B Chromosomes In The Eukaryote Genome (Cytogenetic & Genome Research)

Single topic volume B Chromosomes in the Eukaryote Genome Editor Juan Pedro M. Camacho, Granada 107 figures, 17 in c...

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Single topic volume

B Chromosomes in the Eukaryote Genome

Editor

Juan Pedro M. Camacho, Granada

107 figures, 17 in color, and 77 tables, 2004

Basel • Freiburg • Paris • London • New York • Bangalore • Bangkok • Singapore • Tokyo • Sydney

Cover illustration DAPI-stained primary spermatocyte of the grasshopper Eyprepocnemis plorans, showing the standard A chromosomes (11 bivalents plus the X univalent) and a brightly stained small B chromosome (on the left).

S. Karger Medical and Scientific Publishers Basel • Freiburg • Paris • London New York • Bangalore • Bangkok Singapore • Tokyo • Sydney

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

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.

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 2004 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel

Vol. 106, No. 2–4, 2004

Contents

215 B chromosomes in Crustacea Decapoda Coluccia E, Cannas R, Cau A, Deiana AM, Salvadori S

147 Preface Camacho JP

222 Current knowledge on B chromosomes in natural

A Valuable Tool 149 The B chromosome database Jones RN, Díez M 151 The distribution of B chromosomes across species Palestis BG, Trivers R, Burt A, Jones RN

What Are B Chromosomes? 159 Are the dot-like chromosomes in Trinomys iheringi

(Rodentia, Echimyidae) B chromosomes? Fagundes V, Camacho JPM, Yonenaga-Yassuda Y

populations of helminth parasites: a review Špakulová M, Casanova JC 230 B chromosomes in the fish Astyanax scabripinnis

(Characidae, Tetragonopterinae): An overview in natural populations Moreira-Filho O, Galetti Jr. PM, Bertollo LAC 235 Structure and evolution of B chromosomes in

amphibians Green DM 243 Occurrence of B chromosomes in lizards: a review Bertolotto CEV, Pellegrino KCM, Yonenaga-Yassuda Y

165 Human supernumeraries: are they B chromosomes? Fuster C, Rigola MA, Egozcue J

247 B chromosomes in populations of mammals Vujošević M, Blagojević J

173 Is the aneuploid chromosome in an apomictic

257 B chromosomes in Brazilian rodents Silva MJJ, Yonenaga-Yassuda, Y

Boechera holboellii a genuine B chromosome? Sharbel TF, Voigt M-L, Mitchell-Olds T, Kantama L, de Jong H

264 The mammalian model for population studies of

B chromosomes: the wood mouse (Apodemus) Report of New B Chromosomes 184 The occurrence of different Bs in Cestrum intermedium

and C. strigilatum (Solanaceae) evidenced by chromosome banding

Wójcik JM, Wójcik AM, Macholán M, Piálek J, Zima J 271 A complex B chromosome system in the Korean field

mouse, Apodemus peninsulae Kartavtseva IV, Roslik GV

Fregonezi JN, Rocha C, Torezan JMD, Vanzela ALL 189 Distribution and stability of supernumerary

microchromosomes in natural populations of the Amazon molly, Poecilia formosa Lamatsch DK, Nanda I, Schlupp I, Epplen JT, Schmid M, Schartl M 195 B chromosomes in Amazonian cichlid species Feldberg E, Porto JIR, Alves-Brinn MN, Mendonça MNC, Benzaquem DC

Review on B Chromosomes 199 The B chromosomes in Brachycome Leach CR, Houben A, Timmis JN 210 B chromosomes in Sternorrhyncha (Hemiptera, Insecta) Maryañska-Nadachowska A


Structure and Origin of B Chromosomes 279 A RAPD marker associated with B chromosomes in

Partamona helleri (Hymenoptera, Apidae) Tosta VC, Fernandes-Salomão TM, Tavares MG, Pompolo SG, Barros EG, Campos LAO 284 Comparative FISH analysis of distribution of B

chromosome repetitive DNA in A and B chromosomes in two subspecies of Podisma sapporensis (Orthoptera, Acrididae) Bugrov AG, Karamysheva TV, Rubtsov DN, Andreenkova OV, Rubtsov NB

Population Dynamics and Evolution of B Chromosomes

289 Comparative analysis of micro and macro

B chromosomes in the Korean field mouse Apodemus peninsulae (Rodentia, Murinae) performed by chromosome microdissection and FISH

351 Cytogeography and the evolutionary significance of

Rubtsov NB, Karamysheva TV, Andreenkova OV, Bochkaerev MN, Kartavtseva IV, Roslik GV, Borissov YM

B chromosomes in relation to inverted rearrangements in a grasshopper species Colombo P, Confalonieri V 359 Mitotically unstable B chromosome polymorphism in

295 FISH detection of ribosomal cistrons and

the grasshopper Dichroplus elongatus

assortment-distortion for X and B chromosomes in Dichroplus pratensis (Acrididae)

Remis MI, Vilardi JC

Bidau CJ, Rosato M, Martí DA

365 Geographic and seasonal variations of the number of

B chromosomes and external morphology in Psathyropus tenuipes (Arachnida: Opiliones)

302 X and B chromosomes display similar meiotic

characteristics in male grasshoppers

Tsurusaki N, Shimada T

Viera A, Calvente A, Page J, Parra MT, Gómez R, Suja JA, Rufas JS, Santos JL

376 Spatio-temporal dynamics of a neutralized

size for the maize B chromosome

B chromosome in the grasshopper Eyprepocnemis plorans

Phelps-Durr TL, Birchler JA

Perfectti F, Pita M, de la Vega CG, Gosálvez J, Camacho JPM

309 An asymptotic determination of minimum centromere

386 The parasitic effects of rye B chromosomes might be

beneficial in the long term

Effects of B Chromosomes on the A Genome

González-Sánchez M, Chiavarino M, Jiménez G, Manzanero S, Rosato M, Puertas MJ

314 B chromosomes in hybrids of temperate cereals and

grasses Jenkins G, Jones RN

Integration of B Chromosomes into the A Genome

320 Different numbers of rye B chromosomes induce 394 Interaction of B chromosomes with A or

identical compaction changes in distinct A chromosome domains

B chromosomes in segregation in insects Nokkala S, Nokkala C

Delgado M, Caperta A, Ribeiro T, Viegas W, Jones RN, Morais-Cecílio L

398 Imitate to integrate: Reviewing the pathway for

325 The odd-even effect in mitotically unstable

B chromosomes in grasshoppers

B chromosome integration in Trypoxylon (Trypargilum) albitarse (Hymenoptera, Sphecidae)

Camacho JPM, Perfectti F, Teruel M, López-León MD, Cabrero J

Rocha-Sanchez SMS, Pompolo SG 402 B chromosomes: the troubles of integration Granado N, Rebollo E, Sánchez FJ, Arana P

Transmission of B Chromosomes 332 The B chromosome polymorphism of the grasshopper

Eyprepocnemis plorans in North Africa. IV. Transmission of rare B chromosome variants Bakkali M, Camacho JPM

411 Author Index Vol. 106, No. 2–4, 2004 412 Author Index Vol. 106, 2004 after 412 Contents Vol. 106, 2004

338 Rapid suppression of drive for a parasitic

B chromosome Perfectti F, Corral JM, Mesa JA, Cabrero J, Bakkali M, López-León MD, Camacho JPM 344 Transmission analysis of B chromosomes in

Rattus rattus from Northern Africa Stitou S, Zurita F, Díaz de la Guardia R, Jiménez R, Burgos M 347 B chromosomes and Robertsonian fusions of

Dichroplus pratensis (Acrididae): intraspecific support for the centromeric drive theory Bidau CJ, Martí DA

146

Contents

Contents Vol. 106, 2004

82 A comparative karyological study of the blue-breasted quail

No. 1

(Coturnix chinensis, Phasianidae) and California quail (Callipepla californica, Odontophoridae)

Abstracts 1 16th European Colloquium on Animal Cytogenetics and Gene

Mapping National Institute for Agronomic Research (INRA) Jouy-en-Josas, France, July 6–8, 2004 25 Satellite Meeting: 40th anniversary of the discovery of the

t(1;29) in cattle National Institute for Agronomic Research (INRA) Jouy-en-Josas, France, July 9, 2004

Shibusawa M, Nishida-Umehara C, Tsudzuki M, Masabanda J, Griffin DK, Matsuda Y

91 Cloning and characterization of the mouse Arht2 gene which

encodes a putative atypical GTPase Shan Y, Hexige S, Guo Z, Wan B, Chen K, Chen X, Ma L, Huang C, Zhao S, Yu L

98 Molecular characterization of porcine hyaluronidase genes 1,

2, and 3 clustered on SSC13q21 Gatphayak K, Knorr C, Beck J, Brenig B

107 Gene mapping of 5S rDNA sites in eight fish species from the

Paraíba do Sul river basin, Brazil

Original Articles

Kavalco KF, Pazza R, Bertollo LAC, Moreira-Filho O

28 Analysis of the cytogenetic stability of the human embryonal

kidney cell line 293 by cytogenetic and STR profiling approaches Bylund L, Kytölä S, Lui W-O, Larsson C, Weber G

33 Retained heterodisomy for chromosome 12 in atypical

lipomatous tumors: implications for ring chromosome formation Mertens F, Panagopoulos I, Jonson T, Gisselsson D, Isaksson M, Domanski HA, Mandahl N

39 The effect of cold storage on recombination frequencies in

human male testicular cells Sun F, Trpkov K, Rademaker A, Ko E, Barclay L, Mikhaail-Philips M, Martin RH

43 The most common chromosome aberration detected by

high-resolution comparative genomic hybridization in vulvar intraepithelial neoplasia is not seen in vulvar squamous cell carcinoma Bryndorf T, Kirchhoff M, Larsen J, Andreasson B, Bjerregaard B, Westh H, Rose H, Lundsteen C

49 Evolution of unbalanced gain of distal chromosome 2p in

neuroblastoma Stallings RL, Carty P, McArdle L, Mullarkey M, McDermott M, O’Meara A, Ryan E, Catchpoole D, Breatnach F

55 Mosaicism for an ectopic NOR at 8pter and a complex

rearrangement of chromosome 8 in a patient with severe psychomotor retardation Felbor U, Knötgen N, Schams G, Buwe A, Steinlein C, Schmid M

111 Karyotypic evolution in the Galliformes: An examination of the

process of karyotypic evolution by comparison of the molecular cytogenetic findings with the molecular phylogeny Shibusawa M, Nishibori M, Nishida-Umehara C, Tsudzuki M, Masabanda J, Griffin DK, Matsuda Y

Abstracts 120 38th Biennial American Cytogenetics Conference

April 22–25, 2004 Skamania Lodge, Stevenson, Washington Brief Gene Mapping Reports – Internet Publication 142 A Assignment of two isoforms of the AMP-activated protein

kinase ␥ subunits, PRKAG1 and PRKAG2 to porcine chromosomes 5 and 18, respectively by radiation hybrid panel mapping Haberkern G, Regenhard P, Ottzen-Schirakow G, Kalm E, Looft C

142 B Assignment of the ovine uroporphyrinogen decarboxylase

(UROD) gene to chromosome 1p34tp36 by fluorescence in situ hybridization Nezamzadeh R, Habermann J, Fries R, Brenig B

142 C Assignment of the surfactant protein A gene (SFTPA) to

bovine chromosome 28q1.8tq1.9 by radiation hybrid mapping Gjerstorff M, Dueholm B, Bendixen C, Holmskov U, Hansen S

142 D Physical mapping and marker development for the porcine

Gene Mapping, Cloning and Sequencing

glial cells missing homolog 1 (Drosophila) (GCM1) gene Spötter A, Drögemüller C, Kuiper H, Hamann H, Distl O

61 Cloning and characterization of an inversion breakpoint at

6q23.3 suggests a role for Map7 in sacral dysgenesis

142 E Mapping of three porcine 20S proteasome genes using the

IMpRH panel

Sood R, Bader PI, Speer MC, Edwards YH, Eddings EM, Blair RT, Hu P, Faruque MU, Robbins CM, Zhang H, Leuders J, Morrison K, Thompson D, Schwartzberg PL, Meltzer PS, Trent JM

Wu X, Yu M, Liu B, Yerle M, Zhao SH, Wang YF, Fan B, Li K

Animal Cytogenetics and Comparative Mapping

No. 2–4

68 Isolation and characterization of the Xenopus laevis orthologs

of the human papillary renal cell carcinoma-associated genes PRCC and MAD2L2 (MAD2B)

147 Preface Camacho JP

van den Hurk WH, Martens GJM, Geurts van Kessel A, van Groningen JJM

74 Identifying differentially expressed genes in the mammalian

retina and the retinal pigment epithelium by suppression subtractive hybridization Schulz HL, Rahman FA, Fadl El Moula FM, Stojic J, Gehrig A, Weber BHF

A Valuable Tool 149 The B chromosome database Jones RN, Díez M 151 The distribution of B chromosomes across species Palestis BG, Trivers R, Burt A, Jones RN


© 2004 S. Karger AG, Basel

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

What Are B Chromosomes?

302 X and B chromosomes display similar meiotic characteristics

in male grasshoppers 159 Are the dot-like chromosomes in Trinomys iheringi (Rodentia,

Echimyidae) B chromosomes? Fagundes V, Camacho JPM, Yonenaga-Yassuda Y

165 Human supernumeraries: are they B chromosomes? Fuster C, Rigola MA, Egozcue J 173 Is the aneuploid chromosome in an apomictic Boechera

holboellii a genuine B chromosome? Sharbel TF, Voigt M-L, Mitchell-Olds T, Kantama L, de Jong H

Report of New B Chromosomes

Viera A, Calvente A, Page J, Parra MT, Gómez R, Suja JA, Rufas JS, Santos JL

309 An asymptotic determination of minimum centromere size for

the maize B chromosome Phelps-Durr TL, Birchler JA

Effects of B Chromosomes on the A Genome 314 B chromosomes in hybrids of temperate cereals and grasses Jenkins G, Jones RN 320 Different numbers of rye B chromosomes induce identical

184 The occurrence of different Bs in Cestrum intermedium and

C. strigilatum (Solanaceae) evidenced by chromosome banding Fregonezi JN, Rocha C, Torezan JMD, Vanzela ALL

189 Distribution and stability of supernumerary

microchromosomes in natural populations of the Amazon molly, Poecilia formosa

compaction changes in distinct. A chromosome domains Delgado M, Caperta A, Ribeiro T, Viegas W, Jones RN, Morais-Cecílio L

325 The odd-even effect in mitotically unstable

B chromosomes in grasshoppers Camacho JPM, Perfectti F, Teruel M, López-León MD, Cabrero J

Transmission of B Chromosomes

Lamatsch DK, Nanda I, Schlupp I, Epplen JT, Schmid M, Schartl M

195 B chromosomes in Amazonian cichlid species Feldberg E, Porto JIR, Alves-Brinn MN, Mendonça MNC, Benzaquem DC

Review on B Chromosomes 199 The B chromosomes in Brachycome Leach CR, Houben A, Timmis JN 210 B chromosomes in Sternorrhyncha (Hemiptera, Insecta) Maryañska-Nadachowska A 215 B chromosomes in Crustacea Decapoda Coluccia E, Cannas R, Cau A, Deiana AM, Salvadori S 222 Current knowledge on B chromosomes in natural populations

of helminth parasites: a review Špakulová M, Casanova JC 230 B chromosomes in the fish Astyanax scabripinnis (Characidae,

332 The B chromosome polymorphism of the grasshopper

Eyprepocnemis plorans in North Africa. IV. Transmission of rare B chromosome variants Bakkali M, Camacho JPM

338 Rapid suppression of drive for a parasitic B chromosome Perfectti F, Corral JM, Mesa JA, Cabrero J, Bakkali M, López-León MD, Camacho JPM 344 Transmission analysis of B chromosomes in Rattus rattus from

Northern Africa Stitou S, Zurita F, Díaz de la Guardia R, Jiménez R, Burgos M

347 B chromosomes and Robertsonian fusions of Dichroplus

pratensis (Acrididae): intraspecific support for the centromeric drive theory Bidau CJ, Martí DA

Population Dynamics and Evolution of B Chromosomes

Tetragonopterinae): An overview in natural populations Moreira-Filho O, Galetti Jr. PM, Bertollo LAC

235 Structure and evolution of B chromosomes in amphibians Green DM 243 Occurrence of B chromosomes in lizards: a review Bertolotto CEV, Pellegrino KCM, Yonenaga-Yassuda Y 247 B chromosomes in populations of mammals Vujošević M, Blagojević J 257 B chromosomes in Brazilian rodents Silva MJJ, Yonenaga-Yassuda, Y 264 The mammalian model for population studies of

B chromosomes: the wood mouse (Apodemus) Wójcik JM, Wójcik AM, Macholán M, Piálek J, Zima J

271 A complex B chromosome system in the Korean field mouse,

Apodemus peninsulae Kartavtseva IV, Roslik GV

Structure and Origin of B Chromosomes 279 A RAPD marker associated with B chromosomes in Partamona

helleri (Hymenoptera, Apidae) Tosta VC, Fernandes-Salomão TM, Tavares MG, Pompolo SG, Barros EG, Campos LAO

284 Comparative FISH analysis of distribution of B chromosome

repetitive DNA in A and B chromosomes in two subspecies of Podisma sapporensis (Orthoptera, Acrididae) Bugrov AG, Karamysheva TV, Rubtsov DN, Andreenkova OV, Rubtsov NB

289 Comparative analysis of micro and macro B chromosomes in

the Korean field mouse Apodemus peninsulae (Rodentia, Murinae) performed by chromosome microdissection and FISH Rubtsov NB, Karamysheva TV, Andreenkova OV, Bochkaerev MN, Kartavtseva IV, Roslik GV, Borissov YM

295 FISH detection of ribosomal cistrons and

351 Cytogeography and the evolutionary significance of

B chromosomes in relation to inverted rearrangements in a grasshopper species Colombo P, Confalonieri V

359 Mitotically unstable B chromosome polymorphism in the

grasshopper Dichroplus elongatus Remis MI, Vilardi JC

365 Geographic and seasonal variations of the number of

B chromosomes and external morphology in Psathyropus tenuipes (Arachnida: Opiliones) Tsurusaki N, Shimada T

376 Spatio-temporal dynamics of a neutralized B chromosome in

the grasshopper Eyprepocnemis plorans Perfectti F, Pita M, de la Vega CG, Gosálvez J, Camacho JPM

386 The parasitic effects of rye B chromosomes might be

beneficial in the long term González-Sánchez M, Chiavarino M, Jiménez G, Manzanero S, Rosato M, Puertas MJ

Integration of B Chromosomes into the A Genome 394 Interaction of B chromosomes with A or B chromosomes in

segregation in insects Nokkala S, Nokkala C

398 Imitate to integrate: Reviewing the pathway for

B chromosome integration in Trypoxylon (Trypargilum) albitarse (Hymenoptera, Sphecidae) Rocha-Sanchez SMS, Pompolo SG

402 B chromosomes: the troubles of integration Granado N, Rebollo E, Sánchez FJ, Arana P 411 Author Index Vol. 106, No. 2–4, 2004 412 Author Index Vol. 106, 2004

assortment-distortion for X and B chromosomes in Dichroplus pratensis (Acrididae) Bidau CJ, Rosato M, Martí DA

IV

Cytogenet Genome Res Vol. 106, 2004

Contents

Cytogenet Genome Res 106:147–148 (2004)

Preface

Subsequently to the ascription of genetic inheritance to chromosomes, the presence of additional passengers in the karyotype of a hemipteran insect was detected (Wilson, 1906); supernumerary chromosomes, also called accessory or B chromosomes in order to distinguish them from the standard A chromosomes, had been discovered. It was Östergren (1945) who first considered B chromosomes as parasitic elements, but the scientific community was reluctant to accept this view until the emergence of the selfish DNA theory (Doolittle and Sapienza, 1980; Orgel and Crick, 1980). In the light of this theory the interpretation of B chromosomes had a remarkable advance, opening new approaches to their study and understanding. B chromosomes have been reported in most eukaryote taxa, with the remarkable exception of birds, where only one species, the zebra finch Taeniopygia guttatta, has been reported to carry a single accessory chromosome restricted to the germ line of both sexes (Pigozzi and Solari, 1998). The last compilation of B chromosomes in eukaryotes was done in the classical book authored by Jones and Rees (1982), which has since been the main reference and inspiration for researchers. In these last 22 years, many new findings have contributed to building an increasing body of knowledge of most aspects of B chromosomes, ranging from origin and molecular nature to population dynamics and long term evolution. The field was demanding an update of these new data. The present single topic issue of Cytogenetic and Genome Research tries to cover this need. It contains 40 contributions by colleagues from 19 countries in most continents, and provides both reviews and new original data on many aspects of B chromosomes. This issue begins with a B chromosome database designed by R.N. Jones and M. Dıéz which reviews all the literature on B chromosomes up to 1994, and is here first made available to the scientific community. The usefulness of this resource is illustrated by the comparative analysis of Palestis and coworkers on the presence of B chromosomes across species. The second section includes articles trying to delimit what is and what is not a B chromosome. It includes cases of dot-like chromosomes in

ABC

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

© 2004 S. Karger AG, Basel

rodents, human supernumerary chromosomes, and aneuploid chromosomes associated with apomictic reproduction in a plant. The third section contains articles providing new discoveries of B chromosomes in plants and fish, whereas the fourth includes reviews on the plant Brachycome, Sternorrhyncha hemipterans, decapodan crustaceans, helminth parasites, the fish Astyanax scabripinnis, amphibians, lizards and mammals. The fifth section is devoted to the structure, composition and origin of B chromosomes, focusing on the isolation of a RAPD marker associated with B chromosomes in a bee, FISH analyses of the distribution of repetitive DNAs in A and B chromosomes of a grasshopper and the Korean field mouse, FISH detection of ribosomal cistrons and X–B assortment distortion in the grasshopper Dichroplus pratensis, and the molecular estimation of the smallest functional maize B centromere. The sixth section contains reports on several kinds of effects of B chromosomes on the host genome, such as meiotic behaviour in inter-generic and inter-specific plant hybrids, rye A chromosome organization with respect to rDNA and satellite DNA, and the odd-even effect in mitotically unstable B chromosomes in grasshoppers. The seventh section reports on the transmission of B chromosomes. It includes articles on the transmission rates of rare B chromosome variants in the grasshopper E. plorans, rapid drive suppression of B chromosomes in the same species, the parasitic nature of B chromosomes in the black rat, and the first intraspecific support for a negative association between B chromosomes and Robertsonian fusions as predicted by the theory of centromeric drive (see Palestis et al., 2004). The eighth section contains articles dealing with several aspects of population dynamics and evolution of B chromosomes in grasshoppers, an arachnid species and maize. The last section includes three contributions with total or partial focus on the possibility of an integration of B chromosomes into the A genome of insects. The present volume on B chromosomes is by no means complete, since it was not possible to include all the data found in many other species. Nevertheless, I hope it will provide new

Accessible online at: www.karger.com/cgr

impulses for studies on B chromosomes and contribute to disentangling the biological meaning of these mysterious components of many eukaryotic genomes. I am very thankful to all authors and coauthors for having prepared their excellent manuscripts within a very short period of time and with relentless enthusiasm, and to all expert reviewers for their excellent assistance. Furthermore, I wish to express my gratitude to the staff of the American and European Editorial Offices of Cytogenetic and Genome Research for their support during the preparation of this single topic issue on B chromosomes. Juan Pedro M. Camacho Granada, June 2004

148 6


References Doolittle WF, Sapienza C: Selfish genes, the phenotype paradigm and genome evolution. Nature 284:601–603 (1980). Jones RN, Rees H: B chromosomes (Academic Press, New York 1982). Orgel LE, Crick FH: Selfish DNA: the ultimate parasite. Nature 284:604–607 (1980). Östergren G: Parasitic nature of extra fragment chromosomes. Bot Notiser 2:157–163 (1945). Palestis BG, Burt A, Jones RN, Trivers R: B chromosomes are more frequent in mammals with acrocentric karyotypes: support for the theory of centromeric drive. Proc R Soc London B (Suppl):S22–S24 (2004). Pigozzi MI, Solari AJ: Germ cell restriction and regular transmission of an accessory chromosome that mimics a sex body in the zebra finch, Taeniopygia guttata. Chromosome Res 6:105–113 (1998). Wilson EB: Studies on chromosomes. V. The chromosomes of Metapodius. A contribution to the hypothesis of genetic continuity of chromosomes. J Exp Zool 6:147–205 (1906).

A Valuable Tool Cytogenet Genome Res 106:149–150 (2004) DOI: 10.1159/000079280

The B chromosome database R.N. Jonesa and M. Dıézb a The

University of Wales Aberystwyth, Institute of Biological Sciences, Aberystwyth, Wales (UK); de Genética, Facultad de Biologıá, Universidad Complutense, Madrid (Spain)

b Departamento

Abstract. The database is compiled from the world literature on B chromosomes published between 1906 and 1994, and has 3,484 records. A brief description is given of the history and structure of the database, which runs in Microsoft ACCESS.

Background and context The idea to build a B chromosome database (DB) originated in 1988 during the early stages of a research collaboration between RNJ and Maria Puertas of the Complutense University of Madrid, and more than a year was spent over discussions on how to construct the DB to maximize its usefulness. The literature, in the form of original papers and photocopies, had been in the process of collection by RNJ since 1964, and this resource was available. Manuel Dıéz undertook to deal with the software, and the initial DB used a Spanish version of dBASE III. This was later upgraded to dBASE IV, and then finally transferred to ACCESS 2000 in 2001. The literature collection was maintained, and data gradually entered into the records by RNJ up to 1994 when other duties led to a suspension of the project. The DB therefore covers the period of literature from 1906, when Bs were first discovered, up to 1994. As far as is known all papers which deal with Bs, or make reference to Bs, during this period are included. The early papers were found by systematically “ploughing” through back numbers of journals and following all references until the story was exhausted. There are 3,484 records, and a record is built around a species – so some papers may lead to more than one record if several species are mentioned; and likewise a species may appear in more than one record where several publications are involved. There are fifteen fields, listed below, which includes two keyword fields, a subject field and a memo field for writing more detailed notes (only a few of these).

A downloadable version is available at http://www.bchromosomes.org/bdb/. Copyright © 2004 S. Karger AG, Basel

Any combination of any of the fields can be accessed, so the complete bibliography can be printed out as a full list of all references, or a complete list of species, or species from one family, or papers by one author, and so on. Keywords, which include a short string of words, can be searched. The subject field can also be searched, for all papers which deal with meiosis, for example, or heterochromatic Bs, or fertility, or structure, or populations, or whatever.

Structure of the records The database contains the fields: – AUTHORS – YEAR – TITLE – JOU – VOL – PA – SPECIES – Animal +/– – VARIETY – CHROMOSOME – No. – Bs – No. – PHYLUM – FAMILY – SUBJECT words – MEMO fields (a few entries with detailed notes) – KEYWORDS I a–n – KEYWORDS II o–z

Keywords Received 29 October 2003; manuscript accepted 17 February 2004. Request reprints from Neil Jones, University of Wales Aberystwyth Institute of Biological Sciences, Aberystwyth SY23 3DD, Wales (UK) telephone: +44 1970 622230; fax: +44 1970 622307; e-mail: [email protected]

ABC


© 2004 S. Karger AG, Basel 0301–0171/04/1064–0149$21.00/0

Keywords are divided into two fields, splitting the alphabet, to reduce search time. Each keyword is followed by a short string of words giving some information about the area covered


by the word. A specified list of keywords is used, listed below, and the meaning of keyword words is provided with the list. Additional keywords can be added provided these are included in the list. Keywords I (a–n) Information on acro additionlines androgenetic banding callus cellsize chromocentres “cuckoo” cycles DNAamount endonucleases fertility flowering fold-back germination holocentric hybrids imprinting inbredlines inter iso lampbrush loss majorgen meiosis meta methylation microcloning midget mitostab model molecular norBs nuclearphen

acrocentric chromosomes refers to Bs of rye as addition lines in hexaploid wheat Bs in plants regenerated from anther culture (e.g. Crepis) species with information on C and G-banding Bs in plant callus cultures cases where Bs affect cell size cases where Bs form condensed chromocentres selfish alien addition chromosomes in wheat (not Bs) mitotic and meiotic cycles times DNA values involving Bs endonuclease which destroy lagging Bs Bs effects on fertility in plants and animals flowering time in plants synaptonemal complex studies on fold-back pairing of Bs seed germination in plants Bs with diffuse centromeres Bs in intergeneric and inter-specific hybrids Bs involved in imprinting effects Bs in inbred lines translocations involving Bs Bs as isochromosomes Bs as lampbrush chromosomes loss of knobbed segments, as in maize Bs with major genes behaviour and influence of Bs at meiosis metacentric Bs Bs and methylation microcloning of Bs midget chromosome of rye (not Bs) mitotic stability/instability models of transmission molecular analysis data Bs with NORs influence of Bs on nuclear phenotype

Keywords II (o–z) occur oddeven origin parasitic phenofit polyB polyteny pops recomb regenerants replitrans review size spreading structure submeta subterm synaptonemal telo telomere transinher transposable woody

150 8

data on the occurrence in individuals and populations cases of the odd/even effect information/theories on the origin of Bs cases of parasitic Bs effects on phenotype and fitness polymorphic forms of the Bs within a species species with polytene Bs data on Bs in populations effects on recombination in A chromosomes regenerated plants with Bs data on replication and transcription review papers on Bs data on the size of Bs (and relative to As) Bs and surface spreading of meiocytes information on the structure of Bs submetacentric Bs subterminal Bs synaptonemal complex data telocentric Bs Bs and telomeres data on transmission and inheritance Bs and transposons Bs in woody plants (trees)


Subject words In addition to keywords there are a number of chosen subject words which indicate the main areas which are dealt with in a publication. It is useful to find all of the papers which deal with a particular main topic, e.g. recombination, or structure. There is some overlap with keywords. Subject word

Information on

meiosis heterochrom euchrom transinher poly-B mitostab AB-inter structure norBs cycles replitrans recomb hybrids majorgen parasitic phenofit fertility flowering germinat nuclearphen oddeven population origin discussion review

meiosis heterochromatin euchromatin transmission and inheritance polymorphic forms of Bs mitotic stability/instability A-B translocations structure of Bs Bs with NORs mitotic and meiotic cycle times replication and transcription recombination Bs in intergeneric and inter-specific hybrids major genes parasitic Bs phenotype and fitness fertility effects flowering time in plants seed germination in plants effects on the nuclear phenotype the odd/even effect populations origin papers with good discussion sections review papers on Bs

Working with the database The database is built in Microsoft ACCESS, and it is necessary to have this program installed in your computer in order to use it. It works in any version or language. Discussions are in progress, for the future prospects, to set up the DB online and make it interactive. Ideally it can then be brought up to date and kept current.

A Valuable Tool Cytogenet Genome Res 106:151–158 (2004) DOI: 10.1159/000079281

The distribution of B chromosomes across species B.G. Palestis,a R. Trivers,b A. Burt,c and R.N. Jonesd a Department

of Biological Sciences, Wagner College, Staten Island, NY (USA); of Anthropology, Rutgers University, New Brunswick, NJ (USA); c Department of Biology, Imperial College, Silwood Park, Ascot, Berkshire (UK); d Institute of Biological Sciences, University of Wales Aberystwyth, Ceredigion (UK) b Department

Abstract. In this review we look at the broad picture of how B chromosomes are distributed across a wide range of species. We review recent studies of the factors associated with the presence of Bs across species, and provide new analyses with updated data and additional variables. The major obstacle facing comparative studies of B chromosome distribution is variation among species in the intensity of cytogenetic study. Because Bs are, by definition, not present in all individuals of a species, they may often be overlooked in species that are rarely studied. We give examples of corrections for differences in study effort, and show that after a variety of such corrections, strong correlations remain. Several major biological factors are associated with the presence of B chromosomes. Among flowering plants, Bs are more likely to occur in outcrossing than in inbred species, and their presence is also positively correlated with genome size and negatively with chromosome number.

They are no more frequent in polyploids than in diploids, nor in species with multiple ploidies. Among mammals, Bs are more likely to occur in species with karyotypes consisting of mostly acrocentric chromosomes. We find no evidence for an association with chromosome number or genome size in mammals, although the sample for genome size is small. The associations with breeding system and acrocentric chromosomes were both predicted in advance, but those with genome size and chromosome number were discovered empirically and we can offer only tentative explanations for the very strong associations we have uncovered. Our understanding of why B chromosomes are present in some species and absent in others is still in its infancy, and we suggest several potential avenues for future research.

B chromosome research is presently focused on two main areas of investigation, molecular organization and transmission genotypes (for review see Camacho et al., 2000; Puertas, 2002; Jones and Houben, 2003). Interest is centered around the idea of host-parasite interaction between selfish Bs and the host genome, and on the origin and evolution of Bs, especially as

analyzed at the molecular level. The earlier phases of work dealt more with the occurrence of Bs in various species, modes of inheritance, effects and ecological and adaptive significance in populations (Jones and Rees, 1982). This extensive phase of research, covering many species and many decades, provided the base of knowledge about B chromosomes, and the platform on which the more recent transmission genetics and molecular studies are now being built. With rare exceptions, attempts to find an adaptive value for Bs at the level of the individual ran into virtual dead-ends and attention was redirected toward two areas. One was the co-evolution of the host-parasite relationship itself and the other was a description of sequence organization on Bs. We have also reached the point now where most studies involve only a handful of species, and for the rest we are leaving behind many unanswered questions. Despite the vast body of knowledge

Supported by the Biosocial Research Foundation. Received 3 October 2004; manuscript accepted 20 January 2004. Request reprints from: Dr. Brian G. Palestis Department of Biological Sciences, Wagner College Staten Island, NY 10301 (USA) telephone: +1-718-390-3237; fax: +1-718-420-4172 e-mail: [email protected]

ABC


© 2004 S. Karger AG, Basel 0301–0171/04/1064–0151$21.00/0

Copyright © 2004 S. Karger AG, Basel


which we now have on Bs within species, we have as yet hardly touched on the question of what factors determine the distribution of B chromosomes across different species. Why do some species and groups of species carry Bs while others do not? Is there some innate property of a genome, or a breeding system, or a taxonomic group, or a karyotype, for instance, which determines whether a species is likely to carry Bs or not? Here we review what is known about the distribution of Bs across species, with special attention to correcting for variation in the intensity with which groups are studied cytogenetically. In addition to reviewing the available studies – which suggest that major genetic and social variables are associated with the distribution of Bs, we also present new analyses using updated data and incorporating additional variables. The questions asked here are appropriate for all classes of selfish genetic elements, which are often maintained despite phenotypic costs by transmission at higher than Mendelian frequencies, but Bs are particularly well suited to answer them. Being so easily visible under the microscope, they have been studied for nearly a century and are known for a large number of species (N F 2000) across a broad range of taxonomic groups (Jones and Rees, 1982; Camacho et al., 2000). The major problem confronting any study of the frequency of B chromosomes across species is that, by definition, they are not present in all individuals of a species. Nor are they always present in all populations – nor all tissues within an individual, e.g., root cells, themselves often used for karyotypic work (Chen et al., 1993) or stems and leaves (Wu, 1992). Due to this variability in B presence among populations, individuals, and tissues, and also due in part to differences among taxa in the ease of chromosomal study, we do not know that a species with no reports of Bs truly lacks them. Bs are especially likely to be overlooked if a karyotype is based on a single individual, which was once true of 17,000 plant species (Darlington and Wylie, 1956). Study intensity (and ease of cytogenetic study) likely also contributes to the apparent distribution of Bs across taxa, as Bs are relatively common in grasses (Gramineae = Poaceae), lilies and allied taxa (Lilianae), and grasshoppers (Orthoptera), all of which have been subject to intensive cytogenetic study (Jones and Rees, 1982; Camacho et al., 2000; Camacho, 2004). In other taxa, such as fungi (Covert, 1998), the identification of Bs depends on the use of recently developed techniques, and thus Bs are known in only a small number of species.

Study intensity There is no simple, single cure for the problem of variation in study intensity. In principle, well studied groups are preferable, if only to improve statistical power. For this reason, Burt and Trivers (1998) chose to analyze B chromosome presence and degree of outcrossing in British flowering plants, a group in which both variables were well studied and there was no reason to expect degree of outcrossing to be associated with amount of cytogenetic work. Since intensity of study also varies within well-studied groups, spurious correlations can occur when the variable of interest co-varies with study effort.

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The ideal solution is to statistically correct for variation in study effort. The best measure of study effort would be the total number of karyotypic studies on each species, but such information is often difficult to find and time consuming to compile. To give perhaps the extreme case, in our work on Bs and genome size in 353 species of British flowering plants, it would have been an enormous task to truly quantify the number of karyotypic studies for each species. For example, Darlington and Wylie’s Chromosome Atlas of Flowering Plants was published in 1956 and typically lists only the most “recent” references, yet this compilation has over 2400 references. The available computer literature databases do not search prior to 1965 and most do not search past 1980. Yet databases listing numbers of karyotypic studies would be very valuable to build, especially for well-studied groups. (A complexity is that the effect of study effort on apparent B frequency may be exaggerated if discovery of Bs in a species causes the number of karytotypic studies on that species to increase.) An alternative is to measure some other aspect of study effort in genetics that is believed to be correlated with karyotypic study effort (and, then, test this assumption on a sub-sample of the data). The underlying assumption is that some species are well studied genetically and others not, so degree of study of genome size will correlate positively with intensity of karyotypic study, for example. The former is relatively easy to measure, since online databases of genome size studies exist (Gregory, 2001a, Bennett and Leitch, 2003). A sub-sample of 25 species of British flowering plants shows that number of estimates of genome size does correlate positively with number of cytogenetic studies (P ! 0.01, Spearman’s Rho = 0.56; Trivers et al., 2004). At the very least, comparative studies must exclude species whose chromosomes have never been counted, since obviously Bs could not be found in such species. This simple correction alone can change the rank order of relative B frequency among plant families (Levin et al., manuscript in preparation). In many cases the influence of study effort will be unbiased, at least within taxa. For example, Palestis and colleagues (2004) demonstrate that Bs are more frequent in mammals with karyotypes consisting of mostly acrocentric autosomes (see Chromosome shape). There is no reason to suspect that species with mainly acrocentric chromosomes are studied more frequently than those with mainly bi-armed chromosomes, and, indeed, there is no correlation between study effort and the percentage of autosomes that are acrocentric across mammals (F1, 944 = 0.427, P = 0.513, r2 = 0.0005). Study effort was indexed by the number of studies listed in an online database of mammalian karyotypes (Institute of Cytology and Genetics, 2000). The effect of study effort on apparent B frequency is enormous. Among species with fewer than three studies cited in the online database (n = 647), 2.5 % have reports of B presence, while 30 % of species with greater than 15 references (n = 27) have Bs. But since there is no apparent bias by percentage of acrocentric As, the fact that many species with few karyotypic studies may be misclassified as non-B species would only decrease the chance that a significant correlation between A chromosome shape and B presence would be found. We have added study effort as a variable in a regression analysis and find highly sig-

Table 1. Logistic model coefficients and logistic likelihood ratio tests for the influence of the proportion of A chromosome arms on acrocentrics, study intensity, and number of A chromosomes on B chromosome presence in mammals

Coefficient

Intercept Acrocentric As No. studies Chromosome no.

–3.83 –2.00 0.08 –0.01

SE

0.60 0.49 0.02 0.01

Partial r

–0.31 0.19 0.21 0.00

Logistic likelihood ratio tests χ2

df

probability

16.73 20.48 0.29

1 1 1

P > 0.01; 0.01 > P > 0.001; 0.001 > P; NS = not significant.

plains these correlations in the multiple regression analysis (Table 4). Several models have been proposed to explain clinal distribution of genetic markers, of which the main ones are ecological selection for broad clines and hybrid zones for narrow ones. Essentially, a hybrid zone is a cline between two parapatric hybridizing taxa for genes and the distinguishing characters they determine (Hewitt, 1988). These clines may originate in two ways, primarily or secondarily. In the former case, the differences evolve in a continuous distribution, for example an environmental gradient that favors different alleles on either side; natural selection progressively sharpens the incipient cline until it becomes a narrow hybrid zone between two internally coadapted genotypes (Endler, 1986). In a secondary zone, the differences evolve while the two populations are geographically isolated, so that when their ranges alter and meet, a steep cline is formed as they hybridize (Hewitt, 1988). In this latter case, deterministic forces may not be involved in the formation of clines, but are necessary for a long continuation of this pattern of variation. In T. pallidipennis, the cytogeographic analysis suggests that selection of some sort is more likely to act because of the repetition patterns of variation observed over a wide geographic area and their stability over time (Confalonieri et al., 1998). If current clines were hybrid zones, selection could be acting against recombinant genotypes based on inversion types. However, as the populations are all polymorphic except for some at the very extremes of the distribution of the species in arid and semiarid regions of Argentina, the width of the hybrid zone would seem to be very extensive, exceeding, in some cases, more than 1,000 km, and involving almost the entire distribution of populations from southern latitudes. Therefore, we proposed the hypothesis of geographically variable selection maintaining the clines, although further evidence was necessary to corroborate these conclusions. Clues came from the analysis of protein (Matrajt et al., 1996) and molecular markers (Confalonieri et al., 1998, 2002). Four populations located along an altitudinal gradient (Puente del Inca, Uspallata, Observatorio and Tunuyań), and others outside the cline (Bariloche and Quilmes) (Table 1, Fig. 1) were phylogeographically analyzed using Restriction Site Variation (RFLP) of mitochondrial DNA (Confalonieri et al., 1998). Phylogeography has introduced a phylogenetic-historical perspective to investigate the evolution of populations, and contributes to the drawing of conclusions regarding se-

356 214


quences of colonization and diversification (Avise, 2000; Lanteri and Confalonieri, 2003). We observed no geographical structuring in the unrooted tree connecting all 17 mitochondrial DNA haplotypes found. Many of them are present in most of the populations analyzed, indicating high levels of gene flow. The fact that there neither is obvious differentiation in haplotype distribution between both extremes of the clines nor between chromosomally differentiated populations, shows that the cline is not the result of a hybrid zone and reinforces the selection hypothesis (Confalonieri et al., 1998).

The geographic distribution of Bs and the parasitic hypothesis In order to gain further insights into this longstanding debate concerning the selfish, adaptive or near-neutral nature of B chromosomes, we performed a cytogeographic study of T. pallidipennis populations from Table 1 (25 samples correspond to those already reported in Confalonieri (1995), and two others (Bariloche and Tunuyań) that are incorporated in this report). Multiple regression analysis of B carrier frequencies – which varied from 0 to 37 % – on altitude, latitude and longitude demonstrate that the supernumerary chromosome shows a different pattern of variation compared with the number of inverted sequences per male (I) (Table 4a). As a matter of fact, the frequency of B carriers in each sample is significantly associated with the latitudinal and longitudinal situation of the sample, but, conversely to most inversions, is not associated with any climatic variable (Table 4b). B chromosomes are associated with geographical variables in such a way that in more eastern longitudes and southern latitudes the supernumerary chromosome tends to disappear (Fig. 3). As previously mentioned, T. pallidipennis is endemic to North America and is one of the few trimerotropines to have successfully extended its distribution to Andean South America (Vaio et al., 1979), being adapted here to a wide altitudinal range. Rain forests and humid grasslands (in eastern localities) are not inhabited by this species and its basic requirements appear to be the prevalence of arid and semi-arid conditions. In fact, samples as Laguna Blanca, Plottier, Chelforo´, ChoeleChoel, La Adela, Chocoń and Bariloche can be considered as marginal, because they are situated at the southern border of the species range (Fig. 1). Therefore, more eastern longitudes

0.40 0.35

B-frequency

0.30 0.26 0.20 0.16 0.10 0.06 0 72 71 44

70 69

40

68

38

67 Longitude

66

32

65

28

64

Latitude

63 24

Fig. 3. B frequencies from 27 samples of T. pallidipennis from Argentina plotted against longitude West and latitude South.

and southern latitudes are most probably marginal environments for T. pallidipennis, just where the frequency of B carriers tends to be lower. This pattern of distribution is better explained by means of the parasitic model: B carriers are obviously more frequent in those areas where the species thrive and disappear in circumstances where the burden on fitness is too heavy to bear: i.e. in marginal environments. Similar situations were found in Myrmeleotettix maculatus, where B carriers are limited to populations in the south and east of Great Britain which are climatically better for grasshoppers (Hewitt and Brown, 1970; Hewitt, 1973) and in Crepis capillaris, where higher B frequencies are also the reflection of the suitability of the habitats (Parker et al., 1991).

Concluding remarks Cytogeography has been demonstrated to be an important tool to be applied in natural populations, either to unravel the adaptive significance of chromosome polymorphisms or to investigate the parasitic nature of some genomic elements. Although it is often the first step to be followed when the evolutionary significance of variation is under investigation, it can also provide concluding evidence supporting laboratory experiments that have been previously performed. For instance, Beukeboom and Werren (2000) have recently analyzed the geographic distribution of the paternal-sex-ratio (PSR) chromosome in natural populations of the parasitic wasp Nasonia vitripennis. PSR is a B chromosome that is considered to be an extreme example of selfish (or parasitic) DNA, because it causes all-male families in the parasitic wasp by inducing paternal genome loss in fertilized eggs. The authors demonstrated that PSR has a very limited geographic distribution, agreeing with the fact that PSR completely destroys the genome of its host in each generation, reducing host fitness to zero. If PSR

became common in natural populations, it could drive such populations to extinction. In T. pallidipennis several lines of evidence that issue from the cytogeographic analysis suggest that inverted chromosomes are special sequences that are maintained by deterministic forces while B chromosomes are genomic elements that clearly fit the parasitic model. We also presented the analysis of a single large sample from Uspallata, and demonstrated that Bs have some influence on body size, enlarging many of the morphometric characters of individuals. We know that increased body size often appears to confer significant advantages for adult fitness components in Drosophila (Santos et al., 1988; Hasson et al., 1993; Partridge and Fowler, 1993) and in other insects (Norry and Colombo, 1999). It has been proposed that exotermic organisms tend to be larger in colder climates, however, this does not seem to apply to T. pallidipennis, where body size in cooler climates is smaller (Colombo and Confalonieri, 1996). It is known that, in Uspallata, inversion 4SM1 increases body size (Colombo, 2002). This chromosome sequence has its highest incidence in this population, most probably as a consequence of its adaptive significance. It could be possible that, given that this inversion and the B chromosome are positively associated, the increase of body size in B carriers is due to the increased size of inversion 4SM1 carriers. If this genotypic disequilibrium is demonstrated to be genuine, then it can be considered as a very original way of “accumulation” for this selfish element, through a “hitchhiking” process with adaptive elements. Other aspects that deserve attention in relation to the parasitic model are the effects exerted by B elements on chiasma conditions. It was found that in some samples a B brings about a reduction in total chiasma frequency, although not affecting interstitial chiasma frequencies. Effects on recombination are frequent among B chromosomes, with a tendency to the increase of chiasma frequency rather than to a reduction of it. This was interpreted, in the grasshopper Eyprepocnemis plorans, in terms of increased recombination aroused by the presence of the B chromosome, that would enhance genetic variability in the offspring, thus leading to an increased tolerance to the “parasite” (i.e. the B chromosome; see Camacho et al., 2002). In E. plorans there are many varieties of B chromosomes, some of them parasitic (with drive) and some of them neutralized (without drive); the parasitic Bs produce a greater increase of chiasma frequency than the non-parasitic ones (Camacho et al., 2002). In the light of this hypothesis it is difficult to square parasitism with reduction of recombination in T. pallidipennis. A possible explanation is that interstitial chiasmata, which lead to more recombinant genotypes than terminal or proximal ones, are indeed not affected. Another possibility is that respecting the degree of parasitism which may vary among populations, this would help explain the fact that in other populations the B has no effects on chiasma conditions or phenotype. These variable characteristics of B chromosomes among different samples can be readily conciliated with a more dynamic model in which Bs are evolving through different stages (neutrality, selfishness and even heterosis) in a nonsynchronic fashion, showing then somewhat different results among populations.


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Population Dynamics and Evolution of B Chromosomes Cytogenet Genome Res 106:359–364 (2004) DOI: 10.1159/000079313

Mitotically unstable B chromosome polymorphism in the grasshopper Dichroplus elongatus M.I. Remis and J.C. Vilardi Departamento de Ecologıá, Genética y Evolucioń, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires (Argentina)

Abstract. Dichroplus elongatus, a widespread South American phytophagous grasshopper, exhibits polymorphisms for supernumerary chromosomes and segments (SS) in natural populations in Argentina. In this paper we review the available information on B chromosome polymorphism in D. elongatus related to geographic distribution, patterns of chromosome variation and influence on sperm formation. In D. elongatus the different forms of supernumerary variants are not independent. The proportion of B-carrying individuals (B prevalence) is negatively correlated with SS10 and positively with SS6 frequencies. The analysis of population structure considering the different supernumerary variants would suggest that the patterns of chromosome variation can not be explained only by random factors. Geographic distribution was analyzed scoring the prevalence of B chromosomes in 13 natural populations collected in three different biogeographical provinces from Northwest (Las Yungas province) and East (Espinal and Pam-

peana provinces) of Argentina. The detected heterogeneity may be explained by significant differentiation between Northwest and East regions and among populations within Las Yungas and Pampeana provinces. Correlation analysis suggested that B chromosome prevalence is associated with maximum temperature and with latitude. Additional information about the nature of the patterns of B chromosome variation was obtained comparing them with those obtained at the mitochondrial DNA level. The hierarchical analysis of molecular differentiation revealed discrepancy with respect to chromosome differentiation and also suggested that the pattern of B chromosomes may not be explained by historical factors. We also discussed the probable influence on fertility of carriers considering the production of abnormal sperm formation (macro and microspermatids) in relation to the number of Bs per follicle.

In Orthoptera, as in all main groups of animals and plants, supernumerary chromosome polymorphisms have frequently been found. To analyze the origin, establishment and maintenance of these dispensable elements, several aspects of their biology have been studied exhaustively (Jones and Rees, 1982; Jones, 1995; Camacho et al., 2000).

B-chromosome evolution may be considered as the result of a series of interactions between Bs and the A complement. These interactions may also involve other supernumerary elements (knobs, supernumerary segments or C-bands) (Rhoades and Dempsey, 1973; Lo´pez Leoń et al., 1991). Thus, the prevalence of B chromosomes is generally associated with ability of the population to tolerate this polymorphism coupled with probable accumulation mechanisms (Beukeboom, 1994; Camacho et al., 2000). In several cases, spatial chromosome differentiation is associated with B chromosome clines (Hewitt and Brown, 1970; Shaw, 1983; Parker et al., 1991; Cabrero et al., 1997). In the grasshopper Myrmeleotettix maculatus and the plant Crepis capillaris, B chromosomes were prevalent in regions of the geographical distribution of species more favorable climatically (Hewitt and Brown, 1970; Parker et al., 1991). On the contrary, in the grasshopper Eyprepocnemis plorans, the

Financial support from the Agencia Nacional de Promocioń Cientı´fica y Técnnolo´gica (ANPCYT) (PICT 6628) and Consejo Nacional de Investigaciones Cientı´ficas y Técnicas (CONICET) (PIP N# 0722/98 and PIP No 02442) is gratefully acknowledged. Received 17 September 2003; manuscript accepted 3 March 2004. Request reprints from Maria Isabel Remis, Depto. Ecologıá, Genética y Evolucioń Fac. Cs. Exactas y Naturales, Univ. Buenos Aires 1428 Buenos Aires (Argentina); telephone: 54 11 45763349 fax: 54 11 45763384; e-mail: [email protected]

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were analyzed by means of partial correlation analysis using the STATISTICA program (Statistica Statsoft Inc. Statistica version 4.5. Tulsa, OK). Frequency data were transformed according to Christiansen et al. (1976) as: Xi = ( p1 – p1)

Fig. 1. Meiotic cells of the grasshopper Dichroplus elongatus in diplotene (A) and metaphase I (B) showing a B univalent (arrow). Bar: 10 Ìm.

冪冉

N1 p0 (1 – p0)

冊

where p0 is the mean B-prevalence, p1 represents the B prevalence in the ith population and N1 is the number of individuals sampled per population. This transformation normalizes the distribution and weights sampling sizes.

Interaction among supernumerary variants negative correlation between B chromosome frequency and altitude may be explained historically (Cabrero et al., 1997). The ability of a population to tolerate particular chromosome polymorphisms may be related to their harmful effects. B chromosomes tend to show detrimental effects on fertility (Jones, 1995). In grasshoppers, the effect of Bs on spermiogenesis leads to an increase in the production of both macro and microspermatids (e.g. Nur, 1969; Bidau, 1986; Suja et al., 1986). Dichroplus elongatus (2n = 22 + X0 in males), a widespread South American phytophagous grasshopper, exhibits polymorphisms for supernumerary chromosomes and segments in natural populations of Argentina (Remis and Vilardi, 1986; Loray et al., 1991; Clemente et al., 1994). The B chromosome of D. elongatus is acrocentric, heteropycnotic, and mitotically unstable. Its size is slightly larger than the smallest pair of the A complement (Fig. 1). In B bearing individuals, B number varies from 0 to 6 among testis follicles (Remis and Vilardi, 1986). Supernumerary polymorphic heterochromatic segments are also present in the pairs M6 (SS6), S9 (SS9) and S10 (SS10) (Loray et al., 1991; Clemente et al., 1994). In this paper we review the available results of B chromosome polymorphism analyses in D. elongatus related to geographic distribution, patterns of chromosome variation and influence on sperm formation. Population structure revealed at the chromosome level is also discussed in the light of mitochondrial DNA variation.

Data analysis The comparison of the proportion of B-carrying individuals (B prevalence) among populations was performed through Monte Carlo simulations, as described by Roff and Bentzen (1989). This method compares the extent of heterogeneity (assessed through chi-squared analysis) in the original data matrix to that estimated from repeated randomization of the original matrix. This procedure is designed to minimize the effect of empty cells on the validity of the chi-square test. The relationships between B prevalence and geographical (latitude, altitude and longitude) and climatic (medium temperature, maximum temperature, relative humidity) variables

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B chromosomes are assumed to spread and maintain in populations through accumulation mechanisms in mitosis and meiosis (Jones and Rees, 1982). However, these phenomena may be affected when different forms of supernumerary variants are present in the same population (Rhoades and Dempsey, 1973; Lo´pez Leoń et al., 1991). One of the most interesting features encountered in chromosomally polymorphic populations of D. elongatus is that the different forms of supernumerary variants (chromosomes and segments) are not independent. Remis et al. (1998) analyzed seven populations of this species, five from Northwest (Raco, Tafı´ Viejo, Horco Molle, Famailla´, Campo Quijano), and two from East Argentina (Ing. Maschwitz and San Clemente) and demonstrated that the B prevalence in these populations is correlated negatively with SS10 (r = – 0.89, P ! 0.05) and positively with SS6 (r = 0.88, P ! 0.05) frequencies (Remis et al., 1998). Moreover, analysis of population structure showed different trends according to the supernumerary variant considered. The differentiation for SS9 and SS10 may be explained mainly by heterogeneity among Northwestern populations. By contrast, the frequency of SS6 and the prevalence of B’s are homogeneous within the Northwest but differ between this region and East Argentina. The distribution of neutral variants might be the consequence of gene flow among populations coupled with genetic drift generating similar differences in chromosome frequencies. This explanation seems to be untenable for D. elongatus. Our results suggest that the interactions between different forms of supernumerary variants may establish limits to the effects of gene flow and genetic drift and may be involved in non-random distribution patterns of supernumerary segments and B chromosomes in this species.

Geographical distribution of B chromosomes B chromosome prevalence in 13 natural Argentinean populations of D. elongatus collected in three different biogeographical provinces (sensu Cabrera and Willink, 1980) was considered to analyze geographic distribution of B polymorphism (Remis and Vilardi, 1986; Loray et al., 1991; Clemente et al., 1994; Remis et al., 1998) (Table 1). Four populations belong to “Las Yungas” biogeographic province, (Raco: RA, Horco

Table 1. Proportion of B-carrying individuals and geographic and climatic variables of natural populations of Dichroplus elongatus collected in Argentina. Data from Remis and Vilardi (1986), Loray et al. (1991), Clemente et al. (1994), Remis et al. (1998), Remis et al. (submitted). B

N

Latitude

Longitude

Altitude

Medium Temp. (ºC)

Maximun Temp. (ºC)

Relative Humidity (%)

a) Las Yungas Province HM RA FA CQ

0.139 0.209 0.250 0.065

79 91 20 31

26º48 26º40 27º03 24º55

65º19 65º22 65º23 65º37

550 1172 340 1500

19 16 19 16.1

25.2 20.5 25.3 20.5

76 73 80 73

b) Espinal Province CO DA PA

0.214 0.2 0

14 15 20

31º59 31º53 31º44

60º55 60º53 60º29

18 18 78

19 19 18.2

24.8 24.8 23.8

81 81 73

0 0.263 0 0.07 0.08 0

15 19 17 28 13 22

32º52 33º01 33º30 34º22 34º55 36º39

58º03 58º30 58º47 58º46 57º59 56º42

25 21 5 5 19 0

18 17.8 18 17.4 15.9 14.5

23.8 24 22.1 22.6 21.4 19.4

75 75 73 69 80 85

c) Pampeana Province Uruguayensis District

Oriental District

EP GU CE ES LP LU

Molle: HM, Famailla´: FA and Campo Quijano: CQ) and are located in Northwest Argentina. The remaining populations are located in East Argentina. Three of them belong to “Espinal” (Desvıó Arijoń: DA, Coronda: CO, Parana´: PA) and six to “Pampeana” biogeographic province (El Palmar: EP, Gualeguaychu´: GU, Ceibas: CE, La Plata: LP, Escobar: ES, and La Lucila: LU). The geographical distribution of B chromosomes indicates that they are widespread in the Northwest region of Argentina where all populations of Las Yungas province display this polymorphism and the average prevalence of Bs (0.167) was higher. In the East, by contrast, B chromosomes may be absent or show an extremely low prevalence, excepting the GU population. Within this region, populations of Pampeana province possess lower average prevalence of B chromosomes (0.07) with respect to populations of Espinal province (0.12). There is significant heterogeneity in B distribution when all populations were compared (Table 2a). To gain deeper insight into chromosome variation in this species we performed a hierarchical analysis of population differentiation. The heterogeneity among populations in B chromosome prevalence may be explained by significant differentiation between two main geographic regions (Northwest vs. East) (Table 2b) and within one of them (East). B chromosome prevalence does not differ significantly among populations of Las Yungas province (Table 2c). By contrast, there is a significant heterogeneity in the proportion of B-carrying individuals among populations located in the East (Table 2d). Within the East region, populations of Espinal province were homogeneous in B chromosome distribution (Table 2e) while in Pampeana province chromosome differentiation was highly significant (Table 2f). The last result may be explained by the significant differentiation among populations of Uruguayensis district (Table 2f1) since populations of the Oriental district exhibit similar B prevalence (Table 2f2). The spatial distribution of B chromosome polymorphism in the Eastern geographic region was analyzed with respect to

Table 2. Hierarchical analysis of chromosome and molecular (mitochondrial DNA) differentiation in 13 populations of Dichroplus elongatus belonging to three biogeographic provinces located in Eastern and Northwestern Argentina. The significance of chi-squared values observed was obtained using the randomization method of Monte Carlo according to Roff and Bentzen (1989). a

Comparison a) All Populations b) Northwest vs East region c) Among Northwest populations d) Among Eastern populations e) Among Espinal populations f) Among Pampeana populations f1) Among Uruguayensis populations f2) Between Oriental populations a b

2

χ P 2 χ P 2 χ P 2 χ P 2 χ P 2 χ P 2 χ P 2 χ P

b

B

MtDNA

25.36 0.01 5.62 0.02 4.90 0.18 20.36 0.01 4.73 0.11 15.17 0.008 10.69 0.008 1.74 0.36

87.0 -5 10 72.22 -5 < 10 38.6 0.004 87.3 0.0002 2.90 0.98 64.7 -5 10 18.4 0.24 2.05 0.69

Data from Remis et al. (submitted). Data from Clemente et al. (2000).

some climatic and geographic variables. Partial correlation analysis using the step-wise procedure suggested that B chromosome prevalence is associated with maximum temperature (r = 0.87, P = 0.004) and with latitude (r = 0.82, P = 0.01). In a previous work, genetic variation and phylogeography of this species were studied at mitochondrial DNA level in the same populations as those analyzed chromosomally using RFLP technique (Clemente et al., 2000). This study detected twelve different composite haplotypes for 16 polymorphic sites in the analyzed populations (Fig. 2). Haplotype 1 (H1) is


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entiation between districts because each district is homogeneous for mtDNA patterns. In Las Yungas there is no significant correlation between genetic and geographic distances (r = –0.48, P = 0.88). Thus, the distribution patterns of mtDNA variation observed in D. elongatus may be explained by isolation by distance when referring to the main biogeographic provinces while within Las Yungas and Pampeana provinces ecological factors seem to have contributed to the genetic differentiation among populations. The hierarchical analysis of molecular differentiation revealed discrepancy with respect to B chromosome distribution patterns, mainly in Las Yungas and Pampeana provinces. Populations from Las Yungas, with similar B chromosome prevalence, show significant heterogeneity at the mtDNA level. Moreover, populations of Uruguayensis district of Pampeana province are heterogeneous at chromosome level, but do not show evidence of restriction of gene flow analyzing mtDNA haplotype distribution.

Effects

Fig. 2. Geographic location, proportion of B-carrying individuals (pie charts), and haplotype frequencies (histograms) of the analyzed populations of Dichroplus elongatus. Raco = RA, Horco Molle = HM, Famailla´ = FA, Campo Quijano = CQ, El Palmar = EP, Gualeguaychu´ = GU, Ceibas = CE, Desvıó Arijoń = DA, Coronda = CO, Parana´ = PA, La Plata = LP, Escobar = ES, La Lucila = LU (Hi = i haplotype). Bar: 1000 km.

present in all sampled populations. H5 and H6 are widely distributed, whereas H12 was detected only in Famailla´. The comparison of absolute haplotype frequencies showed a significant heterogeneity of the mtDNA distribution when all thirteen populations were compared (Table 2a). The detected heterogeneity may be explained at two levels: i) by significant differentiation between Northwest and East regions (Table 2b) and ii) by significant differentiation among populations within Las Yungas (Table 2c) and Pampeana (Table 2f) provinces. In the Pampeana province two groups of populations are recognized, each of them homogeneous for mtDNA patterns. One of them includes the populations of the Uruguayensis district (EP, GU, CE, and ES) and the other is formed by those of the Oriental district (LP and LU) (Table 2f1, f2). At the levels where mtDNA heterogeneity was observed, the hypothesis of isolation by distance was tested evaluating the correlation between genetic and geographic distance matrices by the Mantel test. When all populations were considered the correlation was highly significant (r = 0.42, P = 0.001). In the Pampeana province the correlation is also significant (r = 0.54, P = 0.02), but this result is a consequence of a significant differ-

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There is compelling evidence that B chromosomes can show detrimental influence affecting development, fertility and fecundity of carriers (Jones and Rees, 1982; Camacho et al., 2000). In Orthoptera, the basal level of abnormal spermatids is usually very low (Suja et al., 1986). Several examples where Bs can increase the frequency of macro and microspermatids have been reported (Nur, 1969; Bidau, 1986; Suja et al., 1986; Bidau and Confalonieri, 1988). One of the significant effects of B chromosomes detected in D. elongatus, is the influence on spermiogenesis (Loray et al., 1991; Clemente et al., 1994). Due to B mitotic instability, the number of supernumerary chromosomes varies among testicular follicles. Cytogenetic analysis of individual follicles of each B-carrying individual allowed us to analyze the effect of different numbers of Bs on cells with the same genetic background. In both analyzed populations (Raco and Tafı´ Viejo) the results were consistent (Clemente et al., 1994). In each B carrier the trend was similar: the proportion of abnormal spermatids (macro- and microspermatids) was dependent on the number of Bs in the cell. Mean frequencies of macro- and microspermatids in cells with different number of Bs for both populations are summarized in Fig. 3. In Raco, the frequency of macrospermatids was significantly lower in follicles with 0 B, 2 Bs and 3 Bs with respect to 1B and 4B follicles (¯21 = 94.96, P ! 10–5 ). 0B, 2B and 3B follicles showed similar macrospermatid frequencies (¯22 = 0.83, P = 0.66). In the same way, frequencies of macrospermatids in follicles with 1 B and 4 Bs did not differ statistically (¯21 = 3.18, P = 0.08) (Fig. 3A). In Tafı´ Viejo, the frequency of macrospermatids remains similar among follicles with 1 B, 3 Bs and 4 Bs (¯22 = 0.95, P = 0.62) and among 0B and 2B follicles (¯21 = 0.60, P = 0.44). In agreement with the results found in Raco, in Tafı´ Viejo, macrospermatid frequencies were higher in follicles belonging to the first group (¯21 = 88.23, P ! 10–5 ) (Fig. 3A).

Fig. 3. Mean frequencies (in %) of macro- (A) and microspermatids (B) in follicles with different numbers of Bs in two populations of Dichroplus elongatus, Raco (RA) and Tafı´ Viejo (TV). Data from Clemente et al., 1994.

As a general feature, we observed a significant increase in macrospermatid frequencies in follicles with odd or high numbers of Bs that can be explained on mechanical grounds related to the presence of B univalent. In Raco, microspermatid frequencies were significantly different among all classes of follicles (¯24 = 333, P ! 10–5) (Fig. 3B). In follicles with odd numbers of Bs (1 or 3) the frequency of microspermatids is significantly higher than in follicles with even numbers (2 or 4) (¯21 = 157.5, P ! 10–5). Within follicles with even number of Bs the frequency of microspermatids increases with the numbers of supernumeraries (¯21 = 6.10, P = 0.01) while the opposite occurs in follicles with odd numbers of B’s (1 B vs. 3 Bs, ¯21 = 21.40, P ! 10–5 ). In Tafı´ Viejo, the frequency of microspermatids was higher in 1B follicles with respect to the other classes (¯21 = 85.24, P ! 10–5 ) which have similar microspermatid frequencies (¯23 = 1.66, P = 0.64) (Fig. 3B). As in the case of macrospermatids, significant increase in microspermatid frequencies was associated with follicles with odd or high number of Bs. The existence of microspermatids may be explained by the lagging B chromosomes or chromatids which may be excluded in the micronucleus. A cytophotometric analysis of DNA content revealed that the most frequent macrospermatids had a DNA content equivalent to diploid cells (2C) (Table 3). However, B-carrying individuals show significant increase in the frequencies of triploid (3C) and decrease in the frequency of tetraploid (4C) macrospermatids. Moreover pentaploid (5C) macrospermatids were also detected in individuals with Bs. The existence of macrospermatids with odd DNA content suggests that physiological effects related to post meiotic processes of spermatid formation including cell fusion may also be involved in the production of abnormal sperm. The effects of Bs on spermiogenesis in D. elongatus are consistent over the analyzed populations though the influence is more striking in Raco. The higher influence of Bs in this population may be considered as evidence that the environmental conditions and/or the genetic background can modify the effects of supernumerary chromosomes and hence the ability of a population to tolerate this polymorphism.

Table 3. Percent of macrospermatids with different DNA content in Band non-B-carrying individuals of Dichroplus elongatus (Clemente et al., 1994). N = number of analysed cells. DNA content macrospermatid comparison between B- and non-B-carrying individuals (¯22 = 10.37; P = 0.006) (5C macrospermatids were excluded in the analysis by the small sample size).

B-carrying Non-carrying

2C

3C

4C

5C

N

70 82.70

20 1.92

6.67 15.38

3.33 0

60 52

Whether this increase in abnormal sperm production can affect fertility of carriers in species with great sperm production or sperm replacement, as can occur in grasshoppers, remains an interesting question.

Evolutionary dynamics Interpopulation B chromosome differences depend on selective, historical, transmission and random factors (see Camacho et al., 2000). The relative importance of these factors may be obtained by analyzing the distribution patterns of B chromosomes and by comparing the population differentiation of different data sets. In D. elongatus the patterns of variation of different supernumerary variants (chromosomes and segments) do not fit expected patterns under hypotheses based on interaction between genetic drift and migration only (Remis et al., 1998). This condition might be attributed to the mitotic instability of B chromosomes that may yield a probable accumulation mechanism, but the covariation between different supernumerary variants may also contribute to the observed non-random pattern. Additional information about the nature of the patterns of B chromosome variation was obtained by comparing them with those obtained at the mitochondrial DNA level. mtDNA con-


363 221

stitutes a macromolecule of fast evolution that lacks recombination and has maternal inheritance in most animals. Because of these properties, it has been used to analyze population structure related to historical factors and to propose colonization routes (Avise, 1994). In D. elongatus, the distribution of B chromosome variation is not consistent with the gene flow patterns expected according to a phylogeographic analysis based on mtDNA. Our results suggest that the non-random pattern in the frequency of B chromosomes is not explained by historical factors. In a previous paper, based on a lower number of populations from Northwest and East Argentina, we observed differences between regions that suggested a clinal pattern (Sequeira et al., 1995). In the present wider survey, we were able to show that these two regions have different characteristics with respect to B polymorphism. The partial correlation analysis indicated that B chromosome distribution in the East region is

complex because it is positively related to two variables, mean maximum temperature and latitude, which are negatively correlated with each other. The effects of Bs on spermiogenesis, the differences between regions with respect to B distribution, the non random pattern of B distribution with respect to supernumerary segments, the differences between the patterns of B chromosome and mtDNA variation, and the association of B chromosome frequency and climatic and geographical variables in the eastern region indicate that the distribution of B chromosomes in D. elongatus are not to be explained exclusively by random and historical factors. The ability of populations to bear this polymorphism may depend on the interaction between a probable non Mendelian transmission mechanism, negative effect on fertility, environmental variables and the genetic background.

References Avise JC: Molecular Markers, Natural History and Evolution (Chapman and Hall, New York 1994). Beukeboom LW: Bewildering Bs: an impression of the 1st B-chromosome conference. Heredity 73:328– 336 (1994). Bidau CJ: Effects of cytokinesis and sperm formation on a B-isochromosome in Metaleptea brevicornis (Acridinae, Acrididae). Caryologia 39:165–177 (1986). Bidau CJ, Confalonieri V: Cytophotometric study of micro and macrospermatids in three species of grasshoppers. Cytobios 53:31–41 (1988). Cabrera AL, Willink A: Biogeografıá de América Latina, in Monografıás cientı´ficas de la OEA. Buenos Aires, Argentina, vol. 13 (1980). Cabrero J, Lo´pez Leoń MD, Go´mez R, Castro AJ, Martıń Alganza A, Camacho JPM: Geographical distribution of B chromosomes in the grasshopper Eyprepocnemis plorans, along river basin, is mainly shaped by historical non-selective historical events. Chrom Res 5:194–198 (1997). Camacho JPM, Sharbel TF, Beukeboom LW: B-chromosome evolution. Phil Trans R Soc Lond B 355:163–178 (2000). Christiansen FB, Frydenberg O, Hjorth JP, Simonsen V: Genetics of Zoarces populations. IX Geographic variation at the three phosphoglucomutase loci. Hereditas 83:245–256 (1976). Clemente M, Remis MI, Vilardi JC, Alberti A: Supernumerary heterochromatin, chiasma conditions and abnormal sperm formation in Dichroplus elongatus (Orthoptera): Intra and interpopulation analysis. Caryologia 46:321–335 (1994).

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Clemente M, Remis MI, Vilardi JC: Mitochondrial DNA variation and phylogeographic relationships among Argentinian populations of Dichroplus elongatus. Ann Entomol Soc Am 93:653–663 (2000). Hewitt GM, Brown FM: The B-chromosome system of Myrmeleotettix maculatus V. A steep cline in East Anglia. Heredity 25:363–371 (1970). Jones RN: B chromosomes in plants: escapees from the A chromosome genome. New Phytol 131:411–434 (1995). Jones RN, Rees H: B Chromosomes (Academic Press, London 1982). Lo´pez Leoń MD, Cabrero J, Camacho JPM: Meiotic drive against an autosomal supernumerary segment promoted by the presence of a B chromosome in females of the grasshopper Eyprepocnemis plorans. Genome 37:705–709 (1991). Loray MA, Remis MI, Vilardi JC: Parallel polymorphisms for supernumerary heterochromatin in Dichroplus elongatus (Orthoptera): Effects on recombination and fertility. Genetica 84:155–163 (1991). Nur U: Mitotic unstability leading to an accumulation of B-chromosomes in grasshopper. Chromosoma 27:1–19 (1969). Parker JS, Jones GH, Edgar LA, Whitehouse C: The population cytogenetics of Crepis capillaris IV. The distribution of B chromosomes in British populations. Heredity 66:211–218 (1991).


Remis MI, Vilardi JC: Meiotic behaviour and dosage effect of B-chromosomes on recombination in Dichroplus elongatus (Orthoptera: Acrididae). Caryologia 39:287–301 (1986). Remis MI, Clemente M, Pensel S, Vilardi JC: Non random distribution patterns of supernumerary segments and B chromosomes in Dichroplus elongatus (Orthoptera). Hereditas 129:207–213 (1998). Rhoades MM, Dempsey E: Chromatin elimination induced by B chromosomes of maize. Heredity 64:13–18 (1973). Roff DA, Bentzen P: The statistical analysis of mitochondrial DNA polymorphisms: ¯2 and the problem of small samples. Mol Biol Evol 6:539–545 (1989). Ronderos RA: Consideraciones sobre la biogeografıá de los Melanoplinae en Sudamerica (Orthoptera), in Proc 3rd Triennial Meeting Pan American Acridid Society (1985). Sequeira AS, Confalonieri VA, Remis MI, Vilardi JC: B-chromosome and enzyme polymorphisms in the grasshopper Dichroplus elongatus: geographical gradients that are not explained by historical factors. Evolucioń Biolo´gica 9:283–299 (1995). Shaw MW: Rapid movement of a B chromosome frequency cline in Myrmeleotettic maculatus. (Orthoptera: Acrididae). Heredity 50:1–14 (1983). Suja JA, Gosa´lvez J, Lo´pez-Fernańdez C, Rufas JS: A cytogenetic analysis in Psophus stridulus (Orthoptera: Acrididae): B-chromosomes an abnormal spermatids nuclei. Genetica 70:217–224 (1986).

Population Dynamics and Evolution of B Chromosomes Cytogenet Genome Res 106:365–375 (2004) DOI: 10.1159/000079314

Geographic and seasonal variations of the number of B chromosomes and external morphology in Psathyropus tenuipes (Arachnida: Opiliones) N. Tsurusaki and T. Shimada Laboratory of Biology, Faculty of Education and Regional Sciences, Tottori University, Tottori (Japan)

Abstract. Psathyropus tenuipes (= Metagagrella tenuipes) is a harvestman that harbors B chromosomes with extremely high frequency (individuals without Bs are only 1 % of the total number of specimens so far examined) and high numbers (mean number of Bs per individual is about 4). Geographic variations of the number of Bs and external morphology of the species and the relationship between them were studied. A northward increase in the number of Bs was detected throughout the Japanese Islands, though the number also varied considerably locally. Latitudinal gradients were also found in some external characters, while there were no correlations between those external morphologies and the number of Bs. Principal

component analysis using eight morphological data for 21 populations revealed four geographical groups that reflect actual location of the populations. Populations along the Seto Inland Sea were characterized by a lower number of Bs than those in other areas. Seasonal change was also found in a population (Yatsukami in western Honshu) in both 1994 and 1995 for the number of Bs, though the number in the same population was stable at least throughout later postembryonic stages in both 1997 and 1998. Embryos contained fewer numbers of Bs than adults, suggesting that females of the species tend to lay eggs with fewer numbers of Bs.

B or supernumerary chromosomes are extra dispensable chromosomes whose number varies within populations of many organisms. The raison d’etre of B chromosomes is not yet fully understood. Some studies have proposed a selective advantage of individuals with B chromosomes or of certain frequencies of B chromosomes within a population (Robinson and Hewitt, 1976; Plowman and Bougourd, 1994), while most stud-

ies failed to show such adaptive effects. On the contrary, quite a few studies have demonstrated deleterious effects on their carrier and suggested their nature as selfish DNA (Hewitt et al., 1987; Werren et al., 1988; Shaw and Hewitt, 1990; Jones, 1991; Camacho et al., 1997; Muñoz et al., 1998). B chromosome frequency often shows differential geographical distribution (Hewitt and John, 1967; Hewitt and Brown, 1970; Kayano et al., 1970; Bourgourd and Parker, 1975, 1979; John, 1976; Jones and Rees, 1982; Semple, 1989; Parker et al., 1991; Tsurusaki, 1993; Confalonieri, 1995; Cabrero et al., 1997). A classical example is that of Myrmeleotettix maculatus in Great Britain. In this species, the frequency of individuals containing one or more B chromosomes is broadly correlated with latitude, and distribution of individuals with Bs is limited to the southern part of Great Britain (Hewitt and John, 1967; Hewitt and Brown, 1970). A similar cline was found in the plant Crepis capillaris (Asteraceae), in which B chromosomecontaining plants are only found in southern Great Britain

Supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan, and Japan Society for the Promotion of Science to N.T. (06640901 and 13640696). Received 2 October 2003; manuscript accepted 17 March 2004. Request reprints from Nobuo Tsurusaki, Laboratory of Biology Faculty of Education and Regional Sciences Tottori University, Tottori, 680-8551 (Japan) telephone and fax: 81-857-31-5110, e-mail: [email protected]

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© 2004 S. Karger AG, Basel 0301–0171/04/1064–0365$21.00/0



Materials and methods Psathyropus tenuipes is distributed in Japan and the Maritime Province of Far East Russia. This species is typically coastal in western Japan and usually inhabits abrasion cliffs in sandy beaches. On the other hand, the species penetrates inland in northern Japan and can be found in open habitats such as parks with lawns and shady groves in urban areas (Fig. 2). The species is univoltine and overwinters as eggs (Tsurusaki, 2003). Adults appear from late June and can be collected till November in southwestern Japan. Some survive until January to March of the next year. A low level of synchronization in the life cycle is one of the prominent features of the species (Tsurusaki, 2003) and there are some individuals still juvenile even in September. This species is gregarious and is usually found in clumps, each clump consisting of dozens of harvestmen, on the lower surface of overhanging rocks on cliff walls or hollows of tree trunks. This habit makes collecting the species very easy.

Fig. 1. B chromosomes (arrows) of Psathyropus tenuipes in a C-banded spermatogonial metaphase (A) and meiotic metaphase I (B) in the same single male with 2n = 20 ( = 18 + 2Bs).

(Parker et al., 1991). Geographic variation in the frequency of Bs has attracted many researchers because those patterns may provide a clue as to why B chromosomes are maintained in those organisms in spite of their deleterious effects. Geographic variation in the number and frequency of B chromosomes occurs in a Japanese harvestman, Psathyropus tenuipes L. Koch 1878 (Sclerosomatidae: Gagrellinae), which has formerly been known as Metagagrella tenuipes (L. Koch) (Tsurusaki, 1993). B chromosomes (Fig. 1) of the species show the following characteristics (Tsurusaki, 1993; Gorlov and Tsurusaki, 2000a, b): (1) The Bs are widespread over the whole geographic range of the species, nearly every individual possesses at least one B and the population mean for the number of Bs often exceeds six; (2) The number of Bs often fluctuates to some extent among cells from the same individual; (3) Bs vary considerably in size and morphology; (4) Bs are almost completely heterochromatic (Fig. 1), though some of them carry euchromatic segments located on terminal regions or rarely in the middle of the chromosome; (5) Bs behave as univalents at meiosis (Fig. 1); (6) Presence of a peculiar odd-even effect, suggesting differences in susceptibility to parasites between B-odd and B-even harvestmen (Gorlov and Tsurusaki, 2000a; but see discussion section). Although the B chromosomes of P. tenuipes seem to be widespread across the species range, there is also geographic variation in the average number of Bs per population with a rather wide range (Tsurusaki, 1993). Species with such high levels of B chromosome polymorphism are suitable as material to study phenotypic effects of Bs or dynamics of B chromosome polymorphism. The aims of the present study were to assess a more detailed pattern of geographic variation in the number of Bs of this species in Japanese Islands and examine possible relationships between the number of Bs and external morphology or environmental factors.

366 224


Chromosome survey We surveyed chromosomes of adult males collected from a total of 14 populations in Honshu including 10 populations in Tottori Prefecture (Table 1, Fig. 2). In the Yatsukami population, at the Hamamura Coast in eastern Tottori Prefecture which faces the Sea of Japan, chromosomes were surveyed monthly from July to September in 1994 to ascertain whether the number of B chromosomes fluctuates seasonally. For this population we also surveyed chromosomes of developing embryos by using eggs laid by female adults in captivity in the autumns of 1994 and 1995. Females, collected in October and kept separately in plastic containers (24 cm in diameter, 12 cm high), laid eggs in sand beds moistened with water. Eggs dug out from the sand were checked for developmental state and if they were in a stage with limb buds (stages illustrated in Figs. 3–4 of plate 1 in Holm, 1947) they were used for chromosome preparations. We used an air-drying method with a cell dissociation process using lactic acid (see Tsurusaki, 1985; Tsurusaki and Cokendolpher, 1990) for chromosome preparations of testes. For the eggs, we used another air-drying technique with 30 % acetic acid treatment for cell dissociation (Dietrich and Mulder, 1981), which includes the following steps: (1) Hypotonic treatment: we placed a developing egg in a hollow of the depression slide filled with hypotonic solution (1 % citric acid) and left it for 15 min after removing the chorion using a dissecting needle. (2) Fixation: we removed the hypotonic solution with a Pasteur pipette and fixed the embryo immediately with methanol:acetic acid (3:1). We transferred the fixed embryo into a plastic 1.5-ml microtube with 1 ml of fixative and stored it in a freezer (–20 ° C) for more than 24 h. (3) Dissociation of cells: we replaced the fixative with 30 % acetic acid and shook the microtube vigorously with a Vortex mixer. The cell suspension was rinsed three times with fixative by resuspension and recentrifugation. (4) Slide preparation: we dropped a small amount of the cell suspension onto a slide using a micropipette and allowed it to air-dry. The number of Bs in this species fluctuates considerably among cells of the same individual (cf. Table 2), thus, chromosome number of each harvestman was represented by its modal number. Modal number of B chromosomes was closely correlated to median B number, especially when the sample size was large (see data on 20 males in Table 2, r = 0.88, P ! 0.001). Morphometrical analysis of external morphology For the morphological analyses, adult male specimens preserved in 80 % ethanol from various localities that cover the entire distribution range of the species in Japan were used. We selected 21 populations considering geographical locations and number of samples available (we tried to select populations with more than ten individuals as far as possible) for the detailed analyses. All the specimens used for chromosome preparation were also measured and counted, although data on some characters such as body length were unavailable due to severe damage of body during dissection. We selected eight morphometrical characters: (1) body length (BL), (2) cephalothorax length (CL), (3) lengths of femora of the first pair of legs (FIL), (4) width between the scent gland pores (WSG), (5) length of spine on the second abdominal tergite (SPL), (6) number of noduli on the femur of the second legs (NN), (7) number of denticles on dorsal surface of the basal segment of chelicera (NCD), (8) degree of melanism of body (DM). We measured or counted both right and left counterparts for femur I length (FIL), number of noduli (NN), and cheliceral denticles (NCD) to estimate degree of fluctuating asymmetry (FA) as a measure of developmental instability. FA

1. Is. Rishiri

Populations karyotyped

2. Cape SoyaH

Wakasakanai

Populations measured for external characters

SunagawaH

3. Toyotomi Spa

Populations karyoptyped and measured for external characters

Hokkaido Univ. CampusH

26. Yatsukami

4. MaruyamaH 12. Kamogaiso 24. Shirawara 13. Kozomi 23. Higashihama

27. Nagaobawa 28. Uno 29. R. Kaseichi 30. R. Araigawa

Botanical GardenH 5. Oirase

25. Tatsumidai

N 0 250 500 km Populations in Tottori Prefecture

6. Obanazawa

12. Uradome Coast (Kamogaiso) 10. Kurosaki

13. Kozomi 20. Is. Kokuno-jima 19. Is. Noumi-jima 14. Kawatana

Fig. 2. Map showing populations of Psathyropus tenuipes surveyed for karyotypes (solid circles) and for external characters (open circles) with records of the species (smaller circles). Population numbers correspond to those in Figs. 4 and 6. Chromosomal data on the asterisked populations were derived from Tsurusaki (1993).

Yatsukami

11. Amagozen 9. Yokohama

22. ShirahamaH 21. Ushimado

7. Awa-AmatsuH

N 8. Is. Hachijo

18. Is. NakajimaH 0

250

16. Is. Amakusa Kamishima

15. Is. Amakusa Shimojima

500

km

17. Is. Kuchinoerabu

Table 1. The number of B chromosomes of Psathyropus tenuipes in various localities surveyed in the present study. All the statistics presented here are based on the modal numbers of Bs in respective individuals, thus the number presented under “Specimens examined” equals the sample size (n). Populationa

Amagozen Point, ISHIKAWA(11) Higashihama Beach, TOTTORI(23) Shirawara Beach, TOTTORI(24) Kamogaiso Beach, TOTTORI(12) Kozomi Beach, TOTTORI(13) Tatsumidai, TOTTORI (25) Yatsukami, TOTTORI(26) Yatsukami, TOTTORI(26) Yatsukami, TOTTORI(26) Yatsukami, TOTTORI(26) Yatsukami, TOTTORI(26) Yatsukami, TOTTORI(26) Yatsukami, TOTTORI(26) Nagaobana Point, TOTTORI(27) Uno Beach, TOTTORI (28) River Kaseichi, TOTTORI (29) River Arai, TOTTORI (30) Ushimado, OKAYAMA (21) Is. Kokuno-jima, HIROSHIMA (20) Kawatana, YAMAGUCHI (14) a b

Date

25 Jul 1994 22 Sept 1994 22 Sept 1994 12 Jul 1994 1 Aug 1994 19 Aug 1994 12 Jul 1994 5 Aug 1994 9 Sept 1994 11 Oct 1994 Nov 94 – Jan 95 5 Aug 1995 11-23 Nov 95 12 Jul 1994 30 Jul 1994 25 Jul 1994 25 Jul 1994 24 Aug 1995 27 Sept 1995 27 Sept 1995

Specimens examinedb

Number of B-chromosomes min.

max.

mean

SD

median

38 6 24 1 4 12 7 17 34 10 39 eggs 20 54 eggs 2 4 1 7 35 8 4

2 2 2 4 1 1 1 0 1 4 0 1 0 4 1 2 0 0 0 4

8 7 6 4 4 7 7 7 8 8 6 7 4 6 4 2 4 5 2 7

4.5 4.3 3.9 4 2.3 2.8 3.9 2.7 3.6 6 1.4 4.7 1.1 5 1.8 2 1.6 2.2 1.2 5.5

1.29 1.63 1.16 – – 1.6 2.43 2.12 1.37 1.15 1.58 1.93 1.16 – – – 1.4 1.2 1.31 0

4 4 4 4 2 2.5 3 2 3 6 1 5 1 5 1 2 2 2 1 5.5

Numbers in parentheses correspond to those in Fig. 2. All the specimens are males except for eggs.


367 225

Fig. 4. Geographic variation of the number of B chromosomes among populations. Original data for populations asterisked are in Tsurusaki (1993). Explanations for each box plot are in Fig. 3. Some dots deviated from each range bar are outliers. The difference in the number of B-chromosomes among populations is significant (Kruskal-Wallis test, P ! 0.001).

Results

Fig. 3. Seasonal variations of the number of B chromosomes retained in the Yatsukami population of Psathyropus tenuipes in 1994, 1997 and 1998. Data for 1997 and 1998 are based on Gorlov and Tsurusaki (2000a). A line in the body of the box and the ends of the box locate median and the 25th and 75th quantiles, respectively. Bars represent the range. The number of B-chromosomes significantly fluctuated among months in 1994 (Kruskal-Wallis test, P ! 0.001. The difference was significant at the same level, even when data set for July, sample size of which is less than 10, was eliminated in the analysis), while they were rather stable throughout the season in both 1997 and 1998.

was assessed by the value obtained from the following formula: (R – L)/ 0.5(R + L), where R and L represent values for the right and left sides of an individual, respectively (Müller and Swaddle, 1997). All the measurements and counts were made under a stereo-microscope with eyepiece graticule. Principal component analyses were performed by using standardized data. All the continuous measurements (5 characters) were log-transformed. All the statistical analyses were carried out with JMP (Ver. 4, SAS Institute 2000).

368 226


Seasonal change of the number of Bs at Yatsukami population Mean numbers of B chromosomes at the Yatsukami population in four successive months in 1994 and in embryos of the next generation are presented in Table 1 and are shown in Fig. 3 together with data obtained in 1997 and 1998 at the same population (Gorlov and Tsurusaki, 2000a). In 1994, the number of Bs increased from August (2.7) to October (6.0). On the other hand, mean number of Bs dropped to 1.4 in embryos laid by females matured in summer 1994 and collected in October (Table 2). Unfortunately, the numbers of chromosomes retained by those females are unknown due to the difficulty in obtaining good chromosomal spreads from females with fully matured ovaries. However, there was a significant difference between the number of Bs retained in those embryos (mean 2.7, n = 39) and those exhibited by males collected in October (mean 6.0, n = 10) when females used for the rearing experiments were also collected (Mann-Whitney U test, P ! 0.0001). It is unlikely that the number of Bs differed between sexes in this generation, since it has been shown that the number of Bs does not differ between sexes, at least in subadults and younger adults just after the final molting (Tsurusaki, 1993; Gorlov and Tsurusaki, 2000a). Decrease of the number of Bs in embryos was also ascertained in additional rearing experiments using females from the same population in 1995 (Tables 1 and 2). In the experiment, a total of 101 eggs laid by six females showed a very low level of B chromosome frequency (mean with SD: 1.07

Table 2. Intraindividual variation in the number of B chromosomes in males (based on spermatogonial metaphases) and embryos (mitotic metaphases in developing eggs) of Psathyropus tenuipes represented by the number of cells showing different numbers of B chromosomes in each individual surveyed in the Yatsukami population in 1994 (for embryos, 1995 data were also included). Only individuals with more than 30 cells counted are shown (for embryos more than 10 cells). Indiv. No.a

Number of B chromosomes 0

1

2

940805-6 940805-13 940805-30juv 940909-1 940909-2 940909-8 940909-9 940909-23 940909-24 940909-26 940909-27 940909-29 940909-31 940909-33 940909-34 940909-35 941011-1 941011-6 941011-7 941011-9

10

7 5 18 2

12

941114-9egg 950126-1egg 951114-10egg

7 8 3

9 11

1

1 1

11 2

5 18 6 18 2 15 11 11 8 19 8 3 2 1

1 5 7

3

4

10 15 21 14 13 15

2 10 1 3 3 5 5 12 4 5

5 25 10 9 18 1 1 2 1 15

2 19 1 14 2 39 5 12 9

1

2 4 1

5

6

16 1 5 8 2 3 1 9 3 6 2 13

4 11 2 1 2 1 4 16 4 1 3 7

2 7 34 9 3 29

6 6 5 8 3 3

4

1 1

7

8

9

1

10

11

1

3

12

4

2 1

2 5

13 11

12 14 3 2 2

8 9 4 8 1

5

9

2

3 2 3

3

6 2 1

n

mean

SD

CV

mode

median

31 34 41 34 51 67 51 35 34 32 43 43 78 38 31 40 103 36 33 58

1.26 4.21 2.88 3.62 3.08 5.60 2.49 3.94 5.68 3.25 3.09 4.79 5.17 2.53 3.90 5.93 5.07 5.89 6.06 4.45

1.12 1.47 2.06 1.79 1.11 3.65 1.22 1.43 0.94 1.46 1.02 2.52 2.35 0.56 1.40 1.90 1.45 1.94 2.14 1.05

0.89 0.35 0.72 0.50 0.36 0.65 0.49 0.36 0.17 0.45 0.33 0.53 0.45 0.22 0.36 0.32 0.29 0.33 0.35 0.24

2 5 1 3 3 3 2 3 6 2 3 8 4 2 4 7 4 5 4 5

1 5 3 3 3 4 2 4 6 3 3 7 5 2.5 4 6.5 5 6 6 5

16 29 14

2.75 1.48 1.64

2.77 1.55 1.15

1.01 1.05 0.70

0 1 2

3 1 2

12

a

Except for one juvenile male labeled “940805-30juv” and three embryos, all the individuals are adults. First six digits of individual number represent year-month-date surveyed (e.g. 940805 denotes August 5, 1994).

B 1.16). When compared with the data obtained from males collected in August 1995 (n = 20, mean with SD: 4.7 B 1.93), the difference was significant again (Mann-Whitney U test, P ! 0.0001). Contrary to this, the number of Bs was stable (mean 6) throughout later postembryonic stages (later stages of juveniles and long period of adults) in 1997 and 1998 (Fig. 3, middle and bottom) as already reported by Gorlov and Tsurusaki (2000a, b). Gorlov and Tsurusaki (2000a) found an interesting trend that the higher frequency of males with even modal number of Bs in June–July decreased with time in 1997. However, no such trend was observed in the 1994 samples. The frequencies of B-even individuals in 1994 did not deviate much from 50 % and remained stable throughout the adult season; the frequencies of B-even individuals including 0B (excluding 0B are in parentheses) were 40.0 (40.0), 50.0 (46.7), 51.6 (51.6), and 60.0 (60.0) % in July, August, September, and October, respectively. No significant difference between months was found (¯2 test, P = 0.90). Contrary to this, frequency of B-even individuals including 0B reached as much as 84.2 % (71.4 % when 0B individuals were excluded) in embryos produced by the 1994 generation (the difference in the frequency between all the adults combined and embryos was significant, ¯2 test, P ! 0.001; though not significant when 0B individuals were excluded, ¯2 test, P 1 0.096). Dominance of B-even embryos over B-odd was also observed in eggs laid in the autumn of 1995 (frequencies of B-even including or excluding 0B individuals

are 68.5 and 48.5 %, respectively). Difference between this frequency and that obtained for males surveyed in August 1995 (40 %, n = 20) was also significant (¯2 test, P = 0.03) when 0B individuals were counted as B-even. However, it is highly probable that this high frequency of B-even in embryos is an inevitable corollary that comes from lower number of Bs (0, 1 or 2) retained in those embryos; that is, if the modal numbers of Bs are less than three in all the embryos, two of the three classes (0B, 1B, 2Bs) are even. When 0B individuals were excluded, no significant difference was found between adults and embryos in the frequency of B-even individuals in both 1994 and 1995. Geographic variation of the number of B chromosomes Data on the number of B chromosomes of P. tenuipes in various populations surveyed in the present study are summarized in Table 1. Population mean of B chromosome number varied from 1.2 of Is. Kokuno-jima in the Seto Inland Sea to 5.5 exhibited by Kawatana (Yamaguchi Pref.) facing the Sea of Japan. Variation among populations was significant (KruskalWallis test, P ! 0.001) even when only populations with more than ten specimens were used (i.e., Amagozen Point, Shirawara Beach, Tatsumidai, Yatsukami [September, 1994 data], and Ushimado) in the analysis to reduce influence of sampling errors. Population mean in the number of Bs ranged from 1.6 in River Arai (Tottori Pref.) to 5.5 in Kawatana (Yamaguchi Pref.) in the populations along the Sea of Japan coast. On the


369 227

Fig. 5. Latitudinal gradients found in mean number of B chromosomes per population (A) and (B–C) five external characters (CL, FIL, SPL, NCD, NN) to latitude. In a graph (B) for CL, correlation coefficient and probability are calculated for the data excluding Is. Hachijo. When Is. Hachijo was included, correlation was not statistically significant in CL.

contrary, in populations along the Seto Inland Sea, it ranged from 1.2 in Is. Kokuno-jima (Hiroshima Pref.) to 2.2 in Ushimado (Okayama Pref.). The results in the two populations from Seto Inland Sea were close to the number (1.7) already known from Is. Nakajima (an island of the Seto Inland Sea, Ehime Pref.) (Tsurusaki, 1993). When 11 populations from the Sea of Japan side and three from the Seto Inland Sea side were compared, the difference in the number of Bs retained in a population was statistically significant (Mann Whitney U test, P ! 0.001). To analyse the general trend of the geographic variation across Japanese Islands, the present data were combined with those formerly obtained in eight populations (Tsurusaki, 1993) (Figs. 4 and 7). Although not straightforward, there was a trend for the number of Bs to be larger in the more northerly populations (correlation between latitudes of populations and the number of Bs was significant, n = 20, r = 0.58, P ! 0.01, Fig. 5A; this was still significant even when a population “Hokkaido Univ. campus, Sapporo” which is represented by only a single male with an extraordinarily high number, 18 Bs; n = 19, r = 0.50, P = 0.03). The numbers of Bs in three populations (Is. Nakajima, Is. Kokuno-jima, and Ushimado) from the Seto Inland Sea were lower (population mean ranges from 1.2 to 2.2) than almost all populations from other areas. Geographic variation of external characters Since latitudinal clines for morphological characters such as body size or length of legs, which are often exhibited as reverse to Bergmann’s rule, are known in some species of harvestmen (Suzuki, 1973a) and other various arthropods (Masaki, 1967; Johansson, 2003; Bidau and Marti, 2004), we examined latitudinal variation for eight morphological characters. Of the eight characters surveyed, FIL, SPL, NCD, NN showed significant

370 228


correlations with latitude and their values decreased with increasing latitude (Fig. 5C–F). CL also tended to decrease with latitude and correlation between them was statistically significant when Is. Hachijo population, which may be a case of insular dwarfism often typical for populations in small isolated islands, was excluded (Fig. 5B, when included, correlation was not statistically significant). We conducted principal component analyses using all the morphological characters. Table 4 presents the loadings on the first two principal components. The first two components account for 65 % of the total variance. The first axis had high positive loadings for CL, FIL, and WSG. We interpreted the first component as a “size” factor. On the other hand, the second principal component showed high positive loadings to SPL, NN, and NCD and which may be summarized as a “projection-denticulation” factor. Figure 6A plots the positions of populations on the first two principal components. Four groups, which broadly correspond to actual geographical locations, were recognized for their positions on the plots, though there was a slight overlap between the “Pacific coast group” and the “Sea of Japan coast group”. When only metric characters (BL, CL, FIL, WSG, and SPL) were employed for the same analysis, a slight gap was generated between the two groups (Fig. 6B), though Kawatana (No. 14), which is located on the Sea of Japan side, was included in the “Pacific ocean group” in the plots. The first and the second principal components in the latter procedure also showed higher positive loadings to the “size”-related and “projection”-related characters, respectively (Table 4). Degree of melanism (DM), which showed only moderate or low loadings to the first two principal components, showed prominently high positive loadings (0.64) to the third principal component, which is also characterized by a high loading (0.58)

Fig. 6. Projection of 21 populations on the first two principal components based on the eight characters measured, counted, and scored (A) and only on five measured characters (B). Three populations in the Seto Inland Sea are connected by dashed lines. 1, Is. Rishiri (Hokkaido, n = 1); 2, Cape Soya (Hokkaido, n = 2); 3, Toyotomi (Hokkaido, n = 2); 4, Maruyama (Sapporo, Hokkaido, n = 12); 5, Oirase (Aomori Pref., n = 3); 6, Obanazawa (Yamagata Pref., n = 6); 7, Awa-Amatsu (Chiba Pref., n = 20); 8, Is. Hachijo (Tokyo Pref., n = 2); 9, Yokohama (Kanagawa Pref., n = 2); 10, Kurosaki

(Toyama Pref., n = 2); 11, Amagozen Point (Ishikawa Pref., n = 43); 12, Shirawara (Uradome Coast, Tottori Pref., n = 4); 13, Kozomi (Tottori city, Tottori Pref., n = 10); 14, Kawatana (Toyoura-cho, Yamaguchi Pref., n = 4); 15, Is. Shimojima (Amakusa, Kumamoto Pref., n = 3); 16, Is. Kamijima (Amakusa, Kumamoto Pref., n = 4); 17, Is. Kuchinoerabu (Kagoshima Pref., n = 1); 18, Is. Nakajima (Ehime Pref., n = 40); 19, Is. Noumi-jima (Hiroshima Pref., n = 1); 21, Ushimado (Okayama Pref., n = 36); 22, Shirahama (Wakayama Pref., n = 29).

for BL. Individuals with dark bodies predominated in the northeastern part of Japan.

was observed between population means of the number of Bs and those of any of the eight morphological characters (e.g. for the number of Bs and FIL, r = 0.28, P = 0.47, n = 9. Other morphological characters showed lower correlation than this with the number of Bs).

Relationship between the number of B chromosomes and external morphology We chose a set of data obtained for the September samples of the Yatsukami population, to analyze the relationship between the number of Bs and external morphology, since this was the sample with a sufficient number of individuals (34 males) to remove the effects of geographical and seasonal variations. There were no significant correlations between the number of Bs and each of the eight morphological characters analyzed: FIL (r = 0.12, P = 0.53), WSG (r = 0.03, P = 0.88), SPL (r = 0.19, P = 0.27), NN (r = 0.20. P = 0.39), NCD (r = nearly 0, P = nearly 1), FA of FIL (r = 0.02, P = 0.91), FA of NN (r = 0.06, P = 0.78), FA of NCD (r = 0.28, P = 0.14). Gorlov and Tsurusaki (2000a) reported that individuals of P. tenuipes with moderate numbers of Bs (3–6) tend to possess a larger body than individuals with extreme numbers of Bs (0–2 or 7–12). We failed to detect such reverse U-shaped relationships between the B frequency and external morphology in any of the eight morphological characters including body size in the 1994 specimens. Unfortunately, the number of males in the September 1994 sample was not sufficient to detect such a trend, if there was any. There were only nine populations in which both the number of Bs and external morphology were available. No correlation

Discussion It has been reported that Psathyropus tenuipes shows considerable geographic variations in coloration of body, length of a dorsal spine on the second abdominal tergite, or number of noduli on femora of legs, etc. (Suzuki 1949, 1973a, 1973b; Suzuki and Tsurusaki, 1983). Suzuki (1949) distinguished populations of the species in Hokkaido, from those in Honshu and other southern Japanese islands by having a short dorsal spine, extremely darkened body, and low number of dorsal denticles on the basal segments of chelicera, and described them as a different subspecies P. tenuipes yezoensis (originally “Gagrella japonica yezoensis”). However, analyses of external characters in the present study revealed that body size of the species represented by the lengths of cephalothorax (CL) and femora of the first legs (FIL) gradually decreases with increasing latitude (Fig. 5B, C). These trends, which conform to the so-called “reverse Bergmann’s rule”, is rather general among invertebrates with univoltine life cycles (Masaki, 1967; Bidau and Martı´, in press) and probably relates to a shorter period available for


371 229

Table 3. Summary of the number of B chromosomes checked for embryos laid by respective mothers of Psathyropus tenuipes reared in the laboratory in two consecutive years (1994 and 1995)

Mother

Number of eggs prepared

min.

max.

mean

SDa

mode

median

0 0 0 0 0

2 4 3 6 2

0.55 1.00 1.50 1.97 1.33

0.82 – – 1.88 –

0 0 0/3 0 2

0 0 1.5 2 2

1.37

1.63

2.00 1.80 0.60 0.20 1.31 0.88

– 1.14 0.70 – 1.38 –

2 2 0 0 1 0/1

2 2 0.5 0 1 1

1.07

1.16

94-No.4 94-No.5 94-No.7 94-No.8 94-No.9

19 5 4 31 3

11 5 2 18 3

Total

62

39

95-No.1 95-No.4 95-No.14 95-No.19 95-No.20 95-No.24

2 14 21 21 24 19

1 10 10 7 13 8

101

54

Total a

Number of Bs in embryos

2 0 0 0 0 0

2 4 2 4 4 2

Data with more than 10 embryos were used to calculate SD.

growth in areas of higher latitudes. Three other characters, including “dorsal spine length (SPL)” and “number of dorsal denticles (NCD)” which were used for the separation of the “two subspecies” in Suzuki (1949) also showed the same trend, without any indication of discontinuities. Thus, there are no external characters that support the separation of the species into two subspecies. Instead of two groups, principal component analyses using eight morphological data of 21 populations unveiled the occurrence of four geographical groups that roughly reflect actual geographical locations: 1) northern Hokkaido, 2) Sea of Japan coast, 3) Pacific coast, and 4) Is. Hachijo. Of these, two major groups, “Sea of Japan coast” and “Pacific coast” were mainly segregated along the second principal component axis, which can be interpreted as a “projection-denticulation” factor. The number of B chromosomes in this species also varied considerably among populations with a range from 1.2 to 7 (a single male from the campus of Hokkaido University, Sapporo, showed 18 Bs) (Figs. 3 and 7). The mean number of Bs per population significantly correlated with latitude. As already stated, negative latitudinal gradients were also found for five morphological characters (Fig. 5B–F). However, there were no correlations between the mean number of Bs per population and mean values of morphological characters. Furthermore, mean number of Bs did not correlate with any of the eight morphological characters examined when they were analyzed within a single population, Yatsukami. These findings conform to the expectation that the Bs in this species might be almost genetically inactive (Tsurusaki, 1993; Gorlov and Tsurusaki, 2000b) since they are mostly heterochromatic. However, if they were completely inactive and had no effect on phenotypes, what determines the northward increase of their number? Gorlov and Tsurusaki (2000a) found that susceptibility to gregarines, which are protozoan parasites frequently found in mid-gut of harvestmen, may be different between individuals with odd and even numbers of Bs. For example, infection rate by gregarines was lower in individuals with moderate numbers

372 230

Number of eggs with countable mitotic spreads


Table 4. Loadings of the parameters on the first two principal components (PC) in two different procedures: all the characters measured, counted, scored were used (8 characters) and only measured characters were analyzed (5 characters). Parameter

1 2 3 4 5 6 7 8

8 characters

5 characters

PC1

PC2

PC1

PC2

Femur I length (FIL) Body length (BL) Cephalothorax length (CL) Width between scent gland pores (WSG) Length of 2nd tergite spine (SPL) Number of denticles on chelicera (NCD) Number of noduli on femur II (NN) Degree of melanism of body (DM)

0.44 0.33 0.53 0.46 –0.05 0.27 –0.11 –0.35

0.22 –0.14 0.01 –0.18 0.63 0.45 0.56 0.01

0.42 0.43 0.58 0.54 –0.08

0.42 –0.14 0.13 –0.23 0.85

Explained variance (%)

41.1

23.9

55.0

23.2

of Bs (4–6) than in those with both extremes (0–2 or 8–12) in B-even individuals, whereas no such trend was detected in Bodd individuals (Gorlov and Tsurusaki, 2000a). The conceptual background that may generate this type of odd-even effect is rather weak, since each of these harvestmen is actually a mosaic of B-even and B-odd cells and how a particular individual is classified into B-odd or B-even simply depends on the point that whether modal number of Bs among cells was odd or even. However, if there was any relationship between having a moderate number of Bs and resistance to gregarine infection in this species as inferred by Gorlov and Tsurusaki (2000a), a northward increase in B number might be explained in the context of possible geographic variation in the abundance of gregarines. This supposition would expect a lower level of gregarine infection in populations with fewer Bs along the Seto Inland Sea coast than in the other populations, though no data have been available for the frequencies of gregarine infection in populations other than Yatsukami.

Northern Hokkaido group Populations mean of B-chromosome numbers >5

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