predicted based on homologies observed between G-banded chromosomes. Although chromosome painting is valuable for detecting global regions of homology, small-scale rearrangements, particularly within a conserved segment, are undetectable. From the comparisons of gene order for just 3 species, it is evident that inversions, large and small, have featured heavily in the evolution of marsupial chromosomes. The generation of gene maps for additional marsupial species would be valuable. Fortunately, BAC libraries are currently available for 2 additional species, Isoodon macroura (northern brown bandicoot, 2n = 14) and D. virginiana (2n = 22), making it possible for gene maps to be constructed for these species. These 2 species belong to lineages that are important for accurate predictions of gene order on ancestral marsupial chromosomes and will provide higher resolution of the rearrangements that have shaped marsupial chromosomes.

Molecular Cytogenetics and Functional Aspects of the Ever Surprising Monotreme Genome

Monotreme chromosomes have captivated cytogeneticists since their first controversial descriptions in the 1940s [reviewed in Murtagh and Sharman, 2009]. The presence of several unpaired chromosomes in mitosis and multivalent chains in male meiosis created an aura of mystery surrounding monotreme chromosomes. Attempts to characterize monotreme chromosomes by various banding patterns failed to conclusively distinguish the many small chromosomes [Wrigley and Graves, 1988a, b; Murtagh and Sharman, 2009]. It was only with 122


the aid of molecular cytogenetics that the puzzle of monotreme chromosomes was finally solved. The Monotreme Karyotypes In contrast to the few large and readily identifiable chromosomes characteristic of marsupial karyotypes, the monotreme genome consists of many smaller chromosomes. There are 6 large, easily distinguished chromosome pairs and many small chromosomes that are hard to differentiate. All platypus and echidna autosomes are either metacentric, submetacentric or subtelocentric; none of them are acrocentric. Platypus (Ornithorhynchus anatinus) somatic cells contain 26 pairs of chromosomes (2n = 52) [Warren et al., 2008, supp data]; the short-beaked echidna (Tachyglossus aculeatus) somatic nuclei contain 63 chromosomes in the male and 64 chromosomes in the female. The platypus genome has been sized at only 1.92 Gb DNA (measured by flow cytometry), a size more typical of reptile than mammal genomes. The autosomes are ordered according to their size measured by flow cytometry, which has the advantage of being a non-arbitrary measuring method important for comparative genomics. Sex chromosomes are complex and are discussed in detail below. In piecing together the puzzle of monotreme karyotypes, flow-sorted platypus chromosome paints were used to identify autosome pairs [Rens et al., 2004]. However, not all chromosomes could be physically separated into individual chromosomes, resulting in 4 paints that hybridized to more than one chromosome (e.g. chromosomes 9 and 12). To aid in the unambiguous identification of each platypus chromosome, a set of platypus anchor BAC clones (one for each chromosome) was developed [McMillan et al., 2007]. The combination of these resources has been a major step forward for monotreme cytogenetics, enabling the unambiguous localisation of genes to the smaller platypus chromosomes [Edwards et al., 2007; Grafodatskaya et al., 2007]. Unfortunately, it is difficult to establish the monotreme ancestral karyotype because there are only 2 extant families of monotremes, platypus and several closely related echidna species. Being basal to the mammalian radiation, there are no mammalian outgroup species. Chromosome rearrangements and homologies have been established between the platypus and short-beaked echidna [Rens et al., 2007]. Platypus chromosomes 1, 4, 5, 9, 11, 14, 16, and 19 are homologous to echidna chromosomes 1, 4, 3, 10, 11, 14, 19, and 22, respectively. All other chromosomes are derived from new combinations by centric fusion or fission in either species, showing that the monoDeakin /Graves /Rens

treme karyotypes have not been stable after the platypusechidna divergence around 30 mya [Phillips et al., 2009], but have continued to evolve, largely by Robertsonian fissions and fusions. How do monotreme karyotypes relate to those of placental mammals? Ruiz-Herrera et al. [2012] detected 7 syntenic segmental associations characteristic for the placental ancestral karyotype in Ultracontigs of the platypus genome database, which suggests that these placental ancestral associations are ancestral for all mammalian species. The monotreme karyotypes are highly rearranged when compared to the placental ancestral karyotype, meaning that the monotreme karyotypes have their own dynamics of chromosome evolution. The platypus assembly is still too incomplete to define the chromosome rearrangements that occurred during the evolution of the monotreme karyotypes from the mammalian ancestral karyotype. Monotreme Chromosomes Sequencing of the platypus genome revealed a relatively high GC content of 45.5% compared to 41% for most eutherian species [Warren et al., 2008]. It is uncertain whether a high GC content is characteristic of all monotremes because the echidna genome is not fully sequenced yet. However, the platypus and echidna flow karyotypes provide information on relative GC contents of the individual chromosomes of both species [Rens et al., 2004, 2007]. In both species, most of the chromosome peaks do not deviate into AT or GC richer regions, implying that most monotreme chromosomes have similar GC content. In platypus, chromosomes 6, 11, 12, 15, X 2, X4, and Y4 have a somewhat higher GC content, whereas in echidna the larger chromosome 1, 2 and 4 have a relative lower GC content, and chromosome 3, 6 and X5 have a relative higher GC content. The higher GC content for platypus chromosome 6 and echidna chromosome 3, 6 and X5 can be explained by their inclusion of the NOR. (Note that although both platypus and echidna chromosome 6 are NOR-bearing, chromosome painting reveals that they are non-homologous). The difference between the relative GC content of platypus and echidna chromosome 1, which are homologous, can be explained by an apparently AT-rich region that is identified on echidna chromosome 1 and 2 [Rens et al., 2007] and contains blocks of repetitive sequence [Kirby et al., 2007]. Similarly, the difference between platypus and echidna chromosome 4, which are also homologous, can be explained by another apparently ATrich region on echidna chromosome 4 [Rens et al., 2007]. Marsupial and Monotreme Chromosomes

These examples show that homologous chromosomes, as determined by cross-species chromosome painting, may be different at a sequence level, depending on acquisition and amplification of repetitive sequences. Centromeres are a crucial functional feature of chromosomes that appear as constrictions on metaphase chromosomes. In contrast to chromosomes of therian species, the centromeres of platypus chromosomes are not characterised by satellite DNA [Alkan et al., 2011]. Platypus chromosomes show nearly equal amounts of the retrotransposon LINE2 elements, but no LINE1 elements [Warren et al., 2008], in contrast to eutherian species in which the X chromosome contains a relatively large fraction of LINE1 elements. MON1 (SINE) elements are also abundant on platypus chromosomes [Kirby et al., 2007]. Young MON1 and LINE2 insertions are over-represented on the platypus Y chromosomes, a feature also seen in other species [Kortschak et al., 2009]. Platypus chromosomes do resemble reptilian chromosomes with respect to the low microsatellite content, with a high fraction of AT-rich microsatellites. The most common motifs in these microsatellites are also highly similar to the motifs used in lizard [Warren et al., 2008]. Monotreme Sex Chromosomes The evolution of the therian sex chromosome system has been studied in detail by researchers with interests ranging from sex determination and Y chromosome evolution to the mechanism of X inactivation. However, they are studying the complexity and functional implications of just 1 set of sex chromosomes among many different ones in vertebrates, and it is clear that the therian XY system evolved recently [Graves, 2008; Graves and Peichel, 2010]. The phylogenetic position of monotremes between mammals and reptiles means that sex chromosomes of monotremes can fill an important gap in our understanding of how the therian XY system evolved. Monotremes have multiple sex chromosomes with different sizes and genetic contents. The raison d’être and evolutionary history of the monotreme sex chromosome complement is still very puzzling. A first step towards its understanding is the characterisation of the sex chromosomes and the comparison with genomes of other species. The male platypus sex chromosome system consists of 5 different X and 5 different Y chromosomes. The female platypus has the 5 X chromosomes in pairs and contains no Y-specific sequences. The segregation of the 5 X chromosomes from the 5 Y chromosomes during meiosis into separate X- and Y-bearing sperm must occur via a polarised meiosis and be very accurate, since painting revealed Cytogenet Genome Res 2012;137:113–129

123

Fig. 5. The platypus sex chromosome chain showing the pseudoautosomal regions (indicated by grey crosses), homology to the chicken Z (green), the location of MHC genes, and candidate sex determining genes DMRT1, CRSPX and CRSPY.

no sperm or spermatogonia with mixtures of X and Y chromosomes [Grützner et al., 2004]. All 10 platypus sex chromosomes were unambiguously identified by chromosome sorting followed by chromosome painting. The sizes of these chromosomes differ considerably. X1 is the largest sex chromosome with a DNA content of 106 Mb (measured by flow cytometry), which is between the DNA content of platypus chromosome 4 and 5 (123 and 92 Mb, respectively). Platypus X4 is the smallest X chromosome (19 Mb). Y1 is the largest Y chromosome with a DNA content of 48 Mb and Y5 is the smallest Y-chromosome (DNA content too small to be measured by flow cytometry). The order of the sex chromosomes in the karyotype is not determined by size (as for autosomes), but according to their position in the male meiotic chain (fig. 5), which was ascertained by chromosome painting [Grützner et al., 2004]. The sex chromosome system evidently originated by serial translocation of a single XY pair with autosomes [Grützner et al., 2003, 2004; Rens et al., 2004]. Each sex chromosome has a region that pairs with its neighbour during meiosis. Thus a terminal region of X1 pairs with one end of the Y1, and the other end of Y1 pairs with X 2; the X 2 pairs with Y2 etc. The pairing regions occupy substantial pseudoautosomal regions at the tips of sex chromosomes, but the Y-specific regions are heterochromatic and much shorter than the comparable regions of the X, implying that the Y-chromosomes have 124


degenerated during platypus evolution. For instance, Y1 has lost around 50 Mb of DNA, and Y5 has lost around 65 Mb of DNA. In comparison, chromosome size difference between human X and Y is around 100 Mb. Because of the complexity of the platypus sex chromosome system, one may expect that the echidna has either a ‘normal’ one-X-one-Y system or exactly the same system as the platypus. A comparison performed by crossspecies chromosome painting revealed that neither is the case [Rens et al., 2007]. The short-beaked echidna also has a multiple sex chromosome system, but the male has only 9 sex chromosomes: 5 X and 4 Y chromosomes. The missing Y chromosome is the tiny Y5; however, chromosome painting with platypus Y5 reveals that this chromosome has fused with Y3. The female has the 5 X chromosomes in pairs. Surprisingly, not all of the sex chromosomes of platypus and echidna are homologous. Platypus and echidna have X1, X 2 and X3 in common, but platypus X4 is homologous to an autosome (chromosome 27) in echidna, and echidna X5 corresponds to a platypus autosome (chromosome 12p). The best explanation for this difference is that the original sex pair fused with an autosome in a common ancestor, producing a chain of 4. Two other fusions followed, producing a chain of 8, before platypus and echidna diverged 30 mya. Then an additional fusion occurred with a different autosome in the 2 lineages. The platypus-echidna difference shows that the translocation chain still evolved after platypus and echidna diverged around 30 mya and supports the hypothesis of consecutive translocations. The homology of platypus X4 to an echidna autosome is significant for the location of the MHC cluster in platypus. MHC class I and II genes are found on platypus X3 and Y3, and MHC class III is located on platypus Y4 and X5 (fig. 5) [Dohm et al., 2007]. Thus, these 2 clusters were separated by translocation with X4. However, in echidna the MHC cluster is on neighbouring sex chromosomes because platypus X4 is homologous to an echidna autosome. Attempts to Reconstruct the Ancestral Monotreme Karyotype It would be an advantage to know which chromosomes of the sauropsid ancestral karyotype became caught up in the sex chromosome system. The ancestral monotreme sex chromosomes could potentially be deduced by comparing homologous regions with an extant reptile outgroup. The first important and surprising finding was that the orthologue of the chicken DMRT1 gene, a strong canDeakin /Graves /Rens

didate for the bird sex determining gene, is located on X5, the last X chromosome in the platypus sex chromosome chain [Grützner et al., 2004]. This prompted the search for other chicken Z orthologues. Many chicken Z genes can be found on the platypus sex chromosomes. Platypus X5 has a large coverage of chicken Z genes, but X1, X 2 and X3 also contain chicken Z genes [Rens et al., 2007; Veyrunes et al., 2008]. No chicken Z genes have so far been mapped to X4, perhaps because platypus X4 is homologous to an autosome in echidna, so it was a late recruit to the chain. Do the platypus sex chromosomes relate to the eutherian sex chromosomes? The answer is no. The platypus multiple XY system does not correspond to the therian XY- system. Early mapping using heterologous cDNA probes suggested that platypus X1 is homologous to the eutherian X [Wrigley and Graves, 1988a]. However, by investigating the content of platypus X1 and by comparative mapping of eutherian X orthologues, it was discovered that X1 corresponds to eutherian autosome regions [Rens et al., 2007], and new gene mapping methods established that orthologues of genes within the conserved region of the X shared by eutherians and marsupials are all located on platypus chromosome 6 [Waters et al., 2005; Veyrunes et al., 2008]. The location of some chicken Z genes on platypus autosomes and the location of some chicken autosome genes on platypus Z chromosomes probably resulted from serial reciprocal translocations between Z-containing members of the chain and newly recruited autosomes [Rens et al., 2007; Veyrunes et al., 2008]. This is consistent with serial translocations of ancestral autosomes into the chain. Thus, the sex chromosomes of platypus have no homology to the XY system of therian mammals, but very substantial homology with the ZW system of birds. This is a very significant finding for the study of eutherian sex chromosome evolution, implying that the eutherian X is much younger (166 my) than had been thought, and so must have evolved in a faster rate [Graves, 2006]. Monotreme Sex Determination The non-homology of the platypus and eutherian sex chromosomes, and the position of SOX3 (from which SRY evolved) on platypus chromosome 6, indicates that monotreme sex determination is not initiated by SRY, but by a different (unknown) sex determining gene(s). The most obvious candidate is DMRT1, now known to be sex determining in birds: the expectation was that DMRT1 became mutated or amplified on a Y-chromosome in order to initiate male development, in much the same way as in MeMarsupial and Monotreme Chromosomes

daka [Kondo et al., 2009]. However, DMRT1 lies on X5, so it is present in 2 copies in females and a single copy in males, quite the reverse of its dosage in birds [El-Mogharbel et al., 2007]. Thus, it is thought that some other gene, probably located on Y5, is responsible for testis determination. It is tempting to propose that the chicken Z chromosome is the ancestral sauropsid sex chromosome, given the homology between bird and monotreme sex chromosomes, and the more recent finding that a gekko shares an almost identical Z chromosome [Kawai et al., 2009], although this seems not to be the ancestral gekko sex pair. In a large number of representative sauropsid species, the chromosome that became the Z in birds is conserved as one entity, although it is not a sex chromosome in all these studied species [Pokorna et al., 2011]. The conservation is a reflection of the high degree of general chromosome conservation in sauropsids [Pokorna et al., submitted]. Thus, the homology between the bird ZW and platypus sex chromosome chain might be an example of homoplasy that this autosome evolved into a sex chromosome in platypus, gekko and chicken. It is an interesting thought, however, that this autosome may have been particularly suitable to become a sex chromosome [O’Meally et al., 2012]. Vertebrate sex determination is regulated by a complex genetic network comprising several genes; the cascade is initiated by the SRY gene. There is no SRY in monotremes, and the gene from which it evolved (SOX3) is autosomal [Wallis et al., 2007]. Each of the genes in the therian testis determining pathway may be a candidate(s) for the primary sex determining gene(s) in monotremes. A first indication will be the location of this gene(s) on just one of the sex chromosomes (either a dose-dependent gene on the X, or a male-dominant gene on the Y). Several orthologues of genes active early in eutherian sex determination/differentiation have been mapped by FISH or PCR mapping using sorted chromosomes as templates. Possible candidates are the genes WT1, SF1, LHX1, LHX2, FGF9, ATRX, and DAX1, which are active in eutherian male sex determination/ differentiation. SOX9 has a critical conserved role in vertebrate sex determination, since its activation and maintenance is needed for male development. However, SOX9 is located on platypus chromosome 15, so it is not the primary sex determining gene [Wallis et al., 2007]. SF1 (NR5A1) has a crucial role early in eutherian sex determination by aiding SRY to bind to the TESCO enhancer of SOX9; however, its autosome location excludes such a role in platypus. SOX3 is also sex-reversing when expressed ectopically in somatic Cytogenet Genome Res 2012;137:113–129

125

cells of testis [Sutton et al., 2011], but its location on platypus autosome 6 excludes it from this primary role [Wallis et al., 2007]. GATA4 is located on Y1 and X 2, so it is unlikely to function as the primary sex determining gene [Grafodatskaya et al., 2007]. Only 2 genes active in female sex determination have been mapped so far, WNT4 and RSPO1. These 2 genes are located on platypus chromosome 5 and 16, positions that make a primary sex determining role unlikely [Grafodatskaya et al., 2007]. Recently, homologues of human CRSP7 were localised on platypus X1 and Y5. The X1 homologue (CRSPX) and the Y5 (CRSPY) homologue are diverged with different expression levels in adult testis. However, neither activated mouse Sox9 in a reporter assay [Tsend-Ayush et al., 2012]. The hunt for the monotreme sex determining gene continues focusing on the tiny Y5 of platypus, which is the smallest and most degraded Y, so it is probably the oldest and most original. This chromosome was first dismissed as a candidate because it is absent in echidna; however, it was discovered to have been translocated onto Y3 in this species [Rens et al., 2007]. In eutherians, it is likely that the acquisition of SRY initiated the degeneration of the eutherian Y chromosome. Specialisation of adjacent genes to perform maleadvantage functions selected for restriction of recombination at this site. The simplest assumption is that a primary sex determinant is localised on just one Y chromosome, rather than multiple factors being spread between all the Y chromosomes in the monotreme sex chromosome system. However, male-specific (e.g. spermatogenesis) genes other than the primary sex determinant may be located on the differential regions of the Y chromosomes and were also initiators of restriction of recombination at their sites. This led to degeneration of all Y chromosomes so that most genes on the X chromosomes (amounting to about 15% of the genome) lost their active partner and are therefore present in different dosages in XY males and XX females. Conclusions and Future Directions The above discourse shows that the monotreme genome has enriched our knowledge on mammalian genome evolution. Monotreme genomes will no doubt continue to be important for investigations into the appearance and evolution of specific mammalian traits, such as lactation, essential for an understanding of mammalian biology and evolution. At present, the patchy assignment of the platypus genome assembly to chromosomes makes it difficult to de126


termine the ancestral karyotype of all mammals. The genome of the short-beaked echidna (T. aculeatus) is currently being sequenced, and it would be advantageous for genome evolution studies to anchor this sequence to chromosomes, which will enable the demarcation of rearrangements between platypus and echidna to be determined. It is hoped that future research will make it possible to compare the platypus genome arrangement to that of outgroup species such as chicken and anole lizard.

Concluding Remarks

Our understanding of marsupial and monotreme chromosomes and their evolution has greatly advanced due largely to the molecular cytogenetic techniques and the availability of genome sequence data for representative species. Comparisons of gene arrangement between all 3 major mammalian lineages and other amniotes have provided a new perspective on the evolution of mammalian chromosomes, particularly the sex chromosomes. The inclusion of these species in comparative studies has and will continue to greatly impact on our understanding of the evolutionary events that have shaped mammalian genomes.

Acknowledgement J.E.D. is supported by an Australian Research Council Future Fellowship.

References

Al Nadaf S, Waters PD, Koina E, Deakin JE, Jordan KS, Graves JA: Activity map of the tammar X chromosome shows that marsupial X inactivation is incomplete and escape is stochastic. Genome Biol 11:R122 (2010). Alkan C, Cardone MF, Catacchio CR, Antonacci F, O’Brien SJ, et al: Genome-wide characterization of centromeric satellites from multiple mammalian genomes. Genome Res 21: 137–145 (2011). Alsop AE, Miethke P, Rofe R, Koina E, Sankovic N, et al: Characterizing the chromosomes of the Australian model marsupial Macropus eugenii (tammar wallaby). Chromosome Res 13:627–636 (2005). Beck RM: A dated phylogeny of marsupials using a molecular supermatrix and multiple fossil constraints. J Mammal 89:175–189 (2008). Bulazel KV, Ferreri GC, Eldridge MD, O’Neill RJ: Species-specific shifts in centromere sequence composition are coincident with breakpoint reuse in karyotypically divergent lineages. Genome Biol 8:R170 (2007).

Deakin /Graves /Rens

Davidow LS, Breen M, Duke SE, Samollow PB, McCarrey JR, Lee JT: The search for a marsupial XIC reveals a break with vertebrate synteny. Chromosome Res 15: 137–146 (2007). Deakin JE: Physcial and comparative gene maps in marsupials, in Deakin JE, Waters PD, Graves JA (eds): Marsupial Genetics and Genomics, pp 101–115 (Springer, Dordrecht 2010). Deakin JE, Koina E, Waters PD, Doherty R, Patel VS, et al: Physical map of two tammar wallaby chromosomes: a strategy for mapping in non-model mammals. Chromosome Res 16: 1159–1175 (2008). Deakin JE, Chaumeil J, Hore TA, Graves JA: Unravelling the evolutionary origins of X chromosome inactivation in mammals: insights from marsupials and monotremes. Chromosome Res 17:671–685 (2009). Deakin JE, Bender HS, Pearse AM, Rens W, O’Brien PC, et al: Genomic restructuring in the Tasmanian devil facial tumour: chromosome painting and gene mapping provide clues to evolution of a transmissible tumour. PLoS Genet 8:e1002483 (2012). De Leo AA, Guedelha N, Toder R, Voullaire L, Ferguson-Smith MA, et al: Comparative chromosome painting between marsupial orders: relationships with a 2n = 14 ancestral marsupial karyotype. Chromosome Res 7: 509–517 (1999). Delgado CL, Waters PD, Gilbert C, Robinson TJ, Graves JA: Physical mapping of the elephant X chromosome: conservation of gene order over 105 million years. Chromosome Res 17: 917–926 (2009). Dohm JC, Tsend-Ayush E, Reinhardt R, Grützner F, Himmelbauer H: Disruption and pseudoautosomal localization of the major histocompatibility complex in monotremes. Genome Biol 8:R175 (2007). Duke SE, Samollow PB, Mauceli E, Lindblad-Toh K, Breen M: Integrated cytogenetic BAC map of the genome of the gray, short-tailed opossum, Monodelphis domestica. Chromosome Res 15:361–370 (2007). Edwards CA, Rens W, Clarke O, Mungall AJ, Hore T, et al: The evolution of imprinting: chromosomal mapping of orthologues of mammalian imprinted domains in monotreme and marsupial mammals. BMC Evol Biol 7:157 (2007). Eldridge MD, Close RL: Radiation of chromosome shuffles. Curr Opin Genet Dev 3: 915– 922 (1993). Eldridge MD, Johnston PG: Chromosomal rearrangements in rock wallabies, Petrogale (Marsupialia, Macropodidae).VIII. An investigation of the nonrandom nature of karyotypic change. Genome 36: 524–534 (1993).


Eldridge MD, Pearson DJ: Chromosomal rearrangements in rock wallabies, Petrogale (Marsupialia: Macropodidae).IX. Further G-banding studies of the Petrogale lateralis complex: P. lateralis pearsoni, the west kimberley race, and a population heterozygous for a centric fusion. Genome 40: 84–90 (1997). Eldridge MD, Johnston PG, Close RL, Lowry PS: Chromosomal rearrangements in rock wallabies, Petrogale (Marsupialia, Macropodidae).II. G-banding analysis of Petrogale godmani. Genome 32:935–940 (1989). Eldridge MD, Close RL, Johnston PG: Chromosomal rearrangements in rock wallabies, Petrogale (Marsupialia, Macropodidae).III. G-banding analysis of Petrogale inornata and P. penicillata. Genome 33: 798–802 (1990). Eldridge MD, Close RL, Johnston PG: Chromosomal rearrangements in rock wallabies, Petrogale (Marsupialia, Macropodidae).IV. G-banding analysis of the Petrogale lateralis complex. Aust J Zool 39:621–627 (1991). Eldridge MD, Johnston PG, Close RL: Chromosomal rearrangements in rock wallabies, Petrogale (Marsupialia, Macropodidae).VI. Determination of the plesiomorphic karyotype: G-banding comparison of Thylogale with Petrogale persephone, P. xanthopus, and P. l. lateralis. Cytogenet Cell Genet 61:29–33 (1992a). Eldridge MD, Johnston PG, Lowry PS: Chromosomal rearrangements in rock wallabies, Petrogale (Marsupialia, Macropodidae).VII. G-banding analysis of Petrogale brachyotis and P. concinna: species with dramatically altered karyotypes. Cytogenet Cell Genet 61: 34–39 (1992b). El-Mogharbel N, Wakefield M, Deakin JE, Tsend-Ayush E, Grützner F, et al: DMRT gene cluster analysis in the platypus: new insights into genomic organization and regulatory regions. Genomics 89:10–21 (2007). Glas R, De Leo AA, Delbridge ML, Reid K, Ferguson-Smith MA, et al: Chromosome painting in marsupials: genome conservation in the kangaroo family. Chromosome Res 7: 167–176 (1999a). Glas R, Marshall Graves JA, Toder R, FergusonSmith M, O’Brien PC: Cross-species chromosome painting between human and marsupial directly demonstrates the ancient region of the mammalian X. Mamm Genome 10:1115–1116 (1999b). Grafodatskaya D, Rens W, Wallis MC, Trifonov V, O’Brien PC, et al: Search for the sex-determining switch in monotremes: mapping WT1, SF1, LHX1, LHX2, FGF9, WNT4, RSPO1 and GATA4 in platypus. Chromosome Res 15:777–785 (2007). Graves JA: Sex chromosome specialization and degeneration in mammals. Cell 124:901–914 (2006). Graves JA: Weird animal genomes and the evolution of vertebrate sex and sex chromosomes. Annu Rev Genet 42:565–586 (2008).

Graves JA, Peichel CL: Are homologies in vertebrate sex determination due to shared ancestry or to limited options? Genome Biol 11: 205 (2010). Grützner F, Deakin J, Rens W, El-Mogharbel N, Graves JA: The monotreme genome: a patchwork of reptile, mammal and unique features? Comp Biochem Physiol A Mol Integr Physiol 136:867–881 (2003). Grützner F, Rens W, Tsend-Ayush E, El-Mogharbel N, O’Brien PC, et al: In the platypus a meiotic chain of ten sex chromosomes shares genes with the bird Z and mammal X chromosomes. Nature 432: 913–917 (2004). Hayman DL: Marsupial cytogenetics. Aust J Zool 37:331–349 (1990). Hayman DL, Martin PG: Cytogenetics of marsupials, in Benirschke K (ed): Comparative Mammalian Cytogenetics, pp 191–217 (Springer, New York 1969). Hayman DL, Martin PG: Mammalia I: Monotremata and marsupialia, in John B (ed): Animal Cytogenetics (Gebrüder Borntraeger, Berlin and Stuttgart 1974). Hore TA, Koina E, Wakefield MJ, Marshall Graves JA: The region homologous to the Xchromosome inactivation centre has been disrupted in marsupial and monotreme mammals. Chromosome Res 15: 147–161 (2007). Kawai A, Ishijima J, Nishida C, Kosaka A, Ota H, et al: The ZW sex chromosomes of Gekko hokouensis (Gekkonidae, Squamata) represent highly conserved homology with those of avian species. Chromosoma 118: 43–51 (2009). Kirby PJ, Greaves IK, Koina E, Waters PD, Marshall Graves JA: Core-SINE blocks comprise a large fraction of monotreme genomes; implications for vertebrate chromosome evolution. Chromosome Res 15: 975–984 (2007). Kondo M, Nanda I, Schmid M, Schartl M: Sex determination and sex chromosome evolution: insights from medaka. Sex Dev 3:88–98 (2009). Kortschak RD, Tsend-Ayush E, Grützner F: Analysis of SINE and LINE repeat content of Y chromosomes in the platypus, Ornithorhynchus anatinus. Reprod Fertil Dev 21: 964–975 (2009). Luo ZX, Yuan CX, Meng QJ, Ji Q: A Jurassic eutherian mammal and divergence of marsupials and placentals. Nature 476: 442–445 (2011). Martin PG, Hayman DL: Quantitative comparisons between the karyotypes of Australian marsupials from three different superfamilies. Chromosoma 20:290–310 (1967). McMillan D, Miethke P, Alsop AE, Rens W, O’Brien P, et al: Characterizing the chromosomes of the platypus (Ornithorhynchus anatinus). Chromosome Res 15: 961–974 (2007).


127

Meredith RW, Westerman M, Case JA, Springer MS: A phylogeny and timescale for marsupial evolution based on sequences for five nuclear genes. J Mamm Evol 15: 1–36 (2008). Metcalfe CJ, Eldridge MD, McQuade LR, Johnston PG: Mapping the distribution of the telomeric sequence (T2AG3)n in rock-wallabies, Petrogale (Marsupialia: Macropodidae), by fluorescence in situ hybridization.I. The penicillata complex. Cytogenet Cell Genet 78:74–80 (1997). Metcalfe CJ, Eldridge MD, Toder R, Johnston PG: Mapping the distribution of the telomeric sequence (T2AG3)n in the Macropodoidea (Marsupialia), by fluorescence in situ hybridization. I. The swamp wallaby, Wallabia bicolor. Chromosome Res 6:603–610 (1998). Metcalfe CJ, Eldridge MD, Johnston PG: Mapping the distribution of the telomeric sequence (T2AG3)n in rock wallabies, Petrogale (Marsupialia: Macropodidae), by fluorescence in situ hybridization. II. The lateralis complex. Cytogenet Genome Res 96:169–175 (2002). Metcalfe CJ, Eldridge MD, Johnston PG: Mapping the distribution of the telomeric sequence (T2AG3)n in the 2n = 14 ancestral marsupial complement and in the Macropodines (Marsupialia: Macropodidae) by fluorescence in situ hybridization. Chromosome Res 12:405–414 (2004). Metcalfe CJ, Eldridge MD, Johnston PG: Mapping the distribution of the telomeric sequence (T2AG3)n in the Macropodoidea (Marsupialia) by fluorescence in situ hybridization. II. The ancestral 2n = 22 macropodid karyotype. Cytogenet Genome Res 116: 212– 217 (2007). Mikkelsen TS, Wakefield MJ, Aken B, Amemiya CT, Chang JL, et al: Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences. Nature 447: 167–177 (2007). Miller W, Hayes VM, Ratan A, Petersen DC, Wittekindt NE, et al: Genetic diversity and population structure of the endangered marsupial Sarcophilus harrisii (Tasmanian devil). Proc Natl Acad Sci USA 108:12348–12353 (2011). Mohammadi A, Delbridge ML, Waters PD, Graves JA: Conservation of a chromosome arm in two distantly related marsupial species. Cytogenet Genome Res 124: 147–150 (2009). Murchison EP, Schulz-Trieglaff OB, Ning Z, Alexandrov LB, Bauer MJ, et al: Genome sequencing and analysis of the Tasmanian devil and its transmissible cancer. Cell 148:780– 791 (2012). Murtagh CE, Sharman GB: Monotreme chromosomes: an introductory review. Aust J Zool 57:149–155 (2009). Murtagh VJ, O’Meally D, Sankovic N, Delbridge ML, Kuroki Y, et al: Evolutionary history of novel genes on the tammar wallaby Y chromosome: implications for sex chromosome evolution. Genome Res 22:498–507 (2012).

128

Ohno S: Sex Chromosomes and Sex-Linked Genes (Springer, Berlin 1967). O’Meally D, Ezaz T, Georges A, Sarre SD, Graves JA: Are some chromosomes particularly good at sex? Insights from amniotes. Chromosome Res 20:7–19 (2012). O’Neill RJ, Eldridge MD, Toder R, FergusonSmith MA, O’Brien PC, Graves JAM: Chromosome evolution in kangaroos (Marsupialia: Macropodidae): cross species chromosome painting between the tammar wallaby and rock wallaby spp. with the 2n = 22 ancestral macropodid karyotype. Genome 42: 525–530 (1999). Pagnozzi JM, De Jesus Silva MJ, Yonenaga-Yassuda Y: Intraspecific variation in the distribution of the interstitial telomeric (T2AGGG)n sequences in Micoureus demerarae (Marsupialia: Didelphidae). Chromosome Res 8:585–591 (2000). Pagnozzi JM, Ditchfield AD, Yonenaga-Yassuda Y: Mapping the distribution of the interstitial telomeric (T2AG3)n sequences in eight species of Brazilian marsupials (Didelphidae) by FISH and the correlation with constitutive heterochromatin. Do ITS represent evidence for fusion events in American marsupials? Cytogenet Genome Res 98: 278–284 (2002). Pearse AM, Swift K: Allograft theory: transmission of devil facial-tumour disease. Nature 439:549 (2006). Phillips MJ, Bennett TH, Lee MS: Molecules, morphology, and ecology indicate a recent, amphibious ancestry for echidnas. Proc Natl Acad Sci USA 106:17089–17094 (2009). Pokorna M, Giovannotti M, Kratochvil L, Kasai F, Trifonov VA, et al: Strong conservation of the bird Z chromosome in reptilian genomes is revealed by comparative painting despite 275 million years divergence. Chromosoma 120:455–468 (2011). Reig OA, Gardner AL, Bianchi NO, Patton JL: The chromosomes of the Didelphidae (Marsupialia) and their evolutionary significance. Biol J Linnean Soc 9:191–216 (1977). Renfree MB, Papenfuss AT, Deakin JE, Lindsay J, Heider T, et al: Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development. Genome Biol 12: 414 (2011). Rens W, Ferguson-Smith MA: The conserved marsupial karyotype: chromosome painting and evolution, in Deakin JE, Waters PD, Graves JAM (eds): Marsupial Genetics and Genomics, pp 37–53 (Springer, Dordrecht 2010). Rens W, O’Brien PC, Yang F, Graves JA, Ferguson-Smith MA: Karyotype relationships between four distantly related marsupials revealed by reciprocal chromosome painting. Chromosome Res 7:461–474 (1999).


Rens W, O’Brien PC, Yang F, Solanky N, Perelman P, et al: Karyotype relationships between distantly related marsupials from South America and Australia. Chromosome Res 9:301–308 (2001). Rens W, O’Brien PC, Fairclough H, Harman L, Graves JA, Ferguson-Smith MA: Reversal and convergence in marsupial chromosome evolution. Cytogenet Genome Res 102: 282– 290 (2003). Rens W, Grützner F, O’Brien PC, Fairclough H, Graves JA, Ferguson-Smith MA: Resolution and evolution of the duckbilled platypus karyotype with an X1Y1X2Y2X3Y3X4Y4X5Y5 male sex chromosome constitution. Proc Natl Acad Sci USA 101:16257–16261 (2004). Rens W, O’Brien PC, Grützner F, Clarke O, Graphodatskaya D, et al: The multiple sex chromosomes of platypus and echidna are not completely identical and several share homology with the avian Z. Genome Biol 8:R243 (2007). Rofe R, Hayman D: G-banding evidence for a conserved complement in the Marsupialia. Cytogenet Cell Genet 39:40–50 (1985). Rofe RH: G-banded chromosomes and the evolution of Macropodidae. Aust Mammal 2: 53–63 (1978). Sankovic N, Delbridge ML, Grützner F, Ferguson-Smith MA, O’Brien PCM, Marshall Graves JA: Construction of a highly enriched marsupial Y chromosome-specific BAC sublibrary using isolated Y chromosomes. Chromosome Res 14: 657–664 (2006). Sharman GB: The chromosomes of non-eutherian mammals, in Chiarelli AN, Capanna E (eds): Cytotaxonomy and Vertebrate Evolution, pp 485–530 (Academic Press, New York 1973). Sharman GB: Marsupial taxonomy and phylogeny. Aust Mammal 1:137–154 (1974). Shevchenko AI, Zakharova IS, Elisaphenko EA, Kolesnikov NN, Whitehead S, et al: Genes flanking Xist in mouse and human are separated on the X chromosome in American marsupials. Chromosome Res 15:127–136 (2007). Sinclair AH, Foster JW, Spencer JA, Page DC, Palmer M, et al: Sequences homologous to ZFY, a candidate human sex-determining gene, are autosomal in marsupials. Nature 336:780–783 (1988). Sutton E, Hughes J, White S, Sekido R, Tan J, et al: Identification of Sox3 as an XX male sex reversal gene in mice and humans. J Clin Invest 121:328–341 (2011). Svartman M: American marsupials chromosomes: why study them? Genet Mol Biol 32: 675–687 (2009). Svartman M, Vianna-Morgante AM: Karyotype evolution of marsupials: from higher to lower diploid numbers. Cytogenet Cell Genet 82: 263–266 (1998). Svartman M, Vianna-Morgante AM: Comparative genome analysis in American marsupials: chromosome banding and in-situ hybridization. Chromosome Res 7: 267–275 (1999).

Deakin /Graves /Rens

Toder R, O’Neill RJ, Wienberg J, O’Brien PC, Voullaire L, Marshall-Graves JA: Comparative chromosome painting between two marsupials: origins of an XX/XY1Y2 sex chromosome system. Mamm Genome 8: 418–422 (1997). Toder R, Wakefield MJ, Graves JA: The minimal mammalian Y chromosome - the marsupial Y as a model system. Cytogenet Cell Genet 91:285–292 (2000). Tsend-Ayush E, Kortschak RD, Bernard P, Lim SL, Ryan J, et al: Identification of mediator complex 26 (Crsp7) gametologs on platypus X1 and Y5 sex chromosomes: a candidate testis-determining gene in monotremes? Chromosome Res 20: 127–138 (2012). Veyrunes F, Waters PD, Miethke P, Rens W, McMillan D, et al: Bird-like sex chromosomes of platypus imply recent origin of mammal sex chromosomes. Genome Res 18: 965–973 (2008).


Wallis MC, Waters PD, Delbridge ML, Kirby PJ, Pask AJ, et al: Sex determination in platypus and echidna: autosomal location of Sox3 confirms the absence of Sry from monotremes. Chromosome Res 15: 949–959 (2007). Wang C, Deakin JE, Rens W, Zenger KR, Belov K, et al: A first-generation integrated tammar wallaby map and its use in creating a tammar wallaby first-generation virtual genome map. BMC Genomics 12: 422 (2011). Warren WC, Hillier LW, Marshall Graves JA, Birney E, Ponting CP, et al: Genome analysis of the platypus reveals unique signatures of evolution. Nature 453:175–183 (2008). Waters PD, Delbridge ML, Deakin JE, ElMogharbel N, Kirby PJ, et al: Autosomal location of genes from the conserved mammalian X in the platypus (Ornithorhynchus anatinus): implications for mammalian sex chromosome evolution. Chromosome Res 13:401–410 (2005).

Westerman M, Meredith RW, Springer MS: Cytogenetics meets phylogenetics: a review of karyotype evolution in diprotodontian marsupials. J Hered 101:690–702 (2010). Wrigley JM, Graves JA: Sex chromosome homology and incomplete, tissue-specific X-inactivation suggest that monotremes represent an intermediate stage of mammalian sex chromosome evolution. J Hered 79: 115–118 (1988a). Wrigley JM, Graves JA: Karyotypic conservation in the mammalian order monotremata (subclass Prototheria). Chromosoma 96:231–247 (1988b). Young GJ, Graves JA, Barbieri I, Woolley PA, Cooper DW, Westerman M: The chromosomes of dasyurids (Masupialia), in Archer M (ed): Carnivorous Marsupials, pp 783–795 (Royal Zoological Society, NSW 1982).


129


Published online: June 7, 2012

Chromosome Evolution in Xenarthra: New Insights from an Ancient Group M. Svartman Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

Key Words Banding patterns ⴢ Chromosome evolution ⴢ Chromosome painting ⴢ Karyotypes ⴢ Xenarthra

Abstract The Magnaorder Xenarthra is one of the four main supraordinal eutherian clades, together with Afrotheria, Euarchontoglires and Laurasiatheria. Xenarthra is an eminently Central and South American group of special interest in phylogenetic studies due to its possible position at the base of the eutherian tree. The use of modern cytogenetic techniques in some species of Xenarthra has provided important insights into the karyotypic evolution of mammals. Nevertheless, chromosome analyses in the group are still restricted, with only a few individuals of each species studied and karyotype descriptions mostly without banding patterns. In addition, it is likely that still unknown species exist and that the chromosome variability in the group is underestimated. We present a review of the currently available data on Xenarthra chromosomes and genomes and on the impact that their study has had in the understanding of mammalian genome evolution. It is clear that further cytogenetic analyses in Xenarthra, including banding patterns and molecular approaches, are likely to help in the identification of new species, reveal still undetected chromosome variations, provide information to support conservation strategies planning, and greatly contribute to a better understanding of mammalian genome evolution. Copyright © 2012 S. Karger AG, Basel



Xenarthra: An Overview

Xenarthra is the group of eminently Central and South American placental mammals represented by the living armadillos, anteaters and sloths. Together with Euarchontoglires, Laurasiatheria and Afrotheria, Xenarthra is considered one of the four main supraordinal clades of Eutheria. Despite the modest number of 31 living species [Gardner, 2008], this group of Gondwanan origin was very conspicuous in South America from the end of the Cretaceous, around 80–65 million years ago (mya), until the end of the Tertiary (3–2.5 mya) and over 200 fossil genera have been reported. This period marked the time when South America was an isolated landmass, which allowed the development of many forms of Xenarthra that explored varied ecological niches and were very abundant in this period of ‘splendid isolation’ [Simpson, 1980]. A severe decrease in their diversity took place during the mass extinction at the end of the Pleistocene, around 10,000 years ago [Delsuc et al., 2004]. Among the numerous fossil Xenarthra, some reached gigantic proportions and played important roles in the history of biology, as the giant sloth Megatherium americanum described by Cuvier in 1796 and the specimens collected by Darwin between 1832 and 1833 during his visit to South America on his famous voyage on board of the HSM Beagle [Vizcaíno et al., 2008]. The living Xenarthra are distributed in 2 orders: Cingulata, which reunites the species with armor-covered Marta Svartman Departamento de Biologia Geral, Instituto de Ciências Biológicas Universidade Federal de Minas Gerais Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-910 (Brazil) Tel. +55 31 3409 2612, E-Mail svartmanm @ icb.ufmg.br

bodies (armadillos), and Pilosa, which comprises the 2 groups with coated bodies (anteaters and sloths). Cingulata contains all the 21 known armadillo species in the single family Dasypodidae, the most specious among Xenarthra. This family reunites some widely distributed taxa, as the 9-banded armadillo D. novemcinctus, and endemic species which are among the rarest known mammals, as the fairy armadillos Calyptophractus retusus and Chlamyphorus truncatus. These species are distributed into 3 subfamilies: Dasypodinae, with Dasypus as its single genus; Euphractinae, with 3 species of Chaetophractus and the monospecific genera Euphractus, Zaedyus, Chlamyphorus, and Calyptophractus; and Tolypeutinae, with 4 species of Cabassous, 2 of Tolypeutes and the monospecific Priodontes maximus [Gardner, 2008]. The fairy armadillos, currently allocated to Euphractinae, may have diverged enough from the other genera to deserve allocation in their own subfamily, Chlamyphorinae [Delsuc et al., 2011]. Pilosa is divided into 2 suborders. The first of them, Vermilingua, reunites the anteaters, with 2 extant families: the monospecific Cyclopedidae, represented by the silky anteater Cyclopes didactyla, for which there may be up to 7 subspecies, and Myrmecophagidae that reunites the giant anteater Myrmecophaga tridactyla and 2 currently recognized species of Tamandua, the northern tamandua T. mexicana, with 3 subspecies, and the southern tamandua T. tetradactyla, with 4 subspecies [Gardner, 2008]. The second suborder of Pilosa, Folivora, reunites the families Bradypodidae, with 4 species of 3-toed sloths of the genus Bradypus, and Megalonychidae, with 2 species of 2-toed sloths, Choloepus hoffmanni and C. didactylus. B. variegatus may have up to 7 subspecies and C. hoffmanni up to 5, besides the existence of 2 disjunct populations that are possibly different subspecies [Aguiar and Fonseca, 2008; Gardner, 2008]. The maned sloth B. torquatus, endemic to a coastal strip of Atlantic Forest in southeastern Brazil, is listed as vulnerable [IUCN, 2011], and it has been suggested on the basis of morphological and molecular data that it should be allocated to a different genus, Scaeopus torquatus [Wetzel and Ávila-Pires, 1980; Barros et al., 2003; Gardner, 2008; Moraes-Barros et al., 2011]. Morphological analyses demonstrated the monophyly of the 3 morphotypes of Xenarthra (armadillos, anteaters and sloths). The presence of extra intervertebral articulations in their lumbar vertebrae, or ‘xenarthrales’, is a synapomorphy that unites all the living and extinct species and that was used to name the group [Engelman, 1985;

Glass, 1985; Delsuc and Douzery, 2009]. Besides the xenarthrous vertebrae, poor regulation of body temperature, the lack of clear differentiation between uterus and vagina, reduction of teeth by loss of enamel, and intra-abdominal testes are characteristics of the group also considered plesiomorphic for placental mammals, which led to the early idea that they were an early offshoot of the placental radiation [Delsuc and Douzery, 2008]. The monophyly of Xenarthra and of each of its 5 recognized extant families is well supported by molecular studies [Murphy et al., 2001a, b; Delsuc et al., 2001, 2002, 2011; Barros et al., 2003; Möller-Krull et al., 2007]. Molecular data estimates suggest that the group originated around 105 mya [Delsuc et al., 2004; Springer et al., 2004; Delsuc and Douzery, 2008], and their radiation took place during the Cretaceous/Tertiary transition. The most basal Xenarthra group, the armadillos, diverged around 70 mya, followed by the split between anteaters and sloths around 60 mya. The split between the 2 lineages of anteaters was estimated at 40 mya, and the 2 living genera of sloths, which are allocated to separate families, diverged around 20 mya (fig. 1). Paleoenvironmental changes were attributed an important role in the diversification of Xenarthra [Delsuc et al., 2004; reviewed by Delsuc and Douzery, 2008, 2009]. The oldest recorded Xenarthra fossil is represented by armadillo scutes from the late Paleocene of Brazil and is around 58 million years old [Bergqvist et al., 2004], which leaves an interval of almost 50 million years between the estimated origin of Xenarthra based on molecular data and their appearance in the fossil record. This observation concurs with the notion that Xenarthra represent a paleontological and biogeographical enigma that will only be solved by the finding of additional and more ancient fossils [Delsuc and Douzery, 2008]. In spite of their importance, most Xenarthra have been poorly studied, and there is a lack of available knowledge on all aspects of their biology. This is mainly due to their rarity, restricted geographical distribution and poor sampling, which results from the difficulty in spotting them because they may be burrowers, as the armadillos, or dwell in forest canopies, as the sloths. The existence of undescribed taxa is also very likely [Pereira et al., 2004; Gardner, 2008; IUCN, 2011]. The 9-banded armadillo D. novemcinctus, the only Xenarthra species to have reached North America, has been exceptionally well studied. Besides its broad geographic distribution, it has been used as a model organism for many years because of its polyembryony [Boily, 2008] and for being the only known species besides man

Chromosome Evolution in Xenarthra


131

Armadillos

Anteaters Myrmecophagidae

Sloths

Cyclopedidae

Bradypodidae

Megalonichidae

20 mya 40 mya

60 mya

Fig. 1. Phylogenetic relationships and estimated divergence times of the main xenarthran clades. The divergence dates are based on Delsuc and Douzery [2009] and the lengths of the branches are not drawn to scale. Mya = Million years ago.

to naturally host Mycobacterium leprae, the causing agent of leprosy [Truman, 2008]. Not surprisingly, D. novemcinctus has been chosen, together with the 2-toed sloth C. hoffmanni, to have its genome sequenced. Although the sequencing of these 2 Xenarthra representative genomes is still incomplete, this effort has already yielded some interesting data [Waters et al., 2004, 2007; Kriegs et al., 2006; Katzoukaris et al., 2009; Alkan et al., 2011].

Xenarthra on the Spotlight: The Phylogenetic Tree of Eutheria

After a long period of neglect, the interest in genetic studies of Xenarthra has been fueled during the last decade because of their possible basal position in the eutherian phylogenetic tree. The relationships among some placental mammalian orders are still debated, but the division into 4 supraordinal clades is now widely accepted [reviewed in Springer et al., 2004]. This classification was suggested based on mitochondrial and nuclear sequences analyses and by rare genomic changes [Murphy et al., 2001a, b, 2004; Scally et al., 2001; Delsuc et al., 2002; Springer et al., 2004; Kriegs et al., 2006]. The eutherian Magnaorders are represented by 2 clades of southern hemisphere origin (Gondwana) – Afrotheria (which includes 6 orders, Tubulidentata, Macroscelidae, Afrosoricida, Sirenia, Hyra132


70 mya

coidea, and Proboscidea) and Xenarthra (with 2 orders, Pilosa and Cingulata) – and 2 northern hemisphere (Laurasia) sister clades reunited as Boreoeutheria – Euarchontoglires (constituted by the orders Dermoptera, Scadentia, Primata, Rodentia, and Lagomorpha) and Laurasiatheria (which includes the orders Eulipotyphla, Carnivora, Pholidota, Cetartiodactyla, Perissodactyla, and Chiroptera) [Springer et al., 2004]. This new phylogenetic arrangement has received overwhelming support in dozens of studies, with the latest one extending analyses to the family level [Meredith et al., 2011]. Nevertheless, the question of which is the most basal eutherian group has proven to be hard to solve, and there are currently 3 alternatives: (1) Epitheria, in which Xenarthra would be the sister group to all other eutherians; (2) Exafroplacentalia, in which Afrotheria would be at the tree base; and (3) Atlantogenata, in which Xenarthra and Afrotheria together would form a sister taxon that split from all the other placentals (fig. 2). Phylogenies based on morphological and some molecular data usually place Xenarthra at the most basal position, lending support to Epitheria [Shoshani and Mckenna, 1998; Kriegs et al., 2006]. The Exafroplacentalia hypothesis is supported by a wealth of molecular data [Murphy et al., 2001a; Scally et al., 2001; Amrine-Madsen et al., 2003; Nikolaev et al., 2007; Nishihara et al., 2007], whereas Atlantogenata received support from some molecular and phylogenomic analyses [Waddell et al., 1999; Svartman

Epitheria Boreoeutheria Afrotheria Xenarthra

Exafroplacentalia

Atlantogenata

Boreoeutheria

Afrotheria

Xenarthra

Xenarthra

Afrotheria

Boreoeutheria

Fig. 2. The 3 current phylogenetic hypoth-

eses for the root of the eutherian tree.

Madsen et al., 2001; Delsuc et al., 2002; Lin et al., 2002; Hallström et al., 2007; Murphy et al., 2007; Waters et al., 2007; Prasad et al., 2008]. Recently, some studies pointed to the impossibility of solving this issue with current methodology, as incomplete lineage sorting, introgression and other unknown processes may have produced mosaic genomes, with each part having different phylogenetic histories. As a result, a trifurcation instead of a dichotomy would characterize the base of the placental tree, and the search for a bifurcating tree would be futile [Hallström and Janke, 2008, 2010; Churakov et al., 2009; Nishihara et al., 2009]. The origin of Xenarthra and Afrotheria around the same time (100 mya) and coinciding with the separation of South America (where Xenarthra originated) and Africa (the place of Afrotheria origin) led to the proposal of a causal connection between the tectonic events and the diversification of the placental mammals [Eizirik et al., 2001; Murphy et al., 2001b; Delsuc et al., 2004; Springer et al., 2004; Wildman et al., 2007]. On the other hand, it has also been suggested on the basis of fossil, tectonic and molecular data that the early radiation of eutherians was triggered rather by climate changes than by plate tectonics and occurred in Laurasia, from where there would have been an immediate dispersal, with Xenarthra later reaching South America [Hallström and Janke, 2010].


Classical Cytogenetics of Xenarthra

Although the first reports on Xenarthra chromosomes are a century old and the number of species is relatively small, the current karyotypical data on these animals are still poor. While analyzing meiotic cells of the 9-banded armadillo D. novemcinctus, Newman and Patterson [1910] and Newman [1912] made the first observations of Xenarthra chromosomes and concluded that the diploid number of the species was 2n = 32 in females and 2n = 31 in males and that the sex determination was of the XX/ X0 type. Over a decade later, Painter [1925] reported 2n = 60 in amniotic cells of the same species, and only much later, the right chromosome number of 2n = 64 and a XX/XY sex determination system were reported in peripheral blood cells [Beath et al., 1962]. Since then, progress in this area has been slow and irregular, with scarce literature and long intervals between new publications. Until now, only 19 of the 31 living species of Xenarthra have been karyotyped. Their diploid numbers range from 2n = 38 in the armadillo Tolypeutes matacus to 2n = 65 in the 2-toed sloth C. didactylus [Jorge et al., 1977; Dobigny et al., 2005]. For most species, only a few individuals have been analyzed, and therefore the presence of chromosome variations is probably underestimated. Besides, banding patterns have been applied in just a few studies and only conventionally stained chromosomes are known for several species. Most studies are Cytogenet Genome Res 2012;137:130–143

133

decades old, and the degree of resolution of the reported banding patterns is usually low, preventing further comparisons. A summary of the available karyotypic data of Xenarthra is presented in table 1. Chromosome rearrangements, such as pericentric inversions, Robertsonian fusions/fissions and heterochromatin additions/deletions, have been suggested to explain the karyotypic evolution in Xenarthra [Jorge et al., 1977, 1985a, b; Barroso and Seuánez, 1991; Pereira et al., 2004]. Nevertheless, their occurrence remains speculative, as the suggestions were based on conventionally stained chromosomes or low resolution banding patterns. The application of modern cytogenetic techniques and the inclusion of high resolution banded karyotypes will be necessary to ascertain how chromosome changes affected the complements of these animals. The chromosome features are somewhat typical of each of the 3 morphotypes of Xenarthra and will be presented separately in more detail below. Armadillos The karyotypes of 12 of the 21 armadillo species have been described, and they showed the lowest and one of the highest diploid numbers among Xenarthra: 2n = 38 in T. matacus and 2n = 64 in Dasypus species (table 1). Three of the 7 Dasypus species were studied, and all showed karyotypes with 2n = 64, with minor differences in the morphology of a few chromosome pairs. Six out of the 7 species of Euphractinae have been karyotyped and showed diploid numbers ranging from 2n = 58 to 62. Banding patterns were only reported for Chlamyphorus truncatus and the suggestions of rearrangements such as Robertsonian fusions/fissions and pericentric inversions in the group [Jorge et al., 1985a] awaits confirmation through banding patterns based comparative analysis. From the 4 species of Tolypeutinae karyotyped, only T. matacus had GTG-banding patterns described [Jorge et al., 1977; Benirschke, 2006]. This armadillo, with 2n = 38, is the only Xenarthra with less than 50 chromosomes. Two males identified as Cabassous sp. presented a newly described complement with 2n = 46 [Jorge and Pereira, 2008]. Lizarralde et al. [2005] analyzed the chromosomes of 4 armadillo species (D. hybridus 2n = 64, Chaetophractus vellerosus 2n = 62, C. villosus 2n = 60 and Zaedyus pichiy 2n = 62) after fluorescent in situ hybridization (FISH) with a telomeric probe. The interstitial telomeres found in C. villosus and Z. pichiy were interpreted as remnants of chromosome fusions between acrocentrics, which would support the hypothesis of chromosome evolution 134


through reduction of diploid numbers in the Dasypodidae. Further proposals of chromosome evolution in the group will depend on comparative data with higher resolution banding patterns and/or molecular cytogenetic approaches. Anteaters The reported diploid numbers for anteaters varied from 2n = 54 to 2n = 64 (table 1) and, with the exception of T. mexicana, all the recognized Vermilingua have been karyotyped (but see below). The karyotypic data on the genus Tamandua need to be reviewed, partly because of taxonomic changes. For example, the previously accepted T. longicaudata (described in some cytogenetic studies) and T. tetradactyla are now considered a single species (i.e. T. tetradactyla, with 4 subspecies), mainly distributed in South America, and T. mexicana is the taxon found in Central America, Mexico and some parts of northern South America [Gardner, 2008]. A new karyotype with 2n = 56 was recently reported in 1 male anteater from southeastern Brazil and suggested to belong to a still undescribed third species of Tamandua [Pereira Jr. et al., 2004]. On the other hand, Gardner [2008] suggested that this new complement is likely to be that of T. tetradactyla and that the karyotype with 2n = 54 [Hsu and Benirschke, 1969; Jorge et al., 1977] actually belongs to T. mexicana. If this proves to be correct, some recent data from the literature would have to be reviewed as the species studied, identified as T. tetradactyla, may actually be T. mexicana [Benirschke, 2006; Svartman et al., 2006; Yang et al., 2006]. Considering the small number of individuals of each species analyzed, the fact that subspecies were attributed to each of them [Gardner, 2008] and the recent description of a new karyotype [Pereira Jr. et al., 2004], it is clear that additional karyotypic data on anteaters are likely to disclose new chromosome variations and that comparative cytogenetic analyses are needed in order to discuss the possible relationships among the different complements found in anteater species. Sloths Three of the 4 species of Bradypodidae have been karyotyped and the exception is the critically endangered and recently described pigmy sloth B. pygmaeus, endemic to a small Caribbean island on the coast of Panama [Anderson and Handley Jr, 2001].

Svartman

Table 1. Karyotypic data of Xenarthra

Species (according to Gardner, 2008)

2n and FN

Banding patterns

Order Cingulata, Family Dasypodidae, Subfamily Dasypodinae Dasypus novemcinctus 2n = 64, FN = 78 GTG, CBG, Ag-NORs nine-banded armadillo Dasypus hybridus 2n = 64, FN = 78 southern long-nosed armadillo Dasypus septemcinctus 2n = 64; FN = 76 Ag-NORs yellow armadillo Order Cingulata, Family Dasypodidae, Subfamily Euphractinae Chlamyphorus truncatus 2n = 58; FN = ? (only GTG, CBG pink fairy armadillo one female analyzed) Chaetophractus vellerosus 2n = 62; FN = 88 IT screaming hairy armadillo Chaetophractus villosus 2n = 60, FN = 88 IT big hairy armadillo Euphractus sexcinctus 2n = 58, FN = 98 GTG, CBG six-banded armadillo Zaedyus pichiy 2n = 62, FN = 94 IT pichi

References

Beath et al., 1962; Benirschke et al., 1969; Jorge et al., 1977, 1985a; Barroso and Seuánez, 1991; Svartman et al., 2006 Benirschke et al., 1969; Jorge et al., 1977, 1985a; Lizarralde et al., 2005 Barroso and Seuánez, 1991

Jorge et al., 1985a Lizarralde et al., 2005 Benirschke et al., 1969; Jorge et al., 1977; Lizarralde et al., 2005 Benirschke et al., 1969; Jorge et al., 1977; Barroso and Seuánez, 1991; Liu et al., 2011 Meritt et al., 1973; Jorge et al., 1977; Lizarralde et al., 2005

Order Cingulata Family Dasypodidae, Subfamily Tolypeutinae Cabassous centralis 2n = 62, FN = 74 northern naked-tailed armadillo Cabassous tatouay 2n = 50, FN = 68 greater naked-tailed armadillo Cabassous sp. 2n = 46; FN = 66 Priodontes maximus 2n = 50, FN = 76 giant armadillo Tolypeutes matacus 2n = 38 GTG southern three-banded armadillo

Benirschke et al., 1969; Hsu and Benirschke, 1969 Barroso and Seuánez, 1991 Jorge and Pereira Jr., 2008 Benirschke and Wurster, 1969 Jorge et al., 1977; Benirschke, 2006

Order Pilosa, Suborder Folivora, Family Bradypodidae Bradypus torquatus 2n = 50, FN = 64 Ag-NORs maned sloth Bradypus tridactylus 2n = 52, FN = 56 GTG, CBG pale-throated sloth Bradypus variegatus 2n = 54–55, FN = 56– GTG, CBG, Ag-NORs brown-throated sloth 58

Jorge et al., 1985a*; Goldschmidt and Almeida, 1993; Goldschmidt et al., 1995

Order Pilosa; Suborder Folivora, Family Megalonychidae Choloepus didactylus GTG, CBG 2n = 52–65** Linnaeus’s two-toed sloth Choloepus hoffmanni GTG, CBG 2n = 49–53** Hoffmann’s two-toed sloth

Sonta, 1980; Jorge et al., 1985a; Dobigny et al., 2005; Benirschke, 2006; Yang et al., 2006 Corin-Frederic, 1969; Jorge et al., 1977, 1985a; Svartman et al., 2006

Order Pilosa, Suborder Vermilingua, Family Cyclopedidae Cyclopes didactylus 2n = 64, FN = 98 silky anteater Order Pilosa, Suborder Vermilingua, Family Myrmecophagidae Myrmecophaga tridactyla 2n = 60, FN = 104 GTG giant anteater Tamandua tetradactyla 2n = 54, FN = 104*** GTG, CBG southern tamandua Tamandua sp. 2n = 56; FN = 106****

Jorge and Pinder, 1990; Goldschmidt et al., 1995 Jorge et al., 1985a; Dobigny et al., 2005

Jorge, 2000; Jorge et al., 1985b

Pereira Jr. et al., 2004; Benirschke, 2006 Hsu and Benirschke, 1969; Jorge et al., 1977; Dobigny et al., 2005; Benirschke, 2006 Pereira Jr. et al., 2004

2n = Diploid number; FN = fundamental number (number of autosomal arms); IT = interstitial telomeres. * Described as B. infuscatus, ** FNs not recorded due to unpaired chromosomes and Y/autosome translocations; according to Gardner [2008], *** this chromosome formula belongs to T. mexicana and **** this is the complement of T. tetradactyla.



135

The most widespread of the 3-toed sloths, the brownthroated sloth B. variegatus presented 2n = 54, but 4 males with 2n = 55 were described, and the extra chromosome was interpreted as an additional Y [Jorge et al., 1985a; Goldschmidt and Almeida, 1993; Goldschmidt et al., 1995]. Although this species has several subspecies and separate populations have been identified [MoraesBarros et al., 2007; Gardner, 2008], no detectable chromosome differences, besides the extra Y chromosome, were reported. The pale-throated sloth B. tridactylus (2n = 52) had its GTG- and CBG-banding patterns only recently reported [Jorge et al., 1985a; Dobigny et al., 2005]. The apparent similarity of the complements of B. variegatus and B. tridactylus led Jorge et al. [1985a] and Jorge and Pereira [2008] to suggest that an event of Robertsonian fusion could explain the differences between these karyotypes. This suggestion was based on conventionally stained chromosomes and awaits verification. The maned sloth B. torquatus presents the smallest diploid number among sloths (2n = 50) and, although its diploid number is close to those of the other 2 Bradypus species studied, its karyotype presents some morphological chromosome differences [Jorge and Pinder, 1990; Goldschmidt et al., 1995; Azevedo et al., 2012], which is especially interesting in view of its uncertain taxonomic status [Wetzel and Ávila-Pires, 1980; Gardner, 2008] In addition, 2 distinct populations of B. torquatus have been identified by the use of molecular markers [Moraes-Barros, 2006; Lara-Ruiz et al., 2008], but all the karyotyped individuals can be traced to the southernmost population and further sampling from different areas would be interesting to verify if chromosome variations exist between the populations. The second family of sloths, Megalonychidae (genus Choloepus), revealed the most complex and confusing karyotypic picture among all the Xenarthra. The few cytogenetic studies performed in these animals unveiled great chromosome variability, demonstrating the need of further systematic and cytogenetic analyses. These sloths are characterized by the occurrence of Y/autosome translocations and by the presence of a single X chromosome in females. Unpaired chromosomes, supposedly B chromosomes, were also reported. Because the number of specimens studied is small and most were analyzed without banding patterns, it is very hard to ascertain their karyotypic constitution. Specimens of C. hoffmanni from the 2 recognized disjunct populations have been analyzed. Thirteen of them were part of the northern population. Among those, 10 136


animals had 2n = 49 in both sexes, and meiotic analyses revealed that the females were X0 and the males had a Y/23 translocation. The absence of a second X chromosome in females was confirmed by the lack of Barr bodies and of a late replicating chromosome after tritiated timidine incorporation [Corin-Frederic, 1969]. In a sibling pair with 2n = 50, the male had an extremely large Y chromosome interpreted to result from a translocation, and the female showed very different X chromosomes [Jorge et al., 1977]. A male with 2n = 50 and a 2/24 translocation (thus trisomic for the smallest autosome pair) was also reported, but the rearrangement may have arisen in the cell culture [Svartman et al., 2006]. Four females from the southern population of C. hoffmanni presented 2n = 51 and 3 unpaired chromosomes or 2n = 53 and 5 unpaired elements. Because no Barr body was detected, they were considered to be X0, and 1 of the unpaired elements was supposed to be the X chromosome [Jorge et al., 1985a]. The other unpaired elements could correspond to B chromosomes or to heteromorphic pairs, but without banding patterns it is hard to assess their nature. The chromosome data already obtained in C. didactylus suggest that the specimens analyzed represent more than one taxa and point to the need of a taxonomic revision of this species, as already suggested [Gardner, 2008]. Similarly to C. hoffmanni, Y/autosome translocations, X0 females and unpaired chromosomes were found in the few C. didactylus karyotyped. In addition, banding patterns were described for only 3 specimens, 2 with GTGand 1 with CBG-banding, with different karyotypes [Sonta, 1980; Jorge, 1985a; Dobigny et al., 2005; Benirschke, 2006]. Sonta [1980] found 2n = 53 in C. didactylus of both sexes with X0 females and a Y/21 translocation in males, similar to the findings on C. hoffmanni [Corin-Frederic, 1969]. A male with 2n = 53 and 7 unpaired chromosomes and an X0 female with 2n = 53 and 2 unpaired elements were reported [Jorge et al., 1985a; Benirschke, 2006] as well as specimens with 2n = 64 and 2n = 65, with a Y/32 translocation [Jorge et al., 1985a; Dobigny et al., 2005]. The great discrepancy in diploid numbers suggests that at least 2 different taxa are being currently identified as C. didactylus, one with diploid numbers around 2n = 52–54 and another with 2n = 64–65. The few studies performed in this species highlight its current confusing cytotaxonomy and the need of further cytogenetic studies. The detailed analysis of the underlying mechanisms involved in the frequent occurrence of Y/autosome translocations, X0 females and unpaired chromosomes in the Svartman

karyotypes of Choloepus is a promising avenue of research that is likely to provide useful data, especially on sex chromosome evolution.

The Ancestral Eutherian and Xenarthran Karyotypes The currently most accepted hypothetical AEK has 2n = 46 and is composed of human chromosomes HSA1, 2p, 2q, 3/21, 4/8p, 5, 6, 7a, 7b/16p, 8q, 9, 10q, 10p/12pq/22qt, 11, 12qt/22q, 13, 14/15, 16q/19q, 17, 18, 19p, 20, and X [reviewed in Ferguson-Smith and Trifonov, 2007; Graphodatsky et al., 2011]. More recently, the availability of sequence data from 2 outgroup species, the marsupial Monodelphis domestica and the chicken Gallus gallus, has shed new light into the effort of determining the AEK, by providing means to distinguish between genomic symplesiomorphies and synapomorphies. For instance, the ancestral syntenic associations HSA3/21, 4/8p, 7a/16p, 10p/12pq/22qt, 14/15, 16q/19q and 12qt/22q were found in one or both of the outgroup species and may thus be symplesiomorphies, whereas the synapomorphies of eutherians would be 8 intact chromosome pairs (HSA1, 5, 6, 9, 11, 17, 20, and X) and 3 chromosome segments (7b,

2p–q1.3 and 2q1.3–qter) [Robinson and Ruiz-Herrera, 2008; Ruiz-Herrera et al., 2012]. As for classical chromosome analyses, molecular cytogenetic studies in Xenarthra are still somewhat restricted. Nevertheless, their powerful resolution has provided useful insights into the chromosome architecture of the group, especially in relation to the AEK. Importantly, the possible basal position of Xenarthra in the eutherian phylogenetic tree made the inclusion of this taxon obligatory in cross-species chromosome painting experiments aiming at disclosing the AEK and, consequently, fostered new studies of this previously neglected group. In the first comparative molecular cytogenetic study performed in Xenarthra, chromosome-specific probes isolated from the 2-toed sloth C. didactylus (2n = 65) were hybridized to metaphases of the 3-toed sloth B. tridactylus (2n = 52), of the southern anteater T. tetradactyla (2n = 54) and of the 6-banded armadillo Euphractus sexcinctus (2n = 58). The experiments showed that both sloths had similar karyotypes, which significantly differed from the anteater complement. In addition, the rate of chromosome rearrangements in Xenarthra was found to be relatively low and some chromosome combinations were advanced as ancestral for Xenarthra [Dobigny et al., 2005]. Chromosome painting with human chromosomespecific probes has been performed in 7 species of Xenarthra: 2 species of armadillos, D. novemcinctus and E. sexcinctus, the southern anteater T. tetradactyla (which may actually be T. mexicana, as discussed above), the 2 species of 2-toed sloths, C. didactylus and C. hoffmanni, and 2 species of 3-toed sloths, B. variegatus and B. torquatus. In addition, based on literature data, the chromosome segments that would be homologous between human and B. tridactylus could be deduced [Svartman et al., 2006; Yang et al., 2006, Liu et al., 2011; Azevedo et al., 2012]. A summary of the results of these studies is presented in table 2. Taken together, the data of chromosome painting with human probes in Xenarthra revealed some common features to all the species studied: (a) the presence of the ancestral eutherian associations HSA3/21, 4/8, 7/16, 12/22, and 14/15; (b) the conservation of HSA9, 13, 17, 18, 20, and X; (c) the division of HSA19 and 22 in 2 blocks; and (d) 3 chromosome pairs labeled by HSA8 (table 2). The presence of the association HSA7/10 in C. didactylus, T. tetradactyla, E. sexcinctus, B. variegatus, B. torquatus and possibly also in B. tridactylus led to the suggestion that this would be a chromosome signature of Xenarthra [Yang et al., 2006; Liu et al., 2011; Azevedo et al., 2012]. This association was not detected in C. hoff-



Molecular Cytogenetics of Xenarthra

Among the modern cytogenetic techniques, interspecific chromosome painting with species-specific chromosome probes is one of the most used to resolve phylogenetic issues. It has been applied to different mammalian groups and proved to be a very useful approach to examine relationships among species, families and orders, with the potential of revealing characteristic chromosome features, or signatures, of each lineage. Human chromosome probes have been the most widely used to paint the chromosomes of mammals from all recognized eutherian mammal orders in an attempt to determine the ancestral eutherian karyotype (AEK), the complement of the common ancestor of all placental mammals [Ferguson-Smith and Trifonov, 2007; Graphodatsky et al., 2011]. Besides allowing the reconstruction of the hypothetical AEK, these studies were very important in clarifying other issues. For instance, they provided an independent demonstration of the monophyly of Afrotheria, in which the synapomorphic associations HSA1/19 and 5/21 were identified as chromosome signatures, and also revealed chromosome features that reinforced some previously proposed afrotherian subordinal clades [Frönicke et al., 2003; Svartman et al., 2004; Kellogg et al., 2007; RuizHerrera and Robinson, 2007].

137

Table 2. Results from chromosome painting with human chromosome-specific probes in Xenarthra Species

Number of Associations of conserved human autosomes segments

Human chromosomes conserved

References 2 blocks

3 or more blocks

Choloepus hoffmanni (2n = 50)

32

3/21, 4/8, 7/16, 12/22(2x), 14/15, 1, 3, 4, 5, 6, 9, 10, 11, 13, 2, 7, 12, 19, 22 16/19 14, 15, 17, 18, 20, 21, X

8?, 16

Svartman et al., 2006

Choloepus didactylus (2n = 65)

43

2/8, 3/21, 4/8, 7/10, 7/16, 12/22 (2x), 14/15

9, 13, 15, 17, 18, 20, 21, X

1, 3, 4, 5, 6, 10, 11, 12, 14, 16, 19, 22

2, 7, 8

Yang et al., 2006

Dasypus novemcinctus (2n = 64)

41

3/21(2x), 4/8, 7/16, 10/12, 12/22(2x), 14/15, 16/19

5, 9, 13, 14, 15, 17, 18, 20, X

1, 4, 6, 7, 10, 11, 16, 2?, 3, 8, 12 19?, 21, 22

Svartman et al., 2006

Euphractus sexcinctus (2n = 58)*

39

2/8, 2/11, 3/21, 4/8, 7/10, 7/16, 12/22(2x), 14/15, 16/19

5, 9, 13, 14, 15, 17, 18, 20, 21, X

1, 3, 4, 6, 7, 10, 11, 16, 19, 22

2, 8, 12,

Liu et al., 2011

2, 6, 7, 10, 11, 12, 14, 15, 16, 19, 22

1, 3, 4, 5, 8

Svartman et al., 2006; Yang et al., 2006

Tamandua tetradactyla 45 (2n = 54)**

1/9, 1/13, 1/19, 2/8, 3/6, 3/21, 9, 13, 17, 18, 20, 21, X 3/22, 4/8, 5/11, 7/10, 7/16, 7/20, 8/17, 12/22(2x), 14/15(2x), 16/19

Bradypus torquatus (2n = 50)

32

3/21, 4/8, 7/10, 7/16, 12/22(2x), 1, 3, 4, 5, 6, 9, 11, 13, 2, 7, 10, 12, 16, 19, 14/15, 17/19 14, 15, 17, 18, 20, 21, X 22

8

Azevedo et al., 2012

Bradypus variegatus (2n = 54)

36

3/21?, 4/8, 7/10, 7/16, 12/22, 12/22/16, 14/15, 17/19

5, 6, 9, 11, 13, 14, 15, 17, 18, 20, 21?, X

1, 2, 3, 4, 7, 10, 12, 19, 22

8, 16


Bradypus tridactylus (2n = 52)

35

2/6, 3/21, 4/8, 7/10, 7/16, 11/19, 1, 5, 9, 11, 13, 14, 15, 12/22, 12/22/16, 14/15, 17/19 17, 18, 20, 21, X

2, 3, 4, 8, 7, 10, 12, 16, 19, 22

6, 8?


In bold, the features present in all the species studied. The question mark indicates uncertainties in the labeling patterns. * Liu et al. [2011] did not consider the association 2/11 because the segments corresponding to each human chromosome were sepa rated by heterochromatin; they also considered HSA3, HSA4 and HSA19 divided into

three segments (and we, only in two segments) because the associated probe painted a whole biarmed chromosome and a segment of a second chromosome. As a result, we counted 39 conserved segments in E. sexcinctus, and not the 42 reported by the authors. ** The results indicated are a compilation of both studies performed in this species.

manni and D. novemcinctus, but its absence may have resulted from technical difficulties. In addition, a distal segment of the C. hoffmanni chromosome painted by HSA7 was not painted by any human probe and may correspond to HSA10. The association HSA2/8, found in C. didactylus, T. tetradactyla and E. sexcinctus has also been considered a chromosome signature of Xenarthra [Yang et al., 2006; Liu et al., 2011]. Nevertheless, its absence in all the other studied species of the group led Azevedo et al. [2012] to conclude that it may be a result of convergence, instead of a Xenarthra synapomorphy. Besides these 2 chromosome associations, the splitting of HSA8 into 3 blocks was proposed as a further synapomorphy of the group [Svartman et al., 2006; Azevedo et al., 2012]. The other syntenic associations found in Xenarthra are also part of the putative AEK and would thus constitute symplesiomorphies. The 2 supposed xenarthran synapomorphic associations HSA2/8 and 7/10 were also detected in the marsupial and chicken genomes [Graphodatsky et al., 2011] and further comparative anal-

yses between Xenarthra and human will be necessary to establish if these combinations are homologous in the different species. A very interesting result was obtained when human chromosome-specific probes were hybridized to metaphases of the 2-toed sloth C. hoffmanni (2n = 50). This species showed a karyotype extremely similar to the alternative hypothetical AEK with 2n = 48, which differs from the 2n = 46 AEK only by the separation of 10p from the HSA10p/12/22 association [Murphy et al., 2001c; Svartman et al., 2006]. In total, 21 chromosomes were conserved in both complements: HSA1, 2a, 2b, 3/21, 4/8a, 5, 6, 7a, 7b/16a, 9, 11, 12/22 (2!), 13, 14/15, 16b/19a, 17, 18, 19b, 20, and the X. Only 3 rearrangements, i.e. further dissociations of human chromosomes 8 and 16 and the conservation in toto of HSA10 in the sloth, explained the differences between its complement and the AEK with 2n = 48. These results were interpreted as a reinforcement of the hypothetical AEK with 48 chromosomes and as a further support to the basal position of Xenarthra in the eutherian tree [Svartman et al., 2006].

138


Svartman

8p

21

4

1

3

14

5

10p

6

X 12pq

2q

7a

2p

9

15

10q

11

13

8q

17

19q

18

16p

12qt

7b

22q

20 16q

22qt

19p

AEK

21

3

5

10p

14

X

4a

6a

7a

15

1p

1q

2a

2b

9

11

10q

12pq 13

*

*

*

*

*

*

18

*

19q

8a

4b

22qt

*

17

8b

16q

*

20

16p

12qt

7b

22q

*

6b

19p

*

2c 8c

*

AXK

Fig. 3. Putative AEK and the proposed AXK, reconstructed based on data from Liu et al. [2011]. The color code

corresponds to human chromosomes and the asterisks denote the chromosomes of the AXK that differ from their counterparts in the AEK.

More recently, based on the available data from 5 Xenarthra species already painted with human chromosomespecific probes and using the conserved syntenies between the sequenced genomes of human and of the marsupial to represent symplesiomorphies, Liu et al. [2011] proposed an ancestral Xenarthra karyotype (AXK) with 2n = 54. This AXK would be similar to the karyotype of the 6-banded armadillo E. sexcinctus and composed of: the associations HSA2c/8c, 3/21, 4b/8b, 7a/10p, 7b/16p, 12/22 (2!), 14/15, 16q/19q; the conserved chromosomes HSA5, 9, 11, 13, 17, 18, 20, and X; 2 pairs homologous to each HSA1 (1p and 1q), 6 (6a and 6b) and the remaining of HSA2 (2a and 2b); and 4 chromosome pairs corresponding to HSA4a, 8a, 10q, and 19p (fig. 3). Some of these chromosome features correspond to previous suggestions of ancestral xenarthran arrangements made on the basis of intraordinal chromosome painting results [Dobigny et al., 2005], which reinforced the conclusions of Liu et al. [2011]. An alternative AXK with 2n = 48 was recently advanced by Azevedo et al. [2012] after the inclusion of

painting data from the Bradypus species. This AXK differs from the 2n = 54 AXK by: the conservation of chromosomes HSA1, 4 and 6; the absence of the association HSA2/8; and the division of HSA2 into 2 blocks, instead of 3. The presence of the association HSA7/10 and the split of HSA8 into 3 blocks, instead of the 2 found in the eutherian ancestor, would be the differences between the hypothesized 2n = 48 AXK and the AEK. These 2 chromosome features would also support the monophyly of Xenarthra, making this the second supraorder of eutherians, besides Afrotheria, to have a chromosome signature recognized. The painting data obtained in 3-toed sloths also allowed the identification of the HSA17/19 association as a chromosome signature of the genus Bradypus [Azevedo et al., 2012]. The species that already had their karyotypes painted with human chromosome probes represent 4 of the 5 recognized living families of Xenarthra. The inclusion of the remaining family, Cyclopedidae, in these analyses will allow further testing of the interfamilial relationships, the validity of the proposed AXK and of the currently ac-



139

cepted AEK. The incorporation of genome sequencing data from outgroups will also be indispensable in this effort. Until now, only 2 of the 21 armadillo species have been included in chromosome painting studies, and the only representative of anteaters analyzed was T. tetradactyla (that may actually be T. mexicana), which presents a relatively highly rearranged karyotype. Therefore, analyses of additional taxa from both Cingulata and Vermilingua have the potential to add to the understanding of karyotypic evolution in Xenarthra and in mammals as a whole.

Repetitive Sequences in Xenarthra

Xenarthran genomes are significantly larger than those of other Eutheria, with an average of 4.5 pg (4,400 Mb) against 3 pg (2,900 Mb), suggesting an accumulation of repetitive elements [Redi et al., 2005]. In line with this observation, the chromosome painting studies performed in Xenarthra revealed several chromosome segments that were not painted by any human chromosome probe and usually corresponded to CBG-banded regions [Svartman et al., 2006; Yang et al., 2006; Liu et al., 2011; Azevedo et al., 2012]. Repetitive DNA sequences in constitutive heterochromatin are being increasingly viewed as essential genomic components with indispensable roles, an idea that replaced their past portrayal of ‘junk DNA’. Therefore, it is important to understand how these sequences evolved and what the implications are for genome evolution and species diversification in Xenarthra. Among the abundant mammalian non-coding sequences, the long and short interspersed elements (LINEs and SINEs) correspond to, respectively, 21% and 13% of the human genome [Churakov et al., 2004] and have also been detected in some Xenarthra. In this group, SINEs were first reported in a study of the 9-banded armadillo D. novemcinctus, which resulted in the finding of a family named DAS-SINE and extended the observation of SINEs to all mammalian orders. The DAS-SINE from D. novemcinctus included 687 copies divided into 6 subfamilies. It has not been found in any other species, although its presence remains to be tested in other xenarthran genomes [Churakov et al., 2004]. The identification and FISH mapping of LINE-1 in the chromosomes of the aardvark (an Afrotheria) and of 3 species of Xenarthra (B. tridactylus, E. sexcinctus and T. tetradactyla) showed LINE-1 accumulation on the X chromosomes. This indicated the involvement of these 140


elements in the propagation of X-inactivation in mammals, as previously suggested by Lyon [1998], and showed that LINE-1 accumulation on the X is an ancient feature predating the separation of the eutherian lineages [Waters et al., 2004]. Liu et al. [2011] isolated 7 novel repetitive sequences from the 6-banded armadillo E. sexcinctus genome and mapped their chromosome distribution using FISH. These sequences were shown to contain at least 3 different types of repeats: 2 were mainly centromeric and composed of 116 bp repeats, 3 were related to chromosomespecific centromeric heterochromatin, and the remaining 2 had regions highly similar to human LINE-1 elements. Although the latter sequences hybridized to the X chromosomes, similarly to previous results obtained in other Xenarthra and Afrotheria [Waters et al., 2004], these sequences also labeled most non-centromeric heterochromatic regions revealed by CBG-banding, a pattern not observed in some other xenarthran species. Sequencing data from the 9-banded armadillo D. novemcinctus and from the 2-toed sloth C. hoffmanni have been used to search for repetitive elements of interest in 2 recent studies. Genome mining of C. hoffmanni allowed the identification of hundreds of foamy virus insertions termed sloth endogenous foamy viruses. These sequences were subsequently found in the genomes of 2- and 3-toed sloths, but were absent from the anteaters and armadillos genomes, as well as from those of other mammals. Sloth endogenous foamy viruses sequences appear to be old components of the sloth genome (around 39 mya), suggesting that they entered the xenarthran genome after the divergence of anteaters and sloths (around 55 mya), but before the split between 2- and 3-toed sloths (around 21 mya) [Katzourakis et al., 2009]. The satellite repeat sequences that constitute the centromeres of D. novemcinctus have been recently identified using a new bioinformatics approach to analyze the genome sequencing data of this species. The single consensus sequence identified, composed of 173 bp units, was mapped to all the centromeres of the armadillo by FISH [Alkan et al., 2011]. Although repetitive DNA analyses in Xenarthra have just recently started and the available data are still limited, the study of these sequences is likely to experience an increase soon, which will certainly contribute to the understanding of several aspects related to genome evolution, including the factors responsible for genomic size changes among mammals.

Svartman

Conclusions and Prospects

The study of mammalian chromosome evolution has greatly benefited from the application of molecular cytogenetics, genome sequencing and the development of new bioinformatics tools, especially in the last years. The inclusion of xenarthran representatives in the genome analyses was pivotal in providing valuable insights into questions such as the reconstruction of the AEK. Nevertheless, much remains to be gained by further studies of this amazing group, not only by the application of modern techniques, but also by classical cytogenetic analyses, which are still lacking in Xenarthra, and seem to progress at a sloth’s pace. Further analyses of more individuals and of additional species, including high resolution banded karyotypes and molecular cytogenetics, promise to help in the understanding of genome structure and evolution not only of this important group, but for mammals in general. The inclusion of more taxa, especially of those still underrepresented, is essential. Besides the insights into mammalian genome evolution, chromosome analyses of Xenarthra may subsidize conservation strategy planning, providing information

to help in the identification of populations and species. Very little attention has been given to the chromosome variability in conservation genetics studies, which is surprising, as chromosome rearrangements may have important consequences affecting fertility and chromosome differences may accompany speciation events [Allendorf and Luikart, 2006]. Cytogenetic analyses may also help to elucidate taxonomic issues and would be very useful in cases such as that of Choloepus, which is presently rather confusing. Finally, the need of studies in Xenarthra is further urged by the vulnerable or endangered status of several of its few living species that may not be around for very long.

Acknowledgements I am indebted to Dr. Roscoe Stanyon and Dr. Alexander Graphodatsky for the opportunity to write this review and to Dr. Mônica Bucciarelli Rodriguez and Dr. Gustavo Kuhn for the helpful suggestions. This work was possible due to grants received from Fundação de Amparo à Pesquisa do Estado de Minas Gerais (processes CRA-APQ-00170-09 and APQ-00336-09).

References Aguiar JM, Fonseca GA: Conservation status in Xenarthra, in Vizcaíno SF, Loughry WJ (eds): The Biology of the Xenarthra, pp 215– 231 (University Press of Florida, Gainesville, 2008). Alkan C, Cardone MF, Catacchio CR, Antonacci F, O’Brien SJ, et al: Genome-wide characterization of centromeric satellites from multiple mammalian genomes. Genome Res 21: 137–145 (2011). Allendorf FW, Luikart G: Conservation and the Genetics of Populations. (Blackwell Publishing, Malden 2006). Amrine-Madsen H, Koepfli KP, Wayne RK, Springer MS: A new phylogenetic marker, apolipoprotein B, provides compelling evidence for eutherian relationships. Mol Phylogenet Evol 28:225–240 (2003). Anderson RP, Handley Jr CO: A new species of three-toed sloth (Mammalia: Xenarthra) from Panama, with a review of the genus Bradypus. Proc Biol Soc Wash 114:1–33 (2001). Azevedo NF, Svartman M, Manchester A, de Moraes-Barros N, Stanyon R, Vianna-Morgante AM: Chromosome painting in threetoed sloths: a cytogenetic signature and ancestral karyotype for Xenarthra. BMC Evol Biol 12: 36 (2012).


Barros M, Sampaio I, Schneider H: Phylogenetic analysis of 16S Mitochondrial DNA data in sloths and anteaters. Genet Mol Biol 26:5–11 (2003). Barroso CM, Seuánez H: Chromosome studies on Dasypus, Euphractus and Cabassous genera (Edentata: Dasypodidae). Cytobios 68: 179–196 (1991). Beath MM, Benirschke K, Brownhill LE: The chromosomes of the nine-banded armadillo, Dasypus novemcinctus. Chromosoma 13:27– 38 (1962). Benirschke K: Xenarthra, in O’Brien SJ, Menninger JC, Nash WG (eds): Atlas of Mammalian Chromosomes, pp 81–93 (John Wiley and Sons, Hoboken 2006). Benirschke K, Wurster DH: The chromosomes of the giant armadillo, Priodontes giganteus Geoffroy. Acta Zool Pathol Antverp 49: 125– 130 (1969). Benirschke K, Low RJ, Ferm VH: Cytogenetic studies of some armadillos, in Benirschke K (ed): Comparative Mammalian Cytogenetics, pp 330–345 (Springer-Verlag, New York 1969). Bergqvist LP, Abrantes ÉAL, Avilla LS: The Xenarthra (Mammalia) of São José de Itaboraí Basin (upper Paleocene, Itaboraian), Rio de Janeiro, Brazil. Geodiversitas 26: 323–337 (2004).

Boily P: The use of armadillo clones from the genus Dasypus as experimental models to investigate the source of physiological variability, in Vizcaíno SF, Loughry WJ (eds): The Biology of the Xenarthra, pp 126–129 (University Press of Florida, Gainesville 2008). Churakov G, Smit AF, Brosius J, Schmitz J: A novel abundant family of retroposed elements (DAS-SINEs) in the nine-banded armadillo (Dasypus novemcinctus). Mol Biol Evol 22:886–893 (2004). Churakov G, Kriegs JO, Baertsch R, Zemann A, Brosius J, Schmitz J: Mosaic retroposon insertion patterns in placental mammals. Genome Res 19:868–875 (2009). Corin-Frederic J: So-called aberrant gonosomic formulas in placental mammals. The special case of the sloth Choloepus hoffmanni Peters (Edentata, Xenartha, family Bradypodidae) [in French]. Chromosoma 27:268–287 (1969). Delsuc F, Douzery EJ: Recent advances and future prospects in xenarthran molecular phylogenetics, in Vizcaíno SF, Loughry WJ (eds): The Biology of the Xenarthra, pp 11–23 (University Press of Florida, Gainesville 2008). Delsuc F, Douzery EJ: Armadillos, anteaters and sloths (Xenarthra), in Hedges SB, Kumar S (eds): The Timetree of Life, pp 475–478 (Oxford University Press, Oxford 2009).


141

Delsuc F, Catzeflis FM, Stanhope MJ, Douzery EJ: The evolution of armadillos, anteaters and sloths depicted by nuclear and mitochondrial phylogenies: implications for the status of the enigmatic fossil Eurotamandua. Proc Biol Sci 268:1605–1615 (2001). Delsuc F, Scally M, Stanhope MJ, de Jong WW, Catzefelis FM, et al: Molecular phylogeny of living xenarthrans and the impact of characters and taxon sampling on the placental tree rooting. Mol Biol Evol 19:1656–1671 (2002). Delsuc F, Vizcaíno SF, Douzery EJ: Influence of Tertiary paleoenvironmental changes on the diversification of South American mammals: a relaxed molecular clock study within Xenarthrans. BMC Evol Biol 4: 11 (2004). Delsuc F, Superina M, Tilak M, Douzery EJ, Hassanin A: Molecular phylogenetics unveils the ancient evolutionary origins of the enigmatic fairy armadillos. Mol Phylogenet Evol 62: 673–680 (2011). Dobigny G, Yang F, O’Brien PC, Volobouev V, Kovács A, et al: Low rate of genomic repatterning in Xenarthra inferred from chromosome painting data. Chromosome Res 13: 651–663 (2005). Eizirik E, Murphy WJ, O’Brien SJ: Molecular dating and biogeography of the early placental mammal radiation. J Hered 92: 212–219 (2001). Engelman GF: The phylogeny of Xenarthra, in Montgomery GG (ed): The Evolution and Ecology of Armadillos, Sloths and Vermilinguas, pp 51–64 (Smithsonian University Press, Washington 1985). Ferguson-Smith MA, Trifonov V: Mammalian karyotype evolution. Nat Rev Genet 8: 950– 962 (2007). Frönicke L, Wienberg J, Stone G, Adams L, Stanyon R: Towards the delineation of the ancestral eutherian genome organization: comparative genome maps of human and the African elephant (Loxodonta africana) generated by chromosome painting. Proc Biol Sci 270: 1331–1340 (2003). Gardner AL: Magnaorder Xenarthra, in Gardner AL (ed): Mammals of South America, Vol 1: Marsupials, Xenarthrans, Shrews, and Bats, pp 127–176 (The University of Chicago Press, Chicago 2008). Glass BP: History of classification and nomenclature in Xenarthra (Edentata), in Montgomery GG (ed): The Evolution and Ecology of Armadillos, Sloths and Vermilinguas, pp 1–3 (Smithsonian University Press, Washington 1985). Goldschmidt B, Almeida JC: Cytogenetic studies in sloths (Bradypus variegatus). Rev Bras Genet 16:939–948 (1993). Goldschmidt B, Almeida JC, Oliveira VL: Visualization of nucleolar organizer regions in sloths (Bradypus variegatus and Scaeopus torquatus). Rev Bras Genet 18: 111–113 (1995). Graphodatsky AS, Trifonov VA, Stanyon R: The genome diversity and karyotype evolution of mammals. Mol Cytogenet 4:22 (2011).

142

Hallström BM, Janke A: Resolution among major placental mammal interordinal relationships with genome data imply that speciation influenced their earliest radiations. BMC Evol Biol 8: 162 (2008). Hallström BM, Janke A: Mammalian evolution may not be strictly bifurcating. Mol Biol Evol 12:2804–2816 (2010). Hallström BM, Kullberg M, Nilsson MA, Janke A: Phylogenomic data analyses provide evidence that Xenarthra and Afrotheria are sister groups. Mol Biol Evol 24:2059–2068 (2007). Hsu TC, Benirschke K: Tamandua tetradactyla. An Atlas of Mammalian Chromosomes, vol 3, pp 108–109 (John Wiley and Sons, Hoboken 1969). IUCN: IUCN Red List of Threatened Species. Version 2011.2. 2011. http://www.iucnredlist.org (accessed January 11, 2012). Jorge W: Mitotic and meiotic chromosome studies in the silky anteater Cyclopes didactylus (Myrmecophagidae: Xenarthra). Cytobios 101:95–100 (2000). Jorge W, Pinder L: Chromosome study on the maned sloth Bradypus torquatus (Bradypodidae, Xenarthra). Cytobios 62:21–25 (1990). Jorge W, Pereira HR Jr: Chromosomal studies in Xenarthra, in Vizcaíno SF, Loughry WJ (eds): The Biology of the Xenarthra, pp196– 204 (University Press of Florida, Gainesville 2008). Jorge W, Meritt DA Jr, Benirschke K: Chromosome studies in Edentata. Cytobios 18: 157– 172 (1977). Jorge W, Orsi-Souza AT, Best RC: The somatic chromosomes of Xenarthra, in Montgomery GG (ed): The Evolution and Ecology of Armadillos, Sloths, and Vermilinguas, pp121– 129 (Smithsonian Institution Press, Washington 1985a). Jorge W, Best RC, Wetzel RM: Chromosome studies on the silky anteater Cyclopes didactylus L. (Myrmecophagidae: Xenarthra, Edentata). Caryologia 38:325–329 (1985b). Katzourakis A, Gifford RJ, Tristem M, Gilbert MT, Pybus OG: Macroevolution of complex retroviruses. Science 325:1512 (2009). Kellogg ME, Burkett S, Dennis TR, Stone G, Gray BA, et al: Chromosome painting in the manatee supports Afrotheria and Paenungulata. BMC Evol Biol 7: 6 (2007). Kriegs JO, Churakov G, Kiefmann M, Jordan U, Brosius J, Schmitz J: Retroposed elements as archives for the evolutionary history of placental mammals. PLoS Biol 4:e91 (2006). Lara-Ruiz P, Chiarello AG, Santos FR: Extreme population divergence and conservation implications for the rare endangered Atlantic Forest sloth, Bradypus torquatus (Pilosa: Bradypodidae). Biol Conserv 141: 1332–1342 (2008). Lin YH, McLenachan PA, Gore AR, Phillips MJ, Ota R, et al: Four new mitochondrial genomes and the increased instability of evolutionary trees of mammals from improved taxon sampling. Mol Biol Evol 19:2060–2070 (2002).


Liu Y, Ye J, Fu B, Ng B, Wang J, et al: Molecular cytogenetic characterization of the genome organization of the 6-banded armadillo (Euphractus sexcinctus). Cytogenet Genome Res 132:31–40 (2011). Lizarralde MS, Bolzán AD, Poljak S, Pigozzi MI, Bustos J, Merani MS: Chromosomal localization of the telomeric (TTAGGG)n sequence in four species of armadillo (Dasypodidae) from Argentina: an approach to explaining karyotype evolution in the Xenarthra. Chromosome Res 13: 777–784 (2005). Lyon MF: X-Chromosome inactivation: a repeat hypothesis. Cytogenet Cell Genet 80: 133– 137 (1998). Madsen O, Scally M, Douady CJ, Kao DJ, DeBry RW, et al: Parallel adaptive radiations in two major clades of placental mammals. Nature 409:610–614 (2001). Meredith RW, Janečka JE, Gatesy J, Ryder OA, Fisher CA, et al: Impacts of the Cretaceous Terrestrial Revolution and KPg extinction on mammal diversification. Science 334: 521–524 (2011). Meritt DA, Low RJ, Benirschke K: The chromosomes of Zaedyus pichiy. Mamm Chrom Newsletter 14:108–109 (1973). Möller-Krull M, Delsuc F, Churakov G, Marker C, Superina M, et al: Retroposed elements and their flanking regions resolve the evolutionary history of xenarthran mammals (armadillos, anteaters, and sloths). Mol Biol Evol 24:2573–2582 (2007). Moraes-Barros N, Silva JA, Miyaki CY, Morgante JS: Comparative phylogeography of the Atlantic forest endemic sloth (Bradypus torquatus) and the widespread three-toed sloth (Bradypus variegatus) (Bradypodidae, Xenarthra). Genetica 126:189–198 (2006). Moraes-Barros N, Miyaki CY, Morgante JS: Identifying management units in non-endangered species: the example of the sloth Bradypus variegatus Schinz, 1825. Braz J Biol 67 Suppl 4:829–837 (2007). Moraes-Barros N, Silva JA, Morgante JS: Morphology, molecular phylogeny, and taxonomic inconsistensies in the study of Bradypus sloths (Pilosa: Bradypodidae). J Mamm 92:86–100 (2011). Murphy WJ, Eizirik E, O’Brien SJ, Madsen O, Scally M, et al: Resolution of the early placental mammal radiation using Bayesian phylogenetics. Science 295:2348–2351 (2001a). Murphy WJ, Eizirik E, Johnson WE, Ping Zhang YP, Ryder OA, O’Brien SJ: Molecular phylogenetics and the origin of placental mammals. Nature 409:614–618 (2001b). Murphy WJ, Stanyon R, O’Brien SJ: Evolution of mammalian genome organization inferred from comparative gene mapping. Genome Biol 2:REVIEWS0005 (2001c). Murphy WJ, Pevzner PA, O’Brien SJ: Mammalian phylogenomics comes of age. Trend Genet 20:631–639 (2004).

Svartman

Murphy WJ, Pringle TH, Crider TA, Springer MS, Miller W: Using genomic data to unravel the root of the placental mammal phylogeny. Genome Res 17:413–421 (2007). Newman HH: The ovum of the nine-banded armadillo: growth of the oocytes, maturation and fertilization. Biol Bull 23:100–141 (1912). Newman HH, Patterson JT: The development of the nine-banded armadillo from primitive streak to birth, with special reference to the question of polyembryony. J Morphol 21: 359–423 (1910). Nikolaev S, Montoya-Burgos JI, Margulies EH; NISC Comparative Sequencing Program, Rougemont J, et al: Early history of mammals is elucidated with the ENCODE multispecies sequencing data. PLoS Genet 3:e2 (2007). Nishihara H, Okada N, Hasegawa M: Rooting the eutherian tree: the power and pitfalls of phylogenomics. Genome Biol 8:R199 (2007). Nishihara H, Maruyama S, Okada N: Retroposon analysis and recent geological data suggest near-simultaneous divergence of the three superorders of mammals. Proc Natl Acad Sci USA 106:5235–5240 (2009). Painter TS: Chromosome number in mammals. Science 61:423–424 (1925). Pereira HR Jr, Jorge W, Costa MELT: Chromosome study of anteaters (Myrmecophagidae, Xenarthra) – a preliminary report. Genet Mol Biol 27: 391–394 (2004). Prasad AB, Allard MW; NISC Comparative Sequencing Program, Green ED: Confirming the phylogeny of mammals by use of large comparative sequence data sets. Mol Biol Evol 25:1795–1808 (2008). Redi CA, Zacharias H, Merani S, Oliveira-Miranda M, Aguilera M, et al: Genome sizes in afrotheria, xenarthra, euarchontoglires, and laurasiatheria. J Hered 96:485–493 (2005).


Robinson TJ, Ruiz-Herrera A: Defining the ancestral eutherian karyotype: a cladistic interpretation of chromosome painting and genome sequence assembly data. Chromosome Res 16:1133–1141 (2008). Ruiz-Herrera A, Robinson TJ: Chromosomal instability in Afrotheria: fragile sites, evolutionary breakpoints and phylogenetic inference from genome sequence assemblies. BMC Evol Biol 7: 199 (2007). Ruiz-Herrera A, Farré M, Robinson TJ: Molecular cytogenetic and genomic insights into chromosomal evolution. Heredity 108:28–36 (2012). Scally M, Madsen O, Douady CJ, de Jong WW, Stanhope MJ, Springer MS: Molecular evidence for the major clades of placental mammals. J Mammal Evol 8:239–277 (2001). Shoshani J, McKenna MC: Higher taxonomic relationships among extant mammals based on morphology, with selected comparisons of results from molecular data. Mol Phylogenet Evol 9:572–584 (1998). Simpson GG: Splendid Isolation: The Curious History of South American Mammals (Yale University Press, New Haven 1980). Sonta S, Hayata I, Sasaki M, Kondo N: Karyotype and sex determining in the two-toed sloth, Choloepus didactylus. Chromosome Information Service 28:15–17 (1980). Springer MS, Stanhope MJ, Madsen O, de Jong WW: Molecules consolidate the placental mammal tree. Trends Ecol Evol 19: 430–438 (2004). Svartman M, Stone G, Page J, Stanyon R: A chromosome painting test of the basal eutherian karyotype. Chromosome Res 12: 45–53 (2004). Svartman M, Stone G, Stanyon R: The ancestral eutherian karyotype is present in Xenarthra. PLoS Genet 2:e109 (2006).

Truman RW: Leprosy, in Vizcaíno SF, Loughry WJ (eds): The Biology of the Xenarthra, pp 111–119 (University Press of Florida, Gainesville 2008). Vizcaíno SF, Bargo MS, Fariña RA: Form, function and paleobiology in xenarthrans, in Vizcaíno SF, Loughry WJ (eds): The Biology of the Xenarthra, pp 86–99 (University Press of Florida, Gainesville 2008). Waddell PJ, Cao Y, Hauf J, Hasegawa M: Using novel phylogenetic methods to evaluate mammalian mtDNA, including amino acidinvariant sites-LogDet plus site stripping, to detect internal conflicts in the data, with special reference to the positions of hedgehog, armadillo, and elephant. Syst Biol 48: 31–53 (1999). Waters PD, Dobigny G, Pardini AT, Robinson TJ: LINE-1 distribution in Afrotheria and Xenarthra: implications for understanding the evolution of LINE-1 in eutherian genomes. Chromosoma 113:137–144 (2004). Waters PD, Dobigny G, Waddell PJ, Robinson TJ: Evolutionary history of LINE-1 in the major clades of placental mammals. PLoS One 2:e158 (2007). Wetzel RM, Ávila-Pires FD: Identification and distribution of the recent sloths of Brazil (Edentata). Rev Bras Biol 40: 831–836 (1980). Wildman DE, Uddin M, Opazo JC, Liu G, Lefort V, et al: Genomics, biogeography, and the diversification of placental mammals. Proc Natl Acad Sci USA 104:14395–14400 (2007). Yang F, Graphodatsky A, Li T, Fu B, Dobigny G, et al: Comparative genome maps of the pangolin, hedgehog, sloth, anteater and human revealed by cross-species chromosome painting: further insight into the ancestral karyotype and genome evolution of eutherian mammals. Chromosome Res 14: 283– 296 (2006).


143


Published online: August 3, 2012

The Chromosomes of Afrotheria and Their Bearing on Mammalian Genome Evolution M. Svartman a R. Stanyon b a

Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; b Department of Evolutionary Biology, University of Florence, Florence, Italy

Key Words Ancestral eutherian karyotype ⴢ Chromosome painting ⴢ Mammalian phylogeny ⴢ Paenungulata ⴢ Afroinsectiphilia

Abstract Afrotheria is the clade of placental mammals that, together with Xenarthra, Euarchontoglires and Laurasiatheria, represents 1 of the 4 main recognized supraordinal eutherian clades. It reunites 6 orders of African origin: Proboscidea, Sirenia, Hyracoidea, Macroscelidea, Afrosoricida and Tubulidentata. The apparently unlikely relationship among such disparate morphological taxa and their possible basal position at the base of the eutherian phylogenetic tree led to a great deal of attention and research on the group. The use of biomolecular data was pivotal in Afrotheria studies, as they were the basis for the recognition of this clade. Although morphological evidence is still scarce, a plethora of molecular data firmly attests to the phylogenetic relationship among these mammals of African origin. Modern cytogenetic techniques also gave a significant contribution to the study of Afrotheria, revealing chromosome signatures for the group as a whole, as well as for some of its internal relationships. The associations of human chromosomes HSA1/19 and 5/21 were found to be chromosome signatures



for the group and provided further support for Afrotheria. Additional chromosome synapomorphies were also identified linking elephants and manatees in Tethytheria (the associations HSA2/3, 3/13, 8/22, 18/19 and the lack of HSA4/8) and elephant shrews with the aardvark (HSA2/8, 3/20 and 10/17). Herein, we review the current knowledge on Afrotheria chromosomes and genome evolution. The already available data on the group suggests that further work on this apparently bizarre assemblage of mammals will provide important data to a better understanding on mammalian genome evolution. Copyright © 2012 S. Karger AG, Basel

Afrotheria as a Natural Clade

The 18 recognized orders of eutherian mammals are divided into 4 supraordinal clades: Afrotheria, Xenarthra, Euarchontoglires and Laurasiatheria. Afrotheria (orders Tubulidentata, Macroscelidea, Afrosoricida, Sirenia, Hyracoidea and Proboscidea) and Xenarthra (orders Pilosa and Cingulata) originated in the southern hemisphere (Gondwana), whereas Euarchontoglires (orders Dermoptera, Scandentia, Primates, Rodentia and Lagomorpha) and Laurasiatheria (orders Eulipotyphla, CarMarta Svartman Departamento de Biologia Geral, Instituto de Ciências Biológicas Universidade Federal de Minas Gerais Av. Antônio Carlos, 6627 – Pampulha, 31270-910 Belo Horizonte (Brazil) Tel. +55 31 3409 2612, E-Mail svartmanm @ hotmail.com

nivora, Pholidota, Cetartiodactyla, Perissodactyla and Chiroptera) are both of northern hemisphere (Laurasia) origin and reunited under Boreoeutheria [reviewed in Springer et al., 2004]. Among the valuable insights of molecular data applied to the phylogenetic reconstructions of the tree of life, the recognition of the Afrotheria as a clade is an outstanding example of how this approach has changed some longstanding biological perceptions. Afrotheria reunites 1 third of the living placental mammalian orders of Eutheria and is divided into: Proboscidea, Sirenia, Tubulidentata, Macroscelidea, Hyracoidea and Afrosoricida (suborders Tenrecomorpha and Chrysochloridea). This seemingly improbable biological grouping, now widely recognized as a natural clade, would probably have not been suspected based on morphological traits alone. This comes as no surprise when one appreciates the morphological diversity contained in Afrotheria, which reunites elephants, manatees, dugongs, hyraxes, golden moles, elephant shrews (or sengis) and the aardvark. The first hint of the existence of a clade composed of African mammals came from the analysis of the amino acid sequence of the ␣-crystalline A protein in several mammals. The similarities of this protein in the aardvark, manatee, hyrax and elephant led to the suggestion that they were closely related and could be basal in the placental tree [de Jong et al., 1981]. Nevertheless, it was not until the late 1990s that this clade of African mammals was formally proposed and named Afrotheria [Springer et al., 1997; Stanhope et al., 1998]. At the time, the currently recognized afrotherians were viewed in quite a different light. The sengis were thought to be related to rodents, the aardvark was grouped with pangolins and xenarthrans in the no longer existing order Edentata, tenrecs and golden moles were considered Insectivores and hyraxes were grouped with Perissodactyla [reviewed in Asher et al., 2009]. Contrasting with the huge amount of molecular data currently supporting the monophyly of Afrotheria, common morphological traits linking these animals are still elusive. Among the few possible synapomorphies of the group are their late dental eruption, high interspecific variability in vertebral count, intra-abdominal testis (testicondy) and placental morphology [Werdelin and Nilsonne, 1999; Carter et al., 2006; Sánchez-Villagra et al., 2007; Asher and Lehmann, 2008]. Moreover, these traits may also be present in Xenarthra, which would instead give support to Atlantogenata, a group reuniting both Afrotheria and Xenarthra [Asher et al., 2009].

The orders of Afrotheria are usually divided into 2 main clades: Paenungulata, which reunites Proboscidea, Sirenia and Hyracoidea; and Afroinsectiphilia that comprises Afrosoricida (families Tenrecidae and Chrysochloridae), Macroscelidea and Tubulidentata [Asher et al., 2009]. The position of Tubulidentata in this arrangement is still questioned, with some considering it closer to Paenungulata [Seiffert, 2007]. Paenungulata contains only a dozen species, among them the largest terrestrial mammals, the 3 recognized elephant species (Proboscidea), 4 species of herbivorous aquatic mammals, the dugong and manatees (Sirenia), and 5 species of hyraxes (Hyracoidea). The more specious Afroinsectiphilia reunites the Afrosoricida, with 21 golden mole and 33 tenrec species, and Macroscelidea, with 18 species of sengis or elephant shrews. Tubulidentata is represented by the only living species of aardvark Orycteropus afer. Most Afrotheria taxa are endemic to continental Africa. However, tenrecs are mainly found in Madagascar, 1 elephant species occurs in Asia and the aquatic manatees and dugongs are found in the American, African and Australasian coastal waters [Roca et al., 2001; Olson et al., 2004; Wilson and Reeder, 2005; Goodman et al., 2006; Dumbacher et al., 2012]. While Afrotheria is now overwhelmingly accepted as a natural superordinal clade, there is no consensus concerning the relationships among the orders that compose it. Paenungulata has been recognized on morphological grounds for decades [Simpson, 1945], but the long-accepted grouping of Sirenia and Proboscidea in Tethytheria has been recently questioned, with some analyses favoring a closer relationship between Proboscidea and Hyracoidea [Waddell and Shelley, 2003; Roca et al., 2004; Nishihara et al., 2006; Murphy et al., 2007; Kuntner et al., 2011], Sirenia and Hyracoidea [Poux et al., 2008], or still others lending support to Tethytheria [Kjer and Honeycutt, 2007; Arnason et al., 2008]. Most studies point to Tubulidentata as the sister taxon to Afroinsectivora [Poux et al., 2008; Asher et al., 2009; Kuntner et al., 2011], but there have been suggestions of its closer relationship to Chrysochloridae [Kjer and Honeycutt, 2007], to Tenrecidae [Waddell and Shelley, 2003] or to Afrosoricida as a whole [Arnason et al., 2008].

Chromosome Evolution in Afrotheria


The Position of Afrotheria in the Tree of Life

The arrangement of eutherian orders into the 4 main supraordinal clades is now overwhelmingly accepted, but some questions still persist, among them interordinal re145

lationships as outlined above and, more fundamental, the identification of the basal placental clade. Afrotheria (Exafroplacentalia) was early on often suggested as a good candidate. A frequent alternative hypothesis is that Xenarthra would be basal to all other eutherians (the Epitheria hypothesis). The third hypothesis takes a middle ground and suggests that Xenarthra and Afrotheria would form a sister taxon to the other placentals (Atlantogenata). Evidence has been found supporting each of these 3 alternative hypotheses. Morphological and some molecular data favored the Epitheria hypothesis [Shoshani and McKenna, 1998; Kriegs et al., 2006]. Many molecular studies lent support to Exafroplacentalia [Murphy et al., 2001; Scally et al., 2001; Amrine-Madsen et al., 2003; Nikolaev et al., 2007; Nishihara et al., 2007] and Atlantogenata was suggested on the basis of some molecular and phylogenomic analyses [Waddell et al., 1999; Madsen et al., 2001; Delsuc et al., 2002; Lin et al., 2002; Hallström et al., 2007; Murphy et al., 2007; Waters et al., 2007; Prasad et al., 2008]. Recently, some authors have speculated that the exact divergence sequence of the base of the eutherian tree may never be satisfactorily resolved, at least with current methodology. They contend that phylogenetic resolution is limited by incomplete lineage sorting, introgression and other unknown processes that may have produced mosaic genomes with various parts pointing to different phylogenetic histories. In that case, the placental tree would have a trifurcation instead of a dichotomy at its base, which would explain why a consensus on a bifurcating tree has so far been elusive [Hallström and Janke, 2008, 2010; Churakov et al., 2009; Nishihara et al., 2009]. The origin of Afrotheria and Xenarthra is thought to have occurred at around 100 million or more years ago. This time coincides with the tectonic events that separated South America and Africa, where Xenarthra and Afrotheria originated, respectively. A causal connection was thus proposed between the diversification of eutherians and the tectonic events with Gondwana playing an important role in the origin of eutherians [Eizirik et al., 2001; Murphy et al., 2001; Springer et al., 2004; Wildman et al., 2007]. A second possibility raised on the basis of fossil, tectonic, and molecular data is that the early radiation of eutherians was triggered in Laurasia by climate changes instead of plate tectonics and would have been shortly followed by dispersal from that continent [Hallström and Janke, 2010].

146


The Chromosomes of Afrotheria

Shortly after the attention-gathering paper of Murphy et al. [2001], chromosome painting of Afrotherian species began, initially as an independent test of the validity of this superordinal clade. At that point there was still considerable skepticism about the validity of the molecular conclusion. Firstly, it was thought that it might be possible to identify chromosome landmarks which could lend support to the assemblage. Secondly, if confirmed, it was hoped that more complete cytogenetic information might be informative about the internal phylogenetic relationships of Afrotheria taxa. However, even prior to the use of chromosome painting some basic cytogenetic information was already present for Afrotheria species. Diploid Numbers and Chromosome Banding Patterns Chromosome studies performed in Afrotheria showed a trend to higher diploid numbers in Paenungulata compared to Afroinsectiphilia. In the former group, the reported karyotypes presented a minimum of 2n = 48 chromosomes, whereas 2n = 40 was the highest diploid number found in the second group. Paenungulata is better known cytogenetically, as the majority of its species have been karyotyped, mostly with banding methods. The banded chromosomes have already been described for 2 of the 4 known living Sirenia: the Amazonian manatee Trichechus inunguis (2n = 56) and the Florida manatee T. manatus latirostris (2n = 48) [Assis et al., 1988; Gray et al., 2002]. GTG-banded karyotypes of 2 Proboscidea, the African and Asian elephants (Loxodonta africana and Elephas maximus, respectively) were also described and compared, allowing the detection of a high homology between both karyotypes [Houck et al., 2001]. Three of the 5 recognized hyrax species had their complements reported after GTG-banding: Procavia capensis, Dendrohyrax arboreus and Heterohyrax brucei. The 3 species presented 2n = 54, which was suggested as the ancestral 2n for Hyracoidea [reviewed in Frönicke, 2006]. Afroinsectiphilia is less known cytogenetically, as only about half of the species of sengis, golden moles and tenrecs have been karyotyped. The only living Tubulidentata species, the aardvark, has the lowest diploid number known among Afrotheria (2n = 20) and its GTG- and CBG-banding patterns have been described [reviewed in Yang, 2006]. The karyotypes of 14 out of the 21 known golden moles representing 8 of the 9 genera of the family Chrysochloridae have Svartman /Stanyon

been described and GTG-banded chromosomes have been reported for 10 species. All species had 2n = 30, except for Eremitalpa granti (2n = 26), Calcochloris obtusirostris (2n = 28), Amblysomus robustus (2n = 36) and A. septentrionalis (2n = 34) [Bronner, 1995; Gilbert et al., 2006, 2008]. Tenrecs are the least cytogenetically studied Afrotheria. Until recently, only 3 among the 33 recognized species had their karyotypes reported: Echinops telfairi (2n = 40), Hemicentetes nigriceps (2n = 38) and Micropotamogale dobsoni (2n = 30), all of them without banding patterns [reviewed in Robinson, 2006]. This situation has been recently improved with a study that presented GTG-banded karyotypes of Oryzorictes hova (2n = 32) and of 10 Microgale species, with diploid numbers ranging from 2n = 30 to 56 [Gilbert et al., 2007]. Nine of the 18 species of sengis (Macroscelidea) representing 3 of the 4 recognized genera have been karyotyped and GTG-banded chromosomes have been reported for Elephantulus proboscideus, E. edwardii, E. intufi, E. pilicaudus, Macroscelides proboscideus, and Petrodromus tetradactylus [Svartman et al., 2004; Smit et al., 2008, 2011]. All the sengis analyzed presented a karyotype with 2n = 26, with the exception of E. myurus (2n = 30), E. rozeti and P. tetradactylus (both with 2n = 28) [reviewed in Smit et al., 2008]. From the above data, it becomes clear that further basic cytogenetic analyses in Afrotheria, especially in Afroinsectiphilia, including the application of banding patterns, are needed. This kind of studies could be helpful in the understanding of chromosome evolution in this group and provide clues on the rearrangements involved in their karyotype differentiation.

Molecular Cytogenetics in Afrotheria

Chromosome painting, in which species-specific chromosome probes are used in interspecific FISH experiments, has been widely applied to the investigation of phylogenetic issues in mammals. Most of these studies relied on human chromosome probes that, applied to various mammalian groups, allowed the determination of chromosome features specific for different taxa. This approach has proved to be powerful in testing phylogenetic relationships and results from chromosome painting with human probes are currently available for most of the 18 recognized eutherian orders, Hyracoidea being the only exception.


Chromosome Landmarks Supporting Afrotheria The first publications on chromosome painting in Afrotheria were those on the reciprocal painting between humans, elephants and aardvark [Frönicke et al., 2003; Yang et al., 2003]. Yang et al. [2003] contended that their data on aardvark and elephants did not provide any unique cytogenetic signatures that supported the monophyly of the Afrotheria. Instead they interpreted their data to suggest that the Afrotheria provided strong evidence for the content of the mammalian ancestral karyotype which they concluded was composed of 2n = 44 chromosomes. They arrived at this conclusion due to the conservation of numerous human syntenies in these karyotypes and the supposed basal position of Afrotheria in the placental mammalian tree. A remarkable fact was that the synteny to human chromosome 1 was found intact in the aardvark. Previous chromosome painting studies had revealed that in most mammals chromosome 1 was divided into 2 or more segments, with the exception of dolphins and whales [see Murphy et al., 2003]. The aardvark and later elephant shrew data showed instead that chromosome 1 was intact in 3 highly divergent eutherian clades [Yang et al., 2003; Svartman et al., 2004; Robinson et al., 2004]. Multidirectional painting and mapping strongly supported the hypothesis that chromosome 1 was intact in the eutherian ancestor and was variously fissioned in different phylogenetic lines. Further it was associated with a part of chromosome 19 to form a HSA1/19 association. Yang et al. [2003] interpreted that the 1/19 synteny found in both elephants and aardvark was ancestral for eutherians also due to the fact that a 1/19 association had also been previously found in a primate, the galago [Stanyon et al., 2002]. There were slight but significant differences with the data for L. africana reported in the Frönicke et al. [2003] publication. Yang et al. [2003] had found a 5/21 association in the aardvark but not in elephants. Instead, Frönicke et al. [2003] found this association clearly present in the African elephant and interpreted the HSA5/21 association along with the 1/19 association as derived landmarks uniting these 2 distant Afrotheria taxa. Both studies significantly found the presence of a HSA10/12/22 association and the lack of a 4/8 association in the elephants. The following year the publication of Svartman et al. [2004] reported the presence of the HSA1/19, 4/8, 5/21 and 10/12/22 associations in a species of elephant shrew, as did Robinson et al. [2004] for the golden mole and an additional elephant shrew genus. These data strongly supported the hypothesis of the linking association of HSA5/21 for the Afrotheria. Thus, chromosome painting Cytogenet Genome Res 2012;137:144–153

147

was especially important in Afrotheria studies, as it was the first non-sequencing-based approach to provide an independent demonstration of the group’s monophyly. The identification of the HSA1/19 and 5/21 associations as chromosome signatures for the group made Afrotheria the first of the major mammalian superordinal clades to have synapomorphic karyotypical features recognized [Frönicke et al., 2003; Yang et al., 2003; Robinson et al., 2004; Svartman et al., 2004; Kellogg et al., 2007; RuizHerrera and Robinson, 2007]. Afrotheria and the Ancestral Eutherian Karyotype (AEK) One of the primary goals of interspecies chromosome painting in mammals has been the reconstruction of the karyotype present in the common ancestor of all placental mammals or the AEK [reviewed in Ferguson-Smith and Trifonov, 2007 as well as in Graphodatsky et al., 2011]. However, the question of whether the association HSA1/19 was ancestral for eutherians giving a 2n = 44 karyotype or whether, instead, HSA1 and 19p were independent chromosomes giving a 2n = 46 diploid number persisted for a while. Reciprocal painting between galagos and humans also demonstrated that the HSA1/19 association in primates was due to convergence: it was 1/19q [Stanyon et al., 2002]. Yet the choice between 2n = 44 or 46 was difficult to resolve because of a lack of an appropriate outgroup for the eutherian mammals [Svartman et al., 2004]. Another possible diploid number that could not be ruled out was 2n = 48. This diploid number was possible if the HSA10/12/22 was considered as 10 and 12/22 in the ancestor. Simply, chromosome painting between eutherian and other mammals had not provided results, and before the genome assemblies of these mammals were available, it was not possible to have a definitive test. Now it seems that there is more data to support the hypothesis presently most accepted for an AEK complement with 46 chromosomes, composed by human chromosomes HSA1, 2p, 2q, 3/21, 4/8p, 5, 6, 7a, 7b/16p, 8q, 9, 10q, 10p/12pq/22qt, 11, 12qt/22q, 13, 14/15, 16q/19q, 17, 18, 19p, 20 and X. Comparisons with the available sequencing data from the marsupial Monodelphis domestica and the chicken Gallus gallus, used as outgroups, showed that HSA3/21, 4/8p, 7a/16p, 10p/12pq/22qt, 14/15, 16q/19q and 12qt/22q were present in one or both of these species, suggesting that they may be symplesiomorphies, already present before the divergence of eutherians. A 1/19 association was also found, but closer examination shows that it is 1/19p in Afrotheria and 1/19q in marsupi148


als (fig. 1). The synapomorphies present in eutherians would be chromosomes HSA1, 5, 6, 9, 11, 17, 20 and X and 3 chromosomal segments (7b, 2p–q1.3 and 2q1.3–qter) [Robinson and Ruiz-Herrera, 2008; Ruiz-Herrera et al., 2012]. Phylogenetic Relationships within Afrotheria As yet there is no consensus for the phylogenetic branching order within Afrotheria. Murphy et al. [2001] placed the triumvirate of sirenians, elephants and hyraxes (Paenungulata) as basal, but other authors have viewed Macroscelidea, elephant shrews, as the most basal and early divergent order within Afrotheria. There are other intriguing questions within Afrotheria, among them the phylogenetic order within Paenungulata, even if it turns out to be basal, and the relationships within Afrosoricida. Chromosome painting has not resolved these questions, partly because until now, painting with human chromosome-specific probes has only been applied to 7 species of Afrotheria belonging to 5 of the 6 recognized orders. For instance, data with this approach are still missing for hyraxes (order Hyracoidea) and tenrecs (suborder Tenrecomorpha, order Afrosoricida). As for conventional cytogenetic techniques, Paenungulata are relatively better represented in the molecular cytogenetic studies than Afroinsectiphilia. A summary of the main findings obtained after painting with human chromosome-specific probes in different Afrotheria species is presented in table 1. In all species analyzed 6 human chromosomes (HSA5, 9, 17, 18, 20 and X) were conserved, as were 6 of the combinations thought to be present in the AEK (HSA3/21, 7/16, 10/12, 12/22 twice, 14/15 and 16/19). Missing among these is the HSA4/8 association, which is absent in elephants and in the Florida manatee, a possible characteristic linking Paenungulata [Kellogg et al., 2007]. Chromosome Painting in Elephants and Manatees As in the other Afrotheria the Florida manatee also has a 5/21 and 1/19 association (fig. 2) [Kellogg et al., 2007]. Along with the elephant it lacks the HSA4/8 association, and there are 4 additional derived cytogenetic characters linking elephants with manatees: HSA2/3, 3/13, 8/22 and 18/19 (table 1). These 5 synapomorphies unite elephants and manatees in Tethytheria and also implicitly support Paenungulata. The only molecular cytogenetics study until now to include hyraxes was performed by Pardini et al. [2007]. These authors applied reciprocal chromosome painting among species representing each of the 3 orders of Paenungulata (L. africana, Svartman /Stanyon

Fig. 1. Diagram showing the HSA1/19 association in the marsupial Monodelphis domestica deduced from sequencing data (left) showing the coding gene lying adjacent to the association, and in the elephant Loxodonta africana (Afrotheria), demonstrated after reciprocal chromosome painting (idiograms modified from Frönicke et al. [2003]).

Table 1. Chromosome painting with human chromosome-specific probes in Afrotheria Species

Number of conserved segments

Associations of human autosomes

54

Trichechus manatus latirostris Florida manatee (2n = 48)

Human chromosomes

References

conserved

two blocks

three or more blocks

1/2?, 1/3, 1/11?, 1/19, 1/21, 2/3, 2/11, 2/16, 3/6, 3/13, 3/21, 4/15, 4/16, 5/21, 6/13?, 7/10, 7/16, 8/22, 10/12, 11/16, 12/22 (2!), 14/15, 16/19, 18/19

5, 9, 17, 18, 20, X

6?, 7, 8, 10, 12, 13, 14, 16, 21, 22

1, 2, 3, 4, 11, 15, 19

Yang et al. [2003], Frönicke et al. [2003]

44

1/15, 1/19, 2/3 (2!), 3/7 (3!), 3/13, 3/21, 5/21, 7/16, 8/22, 10/12 (2!), 11/20, 12/22 (3!), 14/15, 16/19, 18/19

4, 5, 6, 9, 11, 14, 17, 18, 20, X

1, 7, 8, 10, 12, 13, 15, 16, 21, 22

2, 3, 12, 19

Kellogg et al. [2007]

Orycteropus afer Aardvark (2n = 20)

31

1/6, 1/19, 2/7, 2/8, 2/13, 2/18, 3/20, 3/21, 4/8, 5/21, 7/15, 7/16, 8/9, 10/11, 10/12, 10/17, 12/22 (2!), 13/16, 14/15, 16/19

1, 3, 4, 5, 6, 9, 11, 13, 14, 15, 17, 18, 20, 21, X

2, 7, 8, 10, 12, 16, 19, 22

Macroscelides proboscideus Short-eared elephant shrew (2n = 26)

37

1/2, 1/19, 2/8, 2/9, 2/10, 3/13, 3/15, 3/18, 1, 4, 5, 6, 9, 11, 13, 14, 3/20, 3/21, 4/8, 5/21, 6/7, 7/16, 8/11, 10/12, 15, 17, 18, 20, 21, X 10/17, 12/22 (2!), 14/15, 16/19 (2!), 16/22

7, 8, 10, 12, 22

2, 3, 16, 19

Svartman et al. [2004]

Elephantulus edwardiib, c Western Rock elephant shrew (2n = 26)

35

1/2, 1/19, 2/8, 2/9, 2/10, 3/13, 3/15, 3/18, 3/20, 3/21, 4/8, 5/21, 6/7, 7/16, 8/11, 10/12, 10/17, 12/16, 12/22 (2!), 14/15, 16/19

1, 4, 5, 6, 9, 11, 13, 14, 15, 17, 18, 20, 21, X

7, 8, 10, 12, 16, 19, 22

2, 3

Robinson et al. [2004]

Chrysochloris asiaticab Cape golden mole (2n = 30)

32

1/8, 1/19, 2/8, 2/20, 3/21, 4/8, 5/21, 7/16, 9/11, 10/12, 10/22, 12/22 (2!), 13/18, 14/15, 16/19

1, 3, 4, 5, 6, 9, 11, 13, 14, 15, 17, 18, 20, 21, X

2, 8, 10, 12, 16, 19, 22

7

Robinson et al. [2004]

Loxodonta africanaa African elephant Elephas maximus Asian elephant (2n = 56)

Yang et al. [2003]

In bold, the features present in all the species studied. The question mark indicates uncertainties in the labeling patterns. The associations uniting Paenungulata are highlighted in green (note the absence of the ancestral eutherian association 4/8 in this group); those uniting Afroinsectiphilia are highlighted in red and those linking Tubulidentata and elephant shrews, in blue. a These results are a compilation of the two reports on elephants. b We considered associations between segments separated by the centromere and heterochromatic blocks, which resulted in some combinations not considered in the original report and in differences in the count of conserved segments. c Originally reported as E. rupestris and later identified as E. edwardii [Smit et al., 2008].



149

with 2n = 56, the Cape rock hyrax P. capensis, with 2n = 54, and T. m. latirostris, with 2n = 48) and used the aardvark (2n = 20) as an outgroup in an effort to deduce synapomorphies of the Paenungulata. Their analysis provided further support for the 4 previously suggested chromosome signatures of Paenungulata (corresponding to HSA2/3, 3/13, 8/22 and 18/19), but no further associations indicating a relationship between any 2 pairs of Paenungulata species were identified. A hypothetical ancestral Paenungulata karyotype with 2n = 58 was also advanced in this study. It might be helpful to directly study the hyrax genome using human chromosome paints, but it is unknown if this additional cytogenetic information could resolve the phylogenetic branching order. The molecular data indicate that these taxa probably diverged rapidly from each other. For example, retroposon data also confirmed Paenungulata, but could not resolve the phylogenetic relationships among elephants, sirenians and hyraxes. In a recent study using molecular data, Kuntner et al. [2011] found that Sirenia was the sister group to Hyracoidea plus Proboscidea, a relationship that still needs confirmation. Chromosome Phylogeny within Afroinsectiphilia As reported above, the HSA5/21 and 1/19 associations were present in the elephant shrew and found in all Afrotheria studied so far. Thus, the chromosome data helped resolve any initial uncertainty as to the placement of the Macroscelidea within the Afrotheria. Chromosome painting with human probes also revealed some chromosome features that reinforced previously proposed Afrotherian interordinal clades. The associations HSA2/8, 3/20 and 10/17, present in the aardvark and the elephant shrew M. proboscideus, but not in the elephants, led to the suggestion that they were synapomorphic traits of Afroinsectiphilia and there is no doubt that these 3 associations strongly link aardvarks and elephant shrews after the divergence of the line leading to elephants [Frönicke et al., 2003; Yang et al., 2003; Svartman et al., 2004] (table 1). The presence of the HSA2/8 also in a golden mole species lent further support to this idea, but the absence of HSA3/20 and 10/17 from the golden mole was interpreted as an indication that Macroscelidea and Tubulidentata might be sister taxa. No common chromosome features uniting Afroinsectivora have been found as yet [Robinson et al., 2004]. There are also research reports on Afroinsectiphilia that are not based on painting with human chromosome probes, but instead used interordinal painting with 150


Fig. 2. Metaphase of the manatee Trichechus manatus latirostris (2n = 48) after fluorescence in situ hybridization with specific probes for human chromosomes 1 (in red) and 19 (in green).

probes isolated from Afrotheria species. Gilbert et al. [2006, 2008] used painting with Chrysochloris asiatica chromosome-specific probes in a comparative analysis of golden mole karyotypes, including 8 species from 7 genera. The results from these experiments were complemented by comparative GTG-banding and FISH with a telomere probe. Overall, golden moles were shown to be karyotypically conserved, with only a few fusion/fission rearrangements and heterochromatic changes differentiating their complements, which would have derived from an ancestral karyotype for the family Chrysochloridae with 2n = 30. Painting with individual chromosomes isolated from the tenrec O. hova (2n = 32) was performed in the cells of 10 Microgale species, with diploid numbers ranging from 2n = 30 to 56. The results, allied to those obtained by the comparison of GTG-banded karyotypes, led to the conclusion that the common ancestor of both tenrec genera had a karyotype virtually identical to that observed in some extant species with 2n = 32 and that chromosome evolution in this group proceeded mainly by chromosome fusion/fission events [Gilbert et al., 2007]. Four species of elephant shrews had their chromosomes painted with probes isolated from E. edwardii (2n = 26) and, combined with data from banding patterns, the results of these experiments revealed extremely conserved complements that basically differed between species by their constitutive heterochromatin content and location. The karyotype of P. tetradactylus (2n = 28), the only species among those analyzed which did not Svartman /Stanyon

have 2n = 26, differed from those of the other species by a Robertsonian rearrangement [Smit et al., 2011]. The Base of the Tree: Afrotheria or Xenarthra? Chromosome painting with human probes resulted in the delimitation of 31 conserved segments in the aardvark, the lowest number identified in a non-primate species. This observation was interpreted by Yang et al. [2003] as an indication that the aardvark retained a complement very close to the AEK and led to the now rejected proposal that the HSA1/19 association was ancestral for eutherians. Sequencing data on the marsupial M. domestica also detected an HSA1/19 association, but we now know that the segments of chromosome 19 involved in the rearrangement are not the same in both taxa. The karyotype of the 2-toed sloth Choloepus hoffmanni (2n = 50), a xenarthran, was shown to be very similar to the AEK, from which it differed by only 3 rearrangements [Svartman et al., 2006]. Taken together, these data show that both Afrotheria and Xenarthra present chromosome features that may indicate their basal position in the eutherian tree, but they do not allow deciding between the 3 current hypotheses for the base of the tree. The finding that Afrotheria present a genome size much larger than those of other eutherians prompted Redi et al. [2007] to suggest that this is a further genomic signature of the group. Measurements of the genome sizes of 6 Afrotheria species resulted in a mean genome size value of 5.3 8 0.7 pg, the highest reported for eutherians. The average genome size of Xenarthra was 4.6 pg, whereas Euarchontoglires and Laurasiatheria showed mean values of 3.58 and 2.72 pg, respectively. Based on these observations, a correlation was suggested between larger genome sizes and more basal positions in the eutherian phylogenetic tree. Additionally, it was speculated that larger genomes would contain an excess of repetitive sequences [Redi et al., 2005, 2007]. The development of a new computational method has recently allowed the identification of centromere-related repeat structures from unassembled genomes of species spanning the diversity of mammals (horse, dog, African elephant, 9-banded armadillo, opossum and platypus). Horse and dog presented the greatest diversity of centromeric sequences, contrasting with the highly homogeneous centromeric sequences of the armadillo (a xenarthran) and the elephant. This approach allowed the characterization of a previously unknown satellite DNA from L. africana, which was localized by FISH to all the centromeres. It consisted of a single 1.800-bp fragment that could represent a single centromeric unit or a monomerChromosome Evolution in Afrotheria

ic unit part of an undetected higher-order organization. The centromeres of the armadillo Dasypus novemcinctus were also shown to be constituted from a satellite repeat composed by a single consensus sequence with 173-bp units [Alkan et al., 2011]. In conclusion, this new information points to a seemingly less complex centromeric structure for the 2 representatives of the most basal eutherian clades when compared to other eutherian taxa. Both cytogenetic and biomolecular approaches strongly support that Afrotheria is a natural clade. Morphological analyses are now beginning to support this view. However, a series of problems remain unresolved. Different approaches have been used to determine which is the most basal taxon in the eutherian tree, the exact composition of the AEK and the phylogenetic order of divergence within Afrotheria. None of these problems yet have a definitive answer. Recently, it has been proposed that many of these questions are destined to remain unsolved, at least with current methods. This state of affairs may be due to rapid divergence, incomplete lineage sorting, introgression and perhaps other unknown processes. Nevertheless, it is important to observe that the amount of data accumulated in these studies is impressive. However, we note that there is as yet little cross-talk between different disciplines. Future progress will depend on more fully integrating cytogenetics with other phylogenomic methods to develop a more complete picture of both Afrotheria and mammalian evolution in general. For instance, association, rearrangement homology or understanding the mechanism of chromosome rearrangements must ultimately be tested or understood by integrating sequencing data. Indeed, understanding the mechanisms underpinning mammalian genome architecture and the forces that cause them to evolve is inherently more satisfying than just mapping homology or drawing phylogenetic trees. However, this goal will require an ever tighter integration of cytogenetics and other molecular methods. The Afrotheria may well constitute a testing ground, as it has up to now, for future integrated research programs.

Acknowledgements This work was possible due to grants received by M.S. from Fundação de Amparo à Pesquisa do Estado de Minas Gerais (processes CRA – APQ-00170-09 and APQ-00336-09). This work was also supported by a grant PRIN and from the University of Florence to R.S.


151

References Alkan C, Cardone MF, Catacchio CR, Antonacci F, O’Brien SJ, et al: Genome-wide characterization of centromeric satellites from multiple mammalian genomes. Genome Res 21: 137–145 (2011). Amrine-Madsen H, Koepfli K-P, Wayne RK, Springer MS: A new phylogenetic marker, apolipoprotein B, provides compelling evidence for eutherian relationships. Mol Phylogenet Evol 28:225–240 (2003). Arnason U, Adegoke JA, Gullberg A, Harley EH, Janke A, Kullberg M: Mitogenomic relationships of placental mammals and molecular estimates of their divergences. Gene 421:37– 51 (2008). Asher RJ, Lehmann T: Dental eruption in afrotherian mammals. BMC Biology 6:14 (2008). Asher RJ, Bennett N, Lehmann T: The new framework for understanding placental mammal evolution. BioEssays 31: 853–864 (2009). Assis MF, Best RC, Yonenaga-Yassuda Y: Cytogenetic study of Trichechus inunguis (Amazonian manatee). Braz J Genet 11: 41–50 (1988). Bronner GN: Cytogenetic properties of nine species of golden moles. J Mammal 76:957–971 (1995). Carter AM, Blankenship TN, Enders AC, Vogel P: The fetal membranes of the otter shrews and a synapomorphy for Afrotheria. Placenta 27:258–268 (2006). Churakov G, Kriegs JO, Baertsch R, Zemann A, Brosius J, Schmitz J: Mosaic retroposon insertion patterns in placental mammals. Genome Res 19:868–875 (2009). de Jong WW, Zweers A, Goodman M: Relationship of aardvark to elephants, hyraxes and sea cows from alpha-crystallin sequences. Nature 292:538–540 (1981). Delsuc F, Scally M, Stanhope MJ, de Jong WW, Catzefelis FM, et al: Molecular phylogeny of living xenarthrans and the impact of characters and taxon sampling on the placental tree rooting. Mol Biol Evol 19:1656–1671 (2002). Dumbacher JP, Rathbun GB, Smit HA, Eiseb SJ: Phylogeny and taxonomy of the round-eared sengis or elephant-shrews, genus Macroscelides (Mammalia, Afrotheria, Macroscelidea). PLoS ONE 7:e32410 (2012). Eizirik E, Murphy WJ, O’Brien SJ: Molecular dating and biogeography of the early placental mammal radiation. J Hered 92: 212–219 (2001). Ferguson-Smith MA, Trifonov V: Mammalian karyotype evolution. Nat Rev Genet 8: 950– 962 (2007). Frönicke L: Order Hyracoidea, in O’Brien SJ, Menninger JC, Nash WG (eds): Atlas of Mammalian Chromosomes, pp 78–79 (John Wiley and Sons, Hoboken 2006).

152

Frönicke L, Wienberg J, Stone G, Adams L, Stanyon R: Towards the delineation of the ancestral eutherian genome organization: comparative genome maps of human and the African elephant (Loxodonta africana) generated by chromosome painting. Proc Biol Sci 270: 1331–1340 (2003). Gilbert C, O’Brien PC, Bronner G, Yang F, Hassanin A, et al: Chromosome painting and molecular dating indicate a low rate of chromosomal evolution in golden moles (Mammalia, Chrysochloridae). Chromosome Res 14:793–803 (2006). Gilbert C, Goodman SM, Soarimalala V, Olson LE, O’Brien PC, et al: Chromosomal evolution in tenrecs (Microgale and Oryzorictes, Tenrecidae) from the Central Highlands of Madagascar. Chromosome Res 15: 1075– 1091 (2007). Gilbert C, Maree S, Robinson TJ: Chromosomal evolution and distribution of telomeric repeats in golden moles (Chrysochloridae, Mammalia). Cytogenet Genome Res 121: 110–119 (2008). Goodman SM, Raxworthy CJ, Maminirina CP, Olson LE: A new species of shrew tenrec (Microgale jobihely) from northern Madagascar. J Zool 270:384–398 (2006). Graphodatsky AS, Trifonov VA, Stanyon R: The genome diversity and karyotype evolution of mammals. Mol Cytogenet 4:22 (2011). Gray BA, Zori RT, McGuire PM, Bonde RK: A first generation ideogram for the Florida manatee (Trichechus manatus latirostris) based on multiple chromosome banding techniques. Hereditas 137:215–223 (2002). Hallström BM, Janke A: Resolution among major placental mammal interordinal relationships with genome data imply that speciation influenced their earliest radiations. BMC Evol Biol 8: 162 (2008). Hallström BM, Janke A: Mammalian evolution may not be strictly bifurcating. Mol Biol Evol 12:2804–2816 (2010). Hallström BM, Kullberg M, Nilsson MA, Janke A: Phylogenomic data analyses provide evidence that Xenarthra and Afrotheria are sister groups. Mol Biol Evol 24: 2059–2068 (2007). Houck ML, Kumamoto AT, Gallagher Jr DS, Benirschke K: Comparative cytogenetics of the African elephant (Loxodonta africana) and Asiatic elephant (Elephas maximus). Cytogenet Cell Genet 93:249–252 (2001). Kellogg ME, Burkett S, Dennis TR, Stone G, Gray BA, et al: Chromosome painting in the manatee supports Afrotheria and Paenungulata. BMC Evol Biol 7: 6 (2007). Kjer KM, Honeycutt RL: Site specific rates of mitochondrial genomes and the phylogeny of Eutheria. BMC Evol Biol 7: 8 (2007). Kriegs JO, Churakov G, Kiefmann M, Jordan U, Brosius J, Schmitz J: Retroposed elements as archives for the evolutionary history of placental mammals. PLoS Biol 4:e91 (2006).


Kuntner M, May-Collado LJ, Agnarsson I: Phylogeny and conservation priorities of afrotherian mammals (Afrotheria, Mammalia). Zool Scr 40:1–15 (2011). Lin YH, McLenachan PA, Gore AR, Phillips MJ, Ota R, et al: Four new mitochondrial genomes and the increased instability of evolutionary trees of mammals from improved taxon sampling. Mol Biol Evol 19:2060–2070 (2002). Madsen O, Scally M, Douady CJ, Kao DJ, DeBry RW, et al: Parallel adaptive radiations in two major clades of placental mammals. Nature 409:610–614 (2001). Murphy WJ, Eizirik E, O’Brien SJ, Madsen O, Scally M, et al: Resolution of the early placental mammal radiation using Bayesian phylogenetics. Science 295:2348–2351 (2001). Murphy WJ, Frönicke L, O’Brien SJ, Stanyon R: The origin of human chromosome 1 and its homologs in placental mammals. Genome Res 13:1880–1888 (2003). Murphy WJ, Pringle TH, Crider TA, Springer MS, Miller W: Using genomic data to unravel the root of the placental mammal phylogeny. Genome Res 17:413–421 (2007). Nikolaev S, Montoya-Burgos JI, Margulies EH, Rougemont J, Nyffeler B, Antonarakis SE: Early history of mammals is elucidated with the ENCODE multispecies sequencing data. PLoS Genet 3:e2 (2007). Nishihara H, Hasegawa M, Okada N: Pegasoferae, an unexpected mammalian clade revealed by tracking ancient retroposon insertions. Proc Natl Acad Sci USA 103: 9929– 9934 (2006). Nishihara H, Okada N, Hasegawa M: Rooting the eutherian tree: the power and pitfalls of phylogenomics. Genome Biol 8:R199 (2007). Nishihara H, Maruyama S, Okada N: Retroposon analysis and recent geological data suggest near-simultaneous divergence of the three superorders of mammals. Proc Natl Acad Sci USA 106:5235–5240 (2009). Olson LE, Goodman SM, Yoder AD: Illumination of cryptic species boundaries in longtailed shrew tenrecs (Mammalia: Tenrecidae; Microgale): new insights into geographic variation and distributional constraints. Biol J Linn Soc 83: 1–22 (2004). Pardini AT, O’Brien PC, Fu B, Bonde RK, Elder FF, et al: Chromosome painting among Proboscidea, Hyracoidea and Sirenia: support for Paenungulata (Afrotheria, Mammalia) but not Tethytheria. Proc Biol Sci 274: 1333– 1340 (2007). Poux C, Madsen O, Glos J, de Jong WW, Vences M: Molecular phylogeny and divergence times of Malagasy tenrecs: influence of data partitioning and taxon sampling on dating analyses. BMC Evol Biol 8: 102 (2008).

Svartman /Stanyon

Prasad AB, Allard MW, NISC Comparative Sequencing Program, Green ED: Confirming the phylogeny of mammals by use of large comparative sequence data sets. Mol Biol Evol 25:1795–1808 (2008). Redi CA, Zacharias H, Merani S, Oliveira-Miranda M, Aguilera M, et al: Genome sizes in Afrotheria, Xenarthra, Euarchontoglires, and Laurasiatheria. J Hered 96: 485–493 (2005). Redi CA, Garagna S, Zuccotti M, Capanna E: Genome size: a novel genomic signature in support of Afrotheria. J Mol Evol 64: 484– 487 (2007). Robinson TJ: Order Afrosoricida, order Macroscelidea, in O’Brien SJ, Menninger JC, Nash WG (eds): Atlas of Mammalian Chromosomes, pp 63–70 (John Wiley and Sons, Hoboken 2006). Robinson TJ, Ruiz-Herrera A: Defining the ancestral eutherian karyotype: a cladistic interpretation of chromosome painting and genome sequence assembly data. Chromosome Res 16:1133–1141 (2008). Robinson TJ, Fu B, Ferguson-Smith MA, Yang F: Cross-species chromosome painting in the golden mole and elephant shrew: support for the mammalian clades Afrotheria and Afroinsectiphillia but not Afroinsectivora. Proc Biol Sci 271: 1477–1484 (2004). Roca AL, Georgiadis N, Pecon-Slattery J, O’Brien SJ: Genetic evidence for two species of elephant in Africa. Science 293: 1473–1477 (2001). Roca AL, Bar-Gal GK, Eizirik E, Helgen KM, Maria R, et al: Mesozoic origin for West Indian insectivores. Nature 429: 649–651 (2004). Ruiz-Herrera A, Robinson TJ: Chromosomal instability in Afrotheria: fragile sites, evolutionary breakpoints and phylogenetic inference from genome sequence assemblies. BMC Evol Biol 7: 199 (2007). Ruiz-Herrera A, Farré M, Robinson TJ: Molecular cytogenetic and genomic insights into chromosomal evolution. Heredity 108:28–36 (2012).


Sánchez-Villagra MR, Narita Y, Kuratani S: Thoracolumbar vertebral number: the first skeletal synapomorphy for afrotherian mammals. Syst Biodiv 5: 1–7 (2007). Scally M, Madsen O, Douady CJ, de Jong WW, Stanhope MJ, Springer MS: Molecular evidence for the major clades of placental mammals. J Mamm Evol 8:239–277 (2001). Seiffert ER: A new estimate of afrotherian phylogeny based on simultaneous analysis of genomic, morphological, and fossil evidence. BMC Evol Biol 7: 224 (2007). Shoshani J, McKenna MC: Higher taxonomic relationships among extant mammals based on morphology, with selected comparisons of results from molecular data. Mol Phylogenet Evol 9:572–584 (1998). Simpson GG: The principles of classification and a classification of mammals. Bull Am Mus Nat Hist 85:1–350 (1945). Smit HA, Robinson TJ, Watson J, van Vuuren BJ: A new species of elephant-shrew (Afrotheria: Macroscelidea: Elephantulus) from South Africa. J Mammal 89:1257–1269 (2008). Smit HA, van Vuuren BJ, O’Brien PCM, Ferguson-Smith M, Yang F, Robinson TJ: Phylogenetic relationships of elephant-shrews (Afrotheria, Macroscelididae). J Zool 284: 133– 143 (2011). Springer MS, Cleven GC, Madsen O, de Jong WW, Waddell VG, et al: Endemic African mammals shake the phylogenetic tree. Nature 388:61–64 (1997). Springer MS, Stanhope MJ, Madsen O, de Jong WW: Molecules consolidate the placental mammal tree. Trends Ecol Evol 19: 430–438 (2004). Stanhope MJ, Waddell VG, Madsen O, de Jong W, Hedges SB, et al: Molecular evidence for multiple origins of Insectivora and for a new order of endemic African insectivore mammals. Proc Natl Acad Sci USA 95: 9967–9972 (1998). Stanyon R, Koehler U, Consigliere S: Chromosome painting reveals that galagos have highly derived karyotypes. Am J Phys Anthropol 117:319–326 (2002).

Svartman M, Stone G, Page J, Stanyon R: A chromosome painting test of the basal eutherian karyotype. Chromosome Res 12: 45–53 (2004). Svartman M, Stone G, Stanyon R: The ancestral eutherian karyotype is present in Xenarthra. PLOS Genetics 7:e109 (2006). Waddell PJ, Shelley S: Evaluating placental interordinal phylogenies with novel sequences including RAG1, gamma-fibrinogen, ND6, and mt-tRNA, plus MCMC-driven nucleotide, amino acid, and codon models. Mol Phylogenet Evol 28:197–224 (2003). Waddell PJ, Cao Y, Hauf J, Hasegawa M: Using novel phylogenetic methods to evaluate mammalian mtDNA, including amino acidinvariant sites-LogDet plus site stripping, to detect internal conflicts in the data, with special reference to the positions of hedgehog, armadillo, and elephant. Syst Biol 48: 31–53 (1999). Waters PD, Dobigny G, Waddell PJ, Robinson TJ: Evolutionary history of LINE-1 in the major clades of placental mammals. PLoS ONE 2:e158 (2007). Werdelin L, Nilsonne Å: The evolution of the scrotum and testicular descent in mammals: a phylogenetic view. J Theor Biol 196: 61–72 (1999). Wildman DE, Uddin M, Opazo JC, Liu G, Lefort V, et al: Genomics, biogeography, and the diversification of placental mammals. Proc Natl Acad Sci USA 104:14395–14400 (2007). Wilson DE, Reeder DM (eds): Mammal Species of the World: A Taxonomic and Geographic Reference, 3rd ed (Johns Hopkins University Press, Baltimore, 2005). Yang F: Order Tubulidentata, in O’Brien SJ, Menninger JC, Nash WG (eds): Atlas of Mammalian Chromosomes, pp 80 (John Wiley and Sons, Hoboken 2006). Yang F, Alkalaeva EZ, Perelman PL, Pardini AT, Harrison WR, et al: Reciprocal chromosome painting among human, aardvark, and elephant (superorder Afrotheria) reveals the likely eutherian ancestral karyotype. Proc Natl Acad Sci USA 100:1062–1066 (2003).


153



Chromosome Evolution in Eulipotyphla L. Biltueva N. Vorobieva Institute of Molecular and Cellular Biology, SB RAS, Novosibirsk , Russia

Key Words Comparative chromosome painting ⴢ Insectivora ⴢ Karyotype

Abstract We integrated chromosome painting information on 5 coreinsectivora species available in the literature with new ZooFISH data for Iberian shrew (Sorex granarius) and Altai mole (Talpa altaica). Our analysis of these 7 species allowed us to determine the chromosomal features of Eulipotyphla genomes and to update the previously proposed ancestral karyotype for 2 main groups of the Sorex genus. The chromosome painting evidence with human painting probes (HSA) reveals the presence of the 2 unique associations HSA4/5 and 1/10p/12/22b, which support Eulipotyphla. There are a series of synapomorphies both for Erinaceidae (HSA3/1/5, 3/17, 11/15 and 10/20) and for Soricinae (HSA5/9, 6/7/16, 8/3/21 and 11/12/22). We found associations that link Talpidae/Erinaceidae (HSA7/8, 1/5 and 1/19p), Talpidae/Soricidae (HSA1/8/4) and Erinaceidae/Soricidae (HSA4/20 and 2/13). Genome conservation in Eulipotyphla was estimated on the basis of the number of evolutionary breaks in the ancestral mammalian chromosomes. In total, 7 chromosomes of the boreo-eutherian ancestor (BEA8 or 10, 9, 17, 18, 20–22) were retained in all eulipotyphlans studied; among them moles show the highest level of chromosome conservation. The integration of sequence data into the chromosome painting



information allowed us to further examine the chromosomal syntenies within a phylogenetic perspective. Based on our analysis we offer the most parsimonious reconstruction of phylogenetic relationships in Eulipotyphla. The cytogenetic reconstructions based on these data do not conflict with molecular phylogenies supporting basal position of Talpidae in the order. Copyright © 2012 S. Karger AG, Basel

Eulipotyphla is one of the basic groups for understanding mammalian evolution. Insectivores, based on their retention of a large number of primitive eutherian morphological traits, were often viewed to have diverged early in the radiation of placental mammals. Currently, after comprehensive molecular revision, the order consists of 4 families: Soricidae, Talpidae, Erinaceidae and Solenodontidae [Stanhope et al., 1998; Murphy et al., 2001]. The sister-group relationships at the family level within this order have always been strongly debated [e.g. Symonds, 2005]. According to morphological data, there is a fundamental split into Erinaceomorpha and Soricomorpha, and shrews are grouped with moles [Butler, 1988]. In contrast, most molecular phylogenies support grouping shrews with hedgehogs to the exclusion of moles [Miyamoto and Goodman, 1986; Douady et al., 2002]. Comparative cytogenetics provides a valuable tool for phylogenetic studies in Eutheria [Froenicke, 2005]. PaintLarisa Biltueva Institute of Molecular and Cellular Biology SB RAS Lavrentyeva street, RU–630090 Novosibirsk (Russia) Tel. +7 383 3333 857, E-Mail bilar @ mcb.nsc.ru

H 11 9 X 2 2 X

15 7

* 14 7

1

3 4 10

mole, Talpa altaica. Vertical lines to the right of each pair of chromosomes depict the hybridization of the human and the Iberian shrew (SGR) painting probes. H = Heterochromatic regions. Small asterisks indicate the regions not labeled by any shrew probe.

7

9

18 4 5 16 4 *

ing data can reveal chromosomal homologies between genomes and can aid the reconstruction of phylogenetic relationships at any taxonomic level [Wienberg, 2004]. Most mammalian orders now have phylogenetic reconstructions based on comparative molecular cytogenetics. However, even today chromosomal painting data is available only for 5 core-insectivores: common shrew (Sorex araneus) and Indochinese short-tailed shrew (Blarinella griselda, BGR) from Soricidae [Dixkens et al., 1998; Ye et al., 2006], European mole (Talpa europaea, TEU) from Talpidae [Volleth and Müller, 2006], and 2 Erinaceidae species – hedgehog (Hemiechinus auritus, HAU) and shrew-hedgehog (Neotetracus sinensis, NSI) [Yang et al., 2006; Ye et al., 2006]. The lack of a comprehensive phylogenetic array of studied species as well as a high level of chromosome rearrangements within and among species makes it difficult to find common chromosomal signatures. Here to broaden the number of species that can be compared, we applied human painting probes (HSA) for Zoo-FISH to Iberian shrew (S. granarius, SGR) and Altai mole (T. altaica, TAL) chromosomes. To facilitate the comparison between the karyotypes from different families, we carried out reciprocal (bidirectional) chromosome painting. We compared and reviewed our new results with available chromosome painting data.

14

15 11 22 1 10 17 22 12

5

10 20 6

10 15

3 14

11

H

19 11 19 1 7 16

4 6 4

*

5

12

19 16 12 19 16

16

1

6 5

6

4 8 13 1 7 15

10

12 1 11 22 9

15

1 5 8 1 1 5 8 1

*

13 2

8

13

4

2 X

5 3

7 2

Fig. 1. G-banded karyotype of the Altai

17 6

2 8 1

4 2 8 4 3 1 4 21 1

9 3

X X XX

ing the conventional methods as described previously [Yang et al., 1999; Graphodatsky et al., 2001]. Karyotypes of S. granarius and T. altaica were arranged according to Volobouev [1989] and Graphodatsky et al. [1991], respectively. Painting probes of S. granarius (2n = 36,XX) were characterized previously [Basset et al., 2006]. Painting probes of T. altaica were obtained by P.C.M. O’Brien and V. Trifonov following previously described methods [Telenius et al., 1992; Yang et al., 1995]. FISH was performed following GTG-banding of metaphase chromosomes according to published protocols [Yang et al., 1999; Graphodatsky et al., 2001].

Results

The whole set of human painting probes was hybridized onto mole and shrew chromosomes to delimit the homologous chromosomal segments. Reciprocal painting with the whole chromosome probe sets of the Altai mole and the Iberian shrew provided correspondence between the conserved chromosomal segments found in these 2 species.

Metaphase chromosome spreads were prepared from primary fibroblast cultures of S. granarius and T. altaica. Cell culture, metaphase preparations and G-banding were carried out follow-

Painting the T. altaica Genome with Human and S. granarius Probes The cross-species hybridization results were mapped to the G-banded karyotype of T. altaica (fig. 1). The human and S. granarius painting probes delineated 51 and 42 homologous segments, respectively, in T. altaica. Eight human chromosomes (HSA6, 9, 13, 14, 17, 18, 20 and 21) and 7 Iberian shrew chromosomes (SGR8, 12–17) were entirely conserved in the mole. Our results confirmed

Chromosome Evolution in Eulipotyphla


Materials and Methods

155

15p dis b1 2

a1 12

2q int

b2 13

a2 21

9q

4q 4p dis

4

b3 8 a3 8

7p

a4 6

b4 7

c2 9 2

1 11p pr

i1 10

12p int

15

3q

rian shrew, Sorex granarius. Vertical lines to the right and grey blocks to the left of the chromosomes delimit the hybridization of Talpa altaica and human painting probes, respectively. Italic letters to the left of the grey block indicate the elements of the putative ancestor karyotype of the Sorex genus.

4pq

f3 18

13pq

g1 6

6q int

g2 7

12q dis

16

4

22

j 1 7

5

k1 12

15q dis

5

k2 11

2p

9

8

e X

X

d1 2q

2q

d2 2

9p

11p dis

l2 4

8p

14p

n m2 12

15pq

19 16

16

11

10

o1 1 o2 8

10p

4

12

13 12

p 3

11q

14

previous chromosome painting with human probes on European mole (T. europaea) by Volleth and Müller [2006], with exception of 2 tiny HSA5 and HSA22 segments, which were identified on TEU8 (homologous to TAL5) and TEU10 (homologous to TAL10), respectively. Talpa altaica and T. europaea karyotypes were very similar (online suppl. table 1, for all online suppl. material, see www.karger.com/doi/10.1159/000339889), with differences only in the amount of heterochromatin on TAL1 and 6, and TEU9 and 1, respectively, and small differences in the centromeric position for chromosome 13 of both moles [Kawada et al., 2002]. The discrepancies in hybridization data can probably be explained by the technical limitations of chromosome painting. Painting the S. granarius Karyotype with Human and T. altaica Probes Zoo-FISH with human and T. altaica painting probes delineated 47 and 36 homologous segments in the Gbanded karyotype of S. granarius (fig. 2). Six human chromosomes (HSA9, 13, 14, 17, 18 and 21) and 3 Altai mole chromosomes (TAL1, 5 and 16) were entirely con156

f2 3

3

14

m1 15

Fig. 2. G-banded chromosomes of the Ibe-

12int

1q

6

l1 20

3p

10q pr

i2 h3 17

f1 19

7q

6q pr

h1 20 h2 11

8q

c1 5

3


q 10

10q dis

15

13q int

r 5 16

22 u 10 t 1

pq

14q

17

X

served in S. granarius. These results allowed us to refine the current human-shrew comparative map [Ye et al., 2006]. We added 7 small segments to this more detailed map: HSA4 on SGR2, HSA19p on SGR4, HSA20 on SGR6, HSA10 on SGR7 and SGR17, HSA12/22 on SGR9, and HSA1 on SGR13. HSA19 and 22 on SGR1 and HSA10 on SGR9 were not found. General Description of Painting Data of Eulipotyphlans Including our new data, currently, 7 core-insectivore species have been studied by Zoo-FISH with human painting probes (table 1). To discover common chromosomal associations, we assembled all available painting data in table 2 and arranged the chromosomes of each species in concordance with those of the putative boreoeutherian ancestral (BEA) karyotype proposed by Froenicke [2005]. Species with similar karyotypes like the common and Iberian shrews, or European and Altai moles were described together. All boreo-eutherian ancestral syntenies HSA3/21, 4/8, 14/15, 10p/12/22a, 7/16, 16/19 and 12/22b were retained in studied species. Biltueva/Vorobieva

Table 1. List of Eulipotyphla species analyzed by chromosome painting Taxon

Species

Haploid number Painting probe References

Family Soricidae Subfamily Soricinae Tribe Soricini Tribe Blarinellini

Sorex araneus (common shrew) Sorex granarius (Iberian shrew)

10/11 18/19

human human, mole

Dixkens et al., 1998; Ye et al., 2006 our study, our study

Blarinella griselda (Indochinese short-tailed shrew)

22

human

Ye et al., 2006

Family Talpidae Subfamily Talpinae Tribe Talpini

Talpa europaea (European mole) Talpa altaica (Altai mole)

17 17

human human, shrew

Volleth and Müller, 2006 our study, our study

24

human

Yang et al., 2006

16

human

Ye et al., 2006

Family Erinaceidae Hemiechinus auritus (long-eared hedgehog) Subfamily Erinaceinae Subfamily Galericinae Neotetracus sinensis (shrew-hedgehog)

Table 2. The chromosomes of the Eulipotyphla species arranged according to the putative boreo-eutherian ancestor (BEA) chromo-

somes BEA

Syntenic associa- Soricidae tions of human a chromosome ho- SGR 2n = 36/37, mologs SAR 2n = 20/21

Talpidae BGRb 2n = 44

c

TAL TEU 2n = 34

NSId 2n = 32

HAUe 2n = 48

2-3-1-5 1-11 1-13-10/12/22 8-14-1-5 17-3-22-19-1-20 13-4-20-10 4-5-8/4-18-17-3 4-12

3/21-5-1-3-5 5-4-1-4 6-1 4-1

1

1

1 1-4/8 12/22-10-1

11-12/22-1-4-20 1 1-12/22

1-5-1-5-1-(5)-1 4/8-1-(22)-10 19-11-19-1-7/16 15-22-1-10-12/22

2

4/8p

2-13-4/8-7 20-4 1-4/8

3

3/21

12-3/21-8-6 19-3-18 3

11-12/22-1-4-20 13-10-13-4-5 4-6-16/19 4 11-3 15-19-8-3/21 3

4/8-3/21 4-5 4/8-1-(22)-10 18-5-4 4/8-3/21 20-3

4

5

5-9 5

9-5-7/16-6 13-10-13-4-5 5

1-5-1-5-1-(5)-1 4-5 18-5-4

5

14/15

10-14/15 15-12

14/15 15-22-1-10-12/22

6

6

6-7/16 12-3/21-8-6

7

10p/12/22a

12-3/21-8-6 1-10-12/22 12/22-11 15-12

9-14/15 15-19-8-3/21 14-10-17 9-5-7/16-6 4-6-16/19 6-11 11-12/22-1-4-20 14-10-17 12

8

2q

10 9

2pq 7a

2-13-4/8-7 X(d1) X(d2) 2-13-4/8-7


Erinaceidae

6 12/22 4/8-1-(22)-10

2-3-1-5 4-5-4/8-18-17-3 6-12/22-3/21 17-3-22-19-1-20 2-3-1-5 4-5-4/8-18-17-3 8-14-1-5 15/14/15-6 8-14-1-5 15-11 2-6-2-8-9-8-9 15/14/15-6 6-12/22-3/21 4-12 6-12/22-3/21 17-3-22-19-1-20

16/19-4/8-5-19 4-2 5-4-1-4 4-1 3/21-5-1-3-5 17-3-17-3 10-3 3 16/19-4/8-5-19 5-13 3/21-5-1-3-5 5-4-1-4 14/15-14/15 15-11 14 6 6-1 2-12/22-13-12 12/22-10

10-2 2 2 10-7

2-11 2-13 8-7

1-13-10/12/22 2-6-2-8-9-8-9 2-3-1-5 16/(19)-8-7


2 2-12/22-13-12 4-2 7

157

Table 2 (continued)

BEA

Syntenic associa- Soricidae tions of human a chromosome ho- SGR 2n = 36/37, mologs SAR 2n = 20/21

Talpidae

Erinaceidae

BGRb

TALc

2n = 44

TEU 2n = 34

NSId 2n = 32

HAUe 2n = 48

9-5-7/16-6 9-14/15 11-12/22-1-4-20 11-3 6-11 13-10-13-4-5 10-7 10-2 13-10-13-4-5

9-17

2-6-2-8-9-8-9

9

2-11 19-11-19-1-7/16

1-11 15-11

11 15-11

4/8-1-(22)-10

13-4-20-10

8-10-20-10 10-3

2-13

13-4-20-10 1-13-10/12/22 2-6-2-8-9-8-9 16/(19)-8-7 8-14-1-5 4-5-8-4-18-17-3 17-3-22-19-1-20 17 4-5-8-4-18-17-3 16/(19)-8-7 13-4-20-10 17-3-22-19-1-20 7/16p 1-13-10/12/22 17-3-22-19-1-20

5-13 2-12/22-13-12 8-10-20-10

11

9

5-9

12

11

20-11-17 12/22-11

13

10q

10 10-14/15

14

13

2-13-4/8-7

15

8q

12-3/21-8-6

15-19-8-3/21 8

8-7

16

17

20-11-17

14-10-17

9-17

17 18 19

18 16q/19q 20

18 4-6-16/19 11-12/22-1-4-20

18-5-4 16/19-16/19 20-3

20 21 22

7b/16 12/22b 19p

19-3-18 16/19 20-11-17 20-4 6-7/16 12/22-10-1 19-3-18

9-5-7/16-6 1-12/22 15-19-8-3/21

19-11-19-1-7/16 15-22-1-10-12/22 19-11-19-1-7/16

Putative boreo-eutherian ancestor (BEA) chromosomes are given by Froenicke [2005]. a SGR: 2n = 36/37. Forty-seven conservative segments are revealed using human painting probes. The karyotype of S. granarius can be reconstructed from BEA by 16 fissions and 21 fusions. Eleven BEA chromosomes were conserved without breaks: 2q(2pq), 7a, 9, 13, 8q, 17, 18, 16q/19q, 7/16p, 12/22b, 19p. b BGR: 2n = 44. Fifty-one conservative segments are revealed using human painting probes. The karyotype of B. griselda can be reconstructed from BEA by 22 fissions, 23 fusions and 1 inversion. Ten BEA chromosomes were conserved without breaks: 2q(2pq), 7a, 13, 17, 18, 16q/19q, 20, 7/16p, 12/22b, 19p. c TAL: 2n = 34. Fifty-one (T. europaea: 54) conservative segments are revealed using human painting probes. The karyotype of T. altaica can be reconstructed from the boreo-eutherian ancestor by 12 fissions, 18 fusions and 6 inversions. Fifteen BEA

158


17-3-17-3 7/16-18 16/19-4/8-5-19 8-10-20-10 7/16-18 12/22-10 19

chromosomes were conserved without breaks: 6, 10q, 2q, 2pq, 7a, 9, 13, 8q, 17, 18, 16q/19q, 20, 7/16p, 12/22b, 19p. d NSI: 2n = 32. Fifty-eight conservative segments are revealed using human painting probes. The karyotype of N. sinensis can be reconstructed from the boreo-eutherian ancestor by 25 chromosomal fissions, 32 fusions and 5 inversions. Ten BEA chromosomes were conserved without breaks as 2q, 2pq, 7a, 9, 10q, 18, 16q/19q, 7/16p, 19p and 12/22b. e HAU: 2n = 48. Sixty conservative segments are revealed using human painting probes. The karyotype of H. auritus can be reconstructed from the boreo-eutherian ancestor by 19 chromosomal fissions, 18 fusions and 6 inversions. Eleven BEA chromosomes were conserved without breaks as 2q (or 2pq), 7a, 9, 8q, 17, 18, 16q/19q, 20, 7/16p, 19p and 12/22b. Signatures of Eulipotyphla according to Yang et al. [2006]: 1-5, 1-11, 3-19p, 4-20, 5-19p, 8-10, 8-13.

Biltueva/Vorobieva

Table 3. Syntenic associations of conserved segments revealed by Zoo-FISH with human paint probes

Associations of human chromosome homologs

Soricidae S. araneus S. granarius

1/10p/12/22b 4/5 1/5 1/19p 7/8

+

1/4/8p

+ + + + + + +

2/13 4/20 5/9 6/7/16 8/3/21 11/12/22 3/1/5 3/17 11/15 10/20

Talpidae

Erinaceidae

B. griselda

T. altai T. europaea

H. auritus N. sinensis

(+) +

+ + + + + +

(+) +

+* +

+

+ + +

+* + + + + +

+

+ + + +

+ + + +

Asterisks = Inversions. In brackets = Incomplete associations.

The absence of HSA4/8 in B. griselda and HSA16/19 in N. sinensis may be due to technical limitations in resolving small segments, as almost all preliminary studied species analyzed by chromosome painting with human probes showed these human associations. To examine obtained chromosomal syntenies in phylogenetic perspective, we broadened our comparisons by including available chromosome painting [e.g. Froenicke, 2005; Yang et al., 2006; Ferguson-Smith and Trifonov, 2007] and sequence data (http://www.ensembl.org).

Chromosome Associations and Phylogenetic Implications Erinaceidae Signatures. Analyzing the available chromosome painting data of N. sinensis and H. auritus, we revealed 4 common associations HSA3/1/5, 3/17, 11/15 and 10/20 that united both of them (table 3). The combination HSA3/1/5 expanded the association HSA1/5, which is common for Talpidae and Erinaceidae, by including a segment homologous to human chromosome 3. This association was conserved in NSI2 but it was inverted in HAU7 (table 2). The association HSA17/3/17/3 delineated the entire HAU11 and 2 segments in shrew-

hedgehog (NSI4dist and NSI12prox). The HSA11/15 association was present as a single chromosome in both karyotypes (NSI13 and HAU19). The distal part of chromosome NSI3 was marked by a HSA10/20 association that was present in an inverted form on HAU2q. All revealed associations delineated chromosomes or large chromosomal segments of similar size in both species representing existing Erinaceidae subfamilies. These associations were absent in other mammalian species, which indicates their synapomorphic state for the family. Soricinae Signatures. The comparison of chromosome painting data of B. griselda and two other shrews (S. araneus and S. granarius) revealed 4 common associations HSA8/3/21, 5/9, 6/7/16 and 11/12/22 assembled as single chromosomes in the SGR karyotype (fig. 2) and divided at least into 2 segments in B. griselda (table 2). The HSA8/3/21 combination was proposed as an element of the putative ancestral karyotype of the Soricini genus Sorex [Biltueva et al., 2011]. Its presence in a species of another tribe, Blarinellini (BGR10), suggests that it is a synapomorphic feature of the whole subfamily Soricinae. The HSA5/9 association consists of an entire HSA9 and a large segment of HSA5 on SGR3 in S. granarius, and it is present as 3 segments in the Indochinese short-tailed shrew (BGR1pdist, 3qdist and 6p). It is necessary to note that the remaining part of HSA5 was conserved as a single



Discussion

159

chromosome in both species (SGR16 and BGR20). Most of HSA6 is involved in a syntenic association HSA6/7/16 that delineated SGR5 and 2 BGR chromosomes (1pq and 7q prox or 14prox, some of which later included the remainder of HSA6). The HSA11/12/22 association was identified on SGR9 and was divided into 2 segments in the B. griselda karyotype (2p, 8pprox or 14dist, some of which later included the remainder of HSA11). This association was mentioned as a chromosome signature of Chiroptera and included HSA11dist and 12/22b [Volleth et al., 2002, 2011]. But according to Yang et al. [2006], HSA12/22b associated with HSA10 in long-eared hedgehog chromosome HAU20 which as we proposed is homologous to SGR17. Reciprocal painting of these species would be needed to clarify the origin of this association in other orders. All syntenies found marked large chromosomes or chromosome regions in Soricinae species, therefore suggesting that they are synapomorphic features of the lineage. HSA1/4/8p Is a Common Association in Talpidae and Soricidae To expand our comparisons, we included the painting data from T. europaea [Volleth and Müller, 2006] in our analysis, and as outgroup we used available data from some Erinaceidae species (table 2). We revealed a HSA1/4/ 8 association in S. granarius and T. altaica by Zoo-FISH with human painting probes, which was also confirmed by reciprocal chromosome painting between these species (figs. 1 and 2). This association was present as a single chromosome on SGR13 and as whole arms on TAL10p and TEU10p. Unfortunately, the HSA4/8 segment was not identified in other Soricinae species (B. griselda). The HSA1/4/8 association was not detected in available ZooFISH experiments, but based on syntenic sequence data from ENSEMBLE database (http://www.ensembl.org), it is present in horse (ECA2) and dog (CFA15). It is composed of large segments of HSA1pprox (60 Mb), HSA4qint (70 Mb), and a small segment of HSA8pint (9 Mb) which would be difficult to detect by long-range Zoo-FISH experiments. It is likely that this segment was not detected on the chromosomes carrying HSA1/4 associations in B. griselda and H. auritus (table 2). If additional painting or sequencing data eventually confirms the common origin of this association, then we could suggest that HSA1p/4q/ 8p is a synapomorphic synteny for all Eulipotyphla, or even for all Laurasiatheria. Common Associations in Talpidae and Erinaceidae It is surprising that 3 syntenic associations HSA7/8, 1/5 and 1/19p are common in Talpidae and Erinaceidae. The 160


HSA7/8 association includes 2 ancient chromosomes (HSA8q and 7a) and it is present as single chromosome in moles as TAL7 (fig. 1) and TEU4 and as 2 segments (HSA7/8 and 8) in shrew-hedgehog, NSI6 and 1q, respectively (table 2). Probably, this association was lost in the hedgehog. According to the sequence data, this association is present in rabbit (OCU2) and platypus (OAN4), but it is not overlapped by HSA7 and 8, suggesting that its presence in both species is a result of convergence. The HSA1/5 association is present in inverted state in moles (TAL5, TEU8) and hedgehog (HAU7dist) and as 2 segments in shrew-hedgehog (NSI2q and 11dist). The same association was revealed by Zoo-FISH in pangolin and described as 1 of 7 (HSA1/5, 1/11, 3/19p, 4/20, 5/19p, 8/10, 8/13) Eulipotyphla signatures [Yang et al., 2006]. According to sequence data, this ancient association might be conserved in dog (CFA2 and 4) and some trace might exist in chicken (GGA2). In Artiodactyla there are 2 syntenies including HSA1 and HSA5 (online suppl. fig. 1), the first one including HSA1q/5q/19p is present in pig (SSC2) and cow (BTA7), the second one consisting of HSA1q/5pq was detected in BTA20. Probably, the HSA1q/5q/19p association found by sequence data in pig and cow is the result of an expansion of the HSA5/19p synteny synapomorphic in Cetartiodactyla that includes 2 HSA5qdist segments and was not detected by painting the HSA1 segment (2 Mb). The third association, which unites Talpidae and Erinaceidae, is HSA1/19p. In inverted form it is observed in moles (TAL12pq, TEU11pq) and remained intact in N. sinensis (NSI12dist). It may be part of the ancient synteny HSA1/19p which is often considered apomorphic for Afrotheria [Yang et al., 2003; Frönicke et al., 2003]. Moreover, we can hypothesize that there is an ancient HSA1/5/19p synteny that was partly conserved in genomes of cow, pig, dog and chicken (online suppl. fig. 1) and was disrupted in core-insectivores (HSA1/5 and 1/19p). Further research will be required to clarify the cladistic significance of these associations. Common Associations in Soricidae and Erinaceidae The HSA4/20 association was identified in S. granarius, S. araneus, B. griselda and H. auritus and was proposed as a chromosome signature for Eulipotyphla [Yang et al., 2006; Ye et al., 2006]. The existence of this association in pangolin allows to hypothesize that it is a chromosome feature joining this order to Pholidota [Yang et al., 2006]. Detailed analysis of evolutionary relationships within the Sorex genus demonstrates that HSA4/20 is autapomorphic for the S. araneus group [Biltueva et al., Biltueva/Vorobieva

Table 4. The number of fissions of the putative boreo-eutherian ancestor (BEA) chromosomes revealed by Zoo-FISH with human paint probes

BEA

Syntenic associations of human chromosome homologs

1 1 2 4/8p 3 3/21 4 5 5 14/15 6 6 7 10pq/12/22a 8 2q 10 2pq 9 7a 11 9 12 11 13 10q 14 13 15 8q 16 17 17 18 18 16q/19q 19 20 20 7b/16p 21 12/22b 22 19p Number of conserved chromosomes Number of chromosome breaks Number of chromosome fusions Number of conserved segments Number of inversions

Soricidae

Talpidae

Erinaceidae

S. granarius 2n = 36/37

B. griselda 2n = 44

T. altai T. europaea 2n = 34

H. auritus 2n = 48

N. sinensis 2n = 32

2 2 2 1 1 1 3 1 – – – 1 1 – – – – – 1 – – – 11 16 21 47 –

2 3 2 2 2 2 2 1 – – 1 2 2 – 1 – – – – – – – 10 22 23 51 1

3 3 1 2 1 – 1 – – – – 1 – – – – – – – – – – 15 12 18 54 6

3 3 3 2 2 1 1 1 – – – 1 1 1 – – – – – – – – 11 19 18 60 6

4 2 3 2 2 2 3 – – – – 1 – 1 2 2 – – 1 – – – 10 25 32 58 5

2011]. This interpretation is also supported by the existence of interstitial telomeric sites on the boundary of these segments in the common shrew [Zhdanova et al., 2009]. In accordance with the refined shrew-human comparative map (fig. 2), HSA20 is divided into 2 segments forming SGR6 (HSA20/11/17) and SGR10 (HSA4/20). This association on BGR2dist is composed of the whole HSA20 and a HSA4 segment which is considerably smaller than that in S. granarius. The HSA10/20 association was considered as family synapomorphy for Erinaceidae and includes the entire HSA20 on HAU2 and 2 HSA20 segments in NSI3q (HSA13/4/cent/20/10) and NSI12 (NSI17/3/22/19/1/20). The division of HSA4/20 by a centromere in shrew-hedgehog as well as the inclusion of HSA20 in taxa-specific synteny weakens its position as a lineage signature. Further, its presence in pangolin as

Homologies HSA4/5 and HSA1/10p/12/22b Are Putative Order Signatures A small HSA4/5 segment is common for all studied species except for Sorex shrews (BGR3dist, TAL13dist, TEU13prox and HAU9). The detection of a tiny HSA4 seg-



well as traces of it in the genomes of pig (SSC17), opossum (MDO5) and chicken (GGA4) requires additional research. The HSA2/13 association in Iberian shrew (SGR2) and moles (TAL9, TEU6) was not supported by reciprocal painting between these species (figs. 1, 2, online suppl. fig. 2). The presence of HSA2/13 with inversion on HAU6 as well as some support from sequence data on opossum (MDO4, 7), dog (CFA25) and platypus (OAN20) also require additional study.

161

ment on SGR16 which is homologous to TAL13dist (producing the HSA4/5 association) is currently below the resolution of painting. This association is absent in other mammalian genomes studied by ZOO-FISH data and its presence in dog (CFA3) and platypus (OANX1, X2) as non-overlapping regions (http://www.ensembl.org) is likely to be a convergent event. The HSA1/10/12/22 association was found in homologous segments of moles (TAL14, TEU14) and shrew (SGR17). Probably, it was inverted in NSI10 (table 2). We can propose the existence of this synteny in BGR18 (consisting of HSA1/12/22) and HAU20 (consisting of HSA10/12/22) with undetectable small HSA10 and 1 segments, respectively. Yang et al. [2006] suggested that the HSA10/12/22 association in long-eared hedgehog differs from HSA10p/12/22a association common in Afrotheria and Carnivora and includes 12/22b. Review of sequence data shows that 2 syntenies consist of HSA10/12/22 (online suppl. fig. 3). The first syntenic segment, according to the sequence data, is conserved in genomes of opossum (MDO8) and chicken (GGA1) including HSA10p and HSA12/22a segments. Using ZooFISH it was identified in Canivora and Afrotheria species [Froenicke, 2005]. The second association is entirely conserved in pig (SSC14) and dog (CFA4 and 26). It is composed of a small HSA1qdist segment (11 Mb), whole 10q and 12/22b. Probably, it was disrupted in genomes of horse (ECA1) and cow (BTA28). The question about the presence of these associations in Eulipotyphla requires additional studies. Genome Conservation in Eulipotyphla We made an estimation of the genome conservation in various Eulipotyphla. These species display high diversity in the number of breaks of the ancestral chromosomes (table 4). The moles show the lowest number of evolutionary fissions (12) and highest number of chromosomes conserved in toto (15). Within shrews and hedgehogs we identified a similar number of breaks. The greater number of inversions in Erinaceidae explains the higher number of conserved segments compared to Soricidae species. In total, 7 chromosomes of the boreoeutherian ancestor (BEA8 or 10, 9, 17, 18, 20–22) were retained in all eulipotyphlans studied. Overall, according to Zoo-FISH data moles showed the highest level of chromosome conservation in agreement with comparative cytogenetic data within taxa [Graphodatsky et al., 1991; Kawada et al., 2002; Gornung et al., 2008]. According to molecular clock estimates, T. altaica and T. europaea diverged approximately 11 mya 162


4 l2 20 l1 h1

2 d1

1

13 b2

2 15

1 j

9

1 8p o 4 17

9 c2

10

11

22 18

tu

15

8q a3 5

22 12 k1 19q

m

16p g2 7

n

6 g1

7 b4

18 f3

6

7

8

11 k2 16q

10q i1q

3 p

12

14

15

13

12 a1 19p f1 16

X e

8p 4 b3 19

3 f2

i2

4

3

2 d2

1

a2

3

12

10 12

21 14

b1

11 h2 17 h3

5 c1

5 r 20

6 a4 21

X

Fig. 3. Refined ancestral karyotype of the Sorex genus. The ho-

mologies to human chromosomes or chromosomal segments are indicated in color blocks. Each ancestral element is designated by italic letters corresponding to the respective chromosomes in S. araneus, and the numbers show a position on S. granarius chromosome starting from the centromere (see fig. 2).

[Colangelo et al., 2011], but they have almost identical karyotypes which is not always the case in shrews. Reconstruction of Phylogenetic Relationships in Eulipotyphla The data set allowed us to update the previously proposed ancestral karyotype for 2 main groups of the Sorex genus [Biltueva et al., 2011]. The presence of the associations HSA8q/3/21, 5/9, 6/7/16p and 11/12/22 in the karyotype of B. griselda permits us to conclude that they were intact elements in the ancestral karyotype (fig. 3). Before an appropriate outgroup was available, each of them was considered as 2 ancestral elements as they were found in karyotypes of the S. minutus group. Also, we have to accept that the HSA4/20/11/17 association is a single ancestral element. Altogether, we propose that 4 chromosomes of the Sorex group ancestor (HSA4/20/11/17, 3/18, 12/15 and 12/19p) are synapomorphies for species of this genus. Four associations (HSA5/9, 8q/3/21, 6/7/16 and 11/12/22) represent synapomorphies for the Soricinae subfamily Biltueva/Vorobieva

4/5 1/10/12/22

Neotracus sinensis Hemiechinus auritus

Talpa europaea Talpa altaica

65±5

38–42±5

16.5

Soricidae

Blarinella griselda

1/3/5 3/17 11/15 10/20 1–4/8

1/5 1/19p 7/8 1/4/8

73±5

‘S. minutus’ group

11.5

3.1

Erinacenaidae

2/13 4/20

5+4/8; 6+1/4/8; 1/10 4–20/11/17; 6–7/16; 1*; 11–12/22; 5–9

Talpidae

5/9 6/7/16 8/3/21 11/12/22 1–5 1–19p 7–8

2+2+X 3+18 2/13+4/8+7 2/13/4/8/7+5/9 Sorex araneus 12+15 8/3/21+6+12/19+3/18 12+19p Sorex granarius 11/17–4/20 8/3/21/6–12/19/3/18 4/20+11+17 2*

Million years before present

Fig. 4. The most parsimonious tree from cladistic analysis of syntenies in 7 Eulipotyphla species studied by Zoo-FISH. The time scale is based on molecular divergence estimated by Fumagalli et al. [1999], Dubey et al. [2007], Douady et al. [2003], and Colangelo et al. [2011]. + = Marked fusion; – = marked fission.

(fig. 4). The currently proposed diploid number of the Sorex ancestor is 44 chromosomes. Now we can also suggest sets of synapomorphies for Erinaceidae (HSA1/3/5, 3/17, 11/15 and 10/20). We found that 2 segmental associations (HSA4/5 and 1/10p/12/22b) support Eulipotyphla, 3 segmental associations (HSA1/5, 1/19p and 7/8) unite Talpidae and Erinaceidae, 2 segmental associations (HSA2/3 and 4/20) unite Erinaceidae and Soricidae, and 1 segment association (HSA1/4/8p) is common in Talpidae and Soricidae. Based on our data we offer the most parsimonious reconstruction of phylogenetic relationships in Eulipotyphla (fig. 4). Since the moles have the highest level of chromosome conservation in Eu-

References


lipotyphla, we propose that Talpidae were the first to diverge. This interpretation does not conflict with molecular phylogenies that suggest a more basal position of Talpidae as compared to Soricidae and Erinaceidae.

Acknowledgements We would like to thank V. Volobuev, N. Zhdanova for providing the Iberian shrew chromosome suspension and M. FergusonSmith, P.C.M. O’Brien and V. Trifonov for providing the S. granarius and T. altaica painting probes. This work was supported by MCB, SB RAS and RFBR grants.

Basset P, Yannic G, Yang F, O’Brien PC, Graphodatsky AS, et al: Chromosome localization of microsatellite markers in the shrews of the Sorex araneus group. Chromosome Res 14: 253–262 (2006). Biltueva L, Vorobieva N, Perelman P, Trifonov V, Volobouev V, et al: Karyotype evolution of Eulipotyphla (Insectivora): The genome ho-

mology of seven Sorex species revealed by comparative chromosome painting and banding data. Cytogenet Genome Res 135: 51–64 (2011). Butler P: Phylogeny of the insectivores, in Benton MJ (ed): The Phylogeny and Classification of the Tetrapods. Volume 2: Mammals, pp 117–141 (Clarendon Press, Oxford 1988).


163

Colangelo P, Bannikova AA, Krystufek B, Lebedev VS, Annesi F, et al: Molecular systematics and evolutionary biogeography of the genus Talpa (Soricomorpha: Talpidae). Mol Phylogenet Evol 55:372–380 (2011). Dixkens C, Klett C, Bruch J, Kollak A, Serov OL, et al: Zoo-FISH analysis in insectivores: ‘Evolution extols the virtue of the status quo’. Cytogenet Cell Genet 80:61–67 (1998). Douady CJ, Chatelier PI, Madsen O, de Jong WW, Catzeflis F, et al: Molecular phylogenetic evidence confirming the Eulipotyphla concept and in support of hedgehogs as the sister group to shrews. Mol Phylogenet Evol 25:200–209 (2002). Douady CJ, Douzery EJ: Molecular estimation of eulipotyphlan divergence times and the evolution of ‘Insectivora’. Mol Phylogenet Evol 28:285–296 (2003). Dubey S, Salamin N, Ohdachi S, Barrière P, Vogel P: Molecular phylogenetics of shrews (Mammalia: Soricidae) reveal timing of transcontinental colonizations. Mol Phylogenet Evol 44:126–137 (2007). Ferguson-Smith MA, Trifonov V: Mammalian karyotype evolution. Nat Rev Genet 8: 950– 962 (2007). Frönicke L, Wienberg J, Stone G, Adams L, Stanyon R: Towards the delineation of the ancestral eutherian genome organization: comparative genome maps of human and the African elephant (Loxodonta africana) generated by chromosome painting. Proc R Soc Lond B Biol Sci 270: 1331–1340 (2003). Froenicke L: Origins of primate chromosomes – as delineated by Zoo-FISH and alignments of human and mouse draft genome sequences. Cytogenet Genome Res 108: 122–138 (2005). Fumagalli L, Taberlet P, Stewart DT, Gielly L, Hausser J, Vogel P: Molecular phylogeny and evolution of Sorex shrews (Soricidae: Insectivora) inferred from mitochondrial DNA sequence data. Mol Phylogenet Evol 11:222– 235 (1999). Gornung E, Volleth M, Capanna E, Castiglia R: Comparative cytogenetics of moles (Eulipotyphla, Talpidae): chromosomal differences in Talpa romana and T. europaea. Cytogenet Genome Res 121:249–254 (2008).

164

Graphodatsky A, Radjabli S, Zaitsev M, Sharshov A: The levels of chromosome conservatism in the different groups of insectivores (Mammalia, Insectivora), in Zaitsev EV (ed): Questions of Systematics, Faunistics and Palaeontology of Small Mammals, pp 47–57 (Zoological Institute USSR, St. Petersburg 1991). Graphodatsky AS, Yang F, O’Brien PC, Perelman P, Milne BS, et al: Phylogenetic implications of the 38 putative ancestral chromosome segments for four canid species. Cytogenet Cell Genet 92:243–247 (2001). Kawada S, Harada M, Grafodatsky AS, Oda S: Cytogenetic study of the Siberian mole, Talpa altaica (Insectivora: Talpidae) and karyological relationships within the genus Talpa. Mammalia 66:53–62 (2002). Miyamoto M, Goodman M: Biomolecular systematics of eutherian mammals: phylogenetic patterns and classification. Syst Zool 35: 230–240 (1986). Murphy WJ, Eizirik E, O’Brien SJ, Madsen O, Scally M, et al: Resolution of the early placental mammal radiation using Bayesian phylogenetics. Science 294:2348–2351 (2001). Stanhope MJ, Waddell VG, Madsen O, de Jong W, Hedges SB, et al: Molecular evidence for multiple origins of Insectivora and for a new order of endemic African insectivore mammals. Proc Natl Acad Sci USA 95: 9967–9972 (1998). Symonds MR: Phylogeny and life histories of the ‘Insectivora’: controversies and consequences. Biol Rev 80: 93–128 (2005). Telenius H, Pelmear AH, Tunnacliffe A, Carter NP, Behmel A, et al: Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes Chromosomes Cancer 4: 257–263 (1992). Volleth M, Heller KG, Pfeiffer RA, Hameister H: A comparative Zoo-FISH analysis in bats elucidates the phylogenetic relationships between Megachiroptera and five microchiropteran families. Chromosome Res 10:477– 497 (2002).


Volleth M, Müller S: Zoo-FISH in the European mole (Talpa europaea) detects all ancestral boreo-eutherian human homologous chromosome associations. Cytogenet Genome Res 115:154–157 (2006). Volleth M, Yang F, Müller S: High-resolution chromosome painting reveals the first genetic signature for the chiropteran suborder Pteropodiformes (Mammalia: Chiroptera). Chromosome Res 19: 507–519 (2011). Volobouev VT: Phylogenetic relationships of the Sorex araneus-arcticus species complex (Insectivora, Soricidae) based on high-resolution chromosome analysis. J Hered 80: 284– 290 (1989). Wienberg J: The evolution of eutherian chromosomes. Curr Opin Genet Dev 14: 657–666 (2004). Yang F, Carter N, Shi L, Ferguson-Smith M: A comparative study of karyotypes of muntjacs by chromosome painting. Chromosoma 103: 642–652 (1995). Yang F, O’Brien PC, Milne BS, Graphodatsky AS, Solanky N, et al: A complete comparative chromosome map for the dog, red fox, and human and its integration with canine genetic maps. Genomics 62: 189–202 (1999). Yang F, Alkalaeva EZ, Perelman PL, Pardini AT, Harrison WR, et al: Reciprocal chromosome painting among human, aardvark, and elephant (superorder Afrotheria) reveals the likely eutherian ancestral karyotype. Proc Natl Acad Sci USA 100:1062–1066 (2003). Yang F, Graphodatsky AS, Li T, Fu B, Dobigny G, et al: Comparative genome maps of the pangolin, hedgehog, sloth, anteater and human revealed by cross-species chromosome painting: further insight into the ancestral karyotype and genome evolution of eutherian mammals. Chromosome Res 14: 283– 296 (2006). Ye J, Biltueva L, Huang L, Nie W, Wang J, et al: Cross-species chromosome painting unveils cytogenetic signatures for the Eulipotyphla and evidence for the polyphyly of Insectivora. Chromosome Res 14: 151–159 (2006). Zhdanova NS, Rogozina IuI, Minina IuM, Borodin PM, Rubtsov NB: Telomeric DNA allocation in chromosomes of common shrew Sorex araneus, Eulipotyphla [in Russian]. Cytologia 51:577–584 (2009).

Biltueva/Vorobieva



Chromosome Evolution in Bats as Revealed by FISH: The Ongoing Search for the Ancestral Chiropteran Karyotype M. Volleth a G. Eick b a b

Department of Human Genetics, Otto-von-Guericke-University, Magdeburg, Germany; Institute for Ecology and Evolution, University of Oregon, Eugene, Oreg., USA

Key Words Chromosome painting ⴢ Inversion ⴢ Phylogeny ⴢ Robertsonian translocation

Abstract Chiroptera, the second largest order of mammals, comprises more than 1,000 species in 18 highly morphologically diverse families. Chromosome painting with human probes has been applied to 10 bat species from 8 families. Except for the combination 10/12pq/22q, all syntenic segmental associations proposed for the mammalian ancestor have been found in Chiroptera. Bat-specific painting probes, established from 4 species of 3 families, have been used in whole chromosome painting experiments in 29 species from 8 families. The results show that the prevailing mode of chromosomal evolution in bats is Robertsonian translocation with a large number of convergent events. Given our present knowledge of chiropteran karyotypes, only a few elements of the ancestral chiropteran karyotype can be reconstructed with confidence. Copyright © 2012 S. Karger AG, Basel



Chiroptera is the second largest mammalian order and comprises more than 1,000 described species (1,116 species in Simmons [2005]). Traditionally, bats were divided into 2 suborders, Megachiroptera (non-echolocating flying foxes) and Microchiroptera (echolocating bats). Recent molecular genetic studies, however, revealed that some microchiropteran families are more closely related to the flying foxes than to the remaining microbats [Eick et al., 2005; Teeling et al., 2002, 2005]. In the revised bat nomenclature [Hutcheon and Kirsch, 2006], 2 suborders are recognized: the Pteropodiformes (flying foxes plus 5 former microchiropteran families) and the Vespertilioniformes (the remaining microbats). The diploid chromosome number (2n) and the fundamental number of autosomal arms are known for less than half of all described bat species [Baker, 2006]. The diploid chromosome number ranges between 2n = 14 in Vampyressa melissa [Gardner, 1977] and 2n = 62 in several rhinolophid species [see Zima et al., 1992]. The predominant mode of chromosomal evolution is Robertsonian translocation in either direction (i.e. fusions and fissions). This was already concluded by Capanna and Civitelli [1970] in their review of conventionally stained karyotypes of 160 bat species, based on the observation that intrafamilial fundamental numbers of autosomal arms are highly conserved. Later, karyotype comparisons Marianne Volleth Department of Human Genetics, Otto-von-Guericke-University Leipziger Strasse 44 DE–39120 Magdeburg (Germany) Tel. +49 391 671 5342, E-Mail Marianne.Volleth @ med.ovgu.de

Table 1. Application of human paints in Chiroptera

Family

Species

2n

Segment number a

Reference

Vespertilionidae

Myotis myotis Plecotus auritus Mormopterus planiceps Glossophaga soricina Taphozous melanopogon Rhinolophus mehelyi Hipposideros larvatus Aselliscus stoliczkanus Megaderma spasma Eonycteris spelaea

44 32 48 32 42 58 32 30 38 36

46 47 42 42 43 44 41b 41 43 41

Volleth et al., 2002 Kulemzina et al., 2011 Volleth et al., 2002 Volleth et al., 1999 Mao et al., 2008 Volleth et al., 2002 Volleth et al., 2002 Mao et al., 2007 Mao et al., 2008 Volleth et al., 2002

Molossidae Phyllostomidae Emballonuridae Rhinolophidae Hipposideridae Megadermatidae Pteropodidae a

Autosomal segments plus X. b Two expected segments could not be detected.

Table 2. Mammalian syntenic segments found in Chiroptera

HSA syntenies 3-21b 4-8 16-7 12pq-22qtc 12qt-22qc 14-15 16-19

MMY segmentsa 4 5b+c 7a,c+d 8 proximal 6 23a+c 1 16/17

Corresponding HSA segments 3q12–q21, 3q23–q26, 3q27–qter, 21q 4q33–qter, 8p22, 8p11–p21 4q27–q31, 8p21, 8p23 16p, 7q11.2, 7q21.3–q22 12pter–q23.3, 22q12.3–qter 12q23.3–qter, 22q11.2–q12.3 14q11–q32, 15q11–q24 16q, 19q

a Segments in Myotis are numbered with lowercase letters from

the centromere to the telomere. For further explanations, see Volleth et al. [2011]. b Complete HSA3-21 synteny is conserved in Pteropodidae. c Abbreviation of segment combination as in RuizHerrera et al. [2012].

based on G-banding patterns confirmed that Robertsonian mechanisms are the most important karyotypic changes within a given family [Bickham and Baker, 1979; Baker and Bickham, 1980]. To explain interfamilial differences in G-banding patterns, which in several instances have prevented the unequivocal identification of homologous chromosomal arms [Baker and Bickham, 1980], other types of rearrangements have to be assumed. The advent of the fluorescence in situ hybridization (FISH) technique has facilitated identification of homologous chromosomal arms on the basis of homologous DNA sequences independent of preservation of the Gbanding pattern. In this review, we focus on the results obtained by Zoo-FISH with respect to chromosomal evolution in Chiroptera.

Painting with Human-Specific Whole Chromosome Probes Table 3. Chiropteran specific segment combinations

HSA homology

MMY Corresponding HSA segments segment

1-6a 3 4-10 2 4-8-19 7i 5-7-16 8 11-12qt-22qb 23 13-8-4 5 18-20 10

1pter–p22, 6pter–p22 4pter–q26, 10p 4q27–q31, 8p21, 8p23, 19p 5pter–q23, 16p, 7q11.2, 7q21.3–q22 11q23–qter, 12q23.3–qter, 22q11.2–q12.3 4q33–qter, 8p22, 8p11–p21, 13q 18pter–qter, 20pter–qter

a Complete synteny of HSA6 homologous sequences is conserved in Pteropodidae. b HSA11 is preserved in toto in Pteropodidae and Hipposideridae.

166


Homo sapiens was the first species for which all 23 (22 autosomes plus the X chromosome) whole chromosome painting probes were available. Therefore, Zoo-FISH in bats was initially undertaken with the human probe set. Using this whole chromosome painting probe set, 10 bat species from 8 different families have been studied (table 1). Amplification and labeling of these probes using the degenerate oligonucleotide-primed polymerase chain reaction technique has resulted in better hybridization efficiency than that obtained during the early days of FISH. This likely explains why 3 tiny segments detected recently in Plecotus auritus [Kulemzina et al., 2011] were not observed in earlier studies [Volleth et al., 1999, 2002]. Volleth /Eick

Table 4. Bat paints and applications

Donor species (family)

Target species

2n

Segment number

Reference

Myotis myotis

Homo sapiens Myotis altarium Myotis goudoti Nyctalus velutinus Hypsugo pulveratus Tylonycteris robustula Tylonycteris spp. Glauconycteris beatrix Miniopterus fuliginosus Miniopterus griveaudi Tadarida teniotis Mormopterus jugularis Myzopoda aurita Rhinolophus sinicus Aselliscus stoliczkanus Cynopterus sphinx

46 44 44 36 44 32 30 22 46 46 48 48 26 36 30 34

53 22 22 22 22 22 22 24 23 23 25 24 29 30 25 27

Volleth et al., 2011 Ao et al., 2006 Richards et al., 2010 Ao et al., 2006 Ao et al., 2006 Ao et al., 2006 Ao et al., 2006 Volleth and Heller, 2007 Ao et al., 2006 Richards et al., 2010 Mao et al., 2008 Richards et al., 2010 Richards et al., 2010 Ao et al., 2007 Ao et al., 2007 Ao et al., 2007

Myotis altarium Myotis dasycneme Plecotus auritus Vespertilio murinus Murina hilgendorfi Rhinolophus sinicus Rhinolophus p. pearsoni Rhinolophus ferrumequinum Rhinolophus pusillus Hipposideros larvatus Rousettus leschenaulti

44 44 32 38 44 36 44 58 62 32 34

25 25 24 25 25 30 28 28 30 24 25

Mao et al., 2007 Kulemzina et al., 2011 Kulemzina et al., 2011 Kulemzina et al., 2011 Kulemzina et al., 2011 Mao et al., 2007 Mao et al., 2007 Mao et al., 2007 Mao et al., 2007 Mao et al., 2007 Mao et al., 2007

Carollia brevicauda Diphylla ecaudata Diaemus youngi Desmodus rotundus

21 32 32 28

25 22 21 23

Pieczarka et al., 2005 Sotero-Caio et al., 2011 Sotero-Caio et al., 2011 Sotero-Caio et al., 2011

Phyllostomus hastatus Diphylla ecaudata Diaemus youngi Desmodus rotundus

32 32 32 28

26 27 27 28

Pieczarka et al., 2005 Sotero-Caio et al., 2011 Sotero-Caio et al., 2011 Sotero-Caio et al., 2011

(Vespertilionidae)

Aselliscus stoliczkanus (Hipposideridae)

Phyllostomus hastatus (Phyllostomidae)

Carollia brevicauda (Phyllostomidae)

The results of the Zoo-FISH experiments in representatives from different bat families indicate that the bat karyotype is composed of 26 evolutionary conserved units [Volleth et al., 2002]. Some of these units contain sequences homologous to the human complement in the same combinations proposed to be syntenic segmental associations for Boreoeutheria [Frönicke, 2005] and the mammalian ancestor [Robinson and Ruiz-Herrera, 2008; Ruiz-Herrera et al., 2012]. Other segment combinations have been found only in chiropteran karyotypes up to now, and are therefore most likely bat-specific traits. An overview of mammalian and chiropteran specific segment combinations is provided in tables 2 and 3.

As hybridization efficiency decreases with increasing phylogenetic distance, consistently good results can be obtained with probes originating from a taxon belonging to the same family or at least the same order as the species of interest. The first bat species whose chromosomes were flow-sorted was Myotis myotis (Vespertilionidae) [Ao et al., 2006]. The Myotis set of probes has subsequently been used to characterize the karyotypes of 15 different bat species from 7 families (table 4). In addition, this is the only chiropteran set that has been re-hybridized onto the human chromosomal complement so far

Chromosome Evolution in Bats


Painting with Bat-Specific Probes

167

a

c

b

d

Fig. 1. Comparison of selected G-banded human (HSA, left) and

flying fox (E. spelaea ESP; right) chromosomes. The vertical lines on the left side of each chromosome show the homologous segments revealed by FISH with T. belangeri (TBE) probes. Homology with M. myotis (MMY) painting probes is indicated on the right. a HSA1 and ESP2p, ESP16 and ESP17. b HSA3 and ESP4. c HSA6 and ESP2. d HSA11 and ESP8 (modified from Volleth et al. [2011], with permission of Springer).

[Volleth et al., 2011]. The probe set of the hipposiderid Aselliscus stoliczkanus [Mao et al., 2007] was applied to 11 species from 4 families (for references see table 4). There are also flow-sorted probe sets available for 2 phyllostomid species, Phyllostomus hastatus and Carollia brevicauda [Pieczarka et al., 2005]. Both sets have been used to characterize the karyotypes of 4 phyllostomid taxa each (see table 4). The number of segments obtained using these chiropteran sets ranges between 21 and 30. Rhinolophid species have the highest segment numbers reported to date due to centric fission of certain ancestral bi-armed elements, which is also shown by the high fundamental numbers of autosomal arms observed in this family. In some cases, only selected probes were used to address specific questions. One example is the characterization of the neo-XY body of C. perspicillata by using C. brevicauda probes on meiotic preparations [Noronha et al., 2009]. In the case of the vespertilionid genus Sco168


tophilus, G-band comparison could not resolve the composition of 3 rearranged chromosomal pairs. However, the application of only 7 probes from the Myotis set allowed full characterization of the karyotype [Eick et al., 2007]. The availability of bat-specific paints enables bi-directional comparison of homologous segments. Hybridization of the Myotis paints onto human chromosomes yielded interesting results. Human homology for the small HSA4-8 syntenic segments found in Chiroptera could be defined (see table 2). In addition, the position of the centromere in the HSA6 homologous chromosome of the flying fox Eonycteris was found to be similar to that proposed for the primate ancestor. According to Capozzi et al. [2009], the ancestral centromere is located in the HSA6p22 homologous region. During the course of chiropteran evolution, however, the small HSA6pter–p22 homologous segment was separated by centric fission from the large HSA6 homologous segment in all bats except flying foxes. The complete conservation of HSA11 synteny, proposed as a synapomorphic feature for the Eutheria [Robinson and Ruiz-Herrera, 2008], has been found in the Pteropodidae and Hipposideridae [Volleth et al., 2002; Mao et al., 2007]. Again, centric fission separated a small HSA11 segment from a large segment in other bat families. Reverse painting of Myotis probes onto human chromosome preparations revealed the location of the ancestral chiropteran centromere in 11q23. Balmus et al. [2007] found a similar breakpoint in the camel. A comparison of HSA1, 3, 6, and 11 and their counterparts in the flying fox is provided in figure 1.

High-Resolution Painting

The segmental composition of chromosomal arms or large chromosomal segments that show homology to only 1 human chromosome can be refined further by using painting probes from taxa with fragmented karyotypes. In Chiroptera, the composition of chromosomal segments with homology to HSA1, 3, and 4 was resolved with painting probes from the tree shrew (Tupaia belangeri, Scandentia) [Müller et al., 1997] and the black lemur (Eulemur macaco, Primates) [Müller et al., 1999]. The most interesting finding was a cryptic inversion in members of the Pteropodiformes [Volleth et al., 2011], which could be the first cytogenetic synapomorphy for this suborder (see below).

Volleth /Eick

Modes of Chromosomal Evolution in Bats

Conservation of Chromosomal Arms One of the most obvious characteristics of chromosomal evolution in bats is the conservation of monobrachial homology. This can easily be seen from the results of bi-directional painting between M. myotis and A. stoliczkanus. Of 24 Myotis autosomal arms, only 2 were split into 2 parts in A. stoliczkanus (arms 7 and 8). Vice versa, all Myotis chromosomal arms, except numbers 7 and 8, showed homology to only 1 chromosome or chromosomal arm of A. stoliczkanus [Ao et al., 2007; Mao et al., 2007]. We therefore recommend that the first step during FISH analysis of a bat karyotype should be the use of bat-specific probes to enable quick identification of the different chromosomal arms. Thorough comparison of the Gbanding patterns of the species of interest with a key species should then be performed to detect chromosomal arms with different subchromosomal architectures. Robertsonian Translocation Is the Prevailing Mode of Chromosomal Evolution in Bats A wide range of diploid chromosome numbers is found in nearly every chiropteran family. Taking into account the conservation of chromosomal arms as stable units in chiropteran karyotype evolution, the changes from a higher 2n to a lower 2n can be explained by the occurrence of Robertsonian (centric) fusions, and from a lower 2n to a higher 2n by Robertsonian fissions. A very low 2n, however, can only be explained by the involvement of non-Robertsonian rearrangements, for example tandem fusions. An impressive example is the phyllostomid genus Carollia (C. brevicauda 2n = 21; FISH results by Pieczarka et al. [2005]). Another example is the Malagasy bat Myzopoda aurita (MAU), for which tandem fusions have been proposed to explain the formation of chromosomes MAU4 and MAU5 [Richards et al., 2010]. The number of chromosomal arm combinations for Robertsonian fusion chromosomes is, however, limited. Therefore, convergent evolution of identical arm combinations in unrelated taxa has to be taken into account. This limits the power of karyotype analyses for resolving interfamily relationships in bats [Mao et al., 2008].

Fig. 2. Left: Segment order in chromosomal arm 2 of P. hesperidus

(PHE, Vespertilionidae) chromosome 1/2 (homolog of MMY1/2), upper row, compared with that in R. hipposideros (RHI, Rhinolophidae) chromosome 10, lower row. The proximal part of both chromosomes is homologous to HSA10p, the distal part to HSA4 (last and first image in each row). The results of FISH with 4 black lemur (EMA) chromosomes homologous to HSA4pter–q26 are shown in the centre. Note the inverted segment order in RHI compared to PHE. Right: Homology of EMA chromosomes to human chromosome 4. Starting from the telomere in the short arm, the order is the same as in PHE2 (modified from Volleth et al. [2011], with permission of Springer).

Inversions A small number of peri- and paracentric inversions had been proposed to explain differences in the G-banding patterns observed in some vespertilionid genera [Volleth and Heller, 1994]. These proposed inversions could not be confirmed by FISH experiments, because the rel-

evant chromosomes contain sequences homologous to only 1 human chromosome each. In contrast, Myotis chromosomal arm MMY5 shows homology to 3 human chromosomes, i.e. HSA4, 8, and 13. Interfamilial comparison of the human segment order on the MMY5 counterparts, however, revealed that the small HSA4-8 segment is located distal to the HSA13 segment in taxa belonging to the Vespertilioniformes, in contrast to the location of this small segment within the HSA13 segment in the Pteropodiformes (see character 19 in table 5). These observations can easily be explained by a paracentric inversion. Another small paracentric inversion was detected in the large HSA4 homologous segment located in the MMY2 homologous chromosomal arms. The segmental composition of this large segment was studied using whole chromosome paints of E. macaco (EMA) [Müller et al., 1997], for which homologous sequences to human chromosome 4 have been found on 5 different chromosome pairs (fig. 2). Four of these EMA chromosomes showed homology to MMY2 and corresponding homologs in other bats. The EMA segment order on chromosomal arm 2 of Pipistrellus hesperidus (Vespertilionidae, Vespertilioniformes) is the same as that on human chromosome 4 (fig. 2), but differs from that on chromosome 10 of Rhinolophus hipposideros (Rhinolophidae, Pteropo-



169

Table 5. Shared chromosomal characters in 13 species from 10 bat families

Character no.

description

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20a 20b 21a 21b 22a 22b 23a 23b

RF 1/2 RF 1/16-17 RF 3/4 RF 3/15 RF 4/5 RF 4/18 RF 5/6 RF 7i+7ii RF 7i/19 RF 9/11 RF 13/15 RF 13/23 RF 18/23 fis 8 fis 10 fis 12 fis 22 SC on 21 inv 5 RF 4/6 fus 4-5/6 RF 7i/21 RF 7ii/21 RF 8/14 RF 8ii/14 fus 24+16/17 RF 24/16-17

ESP

CSP

AST

HLA

RME

RPE

MSP

TME

MAU GSO

MFU

MPL

MMY

0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0

0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 1 0 0 nk 1 0 0 0 0 0 0 0

0 1 0 1 0 1 1 0 1 0 0 1 0 1 0 0 0 1 1 0 0 0 0 0 1 0 0

0 0 0 0 1 0 0 0 1 0 0 1 0 1 0 1 0 1 1 0 0 0 1 0 1 0 1

0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 1 1 0 0 0 0 0 0 0 0

0 1 0 1 1 0 0 0 0 0 0 0 0 1 1 1 1 1 nk 0 0 0 0 0 0 0 0

0 0 0 0 0 1 0 0 0 0 0 1a 0 1 1 1 0 0

0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0

0 1 1 0 0 0 0 0 0 1 0 0 1

1 0 0 0 0 0 1

1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 0 1 0 0 0 1

1c 0 0 0 0 0 0 0 0

1 0 0 0 0 0

1a

1a

0 0 1 1 0 0 0 0 0 0 0 0 0

1b 0 0 1 nk nk 0 0 0 0 0 0 0 0

1c 1 0 1 0 0 0

0 0 0 0 0 0 0 0 0 0 nk 0 0 0 0 0 0 0 0

1a 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Characters are numbered according to MMY homology. For the species indicated in italics (CSP, RPE, MAU and MFU), only bat-specific painting probes were applied. All other species were painted with human probes. 0: character not present; 1: character present; numbers in bold: synapomorphies; numbers in italics: symplesiomorphies. Fis = Fission; fus = fusion; inv = inversion; nk = not known; RF = Robertsonian fusion product; SC = secondary constriction (bearing a NOR). Species abbreviations: AST = Aselliscus stoliczkanus; CSP = Cynopterus sphinx; ESP = Eonycteris spelaea; GSO = Glossophaga

soricina; HLA = Hipposideros larvatus; MAU = Myzopoda aurita; MFU = Miniopterus fuliginosus; MMY = Myotis myotis; MPL = Mormopterus planiceps; MSP = Megaderma spasma; RME = Rhinolophus mehelyi; RPE = Rhinolophus p. pearsoni; TME = Taphozous melanopogon. Partial chromosomal arms: 7i = HSA19-8-4 homologous segment; 7ii = HSA5 homologous segment; 8ii = HSA7-5 homologous segment. a Rearranged state. b Breakpoint might differ. c Own unpublished results.

diformes). Therefore, the EMA segment order in Vespertilioniformes is likely the ancestral one [Volleth et al., 2011]. The inversion of the chromosomal segment showing homology to EMA12, EMA19, and EMA20 found in members of the Pteropodiformes could be a synapomor-

phic feature of this suborder. Painting probes from species with fragmented genomes have therefore proven to be extremely useful tools for the detection of small chromosomal changes within Chiroptera. Similarly, in the order Carnivora, dog chromosomal paints have been suc-

170


Volleth /Eick

cessfully used to detect quite a large number of small, mostly family-specific inversions [Nie et al., 2012]. Phylogenetic Relationships Chromosomal characters derived from FISH analyses have been used to investigate evolutionary relationships at various taxonomic levels within Chiroptera [Volleth et al., 2002; Mao et al., 2007, 2008; Richards et al., 2010]. An overview of published data is given in table 5. For families with more than 1 studied taxon, we focused on those painted with human probes. The flying fox Cynopterus sphinx [Ao et al., 2007] was added because of karyotypic differences between this species and the other 2 pteropodid species, Eonycteris and Rousettus, despite a very similar chromosome number (34 versus 36, respectively). Rhinolophus p. pearsoni (2n = 44) was included because this species shares Robertsonian fusion chromosomes with hipposiderids [Mao et al., 2007]. M. myotis was selected from the family Vespertilionidae because its karyotype is very close to the basic karyotype of this family [Volleth and Heller, 1994]. Only these chromosomal features that have been found in more than 1 taxon up to date are presented in table 5 (numbers 1–19). Closer examination and recent results revealed that some characters, which have been previously reported to be shared features [Volleth et al., 2002; Mao et al., 2008; Richards et al., 2010], represent in fact 2 different characters (numbers 20–23). Therefore, we separated these features into ‘a’ and ‘b’ in table 5. Most of the chromosomal characters are Robertsonian fusion chromosomes, which are prone to convergent evolution. One fusion product, 13/23, found in Pteropodiformes, is very likely a symplesiomorphic character. In this chromosome, HSA 11 homologous sequences are found in synteny, a situation proposed to be the ancestral condition for the Eutheria [Robinson and Ruiz-Herrera, 2008]. Six features are very likely synapomorphies. Up to this point, the fusion products 7i/19 and 8ii/14 (characters 9 and 22b) have been found only in members of the family Hipposideridae [Volleth et al., 2002; Ao et al., 2007; Mao et al., 2007, 2008]. Character 19, which is an inversion in arm 5, could be a synapomorphy for the suborder Pteropodiformes. A secondary constriction on arm 21 (character 18) is a diagnostic characteristic of Rhinolophidae and Hipposideridae. Homologs to MMY7 are found only in Vespertilionidae, Miniopterus, and Molossidae; all other taxa have 2 segments, 7i and 7ii. The fusion product has therefore been proposed to be a synapomorphy for Vespertilionoidea [Volleth et al., 2002; Mao et al., 2008]. The largest Myotis chromosome, MMY1/2, has been found in members of the VespertilChromosome Evolution in Bats

ionoidea and the phyllostomid Glossophaga. This character therefore supports the grouping of Vespertilionoidea and Phyllostomidae [Mao et al., 2008]. The fission of the MMY8 homolog into a small and a large segment in Rhinolophoidea appears to be a synapomorphy for this group (character 14 in table 5). This fission separates the HSA16 homologous segment from the HSA7-5 homologous segment, as has been shown for Hipposideros larvatus [Volleth et al., 2002]. Fission of the MMY8 homolog has also been found in Myzopoda [Richards et al., 2010]. Painting with human probes is needed to clarify whether the breakpoint in Myzopoda is similar to that in Rhinolophoidea. At the moment, there are only 2 chromosomal characters that are possibly of interest with regard to the phylogenetic position of the monotypic family Myzopodidae. First, the Robertsonian fusion product 9/11, also found in Glossophaga (Phyllostomidae), could indicate a closer relationship between Myzopoda and Noctilionoidea if increased taxon sampling confirms that this combination is a synapomorphy for this superfamily. Second, the fusion product 3/4, also found in Vespertilionidae, could suggest a closer evolutionary relationship between Myzopodidae and Vespertilionoidea. Molecular genetic analyses placed Myzopodidae either basal to the Vespertilionoidea [Eick et al., 2005; Meredith et al., 2011] or the Noctilionoidea [Miller-Butterworth et al., 2007; Teeling et al., 2012]. The remaining characters in table 5 appear to be homoplasies. Due to the large number of convergent events, phylogenetic analysis of chromosomal characters to resolve interfamily relationships using parsimony resulted in numerous parsimonious trees with poor resolution [Mao et al., 2008]. Therefore, cytogenetic features have been mapped onto the molecular consensus tree [Eick et al., 2005; Teeling et al., 2005] in recent analyses [Mao et al., 2008; Richards et al., 2010]. FISH results for species in additional bat families (e.g. Rhinopomatidae, Nycteridae) and increased taxon sampling in families represented by only a few species (e.g. Emballonuridae, Phyllostomidae) would help to elucidate the phylogenetic value of the chromosomal characters identified to date. Currently, chromosomal evolution in Phyllostomidae is being studied with whole chromosome painting probes from Macrotus californicus [Baker R.J., Sotero-Caio C.G. and Yang F., pers. commun.]. This research should provide significant insights into the karyotype composition of the phyllostomid ancestor and allow the identification of characters synapomorphic for this highly diverse bat family.


171

Suggested Chromosomal Elements of the Chiropteran Ancestor The karyotype composition of the chiropteran ancestor cannot be deduced from our current knowledge of bat karyotypes. The plesiomorphic state can be proposed for only a few chromosomal elements. Amongst these are the elements with conserved synteny of homologous sequences to HSA3, 6, and 11. Robertsonian fusion chromosomes composed of the following Myotis homologs can therefore putatively be considered ancestral: 3/9 (synteny HSA6), 4/19 (synteny HSA3-21), and 13/23 (synteny HSA11). All 3 chromosomes are found in Pteropodidae. In addition, 13/23 is found in Hipposideridae and, in a derived state, in Megadermatidae. Myotis homologs MMY10 and MMY12 may have been present as bi-armed elements in the presumed bat ancestor, because this condition is found in extant pteropodid, phyllostomid, and molossid taxa (a bi-armed MMY10 is also found in Hipposideridae). The ancestral state of MMY7 was very likely 2 separated chromosomes or chromosomal arms, 7i (homologous to HSA19p and a small HSA4-8 segment) and 7ii (comprising HSA5 homologous sequences).

Conclusions

The first step in characterization of the karyotype of a new bat species should be the application of conventional cytogenetic methods. Prior to FISH experiments, a Gbanded karyotype of reasonable quality should be on hand, and the position of heterochromatic blocks and the location of nucleolus organizer regions should be known. The next step in the analysis should then be the application of a bat-specific probe set. To clarify the composition of phylogenetically important segments and further refine the karyotype, selected human probes could then be used, e.g. homologs of MMY5, 7, or 16/17. According to Baker and Bickham [1980], 3 patterns of karyotype evolution can be recognized in bats: conservatism (e.g. Myotis), karyotypic orthoselection (G-banding patterns largely conserved), and karyotypic megaevolution (G-banding pattern severely altered; e.g. Tonatia). FISH with whole chromosome probes is an excellent tool to detect and analyze in detail the chromosomal changes that have occurred in karyotypically megaevolved species.

References Ao L, Gu X, Feng Q, Wang J, O’Brien PC, et al: Karyotype relationships of six bat species (Chiroptera, Vespertilionidae) from China revealed by chromosome painting and Gbanding comparison. Cytogenet Genome Res 115:145–153 (2006). Ao L, Mao X, Nie W, Gu X, Feng Q, et al: Karyotypic evolution and phylogenetic relationships in the order Chiroptera as revealed by Gbanding comparison and chromosome painting. Chromosome Res 15:257–267 (2007). Baker RJ: Order Chiroptera, in O’Brien SJ, Menninger JC, Nash WG (eds): Atlas of Mammalian Chromosomes, pp 378–380 (John Wiley & Sons, Hoboken, NJ 2006). Baker RJ, Bickham JW: Karyotypic evolution in bats: evidence of extensive and conservative chromosomal evolution in closely related taxa. Syst Zool 29:239–253 (1980). Balmus G, Trifonov VA, Biltueva LS, O’Brien PC, Alkalaeva ES, et al: Cross-species chromosome painting among camel, cattle, pig and human: further insights into the putative Cetartiodactyla ancestral karyotype. Chromosome Res 15: 499–514 (2007). Bickham JW, Baker RJ: Canalization model of chromosomal evolution, in Schwartz JH, Rollins HB (eds): Models and Methodologies in Evolutionary Theory, Bulletin of Carnegie Museum of Natural History no. 13, pp 70–84 (Carnegie Museum of Natural History, Pittsburgh 1979).

172

Capanna E, Civitelli MV: Chromosomal mechanisms in the evolution of chiropteran karyotype. Chromosomal tables of Chiroptera. Caryologia 23:79–111 (1970). Capozzi O, Purgato S, D’Addabbo P, Archidiacono N, Battaglia P, et al: Evolutionary descent of a human chromosome 6 neocentromere: a jump back to 17 million years ago. Genome Res 19:778–784 (2009). Eick GN, Jacobs DS, Matthee CA: A nuclear DNA phylogenetic perspective on the evolution of echolocation and historical biogeography of extant bats (Chiroptera). Mol Biol Evol 22:1869–1886 (2005). Eick GN, Jacobs DS, Yang F, Volleth M: Karyotypic differences in two sibling species of Scotophilus from South Africa (Vespertilionidae, Chiroptera, Mammalia). Cytogenet Genome Res 118:72–77 (2007). Frönicke L: Origins of primate chromosomes – as delineated by Zoo-FISH and alignments of human and mouse draft genome sequences. Cytogenet Genome Res 108: 122–138 (2005). Gardner AL: Chromosomal variation in Vampyressa and a review of chromosomal evolution in the Phyllostomidae (Chiroptera). Syst Zool 26:300–318 (1977). Hutcheon JM, Kirsch JA: A moveable face: deconstructing the Microchiroptera and a new classification of extant bats. Acta Chiropt 8: 1–10 (2006).


Kulemzina AI, Nie W, Trifonov VA, Staroselec Y, Vasenkov DA, et al: Comparative chromosome painting of four Siberian Vespertilionidae species with Aselliscus stoliczkanus and human probes. Cytogenet Genome Res 134: 200–205 (2011). Mao X, Nie W, Wang J, Su W, Ao L, et al: Karyotype evolution in Rhinolophus bats (Rhinolophidae, Chiroptera) illuminated by crossspecies chromosome painting and G-banding comparison. Chromosome Res 15: 835–848 (2007). Mao X, Nie W, Wang J, Su W, Feng Q, et al: Comparative cytogenetics of bats (Chiroptera): the prevalence of Robertsonian translocations limits the power of chromosomal characters in resolving interfamily phylogenetic relationships. Chromosome Res 16: 155–170 (2008). Meredith RW, Janečka JE, Gatesy J, Ryder OA, Fisher CA, et al: Impacts of the Cretaceous Terrestrial Revolution and KPg extinction on mammal diversification. Science 334: 521–524 (2011). Miller-Butterworth CM, Murphy WJ, O’Brien SJ, Jacobs DS, Springer MS, Teeling EC: A family matter: conclusive resolution of the taxonomic position of the long-fingered bats, Miniopterus. Mol Biol Evol 24: 1553– 1561 (2007).

Volleth /Eick

Müller S, O’Brien PC, Ferguson-Smith MA, Wienberg J: Reciprocal chromosome painting between human and prosimians (Eulemur macaco macaco and E. fulvus mayottensis). Cytogenet Cell Genet 78:260–271 (1997). Müller S, Stanyon R, O’Brien PC, FergusonSmith MA, Plesker R, Wienberg J: Defining the ancestral karyotype of all primates by multidirectional painting between tree shrews, lemurs and humans. Chromosoma 108:393–400 (1999). Nie W, Wang J, Su W, Wang D, Tanomtong A, et al: Chromosomal rearrangements and karyotype evolution in carnivores revealed by chromosome painting. Heredity 108: 17– 27 (2012). Noronha RC, Nagamachi CY, O’Brien PC, Ferguson-Smith MA, Pieczarka JC: Neo-XY body: an analysis of XY1Y2 meiotic behavior in Carollia (Chiroptera, Phyllostomidae) by chromosome painting. Cytogenet Genome Res 124:37–43 (2009). Pieczarka JC, Nagamachi CY, O’Brien PC, Yang F, Rens W, et al: Reciprocal chromosome painting between two South American bats: Carollia brevicauda and Phyllostomus hastatus (Phyllostomidae; Chiroptera). Chromosome Res 13:339–347 (2005). Richards LR, Rambau RV, Lamb JM, Taylor PJ, Yang F, et al: Cross-species chromosome painting in bats from Madagascar: the contribution of Myzopodidae to revealing ancestral syntenies in Chiroptera. Chromosome Res 18:635–653 (2010).


Robinson TJ, Ruiz-Herrera A: Defining the ancestral eutherian karyotype: a cladistic interpretation of chromosome painting and genome sequence assembly data. Chromosome Res 16:1133–1141 (2008). Ruiz-Herrera A, Farré M, Robinson TJ: Molecular cytogenetic and genomic insights into chromosomal evolution. Heredity 108:28–36 (2012). Simmons NB: Order Chiroptera, in Wilson DE, Reeder DM (eds): Mammal Species of the World. A Taxonomic and Geographic Reference, vol 1, 3rd ed, pp 312–529 (Johns Hopkins University Press, Baltimore 2005). Sotero-Caio CG, Pieczarka JC, Nagamachi CY, Gomes AJ, Lira TC, et al: Chromosomal homologies among vampire bats revealed by chromosome painting (Phyllostomidae, Chiroptera). Cytogenet Genome Res 132: 156–164 (2011). Teeling E, Madsen O, Van Den Bussche R, de Jong WW, Stanhope MJ, Springer MS: Microbat paraphyly and the convergent evolution of a key innovation in Old World rhinolophoid microbats. Proc Natl Acad Sci USA 99:1431–1436 (2002). Teeling E, Springer M, Madsen O, Bates P, O’Brien SJ, Murphy WJ: A molecular phylogeny for bats illuminates biogeography and the fossil record. Science 307: 580–584 (2005). Teeling EC, Dool S, Springer M: Phylogenies, fossils and functional genes: the evolution of echolocation in bats, in Gunnell GF, Simmons NB (eds): Evolutionary History of Bats: Fossils, Molecules and Morphology (Cambridge University Press, Cambridge 2012).

Volleth M, Heller KG: Phylogenetic relationships of vespertilionid genera (Mammalia, Chiroptera) as revealed by karyological analysis. Z Zool Syst Evol 32:11–34 (1994). Volleth M, Heller KG: Chromosome number reduction accompanied by extensive heterochromatin addition in the bat Glauconycteris beatrix (Mammalia; Chiroptera, Vespertilionidae). Cytogenet Genome Res 119: 245–247 (2007). Volleth M, Klett C, Kollak A, Dixkens C, Winter Y, et al: ZOO-FISH analysis in a species of the order Chiroptera: Glossophaga soricina (Phyllostomidae). Chromosome Res 7:57–64 (1999). Volleth M, Heller KG, Pfeiffer RA, Hameister H: A comparative ZOO-FISH analysis in bats elucidates the phylogenetic relationships between Megachiroptera and five microchiropteran families. Chromosome Res 10:477– 497 (2002). Volleth M, Yang F, Müller S: High-resolution chromosome painting reveals the first genetic signature for the chiropteran suborder Pteropodiformes (Mammalia: Chiroptera). Chromosome Res 19: 507–519 (2011). Zima J, Volleth M, Horácek I, Cerveny J, Cervena A, et al: Comparative karyology of rhinolophid bats, in Horácek I, Vorhalik V (eds): Prague studies in Mammalogy, pp 229–236 (Charles University Press, Prague 1992).


173

Published online: August 7, 2012


Comparative Chromosome Painting in Carnivora and Pholidota P.L. Perelman a V.R. Beklemisheva a D.V. Yudkin a T.N. Petrina b V.V. Rozhnov b W. Nie c A.S. Graphodatsky a a

Institute of Molecular and Cellular Biology, Siberian Division of Russian Academy of Sciences, Novosibirsk , and A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow, Russia; c State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, PR China

b

Key Words Ancestral Carnivora karyotype ⴢ Canidae ⴢ Feliformia ⴢ Genet ⴢ Karyotype evolution ⴢ Mustelidae ⴢ Painting probe ⴢ Pangolin

genome sequencing data is discussed in the context of solving the questions that are beyond resolution of conventional banding techniques and chromosome painting.

Abstract The order of Carnivora has been very well characterized with over 50 species analyzed by chromosome painting and with painting probe sets made for 9 Carnivora species. Representatives of almost all families have been studied with few exceptions (Otariidae, Odobenidae, Nandiniidae, Prionodontidae). The patterns of chromosome evolution in Carnivora are discussed here. Overall, many Carnivora species retained karyotypes that only slightly differ from the ancestral carnivore karyotype. However, there are at least 3 families in which the ancestral carnivore karyotype has been severely rearranged – Canidae, Ursidae and Mephitidae. Here we report chromosome painting of yet another Carnivora species with a highly rearranged karyotype, Genetta pardina. Recurrent rearrangements make it difficult to define the ancestral chromosomal arrangement in several instances. Only 2 species of pangolins (Pholidota), a sister order of Carnivora, have been studied by chromosome painting. Future use of whole-

There are 2 major branches on the Carnivora tree – Feliformia and Caniformia. Over 50 species were studied in the order Carnivora by chromosome painting (table 1). In these studies, human painting probes and painting probes from 9 carnivores (table 2) were used to define chromosome homologies between human and carnivores and among different carnivore species. Human painting probes link the painting information in Carnivora to other orders and permit hypotheses about the ancestral karyotype for the entire order. The ancestral carnivore karyotype (ACK) is highly conserved and originated from the proposed boreoeutherian karyotype by the following fusions of human chromosomes/arms: 3+19p, 2+20, and 18+22 along with other associations common to all boreoeutherians (HSA3/21, 4/8p, 7b/16p, 10p/12a/22a, 12b/22b, 14/15, 16q/19q) [Froenicke, 2005]. Comparative G-banding studies predicted, and later chromosome painting studies confirmed, 2 major modes



Copyright © 2012 S. Karger AG, Basel

A.S. Graphodatsky Institute of Molecular and Cellular Biology Siberian Division of Russian Academy of Sciences Lavrentieva 10, RU–630090 Novosibirsk (Russia) Tel. +7 383 333 3857, E-Mail graf @ mcb.nsc.ru

Table 1. Chromosome painting in Carnivora and Pholidota Family

Species

Diploid Painting probes chromosome number

Ailuridae

Ailurus fulgens

36

Canidae

Alopex lagopus

50

Canis familiaris

78

Cerdocyon thous

74

Chrysocyon brachyurus Cuon alpinus Dusicyon vetulus Fennecus zerda

76+B 64 74 66

Nyctereutes procyonoides procyonoides Nyctereutes procyonoides viverrinus Otocyon megalotis Speothus venaticus Urocyon cinereoargenteus

54+В

Vulpes corsac Vulpes macrotis Vulpes vulpes

36 50 34+B

Conepatus leuconotus Mephitis macroura Mephitis mephitis

46 50 50

Spilogale gracilis Ictonyx striatus Martes foina

60 44 38

Notes

Autosomal segments

References

71 21 42 39 68, 67

Tian et al., 2002 Nie et al., 2002 Graphodatsky et al., 2000a Nash et al., 2001 Breen et al., 1999; Yang et al., 1999 Thomas et al., 1999 Nash et al., 2001 Nash et al., 2001 Duke Becker et al., 2011 Duke Becker et al., 2011 Graphodatsky et al., 2008 Duke Becker et al., 2011 Graphodatsky et al., 2008 Duke Becker et al., 2011 Nie et al., 2003 Duke Becker et al., 2011 Graphodatsky et al., 2001 Duke Becker et al., 2011 Duke Becker et al., 2011 Duke Becker et al., 2011 Graphodatsky et al., 2008 Duke Becker et al., 2011 Graphodatsky et al., 2008 Duke Becker et al., 2011 Yang et al., 1999 Duke Becker et al., 2011 Yang et al., 2000 Yang et al., 1999 Graphodatsky et al., 2002 Perelman et al., 2008 Perelman et al., 2008 Perelman et al., 2008 Perelman et al., 2008 Perelman et al., 2008 Graphodatsky et al., 2002 Nie et al., 2012 Nie et al., 2002 Nie et al., 2012 Nie et al., 2002 Graphodatsky et al., 2002 Nie et al., 2002 Graphodatsky et al., 2002 Nie et al., 2002 Graphodatsky et al., 2002 Graphodatsky et al., 2002 Graphodatsky et al., 2002 Graphodatsky et al., 2002 Graphodatsky et al., 2002 Cavagna et al., 2000 Cavagna et al., 2000 Graphodatsky et al., 2000b, 2002 Hameister et al., 1997 Nie et al., 2002 Frönicke et al., 1997

Caniformia

Mephitidae

Mustelidae

Phocidae

38+B 72 74 66

Martes flavigula Martes melampus Meles meles

40 38 44

Melogale moschata Mustela altaica Mustela eversmanni Mustela lutreola Mustela nivalis Mustela putorius Mustela putorius furo

38 38 38 38 42 40 40

Mustela vison

30

Phoca vitulina

32

Comparative Chromosome Painting in Carnivora and Pholidota

CFA MFO CFA NPRv HSA

reciprocal

HSA1, 11, 16, 17 FCA NPRv CFA CFA CFA CFA CFA CFA CFA CFA CFA CFA CFA CFA CFA CFA CFA CFA CFA CFA FCA HSA MVI MFO MFO HSA MFO MFO MVI CFA FCA HSA MFO MVI MFO MVI MFO MVI MVI MVI MVI MVI FCA HSA CFA HSA MFO HSA

CFA5; reciprocal

BACs (selected set) BACs (selected set) BACs (selected set) BACs (selected set) BACs BACs (selected set) BACs (selected set) BACs (selected set) BACs BACs (selected set) reciprocal BACs

reciprocal

reciprocal

4, 4 66 41 n/a n/a 38 n/a 42 n/a 41 42 41 n/a n/a n/a 43 45 33 n/a 42, 42 42 65 73 60 31 30 40 30 36 20 73 21, 21 32 19 20 21 22 18 20 21 21 21 21 22 32 71, 63 32 20 30


175

Table 1 (continued) Family

Species

Painting probes Diploid chromosome number

Procyonidae

Bassariscus astutus

38

Procyon lotor

38

Ailuropoda melanoleuca

42

Ursidae

Helarctos malayanus

74

Tremarctos ornatus

52

Notes

FCA NPRv CFA HSA FCA MFO CFA FCA NPRv HSA CFA AME CFA FCA

reciprocal reciprocal

Autosomal segments

References

21 61 69 31 21 18

Nash et al., 2008 Nash et al., 2008 Nie et al., 2012 Perelman et al., 2008 Perelman et al., 2008 Perelman et al., 2008

74 38, 39 70, 72 44 73 38 72

Nie et al., 2012 Nash et al., 1998 Nash et al., 2001 Nash et al., 1998 Tian et al., 2004 Nash et al., 1998 Yang and Graphodatsky, 2004 Nash et al., 1998

68, 65 4 31 (101 CSO) 30, 30 30, 30 21

Yang et al., 2000 Thomas et al., 1999 Rettenberger et al., 1995; (Wienberg et al., 1997) Perelman et al., 2008 Perelman et al., 2008 Hameister et al., 1997 Graphodatsky et al., 2002 Nash et al., 2001 Nie et al., 2012 Tian et al., 2004 Tian et al., 2004 Nash et al., 2008 Nash et al., 2008 Nie et al., 2012 Perelman et al., 2005 Perelman et al., 2005 Perelman et al., 2005 Nash et al., 2008 Nash et al., 2008 Perelman et al., 2005 Perelman et al., 2005 Perelman et al., 2005 Nie et al., 2012 Nie et al., 2012 this paper

selected probes

Feliformia Felidae

Felis catus

38

Herpestidae

Lynx lynx Neofelis nebulosa Panthera leo Helogale parvula

38 38 38 36

Hyaenidae

Herpestes javanicus Crocuta crocuta

36 40

Eupleridae

Fossa fossana

42

Viverridae

Paguma larvata

44

Paradoxurus hermaphroditus Viverricula indica Genetta pardina

42 36 52

CFA CFA HSA

reciprocal CFA5 reciprocal

MMA MME MVI

reciprocal reciprocal

NPRv CFA CFA CFA FCA NPRv CFA CFA HSA MVI FCA NPRv CFA HSA MVI CFA CFA MFO

reciprocal

67, 65 69 69 69 22 63 68 69 31 23 21 62 67 31 22 67 68 40

Painting of Carnivora probes onto species from other orders Cetartiodactyla Sus scrofa Neophocaena phocaenoides asiaeorientalis

38 44

CFA MFO

Pholidota

Manis javanica

38

Manis pentadactyla

40

HSA MFO MPE HSA

selected probes

105 26 (31 with heterochromatin inserts) 47 35 22 47

Biltueva et al., 2004 Nie et al., 2012

Yang et al., 2006 Yang et al., 2006 Nie et al., 2009 Nie et al., 2009

AME = Ailuropoda melanoleuca; CFA = Canis familiaris; FCA = Felis catus; HSA = Homo sapiens; MFO = Martes foina; MMA = Mephitis macroura; MME = Mephitis mephitis; MPE = Manis pentadactyla; MVI = Mustela vison; NPRv = Nyctereutes procyonoides viverrinus.

176


Perelman /Beklemisheva /Yudkin / Petrina /Rozhnov /Nie /Graphodatsky

Table 2. Carnivora painting probe sets

Species

Abbreviation

2n

Published in

Carnivora Ailuropoda melanoleuca Canis familiaris Felis catus Martes foina Mephitis macroura Mephitis mephitis Mustela vison Nyctereutes procyonoides viverrinus Vulpes vulpes

AME CFA FCA MFO MMA MME MVI NPRv VVU

42 78 38 38 50 50 30 38+B 34+B

Nash et al., 1998 Breen et al., 1999; Yang et al., 1999 Wienberg et al., 1997; Nash et al., 1998 Nie et al., 2002 Perelman et al., 2008 Perelman et al., 2008 Graphodatsky et al., 2002 Nash et al., 2001 Yang et al., 1999

Pholidota Manis pentadactyla

MPE

40

Nie et al., 2009

Primates Homo sapiens

HSA

46

Ferguson-Smith, 1997

of chromosome evolution in Carnivora – slow (species that retained the ancestral carnivore karyotype with few changes) and extremely fast (genomes that were drastically reshuffled to produce chromosome complements of extant species). Feliformia in general have very conserved karyotypes that differ by few interchromosomal rearrangements. Most of the Caniformia families are also characterized by slow rates of chromosomal rearrangements with few exceptions: Canidae, Ursidae and Mephitidae whose genomes required more than 40, 17 and 17 changes, respectively, from the ancestral carnivore karyotype. These are families with a so-called catastrophic rate of chromosomal rearrangements [Grafodatsky and Biltueva, 1987]. Chromosome painting data for the Feliformia and Caniformia branches were recently reviewed [Nie et al., 2012]. A detailed tree depicting chromosomal evolution of both branches of Carnivora was presented in that study. There are some interesting inversions that have been revealed by dog painting probes across the whole order [Nie et al., 2012]. In this review, we discuss the ancestral carnivore karyotype, describing some problematic ACK chromosomes, and the chromosomal evolution of Canidae as revealed by chromosome painting and BAC mapping. We report chromosome painting data on the first species among feliforms with a fast rate of chromosomal evolution (Genetta pardina). We indicate some groups within the Carnivora that might benefit from additional chromosome painting studies. In addition, chromosome evolution in a sister order of Carnivora, the Pholidota, is also reviewed. Comparative Chromosome Painting in Carnivora and Pholidota

Ancestral Carnivore Karyotype

Decades ago extreme karyotypic conservation in Carnivora at the chromosome band level allowed identification of chromosomes ‘shared’ by many Carnivora species and the first attempts to identify the ancestral state of the chromosomes for the whole order [Wurster-Hill and Gray, 1975]. Later, chromosome painting in Carnivora species with conserved karyotypes helped confirm conclusions from classical cytogenetics. On the other hand, without chromosome painting it was impossible to reveal the detailed picture of complex rearrangements in groups with fast chromosome evolution such as Ursidae, Canidae, and Mephitidae. Inversions in conserved chromosomes were also hard to detect using only banding. The ancestral karyotype of Carnivora has been described or discussed several times [Wurster-Hill and Gray, 1975; Dutrillaux and Couturier, 1983; Fronicke et al., 1997; Murphy et al., 2001b; Nash et al., 2008; Perelman et al., 2008; Nie et al., 2012]. Table 3 provides the correspondence of ACK chromosomes with feline (Felis catus, FCA), dog (Canis familiaris, CFA), stone marten (Martes foina, MFO) and human (Homo sapiens, HSA) chromosomes as well as with previously suggested ancestral carnivore karyotypes. Due to the slow rate of chromosome evolution in major Carnivora groups and the conserved pattern of G-bands across many species, even predictions based on banding techniques alone were quite precise. Chromosome painting data allowed an unequivocal deduction of the ancestral state of most chromosomes for the whole order. Cytogenet Genome Res 2012;137:174–193

177

178



A2p+C2 B1 A1q B2 B3 B4 A3 C1q A2q C1p D2 D1 D3 D4 A1p E1 F1 F2 E2 E3 X

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 X

19p+3+21 4+8p+4 5 6 14+15 10p+12pq+22q 20+2p–q 2q 7p–q 1p–q 1qd+10q 115a/21/18b 12q+22+18 9 13 17 1q 8q 19q+16q 7q+16p X

HSA

26a/7b/1a 1c/11b/9b 22/28a 5b/9a 7a/38 34/13a 1d/2c/5e 6a X

20+23/35b/30/27 16b/28b/15c/19a/32/13b/3c 11a/2a2/3a/4b 37/12/1b2 3b/25/8 2a1/29/10a/15b/10b 25/10/17b 19b/36/33/28c 18a/14/16a 17a/6b/5d/15a/2d/5c 4a/31

CFA

x

x

x

x x

x

21 (21q = 62) B1 20 B2 (37) B3 (30–33) B4 (44, 45) A3 22q, 66 26q 63, 25p D2 (74) D1 D3 D4 22p E1 (87) F1 (82) F2 (64), 25q E2 (82) E3 X

21 (19/3/21; sm1) B1 (4/8/4; sm2) 20 (5; sq) B2 (6; sm3) B3 (14/15; sm4) B4 (?/12/22; ?) A3 (20/2; m5) 22q (2; m1q) 64 (7; sm5) 25q (1; m2q) D2 (10; ?m4q) D1 (11, m6) D3 (22/18; m7) D4 (9; m8) 22p (13; m1p) E1/87 (17; sp) F1 (1; m4p) 25p (8; m2p) E2 (19/16; m9) E3 (16; m3p)

Wurster-Hill and Gray Frönicke et al. [1997]c [1975]b 2n = 38 Z-CAR FN > 58 CAR (HSA; PVI) 1 2 5 6 7 8 9 3q 10 4q 11 12 13 14 3p 15 16f 4p 17 18 X

Nash et al. [2008] ACK 2n = 38

ing human and harbor seal chromosomes/associations in parentheses (HSA; PVI). Italic = Homoeology for these chromosomes of harbor seal and Z-CAR elements was not indicated in the paper and is deduced based on the available painting data. d Segment was added based on Ensemble data and was earlier shown by chromosome painting in Yang et al. [2000]. e Inconsistency with modern data. f Corresponding ACK number from Murphy et al. [2001b] is mistakenly given as 18 in Nash et al. [2008].

c CAR chromosome number [based on Wurster-Hill et al., 1975] followed by correspond-

? (19; ?) – 11 (21; C2pdis) – 11(3; C2) 1 (4; B1) ? (8p; ?) 12 (5; A1q) 2 (6; B2) 3 (14–15; B3) 4p (?; B4p) – ? (10p; ?) – 4q (12; B4q) 5p (?; A3p) – ? (20; ?) – 5q(2p; A3q) 14 (2q; C1q) 15 (7; A2q) 13(1p; C1p) 16 (10q; D4e) 6 (11; D1) 8p (?; D3p) – 8q (18; D3q) ? (22; ?) 7 (9; D2e) 17 (13; A1p) 9 (17; E1) 21? (1q; F1) 18 (8q; F2) 10 (?; E2) – ? (19; ?) – ? (16; ?) 23 (?; E3) – ? (16; ?) X

Problematic Dutrillaux and Couturier [1983]a ACK chro- CAR (HSA; FCA) mosomes 2n = 42

ACK = Ancestral carnivore karyotype; CFA = Canis familiaris; FCA =Felis catus; HSA = Homo sapiens; MFO = Martes foina; PVI = Phoca vitulina. Shaded cells indicate chromosomes that are suggested to be present in the ancestor as single chromosome. a Ancestral Carnivora (CAR) chromosome number followed by the corresponding segments of human and cat chromosomes (HSA; FCA) when available. ? = the corresponding segment is not indicated in the paper. b In this paper modified felid homologs were given numbers, indicated in parentheses. In bold are chromosomes that authors suggested to be ‘most ancient and stable chromosomes that are shared by descendants of both the canoid and feloid stock’. Chromosome 25 (italics) was considered by authors as a separate ancient element but in fact (based on corresponding painting data) is homolog of C1q (25p) and F2 (25q).

2 1 5 6 7 9 8 3q 11 4q 12 10 13 14 3p 15 16 4p 17 18 X

FCA

ACK MFO Murphy et al. [2001b] 2n = 42

Table 3. Correspondence of ancestral carnivore karyotype (ACK) chromosomes with stone marten, cat, human and dog chromosomes

Fig. 1. The state of problematic ancestral chromosomes across

Carnivora. ACK chromosome state for each group/species is based on chromosome painting data listed in table 1. * For Nandiniidae and Prionodontidae the chromosomal state is based on G-banding data [Wurster-Hill and Gray, 1975]. Phylogenetic tree topology for Carnivora [Eizirik et al., 2010] and additionally for

viverrids [Gaubert and Cordeiro-Estrela, 2006] is drawn based on modern molecular phylogenies. In grey boxes are taxa with fast rates of genome reorganization. Number of symbols on each branch indicates the number of fragments of the particular chromosome in the karyotype.

However, there are some problematic questions that remain to be resolved and which contribute to the differences in proposed diploid numbers for the ancestral carnivore karyotype, i.e. 2n = 42 and 2n = 38 (fig. 1, table 3). While reconstructing ancestral states one should consider phylogenetic branching of the families [Eizirik et al., 2010]. Nie et al. [2012] provided a detailed figure of karyotype relationships in Carnivora mapped onto a phylogenetic tree. In general, it is agreed that there are 2 major groups in the order, Caniformia and Feliformia. The deduction would include cladistic assumptions if the same chromosomal state occurs in both feliforms and caniforms, or if basal species of either group display the same state of the chromosome as seen in another group, meaning that this state was likely ancestral for the whole order. One has to keep in mind that there are at least 3 families in Carnivora which display severely fissioned karyotypes

relative to the carnivore ancestor, and thus the absence of a particular fusion in their karyotypes has less value than its absence in the conserved karyotype. Here we use the nomenclature of Murphy et al. [2001b] for ACK chromosomes. ACK1 (FCA A2p+C2) represents a long-standing problem in defining the ancestral carnivore karyotype, and it is also one of the signature associations of human chromosomes for the whole order Carnivora (HSA19p+3/21). This ancestral chromosome is present as 2 fragments in all carnivores except for the following families on the Caniformia branch: Pinnipedia, Ailuridae, Procyonidae, Mustelidae (except Meles meles) (fig. 1). Based on G-banding comparative studies in Nandinia binotata (basal branch of Feliformia), the ACK1 chromosome is described as 1 piece. Some authors propose that this state was ancestral [Wurster-Hill and Gray, 1975;



179

Fronicke et al., 1997; Nash et al., 2008]. However, the discussion about whether the fission or fusion of ACK1 represents the ancestral state will not be settled until chromosome painting is done to verify the G-banding data or until another species is identified with FCA A2p+C2 as 1 chromosome within the Feliformia branch. ACK7 (FCA A3; HSA20+2pq prox) is another problematic ancestral chromosome. This case is even more complicated as it is present as 1 piece in all studied Feliformia except Herpestidae, Hyaenidae, Paguma and in all conserved caniforms (except M. meles) (fig. 1). This chromosome may be prone to breakage. It was suggested by Nie et al. [2012] that it could have been present as 2 pieces in the ancestral carnivore karyotype because it occurs as 2 pieces in outgroup species (human, pangolin, cetartiodactyls) as well as in different carnivore families. However, even though ACK7 is split in all outgroups, it is still often present as 1 chromosome on both branches of carnivores and might therefore have been present as single chromosome in the Carnivora ancestor. ACK13 (FCA D3; HSA12qdist/22q prox/18) is present as 2 segments in the highly rearranged genomes of species within the Canidae, Ursidae and Mephitidae and in Genetta; however, it is 1 single element in all other groups. The position of the centromere in ACK13 differs among species; it is at the end of the segment in American mink Neovison (Mustela) vison, in the middle of the segment corresponding to CFA26 in most of the carnivores, and it is in the middle of the segment corresponding to CFA7 in red panda and human. This example shows that even in the branches of the order with highly conserved genomes repositioning of a centromere occurs quite often. The phenomenon of neocentromere formation is absolutely worth studying in Carnivora using a whole-genome sequencing approach in combination with detailed BACmapping and sequencing [Rocchi et al., 2012]. There are also 4 ancestral elements, ACK15 (FCA A1p; HSA13) + ACK8 (FCA C1q; HSA2q) and ACK10 (C1p; HSA1pq) + ACK18 (FCA F2; HSA8q) that appear as 1 or 2 segments throughout the order (not considering groups with highly rearranged genomes). To clarify the ancestral form of these chromosomes will require additional data, especially painting of N. binotata chromosomes (fig. 1). ACK15+ACK8 often appears as 1 chromosome among caniforms (except Mustela and M. meles) and is uniformly present as 2 segments among studied Feliformes (except basal Nandinia). A similar situation is ACK10+ ACK18 which is present as 2 segments in all Feliformes (except Prionodon and Nandinia) and 1 segment in Caniformes (except Mustela). 180


Overall, for ACK1, 7, 15+8, 10+18 it is hard to determine the ancestral state based on current painting data. If the G-banding data on Nandinia is eventually confirmed by chromosome painting, then a single-segment state is more likely for all 4 chromosomes [Wurster-Hill and Gray, 1975]. Another consideration is that the recurrent fusion of the same ancestral segments is less likely than recurrent fissions. Yet another consideration in such complicated cases might be so-called hemiplasy, ancestral polymorphism persisting into modern taxa [Avise and Robinson, 2008; Robinson et al., 2008]. Until today there is no explanation for the sudden disruption of ancestral segments in canids, ursids, skunks and in genet. It will be very important to compare chromosomal breakpoints in these groups once more genomic data with chromosome assignment become available for carnivores. By comparing a series of dog painting probes hybridized to chromosomes of different species within Carnivora, we could reconstruct the ancestral order of dog chromosomal segments on the ancestral Carnivora chromosomes (fig. 2). With additional full-genome sequences of carnivores it will be possible to discern a more detailed order of syntenic segments. With more whole-genome sequencing data appearing every day, one can expect that future comparative chromosome painting will be done computationally through genome comparison [Alekseyev and Pevzner, 2009; Kemkemer et al., 2009; Larkin et al., 2009; Ensemble Genome Browser http://www.ensembl.org/index.html]. However, among carnivores only the domestic dog (C. familiaris) genome assembly currently has full annotation linked with chromosome localizations. There is an urgent need to have additional Carnivora genomes with scaffolds assigned to chromosomes. Although publicly available, the giant panda (Ailuropoda melanoleuca) and the cat (F. catus) genomes do not have chromosome assignments that are integrated into browsers that would allow easy comparison of syntenic segments. For example, Ensemble genome browser has a very useful feature called ‘comparative genomics – synteny’ allowing to view in this browser ‘syntenic regions that are calculated where possible from pairwise (twospecies) whole genome alignments’. We compared results of reciprocal human-dog painting data [Yang et al., 2000] and syntenic blocks order identified in Ensemble and in Evolutionary Highway (that reflects homologous synteny blocks among 10 amniotes (human, chimp, macaque, rat, mouse, pig, cattle, dog, opossum, and chicken) that were identified from pairwise comparisons of all genomes at !1 human-Mb Perelman /Beklemisheva /Yudkin / Petrina /Rozhnov /Nie /Graphodatsky

Fig. 2. Ancestral carnivore karyotype (ACK) with suggested an-

cestral sequence of syntenic segments corresponding to dog chromosomes based on order-wide comparison of chromosome painting data of canid painting probes. Numbers on the left indicate dog chromosomes and segments marked with letters (a, b, c, etc.) starting from the dog chromosome centromere based on recipro-

cal painting data and Ensemble browser data. Numbers on the right of each ancestral chromosome are ACK chromosomes according to Murphy et al. [2001b] nomenclature. ACK chromosome numbers below correspond to Nash et al. [2008] nomenclature. The asterisk indicates a segment added based on analyses of painting and Ensemble data (see discussion).

resolution (http://www.ensembl.org; http://eh-demo. ncsa.uiuc.edu/) (table 4). We aimed to identify if some large syntenic blocks were missed by chromosome painting. We disregarded segments identified in Ensemble that were !1 Mb in human. However, we noticed that there were many small syntenic segments around centromeric and telomeric regions which are shown on Ensemble syntenic view, but are not present in painting data. These might be false-positive blocks that are enriched in repetitive segments. Robust assemblies of repetitive regions are required to clarify whether these syntenies are real. Among larger segments, one was detected in recent painting experiments, HSA5p (CFA35; ACK1 and 3) [Nie et al., 2012] (table 4; fig. 2). Another segment, HSA1q (CFA4; ACK11), was revealed by Yang et al. [2000], but was missed in other painting studies. It forms 1 syntenic group with HSA10q (CFA26; ACK11). One segment on HSA1p (CFA17) was detected by painting and Ensemble, but is absent in Evolutionary Highway. Another segment of CFA6 (HSA2) was only shown by painting and not by other data. Several segments (HSA3p, 3q, 8p, 14q, 20q, 22q) shown in Ensemble were small in size (!4 Mb), had centromere or telomere localization, but were not detected by painting or Evolutionary Highway. Another group of segments were not revealed by painting, but shown in

Ensemble and often in Evolutionary Highway (HSA1q, 4q, 5q, 7p, 8p, 10q, 11p, 15q). These might represent ancestral syntenic segments that were split into small pieces on human chromosomes due to inversions. Analyses showed how precise and robust the chromosome painting tool is in describing overall chromosome evolution and paving the way to a detailed view into evolutionary reorganization of the genome.



Felidae The family Felidae is well-known for its remarkable karyotype conservation [Wurster-Hill and Gray, 1973]. The whole family has 2n = 38, except species from the ocelot lineage that have 2n = 36 [Wurster-Hill and Centerwall, 1982; Johnson et al., 2006]. Three fusions and 1 inversion are common to 4 species studied with chromosome painting (African lion, cat, clouded leopard, Eurasian lynx) and were revealed as ACK1p+9, 3+15, 8+10, inversion ACK2 [Wienberg et al., 1997; Tian et al., 2004; Nie et al., 2012]. Earlier G-banding studies showed that chromosomes of all felids had almost identical G-banding patterns [Wurster-Hill and Centerwall, 1982]. Later chromosome painting studies with high-resolution painting probes did not show any intrachromosomal changes in the few studied felid species (table 1). How181

Table 4. Syntenic blocks of dog (CFA) chromosomes identified by Ensemble and/or Evolutionary Highway on human (HSA) chromo-

somes in addition to painting data CFAa

Size, Mb HSA/CFA

Localization Chromosome Evolutionary Commentc HSA/CFAb painting HSA-CFA Highway [Yang et al., 2000] [Larkin et al., 2009]

1p: 112.0–114.2 1p: 114.2–120.7 1q: 187.0–196.7 1q: 223.7–227.7 3p: 88.2–90.3 3q: 194.1–197.8 4p: 41.3–49.1 4q: 169.3–176.4 5p: 0.1–14.7

17: 54.6–60.2 17: 65.1–67.0 38: 5.9–14.0 7: 40.6–43.4 33: 3.0–4.7 33: 31.7–34.4 13: 41.5–47.7 25: 22.1–28.9 34: 3.1–15.1

2.2/5.6 6.5/1.9 9.7/8.1 3.3/2.8 2.1/1.7 3.7/2.7 7.8/6.2 7.1/6.8 14.6/12.0

tel/tel tel/tel int/int int/int cen/cen tel/tel cen/int int/int cen/tel

+ + – – – – – – –

– – + + – – – + +

5q: 177.5–180.7 7p: 45.7–48.0 8p: 0.5–1.7 8p: 9.7–11.9 10q: 43.3–46.7 10q: 46.9–48.6 10q: 48.7–51.7 10q: 51.9–59.6

11: 3.2–6.5 16: 3.1–4.1 37: 33.1–33.9 25: 28.9–31.2 28: 3.0–7.2 4: 37.9–38.3 28: 3.0–4.9 26: 34.5–40.6

3.2/3.3 2.3/1.0 1.2/0.8 2.2/2.3 3.4/4.2 1.7/0.4 3.0/1.9 7.7/5.1

tel/cen cen/int tel/tel int/int cen/cen int/int int/cen int/int

– – – – – – – –

+ – – – + – – +

10q: 89.2–91.0 11p: 0.8–3.3 14q: 20.2–22.0 15q: 22.8–32.6 20q: 23.6–25.6 22q: 17.5–18.7

26: 40.6–42.0 18: 48.2–50.2 15: 20.2–21.6 3: 34.5–42.2 23: 3.0–4.8 27: 47.8–48.8

1.8/1.4 2.5/2.0 1.8/1.4 9.8/7.7 2.0/1.8 1.2/1.0

int/tel int/int cen/int int/int cen/tel cen/tel

– – – – – –

– – – + – –

HSA

detected by painting detected by painting inversion in human inversion in human small size/distal localization small size/distal localization not separated by centromere in carnivores inversion in human revealed in recent painting experiments [Nie et al., 2012] inversion in human inversion in human small size/distal localization inversion in human inversion in human inversion in human inversion in human this segment HSA1q is present as part of HSA10q (CFA4/31 and CFA 26?) inversion in human inversion in human small size/distal localization inversion in human small size/distal localization small size/distal localization

Only in this table we used the dog chromosome nomenclature of Breen et al. [1999]. a There is a segment of CFA6 on HSA2 according to painting [Yang et al., 2000], but it is not confirmed by Ensemble or Evolutionary Highway data. b Segment is located near the centromere (cen), interstitially (int), or near the telomere (tel). + = Segment is present; – = segment is absent.

c Inversion in human means that the corresponding dog chromosome was split into several small pieces in human (likely due to inversions) and thus was hard to reveal by chromosome painting. Small size/distal localization: those segments are present in Ensemble but not in painting or comparative mapping data. Those fragments are often just slightly bigger than 1 Mb and are located near the centromere or telomere. We hesitate to put them on the map until further evidence is available.

ever, some of the chromosomal differences between felid species were noticed in G-banding comparisons and later received confirmation by molecular methods, such as chromosome F2 in the clouded leopard [Wurster-Hill and Gray, 1973; Wurster-Hill and Centerwall, 1982; Buckley-Beason et al., 2006]. In future studies it will be very interesting to compare genomes of different felid species to reveal whether the level of conservation is also present at the gene order level. Also, it will be important to incorporate species from the ocelot lineage into new chromosome painting studies.

resentatives of Procyonidae, both from the subfamily Procyoninae (Procyon lotor and Bassariscus astutus), have been studied by chromosome painting [Nash et al., 2008; Perelman et al., 2008]. Their karyotypes differ only by 1 inversion, revealed by dog painting probes (ACK13) [Nie et al., 2012], but otherwise they represent well the ancestral musteloid karyotype (AMK). The published banded karyotype of the basal procyonid species kinkajou (Potos flavus) appears to be identical to the karyotype of P. lotor, and this result is expected to be confirmed by chromosome painting [Dutrillaux and Couturier, 1983; Stanyon, 2006]. However, it will be interesting to incorporate representatives of another branch (coati (Nasua, Nasuella) + olingo (Bassaricyon)) into chromosome painting or fine mapping to see if there are cytogenetic signatures for their separate phylogenetic position, as re-

Procyonidae Procyonidae is another carnivore family characterized by a uniform diploid number of 2n = 38 (at least for the species that have been karyotyped so far). Only 2 rep182



Fig. 3. Chromosome painting of female

Pardine genet (Genetta pardina, 2n = 52) with stone marten (Martes foina) painting probes (bars on the right of each chromosome). H indicates the large heterochromatin block on the heteromorphic chromosome pair.

cently discovered by molecular studies [Koepfli et al., 2007]. Interestingly, according to G-banding comparison, the karyotype of Bassaricyon ‘has only three felid homologs that are not considered to be modified’ [Wurster-Hill and Gray, 1975]. Viverridae The family Viverridae includes 16 genera and 36 species [Wilson and Reeder, 2005], but chromosome painting data are very scarce. So far only 4 species from Viverridae (table 1) have been studied by chromosome painting, and there appears to be no common chromosomal signature to join Viverridae with either Felidae/Prionodontidae or Hyaenidae/Herpestidae/Eupleridae. One common fusion joins Paguma larvata and Paradoxurus hermaphroditus (ACK8+20) and could be signature association for the subfamily Paradoxurinae [Nie et al., 2012]. One more species from another subfamily, Viverrinae, has recently been studied by chromosome painting, Viverricula indica [Nie et al., 2012]. Here we present results on another representative from Viverrinae, the Pardine genet (G. pardina, 2n = 52). We studied a female genet from Tchernogolovka biological station of A.N. Severtsov Institute of Ecology and Evolution, RAS, Russia, using stone marten (M. foina) chromosome painting probes described earlier [Nie et al., 2002] (fig. 3). In total, stone Comparative Chromosome Painting in Carnivora and Pholidota

marten probes reveal 40 conserved autosomal segments in the genome of the Pardine genet. In Carnivora species with conserved karyotypes stone marten probes reveal 18–21 conserved segments, in skunks with rearranged karyotypes 30–36, in distant pangolin 35 conserved segments (table 1). Thus chromosome painting demonstrates that G. pardina, and possibly all members of this genus have highly rearranged karyotypes. This is the first report of feliform taxa with karyotypes not showing a high level of conservation of ACK chromosomes. Notably, Wurster-Hill and Gray [1975], based on G-banding comparative data, pointed out that Genetta tigrina has few of ‘shared’ Carnivora chromosomes and thus may represent yet another taxon with rearranged karyotype among otherwise conserved carnivores. Further research mapping dog painting probes will be required to precisely establish the ancestral chromosome exchange events that led to formation of the G. pardina karyotype. For example, ACK7 (FCA A3) is present as 2 segments in genet, but based on banding comparison, the breakpoint is different from other feliform species with fissioned ACK7: Crocuta crocuta, P. larvata, Helogale parvula and Herpestes javanica. We did not reveal any common rearrangement for G. pardina and V. indica. In this regard it would be important to study other species from the subfamily, such as those from genera Poiana and Viverra.


183

Other Feliformes Based on 2 species studied so far, there are 5 common fusions characteristic for Herpestidae (ACK11+9, 17+8, 15+7q, 20+10, 7p+1p) [Nash et al., 2008; Nie et al., 2012]. This large family requires more species to be studied by chromosome painting. For example, the genera Atilax, Bdeogale and Galidia are of special interest because comparative banding analyses revealed that their karyotypes have few ‘shared’ Carnivora chromosomes [Wurster-Hill and Gray, 1975]. A common inversion of ACK9 joins Eupleridae (Fossa fossana) and Herpestidae together in accordance with modern molecular phylogenies [Gaubert and Cordeiro-Estrela, 2006; Eizirik et al., 2010]. Four species of Hyaenidae have the same diploid number and are expected to have similar karyotypes based on G-banding data. The karyotype of the spotted hyena (C. crocuta) evolved from the ancestral carnivore karyotype by 4 fusions (ACK8+11, 7p+10, 3+7q, 6p+20), 1 fission (ACK6) and 3 inversions (ACK1q, 12, 16) [Perelman et al., 2005; Nie et al., 2012]. Some important species that may help to define chromosomal evolution within the Feliformia branch are still lacking chromosome painting data: those are from the sister family of cats, Prionodontidae (Prionodon linsang, P. pardicolor) and the basal branch of Feliformes, Nandiniidae (N. binotata). Mephitidae The skunk family is one of the 3 caniform families with an increased rate of chromosomal rearrangements and includes 4 genera (Mephitis, Conepatus, Spilogale and Mydaus). Representatives of the first 3 genera have been studied by chromosome painting [Perelman et al., 2008]. Twelve fissions and 5 fusions of ancestral Carnivora chromosomes are required to form the karyotype of the skunk ancestor. A further fusion leads to Mephitis karyotypes and 5 and 2 fusions are required to form Spilogale gracilis and Conepatus leuconotus karyotypes, respectively. One common fusion was revealed for Conepatus and Spilogale (ACK14p+4); however, it contradicts the association of Mephitis/Spilogale indicated by molecular phylogeny and requires additional research as the small insertion of ACK14 (FCA D4) on ACK4 may represent repetitive sequences, not revealed by C-banding [Perelman et al., 2008; Eizirik et al., 2010]. Stink badger (genus Mydaus) as basal branch of Mephitidae holds special interest for chromosome painting studies. Also it would be interesting to study South American representatives of the genus Conepatus (C. chinga, C. humboldtii, C. semistriatus).

184


Ursidae Karyotypes of Ursidae were also formed by great fissioning of ancestral elements, namely 16 fissions and 1 inversion (ACK19) [Nie et al., 2012]. Of 8 extant ursid species 2 karyotypes stand out: giant panda (A. melanoleuca, 2n = 42) and spectacled bear (Tremarctos ornatus, 2n = 52), the rest have a high-chromosome, uniform diploid number of 2n = 74. The giant panda karyotype is formed by further 16 fusions and 1 inversion (ACK3). Ursinae and Tremarctinae are joined by 2 fissions (ACK3, 6) and 2 fusions (ACK17+20, 1p+16p) and this forms karyotypes of ursins [Nash et al., 1998, 2001; Tian et al., 2004; Yang and Graphodatsky, 2004]. The karyotype of spectacled bear is further formed by 1 inversion and 11 more fusions that are different from those found in giant panda [Yang and Graphodatsky, 2004]. Musteloidea Mustelidae is one of the families that are best studied by chromosome painting among carnivores (13 species; table 1). It was already known for its karyotype conservation well before chromosome painting was established [Wurster-Hill and Centerwall, 1982; Graphodatsky et al., 1989]. Detailed G-banding comparative analyses even allowed deduction of the ancestral musteloid karyotype; it was suggested that it could be Martes-like [Graphodatsky et al., 1989], which was later confirmed by chromosome painting [Graphodatsky et al., 2002]. Only recent chromosome painting studies with dog paints showed that seemingly conserved musteloid karyotypes do have differences, i.e. inversions. For example, the M. foina karyotype has all the same syntenic groups as the ancestral carnivore karyotype, but differs by 6 inversions revealed by dog painting probes [Nie et al., 2012]. Large heterochromatic blocks, present as whole chromosome arms, contribute to further differences between conserved musteloid karyotypes [Graphodatsky et al., 2002]. In a recent molecular phylogeny of Mustelidae, Koepfli et al. [2008] provided support for 8 subfamilies. Lutrinae is one of the subfamilies with no painting data and only comparative G-banding data available and remarkable uniformity of the chromosome complement: all studied species have 2n = 38. The karyotype of Lutra lutra is very similar to the ancestral musteloid karyotype based on Gbanding analyses. However, it would be interesting to study Lutrinae with dog paint probes. In Mustelinae, the American mink (M. vision) has a basal position and, unusual for Mustelidae, a low diploid chromosome number of 2n = 30 caused by 6 fusions of ancestral chromosomes, accompanied by 3 inversions Perelman /Beklemisheva /Yudkin / Petrina /Rozhnov /Nie /Graphodatsky

(ACK3, 2, 6). Fission of ACK2 is characteristic for the rest of Mustela species and further evolution involves 3 fusions (ACK8+18, 2q+12, 9+11) for M. eversmanni and M. putorius (reverse fission of ACK2q–12) and 1 fusion (ACK11+18) leading to M. altaica. In Galictinae, zorilla (Ictonyx striatus, painting data) and marbled polecat (Vormela peregusna, banding data) have, except heterochromatin additions that are particularly large in zorilla, an unchanged ancestral musteloid karyotype [Graphodatsky et al., 2002]. It would be interesting to incorporate karyotypes of the third genus of this subfamily (neotropical Galictis) into chromosome painting, as it was predicted by G-banding in G. vittata that it has a unique karyotype despite the same diploid number of 2n = 38 [Wurster-Hill and Centerwall, 1982]. In Helictidinae, Melogale moschata also has retained the ancestral musteloid karyotype [Nie et al., 2002]. In Martinae, all Martes species with 2n = 38 are likely to have retained the ancestral musteloid karyotype, as has been confirmed for M. foina (with 6 inversions, painting), M. melampus (painting), M. zibellina, and M. martes (banding). An additional further fission of ACK5 is characteristic for karyotypes of M. flavigula (2n = 40) and Gulo gulo (2n = 42, and also fission of ACK13) [Graphodatsky et al., 2002; Nie et al., 2002]. The tyara (Eira barbara, 2n = 38) chromosomes were also studied by Gbanding and were shown to have some differences with other mustelids (inversions?) [Wurster-Hill and Centerwall, 1982]. The true Melinae species M. meles (2n = 44) has a karyotype that is formed by fission of 3 ‘problematic’ ancestral chromosomes (ACK1, 7, 8+15) [Graphodatsky et al., 2002]. It would be interesting to know whether these 3 fissions are characteristic for the whole subfamily by studying the hog badger (Arctonyx collaris) karyotype. Basal species of Mustelidae, the American badger (Taxidea taxus, 2n = 32) and honey badger (Mellivora capensis, 2n = 40), were shown to have unique karyotypes based on G-banding and are particularly interesting for future chromosome painting studies [Wurster-Hill, 1973; Wurster-Hill and Centerwall, 1982]. The karyotype of the red panda (Ailurus fulgens, 2n = 36, Ailuridae) differs just slightly from the ancestral carnivore karyotype, by 1 fusion (ACK5+20) and 4 inversions revealed by dog painting probes (ACK1, 2, 6, 8+15) [Nie et al., 2002; Tian et al., 2002]. Pinnipedia karyotypes are well-known for their remarkable chromosomal conservation, described by Arnason [1977]. However, just 1 species has been studied by chromosome painting, Phoca vitulina (2n = 32) [Fronicke et al., 1997]. Three fusions of

ancestral chromosomes (ACK3+16, 6+20, 11+17) form its karyotype. The other 2 families, Otariidae and Odobenidae, have not been studied by chromosome painting. It would be interesting to check if there are any inversions in pinniped karyotypes; these could be revealed by hybridizing dog painting probes.



Chromosome Evolution in Canidae

The Canidae family comprises 13 genera and 35 species and has been extensively studied by chromosome painting [Wilson and Reeder, 2005]. Domestic dog, C. familiaris, a member of the family, has been notably difficult to characterize on the chromosomal level because of its karyotype, consisting of 38 acrocentric autosomes. There were early attempts to establish an international standard karyotype for dog, but it was virtually impossible to reliably identify all chromosomes based on the banding alone, especially for the smallest acrocentric chromosomes [Breen, 2008]. Only in 1999, when a combination of painting probes from both dog (2n = 78) and fox (2n = 34+B, easily indefinable chromosomes) was used in cross-species chromosome painting studies (human-dog-fox), the puzzle of dog chromosome identification was solved. Then this nomenclature was linked to radiation-hybrid (RH) mapping and linkage mapping data of the domestic dog [Yang et al., 1999; Sargan et al., 2000]. At the same time, independent reciprocal dog-human painting, later combined with cosmid mapping, also produced molecularly-linked dog chromosome nomenclature, which is currently used in the genomic assembly of the domestic dog [Breen et al., 2001; Lindblad-Toh et al., 2005]. The conversion tables between 2 painting-produced dog chromosomal nomenclatures have been previously described [Graphodatsky et al., 2000a; Breen, 2008]. In this paper we use the nomenclature by Yang et al. [1999]. To date, dog chromosome painting probes have been applied to the chromosomes of 10 canid species, uncovering an incredible picture of chromosomal reshuffling during the evolution of this family (table 1, fig. 4). Although we have learned a great deal from these studies, the story is still far from being complete. Extant canid speciation occurred in the last 10 million years [LindbladToh et al., 2005]. Such recent speciation has created additional difficulties for constructing phylogenies, regardless of the characters being used (sequence, morphology, cytogenetics), and was accompanied by numerous homoplasies. Therefore, phylogenetically there is still no consensus 185

Fig. 4. The state of chromosomal studies in Canidae. The branching of the phylogenetic tree is shown according to LindbladToh et al. [2005] with 2 changes for more parsimonious arrangement based on cytogenetic data (V. corsac is sister branch with A. lagopus/V. velox and Otocyon megalotis is sister branch to Nyctereutes, rather than separate). Diploid chromosome numbers are from http://www.bionet.nsc.ru/labs/ chromosomes/ and Sillero-Zubiri et al. [2004]. Dog chromosome nomenclature follows Yang et al. [1999]. Correspondence with Breen et al. [1999] nomenclature is provided in Breen [2008] and Graphodatsky et al. [2000a]. Recurrent rearrangement is shown in blue. In grey are species with no painting data. Black lines: species was studied by chromosome painting; dotted lines: species was studied by BAC mapping only. FU: fusion of ancestral elements, FI: fission of ancestral elements, BP: breakpoint, Inv: inversion. Arrowheads under chromosome numbers indicate direction of the ancestral segment based on BAC mapping data [Duke Becker et al., 2011]. Painting data are from papers listed in table 1.

on the branching order of species within the canid family. In some aspects all phylogenies agree: there are 3 main groupings: dog-like canids (Canis, Cuon, Lycaon), red foxlike canids (Vulpes, Fennecus, Alopex), South American canids (Pseudoalopex, Lycalopex, Atelocynus, Cerdocyon, Chrysocyon, Speothos), with the most basal branch of North American foxes (Urocyon) [Zrzavy and Ricankova, 2004; Bardeleben et al., 2005; Lindblad-Toh et al., 2005]. There is still a great level of uncertainty about the exact placement of Nyctereutes, Otocyon, Speothos, Chrysocyon, branching within the Holoarctic fox clade, and branching of Canis-Cuon-Lycaon. It is possible that fine mapping across the whole family might provide cytogenetic evidence for species grouping, although it is unlikely to be helpful in case of karyotypically uniform dog-like canids, which will require molecular phylogenies with better species sampling and based on fast evolving sequences. Chromosome painting has revealed interesting patterns of chromosome rearrangements in several branches. All dog-like canids (Canis, Cuon, Lycaon) possess karyotypes with 2n = 78, widely identical to that of the 186


domestic dog. Painting of the full set of dog probes onto chromosomes of Cuon alpinus confirmed that both species have indeed the same chromosomal set without interchromosomal rearrangements [Graphodatsky et al., 2008]. It is highly likely that the rest of the dog-like species with 2n = 78 shares exactly the same set of chromosomes, as expected from G-banding comparative data [Wurster-Hill and Centerwall, 1982; Wayne et al., 1987]. In the red fox-like canids chromosome painting shows that ancestral canid elements were fused into totally different combinations in 3 of the 4 studied species. Arctic fox (Alopex lagopus) has 7 unique fusions, corsac (Vulpes corsac) has 14, and red fox has 16 fusions. Kit fox (Vulpes macrotis) and Arctic fox appear to have identical karyotypes based on G-banding comparisons [Wayne et al., 1987]. Recently, canid chromosome evolution has been studied at a higher level of resolution. Dog BACs with 10-Mb intervals were localized on chromosomes of 3 different canid species (Nyctereutes procyonoides viverrinus, Urocyon cinereoargenteus, Vulpes vulpes). Selected sets of BACs from chromosomes with common canid breakPerelman /Beklemisheva /Yudkin / Petrina /Rozhnov /Nie /Graphodatsky

Table 5. Grey fox (Urocyon cinereoargenteus, UCI) chromosome nomenclature

Graphodatsky et al. [2008]

Duke Becker et al. [2011]





1prox 1dist 2 3 4 5 6 7 8 9 10

12 15dist 8 3 7 1 2 4 5 6 14

11 12 13 14 15 16 17 18 19 20 21

13 17 23 9 11 22 10 31 20 21 27

22 23 24 25 26 27 28 29 30 31 32

24 19 18 16 26 29 30 32 25 15prox 28

points were applied to 8 more species [Duke Becker et al., 2011]. A conversion table for grey fox nomenclature [Graphodatsky et al., 2008] is shown in table 5. BAC mapping also revealed breakpoints that mark intrachromosomal rearrangements and may serve as cytogenetic signatures (fig. 4). The formation of the Cerdocyon thous karyotype is marked by the fusion of 3 dog chromosomes CFA36+37+38 [Nash et al., 2001]. Additionally, an inversion in CFA9 and possibly other rearrangements (because according to BAC-mapping it was split into 3 chromosomes) are characteristic for this karyotype. Different breakpoints of CFA9 homologs are present in Lycalopex vetulus and yet another one in both raccoon dogs (Nyctereutes procyonoides procyonoides and viverrinus). Several breakpoints have been identified in U. cinereoargenteus. Urocyon is found at the basal branch on the canid tree. Until further evidence is made available, the most parsimonious way is to assume that grey fox rearrangements depicted on the tree are specific for this species and not for the canid ancestor. Selected BACs were mapped onto 3 South American species in which no chromosome painting studies had been performed: Chrysocyon brachyurus, L. vetulus and Speothos venaticus [Duke Becker et al., 2011]. The following rearrangements, CFA32–19a, 34–13a, 19a+19b, 13a+13b, 1a+1b, are indeed common for dog-like canids and South American canids, cytogenetically confirming this grouping. Recurrent Fusions of Ancestral Chromosomes in Canidae Two fusions of ancestral elements appear to be homoplasic and have not been resolved by chromosome painting. Fusion of CFA18a+38 occurred both in distantly reComparative Chromosome Painting in Carnivora and Pholidota

lated red fox and Urocyon branches. However, BAC mapping data show that these fusions are likely independent events because 18a and 38 are fused in different directions in these species (fig. 4). This is also true for the complex ancestral chromosome composed of CFA35+36 which is present in V. vulpes and raccoon dogs. Corresponding dog chromosomes are fused by centromeres in fox and by telomeres in raccoon dogs and could have occurred independently. In 2 other instances of assumed homoplasic fusions (CFA18a/38/18b+36 and 19a/32+8) that occurred both in arctic fox (A. lagopus) and corsac (V. corsac), we believe that the likely arrangement for the phylogeny is the sister branching of corsac with arctic fox rather than with red fox, as it is currently suggested by phylogenetic studies. This requires revision of the phylogenetic relationships of these species with more samples and molecular markers [Lindblad-Toh et al., 2005]. Duke Becker et al. [2011] also revealed one more instance of a recurrent rearrangement, CFA31+33, that occurs in the 3 branches Chrysocyon/Speothos, Nyctereutes/ Otocyon and in Urocyon. These authors speculate that it is possible that this fusion was ancestral and was lost in more derived species. It will be very interesting to determine if ancestral segments in this fusion have the same orientation. Canidae Ancestral Karyotype We attempted to refine the structure of the Canidae ancestral karyotype (CAK) (2n = 82) by deducing the centromeric position of the ancestral elements and the orientation of corresponding dog chromosome segments based on the most recent BAC-mapping data of 3 canid Cytogenet Genome Res 2012;137:174–193

187

#

#

$

$

#

#

ancestral elements relative to the centromere in CAK is shown. Empty circles indicate deduced ancestral centromeres, small filled circles centromeres in extant species. Arrows on the left indicate direction of corresponding dog chromosomes. Arrows on the right indicate direction of the ancestral segment in extant species relative to dog. The horizontal bars indicate that this fragment in extant species is fused with another one and not in the centromere. Question mark: it is hard to deduce ancestral direction of the segment based on the currently available data. Actual chromosome size is not reflected in the figure. Dog chromosome

nomenclature follows Yang et al. [1999]. * These chromosomes have breakpoints and have undergone some inversions in U. cinereoargenteus. ** In Otocyon megalotis and N. pr. procyonoides the centromere is repositioned on this chromosome relative to the deduced ancestral state. *** In dog the centromere is repositioned to another end of the ancestral segment. **** In N. pr. procyonoides the centromere is repositioned on this chromosome relative to the deduced ancestral state. # In the branches of true foxes and raccoon dogs the centromere is repositioned to another end of the ancestral segment. $ In the branch of U. cinereoargenteus the centromere is repositioned to another end of the ancestral segment.

species (U. cinereoargenteus, N. procyonoides, V. vulpes) [Graphodatsky et al., 2008; Duke Becker et al., 2011] (fig. 5). In our Canidae ancestral karyotype, 22 ancestral chromosomes would look exactly like extant dog chromosomes. In 5 instances the centromeres on the ancestral chromosomes would be on the opposite end relative to dog. In 8 instances there is not sufficient data to deduce the ancestral position of the centromeres. The remaining chromosomes represent segments of extant dog chromosomes. Our data provides strong evidence that centromere repositioning, recently described in detail in primates, occurred quite often during canid chromosome evolution [Rocchi et al., 2012]. Striking examples of how centromeres can change their position so many times in just 10 million years of evolution are shown in figure 6.

We anticipate that as additional canid genomes are sequenced and anchored to chromosome locations, such information will allow for the detailed reconstruction of the Canidae ancestral karyotype with the orientation of ancestral segments further identified. B chromosomes, supernumerary highly polymorphic elements of the chromosome complement, occur among Carnivora only in canids [Switonski et al., 2009]. The composition, role and origin of B chromosomes are slowly getting uncovered [Camacho et al., 2000; Vujosevic and Blagojevic, 2004; Szczerbal et al., 2005]. Chromosome painting using microdissected probes of B chromosomes has shed light on their basic structure [Trifonov et al., 2002]. Moreover, recently it was discovered that cKIT oncogene sequences are localized on the B chromosomes

Fig. 5. Canidae ancestral karyotype (CAK). The orientation of the

188



Fig. 6. Centromere repositioning in Canidae. Segment orientation is based on BAC mapping data of Duke Becker et al. [2011]. Full circle: Centromere of acrocentric chromosome; half-circle: chromosome has another arm; horizontal bar: syntenic segment does not end at telomere. VVU: Vulpes vulpes; UCI: Urocyon cinereoargenteus; NPRp: Nyctereutes procyonoides procyonoides. Dog chromosome nomenclature follows Yang et al. [1999], UCI nomenclature follows Graphodatsky et al. [2008].

of red fox and raccoon dogs [Graphodatsky et al., 2005; Yudkin et al., 2007], providing new insights into the role of B chromosomes in genome function and evolution. Furthermore, 25 BAC clones (some carrying known genes cKIT, MDN1, RET, LRIG1) have been described with duplicated localizations on both B chromosomes and autosomes in canids (N. procyonoides, V. vulpes) [Duke Becker et al., 2011]. Interesting observations were made about so-called CFA-vacant regions on the chromosomes of different species not covered by any BACs, but also were too large to be represented by centromeric sequences [Duke Becker et al., 2011]. Most of the canid species with contrasting diploid numbers are now studied either by chromosome painting or by BAC mapping. But several species still remain unstudied cytogenetically and may hold surprising rearrangements: fox-like canids Vulpes rueppellii, V. chama and V. cana. BAC mapping may also reveal interesting intrachromosomal rearrangements in species from other genera with the same diploid number and seemingly identical karyotypes: Canis, Lycaon, Lycalopex, Atelocynus, and Urocyon.

Use of Carnivora Painting Probes to Reveal Chromosome Evolution in Other Orders

Carnivora painting probes were also used in several studies to reveal genome-wide chromosome rearrangements in other mammalian orders: dog probes in pig (Sus scrofa, Cetartiodactyla), stone marten probes in Javan Comparative Chromosome Painting in Carnivora and Pholidota

pangolin (Manis javanica, Pholidota) and in Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis, Cetartiodactyla) [Biltueva et al., 2004; Yang et al., 2006; Nie et al., 2012]. These studies showed how informative cross-order painting analyses can be. However, it was made evident that with an increase of phylogenetic distance between species, getting strong hybridization signals became increasingly difficult. It also became apparent that getting strong hybridization signals was easier using chromosome paints derived from a species with conserved karyotype (e.g. the stone marten), than from species with a highly rearranged karyotype like the dog. Pholidota Pholidota is a remarkable group of mammals with striking adaptations, but it remains largely unstudied from the genetic point of view. Recently it was shown by molecular phylogenies to be sister group of Carnivora [Murphy et al., 2001a]. As yet there is no comprehensive molecular phylogeny described for this group; however, there are morphological phylogenetic studies [Gaudin et al., 2009] (fig. 7). There are a total of 8 taxonomically described species of extant pangolins: 4 of African origin (Manis tricuspis, M. tetradactyla, M. gigantea, M. temminckii) and 4 from Asia (M. javanica, M. culionensis, M. pentadactyla, M. crassicaudata) [Gaubert and Antunes, 2005; Wilson and Reeder, 2005; Gaudin et al., 2009]. The genome of M. pentadactyla is being sequenced within the frame of 29 mammalian genome projects (http://genome. wustl.edu/genomes/view/manis_pentadactyla). Complete mitochondrial genomes have been published for 2 pangolin species [Arnason et al., 2008; Qin et al., 2012]. Also, there is a valuable resource of characterized BAC library of M. pentadactyla with numerous clones having been mapped onto chromosomes by FISH [Che et al., 2008]. To the best of our knowledge, the diploid chromosome number for only 3 species of pangolins has been reported: M. javanica (2n = 38), M. pentadactyla (2n = 40, with possible polymorphism 2n = 38–40) and M. crassicaudata (2n = 36) [detailed review and references in Nie et al., 2009]. Chromosomes of M. pentadactyla also display polymorphisms in amounts of heterochromatin and differences in locations of nucleolar organizing regions. Interestingly, one of the nucleolar organizer regions in M. javanica is located on the Y chromosome [Nie et al., 2009]. Chromosome complements of M. pentadactyla and M. javanica differ by 3 centric fissions and 4 centric fusions (with 1 centromere repositioning event) based on chromosome painting using M. pentadactyla painting probes Cytogenet Genome Res 2012;137:174–193

189

[Nie et al., 2009]. A full set of human painting probes was applied to chromosomes of M. javanica and selected human probes to chromosomes of M. pentadactyla [Yang et al., 2006; Nie et al., 2009]. Seven associations of human chromosomes common for the 2 species of pangolin studied by chromosome painting and potentially representing Pholidota cytogenetic signatures are: HSA2q/10q, HSA1q/11, 2p/5, 4p+q/20, 5/13, 6/19p, and 8q/10p [Nie et al., 2009]. Genomes of pangolins contain common mammalian associations: HSA3/21, 4/8, 7/16, 12/22, 14/15 and 16/19. However, chromosome painting revealed 16 other associations of human chromosomes, none of which were common for both pangolins and carnivores or for that matter for pangolins and any other mammalian order. Up to now, only Asian pangolins have been studied by chromosome painting. According to morphological data, the Asian species are more recently derived and likely diverged later than African pangolins [Gaudin and Wible, 1999]. Further studies of more basal pangolin species could potentially reveal ancestral signatures common for pangolins and carnivores.

Other Resources to Complement Chromosome Painting Data

The domestic dog genome is an excellent example of how developing essential genetic tools can advance studies in multiple species. Chromosome painting data has provided a valuable link in assignment between human and dog chromosomes, and allowed unambiguous identification of individual chromosomes in the complex karyotype of the dog [Breen et al., 1999; Yang et al., 1999]. These results have fueled numerous studies for development of more advanced molecular cytogenetics tools for identification of dog chromosomes coupled with the development of rich BAC libraries, dense RH maps, detailed FISH maps, and, most importantly, with high-coverage genomic data [reviewed in Breen, 2008]. This combination of techniques allowed the improvement of the domestic dog genome assembly, and this high-quality genomic resource allowed further expansion to benefit and accomplish comparative studies with other canids [Lindblad-Toh et al., 2005]. With the cost of whole-genome de novo sequencing rapidly coming down, one can expect more Carnivora genomes to be sequenced in the near future. Some of them are already scheduled to be sequenced in frame of an international project to sequence the genomes of 190


Fig. 7. The state of cytogenetic studies in Pholidota. The species

arrangement on the tree is from morphology-based phylogenetic studies of Gaudin et al. [2009] and Gaudin and Wible [1999], modified with the dashed line for Manis culionensis as closely related to M. javanica [Gaubert and Antunes, 2005]. Date of divergence of Pholidota and Carnivora is from calibrated mitogenomic phylogeny [Arnason et al., 2008]. The species distribution and names are from Wilson and Reeder [2005]. Diploid chromosome number references are provided in detail in Nie et al. [2009]. Chromosome rearrangement information is based on chromosome painting data [Yang et al., 2006; Nie et al., 2009]. Grey lines represent species not studied by chromosome painting.

10,000 vertebrate species, Genome10K (http://www.genome10k.org/). For example red fox V. vulpes is on the list to be sequenced shortly. Full-genome sequence of the red fox will allow detailed comparison of the dog and fox genomes and will shed light onto the molecular structure of evolutionary breakpoint regions. Among upcoming or already sequenced genomes that will provide rich ground for comparison are felids (tiger, cheetah, lion, lynx, snow leopard), bears (polar bear, giant panda) and several other Carnivora species. Two poorly studied types of chromosome evolution also occurred in Carnivora. These involve B chromosomes and long stretches of heterochromatic blocks in some chromosomes. Heterochromatin blocks often appear as whole chromosomal arms in many carnivore species (e.g. striped polecat, skunks, mountain weasel, steppe polecat, arctic fox, etc.) [Graphodatsky and Radjabli, Perelman /Beklemisheva /Yudkin / Petrina /Rozhnov /Nie /Graphodatsky

1988]. Future whole-genome sequencing efforts may reveal sequences of those kinds of chromosomal elements that are clearly composed mostly of repeats. Unveiling repeat structures and sequences may identify other unique sequences that are currently overshadowed by repeats, providing more information about the origin and function of B chromosomes and heterochromatic blocks. Another highly anticipated result from the full-genome sequencing of the red fox is providing insight into the sequence composition of B chromosomes, supernumerary elements that often occur in only 1 family among Carnivores, the Canidae [Pienkowska-Schelling et al., 2008; Trifonov et al., 2010; Dementyeva et al., 2012; http:// www.bionet.nsc.ru/labs/chromosomes/]. We also hope that in the framework of the Genome10K project other species with B chromosomes will be sequenced. These data will provide the groundwork for comparison of B chromosome composition in different orders and will shed light on B chromosome origins and the convergent and seemingly random nature of their occurrence in the mammalian tree. Modern molecular cytogenetic techniques based on array and sequencing approaches, such as those recently developed for human, are also paving the way for comparative animal genetics. Soon these methods will be expanded for further study of genomes of many Carnivora

species and will permit more efficient studies of chromosome evolution. The domestic dog SNP chip is now widely used, and a feline (F. catus) SNP chip just became available (www.morrisanimalfoundation.org). Chromosome painting in Carnivora has laid down a solid foundation for describing major chromosome rearrangements and rates of chromosome exchanges, indicating signature associations for many taxa. Now we are coming to the exciting point in the comparative genomics story. In the near future whole-genome sequences for many Carnivora species will become available and hopefully chromosome assignment of scaffolds will not lag too far behind. This will provide rich resources to reveal the actual number of chromosomal rearrangements, especially intrachromosomal changes that occurred during evolution, that surpasses the resolution of chromosome painting and will provide insights into breakpoint region composition and into the origin and function of heterochromatin and B chromosomes. Acknowledgements This study was funded in part by programs MCB, RAS and SB RAS Programs and by research grants of Russian Fund for Basic Research. The authors thank 2 anonymous reviewers for helpful comments.

References Alekseyev MA, Pevzner PA: Breakpoint graphs and ancestral genome reconstructions. Genome Res 19:943–957 (2009). Arnason U: The relationship between the four principal pinniped karyotypes. Hereditas 87: 227–242 (1977). Arnason U, Adegoke JA, Gullberg A, Harley EH, Janke A, Kullberg M: Mitogenomic relationships of placental mammals and molecular estimates of their divergences. Gene 421:37– 51 (2008). Avise JC, Robinson TJ: Hemiplasy: a new term in the lexicon of phylogenetics. Syst Biol 57: 503–507 (2008). Bardeleben C, Moore RL, Wayne RK: A molecular phylogeny of the Canidae based on six nuclear loci. Mol Phylogenet Evol 37:815–831 (2005). Biltueva LS, Yang F, Vorobieva NV, Graphodatsky AS: Comparative map between the domestic pig and dog. Mamm Genome 15: 809–818 (2004). Breen M: Canine cytogenetics – from band to basepair. Cytogenet Genome Res 120: 50–60 (2008).


Breen M, Thomas R, Binns MM, Carter NP, Langford CF: Reciprocal chromosome painting reveals detailed regions of conserved synteny between the karyotypes of the domestic dog (Canis familiaris) and human. Genomics 61:145–155 (1999). Breen M, Jouquand S, Renier C, Mellersh CS, Hitte C, et al: Chromosome-specific singlelocus FISH probes allow anchorage of an 1800-marker integrated radiation-hybrid/ linkage map of the domestic dog genome to all chromosomes. Genome Res 11:1784–1795 (2001). Buckley-Beason VA, Johnson WE, Nash WG, Stanyon R, Menninger JC, et al: Molecular evidence for species-level distinctions in clouded leopards. Curr Biol 16: 2371–2376 (2006). Camacho JP, Sharbel TF, Beukeboom LW: Bchromosome evolution. Philos Trans R Soc Lond B Biol Sci 355:163–178 (2000). Cavagna P, Menotti A, Stanyon R: Genomic homology of the domestic ferret with cats and humans. Mamm Genome 11: 866–870 (2000).

Che J, Wang J, Su W, Ye J, Wang Y, et al: Construction, characterization and FISH mapping of a bacterial artificial chromosome library of Chinese pangolin (Manis pentadactyla). Cytogenet Genome Res 122: 55–60 (2008). Dementyeva P, Makunin A, Graphodatsky AS, Trifonov V: Genes on B chromosomes of vertebrates. Genet Mol Biol, in press (2012). Duke Becker SE, Thomas R, Trifonov VA, Wayne RK, Graphodatsky AS, Breen M: Anchoring the dog to its relatives reveals new evolutionary breakpoints across 11 species of the Canidae and provides new clues for the role of B chromosomes. Chromosome Res 19: 685– 708 (2011). Dutrillaux B, Couturier J: The ancestral karyotype of Carnivora: comparison with that of platyrrhine monkeys. Cytogenet Cell Genet 35:200–208 (1983). Eizirik E, Murphy WJ, Koepfli KP, Johnson WE, Dragoo JW, et al: Pattern and timing of diversification of the mammalian order Carnivora inferred from multiple nuclear gene sequences. Mol Phylogenet Evol 56: 49–63 (2010).


191

Ferguson-Smith MA: Genetic analysis by chromosome sorting and painting: Phylogenetic and diagnostic applications. Eur J Hum Genet 5:253–265 (1997). Froenicke L: Origins of primate chromosomes – as delineated by Zoo-FISH and alignments of human and mouse draft genome sequences. Cytogenet Genome Res 108:122–138 (2005). Frönicke L, Muller-Navia J, Romanakis K, Scherthan H: Chromosomal homeologies between human, harbor seal (Phoca vitulina) and the putative ancestral carnivore karyotype revealed by Zoo-FISH. Chromosoma 106:108–113 (1997). Gaubert P, Antunes A: Assessing the taxonomic status of the Palawan pangolin Manis culionensis (Pholidota) using discrete morphological characters. J Mammalogy 86: 1068– 1074 (2005). Gaubert P, Cordeiro-Estrela P: Phylogenetic systematics and tempo of evolution of the Viverrinae (Mammalia, Carnivora, Viverridae) within feliformians: implications for faunal exchanges between Asia and Africa. Mol Phylogenet Evol 41:266–278 (2006). Gaudin TJ, Wible JR: The entotympanic of pangolins and the phylogeny of the Pholidota. J Mamm Evol 6:39–65 (1999). Gaudin TJ, Emry RJ, Wible JR: The phylogeny of living and extinct pangolins (Mammalia, Pholidota) and associated taxa: a morphology based analysis. J Mammal Evol 16: 235– 305 (2009). Grafodatsky AS, Biltueva LS: Homology of Gstained chromosomes of mammals. Genetika 23:93–103 (1987). Graphodatsky AS, Radjabli SI: Chromosomes of argicultural and laboratory mammals. Atlas. (AN USSR, SB, Institute of Cytology and Genetics, Novosibirsk 1988). Graphodatsky A, Sharshov A, Ternovsky D: Comparative cytogenetics of Mustelidae. Zoologichesky Zhurnal 68:96–106 (1989). Graphodatsky AS, Yang F, O’Brien PC, Serdukova N, Milne BS, et al: A comparative chromosome map of the arctic fox, red fox and dog defined by chromosome painting and high resolution G-banding. Chromosome Res 8:253–263 (2000a). Graphodatsky AS, Yang F, Serdukova N, Perelman P, Zhdanova NS, Ferguson-Smith MA: Dog chromosome-specific paints reveal evolutionary inter- and intrachromosomal rearrangements in the American mink and human. Cytogenet Cell Genet 90: 275–278 (2000b). Graphodatsky AS, Yang F, O’Brien PC, Perelman P, Milne BS, et al: Phylogenetic implications of the 38 putative ancestral chromosome segments for four canid species. Cytogenet Cell Genet 92:243–247 (2001). Graphodatsky AS, Yang F, Perelman PL, O’Brien PC, Serdukova NA, et al: Comparative molecular cytogenetic studies in the order Carnivora: mapping chromosomal rearrangements onto the phylogenetic tree. Cytogenet Genome Res 96:137–145 (2002).

192

Graphodatsky AS, Kukekova AV, Yudkin DV, Trifonov VA, Vorobieva NV, et al: The protooncogene C-KIT maps to canid B-chromosomes. Chromosome Res 13:113–122 (2005). Graphodatsky AS, Perelman PL, Sokolovskaya NV, Beklemisheva VR, Serdukova NA, et al: Phylogenomics of the dog and fox family (Canidae, Carnivora) revealed by chromosome painting. Chromosome Res 16: 129– 143 (2008). Hameister H, Klett C, Bruch J, Dixkens C, Vogel W, Christensen K: Zoo-FISH analysis: the American mink (Mustela vison) closely resembles the cat karyotype. Chromosome Res 5:5–11 (1997). Johnson WE, Eizirik E, Pecon-Slattery J, Murphy WJ, Antunes A, et al: The late miocene radiation of modern Felidae: a genetic assessment. Science 311:73–77 (2006). Kemkemer C, Kohn M, Cooper DN, Froenicke L, Hogel J, et al: Gene synteny comparisons between different vertebrates provide new insights into breakage and fusion events during mammalian karyotype evolution. BMC Evol Biol 9:84 (2009). Koepfli KP, Gompper ME, Eizirik E, Ho CC, Linden L, et al: Phylogeny of the Procyonidae (Mammalia: Carnivora): molecules, morphology and the great American interchange. Mol Phylogenet Evol 43:1076–1095 (2007). Koepfli KP, Deere KA, Slater GJ, Begg C, Begg K, et al: Multigene phylogeny of the Mustelidae: resolving relationships, tempo and biogeographic history of a mammalian adaptive radiation. BMC Biol 6: 10 (2008). Larkin DM, Pape G, Donthu R, Auvil L, Welge M, Lewin HA: Breakpoint regions and homologous synteny blocks in chromosomes have different evolutionary histories. Genome Res 19:770–777 (2009). Lindblad-Toh K, Wade CM, Mikkelsen TS, Karlsson EK, Jaffe DB, et al: Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature 438: 803–819 (2005). Murphy WJ, Eizirik E, O’Brien SJ, Madsen O, Scally M, et al: Resolution of the early placental mammal radiation using Bayesian phylogenetics. Science 294:2348–2351 (2001a). Murphy WJ, Stanyon R, O’Brien SJ: Evolution of mammalian genome organization inferred from comparative gene mapping. Genome Biol 2:REVIEWS0005 (2001b). Nash WG, Wienberg J, Ferguson-Smith MA, Menninger JC, O’Brien SJ: Comparative genomics: tracking chromosome evolution in the family Ursidae using reciprocal chromosome painting. Cytogenet Cell Genet 83: 182–192 (1998). Nash WG, Menninger JC, Wienberg J, PadillaNash HM, O’Brien SJ: The pattern of phylogenomic evolution of the Canidae. Cytogenet Cell Genet 95:210–224 (2001). Nash WG, Menninger JC, Padilla-Nash HM, Stone G, Perelman PL, O’Brien SJ: The ancestral carnivore karyotype (2n=38) lives today in ringtails. J Hered 99:241–253 (2008).


Nie W, Wang J, O’Brien PC, Fu B, Ying T, et al: The genome phylogeny of domestic cat, red panda and five mustelid species revealed by comparative chromosome painting and G-banding. Chromosome Res 10: 209–222 (2002). Nie W, Wang J, Perelman P, Graphodatsky AS, Yang F: Comparative chromosome painting defines the karyotypic relationships among the domestic dog, Chinese raccoon dog and Japanese raccoon dog. Chromosome Res 11: 735–740 (2003). Nie W, Wang J, Su W, Wang Y, Yang F: Chromosomal rearrangements underlying karyotype differences between Chinese pangolin (Manis pentadactyla) and Malayan pangolin (Manis javanica) revealed by chromosome painting. Chromosome Res 17: 321–329 (2009). Nie W, Wang J, Su W, Wang D, Tanomtong A, et al: Chromosomal rearrangements and karyotype evolution in carnivores revealed by chromosome painting. Heredity 108: 17– 27 (2012). Perelman PL, Graphodatsky AS, Serdukova NA, Nie W, Alkalaeva EZ, et al: Karyotypic conservatism in the suborder Feliformia (order Carnivora). Cytogenet Genome Res 108: 348–354 (2005). Perelman PL, Graphodatsky AS, Dragoo JW, Serdyukova NA, Stone G, et al: Chromosome painting shows that skunks (Mephitidae, Carnivora) have highly rearranged karyotypes. Chromosome Res 16: 1215–1231 (2008). Pienkowska-Schelling A, Schelling C, Zawada M, Yang F, Bugno M, Ferguson-Smith M: Cytogenetic studies and karyotype nomenclature of three wild canid species: maned wolf (Chrysocyon brachyurus), bat-eared fox (Otocyon megalotis) and fennec fox (Fennecus zerda). Cytogenet Genome Res 121:25–34 (2008). Qin XM, Dou SR, Guan QX, Qin PS, She Y: Complete mitochondrial genome of the Manis pentadactyla (Pholidota, Manidae): comparison of M. pentadactyla and M. tetradactyla. Mitochondrial DNA 23:37–38 (2012). Rettenberger G, Klett C, Zechner U, Bruch J, Just W, et al: Zoo-FISH analysis: Cat and human karyotypes closely resemble the putative ancestral mammalian karyotype. Chromosome Res 3:479–486 (1995). Robinson TJ, Ruiz-Herrera A, Avise JC: Hemiplasy and homoplasy in the karyotypic phylogenies of mammals. Proc Natl Acad Sci USA 105:14477–14481 (2008). Rocchi M, Archidiacono N, Schempp W, Capozzi O, Stanyon R: Centromere repositioning in mammals. Heredity 108:59–67 (2012). Sargan DR, Yang F, Squire M, Milne BS, O’Brien PC, Ferguson-Smith MA: Use of flow-sorted canine chromosomes in the assignment of canine linkage, radiation hybrid, and syntenic groups to chromosomes: refinement and verification of the comparative chromosome map for dog and human. Genomics 69: 182–195 (2000).


Sillero-Zubiri C, Hoffmann M, Macdonald DW: Canids: Foxes, Wolves, Jackals and Dogs. Status Survey and Conservation Action Plan. 2004. (IUCN/SSC Canid Specialist Group, Gland, Switzerland and Cambridge, UK 2004). Stanyon R: Potos flavus, in O’Brien SJ, Menninger JC, Nash WG (eds): Atlas of Mammalian Chromosomes, p 482 (John Wiley and Sons, New York 2006). Switonski M, Szczerbal I, Nowacka-Woszuk J: Comparative genomics of 3 farm canids in relation to the dog. Cytogenet Genome Res 126:86–96 (2009). Szczerbal I, Kaczmarek M, Switonski M: Compound mosaicism, caused by B chromosome variability, in the Chinese raccoon dog (Nyctereutes procyonoides procyonoides). Folia Biol (Krakow) 53: 155–159 (2005). Thomas R, Breen M, Langford CF, Binns MM: Zoo-FISH analysis of dog chromosome 5:identification of conserved synteny with human and cat chromosomes. Cytogenet Cell Genet 87:4–10 (1999). Tian Y, Nie WH, Wang JH, Yang YF, Yang FT: Comparative chromosome painting shows the red panda (Ailurus fulgens) has a highly conserved karyotype. Yi Chuan Xue Bao 29: 124–127 (2002). Tian Y, Nie W, Wang J, Ferguson-Smith MA, Yang F: Chromosome evolution in bears: reconstructing phylogenetic relationships by cross-species chromosome painting. Chromosome Res 12:55–63 (2004).


Trifonov VA, Perelman PL, Kawada SI, Iwasa MA, Oda SI, Graphodatsky AS: Complex structure of B-chromosomes in two mammalian species: Apodemus peninsulae (Rodentia) and Nyctereutes procyonoides (Carnivora). Chromosome Res 10: 109–116 (2002). Trifonov VA, Dementyeva PV, Beklemisheva VR, Yudkin DV, Vorobieva NV, Graphodatsky AS: Supernumerary chromosomes, segmental duplications , and evolution. Russ J Genet 46:1094–1096 (2010). Vujosevic M, Blagojevic J: B chromosomes in populations of mammals. Cytogenet Genome Res 106:247–256 (2004). Wayne RK, Nash WG, O’Brien SJ: Chromosomal evolution of the Canidae. I. Species with high diploid numbers. Cytogenet Cell Genet 44: 123–133 (1987). Wienberg J, Stanyon R, Nash WG, O’Brien PC, Yang F, et al: Conservation of human vs. feline genome organization revealed by reciprocal chromosome painting. Cytogenet Cell Genet 77:211–217 (1997). Wilson DE, Reeder DM: Mammal Species of the World. A Taxonomic and Geographic Reference, ed 3 (Johns Hopkins University Press, Baltimore 2005). Wurster-Hill DH: Chromosomes of eight species from five families of Carnivora. J Mammal 54:753–760 (1973). Wurster-Hill DH, Centerwall WR: The interrelationships of chromosome banding patterns in canids, mustelids, hyena, and felids. Cytogenet Cell Genet 34:178–192 (1982). Wurster-Hill DH, Gray CW: Giemsa banding patterns in the chromosomes of twelve species of cats (Felidae). Cytogenet Cell Genet 12:388–397 (1973).

Wurster-Hill DH, Gray CW: The interrelationships of chromosome banding patterns in procyonids, viverrids, and felids. Cytogenet Cell Genet 15:306–331 (1975). Yang F, Graphodatsky AS: Integrated comparative genome maps and their implications for karyotype evolution of carnivores, in Schmid M, Nanda I (eds): Chromosomes Today, vol 14, pp 215–224 (Kluwer Academic Publishers, Dordrecht 2004). Yang F, O’Brien PC, Milne BS, Graphodatsky AS, Solanky N, et al: A complete comparative chromosome map for the dog, red fox, and human and its integration with canine genetic maps. Genomics 62: 189–202 (1999). Yang F, Graphodatsky AS, O’Brien PC, Colabella A, Solanky N, et al: Reciprocal chromosome painting illuminates the history of genome evolution of the domestic cat, dog and human. Chromosome Res 8:393–404 (2000). Yang F, Graphodatsky AS, Li T, Fu B, Dobigny G, et al: Comparative genome maps of the pangolin, hedgehog, sloth, anteater and human revealed by cross-species chromosome painting: Further insight into the ancestral karyotype and genome evolution of eutherian mammals. Chromosome Res 14: 283– 296 (2006). Yudkin DV, Trifonov VA, Kukekova AV, Vorobieva NV, Rubtsova NV, et al: Mapping of KIT adjacent sequences on canid autosomes and B chromosomes. Cytogenet Genome Res 116:100–103 (2007). Zrzavy J, Ricankova V: Phylogeny of recent Canidae (Mammalia, Carnivora): relative reliability and utility of morphological and molecular datasets. Zool Scr 33:311–333 (2004).


193



Comparative Molecular Cytogenetics in Cetartiodactyla J. Rubes a P. Musilova a O. Kopecna a S. Kubickova a H. Cernohorska a A.I. Kulemsina b a Veterinary Research Institute, Brno, Czech Republic; b Institute of Molecular and Cellular Biology, SB RAS, Novosibirsk , Russia

Key Words Antilocapridae ⴢ Bovidae ⴢ Camelidae ⴢ Cervidae ⴢ Cetacea ⴢ Chromosome evolution ⴢ Giraffidae ⴢ Hippopotamidae ⴢ Moschidae ⴢ Suidae

Abstract Cetartiodactyla comprises Artiodactyla (even-toed ungulates) and Cetacea (whales, dolphins and porpoises). Artiodactyla is a large taxon represented by about 200 living species ranked in 10 families. Cetacea are classified into 13 families with almost 80 species. Many publications concerning karyotypic relationships in Cetartiodactyla have been published in previous decades. Formerly, the karyotypes of closely related species were compared by chromosome banding. Introduction of molecular cytogenetic methods facilitated comparative mapping between species with highly rearranged karyotypes and distantly related species. Such information is a prerequisite for the understanding of karyotypic phylogeny and the reconstruction of the karyotypes of common ancestors. This study summarizes the data on chromosome evolution in Cetartiodactyla, mainly derived from molecular cytogenetic studies. Traditionally, phylogenetic relationships of most groups have been estimated using morphological data. However, the results of some molecular studies of mammalian phylogeny are discordant with traditional conceptions of phylogeny. Cetartiodactyls provide



several examples of incongruence between traditional morphological and molecular data. Such cases of conflict include the relationships of the major clades of artiodactyls, the relationships among the extant families of the suborder Ruminantia or the phylogeny of the family Bovidae. The most unexpected aspect of the molecular phylogeny was the recognition that Cetacea is a deeply nested member of Artiodactyla. The largest living order of terrestrial hoofed mammals is the even-toed hoofed mammals, or Artiodactyla. The artiodactyls are composed of over 190 living species including pigs, peccaries, hippos, camels, llamas, deer, pronghorns, giraffes, sheep, goats, cattle and antelopes. Cetacea is an order of wholly aquatic mammals, which include whales, dolphins and porpoises. Cetartiodactyla has become the generally accepted name for the clade containing both of these orders. Copyright © 2012 S. Karger AG, Basel

Cetartiodactyla Ancestral Karyotype

Comparative homology maps of cetartiodactyl species from different taxa obtained by cross-species chromosome painting with dromedary (Camelus dromedarius, CDR) and human (Homo sapiens, HSA) probes enabled the proposal of a Cetartiodactyla ancestral karyotype (CAK) [Balmus et al., 2007; Kulemzina et al., 2009]. Jiri Rubes Veterinary Research Institute Hudcova 70 CZ–621 00 Brno (Czech Republic) Tel. +420 5 3333 1401, E-Mail rubes @ vri.cz

The putative CAK (2n = 52) with human and dromedary homologies is shown in table 1. According to the porcine and bovine genome sequence database (www.ensembl.org), the smallest CAK25 was homologous not only to part of HSA8p, but it also included HSA4q, as suggested by Balmus et al. [2007]. However, this small part of HSA4q was not revealed by comparative chromosome painting. The fusion of HSA8 and 9 is characteristic for all cetartiodactyls except camelids. Recent studies suggested Camelidae as the earliest diverged group [Murphy et al., 2007 and references therein]. Therefore, the association of HSA4/8/9 could be a cytogenetic signature for all cetartiodactyls after diverged Camelidae (non-camelid cetartiodactyls). To convert the Boreoeutherian ancestral karyotype [Froenicke et al., 2006] into CAK, only 3 fissions and 1 fusion are necessary.

Suborder Tylopoda

Family Camelidae Extant camelids are classified into 2 tribes: the Old World camelids that contain the dromedary (C. dromedarius) and the bactrian (C. bactrianus); the New World camelids are represented by the vicuña (Lama vicugna), llama (L. glama), guanaco (L. guanicoe) and alpaca (L. pacos, LPA). Hsu and Benirschke [1967] established the diploid number of all camelid species as 74. Balmus et al. [2007] reported the first high resolution comparative mapping of this group using chromosome-specific paints of the dromedary and revealed that all camelid species have almost identical karyotypes. The camelid species have conserved karyotypes formed mainly by fissions of ancestral chromosomes. Therefore, camelids might lack the ancestral synteny homologous to HSA8/9 uniting other cetartiodactyl taxa. In total, 12 fissions, 1 inversion and 1 fusion are required to convert the CAK into the camel karyotype (fig. 1) [Kulemzina et al., 2009]. Dromedary painting probes were used to establish the genome-wide chromosomal homologies between Globicephala melas (Cetacea), Hippopotamus amphibius (Hippopotamidae), Moschus moschiferus (Moschidae) and 2 Giraffidae species Giraffa camelopardalis and Okapia johnstoni [Kulemzina et al., 2009]. The same set of dromedary probes were hybridized onto chromosomes of Capreolus pygargus [Dementyeva et al., 2010] and Tragulus javanicus [Kulemzina et al., 2011]. These comparative maps of major representative cetartiodactyl species, established with the same set of probes, allowed the reconstruction of the Ruminantia ancestral karyotype (RAK). Molecular Cytogenetics in Cetartiodactyla

Table 1. Putative CAK (2n = 52) with dromedary (CDR) and human (HSA) homologies [Kulemzina et al., 2009]

CAK

CDR

HSA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 X

23, 21, 9, 13 1, 17, 1 22, 3 27, 6 2 4 7 5 33, 10 34, 12 8 28, 15 11 29, 25 14 16 24, 30 9pqprox. 20 18 19 32 35 26 31 X

1 3, 21 19p, 5 15, 14 4pq 9 7pprox.qprox. 2qdist. 11 12pqprox., 22qdist. 6q 2pq 10q 8q 13 17 18 19q, 16q 6p 16p, 7 20 12qdist., 22qprox. 10p 4, 8pdist. 8pprox. X

prox. = Proximal part of chromosome arm; dist. = distal part of chromosome arm.

Building up the available human-camel Zoo-FISH data, Avila et al. [2012] developed a cytogenetic map for the alpaca genome by localization of 151 alpaca BAC clones which identified all chromosomes. Their results showed that LPA12, 29 and 36 are submetacentric and not acrocentric as their counterparts in the C. dromedarius karyotype [Balmus et al., 2007]; LPAY is a small acrocentric compared to the submetacentric CDRY, and the banding pattern of LPA24 differed from that of CDR24.

Suborder Suina

The suborder Suina consists of 2 families, Suidae and Tayassuidae. Kulemzina et al. [2009] reported the highest rates of karyotype evolution among Cetartiodactyla during the origin of the ancestral Suina karyotype which differs from the CAK by 11 fissions, 11 fusions and 9 inverCytogenet Genome Res 2012;137:194–207

195

7 fus. 5 fis. 3 inv. 16 fus. 2 inv. 0.3 7 fis. 0.5 4 fus. 1 inv. 15 fus. 0.1 4 inv. 1 fis. CDR1/17 1 inv. 0.5

Fig. 1. Scenario of the karyotype evolution

in Cetartiodactyla with a geological timescale presented on the right. Diploid karyotype numbers are indicated in squares. The rates of chromosomal evolution in different lineages are shown as a quantity of rearrangements per million years (blue numbers). fis. = Fission; fus. = fusion; inv. = inversion.

11 fis. 1 fus. 0.3

Family Suidae This family comprises 5 recent genera and 14 species. Because of its great economic importance, the domestic pig (2n = 38) has been more extensively studied by comparative painting and gene mapping than other suids. The first genome-wide comparative maps were generated using human whole-chromosome painting probes which delineated up to 55 conserved autosomal segments in pig [Rettenberger et al., 1995; Frönicke et al., 1996; Frönicke and Wienberg, 2001]. Bidirectional or reciprocal chromosome painting using sets of probes from both investigated species refined some homologies between pig and human karyotypes [Goureau et al., 1996]. Mapping of 113 goat BAC clones already located in humans to the porcine cytogenetic map enabled us to compare the Cytogenet Genome Res 2012;137:194–207

Bos taurus

Muntiacus reevesi

60 1 fis.

70

3 fis. 11 fus. 7 inv.

20

6 fis. 1 inv.

1 fus. 0.4

30 1 inv.

58

2 fus. 4 inv.

52

40

7 fis. 2 fus. 12 inv. 1.2

48

sions. Since the pig/peccary divergence, evolutionary rates fell and were 7 times slower in Suidae and 3 times lower in Tayassuidae. The karyotype of the domestic pig (Sus scrofa domestica, SSC) can be derived from the Suina ancestral karyotype by 7 fusions and 3 inversions, while 5 fissions, 16 fusions and 2 inversions have occurred in Pecari tajacu (fig. 1) [Kulemzina et al., 2009].

196

10 12 fus. 2 inv. 0.1

52

11 fis. 10 fus. 9 inv. 1.76

46

58

Moschus moschiferus

Giraffa camelopardalis 30

44

Okapia johnstoni

Tragulus javanicus 32

Hippopotamus amphibius 36

Globicephala melas 44

Pecari tajacu 30

Sus scrofa 38

Camelus dromedarius 74

4 fis. 10 fus. 13 fus. 0.8 1 inv. 0.9

0 Million years before present

50

0.4

1 fis. CDR1/17 1 inv.

60

1 fus. CDR4/31

52

genomes with great precision [Pinton et al., 2000]. It was revealed that pig autosomes comprise 84 conserved segments homologous to human chromosomes and 70 conserved blocks homologous to the goat (2n = 60) karyotype. Taking the gene order into account, authors estimated the number of rearrangements between the human and porcine karyotypes and the pig and goat genomes to be 60 and 52, respectively. Further, the pig karyotype was compared with that of cattle (Bos taurus, BTA, 2n = 60) and other bovids. Porcine chromosome painting probes identified a total of 43 autosomal segments of conserved synteny in cattle [Schmitz et al., 1998]. Sheep (2n = 54) painting probes hybridized to pig chromosomes delineated 62 conserved autosomal segments [Frönicke and Wienberg, 2001]. The authors showed that the porcine karyotype experienced many paracentric inversions. Another comparative study of the pig was carried out with dog (2n = 78) painting probes which detected 97 conserved autosomal segments [Biltueva et al., 2004]. Painting probes from 36 autosomal chromosomes of C. dromedarius revealed 55 conserved porcine segments [Balmus et al., 2007; Kulemzina et al., 2009]. Rubes /Musilova /Kopecna /Kubickova / Cernohorska /Kulemsina

Comparative studies of other Suidae species are mainly based on chromosome banding. Due to large well-distinguishable evolutionary conserved chromosomal segments and a limited number of rearrangements, this approach has been productive in identifying homologs between the domestic pig and other suids. Although the diploid number in the domestic pig is 2n = 38, variations have been reported in its wild relative S. scrofa, 2n = 36–38. This polymorphism is due to a centric fusion involving domestic pig chromosomes SSC15/17. Fusion SSC16/17 was reported in Asian wild pigs, 2n = 36 [Tikhonov and Troshina, 1975; Bosma, 1976; Chowdhary, 1998]. Apart from S. scrofa, genus Sus includes 7 more species. G-banding studies revealed a karyotype identical to S. scrofa (2n = 38) in S. salvanius [Bosma et al., 1983], S. verrucosus and S. celebensis [Bosma et al., 1991]. Gbanded karyotypes of the other 2 species S. barbatus and S. cebifrons were presented in the Atlas of Mammalian Chromosomes [O’Brien et al., 2006]. The karyotype of S. barbatus (2n = 38) does not reveal any differences compared to that of the domestic pig. However, the karyotype of S. cebifrons (2n = 34) seems to contain the centric fusions SSC13/16 and 14/18. Although the diploid number of chromosomes in Babyrousa babyrussa (2n = 38) equals that of the domestic pig, their karyotypes differ in 2 fissions (SSC3 and 6), 2 centric fusions (SSC13/16 and 15/17) and 1 pericentric inversion of SSC1p. Fissions were confirmed by comparative painting with whole-chromosome painting probes for SSC3 and 6 as well as by comparative mapping of cosmid probes which enabled determination of segment orientation [Bosma et al., 1996; Thomsen et al., 1996]. The genus Phacochoerus includes 2 living species [Randi et al., 2002]. The chromosomal complement of P. africanus (2n = 34) differs from that of the domestic pig in 2 centric fusions SSC13/16 and 15/17. Orientation of the segments involved in fusions was determined by comparative painting with region-specific painting probes [Musilova et al., 2010]. G-banded karyotype of P. aethiopicus published previously by Bosma [1978] is apparently identical to that of P. aethiopicus. However, formerly, the genus was considered to be monotypic and, therefore, it is not clear if the karyotype really represents the other taxa. Conventional cytogenetic study of the giant forest hog Hylochoerus meinertzhageni revealed 2n = 32 and the chromosomal complement consisting of only (sub)metacentric chromosomes [Melander and Hansen-Melander, 1980]. Unfortunately, no comparative study using banding or in situ hybridization methods has been carried out in this species yet. Molecular Cytogenetics in Cetartiodactyla

Comparative painting with region-specific painting probes revealed that the karyotype of Potamochoerus porcus (2n = 34) differs from that of the domestic pig by the presence of pericentric inversion of a chromosome arm homologous to SSC1p, centric fusion SSC15/17 and tandem fusion of the telomeric part SSC16 with a centromeric part of SSC13 [Musilova et al., 2010]. The G-banded karyotype of P. larvatus was published in the Atlas of Mammalian Chromosomes [O’Brien et al., 2006] and appears to be identical to that of P. porcus. Family Tayassuidae The family Tayassuidae comprises 3 recent genera and 3 species. P. tajacu (2n = 30) and Tayassu pecari (2n = 26) karyotypes were studied by comparative chromosome painting with porcine probes [Bosma et al., 2004]. Thirtyone conserved autosomal segments were delineated in both peccary species. The work revealed extensive karyotypic differences between the 2 Tayassuidae species analyzed. Sheep chromosome paints disclosed at least 62–66 painted segments in P. tajacu [Adega et al., 2006]. Different karyotypes were observed in P. tajacu. Although the diploid numbers are the same, karyotypes of South and North American P. tajacu differ in their ratio of (sub) metacentric to acrocentric chromosomes due to rearrangement involving 2 autosomes. Moreover, there is a difference in the X chromosome centromere position [Adega et al., 2006]. The last living peccary species, Catagonus wagneri (2n = 20) was only studied by G-banding [Benirschke et al., 1985]. Authors of the study found common identity among only a limited number of chromosomes or their segments between P. tajacu and C. wagneri, which implies great rearrangements between the karyotypes.

Suborder Whippomorpha, Infraorder Ancodonta

Family Hippopotamidae The family Hippopotamidae comprises 2 recent genera and 2 living species. A comparative painting study was performed on H. amphibius, 2n = 36 [Kulemzina et al., 2009]. Human and camel painting probes (C. dromedarius, 2n = 74) revealed 45 and 50 conserved autosomal segments in H. amphibius, respectively. No comparative study has been performed in Hexaprotodon (Choeropsis) liberiensis. However, the diploid number of chromosomes, 2n = 36, is similar to H. amphibius [Musilova, pers. commun.]. During evolution, the hippopotamus karyotype underwent multiple reshufflings; some of these rearrangements are very similar to those which occurred in Cytogenet Genome Res 2012;137:194–207

197

man (HSA) homologies [Kulemzina et al., 2011]

Table 3. Putative PAK (2n = 58) with bovine (BTA) and human (HSA) homologies [Slate et al., 2002]

RAK

CDR

HSA

PAK

BTA

HSA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 X

23, 21, 9, 13 32, 2 22, 3, 22, 3, 22, 3 27, 6 31, 4 1 7 5 33, 10 34, 12 8 28, 15 11 25, 29, 25 14 35, 19 17 16 24, 30 9pqprox. 20 18 26 X

1 12qdist., 22qprox., 4pq 19p, 5 15, 14, 15, 14 8pprox., 9 3, 21 7pprox.qprox. 2qdist. 11 12pqprox., 22qdist. 6q 2pq 10q 8q 13 10p, 20 3 17 18 19q, 16q 6p 16p, 7 4, 8pdist. X

A1 A2 B1 B2 C1 C2 D E F G H1 H2 I J K L M N1 N2 O P Q R S T U V W X

3 1 8 2 11 5 4 7 6 10 9 14 12 13 16 15 18 17 19 20 21 22 23 24 25 26, 28 27 29 X

1 3, 21 8, 9 1, 2, 3 2, 9 12, 22 7 5, 19 4 14, 15 6 8 13 10, 20 1 11 16, 19 4, 12, 22 17 5 14, 15 3 6 18 7, 16 1, 10 4, 8 11 X

Table 2. Putative RAK (2n = 48) with dromedary (CDR) and hu-

prox. = Proximal part of chromosome arm; dist. = distal part of chromosome arm.

Pecora karyotypes (2 inversions on ancestral blocks homologous to CDR4/31, 21/13 and fissions of conserved segment CDR2, 21, 22) and must be homoplasies.

Suborder Whippomorpha, Infraorder Cetacea

This taxon of wholly aquatic mammals includes whales, dolphins and porpoises which are classified into 13 families. Formerly, Cetacea was a separate order. However, recent studies suggested a close relationship between Artiodactyla and Cetacea [Graur and Higgins, 1994; Prothero and Foss, 2007; Spaulding et al., 2009]. Only 2 Cetacean species of the family Delphinidae have been studied by comparative painting. Tursiops truncatus and G. melas, 2n = 44, seem to have identical karyotypes [Bielec et al., 1998 and Kulemzina et al., 2009, respectively]. Twenty-two human and 36 camel autosomal paints revealed 34 and 37 conserved segments in the G. melas karyotype, respectively. In cetaceans, the majority of ce198


tartiodactyl ancestral syntenies were conserved without changes; only 4 fusions, 1 fission and 2 inversions were needed to form the G. melas karyotype from a non-camelid ancestral one (fig. 1) [Kulemzina et al., 2009]. Molecular studies suggest that cetaceans and hippopotamuses are closely related [Zhou et al., 2011]. However, the comparative painting study did not reveal any rearrangement common to both taxa. Probably, the period between the origin of Whippomorpha and hippopotamus divergence from whales was short (5–10 million years) and the rate of karyotype evolution was slow at that time.

Suborder Ruminantia

The suborder Ruminantia is the largest within Cetartiodactyla and comprises 2 infraorders, the evolutionary older Tragulina and the plentifully represented Pecora. Rubes /Musilova /Kopecna /Kubickova / Cernohorska /Kulemsina

RAK (table 2) with 2n = 48 appears to be similar to the proposed CAK (2n = 52) only differing from it by 3 fusions, 4 inversions, and 1 fission. The pecoran ancestral karyotype (PAK, table 3) with 2n = 58 proposed by Slate et al. [2002] could be derived from RAK by 7 fissions, 2 fusions and 12 inversions (fig. 1) [Kulemzina et al., 2011]. It is noteworthy that the inversions played a big role in the formation of RAK and PAK as 16 inversions distinguished the PAK from CAK. Some ancestral syntenies (CDR1/17, 3/22, 4/31, 6/27, 10/33, 23/21/9/13) underwent several inversions that could imply the presence of evolutionary hot spots on these chromosomes. Indeed, the same ancestral blocks have inversions in karyotypes of Suidae (CDR3/22, 6/27, 10/33) and Hippopotamidae (CDR4/31, 21/13). But after the divergence of pecoran families, only few inversions were revealed and again some of them occurred in the evolutionary unstable CDR1/17 and 6 [Kulemzina et al., 2009].

Suborder Ruminantia, Infraorder Tragulina

Family Tragulidae The infraorder Tragulina includes only 1 extant family, Tragulidae, comprising 3 mouse deer genera: Hyemoschus, Moschiola and Tragulus. The cytogenetically most studied species is the Java mouse deer (T. javanicus). A conventionally Giemsa-stained karyotype of T. javanicus (2n = 32, NF = 64) was the first described by Yong [1973]. Gallagher et al. [1996] investigated the T. javanicus karyotype by G-, C- and Q-banding and found few putative chromosomal homologies comparing mouse deer and cattle karyotypes. Cytogenetic analysis of T. napu and T. javanicus [Kim et al., 2004] showed that these 2 species have identical diploid numbers (2n = 32) and similar autosomal complements but with notable difference in the size and morphology of the parms of X and Y chromosomes. Kulemzina et al. [2011] published the comparative map of T. javanicus, established by cross-species chromosome painting with human and dromedary probes. According to their results the karyotype evolution of T. javanicus has involved multiple rearrangements, most of which appear to be apomorphic and have not been found in pecoran species. Kulemzina et al. [2011] conclude that the rate of chromosome evolution of the mouse deer was rather low, while the estimated rate of chromosome changes to the lineages leading from Cetartiodactyla ancestor to Ruminantia and from Ruminantia to Pecora were roughly the same. Molecular Cytogenetics in Cetartiodactyla

Suborder Ruminantia, Infraorder Pecora

The infraorder Pecora consists of 5 recent families: Giraffidae, Moschidae, Cervidae, Antilocapridae and Bovidae. There are relatively low numbers of shared chromosomal traits linking Moschidae, Cervidae, Bovidae, and Giraffidae probably because they diverged from a common ancestor at about the same time. After divergence, each lineage independently accumulated apomorphic chromosomal rearrangements [Kulemzina et al., 2009]. The infraorder Pecora has been extensively studied by Zoo-FISH due to the economic value of domesticated species. Family Giraffidae The family Giraffidae comprises only 2 genera, each represented by 1 extant species, the giraffe (G. camelopardalis) and the okapi (O. johnstoni). The idea that the earliest Giraffidae are in close relationship with the families Bovidae and Moschidae/Cervidae is supported by the majority of constructed molecular phylogenetic trees [e.g. Su et al., 1999; Matthee et al., 2001; Fernandez and Vrba, 2005] and the study by Kulemzina et al. [2009]. In the giraffe, studies based on G-banding uncovered a karyotype (2n = 30) with low number of mostly metacentric chromosomes [Gallagher et al., 1994; O’Brien et al., 2006]. Huang et al. [2008] established genome-wide chromosomal homologies among giraffe, Chinese muntjac (Muntiacus reevesi, 2n = 46) and humans (2n = 46) using comparative chromosome painting. Their study revealed that the numerous tandem and Robertsonian fusions account for the reduction in diploid number of chromosomes in giraffes from the PAK. The giraffe karyotype differs from the ancestral Giraffidae karyotype by 13 fusions. Additionally, the pericentric inversion and centromere repositioning occurred in the giraffe lineage [Huang et al., 2008]. The okapi chromosome number varies from 2n = 44–46 due to centric fusion/fission polymorphisms [Petit and de Meurichy, 1986; Vermeesch et al., 1996]. Kulemzina et al. [2009] published the comparative map of okapi using human and dromedary painting probes and found the only common unique association (CDR5;16) of giraffe and okapi species. The authors suggest a minimum of 4 fissions, 10 fusions and 1 inversion needed for transformation of the Giraffidae ancestral karyotype into that of okapi (fig. 1). After the divergence of Giraffidae, 1 inversion arose in the common ancestor of Moschidae, Cervidae and Bovidae [Kulemzina et al., 2009].


199

Family Moschidae The family Moschidae comprises the single genus Moschus, which exhibits a mixture of bovid and cervid characters, and its taxonomic placement remains a topic of hot debates [Hassanin and Douzery, 2003]. The genus has traditionally been placed in the subfamily Moschinae within the family Cervidae [Wang et al., 1993], but there are strong arguments in favor of Moschus being placed in its own family, Moschidae [Groves and Grubb, 1987; Su et al., 2001]. Molecular phylogeny surprisingly places Moschus as the sister group of bovids rather than cervids [Hassanin and Douzery, 2003]. Of the extant Moschidae species, only M. beresovskii and M. moschiferus (both with 2n = 58) were studied by comparative chromosome painting. Dromedary painting probes were used to establish the karyotype of M. moschiferus [Kulemzina et al., 2009]. Chinese muntjac probes were hybridized onto chromosomes of M. beresovskii [Chi et al., 2005a]. Combined FISH and G-banding comparison analyses have demonstrated that both Moschus species have identical karyotypes similar to PAK. Family Cervidae The Cervidae family comprises 51 species [Wilson and Reeder, 2005]. The great diversity of karyotypes among different species with 2n ranging from 6 in M. muntjak vaginalis to 70 in Mazama gouazoubira is a distinctive trait of the Cervidae family. Fontana and Rubini [1990] traced the chromosomal evolution in 30 Cervidae species based on banding data and found that different types of rearrangements have dominated in different cervid subfamilies (i.e. Robertsonian translocations in Cervini and tandem fusions in Muntiacini). These findings have been largely confirmed by more detailed comparative chromosome painting [Yang et al., 1997a; Bonnet-Garnier et al., 2003; Huang et al., 2006a]. Comparative chromosomal analysis has suggested that the ancestor of the Cervidae family could have a karyotype of 2n = 70, including 68 acrocentric autosomes, an acrocentric X chromosome and a small submetacentric Y. This is still retained in Hydropotes inermis and M. gouazoubira. Based on the results of molecular cytogenetic data and comparative chromosome painting, it was proposed that the ancestral Cervidae karyotype could have evolved from the 2n = 58 hypothetical PAK by 6 fissions of chromosomes [Huang et al., 2006a]. Subfamily Capreolinae. The Capreolinae karyotypes (predominantly 2n = 70) have been mostly studied with classical cytogenetic methods. The most widespread karyotype of the subfamily is composed of 66 acrocentric 200


chromosomes, a pair of metacentric autosomes as well as a submetacentric X and a small Y. Both species of the genus Capreolus (C. capreolus and C. pygargus) have an identical 2n = 70 karyotype comprising only acrocentric autosomes. Reduction in chromosome number, presumably due to Robertsonian fusions is found in Ozotoceros bezoarticus, Alces alces (2n = 68), and Blastocerus dichotomus (2n = 66). A high extent of karyotypic differences was described in all species of Mazama [Duarte and Jorge, 2003]. The diploid number in the genus varies from 32 chromosomes in M. bororo to 70 chromosomes in M. gouazoubira. Extensive karyotype variation was delineated in M. americana and many aspects of its taxonomy are uncertain. This neotropical species shows wide cytotype variations with diploid numbers between 42 and 54 [Duarte et al., 2008; Abril et al., 2010]. Tandem and centric fusions detected by G-banding clearly show the pattern of chromosome evolution described for the genus Muntiacus [Abril et al., 2010]. Chromosomes of 1 Capreolus species and M. gouazoubira were characterized using comparative chromosome painting. Dementyeva et al. [2010] have established the comparative chromosome map of C. pygargus by crossspecies chromosome painting with C. dromedarius probes. Subsequently, the chromosomal homologies of C. pygargus and cattle chromosomes were identified indirectly based on the result of previously detected cattle-dromedary homologies [Balmus et al., 2007]. The karyotypes of cattle and Capreolus are separated by 6 fissions in Capreolus and 1 fission in cattle. Homologies between M. gouazoubira and Chinese muntjac (M. reevesi) karyotypes have been established by hybridization of a complete set of Chinese muntjac painting probes onto metaphases of M. gouazoubira and H. inermis [Yang et al., 1997a]. The study confirmed that M. gouazoubira has retained a karyotype closely related to the ancestral 2n = 70 karyotype. Subfamily Cervinae. The subfamily Cervinae comprises the 2 tribes Cervini and Muntiacini. Only Robertsonian translocations were found to have been involved in karyotypic evolution of the Cervini tribe, while repeated tandem chromosome fusions took part in karyotype differentiation of the Muntiacini tribe [Fontana and Rubini, 1990; Huang et al., 2006a]. The diploid number of Cervinae species ranges between 56 and 68 with a constant fundamental number of 70. The most examined member of this tribe is Cervus nippon. Karyotypic evolution of C. nippon (2n = 66) and C. elaphus (2n = 62) was studied by comparative chromosome painting using Chinese muntjac probes [Huang et al., 2006a]. Chromosome homologies between C. nippon Rubes /Musilova /Kopecna /Kubickova / Cernohorska /Kulemsina

and cattle were identified using R-banding and FISH mapping of bovine and caprine marker probes [Bonnet et al., 2001]. Subsequently, karyotypes of another 5 deer species Axis axis (2n = 66), Przewalskium albirostris (2n = 68), Rusa timorensis, (2n = 60), R. unicolor (2n = 56) and Rucervus eldii (2n = 58) were identified by R-banding or by FISH-mapping of bovine and caprine BACs [BonnetGarnier et al., 2003]. The main characteristic in all the species is an identical X chromosome, which is the largest acrocentric chromosome, with a fundamental number of 70 and the presence of 1 common submetacentric chromosome which arose via Robertsonian fusion of segments homologous to the BTA17 and 19. Among 13 Robertsonian translocations, 5 are common in at least 2 species and it was found that equivalents to BTA5, 10, 18, 22, and 27 are more frequently involved in Robertsonian translocations than others. Examination of G- and Rbanded karyotypes shows that the acrocentric chromosomes and the arms of the submetacentric chromosomes are conserved among species of the tribe Cervini but shared biarmed chromosomes are rare [Muir et al., 1997; Bonnet-Garnier et al., 2003]. The genus Muntiacus is famous for its high degree of interspecific karyotype diversity and for variation in chromosomal numbers between closely related species. For instance, M. m. vaginalis has a karyotype of 2n = 6(U)/7(-), the lowest chromosome number known in mammals, and another morphologically similar species, M. reevesi, has the highest diploid number (2n = 46) so far found in Muntiacus [Wurster and Benirschke, 1970]. Comparative chromosomal painting on the M. m. vaginalis chromosomes was accomplished using human [Yang et al., 1997b] and ovine probes [Burkin et al., 1997]. Karyotypes of several muntjac species were compared using painting probes derived from M. m. vaginalis chromosomes [Yang et al., 1995]. Chinese muntjac (M. reevesi) chromosome-specific paints have been prepared and characterized by cross-hybridization with chromosomes of H. inermis and M. gouazoubira, the 2 species retaining the 2n = 70 ancestral karyotype [Yang et al., 1997a]. The same set of Chinese muntjac probes was hybridized onto selected Muntiacini [Yang et al., 1997c, d; Huang et al., 2006a], Cervini [Huang et al., 2006a], Bovini [Chi et al., 2005a] and Caprini [Huang et al., 2005] species. Muntiacus m. vaginalis genomic BAC clones were used for high resolution comparative mapping between M. m. vaginalis and M. reevesi [Chi et al., 2005b], M. crinifrons [Huang et al., 2006b], M. feae and M. gongshanensis [Huang et al., 2006c]. These studies together established that tandem fusions are the major chromosomal rearrangements under-

lying the karyotypic diversification of muntjacs from a 2n = 70 acrocentric ancestral karyotype. The idea that the karyotype of M. reevesi resembles the putative ancestral karyotype of Muntiacus was disproved. The karyotypes of M. m. vaginalis, M. crinifrons, M. feae and M. gongshanensis cannot be reconstructed from the hypothetical M. reevesi-like 2n = 46 ancestral karyotype by chromosome fusions exclusively. The latest common ancestor of extant muntjacs had a karyotype of more than 46 chromosomes. This is perhaps similar to 2n = 70 and the karyotypes of Muntiacus species that could have evolved independently from this ancestral karyotype [Yang et al., 1997d; Chi et al., 2005b; Huang et al., 2006c]. The M. reevesi karyotype has originated from a 2n = 70 ancestral karyotype via 12 tandem fusions. The M. m. vaginalis (2n = 6/7) karyotype is derived by 3 centric and 29 centromere-telomere tandem fusions. In addition to 2 heterozygous centric fusions, 27 centromere-telomere tandem fusions are needed to generate the 2n = 14 male M. feae karyotype, while 28 centromere-telomere tandem fusions and 3 centric fusions are required to reconstruct the 2n = 8 M. gongshanensis karyotype. The genus Elaphodus includes the single species E. cephalophus with 2n = 46 or 47 in females and 47 or 48 in males [Shi et al., 1991]. This karyotype including 4 large and 19 small acrocentric autosomes was characterized using Chinese muntjac painting probes [Huang et al., 2006a]. Three Robertsonian translocations and 9 tandem fusions involving 14 ancestral chromosomal arms have occurred during the speciation of E. cephalophus (2n = 47). The translocation of X chromosome material to an autosome occurs in several Muntiacini species. The X chromosome is fused with chromosomes 1 in M. feae and M. gongshanensis [Huang et al., 2006c], 3 in M. m. vaginalis [Chi et al., 2005b], 4 in M. crinifrons [Huang et al., 2006b] and 17 in E. cephalophus [Huang et al., 2006a]. The aforementioned results demonstrated that extensive centromere-telomere fusions and a few centric fusions are the chromosomal mechanisms underlying the karyotypic evolution of Muntiacini. Although Cervinae and Muntiacinae are reciprocally monophyletic sister groups [Randi et al., 1998], different types of chromosomal rearrangements, which occurred during karyotype evolution, have produced great differences in the genome organization of the 2 groups. Subfamily Hydropotinae. The subfamily Hydropotinae comprises only 1 species H. inermis, the most primitive deer, which has retained the 2n = 70 karyotype closely related to the ancestral. This fact was corroborated by hybridization of painting probes prepared from M. reeve-

Molecular Cytogenetics in Cetartiodactyla


201

si and M. gouazoubira onto H. inermis chromosomes [Yang et al., 1997a]. Family Antilocapridae This family comprises only 1 species, Antilocapra americana (pronghorn), with 5 subspecies [Wilson and Reeder, 2005]. Extensive chromosomal homologies have been reported in the study of Gallagher et al. [1994] among cattle and pronghorn (2n = 58, 54 acrocentric autosomes, 2 biarmed autosomes and sex chromosomes XY) based on QFH-banding. In this study, several chromosomal changes in comparison with the cattle karyotype have been suggested. The telomeric end of the cattle homolog BTA3 in A. americana has been broken and translocated to the telomeric end of chromosome BTA1 or the centromeric end of BTA26. Chromosome BTA28 has been translocated to the telomeric end of BTA1 or the centromeric end of BTA26. Several other chromosomal changes were noted in the study such as 1 or more paracentric inversions of chromosome BTA20, chromatin addition to the centromeric end of BTA20 or paracentric inversion in chromosome BTA27. Antilocapridae is the only cetartiodactyl family that was not investigated by comparative chromosome painting. Mapping of camel paints on A. americana might answer the question whether this species is a sister group to Giraffidae [e.g. Fernandez and Vrba, 2005] or even basal to other pecoran families [e.g. Matthee et al., 2001]. Family Bovidae The Bovidae family, composed of 143 species classified into 50 genera [Wilson and Reeder, 2005], comprises species of considerable economic significance, threatened species, and species on the verge of extinction. Previously, applications of molecular cytogenetics have been predominantly focused on economically important species [reviewed by Iannuzzi et al., 2009 and Rubes et al., 2009] and have been aimed especially at gene and comparative mapping and the identification of chromosome abnormalities. The bovid karyotypes are comprised of primarily acrocentric chromosomes with the primitive karyotype consisting of 58 ancestral acrocentric chromosomal complements and 2 sex chromosomes. Transition from the ancestral pecoran karyotype (2n = 58) to the Bovidae ancestral karyotype occurred by only 1 fission (2n = 60, fig. 1). Evidence indicates that the domestic cattle karyotype (2n = 60) is the primitive karyotype from which all modern karyotypes of bovid species have descended [Gallagher and Womack, 1992]. This is supported by the extensive homologies observed between the chromosome 202


arms of cattle and other bovids [Gallagher and Womack, 1992]. The Bovidae family is subdivided into 2 subfamilies: Bovinae (Bovini, Tragelaphini, Boselaphini) and Antilopinae (Neotragini, Aepycerotini, Cephalophini, Oreotragini, Hippotragini, Alcelaphini, Caprini, Antilopini and Reduncini) [Robinson and Ropiquet, 2011]. The basal split between the subfamilies characterizes the presence/absence of the BTA9;14 simple translocation earlier suggested in the study of Buckland and Evans [1978]. In this case, a small subcentromeric chromosome segment from ‘Bovinae’ chromosome 9 has been translocated to ‘Caprinae’ chromosome 14 [ISCNDB 2000]. This translocation event, which potentially defines Antilopinae, has been demonstrated by both linkage [de Gortari et al., 1998] and FISH-mapping [Iannuzzi et al., 2001] techniques. Bovidae ancestral karyotype was very similar to the present karyotype of the Bovinae subfamily with Bovinae chromosomes 9 and 14, as well as an X chromosome of Bovinae type [Iannuzzi et al., 2009]. Chromosomal evolution in Bovidae is largely attributable to Robertsonian fusions, although the increasing number of cytogenetic reports reveal that tandem fusions also impact karyotype shaping in this family [Rubes et al., 2008; Ropiquet et al., 2010; Cernohorska et al., 2011] by reducing the ancestral diploid number (2n = 60) in derived karyotypes. Recorded chromosome numbers vary from 2n = 30 in gazelles to 2n = 60 in a range of species within the tribes Aepycerotini, Hippotragini, Oreotragini, Cephalophini and Bovini. Robertsonian (centric) fusions of many species were defined with respect to the cattle standard [ISCNDB 2000] using banding and/or FISH methods. However, in spite of the availability of these data, there are doubts whether they are phylogenetically informative. Recently, Robinson and Ropiquet [2011] have analyzed 155 chromosomal characters for 52 taxa which represented all 9 tribes in Bovidae. Of the characters analyzed, only a little less than 27% were exclusive synapomorphies, 44% were autapomorphies, 27% were potentially homoplasic (including 4% of hemiplasic) and the remaining ones were ambiguous. Despite that fact, data from the comparative analysis using molecular cytogenetic methods have been useful in providing support for the phylogenetic relationships of species in some tribes. The methods allow high-resolution comparisons and the detection of subtle interchromosomal rearrangements. A complete set of whole-chromosome painting and subchromosomal probes derived from B. taurus by laser microdissection were used for the first time in the phylogenetic study of spiral-horned antelopes (Tragelaphini) by Rubes et al. [2008]. Fifty-seven different autoRubes /Musilova /Kopecna /Kubickova / Cernohorska /Kulemsina



203

Bovini Bovini Bovini Antilopini Antilopini Antilopini Antilopini Hippotragini Hippotragini Hippotragini Hippotragini Reduncini Reduncini Reduncini Reduncini Reduncini Aepycerotini Caprini Caprini Caprini Caprini Caprini Alcelaphini Alcelaphini Alcelaphini Alcelaphini Alcelaphini Tragelaphini Tragelaphini Tragelaphini Tragelaphini Tragelaphini Tragelaphini Tragelaphini Tragelaphini Neotragini Boselaphini

1 2 3 2 4 4 4 3 3 3 2 5 3 3 2 2 3 2 2 2 2 2 2 2 2 2 2 6 6 6 6 6 6 6 6 7 8 56 52 54–55 56 38 33-/32U 30–33 56 58 58 58 56–57 50–52 52–54 48 52 59–60 52 48 54 58 48 38 40 58 58 58 38 55-/56U 31-/32U 33-/34U 30 33-/34U 31-/32U 31-/32U 46 38

2n 1;29 1;13 1;13 2;25 2;15 2;3 1;25 1;25 1;25 1;25 1;25 2;25 1;19 1;19 1;19 1;19 14;20 1;22 1;27 1;26 1;3 1;17 1;10 1;10 2;25 2;25 2;25 1;5 11;22;2 24;22;2 3;22;2 3;22;2 3;22;2 3;22;2 3;22;2 11;26;23;27;28;1 24;10;5;18 5;28 5;29 2;28 5;25 3;19 3;19

4;7 3;10 1;29 4;12 1;29 5;10 5;10 8;29 15;21;7

2;25 2;28 5;14 2;23 2;25 2;25

2;10 4;5 6;10 5;10 5;10 6;11 6;11 2;20 13;3;19

11;19

6;18

9;26 10;20 7;20

10;23 11;27 8;14

6;20 5;17 8;15 6;21 1;29 1;29 2;22

26;11;12

6;16

4;16

7;18 8;16 6;24 7;28 9;20 9;20

12;18

8;17 11;20 11;18 8;15 8;24 8;24

8;20

22;28 24;29 5;6 7;9 8;17 5;14 7;9 8;17

7;17

7;11 7;29 5;13

8;21 9;17 6;24

1;29 4;15 1;29 4;19 4;12 4;12

3;11

11;27 4;14 6;4

11;23 4;25

6;18 6;18 4;7 6;18

7;22 8;14 4;19

6;25 7;21 3;27

5;17 5;17 3;10 4;7

11;25

5;20 11;25

6;10 2;25 2;25 2;25 2;25

2;28 2;3 2;3 1;29 4;28 4;6 2;29 2;17

Chromosome fusions

13;17 13;19 10;28

14;24 18,29 11;22

12;16 7;28 7;26 9;23 15;16 15;16

9;27

11;23 9;26 16;20 11;20 7;28 7;28

15;22

9;27 23;12 14;19 14;27 18;19 18;19

14;29

11;23 12;16 13;15 11;15 12;16 22;23

12;27 12;16 9;17

19;21 18;21 9;28 16;17 14;26 14;26

20;22

22;25 12;16

14;26 19;25 17;27 18;26 21;23 21;23

23;24 13;18

17;27 17;27

21;23

15;28

26;28 15;23

21;26

smc ac ac ac X;5 X;5 X;5 ac ac ac ac ac ac ac ac ac ac ac ac ac ac ac ac ac ac ac ac X;13 ac ac ac X;13 ac ac ac ac ac

X

mc ac ac ac Y;16 ac ac ac ac ac ac ac ac ac ac ac mc ac ac ac mc mc ac ? ac ac ac Y;13 Y;13 Y;13 Y;13 Y;13 Y;13 Y;13 Y;13 ac ?

Y

R = References: 1 = Ropiquet et al. [2008]; 2 = data unpublished; 3 = Pagacova et al. [2011]; 4 = Cernohorska et al. [2012]; 5 = Rubes et al. [2007]; 6 = Rubes et al. [2008]; 7 = Cernohorska et al. [2011]; 8 = Ropiquet et al. [2010]. ac = Acrocentric; mc = metacentric; smc = submetacentric. Polymorphic fusions are marked in bold.

Bos javanicus birmanicus Syncerus caffer caffer Syncerus caffer nanus Antidorcas marsupialis Nanger dama ruficollis Gazella leptoceros Antilope cervicapra Oryx gazella gazella Oryx leucoryx Oryx dammah Addax nasomaculatus Redunca fulvorufula Kobus ellipsiprymnus ellipsiprymnus Kobus e. defassa Kobus leche kafuensis Kobus megaceros Aepyceros melampus Budorcas taxicolor Hemitragus jemlahicus Pseudois nayaur Ammotragus lervia Ovibos moschatus Damaliscus pygargus phillipsi Alcelaphus lichtensteinii Connochaetes taurinus taurinus Connochaetes gnou Connochaetes taurinus albojubatus Tragelaphus imberbis Tragelaphus angasii Tragelaphus strepsiceros Tragelaphus scriptus Tragelaphus spekii Tragelaphus eurycerus Taurotragus oryx Taurotragus derbianus Madoqua kirkii thomasi Tetracerus quadricornis

Tribes

R Species

Table 4. List of chromosome fusions that have occurred in the Bovidae family identified by comparative painting with whole-chromosome and subchromosomal probes derived from cattle prepared by laser microdissection

somal fusions (53 centric and 4 tandem fusions) were identified among the 8 ingroup taxa. The data support the monophyly of Tragelaphini and a clade comprising Tragelaphus scriptus, T. spekii, T. eurycerus and the eland species Taurotragus oryx and T. derbianus. These findings are largely in agreement with sequence-based molecular phylogenies. The same approach was also used in the Ropiquet et al. [2010] study which reported the outcome of a detailed molecular investigation of the Tetracerus quadricornis (Bovinae) karyotype. In this species (2n = 38), karyotype evolution has been driven by tandem fusions (head to tail) of cattle orthologous chromosomes. By means of bovine painting probes, a further example of the accumulation of tandem fusions in bovids has been described in Madoqua kirkii thomasi (Antilopinae) (2n = 46), as being attributable to cytotype A [Cernohorska et al., 2011]. Populations of M. kirkii have chromosomal variations that have been categorized in 4 distinct cytotypes (A–D) – a situation uncommon in other species of bovids. Chromosome evolution in the subtribe Bovina was described in the study by Ropiquet et al. [2008] suggesting that karyotype differences between B. j. birmanicus (2n = 56) and B. j. javanicus (2n = 60), reflected in their diploid chromosome numbers, may have occurred as a consequence of hybridization between B. javanicus and B. sauveli (2n = ?) during the Pleistocene epoch. In disagreement with previous molecular studies, Nguyen et al. [2008], in their phylogenetic analysis of chromosomal data, suggest that the placement of the saola (Pseudoryx nghetinhensis) should be within the subtribe Bubalina, which contains the species of the Bubalus and Syncerus genera. Chromosome homologies between Damaliscus lunatus and M. reevesi have been established by cross-species painting with Chinese muntjac chromosome paints [Huang et al., 2011]. The chromosomal rearrangements were consistent with published phylogenies based on morphology. The authors suggested that chromosomal rearrangements have played important roles in speciation within Alcelaphini. The studies of chromosomal evolution of Bovidae are also complicated by the polymorphism of centric fusions within the species. Due to the large number of acrocentric chromosomes in bovid karyotypes, there is a predisposition for centric fusion/fission of chromosomes, resulting in the variability of diploid chromosome numbers (2n) of the same species. Chromosomes of 228 captive specimens from 21 species of the family Bovidae have been investigated by Pagacova et al. [2011]. The study showed that 13% of the investigated individuals were polymorphic for the centric fusion. 204


In contrast to the remarkable conservation of autosomal banding patterns, a great variety of X chromosomes, including changes in centromere placement, multiple transpositions of common segments, heterochromatic variation and autosomal translocation have been evolved within bovids [Robinson et al., 1998; Iannuzzi et al., 2000, 2009; Chaves et al., 2004; Rubes et al., 2008]. It was concluded that 3 structural variants of the X chromosome have been evolved among bovids: 1 submetacentric in morphology represented by Bos and Bison (Bovini) and 2 acrocentric types: eland (also referred to as Bubalus type, for example in Tragelaphini) and suni (also referred to as caprine type) [Robinson et al., 1998; Iannuzzi et al., 2000; Nguyen et al., 2008]. All 3 types occur in domestic bovid species. However, only when using the FISH-technique and detailed mapping comparison (gene order) it was possible to clarify the evolution of this chromosome in domestic bovids [Iannuzzi et al., 2009]. Detailed cytogenetic maps of B. taurus, B. bubalis and Ovis aries X chromosomes compared with the HSAX are reviewed in the Iannuzzi et al. [2009] article. The most frequently encountered form of X in the Bovidae family is that of the caprine type represented by Aepycerotini, Alcelaphini, Antilopini, Caprini, Cephalophini, Hippotragini, Reduncini [Robinson et al., 1998; Rubes et al., 2007] and most likely also by Neotragini and Oreotragini, which are also included in the Antilopinae subfamily. This type of X has been suggested as being close to the ancestral state for the Bovidae [reviewed by Robinson et al., 1998]. Several bovid lineages show compound sex chromosomes resulting from gonosome/autosome fusions. Two species within Tragelaphini (T. spekii and T. imberbis) display an X-autosomal translocation involving BTA13. Interestingly, all species of Tragelaphini exhibit Y;BTA13 translocation, which would result in an imbalance in diploid numbers between sexes with the exception of T. spekii (2n = 30) and T. imberbis (2n = 38) [Rubes et al., 2008]. For a clade that includes Antilope, Gazella, Nanger and Eudorcas (Antilopinae), X;BTA5 translocation is a synapomorphy [Cernohorska et al., 2012]. Using region-specific painting probes, the authors revealed that tandem fusion (head-totail) assembled the 2 chromosomes in G. leptoceros and N. dama ruficollis, unlike centric fusion (head-to-head) detected in A. cervicapra. Moreover, the Y chromosome in N. d. ruficollis is submetacentric due to the centric fusion with chromosome BTA16. A similar gonosomal heteromorphism (i.e. X;BTA5 and Y;BTA16 translocations) appears in the genus Eudorcas (E. thomsonii and E. rufifrons) and N. soemmerringii. The Y chromosome has mostly been studied in domestic bovids using chromosome bandRubes /Musilova /Kopecna /Kubickova / Cernohorska /Kulemsina

ing and FISH-mapping comparisons and suggesting differences in shape and size depending on the centromere position and gain or loss of heterochromatin, respectively [reviewed by Iannuzzi et al., 2009].

Conclusion

Recently, the karyotypes of representatives of all cetartiodactyl families (except Antilocapridae) were studied by comparative chromosome painting. The results suggest the karyotypes of common ancestors and a scenario of chromosomal evolution for the main taxa studied. Cetacean karyotypes appear to be very conservative and retained the majority of ancestral syntenies without change. Karyotypes of other families underwent multiple rearrangements. Camelidae karyotypes were formed by fissions, resulting in the highest diploid number of chromosomes within the order. Fusions were the main feature of chromosome evolution in Suidae, Giraffidae, Bovidae and subfamily Cervinae. Inversions also played a big role in forming the cetartiodactyl karyotypes, especially of Suidae chromosomes and PAK. Synteny homologous to HSA4/8/9 that unites Cetacea and other non-camelid cetartiodactyls was revealed.

However, data on comparative chromosome painting turned out to be insufficient to clarify the phylogenetic relationships within Cetartiodactyla due to fast divergence of the main families from a common ancestor. Moreover, the presence of hot spots on some chromosomes led to homoplasies that made phylogenetic analysis based on rearrangements uncertain. Karyotype studies based on banding techniques contained inaccuracies and were verified by molecular methods. A complete set of whole-chromosome painting and subchromosomal probes derived from B. taurus by laser microdissection was used for identification or confirmation of chromosome fusions in the family Bovidae (table 4). The ungulate taxonomy presented by Groves and Grubb [2011] was used in this publication. Acknowledgments This work was supported by grants GA CR P506/10/0421, MZE 0002716202, by the project ‘CEITEC – Central European Institute of Technology’ (CZ.1.05/1.1.00/02.0068) from European Regional Development Funds, and by MCB, SB RAS Programs, research grants of Russian Fund for Basic Research.

References Abril VV, Carnelossi EA, González S, Duarte JM: Elucidating the evolution of the red brocket deer Mazama americana complex (Artiodactyla; Cervidae). Cytogenet Genome Res 128:177–187 (2010). Adega F, Chaves R, Kofler A, Krausman PR, Masabanda J, et al: High-resolution comparative chromosome painting in the Arizona collared peccary (Pecari tajacu, Tayassuidae): a comparison with the karyotype of pig and sheep. Chromosome Res 14: 243–251 (2006). Avila F, Das PJ, Kutzler M, Owens E, Perelman P, et al: Development and application of molecular tools for camelid cytogenetics. J Hered in press (2012). Balmus G, Trifonov VA, Biltueva LS, O’Brien PC, Alkalaeva ES, et al: Cross-species chromosome painting among camel, cattle, pig and human: further insights into the putative Cetartiodactyla ancestral karyotype. Chromosome Res 15: 499–515 (2007). Benirschke K, Kumamoto AT, Meritt DA: Chromosomes of the Chacoan peccary, Catagonus wagneri (Rusconi). J Hered 76: 95–98 (1985).


Bielec PE, Gallagher DS, Womack JE, Busbee DL: Homologies between human and dolphin chromosomes detected by heterologous chromosome painting. Cytogenet Cell Genet 81:18–25 (1998). Biltueva LS, Yang F, Vorobieva NV, Graphodatsky AS: Comparative map between the domestic pig and dog. Mamm Genome 15: 809–818 (2004). Bonnet A, Thévenon S, Claro F, Gautier M, Hayes H: Cytogenetic comparison between Vietnamese sika deer and cattle: R-banded karyotypes and FISH mapping. Chromosome Res 9:673–687 (2001). Bonnet-Garnier A, Claro F, Thévenon S, Gautier M, Hayes H: Identification by R-banding and FISH of chromosome arms involved in Robertsonian translocations in several deer species. Chromosome Res 11: 649–663 (2003). Bosma AA: Chromosomal polymorphism and G-Banding patterns in wild boar (Sus scrofa L.) from the Netherlands. Genetica 46: 391– 399 (1976). Bosma AA: Chromosomal G-banding pattern in wart hog, Phacochoerus aethiopicus (Suidae, Mammalia) and its implications for systematic position of species. Genetica 49: 15–19 (1978).

Bosma AA, Oliver WL, Macdonald AA: The karyotype, including G-banding and Cbanding patterns, of the pigmy hog Sus (Porcula) salvanius (Suidae, Mammalia). Genetica 61:99–106 (1983). Bosma AA, Dehaan NA, Blouch RA, Macdonald AA: Comparative cytogenetic studies in Sus verrucosus, Sus celebensis and Sus scrofa vittatus (Suidae, Mammalia). Genetica 83:189– 194 (1991). Bosma AA, Dehaan NA, Mellink CHM, Yerle M, Zijlstra C: Chromosome homology between the domestic pig and the babirusa (family Suidae) elucidated with the use of porcine painting probes. Cytogenet Cell Genet 75: 32–35 (1996). Bosma AA, de Haan NA, Arkesteijn GJ, Yang F, Yerle M, Zijlstra C: Comparative chromosome painting between the domestic pig (Sus scrofa) and two species of peccary, the collared peccary (Tayassu tajacu) and the white-lipped peccary (T. pecari): a phylogenetic perspective. Cytogenet Genome Res 105:115–121 (2004). Buckland RA, Evans HJ: Cytogenetic aspects of phylogeny in the Bovidae. I. G-banding. Cytogenet Cell Genet 32:64–71 (1978).


205

Burkin DJ, Yang F, Broad TE, Wienberg J, Hill DF, Ferguson-Smith MA: Use of the Indian muntjac idiogram to align conserved chromosomal segments in sheep and human genomes by chromosome painting. Genomics 46:143–147 (1997). Cernohorska H, Kubickova S, Vahala J, Robinson TJ, Rubes J: Cytotypes of Kirk’s dik-dik (Madoqua kirkii, Bovidae) show multiple tandem fusions. Cytogenet Genome Res 132: 255–263 (2011). Cernohorska H, Kubickova S, Vahala J, Rubes J: Molecular insights into X;BTA5 chromosome rearrangements in the tribe Antilopini (Bovidae). Cytogenet Genome Res 136: 188– 198 (2012). Chaves R, Santos S, Guedes-Pinto H: Comparative analysis (Hippotragini versus Caprini, Bovidae) of X-chromosome’s constitutive heterochromatin by in situ restriction endonuclease digestion: X-chromosome constitutive heterochromatin evolution. Genetica 121:315–325 (2004). Chi J, Fu B, Nie W, Wang J, Graphodatsky AS, Yang F: New insights into the karyotypic relationships of Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis). Cytogenet Genome Res 108:310–316 (2005a). Chi JX, Huang L, Nie W, Wang J, Su B, Yang F: Defining the orientation of the tandem fusions that occurred during the evolution of Indian muntjac chromosomes by BAC mapping. Chromosoma 114:167–172 (2005b). Chowdhary BP: Cytogenetics and physical chromosome maps, in Rothschild MF, Ruvinsky A (eds): The Genetics of the Pig, pp 199–264 (CABI Publishing, Wallingford 1998). de Gortari MJ, Freking BA, Cuthbertson RP, Kappes SM, Keele JW, et al: A second-generation linkage map of the sheep genome. Mamm Genome 9:204–209 (1998). Dementyeva PV, Trifonov VA, Kulemzina AI, Graphodatsky AS: Reconstruction of the putative Cervidae ancestral karyotype by chromosome painting of Siberian roe deer (Capreolus pygargus) with dromedary probes. Cytogenet Genome Res 128:228–235 (2010). Duarte JM, Jorge W: Morphologic and cytogenetic description of the small red brocket (Mazama bororo Duarte, 1996) in Brazil. Mammalia 67:403–410 (2003). Duarte JM, González S, Maldonado JE: The surprising evolutionary history of South American deer. Mol Phylogenet Evol 49: 17–22 (2008). Fernandez MH, Vrba ES: A complete estimate of the phylogenetic relationships in Ruminantia: a dated species level supertree of the extant ruminants. Biol Rev Camb Philos Soc 80:269–302 (2005). Fontana F, Rubini M: Chromosomal evolution in Cervidae. Biosystems 24:157–174 (1990).

206

Froenicke L, Caldés MG, Graphodatsky A, Müller S, Lyons AL, et al: Are molecular cytogenetics and bioinformatics suggesting diverging models of ancestral mammalian genomes? Genome Res 156:311–313 (2006). Frönicke L, Wienberg J: Comparative chromosome painting defines the high rate of karyotype changes between pigs and bovids. Mamm Genome 12:442–449 (2001). Frönicke L, Chowdhary BP, Scherthan H, Gustavsson I: A comparative map of the porcine and human genomes demonstrates ZOOFISH and gene mapping-based chromosomal homologies. Mamm Genome 7: 285–290 (1996). Gallagher DS Jr, Womack JE: Chromosome conservation in the Bovidae. J Hered 83:287–298 (1992). Gallagher DS Jr, Derr JN, Womack JE: Chromosome conservation among the advanced pecorans and determination of the primitive bovid karyotype. J Hered 85:204–210 (1994). Gallagher DS Jr, Houck ML, Ryan AM, Womack JE, Kumamoto AT: A karyotypic analysis of the lesser Malay chevrotain, Tragulus javanicus (Artiodactyla: Tragulidae). Chromosome Res 4:545–551 (1996). Goureau A, Yerle M, Schmitz A, Riquet J, Milan D, et al: Human and porcine correspondence of chromosome segments using bidirectional chromosome painting. Genomics 36:252– 262 (1996). Graur D, Higgins DG: Molecular evidence for the inclusion of cetaceans within the order Artiodactyla. Mol Biol Evol 11: 357–364 (1994). Groves C, Grubb P: Relationship of living deer, in Wemmer CM (ed): Biology and Management of the Cervidae, pp 21–59 (Smithsonian Institute, Washington 1987). Groves C, Grubb P: Ungulate Taxonomy (The Johns Hopkins University Press, Baltimore 2011). Hassanin A, Douzery EJ: Molecular and morphological phylogenies of Ruminantia and the alternative position of the Moschidae. Syst Biol 52: 206–228 (2003). Hsu TC, Benirschke K: An Atlas of Mammalian Chromosomes 1: 40 (Springer, New York 1967). Huang L, Nie W, Wang J, Su W, Yang F: Phylogenomic study of the subfamily Caprinae by cross-species chromosome painting with Chinese muntjac paints. Chromosome Res 13:389–399 (2005). Huang L, Chi J, Nie W, Wang J, Yang F: Phylogenomics of several deer species revealed by comparative chromosome painting with Chinese muntjac paints. Genetica 127:25–33 (2006a). Huang L, Chi J, Wang J, Nie W, Su W, Yang F: High-density comparative BAC mapping in the black muntjac (Muntiacus crinifrons): molecular cytogenetic dissection of the origin of MCR 1p+4 in the X1X2Y1Y2Y3 sex chromosome system. Genomics 87: 608–615 (2006b).


Huang L, Wang J, Nie W, Su W, Yang F: Tandem chromosome fusions in karyotypic evolution of Muntiacus: evidence from M. feae and M. gongshanensis. Chromosome Res 14:637– 647 (2006c). Huang L, Nesterenko A, Nie W, Wang J, Su W, et al: Karyotypic evolution of giraffes (Giraffa camelopardalis) revealed by cross-species chromosome painting with Chinese muntjac (Muntiacus reevesi) and human (Homo sapiens) paints. Cytogenet Genome Res 122:132– 138 (2008). Huang L, Jing M, Nie W, Robinson TJ, Yang F: Chromosome homologies between tsessebe (Damaliscus lunatus) and Chinese muntjac (Muntiacus reevesi) facilitate tracing the evolutionary history of Damaliscus (Bovidae, Antilopinae, Alcelaphini). Cytogenet Genome Res 132:264–270 (2011). Iannuzzi L, Di Meo GP, Perucatti A, Incarnato D, Schibler L, et al: Comparative FISH mapping of bovid X chromosomes reveals homologies and divergences between the subfamilies Bovinae and Caprinae. Cytogenet Cell Genet 89:171–176 (2000). Iannuzzi L, Di Meo GP, Perucatti A, Schibler L, Incarnato D, et al: Comparative FISH-mapping in river buffalo and sheep chromosomes: assignment of forty autosomal type I loci from sixteen human chromosomes. Cytogenet Cell Genet 94:43–48 (2001). Iannuzzi L, King WA, Berardino DD: Chromosome evolution in domestic bovids as revealed by chromosome banding and FISHmapping techniques. Cytogenet Genome Res 126:49–62 (2009). ISCNDB 2000: International System for Chromosome Nomenclature of Domestic Bovids. Cytogenet Cell Genet 92:283–299 (2001). Kim KS, Tanaka K, Ismail DB, Maruyama S, Matsubayashi H, et al: Cytogenetic comparison of the lesser mouse deer (Tragulus javanicus) and the greater mouse deer (T. napu). Caryologia 57:229–243 (2004). Kulemzina AI, Trifonov VA, Perelman PL, Rubtsova NV, Volobuev V, et al: Cross-species chromosome painting in Cetartiodactyla: reconstructing the karyotype evolution in key phylogenetic lineages. Chromosome Res 17:419–436 (2009). Kulemzina AI, Yang F, Trifonov VA, Ryder OA, Ferguson-Smith MA, Graphodatsky AS: Chromosome painting in Tragulidae facilitates the reconstruction of Ruminantia ancestral karyotype. Chromosome Res 19:531– 539 (2011). Matthee CA, Burzlaff JD, Taylor JF, Davis SK: Mining the mammalian genome for artiodactyl systematics. Syst Biol 50: 367–390 (2001). Melander Y, Hansen-Melander E: Chromosome studies in African wild pigs (Suidae, Mammalia). Hereditas 92:283–289 (1980).

Rubes /Musilova /Kopecna /Kubickova / Cernohorska /Kulemsina

Muir PD, Semiadi G, Asher GW, Broad TE, Tate ML, Barry TN: Sambar deer (Cervus unicolor) x red deer (C. elaphus) interspecies hybrids. J Hered 88:366–372 (1997). Murphy WJ, Pringle TH, Tess A, Crider TA, Springer MS, et al: Using genomic data to unravel the root of the placental mammal phylogeny. Genome Res 17:413–421 (2007). Musilova P, Kubickova S, Hornak M, Cernohorska H, Vahala J, Rubes J: Different fusion configurations of evolutionarily conserved segments in karyotypes of Potamochoerus porcus and Phacochoerus africanus. Cytogenet Genome Res 129:305–309 (2010). Nguyen TT, Aniskin VM, Gerbault-Seureau M, Planton H, Renard JP, et al: Phylogenetic position of the saola (Pseudoryx nghetinhensis) inferred from cytogenetic analysis of eleven species of Bovidae. Cytogenet Genome Res 122:41–54 (2008). O’Brien SJ, Menninger JC, Nash WG: Atlas of Mammalian Chromosomes (John Wiley & Sons Inc., Hoboken, NJ 2006). Pagacova E, Cernohorska H, Kubickova S, Vahala J, Rubes J: Centric fusion polymorphism in captive animals of family Bovidae. Conserv Genet 12:71–77 (2011). Petit P, de Meurichy: On the chromosomes of the okapi. Okapia johnstoni. Ann Genet 29:232– 234 (1986). Pinton P, Schibler L, Cribiu E, Gellin J, Yerle M: Localization of 113 anchor loci in pigs: improvement of the comparative map for humans, pigs, and goats. Mamm Genome 11: 306–315 (2000). Prothero DR, Foss SE: The Evolution of Artiodactyls (The Johns Hopkins University Press, Baltimore 2007). Randi E, Mucci N, Pierpaoli M, Douzery E: New phylogenetic perspectives on the Cervidae (Artiodactyla) are provided by the mitochondrial cytochrome b gene. Proc Biol Sci 265:793–801 (1998). Randi E, D’Huart JP, Lucchini V, Aman R: Evidence of two genetically deeply divergent species of warthog, Phacochoerus africanus and P. aethiopicus (Artiodactyla: Suiformes) in East Africa. Mamm Biol 67: 91–96 (2002). Rettenberger G, Klett C, Zechner U, Kunz J, Vogel W, Hameister H: Visualization of the conservation of synteny between humans and pigs by heterologous chromosomal painting. Genomics 26:372–378 (1995). Robinson TJ, Ropiquet A: Examination of hemiplasy, homoplasy and phylogenetic discordance in chromosomal evolution of the Bovidae. Syst Biol 60: 439–450 (2011).


Robinson TJ, Harrison WR, Ponce de León FA, Davis SK, Elder FF: A molecular cytogenetic analysis of X chromosome repatterning in the Bovidae: transpositions, inversions, and phylogenetic inference. Cytogenet Cell Genet 80:179–184 (1998). Ropiquet A, Gerbault-Seureau M, Deuve JL, Gilbert C, Pagacova E, et al: Chromosome evolution in the subtribe Bovina (Mammalia, Bovidae): The karyotype of the Cambodian banteng (Bos javanicus birmanicus) suggest that Robertsonian translocations are related to interspecific hybridization. Chromosome Res 16:1107–1118 (2008). Ropiquet A, Hassanin A, Pagacova E, GerbaultSeureau M, Cernohorska H, et al: A paradox revealed: karyotype evolution in the fourhorned antelope occurs by tandem fusion (Mammalia, Bovidae, Tetracerus quadricornis). Chromosome Res 18: 277–286 (2010). Rubes J, Pagacova E, Kopecna O, Kubickova S, Cernohorska H, et al: Karyotype, centric fusion polymorphism and chromosomal aberrations in captive-born mountain reedbuck (Redunca fulvorufula). Cytogenet Genome Res 116:263–268 (2007). Rubes J, Kubickova S, Pagacova E, Cernohorska H, Berardino DD, et al: Phylogenomic study of spiral-horned antelope by cross-species chromosome painting. Chromosome Res 16: 935–947 (2008). Rubes J, Pinton A, Bonnet-Garnier A, Fillon V, Musilova P, et al: Fluorescence in situ hybridization applied to domestic animal cytogenetics. Cytogenet Genome Res 126: 34–48 (2009). Schmitz A, Oustry A, Vaiman D, Chaput B, Frelat G, Cribiu EP: Comparative karyotype of pig and cattle using whole chromosome painting probes. Hereditas 128: 257–263 (1998). Shi L, Yang F, Kumamoto A: The chromosomes of tufted deer (Elaphodus cephalophus). Cytogenet Cell Genet 56:189–192 (1991). Slate J, Van Stijn TC, Anderson RM, McEwan KM, Maqbool NJ, et al: A deer (subfamily Cervinae) genetic linkage map and the evolution of ruminant genomes. Genetics 160: 1587–1597 (2002). Spaulding M, O’Leary MA, Gatesy J: Relationships of Cetacea (Artiodactyla) among mammals: increased taxon sampling alters interpretations of key fossils and character evolution. PLoS One 4:e7062 (2009). Su B, Wang YX, Lan H, Wang W, Zhang Y: Phylogenetic study of complete cytochrome b genes in musk deer (genus Moschus) using museum samples. Mol Phylogenet Evol 12: 241–249 (1999). Su B, Wang YX, Wang QS: Mitochondrial DNA sequences imply Anhui musk deer a valid species in genus Moschus. Zool Res 22: 169– 173 (2001).

Thomsen PD, Hoyheim B, Christensen K: Recent fusion events during evolution of pig chromosomes 3 and 6 identified by comparison with the babirusa karyotype. Cytogenet Cell Genet 73:203–208 (1996). Tikhonov VN, Troshina AI: Chromosome translocations in the karyotypes of wild boars Sus scrofa L. of European and Asian areas of USSR. Theor Appl Genet 45:304–308 (1975). Vermeesch JR, De Meurichy W, Van Den Berghe H, Marynen P, Petit P: Differences in the distribution and nature of the interstitial telomeric (TTAGGG)n sequences in the chromosomes of the Giraffidae, okapai (Okapia johnstoni), and giraffe (Giraffa camelopardalis): evidence for ancestral telomeres at the okapi polymorphic rob(5;26) fusion site. Cytogenet Cell Genet 72:310–315 (1996). Wang YX, Ma SL, Li CY: The taxonomy, distribution and status of forest musk deer in China, in Ohtaishi N, Sheng HL (eds): Deer of China: Biology and Management, pp 22–30 (Elsevier Science, Tokyo 1993). Wilson DE, Reeder DM: Mammal Species of the World (Johns Hopkins University Press, Baltimore 2005). Wurster DH, Benirschke K: Indian muntjac, Muntiacus muntjak: a deer with a low diploid chromosome number. Science 168: 1364– 1366 (1970). Yang F, Carter NP, Shi L, Ferguson-Smith MA: A comparative study of karyotypes of muntjacs by chromosome painting. Chromosoma 103:642–652 (1995). Yang F, O’Brien PC, Wienberg J, Neitzel H, Lin CC, Ferguson-Smith MA: Chromosomal evolution of the Chinese muntjac (Muntiacus reevesi). Chromosoma 106:37–43 (1997a). Yang F, Müller S, Just R, Ferguson-Smith MA, Wienberg J: Comparative chromosome painting in mammals: human and the Indian muntjac (Muntiacus muntjak vaginalis). Genomics 39:396–401 (1997b). Yang F, O’Brien PC, Wienberg J, FergusonSmith MA: Evolution of the black muntjac (Muntiacus crinifrons) karyotype revealed by comparative chromosome painting. Cytogenet Cell Genet 76:159–163 (1997c). Yang F, O’Brien PC, Wienberg J, FergusonSmith MA: A reappraisal of the tandem fusion theory of karyotype evolution in Indian muntjac using chromosome painting. Chromosome Res 5:109–117 (1997d). Yong HS: Complete Robertsonian fusion in the Malaysian Lesser Mouse Deer (Tragulus javanicus). Experientia 29:366–367 (1973). Zhou X, Xu S, Yang Y, Zhou K, Yang G: Phylogenomic analyses and improved resolution of Cetartiodactyla. Mol Phylogenet Evol 61: 255–264 (2011).


207



Chromosome Evolution in Perissodactyla V.A. Trifonov a P. Musilova b A.I. Kulemsina a a

Institute of Molecular and Cellular Biology, SB RAS, Novosibirsk , Russia; b Veterinary Research Institute, Brno, Czech Republic

Key Words Chromosome painting ⴢ Evolutionary new centromeres ⴢ Karyotype evolution ⴢ Perissodactyla

Abstract Comparative painting has provided a wealth of useful information and helped to reconstruct the pathways of karyotype evolution within major eutherian phylogenetic clades. New data have come from gene localizations, BAC mapping and high throughout sequencing projects that enrich and provide new details of genome evolution. Extensive research on perissodactyl genomes has revealed not only increased rates of chromosomal rearrangements, but also an exceptionally high number of centromere repositioning events in equids. Here were combined new physical mapping, comparative painting and genome sequencing data to refine the putative ancestral karyotype maps and to revise the previously proposed scenario of perissodactyl karyotype evolution. Copyright © 2012 S. Karger AG, Basel

The laurasiatherian order Perissodactyla comprises about 16 species of extant equids, tapirs and rhinoceroses as well as many more extinct species. The diversity and abundance of extinct perissodactyl fossil remains has © 2012 S. Karger AG, Basel 1424–8581/12/1374–0208$38.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com


made them very attractive for paleontologists; the morphological evolution of horse has been vividly discussed for over a century and became a classic illustration in textbooks and museum exhibitions. Extensive studies of perissodactyl genomes revealed unprecedentedly rapid rates of chromosomal evolution [Wichman et al., 1991; Trifonov et al., 2008]. Recently, increased interest was devoted to the subchromosomal evolution of equids. These papers have reported on evolutionary new centromeres (ENCs) or centromere repositionings [Carbone et al., 2006; Musilova et al., 2007, 2009] and rapidly evolving pericentromeric satellite DNA [Piras et al., 2010; Alkan et al., 2011]. Sequencing of the horse (Equus caballus, ECA) genome revealed a relatively high level of genome conservation and special attention was given to an ENC on chromosome ECA11 lacking satellite DNA [Wade et al., 2009]. The phylogenetic relationships within Perissodactyla were initially based on morphological characters [Prothero and Schoch, 1989] and later confirmed by molecular and cytogenetic studies [Oakenfull and Clegg, 1998; Norman and Ashley, 2000; Pitra and Veits, 2000; Tougard et al., 2001; Trifonov et al., 2008]; the most recent examination of all extant perissodactyl phylogeny and divergence times was based on comparison of 2 mitochondrial and 9 nuclear genes [Steiner and Ryder, 2011].

Vladimir A. Trifonov Institute of Molecular and Cellular Biology SB RAS RU–630090 Novosibirsk (Russia) Tel. +7 383 333 3857, E-Mail vlad @ mcb.nsc.ru

In this review we integrate recently published data to revise the boreoeutherian ancestral (BEA) karyotype taking into consideration the horse sequencing data. We discuss the general scenario of chromosomal evolution within Perissodactyla and analyze the evolution of individual conserved perissodactyl elements with a special attention to ENC emergence.

Revision of the Putative Boreoeutherian Ancestral Karyotype

New data coming from genome sequencing projects prompted us to revise the existing schemes of ancestral karyotypes. Some associations found in comparative painting experiments turned out to be homoplasies, in some cases we observed different fusion positions. On the other hand, some novel fragments, missing in painting experiments due to their tiny size, appeared to be highly informative as phylogenetic markers. The association HSA1q/10q initially was found in horse, zebra and dog by comparative chromosome painting [Yang et al., 1999, 2003b, 2004]. The boreoeutherian element representing a syntenic association HSA1q/10q (actually including segments HSA1q42.1–q43 and HSA10p11.21–qter) was determined after precise mapping of the breakpoints based on full genome assemblies of pig, dog, cattle, cat, rat and mouse [Murphy et al., 2005]. Comparative painting and genome sequencing data confirmed the presence of the association in perissodactyls [Yang et al., 2003b, 2004; Trifonov et al., 2008; Wade et al., 2009]. Absence of this syntenic association in rabbit (www.ensembl.org) may be due to insufficient data. At the current stage we can hypothesize that this synteny may also be characteristic for chiropterans and insectivorans, but it was not revealed in comparative painting experiments due to the tiny size of the HSA1q segment. The synteny HSA1q/10q is not observed in sequenced genomes of afrotherians and marsupials, and it can be hypothesized that it has originated in the boreoeutherian ancestor after the divergence of afrotherians. Another controversial association is HSA10p/12/22. It was proposed by Yang et al. [2003a] and Froenicke [2005] based on the presence in felids, mustelids and afrotherians. However, this association is missing in most other laurasiatherians studied, and in felids and mustelids the association is linked through the centromere. Besides, this association is missing in Euarchontoglires and Xenarthra, but is present in marsupials. We propose that the fusion found in felids and mustelids represents rather a Chromosome Evolution in Perissodactyla

homoplasic event. The data on the position of the centromere in relation to the association in felids and mustelids as well as detailed fusion analysis in marsupials and carnivores will clarify the situation. We still leave the association HSA4/8p as a continuous syntenic block characteristic for the boreoeutherian ancestor, although a detailed analysis of breakpoints and homologous blocks hints to the appearance of 2 independent segments (HSA4ⴕ/8pⴕ and HSA4ⴕⴕ/8pⴕⴕ; partial chromosomes/chromosome arms are marked by a prime for the first segment and by a double prime for the second segment) corresponding to the elements of the perissodactyl ancestral karyotype (PAK), PAK1 and PAK26, since we find similar elements in canine and cattle genomes (www.ensembl.org). These elements might have fused only in felids and mustelids through the centromere [Graphodatsky et al., 2002; Murphy et al., 2007]. Finally, we made 2 important changes in the BEA karyotype: BEA13 [Froenicke, 2005] could be homologous to HSA10pⴕⴕq/1q part, and BEA7 [Froenicke, 2005] should be split into BEA7 (HSA12pq/22qter) and BEA22 (HSA10pⴕ) (fig. 1). We still fail to find any specific karyotype traits that are characteristic for laurasiatherians, and hypothesize the absence of any gross karyotypic changes during the formation of the laurasiatherian ancestral karyotype.

Ungulata and Perissodactyla Ancestral Karyotypes

As we hypothesized previously, the perissodactyl ancestral karyotype comprised 36 pairs of autosomes [Trifonov et al., 2008], i.e. 2n = 74–78 (there might have been 2 ancestral polymorphisms, PAK1 and PAK18, discussed below). Fourteen fissions and a fusion are necessary to convert the laurasiatherian (boreoeutherian) ancestral karyotype into the perissodactyl ancestral karyotype (fig. 1). The number of fissions was remarkably high (each of the 8 largest elements has been broken at least once, while only 1 of the 11 smallest elements underwent a fission), which made the diploid number of the perissodactyl ancestral karyotype much higher than the ancestral karyotype of any other eutherian order. A similar phenomenon occurred in canids, where the ancestral canid karyotype was derived from the ancestral carnivore karyotype by 42 fissions and 25 fusions [Graphodatsky et al., 2002]. In our previous publications it was proposed that the perissodactyl syntenic association HSA5/19p is common Cytogenet Genome Res 2012;137:208–217

209

Fig. 1. Scheme of the putative BEA karyotype with homologies to the human (HSA; left, black) and perissodactyl ancestral karyotype (PAK; right, red). Black arrows indicate fissions and red asterisks indicate fusion positions.

to artiodactyls and can serve as a signature uniting these 2 mammalian orders [Yang et al., 2006; Balmus et al., 2007]. However, horse genome sequencing data clearly indicate that 2 different segments of HSA5 are involved in association with the HSA19p segment in horse and cattle (HSA5q13.2 and HSA5q35.3, respectively). Therefore, we should consider these events as an apparent homoplasy. The HSA5/19p synteny was also found in a bat species, Mormopterus planiceps [Volleth et al., 2002]. HSA19p/4 is an ancestral synteny characteristic for all representatives of Chiroptera studied, including the basal Pteropodidae family. It is only Vespertilionidae and Molossidae families, where the HSA19p/4 segment was found to be fused with HSA5 homologous element, thus it was considered as a derived character [Volleth et al., 2002]. Another proposed common interordinal feature, the fission of BEA6 (HSA6p/6q), also turned out to be a homoplasy since the breakpoint, although close, is not at the same position in artiodactyls and perissodactyls [Wade et al., 2009]. Further research is needed for Chiroptera. The only specific cytogenetic association landmark for perissodactyls is HSA19p/5q (a closer look reveals 210


that this association is HSA19p13.11/1q44/5q13.2 on the contiguous boundary; www.ensembl.org). Additionally, there are 14 common derived fissions.

An Evolutionary Scenario of Major Chromosomal Rearrangements in Perissodactyls

The ceratomorph ancestral karyotype differs from the perissodactyl ancestral karyotype only by 2 fissions (PAK11 = HSA8q and PAK18 = HSA16q/19q) and comprises 78–80 chromosomes (due to PAK1 polymorphism). The synteny PAK1 (HSA4/8p/4) is characteristic for a common boreoeutherian ancestor (BEA2). It is conserved in Malayan tapir, zebras, kulan and donkey, but disrupted at the same position in all other tapirs, rhinoceroses, onager, kiang and horse. Actually, chromosome painting was only done on a few individuals and it is necessary to add that there is a Robertsonian polymorphism of PAK1 detected in extant populations of Asiatic asses, Somali wild ass and Burchell’s zebra [Myka et al., 2003]. We believe that the current situation represents a typical example of hemiplasy or ancient polymorphism, where the Trifonov /Musilova /Kulemsina

same elements were fixed in fused or disrupted state in different species. The fission of the PAK1 synteny differentiates the New World tapirs from the common ceratomorph ancestor. Karyotypes of Baird’s (Tapirus bairdii, TBA) and lowland tapirs (Tapirus terrestris, TTE) most closely resemble the putative perissodactyl ancestral karyotype. Metacentric elements in these species (TBA1, 2, 7, 8, TTE1) result from either inversions or centromeric shifts of ancestral acrocentrics. The mountain tapir (Tapirus pinchaque, TPI) karyotype has 2 specific fusions (TPI1 = PAK13/35 and TPI3 = PAK11ⴕ/PAK18ⴕ), whereas the PAK13/35 fusion is the only non-Robertsonian translocation among tapirs. In the lineage leading to the only extant Old World tapir (T. indicus) 13 Robertsonian fusions occurred (PAK2/17, 6/27, 21/9, 15/4, 25/3, 24/7, 30/5, 18ⴕⴕ/10, 29/23, 18ⴕ/20, 36/11ⴕ, 8/19, 14/35). The common ancestor of African rhinoceroses had a karyotype identical to the contemporary white rhinoceros karyotype (2n = 82) and it differed from the common ceratomorph ancestor by 2 fissions – PAK1 and PAK7. The fission of PAK2 is characteristic for the black rhinoceros (Diceros bicornis, DBI) karyotype, forming the smallest element (DBI41) and further increasing the diploid number. The putative ancestral equid karyotype had 70–76 chromosomes and differed from the perissodactyl ancestral karyotype by only 2 fusions (ECA1 = PAK4/8 and ECA7 = PAK9/36). These associations were found in all equids and are thus cytogenetic family-specific landmarks. There were probably 3 putative polymorphic associations in the equid ancestor (PAK1, PAK3 and PAK18). Therefore, 2 centric fissions (PAK1 and PAK18) and 5 centric fusions (PAK21/1ⴕ, 18ⴕ/1ⴕⴕ, 32/15, 31/17, 18ⴕⴕ/22 resulting in ECA2, 3, 6, 8, 10, respectively) differentiate the karyotypes of the equid ancestor and domestic horse (2n = 64). The karyotype of Przewalski’s horse has acquired an additional fission (PAK3). A non-Robertsonian fusion (PAK2/34 = ECA4p/ 31) and a centric fission (PAK3 = ECA5p/q) are common for non-caballine equids (zebras and asses). The association PAK2/34 = HSA7/6 could be considered as a cytogenetic signature for this group. According to new molecular phylogenetic data, the donkey takes a basal position within the non-caballine equids [Steiner and Ryder, 2011]. Thus we need to revise our previous data, where it took sister group relationships with zebras, which was supported by a fusion (PAK13/ 31 = ECA8p/20) and globin gene analysis [Oakenfull and Clegg, 1998]. Since the fusion is missing in Asiatic asses,

we assume a polymorphic state of the PAK13/31 element in the non-caballine ancestor (2n = 68–74). Four fusions (PAK10/27, 21/14, 24/17, 19/29) separate the donkey karyotype (2n = 62) from the common non-caballine ancestor, the polymorphic elements PAK1 and PAK13/31 were fixed in a fused state while PAK18 underwent a centric fission. The ancestral karyotype of the Asiatic asses (2n = 54– 56 due to the PAK1 polymorphism) was formed from the common non-caballine ancestor by a centric fission (PAK18) and 9 fusions (PAK10/5, 14/15, 12/31, 27/3ⴕⴕ, 22/26, 18ⴕ/19, 21/25, 20/24, 3ⴕ/32). The elements PAK13 and 31 are not fused. The karyotypes of kulan and onager currently preserved the state ancestral for Asiatic asses, while 2 additional fusions (PAK17/7 = ECA8q/15 and PAK9/36/29 = ECA7/25) occurred in the karyotype of kiang (2n = 52) [Musilova et al., 2009]. The common ancestral karyotype of zebras differs from the common non-caballine ancestral karyotype by 4 fusions: PAK7/21, 32/35, 15/29/6 (besides, PAK1, PAK13/31 and PAK18 are fused). Burchell’s and Grevy’s zebras are in a sister clade, having 3 common fusions: PAK12/20, 22/16 and 5/25. The mountain zebra has a basal position among zebras and its karyotype differs from the common zebras’ ancestral karyotype by 15 fusions and a fission of PAK2 [Yang et al., 2003b]. Interstitial telomeric sites have been preserved at some fusion points in mountain zebra (PAK16/26, PAK30/2, PAK6/29, PAK27/32 and PAK20/28) [Santani et al., 2002]. The phylogenetic relationships within perissodactyls are generally not controversial and consistent [Oakenfull and Clegg, 1998; Norman and Ashley, 2000; Pitra and Veits, 2000; Tougard et al., 2001; Steiner and Ryder, 2011]. An addition to the general scenario of previously unstudied equid species (kiang and kulan) further confirms the tree topology. Divergence times have also been revised recently, what refined our previous data on the rearrangement rates along the phylogenetic lineages [Steiner and Ryder, 2011].

Chromosome Evolution in Perissodactyla


A Short Survey of Perissodactyl Ancestral Elements and Evolutionary New Centromeres

Below we discuss the history of individual ancestral perissodactyl chromosomes and try to trace the history of the revealed centromere repositionings. We superimposed the events of ENC emergence onto the revised perissodactyl phylogenetic tree [Steiner and Ryder, 2011] to estimate the frequency of centromere repositionings in various lineages (summarized in fig. 2). 211

A

B

Fig. 2. The most recent phylogenetic trees of perissodactyls (A) and equids (B) ac-

cording to Steiner and Ryder [2011] against a geological time scale with possible (red circles) and confirmed (green circles) evolutionary new centromeres (ENCs) and ancestral Robertsonian polymorphic elements (blue). Numbers in squares indicate diploid numbers. CSI, white rhinoceros; DBI, black rhinoceros; DSU, Sumatran rhinoceros; EAS, donkey; EBU, Burchell’s zebra; ECA, horse; EGR, Grevy’s zebra; EHE, onager; EHK, kulan; EKI, kiang; EPR, Przewalski’s horse; EZE, mountain zebra; PAK, perissodactyl ancestral karyotype; TBA, Baird’s tapir; TIN, Malayan tapir; TPI, mountain tapir; TTE, lowland tapir; RUN, Indian rhinoceros.

212


Trifonov /Musilova /Kulemsina

PAK1 (the largest part of synteny HSA4/8p = BEA2) might have been the largest element of the perissodactyl ancestral karyotype. Probably it was polymorphic and preserved a fused state in zebras, donkey, kulan and the Malayan tapir. We did not detect any centromere repositioning events on this element. The centromere preserved satellite DNA in donkey and Burchell’s zebra, but not in Grevy’s zebra [Piras et al., 2010]. PAK2 (HSA7pprox,7qmed,7qdist). The synteny corresponds to the largest boreoeutherian ancestral chromosome (BEA9) conserved in toto. This conserved synteny is disrupted in the mountain zebra (the breakpoint corresponds to horse ECA4 centromere) and in the black rhino (the breakpoint is not mapped yet). PAK2 probably was acrocentric and this state (including the conserved centromere position) is observed in the white rhinoceros and all tapirs except the Malayan, where it is fused by Robertsonian translocation. It seems that there was an inversion (or a centromere shift) in the ancestor of equids, followed by the translocation of ECA31 with subsequent centromere inactivation. It is noteworthy that Equus grevyi has lost all pericentromeric repetitive sequences in the centromere, but preserved probably the ancestral inactive block on pter homologous to ECA31, while E. grevyi seems to lack any pericentromeric sequences on PAK2 homologue [Piras et al., 2010]. PAK3 (HSA1pprox,1q prox,1qmedⴕⴕ). The synteny represents the largest part of BEA1. It is disrupted in all equids except domestic horse, but well preserved in all ceratomorphs. The element is acrocentric in rhinos, but metacentric in horse and tapirs except the Malayan tapir. Thus we can assume centromere repositioning events in the ancestor of New World tapirs and in the equid ancestor. There was a polymorphism of ECA5p/5q in the ancestor of horse and Przewalski’s horse, with a fused state fixed in domestic horse and disrupted state in the Przewalski’s horse. An ENC or inversion is evident in submetacentric chromosomes corresponding to ECA5q in donkey and Burchell’s zebra. PAK4 represents the largest part of BEA5 (HSA14/15). It remains autonomous in rhinos and most tapirs except the Malayan tapir and is fused with PAK8 in the equid ancestor. The centromere is located on the terminal part of the HSA15 homologous segment in most ceratomorphs and mountain zebra. A centromere repositioning (or an inversion?) took place only in the Baird’s tapir [Trifonov et al., 2008]. The fragment has lost the centromeric function in most equids. PAK5 is the largest part of BEA4 (HSA5qter). It is quite conserved in perissodactyl evolution. It remained auton-

omous in most ceratomorphs, horse and donkey, but fused with other elements in Asiatic asses, zebras and the Malayan tapir. An ENC has appeared in donkey, although the satellite blocks are still preserved in the old centromeric position [Piras et al., 2010]. The centromere position in ceratomorphs, Asiatic asses and in the Burchell’s zebra is horse-like. In Grevy’s and mountain zebras the fragment has lost the centromeric function due to a tandem fusion. PAK6 is the largest part of BEA3 (HSA3pter,3qmed). It is conserved in all perissodactyls. The centromere position was conserved in horse and donkey (and it looks similar in tapirs and rhinos), while in the Asiatic asses an ENC turned this element into a metacentric [Musilova et al., 2009]. In all zebras the centromere became inactive. PAK7 is homologous to BEA10 (HSA2pq prox). The element maintains synteny in all perissodactyls except rhinocerotids, where it is broken into 2 chromosomes. The centromere position seems to be rather conserved. It may be only changed in donkey, onager and kulan, where either centromeric shift or pericentric inversion occurred and transformed the acrocentric into a subtelocentric element. PAK7 has fused with different elements in all zebras, kiang and Malayan tapir. Fission of PAK7 in the rhinocerotid ancestor has formed the smallest white rhinoceros (Ceratotherium simum, CSI) autosome, CSI40, homologous to the proximal part of ECA15. PAK8 represents a conserved BEA13 (HSA1qsubdist/ 10q part). The synteny is well conserved in all perissodactyls. In the ancestor of equids it has fused with PAK4, followed by a centromeric shift or a pericentric inversion. The ancestral position of the centromere is preserved in the mountain zebra (at the site of PAK4/8 fusion). PAK9 is the largest part of BEA12 (HSA11pmed,11qter). It is conserved in all perissodactyls including the centromere position. This syntenic group is fused with PAK36 in equids and has lost the centromere in some of them (donkey, kiang and probably Burchell’s zebra). A pericentric inversion has occurred in the caballines. A block of centromeric heterochromatin may mark an ancient PAK9/36 fusion in chromosome 14 of Burchell’s zebra. PAK10 is a part of BEA8 (HSA2qmed). It is conserved in all perissodactyls, has acrocentric morphology in ceratomorphs and horse, and has fused with other elements in asses, zebras and Malayan tapir, whereas only in donkey it has lost the centromeric function. PAK11 corresponds to BEA15 (HSA8q). It is conserved in equids as a metacentric with a conserved centromere position which probably represents the ancestral morphology. A centromeric fission has occurred in the ances-



213

tor of ceratomorphs. The resulting elements remained conserved, but the smallest one (ECA9p) underwent a Robertsonian fusion in the Malayan tapir; the largest element (ECA9q) underwent either inversion or centromere repositioning in the Baird’s tapir. PAK12 is the well-conserved BEA14 (HSA13). The element has retained a conserved centromere position in most perissodactyls, but has undergone either inversion or neocentromere activation in Baird’s tapir, and there was a centromere repositioning in donkey [Carbone et al., 2006]. The segment underwent fusions in Asiatic asses, Burchell’s zebra and Grevy’s zebra. PAK13 is a part of BEA6 (HSA6pq prox). The segment is conserved in all perissodactyls. Interestingly, the morphology of the element is acrocentric in horse, Baird’s tapir, lowland tapir and rhinos, and it represents an arm of a metacentric with a conserved centromere position in all zebras. A new centromere occurs in the same (or similar) position in asses, Malayan and mountain tapirs, making this element submetacentric. PAK14 is part of BEA3 (HSA3q prox,3qdist). As most other elements, it is autonomous in horse and all ceratomorphs except the Malayan tapir, but underwent Robertsonian translocations in asses, zebras and the Malayan tapir. The centromere position is conserved except in the donkey where centromere repositioning or inversion took place. PAK15 is the largest part of BEA7 homologous to HSA12pq. The segment seems to retain the centromere position in all perissodactyls. Interestingly, this conserved centromere region has lost heterochromatic blocks in donkey and in Grevy’s zebra, but it was preserved in Burchell’s zebra [Piras et al., 2010]. PAK16 represents a conserved ancestral BEA16 element (HSA17). It is well conserved in all perissodactyls. The centromere of submetacentric ECA11 is the only equine centromere lacking the detectable satellites [Wade et al., 2009; Piras et al., 2010]. In the Asiatic asses 2 different regions (located on ECA11q) took over the centromeric function [Musilova et al., 2009]. Whereas in the kulan, onager and donkey the chromosome is submetacentric (with short p-arms), a terminal centromere position is conserved in kiang, Grevy’s zebra and Burchell’s zebra (centromeric region corresponds to the terminal region of ECA11) and this may represent an ancestral state. The fragment has lost the centromeric function in the mountain zebra. The centromere has been precisely located in horse, revealing a striking example of a newly evolved centromeric region [Wade et al., 2009]. The centromere position is also unstable in ceratomorphs: the el214


ement is acrocentric in rhinos and most tapirs, but submetacentric in the Malayan tapir. PAK17 represents a conserved synteny with BEA17 (HSA18). Horse sequencing data demonstrate that the gene order on HSA18q is identical in horse and human, which is surprising considering a long phylogenetic distance between species and changed gene orders in cattle and dog [Wade et al., 2009]. The centromere position seems to be conserved in all perissodactyls studied except for the donkey where the segment underwent a pericentric inversion [Carbone et al., 2006]. PAK18 represents a conserved synteny with BEA18 (HSA16q/19q). This might be another polymorphic pair in the perissodactyl ancestral karyotype, since it has been preserved in a fused state only in zebras. On the other hand, it might be a case of homoplasy, where 2 separate fragments corresponding to HSA16 and HSA19 have fused in the ancestor zebras. Noteworthy, that a similar breakpoint is characteristic for canids and myomorph rodents. In zebras only PAK18ⴕ (ECA3p) region preserves the centromere. PAK18ⴕⴕ (ECA10p) segment has conserved centromere position in horse, donkey and ceratomorphs, but there is a centromeric repositioning in Asiatic asses [Musilova et al., 2009]. It is difficult to elucidate the centromere evolution of the segment PAK18ⴕ, but at least in horse and Asiatic asses the centromere position is preserved. PAK19 consists of 2 fused fragments of BEA4 and BEA23 (HSA5pq prox/19pprox). It is the result of the only fusion occurring during the formation of the perissodactyl ancestral karyotype. The centromere is located in a region homologous to HSA19p and is conserved in equids and probably ceratomorphs. PAK20 represents a part of BEA11 (HSA9pdist,9q prox). It is conserved in all perissodactyls. Changes of centromeric position have not been detected so far. PAK21 is a part of BEA1 (HSA1pdist). The centromeric position appears to differ between equids and most ceratomorphs according to GTG banding patterns. It seems similar in the Malayan tapir and most equids on the one hand and in donkey and rhinos/most tapirs on the other hand. In Asiatic asses the segment has lost the centromere, being part of the distal q-arm. In Grevy’s and Burchell’s zebras the fragment has retained a block of satellites in the old centromeric position. PAK22 is a part of BEA6 (HSA6q). It is conserved in all perissodactyls (changes of centromeric position have not been detected so far). The segment underwent fusion in all equids except donkey.


PAK23 is a well conserved BEA19 (HSA20), with the gene order perfectly conserved between horse and dog. The fragment is acrocentric in horse, and zebras seem to have a similar centromere position although there is a tiny inversion or a centromere repositioning in Burchell’s zebra according to Carbone et al. [2006]. There was both a centromere repositioning and an inversion in asses, making this autonomous element submetacentric. Again we observe a difference in centromere location in tapirids and rhinos according to GTG banding. PAK24 is a part of BEA5 (HSA14q prox). The centromeric position is highly variable in equids. The horse-like position of the centromere might be ancestral since it looks similar in ceratomorphs and Asiatic asses. However, in the Grevy’s and Burchell’s zebras there was an inversion or a centromeric shift, making this element submetacentric [Musilova et al., 2007]. The segment has lost centromeric function in the mountain zebra and in the donkey [Carbone et al., 2006]. PAK25 is conserved BEA20 (HSA16p/7pdist,7q prox, 7qmed). The element is submetacentric in horse and donkey, and the position of the centromere did not change after fusions with other elements in Asiatic asses. There may be a changed centromeric position in zebras where the chromosome is fused with other segments. There is a difference in centromere location in tapirids (HSA7 segment) and rhinos (HSA16 segment) according to the results of human probe localization. PAK26 is a part of BEA2 (HSA4qter/8pprox,8pmed). The element is conserved in all perissodactyls and represents a separate element in most species (except Asiatic asses and the mountain zebra). There was either an inversion or centromere repositioning in donkey, making this element submetacentric. PAK27 is conserved BEA21 (HSA22qter/12qmed). A centromeric position is conserved and located in the HSA12-homologous region in horse, Asiatic asses, zebras and most ceratomorphs (except the Malayan tapir). As the centromere transposition was excluded in donkey [Carbone et al., 2006], we assume a transposition of a centromere to the distal part of the segment (HSA22) in the Malayan tapir. A large subtelomeric cluster of typically centromeric satellites is located in the distal part of the segment (adjacent to HSA22 homology block) in donkey [Piras et al., 2010]. PAK28 represents the smallest part of BEA3 (HSA3pprox/21). The position of the centromere is conserved in ceratomorphs, horse and Grevy’s and Burchell’s zebras (in a region homologous to HSA3qprox), but there is a common centromere repositioning in asses [Carbone et

al., 2006; Musilova et al., 2009] and centromere function loss in mountain zebra (the ancient centromeric region corresponds to the telomeric part). Interestingly, in donkey the ENC is missing satellite DNAs [Piras et al., 2010]. PAK29 is the smallest part of BEA11 (HSA9pprox,9qdist). The position of the centromere seems to be conserved in horse and Asiatic asses. It is difficult to trace the centromere position in zebras, donkey and ceratomorphs due to the small size of the segment. PAK30 represents a conserved BEA22 segment (HSA10p). The position of the centromere seems to be conserved in horse, donkey and Asiatic asses and Grevy’s zebra, where the element remains autonomous. Additional experiments are needed to trace the centromere position in zebras and ceratomorphs. The centromere was lost in the Burchell’s zebra. PAK31 is conserved BEA21 (HSA12qter/22q prox). In equids the element is fused with other segments by either Robertsonian (horse, Burchell’s zebra, Asiatic asses) or tandem translocations (mountain zebra). It could have been a Robertsonian translocation followed by a centromere shift in the donkey, resulting in the loss of the centromere function in the segment. The position of the centromere corresponds to an HSA12 segment in most equids (except donkey and the mountain zebra) and to an HSA22 segment in ceratomorphs and the mountain zebra. PAK32 is homologous to HSA2qter and corresponds to the smallest part of BEA8. The fragment is autonomous in donkey (although it does not contain any typical satellite DNA) [Piras et al., 2010] and ceratomorphs, constitutes p-arms in horse and Asiatic asses and has lost the centromere in all zebras. Inverted orientation of the segment in fusions implies different centromere positions in horse and Asiatic asses [Musilova et al., 2009]. A centromere repositioning, making this element metacentric, was detected in donkey [Carbone et al., 2006]. PAK33 is the smallest part of BEA1 (HSA1qter). It is autonomous in all perissodactyls except Burchell’s and mountain zebras. The centromere seems to be conserved except in asses, where a centromere shift turned the chromosome into a (sub)metacentric, and Burchell’s zebra, where the segment lost the centromeric function. PAK34 represents the smallest part of BEA6. It is autonomous in all perissodactyls except asses and zebras where it has lost a centromeric function. An additional inversion seems to disrupt this element in donkey [Yang et al., 2004]. PAK35 represents a part of BEA12. It remains autonomous in horse, asses, 2 tapirs and rhinos, but it gets fused



215

with larger elements in the Mayalan tapir and mountain tapir (it is 1 of 2 fusions that recently occurred in the mountain tapir karyotype) and in zebras. The chromosome is metacentric in horse and asses with a conserved centromere position. In addition, the centromeric position is conserved in Grevy’s and Burchell’s zebras [Musilova et al., 2007]; however, the segment has lost the centromere function in mountain zebra. The chromosome is acrocentric in most ceratomorphs. PAK36 (HSA19pⴕ) is the largest part of BEA23. The chromosome remains autonomous in rhinos and all tapirs except for the Malayan tapir. It has fused with PAK9 in the equid ancestor, forming a conserved equid element and retaining the centromere position. Different positions of the centromere can be observed in equids. A pericentric inversion has occurred in horse, disrupting the PAK36 synteny [Yang et al., 2003b; Musilova et al., 2007]. The element represents the smallest autosome in the New World tapirs.

donkey lineage). In fact, the number is probably much higher than that reported in this review because many tiny centromeric repositionings could have escaped our observation and will be revealed only by detailed comparative mapping. The rates of chromosomal rearrangements do not strictly correlate with the rates of ENC emergence, although it seems that in rhinoceroses both processes went very slow, while in asses both rates were exceptionally high. It is noteworthy that we did not detect any ENCs in horse, except the one that was previously intensively studied. As it was shown in the horse sequencing project, neocentromere activation is an ongoing process in equids with some centromeres appearing in generich regions and lacking any characteristic satellite DNA accumulation [Wade et al., 2009]. Such changes might have had a considerable impact on the rapid speciation within the genus. Concerning ceratomorphs, we still lack information on precise centromere localizations, but it seems that the general amount of ENCs was much lower. There might be similar locations of independently evolved ENCs in representatives of some equids and tapirids.

Conclusion

In addition to previous conclusions on genome evolution in perissodactyls, we should emphasize that new centromere activation events have played a large role. Even between recently diverged equids the frequency of chromosome repositioning events is very high (24 ENCs might have emerged in non-caballine equids, i.e. ENC fixation rates were around 0.2 per million years in the

Acknowledgements This work was partially supported by MCB, SB RAS Programs, research grants of Russian Fund for Basic Research and by the Grant Agency of the Czech Republic, project 523/09/1972. Authors thank Roscoe Stanyon and Aleksey Makunin for useful comments on the manuscript.

References Alkan C, Cardone MF, Catacchio CR, Antonacci F, O’Brien SJ, et al: Genome-wide characterization of centromeric satellites from multiple mammalian genomes. Genome Res 21: 137–145 (2011). Balmus G, Trifonov VA, Biltueva LS, O’Brien PC, Alkalaeva ES, et al: Cross-species chromosome painting among camel, cattle, pig and human: further insights into the putative Cetartiodactyla ancestral karyotype. Chromosome Res 15: 499–515 (2007). Carbone L, Nergadze SG, Magnani E, Misceo D, Cardone MF, et al: Evolutionary movement of centromeres in horse, donkey, and zebra. Genomics 87:777–782 (2006). Froenicke L: Origins of primate chromosomes – as delineated by Zoo-FISH and alignments of human and mouse draft genome sequences. Cytogenet Genome Res 108: 122–138 (2005).

216

Graphodatsky AS, Yang F, Perelman PL, O’Brien PC, Serdukova NA, et al: Comparative molecular cytogenetic studies in the order Carnivora: mapping chromosomal rearrangements onto the phylogenetic tree. Cytogenet Genome Res 96:137–145 (2002). Murphy WJ, Larkin DM, Everts-van der Wind A, Bourque G, Tesler G, et al: Dynamics of mammalian chromosome evolution inferred from multispecies comparative maps. Science 309:613–617 (2005). Murphy WJ, Davis B, David VA, Agarwala R, Schaffer AA, et al: A 1.5-Mb-resolution radiation hybrid map of the cat genome and comparative analysis with the canine and human genomes. Genomics 89: 189–196 (2007).


Musilova P, Kubickova S, Zrnova E, Horin P, Vahala J, Rubes J: Karyotypic relationships among Equus grevyi, Equus burchelli and domestic horse defined using horse chromosome arm-specific probes. Chromosome Res 15:807–813 (2007). Musilova P, Kubickova S, Horin P, Vodicka R, Rubes J: Karyotypic relationships in Asiatic asses (kulan and kiang) as defined using horse chromosome arm-specific and regionspecific probes. Chromosome Res 17: 783– 790 (2009). Myka JL, Lear TL, Houck ML, Ryder OA, Bailey E: Homologous fission event(s) implicated for chromosomal polymorphisms among five species in the genus Equus. Cytogenet Genome Res 102:217–221 (2003). Norman JE, Ashley MV: Phylogenetics of Perissodactyla and tests of the molecular clock. J Mol Evol 50:11–21 (2000).


Oakenfull EA, Clegg JB: Phylogenetic relationships within the genus Equus and the evolution of alpha and theta globin genes. J Mol Evol 47:772–783 (1998). Piras FM, Nergadze SG, Magnani E, Bertoni L, Attolini C, et al: Uncoupling of satellite DNA and centromeric function in the genus Equus. PLoS Genet 6:e1000845 (2010). Pitra C, Veits J: Use of mitochondrial DNA sequences to test the Ceratomorpha (Perissodactyla: Mammalia) hypothesis. J Zool Syst Evol Res 38:65–72 (2000). Prothero DR, Schoch RM: Origin and evolution of the Perissodactyla: summary and synthesis; in Prothero DR, Schoch RM (eds): The Evolution of Perissodactyls, pp 504–529 (Oxford University Press, New York 1989). Santani A, Raudsepp T, Chowdhary BP: Interstitial telomeric sites and NORs in Hartmann’s zebra (Equus zebra hartmannae) chromosomes. Chromosome Res 10:527–534 (2002). Steiner CC, Ryder OA: Molecular phylogeny and evolution of the Perissodactyla. Zool J Linn Soc 163:1289–1303 (2011).


Tougard C, Delefosse T, Hanni C, Montgelard C: Phylogenetic relationships of the five extant rhinoceros species (Rhinocerotidae, Perissodactyla) based on mitochondrial cytochrome b and 12S rRNA genes. Mol Phylogenet Evol 19:34–44 (2001). Trifonov VA, Stanyon R, Nesterenko AI, Fu B, Perelman PL, et al: Multidirectional crossspecies painting illuminates the history of karyotypic evolution in Perissodactyla. Chromosome Res 16: 89–107 (2008). Volleth M, Heller KG, Pfeiffer RA, Hameister H: A comparative ZOO-FISH analysis in bats elucidates the phylogenetic relationships between Megachiroptera and five microchiropteran families. Chromosome Res 10:477– 497 (2002). Wade CM, Giulotto E, Sigurdsson S, Zoli M, Gnerre S, et al: Genome sequence, comparative analysis, and population genetics of the domestic horse. Science 326:865–867 (2009). Wichman HA, Payne CT, Ryder OA, Hamilton MJ, Maltbie M, Baker RJ: Genomic distribution of heterochromatic sequences in equids: implications to rapid chromosomal evolution. J Hered 82:369–377 (1991). Yang F, O’Brien PC, Milne BS, Graphodatsky AS, Solanky N, et al: A complete comparative chromosome map for the dog, red fox, and human and its integration with canine genetic maps. Genomics 62: 189–202 (1999).

Yang F, Alkalaeva EZ, Perelman PL, Pardini AT, Harrison WR, et al: Reciprocal chromosome painting among human, aardvark, and elephant (superorder Afrotheria) reveals the likely eutherian ancestral karyotype. Proc Natl Acad Sci USA 100:1062–1066 (2003a). Yang F, Fu B, O’Brien PC, Robinson TJ, Ryder OA, Ferguson-Smith MA: Karyotypic relationships of horses and zebras: results of cross-species chromosome painting. Cytogenet Genome Res 102:235–243 (2003b). Yang F, Fu B, O’Brien PC, Nie W, Ryder OA, Ferguson-Smith MA: Refined genome-wide comparative map of the domestic horse, donkey and human based on cross-species chromosome painting: insight into the occasional fertility of mules. Chromosome Res 12:65–76 (2004). Yang F, Graphodatsky AS, Li T, Fu B, Dobigny G, et al: Comparative genome maps of the pangolin, hedgehog, sloth, anteater and human revealed by cross-species chromosome painting: further insight into the ancestral karyotype and genome evolution of eutherian mammals. Chromosome Res 14: 283– 296 (2006).


217



Low, Complex and Probably Reticulated Chromosome Evolution of Sciuromorpha (Rodentia) and Lagomorpha F. Richard a, b B. Dutrillaux a a

Muséum National d’Histoire Naturelle, Département de Systématique et Evolution, Origine Structure et Evolution de la Biodiversité, Paris, and b Université de Versailles Saint Quentin en Yvelines, UFR de Sciences, Département de Biologie, Versailles, France

Key Words Ancestral karyotypes ⴢ Chromosomes ⴢ Glires ⴢ Lagomorpha ⴢ Leporid ⴢ Reticulated evolution ⴢ Sciurid ⴢ Sciuromorpha

Abstract Lagomorpha (rabbits and pikas) and Sciuromorpha (squirrels) are grouped in the Glires superorder. Their chromosome diversification, since their separation from the eutherian mammalian common ancestor, was characterized by a low rate of chromosome rearrangements. Consequently, the structure of some chromosomes was either conserved or only slightly modified, making their comparison easy at the genus, family and even order level. Interspecific in situ hybridization (Zoo-FISH) largely corroborates classical cytogenetic data but provides much more reliability in comparisons, especially for distant species. We reconstructed common ancestral karyotypes for Glires, Lagomorpha, Sciuromorpha, and Sciuridae species, and then, determined the chromosome changes separating these ancestors from their common eutherian ancestor. We propose that reticulated evolution occurred during the diversification of Glires, which implies that several pericentric inversions and Robertsonian translocations were conserved in the heterozygous status



for an extensive period. Finally, among Lagomorpha and Sciuromorpha, we focused on Leporidae and Sciuridae chromosome evolution. In the various attempts to establish dichotomic evolutionary schemes, it was necessary to admit that multiple homoplasies (convergent and reverse rearrangements) occurred in Sciuridae and in a lesser degree, in Leporidae. In Leporidae, additional rearrangements were sufficient to propose a resolved phylogeny. However, a resolved phylogeny was not possible for Sciuridae because most of the rearrangements occurred in terminal branches. We conclude that a reticulated evolution took place early during the evolution of both families and lasted longer in Sciuridae than in Leporidae. In Sciuridae, most chromosome rearrangements were pericentric inversions involving short fragments. Such rearrangements have only mild meiotic consequences, which may explain the long persistence of the heterozygous status characterizing reticulated evolution. Copyright © 2012 S. Karger AG, Basel

The orders Rodentia and Lagomorpha are grouped in the same taxon, the Glires superorder. They constitute a clade supported by morphological [Luckette and Hartenberger, 1993], chromosomal [Beklemisheva et al., 2011] Florence Richard Muséum National d’Histoire Naturelle, Département de Systématique et Evolution UMR7205 CNRS, Case postale 39, 16 Rue Buffon FR–75005 Paris (France) Tel. +33 140 795 341, E-Mail frichard @ mnhn.fr

and molecular data [Murphy et al., 2001a, b; Huchon et al., 2002; Prasad et al., 2008]. They shared a common ancestor with Primates, Dermoptera and Scandentia, which groups them in the Euarchotonglires clade, as demonstrated by molecular data [Murphy et al., 2001a, b; Huchon et al, 2002; Douzery and Huchon, 2004]. Lagomorpha constitutes a small order, with less than 100 living species, grouped into 2 families, the Ochotonidae (pikas, 30 living species) with a single genus divided in 3 subgenera and Leporidae (hares and rabbits, 91 living species) with 11 genera [Hoffmann and Smith, 2005; Yu et al., 2000]. In contrast, Rodentia constitute by far the largest order of mammals. They comprise about 42% of the mammalian living species, with more than 2,277 living species [Musser and Carleton, 2005]. Lagomorpha share many morphological characters, and constitute a fairly homogenous group, whereas morphotypes are more heterogeneous in rodents, which were classified into 5 suborders (Sciuromorpha, Castorimorpha, Myomorpha, Anomaluromorpha, and Hystrichomorpha). This difference suggests that evolution worked at very different speeds, low in Lagomorpha and fast in Rodentia. However, the diversity of Rodentia does not exclude the possibility that, among this fast-evolving order, some taxa evolved slowly and conserved ancestral characters. Sciuromorpha, divided in 3 families (Sciuridae, Aplodontidae and Gliridae) and composed of only 307 species [Musser and Carleton, 2005] is considered as either the first diverging branch of Rodentia [Huchon et al., 2002] or sharing a basal position with Hystrichomorpha [Montgelard et al., 2008]. Moreover, in the 1970s and early 1980s, chromosome banding comparison showed that the garden dormouse Eliomys quercinus (Gliridae) and, above all, several squirrel species (Sciuridae) shared many chromosomal homoeologies with man and other primates, whereas the karyotype of other rodent species, in particular that of Muridae, appeared to be very distinct [Dutrillaux et al., 1979; Petit et al., 1984]. Thus, among Rodentia, Sciuromorpha have conserved fairly ancestral chromosomes that evolved slowly compared to other rodents. Similar studies were performed in Lagomorpha, which showed that Leporids karyotypes were easy to compare with each other [Stock, 1976; Robinson, 1980; Robinson et al., 1984] and that many chromosomes of Oryctolagus cuniculus could be easily compared with those of man and other primates [Dutrillaux et al., 1980]. Thus, they are fairly conserved. Hence, Lagomorpha and Sciuromorpha, which maintained fairly ancestral characters, might have remained cytogenetically close to some slowly evolving mammals belonging to other or-

ders, favoring comparisons between orders and the reconstruction of ancestral karyotypes at supraorder level. With the development of molecular technologies, chromosome comparisons, by interspecific chromosome painting (Zoo-FISH), became easier and more precise, and the evolution of selected DNA sequences could be investigated with a great efficiency. In interspecific comparisons, homologies could be proposed for almost all chromosomal segments. The high degree of conservation of the rabbit and squirrel chromosomes was confirmed by chromosome painting [Korstanje et al., 1999; Richard et al., 2003a; Stanyon et al., 2003]. Zoo-FISH is an invaluable tool to perform chromosome comparisons between distant taxa of placental mammals. It makes it possible to reconstruct the karyotype of the eutherian ancestor with reasonable accuracy [Richard et al., 2003b; FergusonSmith and Trifonov, 2007; Robinson and Ruiz-Herrera, 2008] and the subsequent chromosome evolution which has led to extant species. In many groups, however, multiple intrachromosomal changes, not detected by chromosome painting, have occurred. A complementary analysis by high-resolution chromosome banding and/or Zoo-FISH with DNA probes (such as BACs) is necessary to provide information on intrachromosomal rearrangements. Currently, there are Zoo-FISH and banding comparisons of a large number of species, and it is now possible to compare representative species of most systematic groups with a great refinement. Taking advantage of these data, we focus on the mechanisms of chromosome evolution in the Glires and the implication of the different kinds of rearrangements. The construction of presumed ancestral karyotypes, at various levels, supraorder (Glires), order (Lagomorpha), suborder (Sciuromorpha) and families (Sciuridae, Gliridae, Leporidae, and Ochotonidae), allowed us to propose which chromosome changes took place between these different stages and their implications. Then, we propose a chromosomal history for Leporidae and Sciuridae which are compared to the phylogenies obtained with molecular data. Finally, we propose that a reticulated evolution is more likely to have occurred in the beginning of the diversification of leporids and sciurids.

Chromosome Evolution in Sciuromorpha and Lagomorpha


Diversity and Stability of Lagomorpha and Sciuromorpha Karyotypes

The diploid numbers of these taxa are relatively stable (table 1). Among Lagomorpha, it is more variable for Ochotonidae than Leporidae and among Sciuromorpha, 219

Table 1. List of Lagomorpha and Sciuromorpha species used for this analysis Species

Code

2n

Number of auto- Karyotype references somal segments conserved

Zoo-FISH references

Lagomorpha Leporidae Brachylagus idahoensis Bunolagus monticularis Lepus europaeus Lepus saxatilis Oryctolagus cuniculus

BID BMO LEU LSA OCU

44 44 48 48 44

38 38 38 38 38

Robinson et al., 1984 Robinson and Skinner, 1983 Richard et al., 2003b Robinson, 1980 Committee for standardized karyotypes, 1981

Robinson et al., 2002 (OCU) Robinson et al., 2002 (OCU)

PRU RDI SFL

42 48 42

38 38 38

Robinson, 1980 Robinson et al., 1981 Robinson et al., 1983

Robinson et al., 2002 (OCU) Korstanje et al., 1999 (HSA); ChantryDarmon et al., 2005 Robinson et al., 2002 (OCU) Robinson et al., 2002 (OCU) Robinson et al., 2002 (OCU)

OHY OFO

40 54

39 41

Romanenko et al., 2010 Ye et al., 2011

Romanenko et al., 2010 (HSA) Ye et al., 2011 (HSA)

EQU EMU EME

48–54 45 46 45 46–48 45

Dutrillaux et al., 1979 Filippucci et al., 1988b Filippucci et al., 1988a; Sannier et al., 2011

Sannier et al., 2011 (HSA)

AGE XER MBA MHI MKA MMO TSI TST SER

38 38 38 38 36 38 38 38 36

35 35 36 36 36 36 35 35 35

Petit et al., 1984 Li et al., 2006 Beklemisheva et al., 2011 Li et al., 2006 Beklemisheva et al., 2011 Petit et al., 1984 Petit et al., 1984 Nadler and Block, 1962; this review Beklemisheva et al., 2011

SMA SUN SSU HGA CER CFI TRO TMA DRU MBE SVU SCA HPH PAL

36 32 34 40 40 40 38 38 38 38 40 40 38 38

35 35 35 35 35 35 35 35 35 35 35 38 35 36

Beklemisheva et al., 2011 Beklemisheva et al., 2011 Beklemisheva et al., 2011 Petit et al., 1984 Petit et al., 1984 Sannier et al., 2011 Sannier et al., 2011 Oshida et al., 2002 Nadler and Hoffmann, 1970, this review Sannier et al., 2011 Petit et al., 1984 Nadler and Sutton, 1967 this review Li et al., 2004

Pronolagus rupestris Romerolagus diazi Sylvilagus floridanus Ochotonidae Ochotona hyperborea Ochotona forresti Sciuromorpha Gliridae Eliomys quercinus Eliomys munbyanus Eliomys melanurus Sciuridae Atlantoxerus getulus Xerus cf. erythropus Marmota baibacina Marmota himalayana Marmota katschenkoi Marmota monax Tamias sibiricus Tamias striatus Spermophilus erythrogenis Spermophilus major Spermophilus undulatus Spermophilus suslicus Heliosciurus gambianus Callosciurus erythraeus Callosciurus finlaysonii Tamiops rodolphii Tamiops maritimus Dremomys rufigensis Menetes berdomrei Sciurus vulgaris Sciurus carolinensis Hylopetes cf pharey Petaurista albiventer

Li et al., 2006 (SCA, HSA) Beklemisheva et al., 2011 (TSI) Li et al., 2006 (SCA, HSA) Beklemisheva et al., 2011 (TSI) Li et al., 2004 (HSA, SCA) Beklemisheva et al., 2011 (TSI) Beklemisheva et al., 2011 (TSI) Beklemisheva et al., 2011 (TSI) Beklemisheva et al., 2011 (TSI) Li et al., 2004 (SCA)

Richard et al., 2003a (HSA) Beklemisheva et al., 2011 (TSI) Stanyon et al., 2003; Li et al., 2004 (HSA) Li et al., 2004 (HSA, SCA)

2n = Diploid number; HSA = human; OCU = rabbit; TSI = chipmunk; SCA = grey squirrel. The autosomal segments homologous to human chromosomes conserved in each species are directly obtained by Zoo-FISH data or deduced after chromosome banding comparisons with painted species. Hylopetes cf

pharey: the attribution of H. pharey is provisional because it may be confused with H. alboniger. In bold type: species whose karyotypes are shown in figure 1. For M. himalayana, the number of autosomal segments conserved is 36 instead of 35; see Li et al. [2006] and comments about HSA12 homologies in Beklemisheva et al. [2011].

it is more stable for Sciuridae than Gliridae. The diploid number for Aplodontidae is known for only one species, Aplodontia rufa with 2n = 46 [Carrasco and Humphrey, 1968]. The diploid number of leporids varies from 42 to 52 chromosomes (Pronolagus rupestris, Sylvilagus transi-

tionalis, respectively), while for Ochotonidae, the variation is greater, from 40 to 68 (Ochotona hyperborea, O. princeps, respectively) [Stock, 1976; Robinson, 1980; Ruedas et al., 1989; Romanenko et al., 2010]. There are different cytotypes for S. transitionalis with a diploid number

220


Richard /Dutrillaux

A

B

C

of 46 or 52 [Robinson et al., 1983; Ruedas et al., 1989]. For Sciurids, the diploid number of 38 is very common (fig. 1), except in some genera (Marmota, Spermophilus, Heliosciurus, Callosciurus and Sciurus) in which small variations exist (table 1). For Spermophilus, the variation is from 32 (S. undulatus) to 42 (S. xanthroprymnus), for Marmota, 36 to 42, and 40 for Sciurus, Callosciurus and Heliosciurus genera [Nadler and Hoffmann, 1970; Nadler et al., 1975; Petit et al., 1984; Li et al., 2004, 2006; Arslan, 2005; Beklemisheva et al., 2011]. In contrast, Gliridae have a diploid number varying from 46 (E. munbyanus) to 62 (Glis glis). There are different cytotypes for some Eliomys species (table 1) with a diploid number varying from 48 to 54 (E. quercinus) or 46–48 (E. melanurus), following Robertsonian fusion or fission events [Zima et al., 1995; Sannier et al., 2011]. The successful comparison on the basis of chromosome banding of some leporid karyotypes with each other and with primates demonstrated that leporids have not experienced rapid chromosome evolution [Stock, 1976; Dutrillaux et al., 1980; Robinson, 1980; Robinson et al., 1984]. The same conclusion was made for Sciuridae and, to a lesser extent, for Gliridae [Dutrillaux et al., 1979; Petit et al., 1984; Petit and Dutrillaux, 1985]. Zoo-FISH using human chromosome probes performed in some Lagomorpha and Sciuromorpha species identified chromosomal segments conserved among species with unknown gene order. Thus, using the chromosomal homologies deduced between human and each species, their chromosomes could be directly compared (fig. 2). ZooFISH was also performed using flow-sorted rodent chromosomes from Sciurus carolinensis, Tamias sibiricus and Castor fiber, and O. cuniculus sorted chromosomes [Robinson et al., 2002; Stanyon et al., 2003; Li et al., 2004, 2006; Beklemisheva et al., 2011]. The number of autosomal segments conserved between human and each species could be directly calculated or deduced from Zoo-FISH (table 1). It varied from 35 to 45 human segments conserved. This number is informative about the speed of chromo-

Fig. 1. A RBG-banded karyotype of Hylopetes cf. pharey, with 2n = 38,XX, showing 15 pairs of submetacentric and 3 pairs of metacentrics (HPH9, 14 and 15) autosomes. The p arm of chromosomes 13, 17 and 18 is completely heterochromatic. B RBG-banded karyotype of Dremomys rufigensis, with 2n = 38,XY, showing 14 submetacentric, 3 metacentric (DRU9, 14, 15) and 1 acrocentric (DRU18) autosomes. Heterochromatin is observed at the end of the p-arm of chromosomes 13, 14, 16 and 17, and in an intercalary position in the p-arm of chromosome 8. C RBG-banded karyotype

of Tamias striatus, with 2n = 38,XX showing 13 submetacentric, 4 acrocentric (TST1, 11, 17 and 18) and 1 submetacentric (TST9). Chromosomes are classified according to Callosciurus sp. karyotype in Sannier et al. [2011], and heterochromatic arms were not taken into consideration for chromosome orientation. h = Heterochromatin. Dots show the positions of the centromeres. The small differences in size or banding patterns observed sometimes between the 2 homologs of a pair are owed to variation in the rate of BrdU incorporation during the S phase of the cells in culture.



221

A

B

Fig. 2. Examples of Zoo-FISH: A Dremo-

mys rufigensis (DRU) partial metaphase (in red) hybridized with human chromosome painting 22, on DRU5 and 7 (in green); same R-banded metaphase. B Hylopetes cf. pharey (HPH) metaphase (in red) hybridized with human chromosome painting 20, on HPH3 (in green); same Rbanded metaphase. C Tamiops rodolphii (TRO) karyotype (in red) hybridized with human chromosome painting 1, on TRO1q and 12q (in green); same R-banded karyotype.

C

some evolution. For instance, for other rodents, this number is 62 in Sicista sp. and increases to 71 in some Cavia species [Romanenko et al., 2012]. Thus, the speed of chromosome evolution was considered as low for Lagomorpha and Sciuromorpha, confirming the conclusion from banding comparisons. Moreover, many human syntenies were conserved during their chromosome evolution from the common eutherian ancestor. Syntenies corresponding to human chromosomes HSA2p, 2q, 8q, 9, 10q, 11, 13, 17, 18, 19p, 20, 16q/19q, 7b/16p, and 12b/22b were totally conserved in all Lagomorpha studied. Those corresponding to HSA1, 5, 6, 7a, 8p/4, 14/15, 10p–12pq– 22qt, and 3/21 were partially conserved. For Sciuromorpha, syntenies corresponding to HSA9, 10q, 11, 13, 17, 19p, 20, 7–16p, and 12b/22b were totally conserved. 222


Reconstruction of Ancestral Karyotypes

Ancestral karyotypes can be reconstructed comparing karyotypes of current species, using Zoo-FISH and/ or banding technique data. With comparisons of banding patterns only, a number of misinterpretations about homologies could not be avoided, but chromosome morphology was often correctly reconstructed. With painting comparisons, most if not all homologies were correctly identified, but chromosome morphology was not always established because inversions were not detected. We consider that the data for Lagomorpha and Sciuromorpha, which have often conserved a stable karyotype, are now quantitatively and qualitatively sufficient to propose banded ancestral karyotypes at the principal stages Richard /Dutrillaux

Fig. 3. Eutherian ancestral R-banded

karyotype (EUT) reconstructed according to Richard et al. [2003b]with modifications according to Ferguson-Smith and Trifonov [2007] and Sannier et al. [2011], with 2n = 46. Chromosomes belong to Carnivora (P. hermaphroditus), Lagomorpha (L. europaeus), Primates (M. murinus and D. madagariensis), and Xenarthra (T. chinensis). The banding pattern of EUT1 remains uncertain. The centromeric position is not well defined for EUT4. The slight differences observed between the 2 homologs of EUT8 come from different amounts of pericentromeric heterochromatin. Beside each pair, human homologies are indicated. Dots show the positions of the centromeres.

Fig. 4. Glires ancestral R-banded karyo-

type (GLI), with 2n = 46–50. Chromosomes belong to Lagomorpha or Sciuridae karyotypes. Pair 4 was either homozygously acrocentric or submetacentric, or heterozygous (see text for details). GLI1 and 5 were homozygously fused or not, or heterozygous (see text for details). The slight differences observed between the 2 homologs of GLI8 come from different amounts of pericentromeric heterochromatin. Beside each chromosome pair, human homologies are indicated. Dots show the positions of the centromeres.

of Glires evolution, starting from the ancestral eutherian karyotype. For these reconstructions, human chromosome homologies were obtained from painting data and deduced by banding comparisons for species not painted. We have compared the species studied with the eutherian ancestral karyotype (EUT) [Dobigny et al., 2004] and considered as ancestral (symplesiomorphic) the chromosomal segments with unknown gene order shared by all these species (table 1). The EUT karyotype, with 2n = 46, was proposed by Sannier et al. [2011]. It is a consensual reconstruction with other authors [Murphy et al., 2001b; Richard et al., 2003b; Ferguson-Smith and Trifonov, 2007;

Robinson and Ruiz-Herrera, 2008], except for the homologous to HSA19p which remains an independent acrocentric chromosome as described in Ruiz-Herrera et al. [2012]. It is shown by R-banded chromosomes in figure 3. Then, we have inferred the position of the centromere as submetacentric if this shape is shared by taxa not directly related. The acrocentric shape is chosen if it is observed in taxa not directly related and/or if there was no common submetacentric shape. With data from the literature and some new karyotype comparisons, we have reconstructed the R-banded karyotypes of the ancestor of Glires, Lagomorpha, Sciuromorpha and Sciuridae, using chromosomes of present species from these clades.



223

Fig. 5. Lagomorpha ancestral R-banded karyotype (LAG), with 2n = 58–60. Chromosomes belong to Lagomorpha (L. europaeus and O. cuniculus) karyotypes. Pairs 1, 3 and 4 were either homozygous for A or B, or heterozygous (see text for details). Beside each chromosome pair, human homologies are indicated. Dots show the positions of the centromeres.

The Glires ancestor karyotype (GLI) is composed of 2n = 46–50 chromosomes (fig. 4). Almost all the eutherian syntenies were conserved in this karyotype: the chromosomes homologous to human chromosomes HSA4–8p, 5, 6, 16q–19q, 7b–16p, 3–21, 14–15, 12qt–22q, 2qt, 8q, 7a, 10q, 13, 2pq, 17, 18, 20, 19p, and X. The synteny was disrupted for EUT1 (homologous to HSA1) and EUT5 (homologous to HSA10p–12pq–22qt), and new syntenies have occurred: HSA9–11 (GLI1) and HSA10p– 1qt (GLI5). We could not decide on the ancestral shape for these 2 chromosomes. For HSA10p and 1qt, Sciuridae and Lagomorpha, but not Gliridae, displayed the fused form; for HSA9 and 11, Sciuridae, except the Sciurus genus, rabbit and one pika (O. hyperborea), displayed the fused form, while Gliridae and most of the Lagomorpha species have 2 acrocentrics. The centromere position was not well defined for the ancestral chromosome homologous to HSA6: a submetacentric form was common to Sciuridae and Leporidae, but not to Gliridae, and one pika has an acrocentric form. A GLI composed of 2n = 50 was proposed by Beklemisheva et al. [2011]. They considered 2 more disruptions, the 8p–4 into 4a and 4b–8p and the 3–21 into 3a–21 and 3b. However, the reassessment of the data of Beklemisheva et al. [2011], who used chipmunk (T. sibiricus) chromosome paintings on rabbit (O. cuniculus) metaphases, indicated that the breakpoints of the fissions differ in Lagomorpha and Sciuromorpha. Thus, they are not inherited from their common ancestor. 224


The reconstruction of the Lagomorpha ancestor karyotype (LAG) was proposed by Beklemisheva et al. [2011] and Ye et al. [2011] with 2n = 56 or 60, respectively. We propose a LAG of 2n = 58–60 chromosomes (fig. 5). The ancestral Glires syntenies are conserved, except for GLI2 (HSA4–8p), GLI3 (HSA5), GLI8 (HSA3–21), GLI9 (HSA14–15), GLI11 (HSA12pq–22qt), and GLI14 (HSA7a). Six fission events have occurred for these chromosomes and 1 inversion for the homologous to HSA14–15. The synteny 10p–1qt (GLI7) is fixed in this ancestral karyotype. The centromeric position remains ambiguous for chromosome homologous to HSA1pq because submetacentric and acrocentric forms are described for this chromosome in various taxa. The lower diploid number in Beklemisheva et al. [2011], compared to our reconstructed Lagomorpha ancestral karyotype, lies in the presence of a small acrocentric chromosome homologous to HSA14b and observed in all the Lagomorpha karyotypes, not considered by these authors. For HSA9 and 11, Ye et al. [2011] proposed the acrocentric forms as ancestral whereas Beklemisheva et al. [2011] preferred the fusion into a single metacentric chromosome. For instance, the rabbit and one pika (O. hyperborea) display the fused form, and most of the Lagomorpha have the acrocentrics. Thus, we preferred to make no formal choice and conserved the 2 possibilities in our figure. No reconstruction of the Sciuromorpha ancestral karyotype was proposed in the literature, but Beklemisheva et al. [2011] proposed that the Sciuridae ancestral Richard /Dutrillaux

Fig. 6. Sciuromorpha ancestral R-banded karyotype (SCI), with 2n = 50–54. Chromosomes belong to Sciuridae or Gliridae (M. berdmorei, T. rodolphii, M. monax and E. melanurus) karyotypes. For SCI3, 5, 7 and 8, two or three chromosomes could represent these ancestral forms, an acrocentric and 1 or 2 different submetacentric ones (see text for details). Pairs 1 and 6 were homozygously fused or not, or heterozygous (see text for details). Beside each chromosome pair, human homologies are indicated. Dots show the positions of the centromeres.

karyotype might be similar to that of the Sciuromorpha (SCI). However, they discarded the Gliridae family which is important for the reconstruction of the SCI karyotype [Sannier et al., 2011]. We propose that the SCI ancestor karyotype is composed of 2n = 48–52 chromosomes (fig. 6). The ancestral Glires syntenies are conserved except for GLI8 (HSA3–21) and GLI2 (HSA4–8p). For 6 SCI chromosomes, some doubts remain (SCI1, 3, 5, 6, 7, and 8). One fission event, different from that in LAG [Beklemisheva et al., 2011], occurred for the homolog to GLI2 (HSA4–8p), followed by an inversion of the 4a–8p segment and a fusion to the GLI11 (HSA12pq–22qt) leading to SCI2. Two fissions different from those of LAG [Beklemisheva et al., 2011] occurred for GLI8 (HSA3–21) leading to 3 acrocentrics or, alternately, to 2 acrocentrics (SCI22 and 24) and 1 submetacentric (SCI5). Uncertainty remains about the centromeric position of SCI5 because SCI5 corresponds to an acrocentric form in the genera Marmota and Spermophilus and different submetacentric forms for the other Sciuridae and Gliridae. For SCI1, homologs to HSA9 and 11 are fused in a metacentric chromosome in Sciuridae except Sciurus sp., and not in Gliridae. For SCI6, homologs to HSA1q and 10p are fused in a submetacentric chromosome in Sciuridae, but not in Gliridae. Therefore, for SCI1 and 6, we cannot decide if the fused form is ancestral or not. For SCI3 (HSA5), submetacentric and acrocentric chromosomes are observed in Sciurids. SCI7, homolog to HSA2pq, has a submetacentric form similar in Gliridae and in some Sciuridae (A. getulus, Callosciurus sp.), an acrocentric form in other Sciuridae (Microphyla berdmorei, Tamias

sp., Hylopetes sp.) and other submetacentric forms in the remaining Sciuridae. SCI8, homolog to HSA18, is submetacentric in Gliridae and a large part of the Sciuridae, and acrocentric in some other Sciuridae (M. berdmorei, Sciurus sp., Petaurista sp.). Hence, the centromeric position for SCI3, 7 and 8 chromosomes remains uncertain, and we maintain both possible forms, acrocentric or submetacentric. The Sciuridae ancestral karyotype (SCIU) was described by Graphodatsky et al. [2008] and Beklemisheva et al. [2011] with a diploid number of 2n = 38. We proposed a SCIU with 2n = 38–40 (fig. 7). In contrast to these authors, we concluded that SCI14 and 19 were fused in the ancestral form, but we think that data is insufficient to decide on the status of these chromosomes. The fused form does not exist in Callosciurus, Heliosciurus, Marmota, and Spermophilus genera. So, we maintain the 2 possibilities, fused and non-fused, in the Sciuridae ancestral karyotype. This reconstructed ancestral karyotype differs from that of Sciuromorpha by 6 fusions specific to the Sciuridae, SCI13+15, SCI14+19, SCI11+20, SCI16+21, SCI17+18, and SCI23+24. SCI6 was fixed in its fused form. The centromeric position for SCIU6, 10, 12, and 13 remain uncertain (see above for explanations). The Gliridae ancestral karyotype was described in Sannier et al. [2011]. It differs from that of Sciuromorpha by 8 fissions, 10 fusions and 14 inversions. We propose a supplementary inversion of the segment of HSA3 fused with HSA9 according to the Sannier et al. [2011] nomenclature.



225

Fig. 7. Sciuridae ancestral R-banded

karyotype (SCIU), with 2n = 38–40. Chromosomes belong to Sciuridae (M. berdmorei, T. rodolphii and M. monax) karyotypes. Pairs 6, 10, 12 and 13 were homozygously acrocentric or submetacentric, or heterozygous (see text for details). Chromosome 4 was homozygously fused or not, or heterozygous (see text for details). Beside each chromosome pair, human homologies are indicated. Dots show the positions of the centromeres.

The Leporidae and Ochotonidae ancestral karyotypes are very close to that of Lagomorpha. Two fusions separate the Leporidae from the Lagomorpha ancestral karyotype, and 1 inversion and 2 fissions separate the Ochotonidae from the LAG (fig. 8).

Chromosome Evolution since the Eutherian Ancestor

The chromosome rearrangements that have occurred between the eutherian ancestor and the GLI, LAG, SCI, SCIU, Gliridae, Ochotonidae, and Leporidae ancestors are shown in figure 8. The rate of rearrangements was very low during the first steps of Glires diversification and in the lineages leading to Ochotinidae, Leporidae and Sciuridae. For instance, about 0.3–0.5 rearrangement per million years would have occurred in the lineages leading to the ancestors of Lagomorpha and Sciuromorpha. The rearrangement rate increased 10-fold in the Gliridae lineage (about 3 rearrangements per million years). This high rate is equivalent to that of the Muridae lineage [Stanyon et al., 1999]. Thus, the clock of chromosome rearrangements varied in the different lineages. It was low in the lineage leading to Glires, Lagomorpha, Sciuromorpha, Ochotonidae, Leporidae, and Sciuridae ancestors, in contrast to the high rate observed in the lineage leading to the Gliridae ancestor. The types of rearrangements that occurred in the branches leading to Lagomorpha and Sciuromorpha ancestors were different, with fissions in the LAG branch and a complex rear226


rangement in the SCI branch (involving 1 fission followed by 1 pericentric inversion and 1 Robertsonian translocation). Few rearrangements occurred during the diversification of Lagomorpha: 2 additional fissions occurred in the branch leading to the Ochotonidae ancestor, whereas 2 Robertsonian translocations took place along the Leporidae branch. During the diversification of Sciuromorpha, the numbers and types of rearrangements were different in the Gliridae and Sciuridae branches. Robertsonian translocations accumulated in the Sciuridae branch, while many fissions, pericentric inversions and complex or Robertsonian translocations occurred in the Gliridae lineage [Sannier et al., 2011]. The time of occurrence for some rearrangements remains uncertain (indicated in white and grey in fig. 8). They might have been present in a homozygous state in the ancestor, but multiple reversions would later then be required in the chromosome evolution of these taxa (not shown in fig. 8). A second hypothesis is that they were heterozygous in ancestral populations. Then, the rearrangements colored in grey would correspond to a progression toward the homozygous state in various phylogenetic lines. This is the interpretation chosen in figure 8. The presence of heterozygosity for some inversions and Robertsonian translocations is proposed during the basal steps of the Glires diversification, which could result from hybridization events between homozygous populations (introgression) or conservation of heterozygous individuals, leading to a reticulated evolution [Chang et al., 2011; Liu et al., 2011].

Richard /Dutrillaux

Fig. 8. Chromosome evolution of Glires since the eutherian (EUT) ancestor with the most parsimonious reconstruction of the rearrangements separating the different ancestral karyotypes. GLI = Glires, LAG = Lagomorpha, SCI = Sciuromorpha. Types of rearrangements: 1 = Arrow: fission, 2 = white square: fusion, 3 = white circle: pericentric inversion, 4 = white and grey circle and 5 = white and grey square: pericentric inversion and fusion present in a heterozygous state, respectively (see text for details). Grey symbols: acquisition of the homozygousity from a previous heterozygous state for pericentric inversions (6 = circle), and fusions (7 = square) (see text for details). On the left of the rearrangements, EUT, GLI, SCI or LAG chromosomes involved and on the right, human homologies. a, b, a’, b’ and c’: segments of ancestral chromosomes which were fissioned during their evolution. Presumed times of divergence are indicated in million years (My) [Huchon et al., 2002; Montgelard et al., 2002, 2008].

Chromosome Evolution in Ochotonidae, Leporidae and Sciuridae

In the Ochotonidae family, 4 karyological groups have been proposed [Vorontsov and Ivanitskaya, 1973; Stock, 1976; Capanna et al., 1991]. However, only 2 species have been compared using Zoo-FISH [Romanenko et al., 2010; Ye et al., 2011] which makes it hazardous to reconstruct chromosome relationships between the species of the Chromosome Evolution in Sciuromorpha and Lagomorpha

family. Molecular phylogenies confirm the 2nd, 3rd and 4th karyological groups including O. macrotis, O. rutila, O. roylei (2n = 62) and O. rufescens (2n = 60) in the 2nd group, O. daurica in the 3rd, and O. hyperborea (2n = 40), O. alpina (2n = 42) and O. pallasi (2n = 38) in the 4th group. In contrast, the first karyological group including O. collaris, O. princeps and O. pusilla (2n = 68) is not supported by molecular data [Capanna et al., 1991; Yu et al., 2000; Niu et al., 2004]. Cytogenet Genome Res 2012;137:218–232

227

Fig. 9. Chromosomal phylogeny of Leporidae. White circle = pericentric inversion; white square = Robertsonian translocation; black square = WART. Grey symbols represent the acquisition of the homozygousity from a previous heterozygous state or homoplasies for pericentric inversions (grey circle) and Robertsonian translocations (grey square) (see text for details). On the left of the rearrangements, LAG chromosomes involved and on the right, human homologies.

During Leporidae diversification, Robertsonian fusions occurred frequently [Robinson, 1980; Robinson and Skinner, 1983; Robinson et al., 1983, 1984, 2002; Ruedas et al., 1989]. A resolved chromosomal phylogeny, reconstructed from species studied with Zoo-FISH [Korstanje et al., 1999; Robinson et al., 2002; Chantry-Darmon et al., 2005] is shown in figure 9. The karyotype of the Lagomorpha ancestor (fig. 5) corresponds to the ancestral conditions. The grey symbols correspond to either homoplasies shared with Ochotonidae or acquisition of a homozygous status. Two branches diverge directly from the Leporidae ancestor: one leading to Bunolagus monticularis and the 2nd to the other leporid genera. B. monticularis has a very derived karyotype obtained by 6 rearrangements specific to this lineage and 3 acquisitions of a homozygous state or homoplastic rearrangements. A common trunk of 4 specific rearrangements and 2 acquisitions of a homozygous state or homoplastic rearrangements leads to the other leporid genera. This situation differs from Robinson et al. 228


[2002] who considered the Lepus karyotype as the closest to the Leporidae ancestor and proposed that 4 fission events occurred in the Bunolagus lineage. After this common trunk, few rearrangements occurred. The 1st diverging branch leads to the rabbit and Brachylagus idahoensis, which share 1 Robertsonian fusion. The 2nd branch leads to Romerolagus, Lepus, Sylvilagus, and Pronolagus. L. saxatilis and R. diazi do not acquire any other rearrangement and seem to share the same karyotype, according to Robinson et al. [2002]. One Robertsonian fusion is shared by Pronolagus rupestris and Sylvilagus floridanus. In the Sylvilagus genus, the diploid number varies mainly following the occurrence of different Robertsonian translocations [data not shown, Ruedas et al., 1989]. Some Sylvilagus sp., such as S. bachmani, which has a karyotype very similar to that of Lepus, did not share the Robertsonian fusion LAG1A+16 [Stock, 1976]. This suggests that either this rearrangement is a convergent event or a population, heterozygous for this rearrangement, has remained in this Richard /Dutrillaux

Fig. 10. Positioning of the chromosome rearrangements having

occurred in Sciuridae, in a published molecular tree [Mercer and Roth, 2003]. Species are named by a 3-letter code (cf. table 1). Circle = inversion; square = Robertsonian translocation; dark square = WART; dark rectangle = complex rearrangement; crossed rearrangement = reversion event. Grey symbols represent the acquisition of the homozygosity from a previous heterozygous

state for pericentric inversions (grey circle) and Robertsonian translocations (grey square) (see text for details). Numbers over the rearrangements: SCIU chromosomes involved; ac = acrocentric; sb = submetacentric; fi = fission; fu = fusion; a–d = different rearrangements involving the same chromosome; 17 = rearrangement of SCIU4, involving the homolog to HSA17.

lineage. Surprisingly, the position of Pronolagus, as sister group of Sylvilagus, is not supported by molecular data, which considered it as the most basal branch of Leporidae [Kriegs et al., 2010]. However, this interpretation is unacceptable because it would require us to ignore about 14 non-homoplastic translocations which separate Sylvilagus-Pronolagus from Bunolagus karyotypes (fig. 9). Moreover, the intergeneric relationships deduced from chromosomal data are not supported by molecular data, but the molecular results remain controversial [Halanych and Robinson, 1997, 1999; Matthee et al., 2004]. If both chromosomal and molecular data were robust, these discrepancies would imply that a quite unusual number of homo-

plasies occurred either at the DNA or at the chromosome level. Finally, the absence of a correlation between the leporid geographical distribution over the world and their genetic (chromosomal and molecular) relationships suggests that either their origin is older than thought or that numerous dispersal events might have occurred during their diversification [Robinson et al., 2002; Matthee et al., 2004]. Whatever the case, their recent evolution has involved few chromosome rearrangements. Sciuridae chromosome evolution is mainly composed of pericentric inversions and some Robertsonian fusions. It is also accompanied by the addition of constitutive heterochromatin in some lineages (for instance: DRU, HPH,



229

SCA, Spermophilus lineages), not considered in figure 10. Forty-five rearrangements are deduced in Sciuridae evolution since their common ancestor SCIU, including 20 autapomorphies (observed only in 1 species). The remaining rearrangements are diversely shared and put together genera which are not closely related according to the systematic classification or molecular phylogenies [Mercer and Roth, 2003; Herron et al., 2004; Steppan et al., 2004; Musser and Carleton, 2005]. As the status of SCIU4, 6, 10, 12, and 13 chromosomes is imprecise (fig. 7), the 5 rearrangements involving these chromosomes (4 pericentric inversions and 1 Robertsonian fission or fusion) could not be polarized. But, whatever ancestral form chosen, multiple homoplasies would need to have occurred (not shown). An alternative hypothesis is that these chromosomes conserved a heterozygous status for many generations before becoming fixed in a homozygous state. This situation makes it impossible to construct a resolved phylogenetic tree based on chromosome rearrangements, in accordance with the systematics: a fast radiation within Sciuridae family coupled to introgression would have eliminated cytogenetic footprints. To illustrate this difficulty, we placed the chromosome rearrangements on a molecular tree [Mercer and Roth, 2003]: obviously, they do not support the basal nodes (fig. 10). The 5 rearrangements involving SCIU4, 6, 10, 12, and 13 chromosomes are homoplastic, whatever the SCIU forms chosen. Thus, the hypothesis of a long persistence of their heterozygous status until they became fixed in a homozygous state is preferred for these characters (grey rearrangements in fig. 10). This conclusion means that several introgression events might have occurred leading to a reticulated evolution during the emergence of various genera [Nosil, 2008; Liu et al., 2011]. The long persistence of heterozygosity implies that these chromosome rearrangements were probably permissive, i.e. without strong

effects on meiosis and reproduction. Interestingly, this is probably the case for the 4 inversions which involve only short pericentromeric segments [Dutrillaux et al., 1986; Morel et al., 2007]. For the remaining rearrangements, 20 are autapomorphies and 20 are informative with 13 shared at the genus level (4 for the Sciurus, 6 for the Tamias, and 3 for Spermophilus, except SUN). Indeed, numerous rearrangements were accumulated at the edge of the branches, leading to genera or species. For instance, inversions occurred in the branch leading to Sciurus, Tamias or Hylopetes genus, Robertsonian translocations and inversions in the branch common to Marmota and Spermophilus genera, and WART in the Spermophilus lineage. Finally, the chromosome evolution remained very low for the genera Callosciurus, Heliosciurus, Dremomys, Tamiops, Atlantoxerus, and Xerus. In conclusion, Leporidae and Sciuridae chromosome evolution are both characterized by an early period of a reticulated evolution with conservation of heterozygosity for several chromosomes. This ‘hybrid’ period was certainly longer for Sciuridae than Leporidae, and it was coupled with a fast radiation within Sciuridae family, eliminating cytogenetic footprints. Then, rearrangements more recently accumulated in some lineages of Sciuridae genera, while the karyotypes of Leporidae remain fairly stable. Thus providing another example of marked difference in the speed of chromosomal change accumulation in 2 close groups, as in Papionini and Cercopithecini among Cercopithecidae [Moulin et al., 2008]. Acknowledgements We thank Michèle Gerbault-Seureau for her excellent technical assistance. This study was partly supported by the Muséum National d’Histoire Naturelle (ATM Biodiversité actuelle et fossile).

References Arslan A: Cytogenetic studies on Spermophilus xanthoprymnus (Rodentia: Sciuridae) in central Anatolia. Folia Zool 54: 278–284 (2005). Beklemisheva VR, Romanenko SA, Biltueva LS, Trifonov V, Vorobieva NV, et al: Reconstruction of karyotype evolution in core Glires. I. The genome homology revealed by comparative chromosome painting. Chromosome Res 19:549–565 (2011).

230

Capanna E, Bonomo M, Civitelli MV, Simonetta A: The chromosomes of Royle’s pika, Ochotona roylei (Mammalia, lagomorpha). Rend Fis Acc Lincei 2:59–67 (1991). Carrasco A, Humphrey D: Chromosomes of the mountain beaver Aplodontia rufa pacifica. Mamm Chr Newsletter 9:238–239 (1968). Chang SW, Oshida T, Endo H, Nguyen ST, Dang CN, et al: Ancient hybridization and underestimated species diversity in Asian striped squirrels (genus Tamiops): inference from paternal, maternal and biparental markers. J Zool 285:128–138 (2011).


Chantry-Darmon C, Bertaud M, Urien C, ChadiTaourit S, Perrocheau M, et al: Expended comparative mapping between man and rabbit and detection of a new conserved segment between HSA22 and OCU4. Cytogenet Genome Res 111:134–139 (2005). Committee for standardized karyotypes of Oryctolagus cuniculus: standard karyotype of the laboratory rabbit. Cytogenet Cell Genet 31:240–248 (1981). Dobigny G, Ducroz JF, Robinson TJ, Volobouev V: Cytogenetics and cladistics. Syst Biol 53: 470–484 (2004).

Richard /Dutrillaux

Douzery EJ, Huchon D: Rabbits, if anything, are likely glires. Mol Phylogenet Evol 33: 922– 935 (2004). Dutrillaux B, Aurias A, Lombard M: Presence of common chromosomes in a rodent (Eliomys quercinus, garden dormouse) and in Primates [in French]. Ann Génét 22: 21–24 (1979). Dutrillaux B, Viegas-Pequignot E, Couturier J: Great homology of chromosome banding of the rabbit (Oryctolagus cuniculus) and primates, including man (author’s transl) [in French]. Ann Génét 23:22–25 (1980). Dutrillaux B, Couturier J, Sabatier L, Muleris M, Prieur M: Inversions in evolution of man and closely related species. Ann Génét 29: 195– 202 (1986). Ferguson-Smith MA, Trifonov V: Mammalian karyotype evolution. Nature Rev Genet 8: 950–962 (2007). Filippucci MG, Nevo SS, Capanna E: The chromosomes of the Israeli garden dormouse, Eliomys melanurus wagner, 1849 (Rodentia, Gliridae). Boll Zool 55:31–33 (1988a). Filippucci MG, Rodino E, Nevo E, Capanna E: Evolutionary genetics and systematics of the garden dormouse, Eliomys wagner 1840.1: allozyme diversity and differentiation of chromosomal races. Boll Zool 55: 47–54 (1988b). Graphodatsky AS, Yang F, Dobigny G, Romanenko SA, Biltueva LS, et al: Tracking genome organization in rodents by Zoo-FISH. Chromosome Res 16: 261–274 (2008). Halanych KM, Robinson TJ: Phylogenetic relationship of cottontails (Sylvilagus, Lagomorpha): congruence of 12s rDNA and cytogenetic data. Mol Phylogenet Evol 3: 294–302 (1997). Halanych KM, Robinson TJ: Multiple substitutions affect the phylogenetic utility of cytochrome b and 12s rDNA data: examining a rapid radiation in Leporid (Lagomorpha) evolution. J Mol Evol 48:369–379 (1999). Herron MD, Castoe TA, Parkinson CL: Sciurid phylogeny and the paraphyly of Holarctic ground squirrels (Spermophilus). Mol Phylogenet Evol 31:1015–1030 (2004). Hoffmann RS, Smith AT: Order Lagomorpha: Mammal Species of the World, pp 185–193 (The John Hopkins University Press, Baltimore 2005). Huchon D, Madsen O, Sibbald MJ, Ament K, Stanhope MJ, et al: Rodent phylogeny and a timescale for the evolution of glires: evidence from an extensive taxon sampling using three nuclear genes. Mol Biol Evol 19: 1053– 1065 (2002). Korstanje R, O’Brien PC, Yang F, Rens W, Bosma AA, et al: Complete homology maps of the rabbit (Oryctolagus cuniculus) and human by reciprocal chromosome painting. Cytogenet Cell Genet 86:317–322 (1999). Kriegs JO, Zemann A, Churakov G, Matzke A, Ohme M, et al: Retroposon insertions provide insights into deep lagomorph evolution. Mol Biol Evol 27: 2678–2681 (2010).


Li T, O’Brien PC, Biltueva L, Fu B, Wang J, et al: Evolution of genome organization of squirrels (Sciuridae) revealed by cross-species chromosome painting. Chromosome Res 12: 317–335 (2004). Li T, Wang J, Su W, Yang F: Karyotypic evolution of the family Sciuridae: inferences from the genome organizations of ground squirrels. Cytogenet Genome Res 112:270–276 (2006). Liu J, Li Y, Arnold ML, Wu CH, Wu SF, et al: Reticulate evolution: frequent introgressive hybridization among Chinese hares (genus Lepus) revealed by analyses of multiple mitochondrial and nuclear DNA loci. BMC Evol Biol 11: 223–236 (2011). Luckette WP, Hartenberger JL: Monophyly or polyphyly of the order of Rodentia, possible conflict between morphological and molecular interpretations. J Mamm Evol 1:124–147 (1993). Matthee CA, van Vuuren BJ, Bell D, Robinson TJ: A molecular supermatrix of the rabbits and hares (Leporidae) allows for the identification of five intercontinental exchanges during the Miocene. Syst Biol 53: 433–447 (2004). Mercer JM, Roth VL: The effects of Cenozoic global change on squirrel phylogeny. Science 299:1568–1572 (2003). Montgelard C, Bentz S, Tirard C, Verneau O, Catzeflis FM: Molecular systematics of Sciurognathi (Rodentia): the mitochondrial cytochrome b and 12S rRNA genes support the Anomaluroidea (Pedetidae and Anomaluridae). Mol Phylogenet Evol 22: 220–233 (2002). Montgelard C, Forty E, Arnal V, Matthee CA: Suprafamilial relationships among Rodentia and the phylogenetic effect of removing fastevolving nucleotides in mitochondrial, exon and intron fragments. BMC Evol Biol 8: 321 (2008). Moulin S, Gerbault-Seureau M, Dutrillaux B, Richard FA: Phylogenomics of African guenons. Chromosome Res 16:783–799 (2008). Morel F, Laudier B, Guérif F, Couet ML, Royère D, et al: Meiotic segregation analysis in spermatozoa of pericentric inversion carriers using fluorescence in situ hybridization. Hum Reprod 22:136–141 (2007). Murphy WJ, Eizirik E, Johnson W, Zkang J, Ryder O, O’Brien: Molecular phylogenics and the origins of placental mammals. Nature 409:614–618 (2001a). Murphy WJ, Stanyon R, O’Brien SJ: Evolution of mammalian genome organization inferred from comparative gene mapping. Genome Biol 2:reviews0005.1–0005.8 (2001b). Musser GG, Carleton MD: Superfamily Muroidea: Mammal Species of the World, pp 894– 1531 (The John Hopkins University Press, Baltimore 2005). Nadler CF, Block MH: The chromosomes of some North American chipmunks (Sciuridae) belonging to the genera Tamias and Eutamias. Chromosoma 13:1–15 (1962).

Nadler CF, Sutton DA: Chromosomes of some squirrels (Mammalia-Sciuridae) from the genera Sciurus and Glaucomys. Experientia 23:249–251 (1967). Nadler CF, Hoffmann RS: Chromosomes of some Asian and South American squirrels (Rodentia: Sciuridae). Experientia 26: 1383– 1386 (1970). Nadler CF, Hoffmann RS, Hight ME: Chromosomes of three species of Asian tree squirrels, Callosciurus (Rodentia: Sciuridae). Experientia 31:166–167 (1975). Niu Y, Wei F, Li M, Liu X, Feng Z: Phylogeny of pikas (Lagomorpha, Ochotona) inferred from mitochondrial cytochrome b sequences. Folia Zool 53:141–155 (2004). Nosil P: Speciation with gene flow could be common. Mol Ecol 17:2103–2106 (2008). Oshida T, Su JF, Lin LK: Chromosomal characterization of the Formosan striped squirrel Tamiops maritimus formosanus (Mammalia, Rodentia). Caryologia 55:213–216 (2002). Petit D, Dutrillaux B: Chromosomal phylogeny of the 7 species of Sciurinae [in French]. Ann Génét 28:13–18 (1985). Petit D, Couturier J, Viegas-Péquignot E, Lombard M, Dutrillaux B: Great degree of homology between the ancestral karyotype of squirrels (rodents) and that of primates and carnivores [in French]. Ann Génét 27: 201– 212 (1984). Prasad AB, Allard MW, NISC Comparative Sequencing Program, Green ED: Confirming the phylogeny of mammals by use of large comparative sequence data sets. Mol Biol Evol 25:1795–1808 (2008). Richard F, Messaoudi C, Bonnet-Garnier A, Lombard M, Dutrillaux B: Highly conserved chromosomes in an Asian squirrel (Menetes berdmorei, Rodentia: Sciuridae) as demonstrated by ZOO-FISH with human probes. Chromosome Res 11: 597–603 (2003a). Richard F, Lombard M, Dutrillaux B: Reconstruction of the ancestral karyotype of eutherian mammals. Chromosome Res 11: 605–618 (2003b). Robinson TJ: Comparative chromosome studies in the family Leporidae (Lagomorpha, Mammalia). Cytogenet Cell Genet 28:64–70 (1980). Robinson TJ, Ruiz-Herrera A: Defining the eutherian ancestral karyotype: a cladistic interpretation of chromosome painting and genome sequence assembly data. Chromosome Res 16:1133–1141 (2008). Robinson TJ, Skinner JD: Karyology of the Riverin rabbit, Bunolagus monticularis, and its taxonomic implications. J Mamm 64: 678– 681 (1983). Robinson TJ, Elder FF, Lopez-Forment W: Banding studies in the Volcano rabbit, Romerolagus diazi, and Crawshay’s hare, Lepus crawshayi: evidence of the leporid ancestral karyotype. Can J Genet Cytol 23: 469–474 (1981).


231

Robinson TJ, Elder FF, Chapman JA: Evolution of chromosomal variation in cottontails, genus Sylvilagus (Mammalia: Lagomorpha): S. aquaticus, S. floridanus and S. transitionalis. Cytogenet Cell Genet 35:216–222 (1983). Robinson TJ, Elder FF, Chapman JA: Evolution of chromosomal variation in cottontails, genus Sylvilagus (Mammalia: Lagomorpha). II Sylvilagus audubonii, S. idahoensis, S. nuttallii and S. palustris. Cytogenet Cell Genet 38:282–289 (1984). Robinson TJ, Yang F, Harrison WR: Chromosome painting refines the history of genome evolution in hares and rabbits (order Lagomorpha). Cytogenet Genome Res 96: 223– 227 (2002). Romanenko SA, Lemskaya NA, Beklemisheva VP, Perelman PL, Serdukova NA, Graphodatsky AS: Comparative cytogenetics of rodents. Russ J Genet 46:1138–1142 (2010). Romanenko SA, Perelman PL, Trifonov VA, Graphodatsky AS: Chromosomal evolution in Rodentia. Heredity 108:4–16 (2012).

232

Ruedas LA, Dowler RC, Aita E: Chromosomal variation in the New England cottontail, Sylvilagus transitionalis. J Mamm 70: 860–864 (1989). Ruiz-Herrera A, Farré M, Robinson TJ: Molecular cytogenetic and genomic insights into chromosomal evolution. Heredity 108:28–36 (2012). Sannier J, Gerbault-Seureau M, Dutrillaux B, Richard FA: Conserved although very different karyotypes in Gliridae and Sciuridae and their contribution to chromosomal signatures in glires. Cytogenet Genome Res 134: 51–63 (2011). Stanyon R, Yang F, Cavagna P, O’Brien PC, Bagga M, et al: Reciprocal chromosome painting shows that genomic rearrangement between rat and mouse proceeds ten times faster than between humans and cats. Cytogenet Genome Res 84:150–155 (1999). Stanyon R, Stone G, Garcia M, Froenicke L: Reciprocal chromosome painting shows that squirrels, unlike murid rodents, have a highly conserved genome organization. Genomics 82:245–249 (2003). Steppan SJ, Storz BL, Hoffman RS: Nuclear DNA phylogeny of the squirrels (Mammalia: Rodentia) and the evolution of arboreality from c-myc and RAG1. Mol Phylogenet Evol 30: 703–719 (2004).


Stock AD: Chromosome banding pattern relationships of hares, rabbits, and pikas (order Lagomorpha). A phyletic interpretation. Cytogenet Cell Genet 17:78–88 (1976). Vorontsov NN, Ivanitskaya EY: Comparative karyology of north Palearctic pikas (Ochotona, Ochotonidae, Lagomorpha). Caryologica 26:213–223 (1973). Ye J, Nie W, Wang J, Su W, Jing M, et al: Genomewide comparative chromosome map between human and the Forrest’s pika (Ochotona forresti) established by cross-species chromosome painting: further support for the Glires hypothesis. Cytogenet Genome Res 132:41–46 (2011). Yu N, Zheng C, Zhang YP, Li WH: Molecular systematics of pikas (genus Ochotona) inferred from mitochondrial DNA sequences. Mol Phylogenet Evol 16:85–95 (2000). Zima J, Macholan M, Filippucci MG: Chromosomal variation and systematics of myoxids. Hystrix 6:63–76 (1995).

Richard /Dutrillaux



Non-Sciuromorph Rodent Karyotypes in Evolution S.A. Romanenko a V. Volobouev b a

Institute of Molecular and Cellular Biology, SB RAS, Novosibirsk , Russia; b Muséum National d’Histoire Naturelle, Département Systématique et Evolution, Paris, France

Key Words Chromosome rearrangements ⴢ Cytogenetics ⴢ Rodents ⴢ Speciation

Abstract Rodents are, taxonomically, the most species-rich mammalian order. They display a series of special genomic features including the highest karyotypic diversity, frequent occurrence of complex intraspecies chromosome variability, and a variety of unusual chromosomal sex determination mechanisms not encountered in other mammalian taxa. Rodents also have an abundance of cytochemically heterogeneous heterochromatin. There are also instances of extremely rapid karyotype reorganization and speciation not accompanied by significant genetic differentiation. All these peculiarities make it clear that a detailed study of rodent genomic evolution is indispensable to understand the mode and tempo of mammalian evolution. The aim of this review is to update the data obtained by classical and molecular cytogenetics as well as comparative genomics in order to outline the range of old and emerging problems that remain to be resolved. Copyright © 2012 S. Karger AG, Basel



Following modern taxonomy, the order Rodentia is divided into 5 suborders: Anomaluromorpha, Castorimorpha, Hystricomorpha, Myomorpha, and Sciuromorpha. Current cytogenetic data further substantiate this division. The suborder Sciuromorpha is well supported as a monophyletic taxon on the basis of both morphological and molecular data [Murphy et al., 2001; Waddell et al., 2001; Churakov et al., 2010]. Recent molecular cytogenetic analyses placed the suborder combined with representatives of Lagomorpha in the cohort ‘core Glires’. A separate review was devoted to a discussion of the evolutionary dynamics in the ‘core Glires’ [Beklemisheva et al., 2011]. Rodents diverged from a common ancestor with Lagomorpha about 65 mya and then underwent a massive radiation. Today, non-sciuromorph rodents comprise 42% of living mammalian species and are found on all continents except Antarctica. Karyotypic and phylogenetic relations between various species have not yet been well studied even at the family level in some taxa. However, scientific interest in rodents has remained intense, and there is a fairly constant high number of articles presenting research on rodents in both comparative cytogenetics and molecular phylogenetics. Among mammals, the geSvetlana A. Romanenko Institute of Molecular and Cellular Biology, SB RAS Lavrentjev Ave., 10 RU–630090 Novosibirsk (Russia) Tel. +7 383 3635 153, E-Mail rosa @ mcb.nsc.ru

nomes of rodents appear to have the greatest plasticity. There are significant variations in diploid number both between and within species as well as variations in the number and distribution of heterochromatin blocks and other cytogenetic features. Many findings of comparative cytogenetic studies using FISH have recently been summarized and analyzed in a review by Romanenko et al. [2012]. Here, we want to look at rodent genomes from various perspectives. We describe the current knowledge about non-sciuromorph genomes from conventional cytogenetics to full genome sequencing. We also discuss in considerable detail the types of chromosome rearrangements identified in rodent genomes and their impact on cladogenetic events.

Chromosomal Rearrangements and Speciation

The massive radiation of rodent superfamilies occurred 55–65 mya [Huchon et al., 2002], leading to the thousands of species known today. Among mammals, rodents are characterized by an extreme variability in diploid chromosome number from 2n = 9, 10 in the South American field mouse Akodon sp. [Silva and YonenagaYassuda, 1998] to the highest known diploid number in mammals, 2n = 102, found in the plains viscacha rat Tympanoctomys barrerae [Contreras et al., 1990]. For more detailed description of 2n variation in rodent, see O’Brien et al. [2006]. It is often argued that chromosomal rearrangements are involved in the processes of speciation [King, 1993]. It is hypothesized that chromosomal rearrangements need to be fixed in a short period of time [Britton-Davidian et al., 2000; Wang and Lan, 2000; Dobigny et al., 2005; Aniskin et al., 2006], so they may create reproductive isolation between populations [Ayala and Coluzzi, 2005]. According to a ‘fragile site’ breakage model of chromosomal evolution, there are some regions in a genome where the frequency of chromosomal rearrangements is very high. Such regions were called ‘evolutionary breakpoint regions’ (EBRs) [Larkin et al., 2009]. It was reported that the EBRs are gene-rich [Ma et al., 2006] but may also contain segmental duplications and SNPs [Murphy et al., 2005; Larkin et al., 2009]. These regions are also associated with centromere and telomere repositions and chromosome fragile sites [Ruiz-Herrera et al., 2006]. EBRs are enriched with genes associated with adaptive functions [Larkin et al., 2009], so at least some evolutionary chromosome rearrangements may have adaptive value by cre234


ating novel configurations of structural and regulatory loci involved in responses to environmental challenges. Brown and O’Neill [2010] recently discussed different theories and studies of speciation.

Intraspecific Chromosome Variability in Rodents

One of the most remarkable features of rodents is their outstanding variability in chromosome numbers and/or chromosome morphology. The origin of diploid number variability may result from Robertsonian or tandem translocations, fissions, or presence of a variable number of supernumerary or B-chromosomes (Bs) occurring separately or together in a given species. Similarly, variable chromosome morphology can result from structural rearrangements such as pericentric inversions, reciprocal translocations or transpositions, or variable amounts of constitutive heterochromatin. Below, we consider these different types of chromosomal changes and their impact on reproductive isolation. Robertsonian Translocations Centric or Robertsonian (Rb) fusions, leading to a reduction in diploid number, are the most common type of structural rearrangements encountered between species or populations in mammals. Extremely large variations of 2n within a species can occur, where, on the one extreme, all chromosomes are one-armed and, on the other extreme, all are bi-armed. The transitory variants (known as Robertsonian fan) were discovered and studied in detail in 2 rodents, the house mouse Mus musculus [Gropp et al., 1982; Bauchau, 1990] and the eastern mole vole Ellobius tancrei [Lyapunova et al., 1980; Bakloushinskaya et al., 2010] and an insectivorous species, the common shrew Sorex araneus [Banaszek et al., 2009]. Studies of these animal models contributed greatly to our understanding of the role played by Rb translocations in genome repatterning. Investigations on the molecular mechanisms of these rearrangements revealed that some particular pericentromeric repeats can mediate centric fusions through heterologous recombination [Garagna et al., 2001; Kalitsis et al., 2006; Gauthier et al., 2010]. Heterologies presumably occur without any effect on the nearby structural genes and thus do not have any genetic effects on individuals (but see John and Miklos [1988] for another opinion). In the mouse, meiotic malsegregation, although low, was documented even in the case of structural heterozygotes for one Rb fusion [Gropp et al., 1975] but increased significantly in double, Romanenko /Volobouev

triple, or higher level heterozygotes [Gropp et al., 1982; Redi and Capanna, 1988; Nachman and Searle, 1995]. On the contrary, no meiotic impairment was detected in the shrews at a low level of heterozygosity [Searle, 1993]. Consequently, 1 or a maximum of 2 chromosome pairs in Mus or 1–4 in Sorex could be simultaneously polymorphic within a population. However, in these species the original polymorphic pairs involved can be different between populations, although they often share the same chromosomal arms, thus resulting in monobrachial homology. Population or a group of populations characterized by the possession of at least one specific Rb metacentric combination forms chromosomal races (CRs, sensu Hausser et al. [1994] or chromosomal population sensu Pialek et al. [2005]). Up to now 75 and 97 CRs were described in S. araneus [Wojcik et al., 2003; Shchipanov et al., 2009; White et al., 2010; Zima and Bulatova, 2011] and in M. musculus [Bauchau, 1990; Pialek et al., 2005], respectively. In the interracial hybrids occurring in the wild or obtained in the lab, monobrachial homology is manifested by the formation in the first meiotic prophase of a ring or chain configuration of varying levels of complexity depending on the number of pairs displaying arm homology. Starting from 2 pairs of shared monobrachial homology, the gametogenesis in hybrid mouse males is affected and can result in their complete sterility or even non-viability in the case of multiple fusions with monobrachial homology [Searle, 1993]. Again, in contrast to mice, normal chromosome segregation forming a chain of 11 chromosomes was documented in the interracial hybrids of the shrew [Pavlova et al., 2007, 2008]. However, fertility effects caused by malsegregation were observed even at lower levels of heterozygosity in the other types of interracial hybrids [Wallace and Searle, 1994]. Taking into account the results from molecular phylogenetic studies, we know that CRs in mice and shrews are of a very recent origin (between 500 and not more than 40,000 years). Therefore, the varying level of sterility in interracial hybrids can be ascribed with confidence to structural heterozygosity for Rb translocations and not to genetic differences which are insignificant at least between some CRs that are reproductively isolated [Britton-Davidian et al., 2000]. In the light of numerous and detailed data obtained on these 2 mammalian species, the model of speciation by multiple centric fusions which share monobrachial homologies put forward by Capanna [1982] has received a further solid support and can be considered as a well established mode of rapid genome repatterning leading to reproductive isolation and cladogenesis.

Inversions Pericentric inversions (PIs) are a second class of rearrangements that are particularly frequent in rodents (Peromyscus: Greenbaum et al. [1994] and references herein; Akodon: Fagundes et al. [1997], Silva and Yonenaga-Yassuda [1998] and references herein; Mastomys: Volobouev et al. [2001, 2002a]; Microtus: Lemskaya et al. [2010]; Sicista: Kovalskaya et al. [2011]; and many other groups: Trifonov et al. [2010a]). PIs change only the number of chromosomal arms and not the diploid chromosome number, one-armed chromosomes can be transformed into bi-armed ones or vice versa. Initially, it was believed that PIs were negatively heterotic, and therefore, it was difficult to explain their frequent occurrence in the karyotype evolution of various groups and the numerous instances of polymorphism for this kind of rearrangements [King, 1993]. Since the discovery of synaptic adjustment that suppresses loop formation thus precluding the production of unbalanced gametes, it is evident that PIs are transmitted and become fixed in a population [Davisson et al., 1981; Greenbaum and Reed, 1984; Hale, 1986]. However, if theoretically there are no meiotic obstacles to their accumulation, it is not clear why the higher levels of PIs heterozygosity (3 or more) remain extremely rare or never observed in some species. Indeed in the case of polymorphism for PIs in Peromyscus boylii among 78 animals karyotyped, 39 (50%) were simple heterozygotes, 5 were double heterozygotes, and 1 was a triple heterozygote [Davis et al., 1986], whereas in the case of Mastomys erythroleucus among 46 specimens karyotyped 17 (37%) were simple and 3 (6.5%) were double heterozygotes [Volobouev et al., 2001]. Perhaps beginning from a certain level of heterozygosity and in the context of a given genotype, the effect of PIs becomes negatively heterotic [Adkins et al., 1991]. Cladistic analysis of cytogenetic data for 8 taxa of Mastomys revealed that practically all dichotomies on the phylogenetic tree were supported by 2 PIs [Volobouev et al., 2001]. It appears that most species of Peromyscus differ from each other by 1–2 PIs [Greenbaum et al., 1994]. Earlier, it was hypothesized that genetic differentiation in Mastomys is initiated by the suppression of recombination in inverted segments. Progressive accumulation of PIs subsequently reinforces the differentiation among cytotypes leading to eventual speciation [Volobouev et al., 2002a] (see also data on Drosophila: Noor et al. [2001] and plant species of the genus Helianthus: Rieseberg [2001]). However, at least in mammals this attractive hypothesis needs experimental support.

Rodent Karyotypes in Evolution


235

Data on the occurrence of paracentric inversions in wild mammalian populations are scanty. However, the recent comparison of sequenced genomes of mouse and rat revealed numerous inversions of varying length inside of conserved blocks, thus suggesting that this kind of chromosomal rearrangements played an important if not leading role in genome repatterning of rodents. Tandem Translocations This kind of structural rearrangements consists in fusion of 2 chromosomes by their telomeres or by centromere and telomere; both variants are considered to be strongly underdominant. In the case of heterozygotes involving 2 acrocentric chromosomes the drastic reduction of fertility, up to 50%, results from the production of unbalanced gametes bearing deletions and duplications [King, 1993]. Among the large number of mammals karyotyped, the occurrence of 1 or 2, but rarely more, tandem fusions has been documented in some dozen of taxa, thus meaning that this kind of chromosomal change occurs and becomes fixed [e.g. Volobouev et al., 2002a; Dobigny et al., 2005; Swier et al., 2009; Ventura et al., 2009]. The relatively rare fixation of tandem translocations in karyotype evolution of mammals and the only documented instance of a polymorphism for this chromosomal change [Kovalskaya et al., 2011] strongly suggests that when they occur and reach a homozygous state, they probably insure an almost instantly and complete reproductive isolation [Dobigny et al., 2005]. For these reasons the fixation of multiple tandem translocations in closely related species remains a rare evolutionary event except in the case of Muntiacus species [Yang et al., 1997; Wang and Lan, 2000]. It was observed only twice: in the gerbils of the genus Taterillus [Dobigny et al., 2002, 2003, 2005] and the birch mice of the genus Sicista [Kovalskaya et al., 2011]. Taking into account that karyotype evolution in these taxa is unusual for a number of aspects, we consider these cases in more details below. B-Chromosomes Compared to obligatory and regular chromosomes in the karyotype of a species (A-chromosomes), supernumerary or B-chromosomes (Bs) as for heterochromatin (see below) are facultative elements in the genome. Except for very small dot-like Bs found in some species, they are undistinguishable from the A-chromosomes by size and morphology. Nevertheless, a series of distinctive features characterize this category of chromosomes [Volobouev, 1981; Cavagna et al., 2002; Vujosevic and Blagojevic, 2004]. Although mostly C-band positive and always late 236


replicating, they display a clear, cytochemical heterogeneity. Some Bs are mitotically unstable, thus resulting in inter- and intraindividual variation in number. There is a pre-meiotic accumulation of Bs probably as a mean against their elimination. In the first prophase, Bs more often are present as univalents but may form bivalents or multivalents sometimes being found in association with centromeric and/or telomeric regions of the A-chromosomes. Among mammals possessing Bs, the majority (about 55 species) belong to rodents with the record number reaching 38 in the Palearctic collar lemming Dicrostonyx torquatus [Chernyavsky and Kozlovsky, 1980]. A detailed review was recently devoted to the investigation of Bs [Vujosevic and Blagojevic, 2004]. There are still many unanswered questions about their origin, molecular composition, and possible role in the cell and in genome evolution. The question why there are so many muroid rodent species with Bs also remains unexplained. Analysis of repetitive sequences in the genome of Apodemus peninsulae suggested that Bs might have a species-specific origin and were derived from A-chromosomes [Matsubara et al., 2008]. The cytogenetic analysis of some populations of A. peninsulae revealed sharply distinct patterns of morphological and numerical variation of Bs even in 2 neighboring populations without any visible geographic barriers between them. These results strongly suggest an adaptive role for the B-chromosome system in mammals [Volobouev, 1979 and references herein]. Molecular analysis of Bs DNA sequences showed the presence of satellite DNAs, ribosomal DNAs, and mobile elements [Camacho et al., 2000; Stitou et al., 2000; Rubtsov et al., 2004; Graphodatsky et al., 2005]. There is a general hypothesis that Bs somehow accompany genome evolution in lineages with rapid chromosome changes as part of genomic plasticity [Trifonov et al., 2002]. Detailed studies of the molecular content of rodent Bs will shed light on their origin and evolution. It is expected that rodent Bs can harbor at least some genes and segment duplications as do Bs of some other mammalian species [Graphodatsky et al., 2005; Trifonov et al., 2010b].

Heterochromatin Variations in Rodents

The term ‘heterochromatin’ is used to describe tightly packed blocks of chromatin. One of the first examples of karyotype evolution by heterochromatic arm additions/ deletions was in the genus Peromyscus [Pathak et al., 1973]. Later, this type of polymorphism was detected in Romanenko /Volobouev

some other rodent species [e.g. Patton and Sherwood, 1982; Graphodatsky, 1989]. However, quantitative heterochromatin variability is indeed omnipresent and is regularly observed between species, between individuals of the same species, and consequently between homologous chromosomes within a cell. It would not be an exaggeration to state that by quantity and distribution patterns there are no 2 identical individuals within a species. Quantitative differences in the content of heterochromatin determine the interspecies variability in the size of mammalian genomes [Graphodatsky, 1989; Graphodatsky et al., 2011]. Sometimes a rather important interspecific or even interindividual difference of the C-heterochromatin amount is limited exclusively to the sex chromosomes where giant blocks of heterochromatin were described in many species of rodents [Nanda et al., 1988; Barome et al., 2001; Marchal et al., 2004; Acosta et al., 2011]. In general, X chromosomes of mammals have a highly conserved genetic content and a similar relative size of about 5% of a haploid set, as was estimated by Ohno [1969] and later confirmed by both comparative gene mapping and comparative chromosome painting. The changes of the ‘standard’ size of an X chromosome due to heterochromatin additions can lead to the formation of giant X chromosomes which make up about 20% of the entire genome as in the case of the vole Microtus agrestis [Pathak and Stock, 1974]. Despite numerous studies there is no evidence for reduced fertility between cytotypes possessing gross structural heterozygosity for the amount and/or location of heterochromatin. Therefore, the direct involvement of this genomic component in the speciation process, although repeatedly claimed [King, 1993; and references herein], seems ungrounded. More often heterochromatic regions are regarded as the main contenders for the role of stabilizing or destabilizing evolution [Graphodatsky, 1989; Hennig, 1999]. It was shown that evolutionary changes of pericentromeric and subtelomeric heterochromatic regions play the role of ‘hot spots’ that facilitate interchromosomal rearrangements. It is worth noting that among 15 species of the genus Gerbillus 10 possess a high amount of heterochromatin (30–50% of total karyotype length). They display a remarkable heterogeneity, and the species of this group differ from each other by a high number of structural interspecies rearrangements (130) and all share a gonosome-autosome translocation (or even 2 in the case of G. gerbillus). They may display large intra- and interpopulation polymorphisms for both structural rearrangements and size of heterochromatic

blocks [Aniskin et al., 2006]. On the contrary, the group of the 5 remaining species is characterized by a low amount of homogeneously stained heterochromatin (!10% of total karyotype length), by a low number of interspecies rearrangements (1–4), and all have conserved the original X-chromosome type. These sharply distinct genomic features, in morphologically and biologically otherwise rather similar species, strongly suggested a causal relationship between abundant amounts of cytologically (and molecularly) heterogeneous heterochromatin and unusually high rates of karyotype repatterning [Aniskin et al., 2006], although the precise mechanisms remain to be elucidated. Up to now, different types of heterochromatic repeat sequences were characterized [e.g. Elisaphenko et al., 1998; Yamada et al., 2006; Matsubara et al., 2008].



Sex Chromosome Systems

The origin and evolution of sex chromosomes in mammals as well as different mechanisms of dosage compensation were thoroughly reviewed in 3 recent reviews [Bakloushinskaya, 2009; Wilson and Makova, 2009; Livernois et al., 2012]. Although some sex chromosome systems other than XX/XY were found within various mammalian taxa, rodents present the largest range of chromosomal mechanisms of sex determination (table 1). It is noteworthy that most of the 34 species listed belong to the Muroidea superfamily inside the suborder Myomorpha. Development of molecular methods has allowed more precise studies of sex determination in some species. In particular, the studies of 2 genera, Ellobius [Bagheri-Fam et al., 2012] and Tokudaia [Murata et al., 2012], showed that in addition to other chromosomal rearrangements a mutation in SRY or its loss could produce instability in sex determination and provide a powerful incentive to speciation. In such cases, other genes might trigger the male sex determination in SRY-negative species. From the available data on rodents it seems that different systems of sex determination serve to maintain genetic diversity. To understand the molecular basis of sex determination in these exceptional cases, a detailed genome sequence analysis will be required. Such studies might be able to demonstrate if genes other than SRY have a role in sex determination cascade triggers or if some other mechanisms, such as sex chromosome dosage, are involved.

237

Table 1. Unusual sex chromosome systems in rodents

System: female/male

Species

Reference

XO/XO

Acomys selousi Ellobius lutescens

Matthey [1965]; Barome et al. [2001] Vorontsov et al. [1980]; Fredga [1983]; Just et al. [1995]; Vogel et al. [1998] Quere et al. [2009] Fredga [1983]; Soullier et al. [1998]; Sutou et al. [2001]; Arakawa et al. [2002]; Kobayashi et al. [2007] Sutou et al. [2001]; Arakawa et al. [2002]; Kobayashi et al. [2007] Meylan [1968] Reig and Kiblisky [1968, 1969] Just et al. [1995] Just et al. [1995] Just et al. [1995] Wahrman et al. [1969] Viegas-Pequignot et al. [1983] Bianchi and Contreras [1967] Sbalqueiro et al. [1984] Raman and Sharma [1976] Gileva [1980]; Fredga [1983] Matthey [1965]; Veyrunes et al. [2010] Zhu et al. [2003] Ohno et al. [1963, 1964]; Fredga [1983] Burgos et al. [1988] Fredga et al. [1976]; Gropp et al. [1975]; Fredga [1983] Fredga [1983] Badenhorst et al. [2009] Viegas-Pequignot et al. [1982] Lay and Nadler [1975] Badenhorst et al. [2012] Lanzone et al. [2011] Volobouev and Granjon [1996] Dobigny et al. [2005] Dobigny et al. [2005] Dobigny et al. [2002] Dobigny et al. [2002] Dobigny et al. [2002] Dobigny et al. [2004]

Eozapus setchuanus Tokudaia osimensis XO/XY XX/XO XX/XX

XX, XY*/XY X1X1X2X2/X1X2Y X1X1X2/X1X2Y XmXm, XmXf, XfO/XmO XX, XX*, X*Y, X1X2/X1X2Y XmXsm, XsmXsm, XmO/XsmY XO/XY, OY XY/XY XX, X*X, X*Y/XY XX, XO, XY/XY XX/XY1Y2

Tokudaia tokunoshimensis Zapus princeps Ctenomys tuconax Ellobius alaicus Ellobius talpinus Ellobius tancrei Microspalax ehrenbergi Uranomys ruddi Akodon azarae Deltamys kempi Vandeleuria oleracea Dicrostonyx torquatus Leggada (Mus) minutoides Microtus mandarinus Microtus oregoni Microtus cabrerae Myopus schisticolor Nesokia indica Bandicota savilei Gerbillus gerbillus Gerbillus gledovi Hapalomys delacouri Salinomys delicatus Taterillus arenarius Taterillus gracilis Taterillus petteri Taterillus pygargus Taterillus sp.1 Taterillus sp.2 Taterillus tranieri

Centromere Repositioning

Centromere repositioning or shift means that the centromeres moved along the chromosome without inversion or other structural rearrangements [Montefalcone et al., 1999]. It implies the inactivation of the old centromere and the rapid progression of the newly seeded centromere toward the complex organization that probably stabilizes its activity [Rocchi and Archidiacono, 2006]. Centromeres that emerge after separation from a common ancestor of the group were called evolutionary-new centromeres [Mlynarski et al., 2010]. This phenomenon is relatively frequent and has been documented in primates, 238


other mammals, and birds. There are only a few welldocumented cases of evolutionary-new centromere formation in rodents [Zhao et al., 2004; Veyrunes et al., 2006; Kobayashi et al., 2008; Mlynarski et al., 2010; Trifonov et al., 2010a; Bakloushinskaya et al., 2012]. Centromere repositioning provides a potentially powerful evolutionary force for reproductive isolation and speciation [Rocchi et al., 2012]. As it was shown by the example of human and other primates, heterozygosity for the neocentromere leads to the formation of dicentric and acentric chromosomes in meiosis. As a consequence, these structural variants could affect the fitness of heterozygous individuals and act as a negative Romanenko /Volobouev

selective pressure. However, 3 healthy human individuals with repositioned but otherwise ‘normal’ chromosomes were recently reported [Amor et al., 2004; Ventura et al., 2004; Liehr et al., 2010], and it was documented that the rearrangement can be inherited without any negative effect. In the future, classical and molecular cytogenetics will continue to have a crucial role in the identification of centromere movements and their input in evolutionary events in rodents.

Repetitive DNA

One of the recent breakthroughs was the discovery of the preferential localization of evolutionary breakpoints in regions enriched in repetitive sequences such as SINEs, LINEs, LTRs, and segmental duplications [Murphy et al., 2005]. It is thought that the presence of repetitive sequences at evolutionary breakpoints can form a substrate for non-homologous recombination [Redi et al., 1990]. Therefore, an important role in chromosomal evolution can be assigned to centromeric and subtelomeric domains because they accumulate repeat sequences. A correspondence between breakpoint reuse and centromere repositioning was also established [Rocchi et al., 2012].

2 rodents, sequences of ITSs were obtained only for some arvicolins [Rovatsos et al., 2011]. Ribosomal DNA Clusters RNA genes coding for the RNAs of ribosomal subunits are essential for the cellular translation machinery. They present a family of tandem repeat sequences which are generally located in pericentromeric and subtelomeric regions. It is now known that rRNA genes are subject to a high rate of intra- and interchromosomal recombination. Chromosomal distribution of rDNA clusters often appears as a useful cytogenetic marker to discriminate between species. An example is the phylogenetic relationship of the 7 Apodemus species inferred from the chromosome rearrangements and the chromosomal distribution patterns of the 18S-28S rRNA genes [Matsubara et al., 2004]. The location of the 5S rRNA genes on the distal telomeric region of chromosome 8 is evolutionarily conserved among Mus species [Matsubara et al., 2003]. The homologous chromosome in the genome of the Apodemus species was also painted with a 5S rRNA probe, indicating that the location of the genes is highly conserved in Apodemus as well as Mus species. The distribution of 18S and 28S rDNA clusters in the genus Mus was highly variable, ranging from 1–21 chromosome pairs. It was shown that more than half of rearrangement breakpoints in the genus were in sites where both rDNA clusters and centromeres were closely present [Cazaux et al., 2011]. Both of these genomic structures coincide with breakpoints in the genus Mus, suggesting that the accumulation of a large number of repeats in the centromeric region may contribute to the high level of chromosome repatterning observed in this group. However, the elevated rate of rDNA change observed in the chromosomally invariant clade indicates that the presence of these sequences was insufficient to lead to genome instability.

Telomeric Repeats A telomere is a functionally defined region of DNA at the molecular end of a linear chromosome that plays an essential role in the maintenance of chromosomal integrity and stability [Cech, 2004]. Besides the location at terminal positions, telomeric repeats have also been detected at internal sites in the genome of several mammalian species, the so-called intrachromosomal (or interstitial) telomeric sequences (ITSs). The first detection of ITSs in rodents was made by FISH in Cricetulus griseus [Meyne et al., 1990; Bertoni et al., 1996]. Later, ITSs were described in many different rodents [Meyne et al., 1990; Bertoni et al., 1996; Fagundes and Yonenaga-Yassuda, 1998; Dobigny et al., 2003; Camats et al., 2006; Castiglia et al., 2006; Veyrunes et al., 2006; Nergadze et al., 2007; Rovatsos et al., 2011]. The detailed mechanisms of origin and role of ITSs in evolution were recently discussed [Ruiz-Herrera et al., 2008]. Sequence analysis of short ITSs in mouse and rat can provide some identification of the age of insertion. It was established that ITSs can accumulate some substitutions in TTAGGG hexamers; thus they may serve as useful polymorphic markers for linkage analysis. Besides of the

Other Repetitive Sequences Information about other types of repetitive sequences has accumulated rapidly. It was postulated that expansion of heterochromatic blocks has occurred subsequently to speciation in the Microtus genus [Elisaphenko et al., 1998]. In some Arvicolinae species it was revealed by FISH that Msat-160 satellite DNA is localized in regions of centromeric heterochromatin and overlaps with regions of ITSs [Rovatsos et al., 2011]. As it was mentioned above, some pericentromeric repeats such as GERB1 and GERB2 can mediate centric fusions in Gerbillus nigeriae through het-



239

erologous recombination [Garagna et al., 2001; Kalitsis et al., 2006; Gauthier et al., 2010]. However, our knowledge about repetitive sequences in genomes of rodents and their role in karyotype evolution is far from being complete.

Simultaneous Fixation of Multiple Chromosome Rearrangements

The possibility of simultaneous multiple chromosomal changes was first advanced by King [1993] as opposing to a, at that time, dominant idea of sequential fixation of rearrangements supported by Capanna et al. [1977] and White [1978]. It should be specified that these opposite views concerned the multiple rearrangements of the same kind whereas below we will consider the instances of simultaneous fixation of various kinds of chromosomal changes. It is well known that together with the cases of karyotype evolution predominantly by fixation of the same type of rearrangement (karyotypic orthoselection sensu White [1978]) accompanied by corresponding polymorphisms (Rbs in Mus, Ellobius, Sorex or PIs in different cytotypes of Mastomys among numerous other instances), there are many examples of polymorphisms for 2 or more types of chromosomal changes. The large numeric chromosomal variation in Gerbillus nigeriae caused by Rb fusions (diploid numbers vary from 62–73) is superposed by extensive variation of Cheterochromatin present as entire chromosomal arms or found in juxtacentromeric or interstitial regions [Volobouev et al., 1988, 1995 and references herein]. In 2n = 48 species of the genus Peromyscus, the intra-population NF variation results in pericentric inversions and entirely heterochromatic arms addition [Pathak et al., 1973]. Simultaneous occurrence of Rb polymorphism, Bs variation, and sex chromosome heteromorphism was found in African water rats of the genus Dasymys [Volobouev et al., 2000]. It should be noted that in all these and numerous other instances [for review, see King, 1993] the rearrangements involved in polymorphisms are characterized by a low or no negative effect on fertility. However, the intrapopulation numeric and/or morphological chromosome variability is regularly much narrower than that observed within the whole species range. In this context, the study of the extensive karyotype reorganization in the species of the genera Taterillus [Dobigny et al., 2005 and references herein] and Sicista [Kovalskaya et al., 2011] contributed significantly to our understanding of the mechanisms of chromosomal evolution and speciation. 240


Both species complexes, although belonging to different superfamilies of Myomorpha with very different biology and ecology, display an astonishing similarity in the mode and rate of karyotype repatterning. Both resulted in the formation of a series of reproductively isolated species by means of numerous highly deleterious tandem translocations and Rb fusions displaying monobrachial homology plus less numerous other types of structural rearrangements. Of the total of 38 rearrangements identified in 7 species of Taterillus there were 18 Rb fusions (numerous displaying monobrachial homology), 11 tandem translocations, 6 PIs, and 3 Rb fissions, and 6–21 structural rearrangements. Molecular phylogenetic analysis shows that these 6 more species all radiated during the last 0.4 myr [Dobigny et al., 2002], providing an average rate of 45 rearrangements per myr. Importantly, these 6 species are characterized by rather narrow geographic ranges and are distributed allopatrically [Dobigny et al., 2005]. It was hypothesized that explosive speciation in Taterillus was caused by fixation of highly underdominant genomic rearrangements possible through severe population bottlenecks which resulted from drastic climate and subsequent environmental changes [Dobigny et al., 2002, 2005]. Genome repatterning within the Sicista subtilis complex of sibling species proceeded mainly by Rb fusions (40% of a total of 56 rearrangements). Most displayed monobrachial homology and tandem translocations (22%); one was found in a polymorphic state representing, as far as we are aware, the first case of an in statu nascendi tandem fusion in wild populations [Kovalskaya et al., 2011]. It appeared that the PIs, also numerous (28%), played a very special role in karyotype reorganization. First, the inversion segment of all inversions correspond to the entire acrocentric chromosomes of the out-group taxon, and second, most of them preceded the in statu nascendi fusions involving 2 bi-armed chromosomes by transforming one or both of them into acrocentrics, thus resulting in either centromere-telomere or Rb translocations. Molecular phylogenetic analysis showed that the emergence of 5 taxa, differing from each other by 10–29 structural rearrangements, dated back to 12,500–18,000 years ago [Nicolas, unpublished data], thus giving an unprecedented rate of 2,500 rearrangements per myr. Except for one pair of species found in parapatry, all species are geographically isolated from each other within approximately 135,000 km2 area of the Russian Plain in the vicinity of the Don River bend. It should be noted that the process of isolation probably started with the Don Glaciation (18,000 BP) and has been exacerbated by increasRomanenko /Volobouev

ing human activity in the region in modern time [Kovalskaya et al., 2011]. When discussing the rate of chromosomal evolution, one should keep in mind that the fixation of strongly underdominant rearrangements, such as tandem translocations and Rb fusions displaying monobrachial homology, are more probably very rapid. In some cases they appear almost instantaneous, such as the 20 Rb fusions which occurred in 500–1,000 years in the small M. musculus populations on the island of Madeira [Britton-Davidian et al., 2005].

Genomic Stress and Speciation

The rare instances of the unprecedented rate of fixation of multiple underdominant chromosomal mutations observed among more than 2,000 mammalian species karyotyped raise a question about the nature of events which led to the rapid and extensive genome reorganization. If we consider data inferred from phylogenetic analysis of cytogenetic and molecular data available for some groups of rodent sibling species (Arvicanthis, Acomys, Mastomys: Volobouev et al. [2002a, b]), it appears that the rate of chromosomal changes during evolution in these genera is irregular. For example, in Arvicanthis it reaches 0.7 rearrangements per myr within a 2.9 myr period and reaches 11.6 in another dichotomic period of 0.6 myr. Indeed, the rate can differ sometimes sensibly at each step of divergence of these rodent taxa. A closer look on the periods of accelerated chromosome evolution in these rodent taxa revealed that they are all related to important well-documented climatic and subsequent environmental changes. Unfortunately, there are not many phylogenetic studies yet which integrate cytogenetic and molecular data. Combining molecular and cytogenetic data is the only approach that allows the rearrangements to be correctly mapped onto a phylogenetic tree and thus to determine how many and what kinds of chromosomal changes occurred in a given period. We thus intentionally included the Muntiacus ‘chromosome story’ because its molecular and biological evolution is documented. Cladistic analysis of chromosomal rearrangements carefully identified by means of classical and molecular cytogenetics in the Muntiacus species (together with molecular phylogenetic data [Wang and Lan, 2000] available from abundant literature) revealed that the highest mutation rate was experienced by the last common ancestor of the 4 low diploid numbers species (M. muntjak, M. feae (MFE), M. crinifrons (MCR), and M. gongshanensis (MGO) within a span Rodent Karyotypes in Evolution

of time at most 0.5 myr being 29 rearrangements per myr compared to 1.25 in the lineage of M. reevesi and M. vuquangensis or to 5.9 and 2.3 in the subsequently diverged (MFE+MCR+MGO and MCR+MGO, respectively) [Volobouev, unpublished data]. Again, it appears that despite the evident biological differences between these animal models, the beginning of the process of tandem fusions in Muntiacus is similar to Taterillus and Sicista and coincides with drastic climatic and subsequent environmental changes (different by nature and duration in each of 3 groups; for details see the above cited corresponding references). These changes were followed by disjunction of the distribution area and a drastic reduction of population density in the rare refuges suitable for survival. These observations strongly suggest that an increase in the number of chromosomal mutations is a characteristic of isolated low-density populations. These mutations can be at origin of new species and are the direct results of the genomic stress inevitably arising as a response to the changing biological and population parameters [McClintock, 1984 and references herein]. As it is well known now, there is a large number of ways in which the genome from bacteria to man is prepared ‘to rapidly respond in a programmed manner’ to various internal or external stress. The transposable elements closely related with heterochromatic components present in abundance in the rodent genome played and play probably the leading role in genome repatterning and the most successful radiation among mammals.

Conclusion

Knowledge about the structure of rodent genomes is critical for understanding genome evolution in mammals. Despite decades of study at the chromosomal and single-gene levels and the evidence for great diversity in karyotypes, B-chromosomes, and sex chromosomes, rodent genomes remain poorly known even in this comparative genomics era. Recent comparative cytogenetic data have begun to clarify the events leading from an ancestral rodent karyotype to the very highly modified karyotypes of living species. These findings provide a significant input to our understanding of the relationships between representatives of the taxon. Rodents have greatly different rates of karyotype evolution in different branches of the phylogenetic tree, e.g. from very slow in sciuromorphs to explosive in some myomorphs. These differences illustrate various mechanisms underlying speciation. Certain types of Cytogenet Genome Res 2012;137:233–245

241

chromosome rearrangements make an undoubted contribution to the processes of cladogenesis. As stated, the formation of various phylogenetic groups must have been accompanied by multiple events. At this moment, comparative cytogenetic data have played a larger role than molecular approaches in defining the main trends of genome evolution in rodents. However, phylogenetic comparisons using a wild range of molecular markers and surely full genome sequence comparisons will provide very important additional information about ‘hidden’ rearrangements and inner mechanisms of genome shuffling that have resulted in massive changes in rodent genome architecture.

Further studies of rodents should follow the path of expanding the list of species involved in the comparative work and in more subtle ways of studying the structure of genomes and search for small but evolutionarily significant events. The promise of rodent genomics lies in the greater diversity of the taxa as compared to other mammals and in the increased definition of ancestral states and the range of genomic possibilities explored through the course of evolution.

Acknowledgments This study was funded in part by programs MCB and SB RAS Programs and research grants of Russian Fund for Basic Research.

References Acosta MJ, Romero-Fernandez I, Sanchez A, Marchal JA: Comparative analysis by chromosome painting of the sex chromosomes in arvicolid rodents. Cytogenet Genome Res 132:47–54 (2011). Adkins RM, Mcbee K, Porter CA, Baker RJ: Hybrid breakdown in Peromyscus leucopus and examination of the recombinational-breakdown model. J Mammal 72: 535–541 (1991). Amor DJ, Bentley K, Ryan J, Perry J, Wong L, et al: Human centromere repositioning ‘In progress’. Proc Natl Acad Sci USA 101:6542– 6547 (2004). Aniskin VM, Benazzou T, Biltueva L, Dobigny G, Granjon L, Volobouev V: Unusually extensive karyotype reorganization in four congeneric Gerbillus species (Muridae: Gerbillinae). Cytogenet Genome Res 112: 131– 140 (2006). Arakawa Y, Nishida-Umehara C, Matsuda Y, Sutou S, Suzuki H: X-chromosomal localization of mammalian Y-linked genes in two XO species of the Ryukyu spiny rat. Cytogenet Genome Res 99:303–309 (2002). Ayala FJ, Coluzzi M: Chromosome speciation: humans, drosophila, and mosquitoes. Proc Natl Acad Sci USA 102(suppl 1): 6535–6542 (2005). Badenhorst D, Herbreteau V, Chaval Y, Pages M, Robinson TJ, et al: New karyotypic data for Asian rodents (Rodentia, Muridae) with the first report of B-chromosomes in the genus Mus. J Zool 279:44–56 (2009). Badenhorst D, Dobigny G, Robinson TJ: Karyotypic evolution of Hapalomys inferred from chromosome painting: a detailed characterization contributing new insights into the ancestral murinae karyotype. Cytogenet Genome Res 136:83–88 (2012). Bagheri-Fam S, Sreenivasan R, Bernard P, Knower KC, Sekido R, et al: Sox9 gene regulation and the loss of the XY/XX sex-determining mechanism in the mole vole Ellobius

242

lutescens. Chromosome Res 20: 191–199 (2012). Bakloushinskaya IY: Evolution of sex determination in mammals. Biol Bull 36: 167–174 (2009). Bakloushinskaya IY, Romanenko SA, Graphodatsky AS, Matveevsky SN, Lyapunova EA, Kolomiets OL: The role of chromosome rearrangements in the evolution of mole voles of the genus Ellobius (Rodentia, Mammalia). Russ J Genet 46:1143–1145 (2010). Bakloushinskaya IYu, Matveevsky SN, Romanenko SA, Serdukova NA, Kolomiets OL, et al: A comparative analysis of mole voles sibling species Ellobius tancrei and E. talpinus (Cricetidae, Rodentia) by chromosome painting and synaptonemal complexes structures of hybrids. Cytogen Genome Res 136:199–207 (2012). Banaszek A, Taylor JR, Ochocinska D, Chetnicki W: Robertsonian polymorphism in the common shrew (Sorex araneus L.) and selective advantage of heterozygotes indicated by their higher maximum metabolic rates. Heredity (Edinb) 102:155–162 (2009). Barome PO, Lymberakis P, Monnerot M, Gautun JC: Cytochrome b sequences reveal Acomys minous (Rodentia, Muridae) paraphyly and answer the question about the ancestral karyotype of Acomys dimidiatus. Mol Phylogenet Evol 18:37–46 (2001). Bauchau V: Phylogenetic analysis of the distribution of chromosomal races of Mus musculus domesticus rutty in Europe. Biol J Linn Soc 41:171–192 (1990). Beklemisheva VR, Romanenko SA, Biltueva LS, Trifonov VA, Vorobieva NV, et al: Reconstruction of karyotype evolution in core Glires. I. The genome homology revealed by comparative chromosome painting. Chromosome Res 19:549–565 (2011). Bertoni L, Attolini C, Faravelli M, Simi S, Giulotto E: Intrachromosomal telomere-like


DNA sequences in Chinese hamster. Mamm Genome 7:853–855 (1996). Bianchi NO, Contreras JR: The chromosomes of the field mouse Akodon azarae (Cricetidae, Rodentia) with special reference to sex chromosome anomalies. Cytogenetics 6:306–313 (1967). Britton-Davidian J, Catalan J, da Graca Ramalhinho M, Ganem G, Auffray JC, et al: Rapid chromosomal evolution in island mice. Nature 403:158 (2000). Britton-Davidian J, Catalan J, da Graca Ramalhinho M, Auffray JC, Claudia Nunes A, et al: Chromosomal phylogeny of Robertsonian races of the house mouse on the island of Madeira: testing between alternative mutational processes. Genet Res 86:171–183 (2005). Brown JD, O’Neill: Chromosomes, conflict, and epigenetics: chromosomal speciation revisited. Annu Rev Genomics Hum Genet 11: 291–316 (2010). Burgos M, Jimenez R, Diaz de la Guardia R: XY females in Microtus cabrerae (Rodentia, microtidae): a case of possibly Y-linked sex reversal. Cytogenet Cell Genet 49: 275–277 (1988). Camacho JP, Sharbel TF, Beukeboom LW: Bchromosome evolution. Philos Trans R Soc Lond B Biol Sci 355:163–178 (2000). Camats N, Ruiz-Herrera A, Parrilla JJ, Acien M, Paya P, et al: Genomic instability in rat: breakpoints induced by ionising radiation and interstitial telomeric-like sequences. Mutat Res 595:156–166 (2006). Capanna E: Robertsonian numerical variation in animal speciation: Mus musculus, an emblematic model. Prog Clin Biol Res 96: 155– 177 (1982). Capanna E, Civitelli MV, Cristaldi M, Noack G: New Robertsonian metacentrics in another 22-chromosome mouse population in Central Apennines. Experientia 33: 173–175 (1977).

Romanenko /Volobouev

Castiglia R, Garagna S, Merico V, Oguge N, Corti M: Cytogenetics of a new cytotype of African Mus (subgenus Nannomys) minutoides (Rodentia, muridae) from Kenya: C- and Gbanding and distribution of (TTAGGG)n telomeric sequences. Chromosome Res 14: 587–594 (2006). Cavagna P, Stone G, Stanyon R: Black rat (Rattus rattus) genomic variability characterized by chromosome painting. Mamm Genome 13: 157–163 (2002). Cazaux B, Catalan J, Veyrunes F, Douzery EJ, Britton-Davidian J: Are ribosomal DNA clusters rearrangement hotspots?: a case study in the genus Mus (Rodentia, Muridae). BMC Evol Biol 11: 124 (2011). Cech TR: Beginning to understand the end of the chromosome. Cell 116:273–279 (2004). Chernyavsky FB, Kozlovsky AI: Species status and history of collared lemmings (Dicrostonyx, Rodentia) from Wrangel Island. Zool Zh 59:266–272 (1980). Churakov G, Sadasivuni MK, Rosenbloom KR, Huchon D, Brosius J, Schmitz J: Rodent evolution: back to the root. Mol Biol Evol 27: 1315–1326 (2010). Contreras LC, Torres-Mura JC, Spotorno AE: The largest known chromosome number for a mammal in a South American desert rodent. Experientia 46:506–508 (1990). Davis KM, Smith SA, Greenbaum IF: Evolutionary implications of chromosomal polymorphism in Peromyscus boylii from southwestern Mexico. Evolution 40:645–649 (1986). Davisson MT, Poorman PA, Roderick TH, Moses MJ: A pericentric inversion in the mouse. Cytogenet Cell Genet 30:70–76 (1981). Dobigny G, Aniskin V, Volobouev V: Explosive chromosome evolution and speciation in the gerbil genus Taterillus (Rodentia, Gerbillinae): a case of two new cryptic species. Cytogenet Genome Res 96:117–124 (2002). Dobigny G, Ozouf-Costaz C, Bonillo C, Volobouev V: Evolution of rRNA gene clusters and telomeric repeats during explosive genome repatterning in Taterillus X (Rodentia, Gerbillinae). Cytogenet Genome Res 103: 94–103 (2003). Dobigny G, Ozouf-Costaz C, Bonillo C, Volobouev V: Viability of X-autosome translocations in mammals: an epigenomic hypothesis from a rodent case-study. Chromosoma 113:34–41 (2004). Dobigny G, Aniskin V, Granjon L, Cornette R, Volobouev V: Recent radiation in West African Taterillus (Rodentia, Gerbillinae): the concerted role of chromosome and climatic changes. Heredity 95:358–368 (2005). Elisaphenko EA, Nesterova TB, Duthie SM, Ruldugina OV, Rogozin IB, et al: Repetitive DNA sequences in the common vole: cloning, characterization and chromosome localization of two novel complex repeats MS3 and MS4 from the genome of the East European vole Microtus rossiaemeridionalis. Chromosome Res 6:351–360 (1998). Fagundes V, Yonenaga-Yassuda Y: Evolutionary conservation of whole homeologous chromosome arms in the Akodont rodents Bolomys and Akodon (Muridae, Sigmodontinae):


maintenance of interstitial telomeric segments (ITBs) in recent event of centric fusion. Chromosome Res 6:643–648 (1998). Fagundes V, Vianna-Morgante AM, YonenagaYassuda Y: Telomeric sequences localization and G-banding patterns in the identification of a polymorphic chromosomal rearrangement in the rodent Akodon cursor (2n = 14, 15 and 16). Chromosome Res 5: 228–232 (1997). Fredga K: Aberrant sex chromosome mechanisms in mammals. Evolutionary aspects. Differentiation 23 Suppl:S23–30 (1983). Fredga K, Gropp A, Winking H, Frank F: Fertile XX- and XY-type females in the wood lemming Myopus schisticolor. Nature 261: 225– 227 (1976). Garagna S, Marziliano N, Zuccotti M, Searle JB, Capanna E, Redi CA: Pericentromeric organization at the fusion point of mouse Robertsonian translocation chromosomes. Proc Natl Acad Sci USA 98:171–175 (2001). Gauthier P, Hima K, Dobigny G: Robertsonian fusions, pericentromeric repeat organization and evolution: a case study within a highly polymorphic rodent species, Gerbillus nigeriae. Chromosome Res 18: 473–486 (2010). Gileva EA: Chromosomal diversity and an aberrant genetic system of sex determination in the Arctic lemming, Dicrostonyx torquatus Pallas (1779). Genetica 52–3:99–103 (1980). Graphodatsky AS: Conserved and variable elements of mammalian chromosomes, in Halnan CRE (ed): Cytogenetics of Animals, pp 95–123 (CAB International Press, Oxon 1989). Graphodatsky AS, Kukekova AV, Yudkin DV, Trifonov VA, Vorobieva NV, et al: The protooncogene C-KIT maps to canid B-chromosomes. Chromosome Res 13:113–122 (2005). Graphodatsky AS, Trifonov VA, Stanyon R: The genome diversity and karyotype evolution of mammals. Mol Cytogenet 4:22 (2011). Greenbaum IF, Reed MJ: Evidence for heterosynaptic pairing of the inverted segment in pericentric inversion heterozygotes of the deer mouse (Peromyscus maniculatus). Cytogenet Cell Genet 38:106–111 (1984). Greenbaum IF, Gunn SJ, Smith SA, McAllister BF, Hale DW, et al: Cytogenetic nomenclature of deer mice, Peromyscus (Rodentia): revision and review of the standardized karyotype. Report of the committee for the standardization of chromosomes of Peromyscus. Cytogenet Cell Genet 66:181–195 (1994). Gropp A, Kolbus U, Giers D: Systematic approach to the study of trisomy in the mouse. II. Cytogenet Cell Genet 14:42–62 (1975). Gropp A, Winking H, Redi CA: Consequences of chromosomal heterozygosity, in Crosignani PG, Rubin BL (eds): Genetic Control of Gamete Production and Function, pp 115–134 (Grune and Stratton, New York 1982). Hale DW: Heterosynapsis and suppression of chiasmata within heterozygous pericentric inversions of the Sitka deer mouse. Chromosoma 94:425–432 (1986). Hausser J, Fedyk S, Fredga K, Searle JB, Volobouev V, et al: Definition and nomenclature

of the chromosome races of Sorex araneus. Folia Zool 43:1–9 (1994). Hennig W: Heterochromatin. Chromosoma 108: 1–9 (1999). Huchon D, Madsen O, Sibbald MJ, Ament K, Stanhope MJ, et al: Rodent phylogeny and a timescale for the evolution of Glires: evidence from an extensive taxon sampling using three nuclear genes. Mol Biol Evol 19: 1053–1065 (2002). John B, Miklos GL: The Eukaryote Genome in Development and Evolution. (Allen & Unwin, London 1988). Just W, Rau W, Vogel W, Akhverdian M, Fredga K, et al: Absence of Sry in species of the vole Ellobius. Nat Genet 11:117–118 (1995). Kalitsis P, Griffiths B, Choo KH: Mouse telocentric sequences reveal a high rate of homogenization and possible role in Robertsonian translocation. Proc Natl Acad Sci USA 103: 8786–8791 (2006). King M: Chromosomal Speciation Revisited (Again). Species Evolution. The Role of Chromosome Change. (Cambridge University Press, Cambridge 1993). Kobayashi T, Yamada F, Hashimoto T, Abe S, Matsuda Y, Kuroiwa A: Exceptional minute sex-specific region in the X0 mammal, Ryukyu spiny rat. Chromosome Res 15:175– 187 (2007). Kobayashi T, Yamada F, Hashimoto T, Abe S, Matsuda Y, Kuroiwa A: Centromere repositioning in the X chromosome of X0/X0 mammals, Ryukyu spiny rat. Chromosome Res 16:587–593 (2008). Kovalskaya YM, Aniskin VM, Bogomolov PL, Surov AV, Tikhonov IA, et al: Karyotype reorganisation in the subtilis group of birch mice (Rodentia, Dipodidae, Sicista): unexpected taxonomic diversity within a limited distribution. Cytogenet Genome Res 132: 271–288 (2011). Lanzone C, Rodriguez D, Cuello P, Albanese S, Ojeda A, et al: XY1Y2 chromosome system in Salinomys delicatus (Rodentia, Cricetidae). Genetica 139:1143–1147 (2011). Larkin DM, Pape G, Donthu R, Auvil L, Welge M, Lewin HA: Breakpoint regions and homologous synteny blocks in chromosomes have different evolutionary histories. Genome Res 19:770–777 (2009). Lay DM, Nadler CF: A study of Gerbillus (Rodentia: Muridae) east of the Euphrates River. Mammalia 39:423–445 (1975). Lemskaya NA, Romanenko SA, Golenishchev FN, Rubtsova NV, Sablina OV, et al: Chromosomal evolution of Arvicolinae (Cricetidae, Rodentia). III. Karyotype relationships of ten Microtus species. Chromosome Res 18: 459–471 (2010). Liehr T, Kosyakova N, Weise A, Ziegler M, Raabe-Meyer G: First case of a neocentromere formation in an otherwise normal chromosome 7. Cytogenet Genome Res 128: 189–191 (2010). Livernois AM, Graves JA, Waters PD: The origin and evolution of vertebrate sex chromosomes and dosage compensation. Heredity 108:50–58 (2012).


243

Lyapunova EA, Vorontsov NN, Korobitsyna KV, Ivanitskaya EY, Borisov YM, et al: A Robertsonian fan in Ellobius talpinus. Genetica 523:239–247 (1980). Ma J, Zhang L, Suh BB, Raney BJ, Burhans RC, et al: Reconstructing contiguous regions of an ancestral genome. Genome Res 16: 1557– 1565 (2006). Marchal JA, Acosta MJ, Nietzel H, Sperling K, Bullejos M, et al: X chromosome painting in Microtus: origin and evolution of the giant sex chromosomes. Chromosome Res 12:767– 776 (2004). Matsubara K, Nishida-Umehara C, Kuroiwa A, Tsuchiya K, Matsuda Y: Identification of chromosome rearrangements between the laboratory mouse (Mus musculus) and the Indian spiny mouse (Mus platythrix) by comparative FISH analysis. Chromosome Res 11:57–64 (2003). Matsubara K, Nishida-Umehara C, Tsuchiya K, Nukaya D, Matsuda Y: Karyotypic evolution of Apodemus (Muridae, Rodentia) inferred from comparative FISH analyses. Chromosome Res 12:383–395 (2004). Matsubara K, Yamada K, Umemoto S, Tsuchiya K, Ikeda N, et al: Molecular cloning and characterization of the repetitive DNA sequences that comprise the constitutive heterochromatin of the A and B chromosomes of the Korean field mouse (Apodemus peninsulae, Muridae, Rodentia). Chromosome Res 16:1013–1026 (2008). Matthey R: A new type of multiple sex chromosome in an African mouse of the group Mus (leggada) minutoides (Mammalia, Rodentia). Male: X1X 2/Y. Female: X1X 2/X1X 2. Chromosoma 16: 351–364 (1965). McClintock B: The significance of responses of the genome to challenge. Science 226: 792– 801 (1984). Meylan A: Chromosome patterns of various small North American mammals. Rev Suisse Zool 75:691–696 (1968). Meyne J, Baker RJ, Hobart HH, Hsu TC, Ryder OA, et al: Distribution of non-telomeric sites of the (TTAGGG)n telomeric sequence in vertebrate chromosomes. Chromosoma 99: 3–10 (1990). Mlynarski EE, Obergfell CJ, O’Neill MJ, O’Neill RJ: Divergent patterns of breakpoint reuse in muroid rodents. Mamm Genome 21: 77–87 (2010). Montefalcone G, Tempesta S, Rocchi M, Archidiacono N: Centromere repositioning. Genome Res 9:1184–1188 (1999). Murata C, Yamada F, Kawauchi N, Matsuda Y, Kuroiwa A: The Y chromosome of the Okinawa spiny rat, Tokudaia muenninki, was rescued through fusion with an autosome. Chromosome Res 20: 111–125 (2012). Murphy WJ, Eizirik E, Johnson WE, Zhang YP, Ryder OA, O’Brien SJ: Molecular phylogenetics and the origins of placental mammals. Nature 409:614–618 (2001). Murphy WJ, Larkin DM, Everts-van der Wind A, Bourque G, Tesler G, et al: Dynamics of mammalian chromosome evolution inferred from multispecies comparative maps. Science 309:613–617 (2005).

244

Nachman MW, Searle JB: Why is the house mouse karyotype so variable? Trends Ecol Evol 10:397–402 (1995). Nanda I, Neitzel H, Sperling K, Studer R, Epplen JT: Simple GATCA repeats characterize the X chromosomal heterochromatin of Microtus agrestis, European field vole (Rodentia, Cricetidae). Chromosoma 96: 213–219 (1988). Nergadze SG, Santagostino MA, Salzano A, Mondello C, Giulotto E: Contribution of telomerase RNA retrotranscription to DNA double-strand break repair during mammalian genome evolution. Genome Biol 8:R260 (2007). Noor MA, Grams KL, Bertucci LA, Reiland J: Chromosomal inversions and the reproductive isolation of species. Proc Natl Acad Sci USA 98:12084–12088 (2001). O’Brien SJ, Menninger JC, Nash WG: Atlas of Mammalian Chromosomes (John Wiley & Sons, Hoboken 2006). Ohno S: Evolution of sex chromosomes in mammals. Annu Rev Genet 3:496–524 (1969). Ohno S, Jainchill J, Stenius C: The creeping vole (Microtus oregoni) as a gonosomic mosaic. I. The 0Y/XY constitution in the male. Cytogenetics 2:232 (1963). Ohno S, Stenius C, Christian L: The X0 as the normal female of the creeping vole (Microtus oregoni), in Darlington CD, Lewis KR (eds): Chromosomes Today, pp 182–187 (Oliver and Boyd, Edinburgh 1964). Pathak S, Stock AD: The X chromosomes of mammals: karylogical homology as revealed by banding techniques. Genetics 78:703–714 (1974). Pathak S, Hsu TC, Arrighi FE: Chromosomes of Peromyscus (Rodentia, Cricetidae). IV. The role of heterochromatin in karyotypic evolution. Cytogenet Cell Genet 12: 315–326 (1973). Patton JL, Sherwood SW: Genome evolution in pocket gophers (genus Thomomys). I. Heterochromatin variation and speciation potential. Chromosoma 85: 149–162 (1982). Pavlova SV, Bulatova N, Shchipanov NA: Cytogenetic control of a hybrid zone of between two Sorex araneus chromosome races before breeding season. Genetika 43: 1619–1626 (2007). Pavlova SV, Kolomiets OL, Bulatova NS, Searle JB: Demonstration of a WART in a hybrid zone of the common shrew (Sorex araneus Linnaeus, 1758). Comp Cytogenet 2:115–120 (2008). Pialek J, Hauffe HC, Searle JB: Chromosomal variation in the house mouse. Biol J Linn Soc 84:535–563 (2005). Quere JP, Raoul F, Aniskin V, Durette-Desset MC, Giraudoux P: Original biological and ecological data on the endemic Chinese jumping mouse Eozapus setchuanus (Pousargues, 1896). Mamm Biol 74: 507–513 (2009). Raman R, Sharma T: Unique multiple sex chromosomes of the tree mouse Vandeleuria o. Oleracea: identification of X1 and X 2. Heredity (Edinb) 37:435–439 (1976).


Redi CA, Capanna E: Robertsonian heterozygotes in the house mouse and the fate of their germ cells, in Daniel A (ed): The Cytogenetic of Mammalian Rearrangements, pp 315– 359 (Alan R Liss, New York 1988). Redi CA, Garagna S, Capanna E: Nature’s experiment with in situ hybridization? A hypothesis for the mechanism of the Rb fusion. J Evol Biol 3:133–137 (1990). Reig OA, Kiblisky P: Chromosomes in four species of rodents of the genus Ctenomys (Rodentia, Octodontidae) from Argentina. Experientia 24:274–276 (1968). Reig OA, Kiblisky P: Chromosome multiformity in the genus Ctenomys (Rodentia, Octodontidae). A progress report. Chromosoma 28: 211–244 (1969). Rieseberg LH: Chromosomal rearrangements and speciation. Trends Ecol Evol 16:351–358 (2001). Rocchi M, Archidiacono N: The centromere repositioning, in Lupski JR, Stankiewicz P (eds): Genome Plasticity in Evolution, pp 153–165 (Humana Press, Totowa 2006). Rocchi M, Archidiacono N, Schempp W, Capozzi O, Stanyon R: Centromere repositioning in mammals. Heredity (Edinb) 108: 59– 67 (2012). Romanenko SA, Perelman PL, Trifonov VA, Graphodatsky AS: Chromosomal evolution in Rodentia. Heredity (Edinb) 108: 4–16 (2012). Rovatsos MT, Marchal JA, Romero-Fernandez I, Fernandez FJ, Giagia-Athanosopoulou EB, Sanchez A: Rapid, independent, and extensive amplification of telomeric repeats in pericentromeric regions in karyotypes of arvicoline rodents. Chromosome Res 19: 869– 882 (2011). Rubtsov NB, Karamysheva TV, Andreenkova OV, Bochkaerev MN, Kartavtseva IV, et al: Comparative analysis of micro and macro B chromosomes in the Korean field mouse Apodemus peninsulae (Rodentia, Murinae) performed by chromosome microdissection and FISH. Cytogenet Genome Res 106: 289– 294 (2004). Ruiz-Herrera A, Castresana J, Robinson TJ: Is mammalian chromosomal evolution driven by regions of genome fragility? Genome Biol 7:R115 (2006). Ruiz-Herrera A, Nergadze SG, Santagostino M, Giulotto E: Telomeric repeats far from the ends: mechanisms of origin and role in evolution. Cytogenet Genome Res 122: 219–228 (2008). Sbalqueiro IJ, Mattevi MS, Oliveira LF: An X1X1X 2X 2/X1X 2Y mechanism of sex determination in a South American rodent, Deltamys kempi (Rodentia, Cricetidae). Cytogenet Cell Genet 38:50–55 (1984). Searle JB: Chromosomal hybrid zones in eutherian mammals, in Harrison RG (ed): Hybrid Zones and the Evolutionary Process, pp 309– 353 (Oxford University Press, New York 1993). Shchipanov NA, Bulatova NS, Pavlova SV, Shchipanov AN: The common shrew (Sorex araneus) as a model species in ecological and evolutionary studies. Zool Zh 88: 975–989 (2009).

Romanenko /Volobouev

Silva MJ, Yonenaga-Yassuda Y: Karyotype and chromosomal polymorphism of an undescribed Akodon from Central Brazil, a species with the lowest known diploid chromosome number in rodents. Cytogenet Cell Genet 81:46–50 (1998). Soullier S, Hanni C, Catzeflis F, Berta P, Laudet V: Male sex determination in the spiny rat Tokudaia osimensis (Rodentia: Muridae) is not Sry dependent. Mamm Genome 9: 590– 592 (1998). Stitou S, Diaz de La Guardia R, Jimenez R, Burgos M: Inactive ribosomal cistrons are spread throughout the B chromosomes of Rattus rattus (Rodentia, Muridae). Implications for their origin and evolution. Chromosome Res 8:305–311 (2000). Sutou S, Mitsui Y, Tsuchiya K: Sex determination without the Y chromosome in two Japanese rodents Tokudaia osimensis osimensis and Tokudaia osimensis spp. Mamm Genome 12: 17–21 (2001). Swier VJ, Bradley RD, Rens W, Elder FF, Baker RJ: Patterns of chromosomal evolution in Sigmodon, evidence from whole chromosome paints. Cytogenet Genome Res 125: 54–66 (2009). Trifonov VA, Perelman PL, Kawada SI, Iwasa MA, Oda SI, Graphodatsky AS: Complex structure of B-chromosomes in two mammalian species: Apodemus peninsulae (Rodentia) and Nyctereutes procyonoides (Carnivora). Chromosome Res 10: 109–116 (2002). Trifonov VA, Kosyakova N, Romanenko SA, Stanyon R, Graphodatsky AS, Liehr T: New insights into the karyotypic evolution in muroid rodents revealed by multicolor banding applying murine probes. Chromosome Res 18:265–275 (2010a). Trifonov VA, Dement’eva PV, Beklemisheva VR, Iudkin DV, Vorob’eva NV, Grafodatskii AS: Supernumerary chromosomes, segmental duplications, and evolution. Genetika 46: 1234–1236 (2010b). Ventura K, O’Brien PC, Yonenaga-Yassuda Y, Ferguson-Smith MA: Chromosome homologies of the highly rearranged karyotypes of four Akodon species (Rodentia, Cricetidae) resolved by reciprocal chromosome painting: the evolution of the lowest diploid number in rodents. Chromosome Res 17: 1063– 1078 (2009). Ventura M, Weigl S, Carbone L, Cardone MF, Misceo D, et al: Recurrent sites for new centromere seeding. Genome Res 14: 1696–1703 (2004). Veyrunes F, Dobigny G, Yang F, O’Brien PC, Catalan J, et al: Phylogenomics of the genus Mus (Rodentia; Muridae): extensive genome repatterning is not restricted to the house mouse. Proc Biol Sci 273:2925–2934 (2006).


Veyrunes F, Chevret P, Catalan J, Castiglia R, Watson J, et al: A novel sex determination system in a close relative of the house mouse. Proc Biol Sci 277: 1049–1056 (2010). Viegas-Pequignot E, Benazzou T, Dutrillaux B, Petter F: Complex evolution of sex chromosomes in Gerbillidae (Rodentia). Cytogenet Cell Genet 34:158–167 (1982). Viegas-Pequignot E, Dutrillaux B, Prod’Homme M, Petter F: Chromosomal phylogeny of muridae: a study of 10 genera. Cytogenet Cell Genet 35:269–278 (1983). Vogel W, Jainta S, Rau W, Geerkens C, Baumstark A, et al: Sex determination in Ellobius lutescens: the story of an enigma. Cytogenet Cell Genet 80:214–221 (1998). Volobouev VT: Karyological analysis of three Siberian populations of the Korean field mouse Apodemus peninsulae (Rodentia, Muridae). Reports of AS USSR 248:1452–1454 (1979). Volobouev VT: B-chromosomes system of the mammals. Caryologia 34: 1–23 (1981). Volobouev VT, Granjon L: A finding of the XX/ XY1Y2 sex-chromosome system in Taterillus arenarius (Gerbillinae, Rodentia) and its phylogenetic implications. Cytogenet Cell Genet 75:45–48 (1996). Volobouev VT, Viegaspequignot E, Petter F, Gautin JC, Sicard B, Dutrillaux B: Complex chromosomal polymorphism in Gerbillus nigeriae (Rodentia, Gerbillidae). J Mammal 69:131–134 (1988). Volobouev VT, Vogt N, Viegas-Pequignot E, Malfoy B, Dutrillaux B: Characterization and chromosomal location of two repeated DNAs in three Gerbillus species. Chromosoma 104:252–259 (1995). Volobouev VT, Sicard B, Aniskin VM, Gautun JC, Granjon L: Robertsonian polymorphism, B chromosomes variation and sex chromosomes heteromorphism in the African water rat Dasymys (Rodentia, Muridae). Chromosome Res 8:689–697 (2000). Volobouev VT, Hoffmann A, Sicard B, Granjon L: Polymorphism and polytypy for pericentric inversions in 38-chromosome Mastomys (Rodentia, Murinae) and possible taxonomic implications. Cytogenet Cell Genet 92: 237– 242 (2001). Volobouev VT, Aniskin VM, Lecompte E, Ducroz JF: Patterns of karyotype evolution in complexes of sibling species within three genera of African murid rodents inferred from the comparison of cytogenetic and molecular data. Cytogenet Genome Res 96:261– 275 (2002a). Volobouev VT, Ducroz JF, Aniskin VM, BrittonDavidian J, Castiglia R, et al: Chromosomal characterization of Arvicanthis species (Rodentia, Murinae) from Western and Central Africa: implications for taxonomy. Cytogenet Genome Res 96:250–260 (2002b).

Vorontsov NN, Lyapunova EA, Borissov YM, Dovgal VE: Variability of sex-chromosomes in mammals. Genetica 52–3:361–372 (1980). Vujosevic M, Blagojevic J: B chromosomes in populations of mammals. Cytogenet Genome Res 106:247–256 (2004). Waddell PJ, Kishino H, Ota R: A phylogenetic foundation for comparative mammalian genomics. Genome Inform 12: 141–154 (2001). Wahrman J, Goitein R, Nevo E: Mole rat spalax: evolutionary significance of chromosome variation. Science 164:82–84 (1969). Wallace BM, Searle JB: Oogenesis in homozygotes and heterozygotes for Robertsonian chromosomal rearrangements from natural populations of the common shrew, Sorex araneus. J Reprod Fertil 100:231–237 (1994). Wang W, Lan H: Rapid and parallel chromosomal number reductions in muntjac deer inferred from mitochondrial DNA phylogeny. Mol Biol Evol 17:1326–1333 (2000). White MJ: Modes of Speciation (Freeman, San Francisco 1978). White TA, Bordewich M, Searle JB: A network approach to study karyotypic evolution: the chromosomal races of the common shrew (Sorex araneus) and house mouse (Mus musculus) as model systems. Syst Biol 59:262–276 (2010). Wilson MA, Makova KD: Genomic analyses of sex chromosome evolution. Annu Rev Genomics Hum Genet 10:333–354 (2009). Wojcik JM, Borodin PM, Fedyk S, Fredga K, Hausser J, et al: The list of the chromosome races of the common shrew Sorex araneus (updated 2002). Mammalia 67: 169–178 (2003). Yamada K, Kamimura E, Kondo M, Tsuchiya K, Nishida-Umehara C, Matsuda Y: New families of site-specific repetitive DNA sequences that comprise constitutive heterochromatin of the syrian hamster (Mesocricetus auratus, Cricetinae, Rodentia). Chromosoma 115: 36–49 (2006). Yang F, O’Brien PC, Wienberg J, Neitzel H, Lin CC, Ferguson-Smith MA: Chromosomal evolution of the chinese muntjac (Muntiacus reevesi). Chromosoma 106:37–43 (1997). Zhao S, Shetty J, Hou L, Delcher A, Zhu B, et al: Human, mouse, and rat genome large-scale rearrangements: stability versus speciation. Genome Res 14:1851–1860 (2004). Zhu B, Gao H, Wang H, Gao J, Zhang Y, et al: The origin of the genetical diversity of Microtus mandarinus chromosomes. Hereditas 139: 90–95 (2003). Zima J, Bulatova N: The astonishing diversity of chromosomal races in the common shrew (Sorex araneus). ECM 2011 VIth European Congress of Mammalogy. Abstract volume: 23 (2011).


245



Molecular Cytogenetic Studies in Strepsirrhine Primates, Dermoptera and Scandentia W. Nie State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, PR China

Key Words Chromosome painting ⴢ Flying lemurs ⴢ Lemurs ⴢ Lorises ⴢ Syntenic associations ⴢ Tree shrews

Abstract Since the first chromosome painting study between human and strepsirrhine primates was performed in 1996, nearly 30 species in Strepsirrhini, Dermoptera and Scandentia have been analyzed by cross-species chromosome painting. Here, the contribution of chromosome painting data to our understanding of primate genome organization, chromosome evolution and the karyotype phylogenetic relationships within strepsirrhine primates, Dermoptera and Scandentia is reviewed. Twenty-six to 43 homologous chromosome segments have been revealed in different species with human chromosome-specific paint probes. Various landmark rearrangements characteristic for each different lineage have been identified, as cytogenetic signatures that potentially unite certain lineages within strepsirrhine primates, Dermoptera and Scandentia. Copyright © 2012 S. Karger AG, Basel



Molecular phylogenetic studies placed Primates, Dermoptera, Scandentia, Rodentia, and Lagomorpha in 1 superordinal clade called Euarchontoglires or Supraprimates, 1 of the 4 superordinal clades for the 18 extant placental orders [Waddell et al., 1999, 2001; Liu et al., 2001; Murphy et al., 2001a, 2001b; Springer et al., 2003, 2004; Bininda-Emonds et al., 2007]. Within the Euarchontoglires, there are 2 subgroups: Euarchonta (=Primates + Dermoptera + Scandentia) and Glires (Rodentia + Lagomorpha). However, molecular and morphological studies so far have not resolved unambiguously the relationships among Primates, Dermoptera and Scandentia. Within Euarchonta, various hypotheses on the inter-ordinal relationships have been proposed by different morphological and molecular phylogenetic studies. These hypotheses included all possible combinations: (1) a closer relationship between Primates and Dermoptera, forming the clade Primatomorpha [Kay et al., 1992; Novacek, 1992; Beard, 1993; Waddell et al., 1999, 2001; Hudelot et al., 2003; Bininda-Emonds et al., 2007]; (2) Dermoptera as a sister group to the anthropoid primates [Murphy et W. Nie State Key Laboratory of Genetic Resources and Evolution Kunming Institute of Zoology, Chinese Academy of Sciences 32 Jiaochang Donglu, Kunming, Yunnan 650223 (PR China) Tel. +86 871 519 5375, E-Mail whnie @ mail.kiz.ac.cn

al., 2001a; Arnason et al., 2002]; (3) Dermoptera as the closest living relative of Primates [Janecka et al., 2007]; (4) a closer relationship between Scandentia and Primates [Martin, 1990; Silcox et al., 2005]; (5) a closer relationship between Scandentia and Dermoptera [Adkins and Honeycutt, 1991; Liu et al., 2001; Murphy et al., 2001b; Springer et al., 2003, 2004]; (6) a sister-group relationship between Scandentia and Lagomorpha [Martin, 1990; Bailey et al., 1992; Schmitz et al., 2000]. The classification of some groups in Euarchonta has long been controversial, e.g. the tree shrews and the tarsiers. The tree shrews had been classified either within the order ‘Insectivora’ or with in the infraorder Lemuriformes of the Primates. Now, the view that tree shrews represent a distinct order, Scandentia, has won general acceptance. Recent studies divide this order into 2 families: Tupaiidae (the true tree shrew, including 4 genera) and Ptilocercidae (the feather-tailed tree shrew, containing only a single genus). Currently, there are about 20 species recognized in Scandentia [Helgen, 2005].The tarsiers have a number of primitive features. They were more traditionally placed with the lemurs and lorisiforms in prosimians. However, several critical characters suggest a closer relationship to the monkeys and apes. According to this view, order Primates is split into 2 suborders: Stresirrhini, or curly-nosed primates, consisting of non-tarsier prosimians, and Haplorhini, or dry-nosed primates, consisting of the tarsiers and the simians [Nowak, 1999; Groves, 2005 and references therein]. The suborder Stresirrhini includes 2 Infraorders: Lemuriformes and Lorisiformes. Lemuriformes consists of 5 families: Cheirogaleidae, Daubentoniidae, Lemuridae, Lepilemuridae, Indriidae. More than 100 species are described in these 5 lemur families, but in Daubentoniidae, there is only 1 living species. All species in Lemuriformes are found exclusively in Madagascar. Lorisiformes contains 2 families – Lorisidae and Galagidae – with 28 species, distributing in both Asia and Africa [Groves, 2005]. Order Dermoptera includes a single family, with 2 living species [Nowak, 1999 and references therein]. Galeopterus variegates, is found in southern parts of Thailand and Indochina, Malay Peninsula, Sumatra, Java, Borneo, and many nearby islands [Corbert and Hill, 1992]. Cynocephalus volans, is restricted to the Mindanao faunal region of the southern Philippines [Heaney et al., 1998]. Karyotypes, as one of the distinctive inherited characters for each species, can provide reliable information for the study of taxonomic and phylogenetic relationships if they are compared across species on the basis of true homologies. As a result, the advances of comparative cyto-

genetics have been propelled by technical innovation in tools that enable a reliable detection of interspecific chromosome homologies. Over the last decades, comparative cytogenetic studies have contributed many data about evolutionary relationships between a great number of mammalian species, demonstrating that despite variations in diploid number and chromosome morphology mammalian genomes are highly conserved in general and that only a limited number of chromosome rearrangements have occurred during the evolution of most species [see Ferguson-Smith and Trifonov, 2007, and references therein]. The karyotype evolution of Primates is among the most well documented. Such studies are in part driven by our curiosity to understand our origin and evolution of our own chromosomes. Chromosome banding homologies have been proposed for 60 species of primates, including man [Dutrillaux, 1979], which have allowed the proposition of a phylogenetic tree and ancestral karyotypes for different suborders or infraorders of Primates [Dutrillaux, 1979; Dutrillaux and Couturier, 1981; Rumpler and Dutrillaux, 1990]. In addition, cytogenetic studies are very useful for species identification in prosimians and New World primates. However, it is very difficult to compare the karyotypes between species with distant relationships or with highly rearranged chromosomes only based on banding patterns. The introduction of fluorescence in situ hybridization, using whole chromosome-specific probes, made it possible to establish chromosome homology between any 2 placental mammalian species based on synteny conservation of homologous DNA sequences [Scherthan et al., 1994; Wienberg and Stanyon, 1995]. Cross-species chromosome painting allows us to track chromosome rearrangements that have occurred during evolution based on the distribution pattern of conserved chromosome segments and verify independently the molecular phylogenetic tree using signature chromosome rearrangements. Chromosome painting has provided reliable data for the reconstruction of genome evolution, the mechanisms involved in speciation and an overview of phylogenetic and taxonomic relationships [Wienberg and Stanyon, 1997; Chowdhary et al., 1998; O’Brien et al., 1999; Murphy et al., 2001c, 2004; Frönicke et al., 2003; Richard et al., 2003; Yang et al., 2003; Ferguson-Smith and Trifonov, 2007]. For the last 2 decades, many species from different orders of the Euarchonta clade, especially Primates, have been analyzed by chromosome painting. Wienberg [2005] reviewed the progress of studying human and primate genome evolution using fluorescence in situ hybridiza-

Chromosome Painting in Strepsirrhini, Dermoptera and Scandentia


247

tion on chromosomes as a technique. Horvath and Willard [2007] reviewed the current state of lemur cytogenetic and phylogenetic analyses. Stanyon et al. [2008] summed the results of determining marker order along primate chromosomes by BAC-FISH and proposed the presumed marker order and centromere position in primate ancestral karyotypes at 5 major branching points on the primate evolutionary tree. Kehrer-Sawatzki and Cooper [2008] discussed molecular mechanisms of chromosome rearrangement during primate evolution. Picone et al. [2011] used molecular systematic methods and demonstrated 161 chromosome associations revealed by chromosome painting as characters to reanalyze the inter-order phylogenetic relationships in Euarchontoglires. Here, I mainly focus on chromosome painting among species of strepsirrhine primates, Dermoptera and Scandentia. I briefly introduce the classical cytogenetic studies of strepsirrhine primates, tree shrews and flying lemurs, and provide an overview of chromosome painting results in this group. The contribution of chromosome painting endeavour to decipher the karyotype evolution and phylogenetic relationships of Strepsirrhini, Dermoptera and Scandentia is also discussed.

Classical Cytogenetic Studies in Species of Strepsirrhini, Dermoptera and Scandentia

During the past 4 decades, karyotypes of more than 60 lemurs have been reported. The diploid chromosome number ranges from 2n = 20 (Lepilemur ruficaudatus) to 2n = 70 (Avahi laniger) [reviewed in Horvath and Willard, 2007]. Banding analyses suggested that some lemur species might have a karyotype close to that of the assumed ancestral primate, and that Robertsonian rearrangements were the most common type of rearrangements in most lemurs, except for one genus wherein tandem fusions were common [Rumpler and Dutrillaux, 1976, 1978, 1979; Dutrillaux, 1979; Rumpler et al., 1983a, 1985]. For Lorisiformes, the diploid chromosome number varies from 2n = 38 to 2n = 62 [Dutrillaux, 1979; Dutrillaux et al., 1982; Rumpler et al., 1983b, 1987]. Banding studies indicated that lorisiform primates probably evolved from a common ancestor with a diploid number of 2n = 62 [Rumpler et al., 1983b]. The karyotype phylogenetic relationships for strepsirrhine primates were proposed by Rumpler et al. [1989]. There are only 2 living species in Dermoptera. The karyotype of G. variegates is 2n = 56 [Hsu and Benirschke, 1973], while that of C. volans is 2n = 38 [Rickart, 2003]. 248


The substantial differences between karyotypes of these 2 living flying lemurs also provide support for placing these 2 species in 2 different genera [Stafford and Szalay, 2000]. In Scandentia, only 7 species from 2 of the 5 currently recognized tree shrew genera have been karyotyped. The diploid number varies from 2n = 44 (Urogale everetti) to 2n = 68 (Tupaia montana) [reviewed in Helgen, 2006]. Chromosome homologies between different tree shrews have been proposed by banding analysis [Toder et al., 1992].


In strepsirrhine primates, Dermoptera and Scandentia, nearly 30 species have been analyzed by chromosome painting [Apiou et al., 1996; Müller et al., 1997, 1999; Vezuli et al., 1997; Cardone et al., 2002; Stanyon et al., 2002, 2006; Warter et al., 2005; Nie et al., 2006, 2008; Rumpler et al., 2008; Dumas et al., 2012]. Chromosome homologies between human (Homo sapiens, HSA) and these studied species have been established in all of these studies by using partial or the whole set of human chromosome-specific probes. Besides human chromosome-specific probes, 7 sets of chromosome-specific probes from 2 lemurs (Eulemur fulvus mayottensis, 2n = 60, and E. macaco macaco, 2n = 44) [Müller et al., 1997], 2 galagos (Galago moholi, 2n = 38) [Nie et al., 2006], (Otolemur garnettii, 2n = 62) [Stanyon et al., 2006], 1 slow loris (Nycticebus coucang, 2n = 50) [Stanyon et al., 2006], 1 tree shrew (Tupaia belangeri, 2n = 62) [Müller et al., 1999], and 1 flying lemur (G. variegates, 2n = 56) [Nie et al., 2008] have been prepared from flow sorted chromosomes and used in some of these studies. In addition, locus-specific probes derived from the human BAC/PAC libraries have also been used in some of these studies to reconstruct ancestral marker order [Cardone et al., 2002; Stanyon et al., 2008 and references therein]. Table 1 lists all the species studied by chromosome painting in Strepsirrhini, Dermoptera and Scandentia. The establishment of comparative chromosome maps enables the precise identification of the homologous segments between human and these species, and the comparison of the chromosome homologies among these species with human chromosomes as references. Table 2 shows homologous chromosome segments of human and 17 species of strepsirrhine primates, Dermoptera and Scandentia. Twenty-six to forty-three conserved chromosome segments homologous to human chromosomes Nie

Table 1. List of species analyzed by chromosome painting among Strepsirrhini, Dermoptera and Scandentia

Order

Species

Method

References

HSA]Propithecus verreauxi HSA]other lemur species

CP, 6 probes CP, 4 probes CP, partial probes CP, 6 probes RP CP, 6 probes CP, 4 probes CP, BAC/PAC CP CP, 4 probes CP, 4 probes CP, 4 probes CP CP, 4 probes CP CP CP CP CP CP CP, partial probes CP CP, partial probes CP CP, partial probes

Apiou et al., 1996 Vezuli et al., 1997 Rumpler et al., 2008 Apiou et al., 1996 Müller et al., 1997 Apiou et al., 1996 Vezuli et al., 1997 Cardone et al., 2002 Warter et al., 2005 Vezuli et al., 1997 Vezuli et al., 1997 Vezuli et al., 1997 Warter et al., 2005 Vezuli et al., 1997 Müller et al., 1997 Müller et al., 1997 Warter et al., 2005 Warter et al., 2005 Warter et al., 2005 Warter et al., 2005 Rumpler et al., 2008 Warter et al., 2005 Rumpler et al., 2008 Warter et al., 2005 Warter et al., 2005

Lorisiformes HSA]Otolemur crassicaudatus HSA]Galago moholi HSA]Nycticebus coucang HSA[]N. coucang G. moholi]N. coucang, HSA Otolemur garnettii]HSA

CP CP CP RP CP CP

Stanyon et al., 2002 Stanyon et al., 2002 Nie et al., 2006; Stanyon et al., 2006 Nie et al., 2006 Stanyon et al., 2006

Dermoptera

HSA[]Galeopterus variegatus

RP

Nie et al., 2008

Scandentia

HSA[]Tupaia belangeri T. belangeri]E. macaco macaco HSA]Tupaia minor

RP CP CP

Müller et al., 1999 Müller et al., 1999 Dumas et al., 2012

Primates Strepsirrhini

Lemuriformes HSA]Eulemur fulvus mayottensis HSA]Eulemur macaco macaco HSA[]E. macaco macaco HSA]Lemur catta

HSA]Varecia variegate HSA]Hapalemur simus HSA]Hapalemur griseus griseus HSA]Hapalemur aureus HSA]E. fulvus mayottensis E. macaco macaco]E. fulvus mayottensis HSA]Avahi laniger HSA]Daubentonia madagascariensis HSA]Indri indri HSA]Lepilemur spp. HSA]Microcebus murinus

HSA = Homo sapiens; CP = chromosome painting; RP = reciprocal chromosomal painting.

] = Unidirectional; [] = bidirectional.

were found in different species from Strepsirrhini, Dermoptera and Scandentia. Chromosome painting data demonstrated that many human chromosome homologues were fragmented in the genomes of these species, and that there were numerous chromosome rearrangements differences between their karyotypes. Since chro-

mosome rearrangements are often considered as unique events with low level of convergence, the use of specific chromosome rearrangements may be helpful in providing independent phylogenetic support for relationships established from morphological or molecular techniques [Murphy et al., 2001c; Wienberg, 2005].



249

Table 2. Chromosomal homologies between human and species from Strepsirrhini, Dermoptera and Scandentia revealed by chromosome painting Lemuriformes

Lorisiformes

HSA

DMA MIM

LCA

1

7

2, 17, 27

HGG

EFU

Dermoptera

Scandentia

IIN

OCR

OGA

GMO NCO

GVA

TBE

TMI

2, 13, 28, 5, 7, 29 19, 21

6, 7, 9, 10

8, 21, 23

8, 21, 23

7, 12, 8, 10, 14 22

6, 10, 16, 27

10, 19

9, 14, 18

EFM

EMA

ALA

3, 22, 25 2, 22, 24

2, 25, 27 2, 22, 23

9, 14, 18

PVE

2

6

4, 8

5, 11

7, 10

4, 8

4, 8

2, 6

4, 11

3, 2

2, 4

2, 4

2, 4

4, 9

1, 11

1, 8

7, 12

5, 11

3 + 21

1

1, 31, 32

1, 3

1, 18

1

1

1

1

1

1

5, 10, 26

5, 10

3, 11

9, 12

1, 6

6, 7, 24, 28

5, 23, 25, 29, 32

4

3

4, 14, 28, 4, 5, 3, 7, 29, 30 19, 23, 26 18, 23, 25

4, 13, 18, 4, 13, 18, 6, 8, 23, 26 28 12, 20

3, 4, 2, 11, 17, 27, 31 13, 20, 23

2, 9, 14, 17, 18

9, 27

9, 27

6, 16

2, 24

12, 22, 26

22, 23, 25, 26

21, 22, 24, 27

5

4

12, 23

4, 12

13, 19

11, 21

7, 25

3, 15

10, 24

4, 6

1, 5

13, 17

13, 17

1, 7

2, 19

14, 17

14, 21

13, 20

6

2

6, 14

2, 4

3, 6

5, 13

5, 13

8, 11

5, 30

1, 11

6, 14

1, 18

1, 18

1, 6

7, 10

4

8, 29

6, 30

7

5, 9

11, 17

2, 7

5, 14

9, 19

12, 27

3, 19

2, 9

2, 7

2, 6

6, 7

6, 7

3, 5

8

10, 13 9

6

12

7

6, 21

8

6

3

4

12, 29

12, (29) 8, 18

9

3

10

10

11

1

1

1

8

10

8, 14

15, 25

1, 14

1, 16

14, 22

14, 26

5, 17

11

1

5

8

8

6

11

4

3

8

11

12 + 22

5, 10

7, 21

9, 21

9, 12

10, 24

10, 19

5, 13

14, 25

10, 17

10, 12

13

8

13

13

15

12

9

4

12

6

3

14

14 + 15

2

3, 9

1, 6

1, 5

3, 7

3, 6

2, 7

6, 7

3, 9

4, 8

16

9, 11

17, 20

2, 19

5, 20

19, 20

16, 24

7, 10

2p, 2qter

7, 13

6, 10

17

9

16

15

4

15

15

10

15

7

18

5

19

16

17

16

16

8

16

14

19

11, 12p 12, 14

1, 19

1, 18

3

3, 18

7, 12

3, 33

20

12p, 12q

18

17

4

18

17

6

HCSs

26

36

35

35

33

38

38

3, 21

4

2, 8

7, 22

13

12, 21

4

5

3

3

2

3

23, 24

1

10

12, 18

3, 13

11, 30

11, 26

9, 10

4, 15

3, 15, 27

1, 16

8, 15

15, 28

15, 28

8, 17

6, 16

5

2, 20

3, 19

2, 24

2, 7, 24 4, 5, 12 1, 4

9, 20

9, 27

7, 28

14

17

13

17

16

1, 3, 16 1, 3, 16 1, 2

3, 6, 7

2, 21

11, 30

1, 31

7, 25

7, 25

5, 15

3, 5

3, 11

1, 5

4, 8

3

19

19

13

14

18

18

17

15

20

20

4

20

22, 25

15

14

13, 15

9, 16

8, 11

8, 11

7, 10

4, 8

11, 21

3, 5

4, 26

20

5

7

22

22

10

21

19

2

3

33

35

35

36

35

38

40

43

40

43

HSA = Homo sapiens; DMA = Daubentonia madagascariensis; MIM = Microcebus murinus; LCA = Lemur catta; HGG = Hapalemur griseus griseus; EFM = Eulemur fulvus mayottensis; EFU = Eulemur fulvus; EMA = Eulemur macaco macaco; ALA = Avahi laniger; PVE = Propithecus verreauxi; IIN = Indri indri; OCR = Otolemur crassicaudatus; OGA = Otolemur garnettii; GMO = Galago moholi; NCO = Nycticebus coucang;

Landmark Rearrangements in the Karyotype Evolution of Strepsirrhini, Dermoptera and Scandentia

With the increasing of chromosome painting data in strepsirrhine primates, Dermoptera and Scandentia, it is possible to compare chromosome rearrangements, which occurred in different lineages and to search for common chromosome rearrangements linking different groups. Table 3 lists chromosome segment associations revealed by chromosome painting in several species of Strepsir250

5, 13 18, 23


5

GVA = Galeopterus variegates; TBE = Tupaia belangeri; TMI = Tupaia minor; HCSs = human chromosome segments. Published chromosome painting data for Lemuriformes (Warter et al., 2005; Müller et al., 1997), Lorisiformes (Stanyon et al., 2002, 2006; Nie et al., 2006), Dermoptera (Nie et al., 2008) and Scandentia (Müller et al., 1999; Dumas et al., 2012) were used in this table.

rhinini, Dermoptera and Scandentia with human chromosomes as a reference. In Lemuriformes, many species have been studied by chromosome painting, but only several comparative chromosome maps between human and lemurs have been published. Due to this reason, only species which have chromosome homologies with human from different lemur families are included in table 3. The associations that are specific for one lemur species or associations formed obviously by centromere fusion are not listed in table 3. Based on the distribution of different chromosome arrangements in different species, some Nie

common chromosome rearrangements are found in different lineages of strepsirrhine primates, Dermoptera and Scandentia. Lemuriformes Chromosome painting studies confirmed most previous conclusions of lemur chromosome evolution based on chromosome banding, but modified some chromosome rearrangements on different branches of the phylogenetic tree. [Vezuli et al., 1997; Cardone et al., 2002; Warter et al., 2005; Rumpler et al., 2008]. Three homologous human chromosome segment associations (HSA 2/4, 4/6 and 8/15) were found to be distinctive cytogenetic signatures for all Lemuriformes, except for Daubentoniidae (table 3). HSA 4/19 association was common to species from Lemuridae, Lepilemuridae and Indriidae, but this chromosome rearrangement was not present in species from Daubentoniidae and Cheirogaleidae [Warter et al., 2005] (table 3). HSA 1/7 association was shared by species from Cheirogaleidae, Lepilemuridae and Indriidae, but it was not found in Daubentoniidae and Lemuridae [Warter et al., 2005] (table 3).These 2 associations each seemed to be a specific character linking 3 different lemur families or to be considered as additional characters supporting the lemuriform group, except for Daubentoniidae. However, more chromosome painting data from species with different diploid numbers in Cheirogaleidae and Lemuridae are needed to clarify the situation. HSA 15/19 association was found in all studied species in Lemuridae. This association could be considered as a common character for Lemuridae. Species in Daubentoniidae had some specific associations differing from those of other lemur families, supporting the hypothesis that Daubentoniidae diverged early from other Lemuriformes. Lorisiformes Only 4 of 28 species in Lorisiformes have been analyzed by chromosome painting [Stanyon et al., 2002, 2006; Nie et al., 2006]. Except for 5 common ancient associations, 6 common chromosome associations (i.e. HSA 1/19, 2/12, 6/14, 7/12, 9/15, and 10/19) were found in these African galagos and Asian slow lorises [Nie et al., 2006; Stanyon et al., 2006] (table 3), providing evidence to support that galagos and lorises form a monophyletic group. Whether they represent cytogenetic signatures characterizing this group remain to be validated by more extensive sampling, as only 4 species in Lorisiformes have been analyzed by chromosome painting.


Strepsirrhini Comparison of homology maps among 2 lemurs and 3 species in Lorisiformes defined by HSA painting probes revealed no common derived syntenic associations shared by Lorisiformes and Lemuriformes, but 7 fissions of homologues to HSA 1, 4, 5, 6, 8, 10, and 15 seemed to be shared by these 2 branches [Nie et al., 2006; Stanyon et al., 2006]. These 7 fissions, thus, could represent common cytogenetic signatures for Strepsirrhini. However, Robertsonian fissions are known to be prone to homoplasy and thus, have low phylogenetic weight. Only 2 fissions (homologues to human chromosomes 1 and 15) were proposed to provide significant cytogenetic evidence that unites Lemuriformes and Lorisiformes [Stanyon et al., 2006, 2008]. When chromosome painting data of more lemur species were added to the comparison, some associations, i.e. HSA 1/19, 4/5 and 19/20, seemed to be shared by 1 lemur species and one or more species in Lorisiformes (table 3). HSA 1/19 association, 1 of the 6 synapomorphies in Lorisiformes, shared by Indri indri and 3 species of Lorisiformes. HSA 4/5 association was common to Lemur catta and N. coucang. HSA 19/20 association shared by Daubentonia madagascariensis and G. moholi. In addition, HSA 16/19 association was found in D. madagascariensis and E. macaco macaco [Müller et al. 1997; Warter et al., 2005]. But the reverse painting with E. macaco macaco probes hybridizing on human chromosomes showed HSA 16/19 association in E. macaco macaco was not homologous to that found in many other mammals [Müller et al., 1997]. The fission of the ancestral association HSA 16q/19q should be a common derived trait in primates. The origin of segments in these so-called common associations in lemurs is still unknown. Further reverse painting with lemur probes hybridizing on human chromosomes will be helpful to verify if Lemuriformes and Lorisiformes share derived syntenies. Dermoptera and Scandentia Up until recently, only 2 species in Scandentia (T. belangeri, 2n = 62 and T. minor, 2n = 66) and 1 in Dermoptera (G. variegates, 2n = 56) had been analyzed by chromosome painting [Müller et al., 1999; Nie et al., 2008; Dumas et al., 2012]. Comparisons of the comparative chromosome maps between human and T. belangeri, T. minor and G. variegatus with the maps between human and other species in Euarchonta revealed that the HSA 2/21 association most likely represents a signature rearrangement that unites Scandentia and Dermoptera orders into 1 sister clade [Nie et al., 2008] (table 3). In ScanCytogenet Genome Res 2012;137:246–258

251

Dermoptera Scandentia

+ + + +

+ +

+

+

+

+

+

+ + +

+ + + +

+

+ + + + + + + +

+ +

+ + +

+

+ + + + + + +

+

+

+ + +

7/10

4/20

4/19

4/18

+ + + + + + + +

+

+ + +

? +

4/6

4/5

3/7

2/21

2/12

2/10

2/4

1/19

1/18

1/10

1/7

1/6

1/3

1/5

+ + + + +

6/14

Lorisiformes

+ + + + + + + + + + + + + + +

+

+ + + + + + + + + + + + + +

5/14

Lepilemuridae Lemuridae

+ + + + + +

16/19

+ + + + + + + + + + + + + + +

14/15

DMA (30) MIM (66) INN (40) PVE (48) ALA (70) LEPI (38) EMA (44) LCA (56) EFM (60) GMO (38) OCR (62) NCO (50) GVA(56) TBE (62) TMI (66)

12/22

Strepsirrhini Lemuriformes Daubentoniidae Cheirogaleidae Indriidae

7b/16p

Human chromosomal segment associations

4/8

Species (2n)

3/21

Suborder/ order/family

1/12

Table 3. Syntenic associations in species of Strepsirrhini, Dermoptera and Scandentia that have been published in comparative chromosome maps with the human

+

+ + +

ALA = Avahi laniger; DMA = Daubentonia madagascariensis; EMA = Eulemur macaco macaco; EFM = Eulemur fulvus mayottensis; GMO = Galago moholi; GVA = Galeopterus variegates; IIN = Indri indri; LCA = Lemur catta; LEPI = presumed ancestor of sportive lemur species; MIM = Microcebus murinus; NCO = Nycticebus coucang; OCR = Otolemur crassicaudatus; PVE = Propithecus verreauxi; TBE = Tupaia belangeri; TMI = Tupaia minor. Data used for this table were cited from Müller et al. (1997, 1999; EMA, EFM, TBE), Cardone et al. (2002; LCA), Stanyon et al. (2002, 2006; GMO, OCR, NCO), Warter et al. (2005; DMA, MIM, IIN, PVE, ALA), Nie et al. (2006; NCO), Rumpler et al. (2008; LEPI) and Dumas et al. (2012; TMI).

dentia, the ancient associations HSA 4/8 and 7/16p were confirmed existing in the genome of T. minor, demonstrating that no derived cytogenetic features link Scandentia and Primates [Dumas et al., 2012]. Three associations (HSA 1/18, 10p/7 and 11/20) should be common features for these 2 species in Tupaia [Dumas et al., 2012] (table 3), although an additional chromosome painting needs to be done to confirm if HSA 1/18 association was missed in previous chromosome painting between human and T. belangeri due to technical problems. Interestingly, Picone et al. [2010] proposed that Dermoptera and Lagomorpha formed a monophyletic clade supported by 3 synapomorphies: HSA 4/18, 7/19 and 1/10. However, these are not true derived associations shared by Dermoptera and Lagomorpha (Oryctolagus cuniculus, 2n = 44). The association of HSA 4/18 was not detected in O. cuniculus, instead it was HSA 3/18 [Korstanje et al., 1999]. Further, the reverse painting results indicated that HSA associations 7/19 and 1/10 detected in G. variegatus and O. cuniculus had different origins [Nie et al., 2008]. Therefore, up to now, HSA 2/21 association is the sole likely derived association linking Dermoptera with other Euarchonta, namely Scandentia. 252


The Revised Presumed Ancestral Karyotypes for All Primates and Different Strepsirrhine Primates

With the accumulation of comparative chromosome painting data in Primates and other orders, the number and complement of the presumed ancestral karyotypes for all primates and different strepsirrhine primates were revised. The diploid number of the earlier assumed ancestral karyotype for primates varied from 2n = 48 [Wienberg and Stanyon, 1998; O’Brien and Stanyon, 1999] to 2n = 50 [Müller et al., 1999; Murphy et al., 2001c]. Based on the painting results that HSA 16/19 association was still present in the tree shrew but was broken up in the lemur with formation of 2 independent chromosomes [Müller et al., 1997, 1999], Müller et al.[1999] proposed a 2n = 50 ancestral primates karyotype, including the following homologous human chromosomes: 3/21, 4, 5, 6, 12/22a, 14/15, 7, 1a, 11, 2a, 8, 10, 9, 2b, 13, 17, 1b, 18, 16a, 20, 16b, 19a, 12/22b, 19b and X and Y. A similar 2n = 50 ancestral primates karyotype was also proposed by Murphy et al. [2001c]. The human 7/16 syntenic association was detected in at least 1 species of most orders studied, and it was conNie

19/20

18/22

15/19

15/16

13/16

12/19

12/16

11/20

11/15

10/19

10/16

9/15

8/15

8/11

7/19

7/16

7/15

7/12

+ + + + + + + + + + + +

+

+

+

+

+ + + + + + +

+ + +

+

+

+

+ +

+

+

+ + +

sidered as 1 chromosome of the presumed eutherian ancestral karyotype [Murphy et al., 2001c; Yang et al., 2003]. However, HSA 7/16 association was not detected in any primate species including lemurs before 2005 and was consequently not considered in the putative primate ancestral karyotype. Warter et al. [2005] used chromosome painting with human chromosome-specific probes to reassess the karytotypes of 22 lemur species or subspecies. They found that HSA 7/16 association existed in all studied species from Lemuriformes, including the species in Daubentoniidae, the early diverged branch in Lemuriformes. Frönicke [2005] proposed a 2n = 50 ancestral primates karyotype including HSA 7/16 association based on unpublished data. Later, HSA 7/16 association was also detected in slow lorises and galagos by multidirectional chromosome painting [Nie et al., 2006; Stanyon et al., 2006]. Reverse painting using chromosome-specific probes from slow lorises and galagos confirmed that the HSA 7/16 association found in slow lorises and galagos was the same as that found in other eutherian species. Thus, they proposed that the HSA 7/16 association should be considered as an ancestral synteny for primates and should be included in the presumed ancestral karyotype Chromosome Painting in Strepsirrhini, Dermoptera and Scandentia

for primates. Together with the previous conclusion that the homolog of chromosome 1 was intact in the mammalian and primate ancestor, and with the BAC-FISH results, a 2n = 50 presumed ancestral primate karyotype, including the following homologous human chromosomes: 1, 2p–q, 2q, 3/21, 4, 5, 6, 7b, 7a/16p, 8, 9, 10p, 10q, 11, 12a/22a, 12b/22b, 13, 14/15, 16q, 17, 18, 19p, 19q, 20, X and Y, was proposed recently by Stanyon et al. [2008] and presumed marker order and centromere position in this ancestral primates karyotype were present. For Lemuriformes, most lemurs from all 5 families were analyzed by R-banding technique, and the karyotype homologies were compared to each other and with those of outgroups. These studies allowed the reconstruction of a 2n = 60 ancestral karyotype for Lemuriformes, which was similar to those of genera Lemur and Hapalemur [Rumpler et al., 1989]. On the basis of the available data from the literature and data from their chromosome painting results, Cardone et al. [2002] and Warter et al. [2005] proposed 2 revised ancestral karyotypes for lemurs: 2n = 58 and 2n = 66. Although the diploid number differed from each other, common derived associations: 2/4, 4/6, 8/15 for lemurs were involved in both ancestral karyotypes for Lemuriformes. The human 7/16 association was also considered as an ancestral synteny for lemurs and included in the 2n = 66 ancestral karyotype for Lemuriformes [Warter et al., 2005]. In addition, Rumpler et al. [2008] compared the R-banding patterns and chromosome painting data of 10 sportive lemur species and proposed a 2n = 38 ancestral karyotype for genus Lepilemur. For Lorisiformes, previous comparative karyotype analyses indicated that Lorisiformes probably evolved from a common ancestor with a 2n = 62 chromosome number similar to that of O. crassicaudatus [Rumpler et al., 1983b]. Chromosome painting results indicated that O. crassicaudatus had a highly derived karyotype which was not close to the primate ancestral karyotype [Stanyon et al., 2002]. Combining their chromosome painting results between human and 2 lorisiform primates (G. moholi and N. coucang) and the available chromosome painting results, Nie et al. [2006] proposed an ancestral karyotype with 2n = 60 for Lorisiformes. The human 7/6 association and 6 common associations (HSA 1/19, 2/12, 6/14, 7/12, 9/15 and 10/19) for Lorisiformes were involved in this ancestral karyotype.


253

Fig. 1. Karyotype relationships among primates, Dermoptera and Scandentia. This illustration is based on the generally accepted AEK (2n = 46), reviewed in Ferguson-Smith and Trifonov (2007). FISH data are cited from Müller et al. (1999), Nie et al. (2008) and Dumas et al. (2012). TBE = Tupaia belangeri; TMI = T. minor; GVA = Galeopterus variegates.

Karyotype Relationships of Strepsirrhini, Dermoptera and Scandentia

Based on syntenic associations that are shared by all orders, an ancestral eutherian karyotype (AEK) of 2n = 46 is generally accepted [reviewed in Ferguson-Smith and Trifonov, 2007]. Using the 2n = 46 AEK as the starting point, we can postulate the karyotype relationships among Strepsirrhini, Dermoptera and Scandentia and define the chromosome rearrangements that have occurred in different lineages (figs. 1, 2). The proposed ancestral primates karyotype (APK, 2n = 50) is very similar to AEK. Only 3 fissions and 1 fusion are needed to derive the APK from the AEK. Although none of sytenic associations are shared by species from Primates, Dermoptera 254


and Scandentia, the fission of HSA 10p/12pq/22qt association is common for these 3 orders. HSA 2q/21/3 association is shared by 1 flying lemur and 2 tree shrews, linking Dermoptera and Scandentia in 1 clade. The 2 tree shrews share 12 fissions and 3 fusions (fig. 1). With the change of the presumed ancestral primate karyotype, the proposed ancestral karyotypes for Strepsirrhini and Lorisiformes are revised (fig. 2). Some components of the revised ancestral karyotypes for Strepsirrhini and Lorisiformes are different from that were proposed before [Nie et al., 2006] although they have the same diploid numbers, 2n = 64 for the ancestral karyotype of Strepsirrhini and 2n = 60 for the ancestral karyotype of Lorisiformes. The presumed ancestral karyotype of Lemuriformes is 2n = 66, the same as the one proposed Nie

Fig. 2. Karyotype relationships among strepsirrhine primates. The ancestral primate karyotype (2n = 50) and the ancestral karyotype of all of the Lemuriformes, except for Daubentoniidae, are from Stanyon et al. (2008) and Warter et al. (2005), with some modifications in order to consist with the AEK. Data used in this figure are cited from Stanyon et al. (2002, 2006), Warter et al. (2005) and Nie et al. (2006). OCR = Otolemur crassicaudatus; OGR = Otolemur garnettii; GMO = Galago moholi; NCO = Nycticebus coucang.



255

by Water et al. [2005]. Different chromosome rearrangements in different lineages of strepsirrhine primates are indicated in figure 2. Considering that only few species in Lorisiformes, Dermoptera and Scandentia have so far been investigated by chromosome painting, more FISH data from these groups will be necessary to reconstruct more precisely the ancestral karyotypes for Strepsirrhini, Lorisiformes and Lemuriformes and to clearly depict the karyotype relationships among Primates, Dermoptera and Scandentia.

Conclusions

(1) The chromosome painting results have demonstrated that strepsirrhine primates have highly derived karyotypes. Numerous chromosome rearrangements have been revealed in different lineages of strepsirrhine primates. Several common syntenic associations have been found for given groups. Three human chromosome segment associations (HSA 2/4, 4/6 and 8/15) are common for all Lemuriformes except for Daubentoniidae. The sole species in Daubentoniidae has distinct chromosome rearrangement, indicating that the branch leading to the Daubentoniidae derived earlier than those of other families of Lemuriformes did. Six chromosome associations (HSA 1/19, 2/12, 6/14, 7/12, 9/15, and 10/19) are common cytogenetic signatures linking Asian and Afri-

can Lorisiformes. However, none of derived syntenic associations appear to be shared by Lorisiformes and Lemuriformes, indicating that lemurs are more closely related to each other than to other strepsirrhine primates. (2) The ancient association HSA 7/16, which was found in many mammal orders, but not in higher primates, was only later detected in strepsirrhine primates. This finding, together with other results, leads to a revision in the proposed ancestral primate karyotype. The presumed ancestral primate karyotype now is 2n = 50, including these homologous human chromosomes 1, 2p–q, 2q, 3/21, 4, 5, 6, 7b, 7a/16p, 8, 9, 10p, 10q, 11, 12a/22a, 12b/22b, 13, 14/15, 16q, 17, 18, 19p, 19q, 20, X and Y. (3) Comparative analysis of the chromosome painting data from representative species of each order within the superordinal clade Euarchontoglires, the syntenic association of HSA 2/21 is found to be common for Dermoptera and Scandentia. This provides new molecular cytogenetic evidence that supports a sister-clade relationship between Dermoptera and Scandentia.

Acknowledgements I would very much like to thank F. Yang (Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge) and R. Stanyon (Department of Evolutionary Biology ‘Leo Pardi’, University of Florence, Florence, Italy) for their kind support and helpful comments.

References Adkins RM, Honeycutt RL: Molecular phylogeny of the superorder Archonta. Proc Natl Acad Sci USA 88:10317–10321 (1991). Apiou F, Rumpler Y, Warter S, Vezuli A, Dutrillaux B: Demonstration of homoeologies between human and lemur chromosomes by chromosome painting. Cytogenet Cell Genet 72: 50–52 (1996). Arnason U, Adegoke JA, Bodin K, Born EW, Esa YB, et al: Mammalian mitogenomic relationships and the root of the eutherian tree. Proc Natl Acad Sci USA 99:8151–8156 (2002). Bailey WJ, Slightom JL, Goodman M: Rejection of the ‘flying primate’ hypothesis by phylogenetic evidence from the epsilon-globin gene. Science 256:86–89 (1992). Beard KC: Phylogenetic systematics of the Primatomorpha, with special reference to Dermopter, in Szalay FS, Novacek MJ, Mckenna MC (eds): Mammal Phylogeny: Placentals (Springer, New York 1993).

256

Bininda-Emonds OR, Cardillo M, Jones KE, MacPhee RD, Beck RM, et al: The delayed rise of present-day mammals. Nature 446: 507–512 (2007). Cardone MF, Ventura M, Tempesta S, Rocchi M, Archidiacono N: Analysis of chromosome conservation in Lemur catta studied by chromosome paints and BAC/PAC probes. Chromosoma 111:348–356 (2002). Chowdhary BP, Raudsepp T, Frönicke L, Scherthan H: Emerging patterns of comparative genome organization in some mammalian species as revealed by Zoo-FISH. Genome Res 8:577–589 (1998). Corbert GB, Hill JE: The mammals of the Indomalayan region: a systematic review. (Oxford University Press, Oxford 1992). Dumas F, Houck ML, Bigoni F, Perelman P, Romanenko SA, et al: Chromosome painting of the Pygmy tree shrew shows that no derived cytogenetic traits link Primates and Scandentia. Cytogenet Genome 136: 175–179 (2012).


Dutrillaux B: Chromosomal evolution in primates: tentative phylogeny from Microcebus murinus (Prosimian) to man. Hum Genet 48:251–314 (1979). Dutrillaux B, Couturier J: The ancestral karyotype of platyrrhine monkeys. Cytogenet Cell Genet 30:232–242 (1981). Dutrillaux B, Couturier J, Warter S, Rumpler Y: Chromosomal evolution in ‘lemurs.’ VI. Chromosomal banding studies of Galago senegalensis, Galago alleni, Galago demidovii and Euoticus elegantulus. Folia Primatol (Basel) 37:280–296 (1982). Ferguson-Smith MA, Trifonov V: Mammalian karyotype evolution. Nature Rev Genet 8: 950–962 (2007). Frönicke L: Origins of primate chromosomes – as delineated by Zoo-FISH and alignments of human and mouse draft genome sequences. Cytogenet Genome Res 108: 122–138 (2005).

Nie

Frönicke L, Wienberg J, Stone G, Adams L, Stanyon R: Towards the delineation of the ancestral eutherian genome organization: comparative genome maps of human and the African elephant (Loxodonta africana) generated by chromosome painting. Proc R Soc Lond B Biol Sci 270: 1331–1340 (2003). Groves C: Order primates, in Wilson DE, Reeder DM (eds): Mammal Species of the World, ed 3 (Johns Hopkins University Press, Baltimore 2005). Heaney L, Balete D, Dolar L, Alcala A, Dans A, et al: A synopsis of the mammalian fauna of the Philippine Islands. Fieldiana: Zool New Ser 88:1–61 (1998). Helgen KM: Order Scandentia, in Wilson DE, Reeder DM (eds): Mammal Species of the World: A Taxonomic and Geographic Reference, ed 3 (Smithsonian Institution Press, Washington 2005). Helgen KM: Order Scandentia, in O’Brien SJ, Menninger JC, Nash WG (eds): Atlas of Mammalian Chromosomes, pp 95–97 (John Wiley and Sons Inc, New York 2006). Horvath JE, Willard HF: Primate comparative genomics: lemur biology and evolution. Trends Genet 23:173–182 (2007). Hsu TC, Benirschke K: An Atlas of Mammalian Chromosomes Volume 7, pp 339–340 (Springer Verlag, Berlin 1973). Hudelot C, Gowri-Shankar V, Jow H, Rattray M, Higgs PG: RNA-based phylogenetic methods: application to mammalian RNA sequences. Mol Phylogenet Evol 28: 241–252 (2003). Janecka JE, Miller W, Pringle TH, Wiens F, Zitzmann A, et al: Molecular and genomic data identify the closest living relative of primates. Science 318:792–794 (2007). Kay RF, Thewissen JG, Yoder AD: Cranial anatomy of Ignacius graybullianus and the affinities of the Plesiadapiformes. Am J Phys Anthropol 89: 477–498(1992). Kehrer-Sawatzki H, Cooper DN: Molecular mechanisms of chromosomal rearrangement during primate evolution. Chromosome Res 16:41–56 (2008). Korstanje R, O’Brien PC, Yang F, Rens W, Bosma AA, et al: Complete homology maps of the rabbit (Oryctolagus cuniculus) and human by reciprocal chromosome painting. Cytogenet Cell Genet 86:317–322 (1999). Liu FGR, Miyamoto MM, Freire NP, Ong PQ, Tennant MR, et al: Molecular and morphological supertrees for eutherian (placental) mammals. Science 291:1786–1789 (2001). Martin RD: Primate Origins and Evolution: A Phylogenetic Reconstruction (Chapman Hall/Princeton University Press, London/ Princeton 1990). Müller S, O’Brien PC, Ferguson-Smith MA, Wienberg J: Reciprocal chromosome painting between human and prosimians (Eulemur macaco macaco and E. fulvus mayottensis). Cytogenet Cell Genet 78:260–271 (1997).


Müller S, Stanyon R, O’Brien PC, FergusonSmith MA, Plesker R, Wienberg J: Defining the ancestral karyotype of all primates by multidirectional chromosome painting between tree shrews, lemurs and humans. Chromosoma 108:393–400 (1999). Murphy WJ, Eizirik E, Johnson WE, Zhang YP, Ryder OA, et al: Molecular phylogenetics and the origins of placental mammals. Nature 409:614–618 (2001a). Murphy WJ, Eizirik E, O’Brien SJ, Madsen O, Scally M, et al: Resolution of the early placental mammal radiation using Bayesian phylogenetics. Science 294:2348–2351 (2001b). Murphy WJ, Stanyon R, O’Brien SJ: Evolution of mammalian genome organization inferred from comparative gene mapping. Genome Biol 2:REVIEWS0005 (2001c). Murphy WJ, Pevzner PA, O’Brien SJ: Mammalian phylogenomics comes of age. Trends Genet 20:631–639 (2004). Nie W, Fu B, O’Brien PC, Wang W, Su W, et al: Chromosome painting between human and lorisiform prosimians: evidence for the HSA 7/16 synteny in the primate ancestral karyotype. Am J Phys Anthropol 129: 250–259 (2006). Nie W, Fu B, O’Brien PC, Wang J, Su W, et al: Flying lemurs – the ‘flying tree shrews’? Molecular cytogenetic evidence for a Scandentia-Dermoptera sister clade. BMC Biol 6: 18 (2008). Novacek MJ: Mammalian phylogeny: shaking the tree. Nature 356:121–125 (1992). Nowak RM: Walker’s Mammals of the World, vol 2, ed 6 (The Johns Hopkins University Press, Baltimore 1999). O’Brien SJ, Stanyon R: Phylogenomics. Ancestral primate viewed. Nature 402: 365–366 (1999). O’Brien SJ, Menotti-Raymond M, Murphy WJ, Nash WG, Wienberg J, et al: The promise of comparative genomics in mammals. Science 286:458–462 (1999). Picone B, Masters J, Silvestro D, Sineo L, DelPero M: A phylogenetic analysis of human syntenies revealed by chromosome painting in euarchontoglires orders. J Mammal Evol 18: 131–146 (2011). Richard F, Lombard M, Dutrillaux B: Reconstruction of the ancestral karyotype of eutherian mammals. Chromosome Res 11: 605–618 (2003). Rickart EA: Chromosomes of Philippine mammals (Insectivora, Dermoptera, Primates, Rodentia, Carnivora). Proc Biol Soc Wash 116:699–709 (2003). Rumpler Y, Dutrillaux B: Chromosomal evolution in Malagasy lemurs. I. Chromosome banding studies in the genuses Lemur and Microcebus. Cytogenet and Cell Genet 17: 268–281 (1976). Rumpler Y, Dutrillaux B: Chromosomal evolution in Malagasy lemurs. III. Chromosome banding studies in the genus Hapalemur and the species Lemur catta. Cytogenet and Cell Genet 21:201–211 (1978).

Rumpler Y, Dutrillaux B: Chromosomal evolution in Malagasy lemurs. IV. Chromosome banding studies in the genuses Phaner, Varecia, Lemur, Microcebus, and Cheirogalus. Cytogenet and Cell Genet 24:224–232 (1979). Rumpler Y, Dutrillaux B: Chromosomal evolution and speciation in Primates. University of the Basque Country, vol 23 (Springer Verlag, Berlin 1990). Rumpler Y, Couturier J, Warter S, Dutrillaux B: Chromosomal evolution in Malagasy lemurs. VII. Phylogenic relationships between Propithecus, Avahi (Indriidae), Microcebus (Cheirogaleidae), and Lemur (Lemuridae). Cytogenet Cell Genet 36:542–546 (1983a). Rumpler Y, Couturier J, Warter S, Dutrillaux B: The karyotype of Galago crassicaudatus is ancestral for lorisiforms. Folia Primatol 40: 227–231 (1983b). Rumpler Y, Ishak B, Warter S, Dutrillaux B: Chromosomal evolution in Malagasy lemurs. VIII. Chromosome banding studies of Lepilemur ruficaudatus, L. leucopus, and L. septentrionalis. Cytogenet Cell Genet 39: 194–199 (1985). Rumpler Y, Warter S, Meier B, Preuschoft H, Dutrillaux B: Chromosomal phylogeny of three Lorisidae: Loris tardigradus, Nycticebus coucang, and Perodicticus potto. Folia Primatologica 48:216–220 (1987). Rumpler Y, Water S, Ishak B, Dutrillaux B: Chromosomal evolution in Prosimians. Hum Evol 4:157–170 (1989). Rumpler Y, Warter S, Hauwy M, Fausser JL, Roos C, Zinner D: Comparing chromosomal and mitochondrial phylogenies of sportive lemurs (Genus Lepilemur, Primates). Chromosome Res 16:1143–1158 (2008). Scherthan H, Cremer T, Arnason U, Weier HU, Lima-de-Faria A, Frönicke L: Comparative chromosome painting discloses homologous segments in distantly related mammals. Nat Genet 6:342–347 (1994). Schmitz J, Ohme M, Zischler H: The complete mitochondrial genome of Tupaia belangeri and the phylogenetic affiliation of Scandentia to other eutherian orders. Mol Biol Evol 17:1334–1343 (2000). Silcox MT, Bloch JI, Sargis EJ, Boyer DM: Euarchonta (Dermoptera, Scandentia, Primates), in Rose KD, Archibald JD (eds): The Rise of Placental Mammals (The Johns Hopkins University Press, Baltimore 2005). Springer MS, Murphy WJ, Eirizik E, O’Brien SJ: Placental mammal diversification and the Cretaceous-Tertiary boundary. Proc Natl Acad Sci USA 100:1056–1061 (2003). Springer MS, Stanhope MJ, Madsen O, de Jong WW: Molecules consolidate the placental mammal tree. Trends Ecol Evol 19: 430–438 (2004). Stafford BJ, Szalay FS: Craniodental functional morphology and taxonomy of dermopterans. J Mammal 81:360–385 (2000).


257

Stanyon R, Koehler U, Consigliere S: Chromosome painting reveals that galagos have highly derived karyotypes. Am J Phys Anthropol 117:319–326 (2002). Stanyon R, Dumas F, Stone G, Bigoni F: Multidirectional chromosome painting reveals a remarkable syntenic homology between the greater galagos and the slow loris. Am J Primatol 68:349–359 (2006). Stanyon R, Rocchi M, Capozzi O, Roberto R, Misceo D, et al: Primate chromosome evolution: ancestral karyotypes, marker order and neocentromeres. Chromosome Res 16:17–39 (2008). Toder R, Vonholst D, Schempp W: Comparative cytogenetic studies in tree shrews (Tupaia). Cytogenet Cell Genet 60:55–59 (1992).

258

Vezuli A, Hauwy M, Warter S, Rumpler Y: Chromosome painting: a method for testing chromosomal changes in lemur evolution. Cytogenet Cell Genet 78:147–152 (1997). Waddell PJ, Okada N, Hasegawa M: Towards resolving the interordinal relationships of placental mammals. Syst Biol 48: 1–5 (1999). Waddell PJ, Kishino H, Ota R: A phylogenetic foundation for comparative mammalian genomics. Genome Inform 12: 141–154 (2001). Warter S, Hauwy M, Dutrillaux B, Rumpler Y: Application of molecular cytogenetics for chromosomal evolution of the Lemuriformes (Prosimians). Cytogenet Genome Res 108:197–203 (2005). Wienberg J: Fluorescence in situ hybridization to chromosomes as a tool to understand human and primate genome evolution. Cytogenet Genome Res 108:139–160 (2005).


Wienberg J, Stanyon R: Chromosome painting in mammals as an approach to comparative genomics. Curr Opin Genet Dev 5: 792–797 (1995). Wienberg J, Stanyon R: Comparative painting of mammalian chromosomes. Curr Opin Genet Dev 7:784–791 (1997). Wienberg J, Stanyon R: Comparative chromosome painting of primate genomes. ILAR J 39:77–91(1998). Yang F, Alkalaeva EZ, Perelman PL, Pardini AT, Harrison WR, et al: Reciprocal chromosome painting among human, aardvark, and elephant (superorder Afrotheria) reveals the likely eutherian ancestral karyotype. Proc Natl Acad Sci USA 100:1062–1066 (2003).

Nie



Chromosome Evolution in New World Monkeys (Platyrrhini) E.H.C. de Oliveira a, b M. Neusser c S. Müller c a

Laboratório de Cultura de Tecidos, SAMAM, Instituto Evandro Chagas, Ananindeua, Pará, and b Faculdade de Ciências Naturais (ICEN), Universidade Federal do Pará, Belém, Brazil; c Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University, Munich, Germany

Key Words Chromosome painting ⴢ Chromosome rearrangement ⴢ Karyotype evolution ⴢ New World monkey ⴢ Platyrrhini

Abstract During the last decades, New World monkey (NWM, Platyrrhini, Anthropoideae) comparative cytogenetics has shed light on many fundamental aspects of genome organisation and evolution in this fascinating, but also highly endangered group of neotropical primates. In this review, we first provide an overview about the evolutionary origin of the inferred ancestral NWM karyotype of 2n = 54 chromosomes and about the lineage-specific chromosome rearrangements resulting in the highly divergent karyotypes of extant NWM species, ranging from 2n = 16 in a titi monkey to 2n = 62 in a woolly monkey. Next, we discuss the available data on the chromosome phylogeny of NWM in the context of recent molecular phylogenetic analyses. In the last part, we highlight some recent research on the molecular mechanisms responsible for the large-scale evolutionary genomic changes in platyrrhine monkeys. Copyright © 2012 S. Karger AG, Basel



New World monkeys (NWM) represent a monophyletic group of higher primates (infraorder Platyrrhini). The currently over 120 recognized species are assigned to at least 16 genera. Today these species are commonly classified in 3 families, the Cebidae, Atelidae and Pitheciidae [Opazo et al., 2006; Wildman et al., 2009; Perelman et al., 2011; Matsui and Hasegawa, 2012 for recent review]. Owl monkeys (genus Aotus) are usually included in the family Cebidae, while Groves [2001] classified Aotus as a fourth family. The Cebidae comprise the capuchins, squirrel monkeys, marmosets, and tamarins (and owl monkeys); the Atelidae include spider monkeys, woolly monkeys, howlers, and muriquis; and the Pitheciidae comprise sakis, uakaris and titis. The majority of studies agreed on the classification of 11 genera in 3 monophyletic clades: (1) the large monkeys with prehensile tails from the family Atelidae with genera Alouatta, Ateles, Lagothrix, and Brachyteles; (2) the seed predator monkeys from the family Pitheciidae, with genera Pithecia, Chiropotes and Cacajao), and (3) the small clawed monkeys from the subfamily Callithrichinae (family Cebidae, genera Saguinus, Leontopithecus, CalliStefan Müller Institute of Human Genetics, University Hospital Ludwig-Maximilians-University, Goethestr. 29 DE–80336 Munich (Germany) Tel. +49 895 160 4463, E-Mail Stefan.Mueller02 @ med.uni-muenchen.de

thrix, and Cebuella) [Canavez et al., 1999; Schneider, 2000; Schneider et al., 2001; Singer et al., 2003; Steiper and Ruvolo, 2003; Ray and Batzer, 2005; Osterholz et al., 2009]. However, some disagreement remained with regard to the branching order between the clades and phylogenetic affiliations of the remaining genera Cebus, Callimico, Saimiri, Callicebus, and Aotus. Recent debate has been concerned with the interfamiliar relationship and the internal arrangements within Atelines and Callithrichines, but 2 recent comparative DNA sequence [Wildman et al., 2009; Perelman et al., 2011] and SINE integration [Osterholz et al., 2009] analyses, each including species from 15 or more genera, shed new light on some of these issues. Together with some earlier studies, these 3 phylogenetic reconstructions agreed on the branching sequence of the 3 families Pitheciidae{Atelidae-Cebidae}, on the linkage of Callicebus to the Pitheciidae and of Aotus to the Cebidae. Despite this, the position of Aotus is still not well resolved in these studies. Furthermore, the species affiliation has been continuously revised, for example the species record of Callicebus has been elevated from 13 [Hershkovitz, 1990] to 28 [van Roosmalen et al., 2002].

New World Monkey Comparative Molecular Cytogenetics

Because of the great karyological variability found in Platyrrhini with chromosome numbers ranging from 16 in a titi monkey to 62 in the woolly monkey, most species of this group have been subject of classical chromosome banding analyses since the 1970s. Due to their Mendelian pattern of inheritance, it is possible to use chromosome rearrangements as cladistic markers, to detect synapomorphies and to clarify sister-group relationships among taxa [Dobigny et al., 2004]. These characteristics, added to the fact that they are rare events, render chromosome rearrangements potentially powerful markers in phylogenetic investigations [Rokas and Holland, 2000]. The introduction of cross-species fluorescence in situ hybridization revolutionized the field of comparative cytogenetics and allowed to establish chromosome homology maps between human and other primate species including NWM at a resolution of 3–5 Mb [Müller, 2006; Wienberg and Stanyon, 1998 for review]. Since the mid1990s, when the first NWM species where analysed by cross-species chromosome painting using human probes [Consigliére et al., 1996; Richard et al., 1996; Sherlock et al., 1996], comparative chromosome maps between hu260


mans and almost 40 species of Platyrrhini from all 16 genera have been published (table 1). In addition, chromosome-specific painting probes from several NWM species were established by fluorescence activated chromosome sorting and DOP-PCR amplification, namely from Saguinus oedipus [Müller et al., 2001], Lagothrix lagotricha [Stanyon et al., 2001], Aotus trivirgatus [Stanyon et al., 2004], Callicebus pallescens [Dumas et al., 2005], Callithrix argentata, Cebuella pygmaea, Callimico goeldii, and Saimiri sciureus [Dumas et al., 2007]. These NWM painting probes were used in reciprocal or multidirectional painting experiments and provided important additional information about sub-chromosome homologies between human and NWM as well as between different NWM. Human-NWM chromosome homology maps established by chromosome painting served as a starting point for various downstream analyses, which will be reviewed in detail below: (a) the ancestral NWM karyotype could be reconstructed, (b) derived chromosome characters unique to individual NWM species or, more importantly, to a particular subgroup of NWM species were identified, (c) the succession of chromosome rearrangements including the various phylogenetic lineages was reconstructed, and landmark rearrangements were identified, (d) phylogenetic inferences could be made using chromosome data as cladistic markers, and (e) detailed analyses were carried out aiming to elucidate the molecular mechanisms of large-scale genome organisation and evolution in NWM.

The Ancestral NWM Karyotype

When the cross-species chromosome painting data on the various NWM (table 1) are integrated, the ancestral NWM karyotype can be inferred. It comprises chromosome forms which were observed throughout NWM. According to several studies [Neusser et al., 2001; Stanyon et al., 2001; and for recent reviews Müller, 2006; Stanyon et al., 2008], the ancestral NWM karyotype had 2n = 54 chromosomes (fig. 1). Human chromosome 4, 6, 9, 11, 12, 13, 17, 19, 20, 22, X, and Y homologs are found entirely conserved as separate chromosomes. Chromosome 5, 14, 18, and 21 homologs show conserved synteny; however, they are in syntenic association (5/7, 14/15, 8/18, and 3/21). The remaining human homologs are fragmented: chromosome 1 (3 fragments), chromosome 2 (2 fragments), chromosome 3 (3 fragments), chromosome 7 (2 fragments), chromosome 8 (2 fragments), chromosome 10 (2 fragments), chromosome 15 (2 fragments), and chromosome 16 (2 fragments). de Oliveira /Neusser /Müller

Table 1. Summary of all NWM species analysed by chromosome painting to date

Family

Genus

Species

Reference

Atelidae

Alouatta

A. belzebul A. sara A. seniculus arctoidea A. s. macconnelli A. caraya A. fusca A. guariba guariba A. geoffroyi A. belzebuth hybridus A. b.h. marginatus A. paniscus B. arachnoides L. lagothricha C. moloch C. donacophilus C. lugens C. cupreus C. pallescens C. personatus C. utahicki C. israelita P. irrorata C. calvus rubicundus C. jacchus C. argentata C. pygmaea C. goeldii S. oedipus L. chrysomelas S. sciureus C. capucinus C. apella C. nigrivitatus C. olivaceus C. albifrons A. nancymae A. sp. A. lemurinus griseimembra

Consigliére et al., 1998 Consigliére et al., 1996 Consigliére et al., 1996 de Oliveira et al., 2002 de Oliveira et al., 2002 de Oliveira et al., 2002 Stanyon et al., 2011 Morescalchi et al., 1997 Garcia et al., 2002 de Oliveira et al., 2005 de Oliveira et al., 2005 de Oliveira et al., 2005 de Oliveira et al., 2005 Stanyon et al., 2000 Barros et al., 2003 Stanyon et al., 2003 Dumas et al., 2005 Dumas et al., 2005 Rodrigues et al., 2011 Stanyon et al., 2004 Stanyon et al., 2004 Finotelo et al., 2010 Finotelo et al., 2010 Neusser et al., 2001; Sherlock et al., 1996 Neusser et al., 2001 Neusser et al., 2001 Neusser et al., 2001 Müller et al., 2001 Gerbault-Serreau et al., 2004 Stanyon et al., 2000 Richard et al., 1996 Garcia et al., 2000 Garcia et al., 2002 Amaral et al., 2008 Amaral et al., 2008 Stanyon et al., 2004 Ruiz-Herrera et al., 2005 Stanyon et al., 2011

Ateles

Pitheciinae

Brachyteles Lagothrix Callicebus

Chiropotes

Cebidae

Pithecia Cacajao Callithrix Cebuella Callimico Saguinus Leontopithecus Samiri Cebus

Aotus

N WM species classification follows Perelman et al. [2011].

By comparison of the NWM data set with the chromosome painting results available on other primate and non-primate mammals, the evolutionary origin of all chromosome forms present in the ancestral NWM karyotype can be readily reconstructed [Müller, 2006; Stanyon et al., 2008 for review]. NWM conserved human chromosome 4, 6, 9, 11, 13, 17, 20, X, and Y homologs were already found as separate entities in the primate ancestral karyotype as well as 2 chromosome 2 homologous segments

and the larger of the 2 chromosome 7 segments. In addition, primate ancestral syntenic associations 14/15 and 3/21 were conserved, although the NWM homologs were further rearranged (see below). The human chromosome 10 and 16 homologs were still found conserved as each 2 independent units in the NWM ancestor, but the primate ancestral syntenic association 7/16 was lost, and both chromosome 16 units and the larger chromosome 10 unit were involved in further rearrangements (see below).

New World Monkey Chromosome Evolution


261

Saimiri 2/15 synapomorphic chromosomal rearrangement in agreement with molecular phylogeny

Cebus

no informative chromosomal character available

Aotus 10/11 Saguinus

homoplasy of chromosomal data

Leontopithecus

Cebidae

chromosomal data not in agreement with molecular phylogeny

Callimico

13/9/22 2/15

Callithrix 13/17 Cebuella

1/10

Lagothrix 1

1

1

2 2/16 3

3 3/21 4

5/7 6

7

8 8/18 9

10 10/16 11

12 13 14/15 15 17 19 20 22

X Y

Ateles

4

Atelidae

Brachyteles

Alouatta

4 4/15 16/10/16/10

Inferred ancestral NWM karyotype (2N=54) 14/15 1

1

Pithecia

5 7/5/7 2/10

Cacajao

Pithecidae

Chiropotes

5 5/7 14/15/20 Callicebus 16/10/16/10

10/11

Fig. 1. The NWM ancestral karyotype and evolutionary landmark rearrangements superimposed on the molecular phylogenetic tree of Platyrrhini presented by Perelman et al. [2011] and Wildman et al. [2009]. Chromosomes are colour coded and numbered according to their homology with human chromosomes.

NWM conserved chromosome 12, 19 and 22 homologs are of more recent evolutionary origin and can be assigned to the simian ancestral karyotype. The remaining NWM ancestral chromosome forms represent exclusive NWM synapomorphies: fragmentation of the primate ancestral human chromosome 1 homolog by 2 fissions and of the 3/21 homolog by 2 fissions with breakpoints in the human chromosome 3 homolog, fission of the chromosome 8 homolog and subsequent fusion of the 8p segment with chromosome 18, fusion of the 2 chromosome 262


16 segments to form 2/16 and 10/16 homologs, respectively, fusion of the smaller chromosome 7 segment resulting in the 5/7 homolog, and fission of a small chromosome 15 homologous segment from the 14/15 homolog. It is of note that the syntenic associations 5/7, 2/16, 10/16, and 8/18 were observed in most, but not in all NWM species. As far as the chromosome 5/7 is concerned, it is likely that for technical reasons the small chromosome 7 segment was missed in some evaluations in species from different families. In contrast, technical de Oliveira /Neusser /Müller

reasons are unlikely the cause for the absence of the association 2/16 in all howler and owl monkeys and associations 10/16 and 8/18 in owl monkeys. It could be speculated that these characters mark a polyphyly of NWM; however, the monophyly of NWM was never questioned in previous studies using molecular, morphological or ecological data sets [Groves, 2001; Matsui and Hasegawa, 2012 for review]. Association 2/16 and 8/18 may rather have been lost secondarily, and the association 10/16 was probably obscured by a derived rearrangement resulting in 10/22/16 (see below for details).

could identify the fission of 2/16, association 3/15 and possibly a Y-autosomal translocation as chromosome signatures, confirming the monophyly of this clade [de Oliveira et al., 2002] (table 2). Associations 2/20, 5/7/5/7 and 4/16 define A. belzebul and A. caraya as sister clades, while A. seniculus, A. sara, A. macconelli, and A. guariba share chromosome form 2/4 [Consigliére et al., 1996, 1998; Stanyon et al., 2011]. Further, only A. seniculus, A. macconelli and A. sara showed synapomorphic chromosome forms 1/20 and 8/7/5/7, and another 6 derived chromosome rearrangements occurred in the last common ancestor of A. seniculus and A. sara.

Atelidae Pitheciidae

The inclusion of Alouatta, Ateles, Brachyteles, and Lagothrix in a monophyletic clade is widely accepted in most phylogenetic proposals, and the chromosome data unequivocally support this classification. Atelidae exclusively share common derived fission of a chromosome 1 homologue, fission of the 5/7 homolog with a break in the chromosome 5 segment and followed by an inversion 7/5/7, an additional fission of the NWM ancestral 14/15 homolog with break in the 15 homologous segment and subsequent fusion 4/15, and fission of the chromosome 4 homolog. Further, a derived inversion of the chromosome form 10/16, resulting in 16/10/16/10 was observed in Alouatta, Brachyteles and Lagothrix, while in Ateles the presumably related chromosome form 16/10/16 was present. The ancestral Atelidae karyotype would therefore be comprised of 2n = 62 chromosomes, similar to the chromosome complement found conserved in extant Lagothrix lagotricha and Brachyteles arachnoides [Stanyon et al., 2001; de Oliveira et al., 2005] (table 2). In Ateles, 17 tandem or Robertsonian type fusions and 3 fissions resulted in a dramatic reduction of the diploid chromosome number to 34 as found conserved in Ateles b. marginatus [Morescalchi et al., 1997; Garcia et al., 2002; de Oliveira et al., 2005]. A shared derived inversion 1/6/1 phylogenetically linked A. p. paniscus, A. geoffroyi and A. b. hybridus, while another synapomorphic inversion 16/2/16/2/1 defined A. geoffroyi and A. b. hybridus as sister taxa (table 2). This chromosome phylogeny would, however, require the reclassification of A. b. marginatus and is in disagreement with a previous molecular phylogeny based on mitochondrial sequence comparison by Collins and Dubach [2001]. In Alouatta, in addition to many autapomorphies found in different species because of extensive chromosome reshuffling, the molecular cytogenetic analyses New World Monkey Chromosome Evolution

From the clade of the seed predator monkeys Pithecia, Chiropotes and Cacajao, members from all 3 genera share a derived fusion resulting in chromosome form 2/10 [Stanyon et al., 2004; Finotelo et al., 2010]. In addition, FISH analyses favour the proximity of Cacajao and Chiropotes by fission of the chromosome 5 homologous segment of the NWM ancestral 5/7 homolog with a breakpoint distinctly different from that observed in Atelidae and by a fusion 20/15/14 (table 3). In Callicebus, molecular cytogenetic analyses [Stanyon et al., 2000, 2003; Barros et al., 2003; Dumas et al., 2005; Rodrigues et al., 2011] confirmed the extensive intrageneric karyotype variability observed in earlier chromosome banding studies. Moreover, despite being the species with the lowest chromosome number among Platyrhhini, Callicebus lugens (2n = 16) showed the evolutionary conservation of 11 human chromosomes [Stanyon et al., 2003]. Syntenic associations 7/15, 16/2/16/2, 10/11, and 22/2/22 were found in all 6 species analysed so far and thus, represent ancestral Callicebus chromosome forms (table 3). Interestingly, syntenic associations 13/17 and 17/20 were present in 4 species of Callicebus as well as in Callithrichinae (see below). However, a more detailed analysis of the translocated segments in Callicebus donacophilus, using S. oedipus paints as subregional probes, indicates the involvement of different chromosome 13 homologous segments in the rearrangement in the 2 NWM species groups and hence, an independent evolutionary origin of the syntenic associations 13/9 and 13/17/20 in Callithrichinae compared to chromosome forms 13/17 and 17/20 in Callicebus [M. Neusser, unpublished results].


263

Table 2. Atelidae-derived chromosome forms observed in at least 2 species, excluding chromosome forms

present in the ancestral NWM karyotype Chromosome trait

Ateles

Lag.

Bra.

Alouatta

AHB ABM APP AGE

LLA

BAR

ABE ACA AGG ASA ASE AMA

1 fission 1/2/16 1/2/16/2/16 1/6/1 1/6/1/6/1 1/7/3 1/20 2/3/15/22 2/4 2/10 2/16 fission 2/20 3/15 6/3/21 4 fission 4/15 4/16 5 fission* 4/7/5/7 5/7/5/7 5/8 5/11 7/5/7 8/7/5/7 9/18/8 10/19 12/15/14/1/4/15 15/16 16/10/16 16/10/16/10 19/20 20/22

+ + + + + +

+ +

+ +

+

+

+

+ +

+ + +

+ + + + + +

+

+

+

+

+

+

+

+ + +

+ + +

+ + +

+ + +

+ + +

+ +

+ +

+ +

+ +

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+ +

+ +

+

+

+

+

+

+

+ + + + + + + +

+

+

+

+

+

+

+

+

+

+

+

+

+

+ + +

+

+

+

+

+

+

+

+

+ + + + + +

+ +

+ +

+ +

+ +

+

+

+

+

+ + + +

+ + + +

+ +

+

+

+

+

+

+

+

+

+

+

+

+

+

* Fission in 5 with a breakpoint distinctly different compared to Pitheciidae, as revealed by L. lagotricha painting probes. ABH = Ateles belzebuth hybridus; ABM = A. b. marginatus; APP = A. paniscus paniscus; AGE = A. geoffroyi; LLA = Lagothrix lagotricha; BAR = Brachyteles arachnoids; ABE = Alouatta belzebul; ACA = A. caraya; AGG = A. guariba guariba (formerly also classified as A. fusca); ASA = A. sara; ASE = A. seniculus arctoidea; AMA = A. macconelli. For references see table 1.

Cebidae

The putative ancestral NWM karyotype of 2n = 54 was found conserved in Cebus capucinus [Richard et al., 1996], from which the Cebus apella karyotype can be derived by an inversion 14/15/14 [García et al., 2000] (table 4). Each one species-specific fusion explains the reduction of diploid number to 52 in C. albifrons (12/15) 264


and C. olivaceus (8/15/8), respectively [Amaral et al., 2008]. The chromosome phylogeny of the smallest NWM, the callithrichines, is resolved reasonably well at the genus level. All Callithrichinae studied so far share synapomorphic associations 2/15, 13/9, 13/22, and 13/17 [Sherlock et al., 1996; Stanyon et al., 2000; Müller et al., 2001; Neusser et al., 2001; Gerbault-Serreau et al., 2004] (table 4). Fusion de Oliveira /Neusser /Müller

Table 3. Pitheciidae-derived chromosome forms observed in at least 2 species, excluding chromosome forms

present in the ancestral NWM karyotype Chromosome trait 2/10 4 fission 5 fission* 7/5/7/5 7/15 9/7/5/7/5 10/11 12/19 13/17 16/2/16/2 16/10/16/10 17/20 20/15/14 22/2/22

Chir.

Cacajao

Pith.

Callicebus

CUT

CIS

CCA

PIR

CDO

+

+

+

+

+

+

+

CLU

+

+

+

+

+

+

+

+

+ + + + + +** + + +** +

CCU

CPA

CMO

CPE

+ +

+

+

+ +

+ +

+

+ +

+

+

+

+ + +

+ + + + + + + + +

+ + + + + +

+

+

+

+ +

* Fission in 5 with a breakpoint distinctly different compared to Atelidae, as revealed by L. lagotricha painting probes. ** Association not observed by Barros et al. [2003], but clearly present in C. donacophilus [M. Neusser, unpublished results]. CUT = Chiropotes utahicki; CIS = Cacajao israelita; CCA = C. calvus rubicundus; PIR = Pithecia irrorata; CDO = Callicebus donacophilus; CLU = C. lugens; CCU = C. cupreus; CPA = C. pallescens; CMO = C. moloch; CPE = C. personatus. For references see table 1.

1/10 is further shared by Cebuella pygmaea, Callithrix argentata, C. jacchus, and Callimico goeldii to the exclusion of Leontopithecus chrysomelas and Saguinus oedipus, placing C. goeldii next to Callithrix and Cebuella. This observation provides further evidence for the taxonomic and phylogenetic integration of Callimico within Callithrichinae [Neusser et al., 2001]. Importantly, the syntenic association 2/15 present in Saimiri sciureus demonstrated that Saimiri shares a synapomorphy otherwise found only in Callithrichinae, thus arguing for a closer relationship of Saimiri to this clade than to any other group of Platyrrhini [Stanyon et al. 2000; Neusser et al., 2001] (table 4). Aotus, which shows a high karyological diversity at the species level, was found not to have retained the 2 NWM ancestral chromosomal associations 2/16 and 10/16. Both associations were absent in A. lemurinus griseimembra, A. nancymae and Aotus sp. [Stanyon et al., 2004, 2011; RuizHerrera et al., 2005]. A closer look at the syntenic group 10/22/16 in A. nancymae observed by multidirectional chromosome painting, using human and Lagothrix lagotricha probes, indicates that the chromosome association may be the result of an insertion or of a fusion of the NWM

ancestral chromosome 10/16 with 22 followed by an inversion. Concerning association 2/16, a fission followed by a fusion with a chromosome 1 homologous segment would explain the absence of this ancestral association in Aotus. When considering chromosome data to try to clarify the position of Aotus with respect to other Platyrrhini, all but one synapomorphy for Aotus found so far, namely the mentioned disruptions of 2/16 and 16/10, but also association 1/3, 1/16, 2/20, 4/15, 7/11, 16/22 and inversion 15/14/15/14 are autapomorphic for this genus (table 4). Importantly, as an exception to this rule, all Aotus and Callicebus species analysed until now exclusively share the derived association 10/11, which would also indicate a phylogenetic link between these 2 clades, but is in contrast to the recent trend to classify Aotus with the Cebidae (fig. 2).



NWM Phylogenetic Reconstructions and Chromosome Evolution

During the last decades, numerous studies have attempted phylogenetic reconstructions in NWM using a broad spectrum of different approaches, including mor265

Table 4. Cebidae-derived chromosome forms observed in at least 2 species, excluding chromosome forms pres-

ent in the ancestral NWM karyotype Chromosome trait 1/3 1/16 1/10 2/12 2/15 2/16 fission 2/20 3/14 4/15 5/15 7/11 9/15 9/22 10/11 10/16 fission 10/22 13/9 13/9/22 13/17 13/17/20 14/15/14 16/22

Cebus CAP

CCA CNI

Callithrix

Callim.

Ceb.

Leon.

Sag.

Saim.

Aotus

CJA

CGO

CPY

LCH

SOE

SSC

ALG ANA Asp.

CAR

+ + +

+

+

+

+

+

+

+

+

+

+ +

+ +

+

+

+ + + + + + +

+ +

+ +

+ + +

+ + +

+

+

+

+ + + + +

+

+ + + + +

+

+

+ + + +

+

+ +

+

+ + + +

+

+ + + +

+ + + +

+

+ + + +

+

CAP = Cebus apella; CCA = C. capucinus; CNI = C. nigrivitatus; CJA = Callithrix jacchus; CAR = C. argentata; CGO = Callimico goeldii; CPY = Cebuella pygmaea; LCH = Leontopithecus chrysomelas; SOE = Saguinus oedipus; SSC = Saimiri sciureus; ALG = Aotus lemurinus griseimembra; ANA = A. nancymae; Asp = Aotus sp. For references see table 1.

phology, biogeography, behaviour, molecular genetics, and cytogenetics. It is beyond the scope of this review to discuss the strengths or limitations of approaches other than comparative cytogenetics. Instead, we will first attempt to integrate the molecular cytogenetic data summarized above in the context of recent trends towards a ‘consolidated branching order’ of NWM [Osterholz et al., 2009] based on molecular genetic data sets. Next, we will propose an alternative NWM phylogenetic tree using chromosome data alone, and finally, we make an attempt to reconcile both approaches. In doing so, we will try to weigh the value of the NWM chromosome landmark rearrangements described above as markers for phylogenetic reconstructions. Most recent molecular phylogenetic reconstructions agree on the classification of NWM in 3 families and also show a clear tendency towards a consensus branching sequence at the level of individual genera [Matsui and Hasegawa, 2012 for recent review]. We will first superimpose the chromosome data onto the tree proposed by Perelman et al. [2011] and Wildman et al. [2009] based 266


on the comparative analysis of nuclear sequence data (fig. 1). Both the molecular and the cross-species chromosome painting data with 7 derived chromosome forms unequivocally agree on the monophyly of Atelidae. At present, no molecular cytogenetic evidence is available to support the internal Atelidae branching sequence (fig. 2) because Brachyteles and Lagothrix have conserved the ancestral Atelidae karyotype. This polytomy could be resolved when also considering comparative G-banding data, which would suggest a common derived inversion of the human chromosome 8 homolog in all Atelidae, except for Alouatta, and inversion of the chromosome 13 homolog in support of a Lagothrix/Ateles clade [de Oliveira et al., 2005]. Here, high-resolution analysis of the marker order on Atelidae chromosome 8 and 13 homologs by FISH, using locus-specific probes (e.g. BAC probes) would be required to verify the G-banding data. Even then the resulting branching sequence {Brachyteles{Lagothrix-Ateles} would not be in agreement with the molecular phylogeny presented in figure 1. In these molecular phylogenies, de Oliveira /Neusser /Müller

Cebus

synapomorphic chromosomal rearrangement in agreement with molecular phylogeny

Saimiri

no informative chromosomal character available

Saguinus chromosomal data not in agreement with molecular phylogeny

Callimico 13/9/22 13/17

Cebidae

Leontopithecus 2/15

homoplasy of chromosomal data

Callithrix

Cebuella 1/10 1

1

1

2 2/16 3

3

3/21 4

Aotus 10/11 Callicebus

5/7

6

7

8 8/18 9

10 10/16 11

10/11

12 1314/15 15 17 19 20 22

X Y Chiropotes

Inferred ancestral NWM karyotype (2N=54)

Pithecidae

Pithecia

Cacajao

2/10 5 5/7 14/15/20

Lagothrix 16/10/16/10

16/22/10 Ateles

Callicebus 4

Atelidae

Brachyteles

Aotus

4 4/15

10/11

Alouatta 14/15 1

1

5 7/5/7

Fig. 2. Chromosomal phylogeny of NWM and evolutionary landmark rearrangements (inset: alternative phylogeny assuming that syntenic association 10/11 represents a phylogenetic link between Aotus and Callicebus, see text for details). Chromosomes are colour coded and numbered according to their homology with human chromosomes.

however, low bootstrap values also provided no unequivocal support for a Lagothrix-Ateles clade [Wildman et al., 2009; Perelman et al., 2011]. In Pithecidae, the DNA sequence and the molecular cytogenetic trees are in agreement when only considering

the genera Pithecia, Chiropotes and Cacajao. Both trees agree on their monophyly, and also each one chromosome landmark rearrangement supports the branching sequence {Pithecia{Chiropotes/Cacajao} [Finotelo et al., 2010]. In contrast, no molecular cytogenetic evidence is



267

presently available in support of Callicebus as the basal Pitheciidae clade (fig. 1). None of the 4 shared derived chromosome forms allocated to the inferred ancestral Callicebus karyotype, nor any other derived chromosome rearrangement observed in one of the 6 Callicebus species so far was found to be shared with other Pitheciidae [Dumas et al., 2005; Finotelo et al., 2010]. Furthermore, the presence of a unique shared derived chromosome association 10/11 found in all Callicebus and Aotus species so far would argue for a different tree topology with Callicebus and Aotus as sister clades without any chromosome affinity to other NWM species (fig. 2). Assuming that the DNA sequence tree reflects the correct phylogeny – which may well be the case since the placement of Callicebus as a basal Pitheciidae is well supported in most molecular analyses [Matsui and Hasegawa, 2012], and only few studies using morphological characters favoured a Callicebus/Aotus clade [Rosenberger, 1981; Ford, 1986] – chromosome form 10/11 would be the product of a convergent fusion (fig. 1). In any case, a detailed FISH analysis of the associated segments 10/11 using locus-specific probes would be required to determine whether the associated syntenic segments in Callicebus and Aotus are truly homologous or not (preliminary, unpublished results indicate they are indeed homologous, M. Neusser). The phylogenetic relationships among the 3 Cebidae subgroups Aotinae, Cebinae and Callithrichinae are much less well understood compared to Pithecidae. Although the placement of Aotus with Cebidae has been confirmed in most recent molecular studies, its position with respect to the other 2 subfamilies received no or only particularly low support in recent SINE insertion and nuclear sequence analyses, respectively [Osterholz et al., 2009; Wildman et al., 2009; Perelman et al., 2011]. Molecular cytogenetic data offer no deeper insight; since so far, Aotus was not found to share any synapomorphic traits with other Cebidae, and the paucity of evolutionary chromosome changes in genus Cebus hampered its phylogenetic classification with respect to other Cebidae. For the same reason, the classification of Cebus and Saimiri as sister clades could not be confirmed (fig. 1). Instead, the shared derived association 2/15 placed Saimiri in a basal position of a clade together with marmosets and tamarins [Stanyon et al., 2000; Neusser et al., 2001]. Hence, this chromosome trait is representing another discrepancy with generally accepted molecular phylogenies where Cebus and Saimiri are sharing a last common ancestor (fig. 1). Again, under the assumption that the present NWM molecular phylogenies are correct, 2 alternative scenarios may explain the cytogenetic findings: as268


sociation 2/15 is an ancestral Cebidae chromosome form which was lost again in Cebus (and possibly also in Aotus when accepting the phylogeny presented in fig. 1) or is a convergent fusion which occurred independently in Saimiri and in the Callithrichinae ancestor. Since this association is a potentially important cladistic marker, highresolution FISH analysis with fusion point flanking BAC probes in Cebus and Aotus may be indicated to clarify if a cryptic association 2/15 below the resolution of chromosome painting was retained in these 2 species. At least 3 derived chromosome rearrangements highlight the monophyly of Callithrichinae. One additional derived chromosomal fusion places Callimico next to Callithrix and Cebuella [Neusser et al., 2001], in agreement with recent molecular phylogenies (fig. 1, 2). Finally, when considering the relationship between the 3 NWM families, molecular approaches favour the branching sequence Pitheciidae{Atelidae-Cebidae} with low– moderate support by sequence analysis [Wildman et al., 2009; Perelman et al., 2011] and by a substantially high number of SINE integrations [Ray and Batzer, 2005; Osterholz et al., 2009], whereas solid molecular cytogenetic data are not available to date. All but one shared derived chromosome rearrangements detected in NWM so far can be either assigned to the NWM common ancestor or are at least confined to the ancestor of 1 of the 3 families. The sole exception is a derived inversion of the chromosome form 10/16, resulting in 16/10/16/10, which was found in all Pitheciidae including 5/6 species of Callicebus and in all Atelidae, except for genus Ateles. It can be speculated that the chromosome form 16/10/16 present in all Ateles species analysed until now is in fact a derivative of the ancestral segment association 16/10/16/10 by translocation of the terminal chromosome 10 homologous segment. Under these presumptions, chromosome form 16/10/16/10 would represent a phylogenetic link between Atelidae and Pitheciidae, leading to a NWM branching sequence Cebidae{Atelidae-Pitheciidae}, as proposed in the molecular phylogeny by Schneider [2000] and Schneider et al. [2001]. From the present cross-species chromosome painting and from G-banding data it is, however, difficult to determine if the inversion breakpoints in all clades are identical and, by consequence, whether chromosome form 16/10/16/10 is truly homologous. In order to substantiate this finding, detailed comparative chromosome maps established by BAC-FISH would be required. If confirmed, this landmark rearrangement would contradict the presumed synapomorphic association 10/11 shared by Aotus and Callicebus and consequently the phylogenetic link between the 2 species [Dumas et al., 2005] (fig. 2). de Oliveira /Neusser /Müller

The Subregional Organisation of NWM Chromosomes

Although cross-species chromosome painting using human probes to delineate interspecies chromosomal homologies provides a comprehensive overview of syntenic segments, the subregional organisation of these segments and also the precise localisation of breakpoints cannot be resolved, and most intrachromosomal rearrangements escape detection. Reverse painting of NWM chromosome-specific probes to human chromosomes was used to map the breakpoints involved in NWM specific chromosome rearrangements with reference to human chromosomes. According to these studies, the ancestral NWM karyotype of 2n = 54 chromosomes would comprise of the 28 syntenic units 1p, 1q32–qter, 1q21–q31, 2q13–qter, 2pter– q13/16q, 3p12–q12/21, 3p24–pter/3p12–p14/3q12–q21/ 3q27–qter, 3p21–p24/3q21–q26, 4, 5/7p22/7q11.2/7q22, 6, 7p11–21/7q11.2–q21/7q22–qter, 8p/18, 8q, 9, 10q/16p, 10p, 11, 12, 13, 14/15q14–24, 15q11–q13/15q25–qter, 17, 19, 20, 22, X and Y [Neusser et al., 2001; Stanyon et al., 2001; Dumas et al., 2007]. Of note, individual NWM ancestral chromosomes showing multiple syntenic segments for chromosomes 3, 7 or 15 homologs in (e.g. 3p21–p24/3q21– q26) resulted at least in part from derived intrachromosomal rearrangements which occurred in the higher Old World primate/human lineage [Müller et al., 2004; Ventura et al., 2004; Stanyon et al., 2008, see below for details]. The ancestral Atelidae karyotype of 2n = 62 chromosomes resulted by additional synteny breaks in human homologous regions 1p36, 4q24, 4q31, 5q31, and 15q21 [Stanyon et al., 2001]. High-resolution comparative maps between human and NWM chromosomes could be established by crossspecies FISH using BAC probes which were sequenced during the course of the human genome project and are anchored in the human reference sequence [Stanyon et al., 2008 for review]. These studies delineated the marker order of human homologs in index species from all major primate clades, including at least 1 species from NWM, in some cases even species from each of the tree NWM families (see table 1 in Stanyon et al. [2008] for a comprehensive reference list). For example, the order of 23 BACFISH markers along chromosome 1 homologs was found conserved between human and the inferred ancestral primate, from which the 4 Lagothrix lagotricha homologs originated by 3 fissions with breakpoints at approximately 84–97 Mb, 1 centromere and 186–195 Mb of the human reference sequence (UCSC build May 2004), respectively. New World Monkey Chromosome Evolution

Strikingly, when comparing the marker order present on chromosomes in the inferred ancestral primate and in the ancestral NWM karyotype, almost all human homologs show completely conserved subregional organisation at a resolution defined by the genomic distance each BAC pair used in the respective studies [Stanyon et al., 2008 and references therein]. Only the human chromosome 3 and 9 homologs showed a large NWM-specific inversion and 2 nested inversions, respectively [Montefalcone et al., 1999; Ventura et al., 2004]. From these studies, it can be concluded that the vast majority of NWM shared derived chromosome forms are the product of the above-mentioned interchromosomal rearrangements and that inversions were rather rare.

Patterns of Large-Scale Genome Rearrangement in NWM

In the 38 NWM species analysed by cross-species chromosome painting to date (table 1), a total of 182 chromosome rearrangements were recorded after correction for changes that occurred in the inferred ancestral NWM or in the inferred ancestor of the various NWM phylogenetic lineages. Of these, 129 changes account for fusions (71%), 39 for fissions (21%) and 13 for inversions (7%). Hence, Robertsonian type fusions, centromere-telomere and tandem fusions are the predominant mechanism of evolutionary change in NWM. Only 2 reciprocal translocations were observed (1%), indicating that reciprocal exchanges occur at exceptionally low rates in Platyrrhini or, more likely, that reciprocal translocations have a very low chance to be fixed in any population of NWM. The number of rearrangements between the NWM ancestor and extant species, however, varies greatly between zero in Cebus capucinus [Richard et al., 1996] and over 20 in Callicebus lugens [Stanyon et al., 2003]. Equally, the direction of changes does not appear to follow a continuous trend in any of the NWM clades. For example, in the ancestor of Atelidae, a series of fissions occurred, increasing the chromosome number from 54 to 62. Since then, the karyotypes in Brachyteles and in Lagothrix remained essentially conserved, whereas in Ateles a dramatic reduction to 2n = 32 chromosomes took place. From the genomics perspective, but also to determine the robustness of chromosome rearrangements as phylogenomic markers, it is of interest to estimate the probability that identical chromosome rearrangements are recurrent. Taking into account the evolutionary changes resulting in the 13 derived chromosome forms present in Cytogenet Genome Res 2012;137:259–272

269

the NWM ancestor and in the 57 different chromosome forms summarized in tables 2, 3 and 4, and assuming further that the molecular phylogeny illustrated in figure 1 is correct, over 90% of these chromosome changes were unique evolutionary events. Only a maximum of 6/57 rearrangements (9%) may represent recurrent and therefore convergent gains: associations 5/8 in Alouatta/Ateles, 10/11 in Aotus/Callicebus, 10/16/10/16 in Atelidae/Pitheciidae, 2/20 in Aotus/Alouatta, 2/15 in Saimiri/Callithrix, and 2/10 in Ateles/Chiropotes. Not surprisingly, the break or fusion points in 5 of these rearrangements are located in centromeric regions. Strikingly, besides the conventional structural changes mentioned above, the detailed FISH analysis of the marker order in NWM revealed the emergence of evolutionary neocentromeres (ENC) in 20 NWM cases so far [Rocchi et al., 2012 for recent review]. The majority of ENC appeared during the process of non-centromeric fission, while some represent ‘classical’ ENC, including the X chromosome centromere of squirrel monkeys (Saimiri sciureus) [Rocchi et al., 2012]. The latter ENC emerged by a yet poorly understood process of inactivation and degeneration of the ancestral centromere and simultaneous epigenetic seeding of the new centromere in a different location of the same chromosome. In addition, several instances of telomere conversion into centromeres were recorded, for example, on a chromosome 3 homolog in Callicebus moloch [Ventura et al., 2004] and on a chromosome 1 homolog in Callithrix jacchus [Neusser et al., 2001]. Further, amplification and rapid sequence divergence of pericentromeric, interstitial and subtelomeric heterochromatin appears to be commonplace also in New World primates. As determined by interspecies comparative genomic hybridization and FISH using a microdissected DNA probe, even in the close related marmosets Callithrix argentata and Cebuella pygmaea, rapid and species-specific amplification of repetitive sequences could be observed when compared to the Calli-

References

270

thrix jacchus genome [Neusser et al., 2005]. The same observation was made when comparing the genomes of Cebus libidinosus with other species of NWM [Fantini et al., 2011; Nieves et al., 2011], where the amplification of Cebus-specific repeat sequences in C. libidinosus accounted for an increase in genome size of around 10%.

Conclusions and Perspectives

During the last decades, NWM comparative classical and, in particular, molecular cytogenetics have highlighted many fundamental aspects of genome organisation and evolution in this fascinating, but also highly endangered group of primates. In summary, NWM; most probably represent the mammalian clade with the most comprehensive molecular cytogenetic data set available at present. These studies have impact in various fields including comparative genomics, taxonomy and phylogeography but also in species conservation and management. This work aspired to gain fundamental insight into the evolutionary principles, which shaped the genomes of extant primates and to solve the jigsaw puzzle of Platyrrhini chromosome reshuffling, but also by the expectation that sequencing the entire genomes of all 120 and more NWM species would not be feasible in the foreseeable future. However, in recent years, with novel timeand cost-effective parallel sequencing techniques emerging, the situation has changed dramatically. It can be envisioned that at least low-coverage whole genome sequence of 1 species per NWM genus will become publicly available within the next few years. This data set will help to resolve the remaining open questions concerning NWM phylogeny and will also complement or even supersede the cytogenetic data available today because it will resolve syntenic segments and structural rearrangement breakpoints at the ultimate level of resolution.

Amaral PJ, Finotelo LF, de Oliveira EH, Pissinatti A, Nagamachi CY, Pieczarka JC: Phylogenetic studies of the genus Cebus (Cebidaeprimates) using chromosome painting and g-banding. BMC Evol Biol 8: 169 (2008). Barros RM, Nagamachi CY, Pieczarka JC, Rodrigues LR, Neusser M, et al: Chromosomal studies in Callicebus donacophilus pallescens, with classic and molecular cytogenetic approaches: multicolour FISH using human and Saguinus oedipus painting probes. Chromosome Res 11:327–334 (2003).


Canavez FC, Moreira MA, Simon F, Parham P, Seuánez HN: Phylogenetic relationships of the Callitrichinae (Platyrrhini, primates) based on beta2-microglobulin DNA sequences. Am J Primatol 48:225–236 (1999). Collins AC, Dubach JM: Nuclear DNA variation in spider monkeys (Ateles). Mol Phylogenet Evol 19:67–75 (2001).

de Oliveira /Neusser /Müller

Consigliére S, Stanyon R, Koehler U, Agoramoorthy G, Wienberg J: Chromosome painting defines genomic rearrangements between red howler monkey subspecies. Chromosome Res 4:264–270 (1996). Consigliére S, Stanyon R, Koehler U, Arnold N, Wienberg J: In situ hybridization (FISH) maps chromosomal homologies between Alouatta belzebul (Platyrrhini, Cebidae) and other primates and reveals extensive interchromosomal rearrangements between howler monkey genomes. Am J Primatol 46: 119–133 (1998). de Oliveira EH, Neusser M, Figueiredo WB, Nagamachi C, Pieczarka JC, et al: The phylogeny of howler monkeys (Alouatta, Platyrrhini): reconstruction by multicolor cross-species chromosome painting. Chromosome Res 10:669–683 (2002). de Oliveira EH, Neusser M, Pieczarka JC, Nagamachi C, Sbalqueiro IJ, Müller S: Phylogenetic inferences of Atelinae (Platyrrhini) based on multi-directional chromosome painting in Brachyteles arachnoides, Ateles paniscus paniscus and Ateles b. marginatus. Cytogenet Genome Res 108:183–190 (2005). Dobigny G, Ducroz JF, Robinson TJ, Volobouev V: Cytogenetics and cladistics. Syst Biol 53: 470–484 (2004). Dumas F, Bigoni F, Stone G, Sineo L, Stanyon R: Mapping genomic rearrangements in titi monkeys by chromosome flow sorting and multidirectional in-situ hybridization. Chromosome Res 13: 85–96 (2005). Dumas F, Stanyon R, Sineo L, Stone G, Bigoni F: Phylogenomics of species from four genera of New World monkeys by flow sorting and reciprocal chromosome painting. BMC Evol Biol 7 Suppl 2:S11 (2007). Fantini L, Mudry MD, Nieves M: Genome size of two Cebus species (primates: Platyrrhini) with a fertile hybrid and their quantitative genomic differences. Cytogenet Genome Res 135:33–41 (2011). Finotelo LF, Amaral PJ, Pieczarka JC, de Oliveira EH, Pissinati A, et al: Chromosome phylogeny of the subfamily Pitheciinae (Platyrrhini, primates) by classic cytogenetics and chromosome painting. BMC Evol Biol 10:189 (2010). Ford SM: Systematics of the New World Monkeys (Alan R. Liss, New York 1986). García F, Nogués C, Ponsà M, Ruiz-Herrera A, Egozcue J, Garcia Caldés M: Chromosomal homologies between humans and Cebus apella (primates) revealed by ZOO-FISH. Mamm Genome 11:399–401 (2000). García F, Ruiz-Herrera A, Egozcue J, Ponsà M, Garcia M: Chromosomal homologies between Cebus and Ateles (primates) based on ZOO-FISH and G-banding comparisons. Am J Primatol 57:177–188 (2002).


Gerbault-Serreau M, Bonnet-Garnier A, Richard F, Dutrillaux B: Chromosome painting comparison of Leontopithecus chrysomelas (Callitrichine, Platyrrhini) with man and its phylogenetic position. Chromosome Res 12: 691–701 (2004). Groves CP: Primate Taxonomy, ed 2 (Smithsonian Institution Press, Washington 2001). Hershkovitz P: Titis, new world monkeys of the genus Callicebus (Cebidae, Platyrrhini): a primary taxonomic review. Fieldiana Zool 55:1–109 (1990). Matsui A, Hasegawa M: Molecular phylogeny and evolution in primates, in Hirai H, Imai H, Go Y (eds): Post-Genome Biology of Primates, pp 243–268 (Springer, Tokyo 2012). Montefalcone G, Tempesta S, Rocchi M, Archidiacono N: Centromere repositioning. Genome Res 9:1184–1188 (1999). Morescalchi MA, Schempp W, Consigliére S, Bigoni F, Wienberg J, Stanyon R: Mapping chromosomal homology between humans and the black-handed spider monkey by fluorescence in situ hybridization. Chromosome Res 5: 527–536 (1997). Müller S: Primate chromosome evolution, in Lupski JR, Stankiewicz P (eds): Genomic Disorders: The Genomic Basis of Disease, pp 111–152 (Humana Press, Totowa 2006). Müller S, Neusser M, O’Brien PC, Wienberg J: Molecular cytogenetic characterization of the EBV-producing cell line B95–8 (Saguinus oedipus, Platyrrhini) by chromosome sorting and painting. Chromosome Res 9: 689– 693 (2001). Müller S, Finelli P, Neusser M, Wienberg J: The evolutionary history of human chromosome 7. Genomics 84:458–467 (2004). Neusser M, Stanyon R, Bigoni F, Wienberg J, Müller S: Molecular cytotaxonomy of New World monkeys (Platyrrhini) – comparative analysis of five species by multi-color chromosome painting gives evidence for a classification of Callimico goeldii within the family of Callitrichidae. Cytogenet Cell Genet 94:206–215 (2001). Neusser M, Münch M, Anzenberger G, Müller S: Investigation of marmoset hybrids (Cebuella pygmaea ! Callithrix jacchus) and related Callitrichinae (Platyrrhini) by cross-species chromosome painting and comparative genomic hybridization. Cytogenet Genome Res 108:191–196 (2005). Nieves M, De Oliveira EH, Amaral PJ, Nagamachi CY, Pieczarka JC, et al: Analysis of the heterochromatin of Cebus (primates, Platyrrhini) by micro-FISH and banding pattern comparisons. J Genet 90:111–117 (2011). Opazo JC, Wildman DE, Prychitko T, Johnson RM, Goodman M: Phylogenetic relationships and divergence times among New World monkeys (Platyrrhini, Primates). Mol Phylogenet Evol 40:274–280 (2006). Osterholz M, Walter L, Roos C: Retropositional events consolidate the branching order among New World monkey genera. Mol Phylogenet Evol 50:507–513 (2009).

Perelman P, Johnson WE, Roos C, Seuanez HN, Horvath JE, et al: A molecular phylogeny of living primates. PLoS Genet 7:e1001342 (2011). Ray DA, Batzer MA: Tracking Alu evolution in New World primates. BMC Evol Biol 5: 51 (2005). Richard F, Lombard M, Dutrillaux B: ZOOFISH suggests a complete homology between human and capuchin monkey (Platyrrhini) euchromatin. Genomics 36: 417–423 (1996). Rocchi M, Archidiacono N, Schempp W, Capozzi O, Stanyon R: Centromere repositioning in mammals. Heredity (Edinb) 108: 59– 67 (2012). Rodrigues LR, Pieczarka JC, Pissinati A, de Oliveira EH, das Dores Rissino J, Nagamachi CY: Genomic mapping of human chromosome paints on the threatened masked Titi monkey (Callicebus personatus). Cytogenet Genome Res 133:1–7 (2011). Rokas A, Holland PW: Rare genomic changes as a tool for phylogenetics. Trends Ecol Evol 15: 454–459 (2000). Rosenberger AL: Systematics: the higher taxa, in Coimbra-Filhó AF, Mittermeier RA (eds): Ecology and Behavior of Neotropical Primates pp 9–27 (Academia Brasiliera de Ciencias, Rio de Janeiro 1981). Ruiz-Herrera A, García F, Aguilera M, Garcia M, Ponsà Fontanals M: Comparative chromosome painting in Aotus reveals a highly derived evolution. Am J Primatol 65: 73–85 (2005). Schneider H: The current status of the New World monkey phylogeny. An Acad Bras Cienc 72:165–172 (2000). Schneider H, Canavez FC, Sampaio I, Moreira MA, Tagliaro CH, Seuánez HN: Can molecular data place each neotropical monkey in its own branch? Chromosoma 109: 515–523 (2001). Sherlock JK, Griffin DK, Delhanty JD, Parrington JM: Homologies between human and marmoset (Callithrix jacchus) chromosomes revealed by comparative chromosome painting. Genomics 33:214–219 (1996). Singer SS, Schmitz J, Schwiegk C, Zischler H: Molecular cladistic markers in New World monkey phylogeny (Platyrrhini, primates). Mol Phylogenet Evol 26:490–501 (2003). Stanyon R, Consigliére S, Müller S, Morescalchi A, Neusser M, Wienberg J: Fluorescence in situ hybridization (FISH) maps chromosomal homologies between the dusky titi and squirrel monkey. Am J Primatol 50: 95–107 (2000). Stanyon R, Consigliére S, Bigoni F, FergusonSmith M, O’Brien PC, Wienberg J: Reciprocal chromosome painting between a New World primate, the woolly monkey, and humans. Chromosome Res 9:97–106 (2001). Stanyon R, Bonvicino CR, Svartman M, Seuánez HN: Chromosome painting in Callicebus lugens, the species with the lowest diploid number (2n = 16) known in primates. Chromosoma 112: 201–206 (2003).


271

Stanyon R, Bigoni F, Slaby T, Müller S, Stone G, et al: Multi-directional chromosome painting maps homologies between species belonging to three genera of New World monkeys and humans. Chromosoma 113: 305– 315 (2004). Stanyon R, Rocchi M, Capozzi O, Roberto R, Misceo D, et al: Primate chromosome evolution: ancestral karyotypes, marker order and neocentromeres. Chromosome Res 16:17–39 (2008).

272

Stanyon R, Garofalo F, Steinberg ER, Capozzi O, Di Marco S, et al: Chromosome painting in two genera of South American monkeys: species identification, conservation, and management. Cytogenet Genome Res 134: 40–50 (2011). Steiper ME, Ruvolo M: New World monkey phylogeny based on X-linked G6PD DNA sequences. Mol Phylogenet Evol 27: 121–130 (2003). van Roosmalen MG, van Roosmalen T, Mittermeier RA: Taxonomic review of the Titi monkey, genus Callicebus, Thomas, 1903, with the description of the two new species, Callicebus bernhardi and Callicebus stephennashi, from Brazilian amazonia. Neotropical Primates 10:1–52 (2002).


Ventura M, Weigl S, Carbone L, Cardone MF, Misceo D, et al: Recurrent sites for new centromere seeding. Genome Res 14: 1696–1703 (2004). Wienberg J, Stanyon R: Comparative chromosome painting of primate genomes. ILAR J 39:77–91 (1998). Wildman DE, Jameson NM, Opazo JC, Yi SV: A fully resolved genus level phylogeny of neotropical primates (Platyrrhini). Mol Phylogenet Evol 53:694–702 (2009).

de Oliveira /Neusser /Müller



Evolutionary Molecular Cytogenetics of Catarrhine Primates: Past, Present and Future R. Stanyon a M. Rocchi b F. Bigoni a N. Archidiacono b a Department of Evolutionary Biology, University of Florence, Florence, and b Department of Genetics and Microbiology, University of Bari, Bari, Italy

Key Words BAC-FISH ⴢ Chromosome flow sorting ⴢ Chromosome painting ⴢ Marker order ⴢ Neocentromeres

Abstract The catarrhine primates were the first group of species studied with comparative molecular cytogenetics. Many of the fundamental techniques and principles of analysis were initially applied to comparisons in these primates, including interspecific chromosome painting, reciprocal chromosome painting and the extensive use of cloned DNA probes for evolutionary analysis. The definition and importance of chromosome syntenies and associations for a correct cladistics analysis of phylogenomic relationships were first applied to catarrhines. These early chromosome painting studies vividly illustrated a striking conservation of the genome between humans and macaques. Contemporarily, it also revealed profound differences between humans and gibbons, a group of species more closely related to humans, making it clear that chromosome evolution did not follow a molecular clock. Chromosome painting has now been applied to more that 60 primate species and the translocation history has been mapped onto the major taxonomic divisions in the tree of primate evolution. In situ hybridization of cloned DNA



probes, primarily BAC-FISH, also made it possible to more precisely map breakpoints with spanning and flanking BACs. These studies established marker order and disclosed intrachromosomal rearrangements. When applied comparatively to a range of primate species, they led to the discovery of evolutionary new centromeres as an important new category of chromosome evolution. BAC-FISH studies are intimately connected to genome sequencing, and probes can usually be assigned to a precise location in the genome assembly. This connection ties molecular cytogenetics securely to genome sequencing, assuring that molecular cytogenetics will continue to have a productive future in the multidisciplinary science of phylogenomics. Copyright © 2012 S. Karger AG, Basel

The catarrhine primates (Old World monkeys, apes and humans) have long captivated the attention of cytogenetics. Clearly, they hold special interest because they include our own species and comparative genomics has inevitably been framed in terms of the human genome. This tendency became even stronger in the genomic era when cytogenetics went molecular. The human genome project provided new DNA tools for comparative cytogenetics based on the human genome. Foremost among Roscoe Stanyon Department of Evolutionary Biology, University of Florence Via del Proconsolo 12 IT–50122 Florence (Italy) Tel. +39 055 274 3013, E-Mail roscoe.stanyon @ unifi.it

these were chromosome paints, first from plasmid libraries [Wienberg et al., 1990] and later from flow-sorted chromosomes [Telenius et al., 1992; Ferguson-Smith et al., 2005]. The first species entirely mapped by whole human chromosome probes were the Japanese macaque [Wienberg et al., 1992] and apes [Jauch et al., 1992]. The range of variability was incredibly vast in just these species. All human syntenies, except chromosome 2 and a reciprocal translocation between gorilla homologs to human chromosomes 5 and 17 [Stanyon et al., 1992b], were conserved in great apes and macaques. Yet, amazingly, just the opposite was found in the lar gibbon, Hylobates lar, where only 1 human chromosome was found intact. Several fundamental results of molecular cytogenetics were established from these data [Wienberg and Stanyon, 1995]. First of all, large segments of the human genome could be conserved over millions of years. Secondly, chromosome changes did not follow a molecular clock. Clearly, vastly different rates of chromosome evolution were needed to explain why the macaque, which diverged from humans at least 25 million years ago (MYA), had conserved human syntenies, while the gibbons, which are closer to humans and classified in the same family Hominoidea, had in a minor time evolved such an incredibly rearranged genome. Many of the conclusions based on banding comparisons were confirmed by chromosome painting in Old World monkeys and great apes [i.e. Dutrillaux, 1980; Dutrillaux et al., 1982]. However, a range of other questions was opened by these studies, and comparison began to be framed on the basis of conserved human chromosomal syntenies and associations. Although all human syntenies were found intact in the macaque, a number of human chromosomes were found together on the same macaque homolog [Wienberg et al., 1992]. Later, human chromosomes mapped contiguously in another species genome were termed ‘associations’. Three macaque homologs showed human associations, 7/21, 14/15 and 20/22, accounting for the difference in diploid number (2n = 46 in humans and 2n = 42 in macaques). These associations were not found in any of the hominoids (great apes and gibbons). The remaining difference from humans was that, instead of a syntenic chromosome 2, this chromosome was always present as 2 independent chromosomes in great apes and macaques and in 5 segments in 4 chromosomes of the lar gibbon genome [Jauch et al., 1992]. These data supported the hypothesis derived from banding comparisons that human chromosome 2 was a recently formed synteny unique to the human line. How274


ever, the polarity to the other associations was not clear. It was not known if the 7/21 and 14/15 associations found in the macaque were derived or inherited from some common ancestor of macaques, apes and humans as yet there was no outgroup to properly compare the human and ape genomes to the macaque. The great number of associations in the lar gibbon on the other hand were clearly derived. In this case, the chromosome painting data in the macaque genome provided a cladistic framework for comparison. The macaque could serve as a proper outgroup to compare the hominoids. The conservation of human syntenies in the macaque indicated that the gibbon genome was apomorphically highly rearranged. In reality, the spectacular differences of the painting pattern found in H. lar were not unexpected. Indeed, human chromosome paints were viewed as a tool to resolve a puzzle of primate chromosome evolution that had appeared with the first banding comparisons of lesser apes with humans and other primates. Whereas the homologs to many human chromosomes could be identified with considerable confidence using banding methods, the gibbon chromosomes were much more difficult to match. This difficulty led a number of researchers to erroneously conclude that the gibbon was phylogenetically further from humans than the macaques [Bernstein et al., 1980] or to make mistakes in matching gibbon chromosomes with those of other primates [Stanyon et al., 1983]. Taxonomic and systematic errors in cytogenetically placing gibbons were also present in the early cytogenetic literature. For instance, Chiarelli [1963], comparing the classically stained karyotypes of gibbons, had made a similar error by removing gibbons from the hominoids and allying them with catarrhine monkeys, in particular with Colobinae. This error was easy to make given that many species of gibbons had a diploid number of 2n = 44, composed of metacentric chromosomes with 1 pair, the ‘marked’ chromosomes, bearing the nucleolar organizing region (NOR), so that the morphology of these chromosomes was practically identical in both taxa. Indeed chromosome painting showed that there is often no chromosomal homology of the NOR-bearing chromosome outside of the NOR itself [Stanyon et al., 1995]. Errors in assigning homology still afflict the cytogenetics of organisms for which chromosome painting is not available. Initial studies using chromosome painting, and others that quickly followed, showed that errors were caused by the very rapid chromosome evolution in lesser apes [Koehler et al., 1995a, b]. The diploid number and chromosome morphology, which led Chiarelli [1963] to classify gibbons among the leaf-eating monkeys, was due to Stanyon /Rocchi /Bigoni /Archidiacono

convergence. The error was that those chromosomes were similar not for homology (inherited from a common ancestor) but for convergence [Stanyon et al., 1995]. The difficulty in matching gibbon chromosomes was owing to the vast number of rearrangements that had rapidly transformed the gibbon karyotypes, diversifying them even on the basis of diploid number alone into karyomorphs with 38, 44, 50 or 52 chromosomes. The immediate impact of chromosome painting was that cytogenetic comparisons could be made not on the basis of similarity in morphology or banding patterns (in reality both equivalent to phenotypes), but on the basis of DNA content. It allowed a fairly foolproof method of distinguishing homology from convergence and conserved versus derived features. Finally cytogenetics had graduated to a reliable, cladistic science that could conceivably make a contribution to phylogenetics and the tree of life. Although chromosome painting continues to provide fruitful results even today (as do all previous techniques such as banding) and is an indispensable technique, we are fortunate that molecular probes come in all sizes. Indeed, chromosome painting is limited due to the fact that these are enormous probes; in fact, chromosome paints cover the entire synteny. They are particularly useful to track translocations (interchromosomal rearrangements), but are not very good at discriminating intrachromosomal rearrangements such as inversions. Cloned DNA probes, such as BACs, PACs and YACs and other probes ranging in size from 150 kb down to 5 kb, can literally dissect each chromosome and be used to establish marker order within each chromosome [Müller et al., 2000]. Comparisons between species can then identify differences in marker order. A cladistic analysis helps to establish the polarity of changes in marker order [Stanyon et al., 2008]. One important outcome of such studies was the discovery that centromere shifts, changes of centromere position without any concurrent change in marker order, were a common feature of chromosome evolution in primates on a par with other types of rearrangements [Ventura et al., 2007]. Today, due to the progress in assembling the genome sequence of humans and other species, the cytogenetic data can be tied and compared to the results from other levels of genome analysis. The precise position of the clones on the assembled human sequence can be easily derived from the 2 end sequences of the clone. Some genome browsers systematically perform this mapping, and the position of each clone is graphically displayed. Therefore, it is possible to select specific clones on the basis of their precise position on the human sequence. However, we should note that

human clones work well in catarrhines, but less well in other primates. There is a noticeable drop off in New World monkeys and in prosimians, and good hybridizations are much more difficult to routinely obtain. In this article we want to summarize the molecular cytogenetic data in catarrhine primate evolution by reviewing the data from chromosome painting and FISH with cloned DNA probes. We will start with the chromosome painting data, and then we will explore the results coming from FISH with BAC probes with particular reference to centromere shifts or evolutionary new centromeres (ENCs).

Catarrhine Chromosome Evolution


Chromosome Painting Results in the Catarrhine Primates

Cercopithecidae The Cercopithecidae comprise Papionini (baboons, macaques, mangabeys) and Cercopithecini (guenons). Chromosome painting confirmed the banding conclusions that Papionini species had very similar and often identical chromosomes, while the Cercopithecini are variable in chromosome number and chromosome painting [Dutrillaux et al., 1982]. Papionini The same pattern of conserved chromosome syntenies was discovered in every Papionini species studied: the 7/21, 14/15 and 20/22 associations are common to all species [Wienberg et al., 1992]. However, some intrachromosomal rearrangements or differences found between Papionini species on the basis of banding have never been confirmed at the molecular level [Stanyon et al., 1988]. For instance, differences at the banding level are found most notably in chromosome 9 (homolog to human chromosome 10) of drills, mandrills and mangabeys [Dutrillaux et al., 1982; Stanyon et al., 1983]. This difference has been taken to phylogenetically ally these taxa, a conclusion supported by limited molecular data [Harris and Disotell, 1998; Page and Goodman, 2001]. A difference in the banding pattern of the Macaca fascicularis chromosome 6 (homologous to human chromosome 5), sometimes interpreted as a paracentric inversion, has never been studied at the molecular level [Stanyon et al., 1988]. Overall, the Papionini illustrate that chromosome rearrangements are not necessarily correlated with speciation due to the fact that over 20 species in this group have conserved essentially identical karyotypes [Stanyon et al., 1988]. 275

Cercopithecini Contrary to the Papionini, most Cercopithecini have different karyotypes. It was known from classical staining that this tribe of Old World monkeys varied remarkably in diploid number, from 48 to 72 [Eckhardt, 1979]. Some researchers thought that the Cercopithecini originally had a high diploid number and evolved by Robertsonian fusions. Others proposed the opposite, that the Cercopithecini started out low and the increasing diploid numbers were due to Robertsonian fissions [Stock and Hsu, 1973; Dutrillaux et al., 1978]. The chromosome painting data showed that the trend went from a low to a high number, but that the mechanisms were more complex [Finelli et al., 1999]. Both Robertsonian and nonRobertsonian fissions were involved. Furthermore, the phylogenetic tree was not a simple trend toward higher diploid numbers and with higher numbered taxa sharing a more recent common ancestor. Instead, the reciprocal chromosome painting data supported another hypothesis based on banding, i.e. that the guenons had diverged early into 2 main branches [Dutrillaux et al., 1982; Stanyon et al., 2005]. This interpretation was also supported by molecular work, which suggested that one branch led to more terrestrial oriented species [Tosi et al., 2004; Xing et al., 2007]. Colobidae The general similarity in Colobinae karyotypes, all except the proboscis monkeys have 2n = 44 with mainly biarmed chromosomes, hides some noteworthy differences of probable phylogenetic significance. Some of these were shown by the few chromosome banding studies in these primates [Dutrillaux et al., 1981, 1984; Ponsa et al., 1983; Stanyon et al., 1992a]. The only African colobine studied up to now with chromosome paints is the black-and-white colobus, Colobus guereza (CGU) [Bigoni et al.,1997a, b, 2003, 2006]. In C. guereza there are 6 human chromosomes (HSA1, 2, 3, 10, 17 and 19) that are fragmented, meaning that the synteny is disrupted. These breaks and translocations have produced CGU5 (HSA1/10), CGU11 (HSA17/1/10), CGU12 (HSA3/19/3/19), CGU13 (HSA1/17), and CGU18 (HSA3/19). The alternating pattern of human segments 3 and 19 on CGU12 is best interpreted as a pericentric inversion that followed a translocation. Bigoni and coworkers also studied 3 species of Asian colobines: Trachypithecus cristatus (2n = 44) [Bigoni et al., 1997a], Nasalis larvatus (2n = 48) [Bigoni et al., 2003] and Pygathrix nemaeus (2n = 44) [Bigoni et al., 2004]. Nie et al. [1998] also reported on the hybridization of the entire 276


set of human chromosome paints to the Asian colobines Trachypithecus francoisi and T. phayrei. In Asian leaf-eaters, the fragmentation and association of human chromosomes 1 and 19 can be explained with a reciprocal translocation. This association was not found in the African colobine C. guereza where different translocations are present [Bigoni et al., 1997a, b]. The most parsimonious explanation is that a reciprocal translocation occurred in the lineage of the Asian colobines and distinguishes this group from the African colobines. The karyotypes of T. cristatus, T. francoisi, T. phayrei, and N. larvatus showed an alternating association of HSA1 and 19 [Nie et al., 1998; Bigoni et al., 2003, 2004], best explained as due to an inversion. Comparisons of painting patterns supported the monophyly of Colobinae and their division in an African and an Asian clade. The chromosome painting also shows that N. larvatus, often considered the most primitive and isolated colobine, is karyologically derived and phylogenetically nested with Asian colobines [Bigoni et al., 2003]. T. cristatus appears to be karyologically the most derived among the Asian colobines. This species has a reciprocal translocation of human chromosomes 6 and 16 and is one of the very few primates and the only catarrhine showing a reciprocal translocation involving the Y chromosome and an autosome [Bigoni et al., 1997a]. The probe specific for human chromosome 6 painted only 1 chromosome in the African colobine species C. guereza [Bigoni et al., 1997b] and in P. nemaeus [Bigoni et al., 2004]. G-banding analyses and comparisons demonstrated that human chromosome 6 is also maintained in some other species of Asian colobines, including Semnopithecus entellus, Presbytis comata and Semnopithecus vetulus. In T. cristatus, T. francoisi and T. phayrei the probe specific for human chromosome 6 painted 2 segments of 2 different chromosomes, but they are associated with a segment homologous to human chromosome 16, following a reciprocal translocation that involved human homologs 6 and 16. Karyological data supporting a closer relationship between S. entellus (which also has a syntenic chromosome 6, unpubl. data) and S. vetulus are not in contrast with geographical distribution of these 2 species (S. entellus in India and Sri Lanka, S. vetulus in Sri Lanka) and with observations on the color of infants, an important and variable character in colobines. In fact, S. vetulus could be excluded from Trachypithecus on the basis that infants are not orange but gray. On the same basis of a blackish color of newborn infants, Groves [1989] argued that S. entellus is more primitive than Trachypithecus. Using a Stanyon /Rocchi /Bigoni /Archidiacono

5-kb mtDNA fragment, Osterholz et al. [2008] found a link between these clusters in S. vetulus and in Semnopithecus. The exceptional diploid number of Nasalis, 2n = 48 [Chiarelli, 1963; Stanyon et al., 1992a], has played a pivotal role in phylogenies which place the proboscis monkey as the most primitive colobine and a long isolated genus of the group [Giusto and Margulis, 1981; Groves, 1989; Peng et al., 1993]. Groves [1989] considered Nasalis primitive for a relevant number of morphological characters, for the most part linked to the lack of masticatory specialization seen in other colobines, and for the diploid number. He considered Nasalis as a sister species to all other African and Asian colobines and he divided the Colobidae into 2 subfamilies: Nasalinae and Colobinae. Harvati [2000] found support for Groves on the basis of colobine dental eruption sequences. Peng et al. [1993] also claimed that Nasalis is the most primitive colobine genus on the basis of morphological measurements and again for the chromosome number. However, Bigoni et al. [2003], as discussed above, showed that the proboscis monkey genome is derived and not primitive. The diploid number of 2n = 48 can be best explained by derived fissions of a segment of human chromosome 14 and the fission of human chromosome 6. Consequently, the higher diploid number found in Nasalis is not, as mistakenly assumed, a primitive character. Zhang and Ryder [1998] supported the existence of a monophyletic Asian clade and suggested the possibility of an odd-nosed lineage including Nasalis, Rhinopithecus and Pygathrix as did a more recent molecular study [Sterner et al., 2006]. If this hypothesis is correct, then the previously hypothesized karyological links of Nasalis with Presbytis [Bigoni et al., 2003] could be erroneous. The alternating pattern of paints to HSA1 and 19 might be due to independent, convergent pericentric inversions on this chromosome.

ments. Reciprocal painting in Old World monkeys is fairly limited and there are only 4 reports available on reciprocal chromosome painting between humans and Old World monkeys: Chlorocebus aethiops [Finelli et al., 1999], Erythrocebus patas, Cercopithecus neglectus [Stanyon et al., 2005] and C. guereza [Bigoni et al., 2006]. These results have been summarized in figure 1. The diverse pattern of hybridization signals on the human chromosomes allows us to conclude that there are 2 lines of evolution within the Cercopithecini. In these 2 lines, even when a particular chromosome is fragmented into the same number of segments, the resulting segments are not homologous because the breakpoints are diverse.

With chromosome sorting, probes can be made from any species and reciprocal chromosome painting is possible between species. Paints from one species are used to hybridize another species’ chromosomes and vice versa. More than 2 paint sets allow multi-directional chromosome painting. Such reciprocal and multi-directional chromosome painting permits the delineation of subchromosomal homology and a much more precise inference of breakpoints involved in chromosome rearrange-

Hylobatidae: The Small Apes As mentioned above the dozen or more species of lesser apes can be divided into 4 karyomorphs: 2n = 38, 44, 50 and 52. The painting data showed that human chromosomes were highly fragmented and reshuffled in each of these karyomorphs [Jauch et al., 1992; Koehler et al., 1995a, b]. For instance, H. lar chromosomes (2n = 44) were composed of up to 5 segments of different human chromosomes. The 24 siamang autosomes (2n = 50), composed of 60 segments homologous to human chromosomes, were the result of at least 33 translocations. In the concolor gibbon, the autosomes were composed of 63–67 segments as the result of at least 31 translocations. Translocations, often reciprocal, played a major role in transforming these genomes. The chromosome painting data suggested that although each genus of small apes differed dramatically from each other, they probably shared a large number of rearrangements that occurred in their last common ancestor. Reciprocal painting confirmed the data from painting small ape metaphases with human paint sets and added important data on the homology of each gibbon chromosome to subchromosomal regions of human chromosomes [Nie et al., 2001; Müller et al., 2003]. Müller et al. [2003] used these data to propose an ancestral karyotype and a chromosome phylogeny for the gibbons. They placed Hoolock at the base of the tree with Nomascus/ Symphalangus diverging last. However, because the rearrangements were so complex, chromosome painting did not allow final conclusions about the steps that led to the 4 existing karyomorphs typical for each genus of small apes or permitted confidence in the exact phylogeny. Later, even more sophisticated molecular cytogenetic techniques and sequencing added precise details on the breakpoints involved in small ape chromosome evolution [Carbone et al., 2006; Roberto et al., 2007; Misceo et al.,



Reciprocal Painting

277

1

Fig. 1. This figure reports, charted on the human ideogram, break points determined by reciprocal chromosome painting between humans and various Old World monkeys. Blue lines to the right represent break points found in the 3 studied Cercopithecini (CAE = Chlorocebus aethiops, CNE = Cercopithecus neglectus, EPA = Erythrocebus patas). Purple lines show breaks found in both CAE and EPA. Yellow lines show breaks found only in CAE and green those found only in CNE. Red lines show breaks found in Colobus guereza, CGU.

2

9

10

16

11

17

Ancestral centromere

2008; Girirajan et al., 2009]. In these studies, the main techniques were array painting FISH using panels of 600–800 BACs which covered the entire genome and limited sequencing. Breakpoints were then defined by spanning or flanking BACs, with an average resolution of about 140 kb. By these refined criteria, the results showed that H. lar and Nomascus leucogenys shared many common breakpoints, and brought convincing data that these rearrangements occurred in their last common ancestor (fig. 2; for online supplementary table 1, www. karger.com/doi/10.1159/000339381). Misceo et al. [2008], also integrating these results with previous reports, proposed a karyotype with a diploid number of 2n = 60 for the Hylobates ancestor. A final ancestral karyotype for the small apes will become available when the remaining 2 genera, Symphalangus and Hoolock are analyzed at a comparable level of resolution. The underlying reason why small apes had very rapid chromosome evolution remains speculative. Over the years many possibilities have been discussed, including meiotic drive, recombination suppression, molecular divergence in rearranged chromosomes as well as the demographic parameter [Misceo et al., 2008; Brown and O’Neill, 2009; Israfil et al., 2011]. Recently it has been 278


3

4

12

18 HSA fusion

5

6

13

19 EPA/CAE

20

7

14

15

21 CNE/EPA/CAE

8

22 CAE

X CGU

Y

CNE

noted that breakpoints in gibbons are enriched in segmental duplications and repeats. Alu elements are very abundant at breakpoints and the Alus at gibbon breakpoints have a higher CpG content than other Alus, with a comparatively low density of methylated cytosine. These findings suggest that gibbon chromosomes are epigenetically distinct and correlate the epigenetic state and structural genome variation with rapid chromosome evolution [Carbone et al., 2009]. However, a comprehensive understanding of small ape evolution will probably need to await the sequencing of one or several small ape genomes. Great Apes As expected, the synteny of most human chromosomes (each human chromosome had an equivalent in the great apes) was found intact in all great apes with only the exception of chromosome 2 and a reciprocal translocation between human homologs 5 and 17 in the gorilla [Yunis and Prakash, 1982; Stanyon et al., 1992b]. However, the chromosome painting data were of no utility to view the remaining rearrangements known from banding which are intrachromosomal as inversions. Later, other molecular methods such as BAC-FISH, comparaStanyon /Rocchi /Bigoni /Archidiacono

2

1

9

3

10

16

17

Ancestral centromere

4

11

18

6

5

12

13

19

20

Hylobatidae ancestral centromere

Hylobatidae breakpoints

7

15

14

21

8

22

Hylobates breakpoints

X

Y

Nomascus breakpoints

Fig. 2. This figure reports, charted on the human ideogram, the breakpoints revealed via BAC-FISH in the small

ape genera Hylobates (blue), Nomascus (green), and those common to both and proposed to have originated in the Hylobatidae ancestor (red) as reconstructed by Misceo et al. [2008] (see online suppl. table 1).

tive genomic hybridization on arrays and sequencing were used to define these chromosome rearrangements [Locke et al., 2003; Kehrer-Sawatzki and Cooper, 2007; Stanyon et al., 2008].

which led to the discovery of the extent of centromere shifts or ENCs.

As we mentioned above, BAC-FISH analysis can provide information on marker order and breakpoint location on a much higher level of resolution. Mapping marker order and defining breakpoints contributes to the understanding of the flow of evolutionary chromosome rearrangements, especially in highly rearranged genomes such as in the small apes. Comparisons of marker order permit more detailed ancestral karyotypes to be inferred for catarrhine evolution. That includes reconstruction of centromere positions, a field of enquiry

Ancestral Karyotype of Catarrhines Stanyon et al. [2008] summarized all available BACFISH data from primates in order to reconstruct ancestral karyotypes for the major nodes on the primate phylogeny. On average, a panel of about 900 BACs was used to compare marker order in an array of primate species. The ancestral catarrhine karyotype (2n = 46) is remarkably similar to the karyotype found in many living species (fig. 3). Certainly this is not unexpected since the conservation of banding pattern and synteny between macaques and humans was already established previously. Most human chromosomes have retained identical marker order to the ancestral catarrhine chromosomes (4, 5, 6, 8, 11, 12, 13, 14, 15, 17, 19, 21 and 22). Only one or very few inversions can explain the marker order of the remaining, present day human chromosomes.



BAC-FISH, Ancestral Karyotypes and Neocentromeres in the Evolution of the Catarrhini

279

U T S R Q P O N M L J

1

Ancestral catarrhine karyotype (ACK)

F G H I

A B C

N 3

4

M

D

L K

K I H G F E D C B A

5

E A 2

E

6

E F G H I J K L M N O P Q R

F G H IJ K L M N O P Q R S

B C D

1

D

E

J

A B C D

A B C

A

B

P K J

C

15

F G

D

H I J K L

E F G

7

14

A B C D E

B

8

I H G F ED O

E

5

6

A

12

B C

Q P O N M EF G H I J K L DB A

G F E I J

K

8

7

R 11

D E I H

H I J K L M N O

T

J

B C

10

G

I

N O

A

F

N M LR S

H

M

4

3

A

9

10

D E F G H I J K 11 A B

O N P Q R S T U V W X Y Z

2q

12

D C B A E F G H I J K

9

13

L M K J I H G F E D C B A

2q

2p

14

A B C D E F G H I J K L M N

13

15

A B C D E F GH I J K PO M N L RQ ST UV

17

16

C D

A B C D

16

E

B A

18

C D E F G

F G H

17

18

A B C D E

19

E D C B A F G H I J

20

F G H IJ K L

19

20

A

22

A

21

S Y

D

21

E

G

B C

22

F

X

Y

Fig. 3. This figure shows the proposed ancestral catarrhine karyotype modified from Stanyon et al. [2008]. The

homology to human chromosomes is indicated to the left. The marker order of each chromosome is indicated to the right of the chromosomes in letters. The BAC clones are lettered according to their order in the human genome. The arrows on the right show the synteny block direction in relationship to the human genome.

Evolutionary New Centromeres The centromere, a fundamental structure of chromosomes, represents a black hole to sequencing efforts. Up until a few years ago centromeres were considered as stable elements of genomes. Indeed, Robertsonian mechanisms, about the only process thought to lead to difference in diploid number, were seen as dividing centromeres or fusing centromeres. In the chromosome banding era, different positions of the centromere along a chromosome considered homologous between species were almost always interpreted as the result of a pericentric inversion or of a complex rearrangement. With the introduction of molecular techniques into cytogenetics, BAC-FISH allows the evolutionary history of a centromere to be tracked. This approach showed that centromeres often shift or reposition without any change in marker order. Montefalcone et al. [1999] were the first to unequivocally demonstrate the existence of the evolutionary centromere repositioning phenomenon. Further, it is not a rare phe280


nomenon (fig. 4). Ventura et al. [2007] showed that comparing macaques and humans, 15 centromere shifts could be found: 9 along the macaque lineage and 6 in the human line. Orangutan chromosome 9, long known to be heterozygous for a complex rearrangement, was discovered to be an ENC polymorphism [Rocchi et al., 2012]. The X chromosome of squirrel monkeys, previously thought to differ from the normal mammalian X for a paracentric inversion, is now known to differ for a centromere shift [Rocchi et al., 2012]. More recently, chromatin immunoprecipitation followed by hybridization on microarrays (ChIP-onchip analysis) allows centromeres to be mapped down to the precise sequence location. However, this method is expensive and has not been widely employed. Comparative data shows that the macaque ENCs are actually found in all studied Old World monkeys; therefore they accumulated during the ⬃14-million-year span from the Hominoidea/Cercopithecoidea split (⬃32 MYA) to the Cercopithecinae/Colobinae divergence (⬃18 MYA) Stanyon /Rocchi /Bigoni /Archidiacono

1

2

3

4

9

10

11

12

16

17

18

Ancestral inactivated centromere OWM centromere

5

19

6

13

7

14

20

21

8

15

22

X

Y

Ancestral centromere Catarrhine centromere Hominoid centromere

Fig. 4. This figure graphically summarizes, on the human chromosome ideogram, all the evolutionary neocentromeres found in diverse hypothesized ancestors of the Catarrhini. Black centromeres are those that were present in the primate ancestral karyotype, green represents centromeres that originated in the catarrhine ancestor, blue centromeres are those that originated in the hominoid ancestor. Red filled circles to the right of the chromosomes represent centromeres that originated in the Old World monkey (OWM) ancestor and dotted ovals to the right of chromosomes 2, 6, and 15 represent inactivated ancestral centromeres in humans. Centromeres that formed apomorphically in non-human primate species are not indicated. Additionally, neocentromeres that are sometimes found in normal individual humans are not reported here, nor are clinical neocentromeres.

other catarrhine primates. These rearrangements have also been accompanied by the formation of ENCs. The ENCs in Nomascus and Hylobates have been reported by Roberto et al. [2007], and a large number of ENCs were discovered by Misceo et al. [2008]. Mature repositioned centromeres are thought to have slowly acquired the large arrays of satellite DNA after their seeding in an anonymous sequence. The macaque repositioning events occurred at least 18 MYA, providing plenty of time to ‘mature’. On the other hand, the orangutan neocentromere on chromosome 9 appears to have a less complex structure than other orangutan centromeres and thus appears to be younger [Rocchi et al., 2012]. Since this ENC was found both in Bornean and Sumatran orangutans, an important point is that the ENC polymorphism survived a fairly recent speciation event [Locke et al., 2011]. Another point is that either the orangutan population was never particularly small or unknown selection factors are maintaining this polymorphism. The process of ENC maturation (and centromere deactivation) is still not clear. Perhaps in the future orangutan chromosome 9 might eventually provide some clues, once the neocentromere region is sequenced.

Mechanisms of Centromere Shift: Connecting the Past with the Present

[Perelman et al., 2011]. By comparison, 6 human centromeres are evolutionarily new. Centromeres of chromosomes 3, 6, and 11 were repositioned along the chromosomes [Ventura et al., 2004; Cardone et al., 2007; Capozzi et al., 2009]. Those of chromosomes 14 and 15 were seeded, as mentioned, in the Hominoidea ancestor following the fission of a chromosome corresponding to chromosome 7 in macaque [Ventura et al., 2003]. A non-centromeric fission of the 3/21 association synteny in the Hominoidea ancestor generated chromosome 21 and its neocentromere. As mentioned above, the small apes have accumulated 10 to 20 times the number of rearrangements found in

The comparative genomic data provide some clues about the mechanisms of centromere shifts. For example, the ENC found in macaque chromosome 18 (HSA18) corresponded in humans to a clone gap, positioned at chr18: 50,313,135–50,360,134 [Carbone et al., 2009]. The gap is composed of non-alpha, satellite-like DNA. Sequence analysis of several primate species suggested that this sequence was present in the Cercopithecidae ancestor at the time of the neocentromere seeding. This satellite DNA was subsequently replaced by alpha satellite DNA. Apparently centromeres most often relocate to such repeat domains which are sometimes referred to as gene deserts [Lomiento et al., 2008]. There appear to be correlations between ENCs and neocentromeres in clinical genetics [Marshall et al., 2008; Rocchi et al., 2012. In human clinical neocentromeres there is a clustering at 15q24–q26. As we discussed above, the association 14/15 was split by a fission in the hominoid ancestor and ENCs were formed in both derivative chromosomes. The ancestral centromere, located in a region corresponding to 15q24–q26, was inactivated. The abundant segmental duplications clustered at this do-



281

main are remains of the pericentromeric segmental duplications that flanked the ancestral inactivated centromere, and this is the domain in which human clinical neocentromeres are found today. In human chromosome 3, a repositioned centromere and a clinical neocentromere were seeded to the 3q26 chromosomal domain, the locus where the centromere repositioned in the Old World monkey ancestor [Ventura et al., 2004]. Recently, Capozzi et al. [2009] reported a similar situation for a clinical neocentromere. The ancestral position of the centromere in hominoids was at 6p22.1. In the clinical case the centromere had shifted back to its location in the Hominoidea ancestor. Therefore, the same domain has been used as seeding point of an ENC and clinical neocentromeres. It is an intriguing thought that knowledge of the evolutionary past provides compelling underlying explicative grounds for contemporary genomic phenomena [Capozzi et al., 2009].

Conclusions

Over the last 2 decades cytogenetics in primates has led the way in the application of molecular tools to study chromosome evolution as well as in providing the theoretical basis to interpret results. Initially efforts were focused on chromosome painting, and later flow-sorting reciprocal hybridization permitted subchromosomal assignment of homologies. Chromosome paints, however, efficient at charting translocation, were very poor at visualizing inversions. Cloned DNA probes then provided high resolution down to a few kb and were able to map marker order along each chromosome. Breakpoints could be narrowed down to spanning or flanking BACs. Panels

of BACs hybridized to an array of primate species provided the first clues about ancestral marker order in chromosomes. The BAC-FISH results and sequencing have a beneficial reciprocal relationship. BACs can aid genome assembly efforts because synteny blocks including orientation can be readily discerned. Cloned DNA is also intimately connected with sequence assemblies. Usually a BAC has a precise location in a genome assembly, providing a fruitful connection of molecular cytogenetics to genome sequencing efforts. Finally, molecular cytogenetics and sequencing are complementary, and genome assembly should best be guided by molecular cytogenetic results and confirmation [Rocchi et al., 2006, 2012; Roberto et al., 2008]. For the shot-gun assembly methods the centromere is a black hole. Evolutionary new centromeres would never have been found with sequencing. Even if primates are the best-studied group from a molecular cytogenetic point of view, the perspective is still that many species remain to be studied even with chromosome painting. Among these are most species of Colobinae. Up to now only 1 African colobine has been studied and many Asian colobines are not yet known. On the other major branch of Old World monkeys, the guenons or Cercopithecini are not well known. Reciprocal painting and BAC-FISH studies are still in their infancy. We predict that molecular cytogenetics, and in particular primate cytogenetics, has much yet to accomplish and will continue to remain on the forefront of phylogenomics in the years to come. Acknowledgements This work was supported by PRIN (Programmi di Ricerca di Interesse Nazionale) to M.R., N.A., and R.S. We thank Oronzo Capozzi for his aid in assembling the supplemental table.

References Bernstein R, Pinto M, Morcom G, Bielert C: A reassessment of the karyotype of Papio ursinus. Homoeology between human chromosome 15 and 22 and a characteristic submetacentric baboon chromosome. Cytogenet Cell Genet 28:55–63 (1980). Bigoni F, Koehler U, Stanyon R, Ishida T, Wienberg J: Fluorescence in situ hybridization establishes homology between human and silvered leaf monkey chromosomes, reveals reciprocal translocations between chromosomes homologous to human Y/5, 1/19, and 6/16, and delineates an X1X2Y1Y2/ X1X1X2X2 sex-chromosome system. Am J Phys Anthropol 102:315–327 (1997a).

282

Bigoni F, Stanyon R, Koehler U, Morescalchi AM, Wienberg J: Mapping homology between human and black and white colobine monkey chromosomes by fluorescent in situ hybridization. Am J Primatol 42: 289–298 (1997b). Bigoni F, Stanyon R, Wimmer R, Schempp W: Chromosome painting shows that the proboscis monkey (Nasalis larvatus) has a derived karyotype and is phylogenetically nested within Asian colobines. Am J Primatol 60: 85–93 (2003).


Bigoni F, Houck ML, Ryder OA, Wienberg J, Stanyon R: Chromosome painting shows that Pygathrix nemaeus has the most basal karyotype among Asian colobines. Int J Primatol 25:679–688 (2004). Bigoni F, Stone G, Perelman PL, Stanyon R: Cytotaxonomy of Colobinae primates with reference to reciprocal painting of Colobus guereza and humans; in Sineo L, Stanyon R (eds): Primate Cytogenetic and Comparative Genomics, pp 19–32 (Firenze University Press, Firenze 2006).

Stanyon /Rocchi /Bigoni /Archidiacono

Brown JD, O’Neill RJ: The mysteries of chromosome evolution in gibbons: Methylation is a prime suspect. PLoS Genet 5:e1000501 (2009). Capozzi O, Purgato S, D’Addabbo P, Archidiacono N, Battaglia P, et al: Evolutionary descent of a human chromosome 6 neocentromere: a jump back to 17 million years ago. Genome Res 19:778–784 (2009). Carbone L, Vessere GM, ten Hallers BF, Zhu B, Osoegawa K, et al: A high-resolution map of synteny disruptions in gibbon and human genomes. PLoS Genet 2:e223 (2006). Carbone L, Harris RA, Vessere GM, Mootnick AR, Humphray S, et al: Evolutionary breakpoints in the gibbon suggest association between cytosine methylation and karyotype evolution. PLoS Genet 5:e1000538 (2009). Cardone MF, Lomiento M, Teti MG, Misceo D, Roberto R, et al: Evolutionary history of chromosome 11 featuring four distinct centromere repositioning events in Catarrhini. Genomics 90:35–43 (2007). Chiarelli B: Comparative morphometric analysis of primate chromosomes. III. The chromosomes of the genera Hylobates, Colobus and Presbytis. Caryologia 16:637–648 (1963). Dutrillaux B: Chromosomal evolution of the great apes and man. J Reprod Fertil Suppl Suppl 28:105–111 (1980). Dutrillaux B, Viegas-Pequignot E, Couturier J, Chauvier G: Identity of euchromatic bands from man to Cercopithecidae (Cercopithecus aethiops, Cercopithecus sabaeus, Erythrocebus patas, and Miopithecus talapoin). Hum Genet 45:283–296 (1978). Dutrillaux B, Couturier J, Ruedi D: The karyotypes of Colobus vellerosus and of C. palliatus: Comparison with Cercopithecidae and man [in French]. Ann Genet 24:78–81 (1981). Dutrillaux B, Couturier J, Muleris M, Lombard M, Chauvier G: Chromosomal phylogeny of forty-two species or subspecies of Cercopithecoids (Primates Catarrhini). Ann Genet 25:96–109 (1982). Dutrillaux B, Webb G, Muleris M, Couturier J, Butler R: Chromosome study of Presbytis cristatus: Presence of a complex Y-autosome rearrangement in the male. Ann Genet 27: 148–153 (1984). Eckhardt RB: Chromosome evolution in the genus Cercopithecus; in Chiarelli B, Koen AL, Ardito G (eds): Comparative Karyology of Primates, pp 39–46 (Mouton, New York 1979). Ferguson-Smith MA, Yang F, Rens W, O’Brien PC: The impact of chromosome sorting and painting on the comparative analysis of primate genomes. Cytogenet Genome Res 108: 112–121 (2005). Finelli P, Stanyon R, Plesker R, Ferguson-Smith MA, O’Brien PC, Wienberg J: Reciprocal chromosome painting shows that the great difference in diploid number between human and African green monkey is mostly due to non-Robertsonian fissions. Mamm Genome 10:713–718 (1999).


Girirajan S, Chen L, Graves T, Marques-Bonet T, Ventura M, et al: Sequencing human-gibbon breakpoints of synteny reveals mosaic new insertions at rearrangement sites. Genome Res 19:178–190 (2009). Giusto JP, Margulis L: Karyotypic fission theory and the evolution of Old World monkeys and apes. Biosystems 13:267–302 (1981). Groves CP: A Theory of Human and Primate Evolution (Clarendon Press, Oxford 1989). Harris EE, Disotell TR: Nuclear gene trees and the phylogenetic relationships of the mangabeys (Primates: Papionini). Mol Biol Evol 15:892–900 (1998). Harvati K: Dental eruption sequence among colobine primates. Am J Phys Anthropol 112: 69–85 (2000). Israfil H, Zehr SM, Mootnick AR, Ruvolo M, Steiper ME: Unresolved molecular phylogenies of gibbons and siamangs (Family: Hylobatidae) based on mitochondrial, Y-linked, and X-linked loci indicate a rapid Miocene radiation or sudden vicariance event. Mol Phylogenet Evol 58:447–455 (2011). Jauch A, Wienberg J, Stanyon R, Arnold N, Tofanelli S, et al: Reconstruction of genomic rearrangements in great apes and gibbons by chromosome painting. Proc Natl Acad Sci USA 89:8611–8615 (1992). Kehrer-Sawatzki H, Cooper DN: Understanding the recent evolution of the human genome: insights from human-chimpanzee genome comparisons. Hum Mutat 28:99–130 (2007). Koehler U, Arnold N, Wienberg J, Tofanelli S, Stanyon R: Genomic reorganization and disrupted chromosomal synteny in the siamang (Hylobates syndactylus) revealed by fluorescence in situ hybridization. Am J Phys Anthropol 97: 37–47 (1995a). Koehler U, Bigoni F, Wienberg J, Stanyon R: Genomic reorganization in the concolor gibbon (Hylobates concolor) revealed by chromosome painting. Genomics 30: 287–292 (1995b). Locke DP, Archidiacono N, Misceo D, Cardone MF, Deschamps S, et al: Refinement of a chimpanzee pericentric inversion breakpoint to a segmental duplication cluster. Genome Biol 4:R50 (2003). Locke DP, Hillier LW, Warren WC, Worley KC, Nazareth LV, et al: Comparative and demographic analysis of orang-utan genomes. Nature 469:529–533 (2011). Lomiento M, Jiang Z, D’Addabbo P, Eichler EE, Rocchi M: Evolutionary-new centromeres preferentially emerge within gene deserts. Genome Biol 9:R173 (2008). Marshall OJ, Chueh AC, Wong LH, Choo KH: Neocentromeres: new insights into centromere structure, disease development, and karyotype evolution. Am J Hum Genet 82: 261–282 (2008).

Misceo D, Capozzi O, Roberto R, Dell’oglio MP, Rocchi M, et al: Tracking the complex flow of chromosome rearrangements from the Hominoidea ancestor to extant Hylobates and Nomascus gibbons by high-resolution synteny mapping. Genome Res 18: 1530– 1537 (2008). Montefalcone G, Tempesta S, Rocchi M, Archidiacono N: Centromere repositioning. Genome Res 9:1184–1188 (1999). Müller S, Stanyon R, Finelli P, Archidiacono N, Wienberg J: Molecular cytogenetic dissection of human chromosomes 3 and 21 evolution. Proc Natl Acad Sci USA 97: 206–211 (2000). Müller S, Hollatz M, Wienberg J: Chromosomal phylogeny and evolution of gibbons (Hylobatidae). Hum Genet 113:493–501 (2003). Nie W, Liu R, Chen Y, Wang J, Yang F: Mapping chromosomal homologies between humans and two langurs (Semnopithecus francoisi and S. phayrei) by chromosome painting. Chromosome Res 6:447–453 (1998). Nie W, Rens W, Wang J, Yang F: Conserved chromosome segments in Hylobates hoolock revealed by human and H. leucogenys paint probes. Cytogenet Cell Genet 92: 248–253 (2001). Osterholz M, Walter L, Roos C: Phylogenetic position of the langur genera Semnopithecus and Trachypithecus among Asian colobines, and genus affiliations of their species groups. BMC Evol Biol 8: 58 (2008). Page SL, Goodman M: Catarrhine phylogeny: noncoding DNA evidence for a diphyletic origin of the mangabeys and for a humanchimpanzee clade. Mol Phylogenet Evol 18: 14–25 (2001). Peng YZ, Pan RL, Jablonski NG: Classification and evolution of Asian colobines. Folia Primatol (Basel) 60:106–117 (1993). Perelman P, Johnson WE, Roos C, Seuánez HN, Horvath JE, et al: A molecular phylogeny of living primates. PLoS Genet 7:e1001342 (2011). Ponsa M, de Boer LEM, Egozcue J: Banding patterns of the chromosomes of Presbytis cristatus pyrrhus and P. obscurus. Am J Primatol 4:165–169. (1983). Roberto R, Capozzi O, Wilson RK, Mardis ER, Lomiento M, et al: Molecular refinement of gibbon genome rearrangements. Genome Res 17:249–257 (2007). Roberto R, Misceo D, D’Addabbo P, Archidiacono N, Rocchi M: Refinement of macaque synteny arrangement with respect to the official rheMac2 macaque sequence assembly. Chromosome Res 16: 977–985 (2008). Rocchi M, Archidiacono N, Stanyon R: Ancestral genomes reconstruction: an integrated, multi-disciplinary approach is needed. Genome Res 16:1441–1444 (2006). Rocchi M, Archidiacono N, Schempp W, Capozzi O, Stanyon R: Centromere repositioning in mammals. Heredity (Edinb) 108: 59– 67 (2012).


283

Stanyon R, Ardito G, Lamberti L, Bigatti P: The banded karyotypes of Macaca fuscata compared with Cercocebus aterrimus. Folia Primatol (Basel) 41:137–146 (1983). Stanyon R, Fantini C, Camperio-Ciani A, Chiarelli B, Ardito G: Banded karyotypes of 20 Papionini species reveal no necessary correlation with speciation. Am J Primatol 16: 3– 17 (1988). Stanyon R, Camperio-Ciani A, Sineo L, Morescalchi MA: The G-banded chromosomes of the proboscis monkey (Nasalis larvatus) compared with the macaque (Macaca mulatta). Antropologia Contemporanea 15: 101–104 (1992a). Stanyon R, Wienberg J, Romagno D, Bigoni F, Jauch A, Cremer T: Molecular and classical cytogenetic analyses demonstrate an apomorphic reciprocal chromosomal translocation in Gorilla gorilla. Am J Phys Anthropol 88:245–250 (1992b). Stanyon R, Arnold N, Koehler U, Bigoni F, Wienberg J: Chromosomal painting shows that ‘marked chromosomes’ in lesser apes and Old World monkeys are not homologous and evolved by convergence. Cytogenet Cell Genet 68:74–78 (1995). Stanyon R, Bruening R, Stone G, Shearin A, Bigoni F: Reciprocal painting between humans, De Brazza’s and patas monkeys reveals a major bifurcation in the Cercopithecini phylogenetic tree. Cytogenet Genome Res 108:175–182 (2005).

284

Stanyon R, Rocchi M, Capozzi O, Roberto R, Misceo D, et al: Primate chromosome evolution: ancestral karyotypes, marker order and neocentromeres. Chromosome Res 16:17–39 (2008). Sterner KN, Raaum RL, Zhang YP, Stewart CB, Disotell TR: Mitochondrial data support an odd-nosed colobine clade. Mol Phylogenet Evol 40:1–7 (2006). Stock AD, Hsu TC: Evolutionary conservatism in arrangement of genetic material. A comparative analysis of chromosome banding between the rhesus macaque (2n equals 42, 84 arms) and the African green monkey (2n equals 60, 120 arms). Chromosoma 43: 211– 224 (1973). Telenius H, Carter NP, Bebb CE, Nordenskjold M, Ponder BAJ, Tunnacliffe A: Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 13:718–725 (1992). Tosi AJ, Melnick DJ, Disotell TR: Sex chromosome phylogenetics indicate a single transition to terrestriality in the guenons (tribe Cercopithecini). J Hum Evol 46: 223–237 (2004). Ventura M, Mudge JM, Palumbo V, Burn S, Blennow E, et al: Neocentromeres in 15q24-26 map to duplicons which flanked an ancestral centromere in 15q25. Genome Res 13: 2059– 2068 (2003).


Ventura M, Weigl S, Carbone L, Cardone MF, Misceo D, et al: Recurrent sites for new centromere seeding. Genome Res 14: 1696–1703 (2004). Ventura M, Antonacci F, Cardone MF, Stanyon R, D’Addabbo P, et al: Evolutionary formation of new centromeres in macaque. Science 316:243–246 (2007). Wienberg J, Stanyon R: Chromosome painting in mammals as an approach to comparative genomics. Curr Opin Genet Dev 5: 792–797 (1995). Wienberg J, Jauch A, Stanyon R, Cremer T: Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization. Genomics 8:347–350 (1990). Wienberg J, Stanyon R, Jauch A, Cremer T: Homologies in human and Macaca fuscata chromosomes revealed by in situ suppression hybridization with human chromosome specific DNA libraries. Chromosoma 101: 265–270 (1992). Xing J, Wang H, Zhang Y, Ray DA, Tosi AJ, et al: A mobile element-based evolutionary history of guenons (tribe Cercopithecini). BMC Biol 5: 5 (2007). Yunis JJ, Prakash O: The origin of man: a chromosomal pictorial legacy. Science 215:1525– 1530 (1982). Zhang YP, Ryder OA: Mitochondrial cytochrome b gene sequences of Old World monkeys: with special reference on evolution of Asian colobines. Primates 39:39–49 (1998).

Stanyon /Rocchi /Bigoni /Archidiacono

Author Index Vol. 137, No. 2–4, 2012

Archidiacono, N. 273 Beklemisheva, V.R. 174 Bigoni, F. 273 Biltueva, L. 154 Capanna, E. 97 Cernohorska, H. 194 Deakin, J.E. 113 de Oliveira, E.H.C. 259 Dutrillaux, B. 218

Neusser, M. 259 Nie, W. 174, 246 Perelman, P.L. 174 Petrina, T.N. 174 Redi, C.A. 97 Rens, W. 113 Richard, F. 218 Rocchi, M. 273 Romanenko, S.A. 233 Rozhnov, V.V. 174 Rubes, J. 194

Eick, G. 165 Ferguson-Smith, M.A. 83 Graphodatsky, A.S. 83, 174 Graves, J.A.M. 113 Kopecna, O. 194 Kubickova, S. 194 Kulemsina, A.I. 194, 208

Stanyon, R. 83, 144, 273 Svartman, M. 130, 144 Trifonov, V.A. 208 Volleth, M. 165 Volobouev, V. 233 Vorobieva, N. 154 Yudkin, D.V. 174

Müller, S. 259 Musilova, P. 194, 208



285

Author Index Vol. 137, 2012

Archidiacono, N. 273

Isobe, S. 60

Bach, L.H. 7 Beklemisheva, V.R. 174 Bigoni, F. 273 Biltueva, L. 154 Birchler, J.A. 50

Kataoka, R. 60 Kato, S. 60 Kent, M.S. 7 Kopecna, O. 194 Král, J. 22 Kubickova, S. 194 Kula, A. 70 Kulemsina, A.I. 194, 208 Kuzminski, H. 1 Kwolek, D. 70

Capanna, E. 97 Cernohorska, H. 194 Cole, S.A. 7 Dall’Agnol, M. 42 Deakin, J.E. 113 de Oliveira, E.H.C. 259 del Priore, L. 15 Dobosz, S. 1 Dutrillaux, B. 218 Eick, G. 165 Ferguson-Smith, M.A. 83 Ferreira, J. 42 Gandolfi, B. 7 Grabowska-Joachimiak, A. 70 Grahn, J.C. 7 Grahn, R.A. 7 Graphodatsky, A.S. 83, 174 Graves, J.A.M. 113



Sandal, N. 42 Sato, S. 42, 60 Šťáhlavský, F. 22 Stanyon, R. 83, 144, 273 Svartman, M. 130, 144

Lyons, L.A. 7 Marciniuk, P. 70 Masonbrink, R.E. 50 Mendes, S. 42 Millon, L.V. 7 Müller, S. 259 Mullikin, J.C. 7 Musilova, P. 194, 208 Narfstrom, K. 7 Neusser, M. 259 Nie, W. 174, 246

Tabata, S. 60 Trifonov, V.A. 208 Volleth, M. 165 Volobouev, V. 233 Vorobieva, N. 154 Xu, C. 31 Yu, W. 31 Yudkin, D.V. 174 Zhang, L. 31

Ocalewicz, K. 1 Ohmido, N. 60 Pedrosa-Harand, A. 42 Perelman, P.L. 174 Petrina, T.N. 174 Pigozzi, M.I. 15

Haddad, C.R. 22 Hara, M. 60 Harvey, M.S. 22

Redi, C.A. 97 Rens, W. 113 Richard, F. 218 Rocchi, M. 273 Romanenko, S.A. 233 Rozhnov, V.V. 174 Rubes, J. 194

Evolutionary Dynamics of Mammalian Karyotypes: Reprint of:'Cytogenetic and Genome Research 2012, Vol. 137, No. 2-4&#039

Meiosis (Genome Dynamics Vol 5)

Chromosome Aberrations (Reprint of Cytogenetic and Genome Research 2004)

Evolutionary Dynamics of Organizations

137

Microbial Pathogenomics (Genome Dynamics)

Evolutionary games and population dynamics

Evolutionary Games and Population Dynamics

Transposons and the Dynamic Genome (Genome Dynamics and Stability)

Karyotypes of Parasitic Hymenoptera

Karyotypes of Parasitic Hymenoptera

No Copyright 2012

Trends in Genome Research

Genome Research Dec 2010

137 секунд

Aesthetical and Philosophical Essays, Vol. 1 of 2 (Classic Reprint)

Evolutionary Dynamics and Extensive Form Games

Evolutionary Dynamics: Exploring the Equations of Life

Evolutionary Dynamics and Extensive Form Games

Evolutionary Dynamics of Forests Under Climate Change

Advances in Chemical Physics, Vol. 137

Atlas of Mammalian Chromosomes

Atlas of Mammalian Chromosomes

Gas Dynamics Vol.1

Proto-Wintun (University of California Publications Linguistics Vol. 137)

Mammalian Evolutionary Morphology: A Tribute to Frederick S. Szalay

2002 (137)

Evolutionary Biology - Concepts, Biodiversity, Macroevolution and Genome Evolution

Mammalian paleofaunas of the world

Microsoft Dynamics AX 2012 Development Cookbook

Evolutionary Dynamics of Mammalian Karyotypes: Reprint of:&#039;Cytogenetic and Genome Research 2012, Vol. 137, No. 2-4&#039

Meiosis (Genome Dynamics Vol 5)

Chromosome Aberrations (Reprint of Cytogenetic and Genome Research 2004)

Evolutionary Dynamics of Organizations

137

Microbial Pathogenomics (Genome Dynamics)

Evolutionary games and population dynamics

Evolutionary Games and Population Dynamics

Transposons and the Dynamic Genome (Genome Dynamics and Stability)

Karyotypes of Parasitic Hymenoptera

Karyotypes of Parasitic Hymenoptera

No Copyright 2012

Trends in Genome Research

Genome Research Dec 2010

137 секунд

Aesthetical and Philosophical Essays, Vol. 1 of 2 (Classic Reprint)

Evolutionary Dynamics and Extensive Form Games

Evolutionary Dynamics: Exploring the Equations of Life

Evolutionary Dynamics and Extensive Form Games

Evolutionary Dynamics of Forests Under Climate Change

Advances in Chemical Physics, Vol. 137

Atlas of Mammalian Chromosomes

Atlas of Mammalian Chromosomes

Gas Dynamics Vol.1

Proto-Wintun (University of California Publications Linguistics Vol. 137)

Mammalian Evolutionary Morphology: A Tribute to Frederick S. Szalay

2002 (137)

Evolutionary Biology - Concepts, Biodiversity, Macroevolution and Genome Evolution

Mammalian paleofaunas of the world

Microsoft Dynamics AX 2012 Development Cookbook

Recommend Documents

Evolutionary Dynamics of Mammalian Karyotypes: Reprint of:'Cytogenetic and Genome Research 2012, Vol. 137, No. 2-4&#039