Reproductive Biology and Phylogeny of Birds: Phylogeny, Morphology, Hormones, Fertilization (Reproductive Biology and Phylogeny, Vol 6A)

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Reproductive Biology and Phylogeny of Birds Phylogeny Hormones

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Morphology Fertilization

Reproductive Biology and Phylogeny Series Series Editor: Barrie G. M. Jamieson

Published: Vol. 1: Reproductive Biology and Phylogeny of Urodela (Volume Editor: David M. Sever) Vol. 2: Reproductive Biology and Phylogeny of Anura (Volume Editor: Barrie G. M. Jamieson) Vol. 3: Reproductive Biology and Phylogeny of Chondrichthyes (Volume Editor: William C. Hamlett) Vol. 4: Reproductive Biology and Phylogeny of Annelida (Volume Editor: G. Rouse and F. Pleijel) Vol. 5: Reproductive Biology and Phylogeny of Gymnophiona (Caecilians) (Volume Editor: Jean-Marie Exbrayat) Vol. 6A: Reproductive Biology and Phylogeny of Birds (Volume Editor: Barrie G. M. Jamieson)

In press/under preparation: Vol. 6B: Reproductive Biology and Phylogeny of Birds (Volume Editor: Barrie G. M. Jamieson) Vol. 7: Reproductive Biology and Phylogeny of Cetacea (Volume Editor: D. Miller)

Reproductive Biology and Phylogeny of Birds Part A

Phylogeny Hormones

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Morphology Fertilization

Volume edited by BARRIE G.M. JAMIESON School of Integrative Biology University of Queensland St. Lucia, Queensland Australia

Volume 6A of Series: Reproductive Biology and Phylogeny Series edited by BARRIE G.M. JAMIESON School of Integrative Biology University of Queensland THE UNIVERSITY St. Lucia, Queensland OF QUEENSLAND AUSTRALIA Australia

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SCIENCE PUBLISHERS An imprint of Edenbridge Ltd., British Isles.

Post Office Box 699 Enfield, New Hampshire 03748 United States of America Website: http://www.scipub.net sales @ scipub.net (marketing department) [email protected] (editorial department) [email protected] (for all other enquiries) ISBN (Series)

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ISBN (Set)

978-1-57808-502-6

ISBN (Vol 6 Pt A) 978-1-57808-386-2 ISBN (Vol 6 Pt B)

978-1-57808-444-9

© 2007, Copyright reserved

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying or otherwise, without the prior permission. This book is sold subject to the condition that it shall not, by way of trade or otherwise be lent, re-sold, hired out, or otherwise circulated without the publisher’s prior consent in any form of binding or cover other than that in which it is published and without a similar condition including this condition being imposed on the subsequent purchaser. Published by Science Publishers, Enfield, NH, USA An imprint of Edenbridge Ltd. Printed in India

Preface to the Series This series was founded by the present series editor, Barrie Jamieson, in consultation with Science Publishers, in 2001 and bears the title ‘Reproductive Biology and Phylogeny’, followed in each volume with the name of the taxonomic group which is the subject of the volume. Each publication has one or more invited volume editors (sometimes the series editor) and a large number of authors of international repute. The level of the taxonomic group which is the subject of each volume varies according, largely, to the amount of information available on the group, the advice of proposed volume editors, and the interest expressed by the zoological community in the proposed work. The order of publication of taxonomic groups reflects these concerns, and the availability of authors for the various chapters, and it is not proposed to proceed serially through the animal kingdom in a presumed “ladder of life” sequence. A second aspect of the series is coverage of the phylogeny and classification of the group, as a necessary framework for an understanding of reproductive biology. Evidence for relationships from molecular studies is an important aspect of the chapter on phylogeny and classification. Other chapters may or may not have phylogenetic themes, according to the interests of the authors. It is not claimed that a single volume can, in fact, cover the entire gamut of reproductive topics for a given group but it is believed that the series gives an unsurpassed coverage of reproduction and provides a general text rather than being a mere collection of research papers on the subject. Coverage in different volumes varies in terms of topics, though it is clear from the first volumes that the standard of the contributions by the authors will be uniformly high. The stress varies from group to group; for instance, modes of external fertilization or vocalization, important in one group, might be inapplicable in another. The first five volumes on Urodela, edited by Professor David Sever, Anura, edited by myself, Chondrichthyes, edited by Professor William Hamlett, Annelida, edited by Dr. Greg Rouse and Professor Fredrik Pleijel, and Gymnophiona, edited by Professor Jean-Marie Exbrayat, reflected the above exacting criteria and the interests of certain research teams. This, the sixth volume, in two parts, has resulted from my interest in the natural history of birds, which was stimulated in childhood by Gilbert White’s incomparable

LE Reproductive Biology and Phylogeny of Birds ‘Natural History of Selborne’, and my good fortune in being joined by a most distinguished group of authors who need no introduction to those familiar with avian studies. A volume in preparation is on Cetacea (Debra Miller). My thanks are due to the School of Integrative Biology, University of Queensland, for facilities, and especially to the Executive Dean, Biological & Chemical Sciences, Professor Mick McManus, for his continuing encouragement. I am everlastingly indebted to Sheila Jamieson, who has supported me indirectly in so many ways in this work. I am grateful to the publishers for their friendly support and high standards in producing this series. Sincere thanks must be given to the volume editors and the authors, who have freely contributed their chapters, in very full schedules. The editors and publishers are gratified that the enthusiasm and expertise of these contributors has been reflected by the reception of the series by our readers.

THE UNIVERSITY OF QUEENSLAND AUSTRALIA

21 February 2006

Barrie G.M. Jamieson School of Integrative Biology University of Queensland

Classification and Phylogeny

LEE

Preface to this Volume There are almost ten thousand known species of birds of which more than half are song birds. They are an ideal subject of study as they are one of the few groups of animals for which almost the total number of species is estimated to be known, they have been comprehensively catalogued and illustrated and are readily identified in the field whereas groups such as annelids (Volume 5) require detailed microscopical work for identification. Besides these practical qualifications for study and the biological questions that they pose, they have endeared themselves to humanity not least for their beauty and their song. This volume, in two parts, attempts to document most of the important aspects of the reproductive biology of birds and places them in a setting of phylogenetic relationships. Aspects of reproduction that comprise this, the first part of volume 6, are classification and phylogeny as revealed by molecular biology; anatomy of the male reproductive tract and organs; anatomy and evolution of copulatory structures; development and anatomy of the female reproductive tract; endocrinology of reproduction; ovarian dynamics and follicle development; spermatogenesis and testicular cycles; avian spermatozoa: structure and phylogeny; testis size, sperm size and sperm competition and, lastly, fertilization. We may here, albeit superficially, sample some of the many topics treated in the ten chapters. As more and more homologous DNA sequences and other genetic characters become available for more and more species, we are gradually resolving the remaining uncertain nodes of the avian phylogenetic tree, and this trend will only accelerate as DNA sequencing becomes both cheaper and more reliable. These are heady times in avian systematics and the present state of our knowledge is authoritatively reviewed. The testes of birds are intra-abdominal, and, in contrast with most mammals, they do not migrate from their site of embryological origin. They are, thus, closely related, topographically, to the kidneys. The anatomy and histology of avian testes and their ducts receive a detailed examination. In birds, unlike most other animal classes, males of some species possess an intromittent organ, whereas males of other species do not. Thus, in birds at least, the organ does not seem to be necessary for internal fertilization. This raises the question whether the avian intromittent organ has evolved as a

LEEE Reproductive Biology and Phylogeny of Birds primary sexual trait simply for the delivery of sperm or as a secondary sexual trait. Its absence in so many species is an evolutionary puzzle investigated here. Only the left genital primordia develop to functional ovaries in birds except in a few cases, particularly birds of prey. The reason for the unilateral development of female genital organs might be to reduce weight for flying. The question then arises why the falconiforms allow themselves the luxury of two genital tracts. To reduce weight their hard-shelled eggs are relatively small and are laid down at an early stage of development. This and other aspects of the female genital tract are examined. The Müllerian and Wolffian ducts receive detailed treatment and a valuable analysis of the function of the oviduct in egg laying is provided. In an innovative chapter hormonal control systems in birds are investigated in four major parts: 1) types of environmental signals that influence reproduction and how they are perceived; 2) the hypothalamus as an integrator of environmental information from the external and internal environments, biological clock, etc., that through neuroendocrine and neural secretions affects all aspects of reproduction; 3) the hypothalamo-pituitary unit that transduces environmental information processed by higher centers into endocrine secretions; 4) the functional gonads (ovary and testis) themselves. A unique morphological and functional aspect of the reproductively active avian ovary, as contrasted with the mammalian counterpart, is that follicles at all stages of development, from resting primordial and primary follicles to the fully differentiated preovulatory stage, exist simultaneously during egglaying. As a consequence, the sequential selection of one undifferentiated follicle into the final rapid growth stage of development provides for ovulation of an oocyte from a fully differentiated follicle on an approximate daily basis. The ovarian follicular hierarchy is a reflection of oviparity, and is a feature held in common with avian predecessors, the reptiles including, probably, some dinosaurs. Other aspects of ovarian function are examined. The seminiferous epithelium in avian testis is made up of germ cells at varying levels of development, and Sertoli cells. Sertoli cells have multiple functions, including formation of the blood-testis barrier by means of junctional complexes, and providing anchorage and nutrition for, as well as regulation of, germ cells during development. The most primitive or immature germ cells lie on the basement membrane and the mature germ cells, the spermatozoa, line the lumen of the seminiferous tubule. Germ cells develop in close association with one another because as they divide they maintain close linkage through intercellular bridges which are the result of incomplete cytoplasmic divisions. A detailed examination of spermatogenesis and factors affecting it is given from spermatogonia to the mature spermatozoa. The chapter on sperm structure and phylogeny is the longest of the book, in keeping with the great amount of information, though often fragmentary, which is available and the fact that the chapter represents the final scientific

Preface to this Volume

EN

contribution of any magnitude by the editor. Former workers recognized the ‘sauropsid’ features of non-passerine spermatozoa. However, the ratite and lower non-passerine spermatozoon, especially the former, are shown to more appropriately be termed crocodiloid. Features of Ostrich sperm which are similar to those of crocodiles are described. These features are also seen in turtles and are basic (symplesiomorphic) to amniotes, only the fibrous sheath and the long distal centriole being amniote synapomorphies. The sole spermatozoal synapomorphy of crocodiles and birds is the dense sheath investing the two central singlets within the elongate distal centriole. In birds this sheath is known only in ratites (Ostrich), galliforms and anseriforms. The literature is reviewed in a phylogenetic context. Almost since the moment that animal semen was first viewed under the microscope, in the seventeenth century, it was clear that the shape and size of spermatozoa varied from one species to the next. Early comparative biologists similarly noted striking differences in the size of the testis across different species. Although the general features of the male reproductive system are rather conservative across extant species of birds, recent work has confirmed large interspecific variation in the sizes of both testes and sperm. The chapter addresses the question why should testes mass and sperm size differ so much from one species to the next? Until recently, the cellular and molecular events comprising fertilization in birds were poorly understood. However, several recent studies in mammals as well as in birds have considerably contributed to our knowledge of the fertilization process. In mammals only a single spermatozoon enters the egg. Monospermy is ensured by a variety of mechanisms, which is collectively known as the block to polyspermy. Unlike mammals, the principal feature of fertilization in birds is physiological polyspermy, penetration of the ovum by many sperm. It has been suggested that polyspermy occurs in animals that produce large yolky (megalecithal) ova and this theme is developed. The structure of the mature ovum and the events of fertilization are discussed. There is an extensive section on assisted reproductive technologies. Many new illustrations are provided throughout the volume. The generosity of authors and publishers who have allowed illustrations to be reproduced is most gratefully acknowledged. The kind collaboration of all the authors, who have borne uncomplainingly the requests of an editor overseeing the gestation of this work, has been greatly appreciated. Finally, the courteous and efficient participation of the publishers was indispensable to production of this volume.

THE UNIVERSITY OF QUEENSLAND AUSTRALIA

21 February 2006

Barrie G.M. Jamieson School of Integrative Biology University of Queensland

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Contents Preface to the Series—Barrie G. M. Jamieson Preface to this Volume—Barrie G. M. Jamieson

v vii

1. Classification and Phylogeny of Birds John Harshman

1

2. Anatomy of the Testis and Male Reproductive Tract Tom A. Aire

37

3. Anatomy and Evolution of Copulatory Structures Robert Montgomerie and James Briskie

115

4. Developmental Anatomy of the Female Reproductive Tract Monika Jacob and Murray R. Bakst

149

5. Endocrinology of Reproduction G. E. Bentley, K. Tsutsui and J. C. Wingfield

181

6. Ovarian Dynamics and Follicle Development A. L. Johnson and Dori C. Woods

243

7. Spermatogenesis and Testicular Cycles Tom A. Aire

279

8. Avian Spermatozoa: Structure and Phylogeny Barrie G. M. Jamieson

349

9. Testis Size, Sperm Size and Sperm Competition James V. Briskie and Robert Montgomerie

513

10. Fertilization Urszula Stepinska and Murray R. Bakst Index

553

589

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About the Series This series bears the title ‘Reproductive Biology and Phylogeny’ followed by the name of the taxonomic group which is the subject of the volume. B. G. M. Jamieson is the founding series editor and each publication has one or more invited volume editors (if not the series editor) and a large number of authors of international repute. The series gives a unique coverage of reproductive biology. The level of the taxonomic group which is the subject of each volume varies according, largely, to the amount of information available, the advice of proposed volume editors, and the interest expressed by the zoological community in the proposed work. Phylogeny and classification is covered as a necessary framework for an understanding of reproductive biology. Evidence for relationships from molecular studies is an important aspect of the phylogenetic section. About the Volume There are almost ten thousand known species of birds of which more than half are song birds. They are an ideal subject of study as they are one of the few groups of animals for which almost the total number of species is estimated to be known, they have been comprehensively catalogued and illustrated and are readily identified in the field. Besides these practical qualifications for study and the biological questions that they pose, they have endeared themselves to humanity not least for their beauty and their song. This volume, in two parts, attempts to document most of the important aspects of the reproductive biology of birds and places them in a setting of phylogenetic relationships. Aspects of reproduction that comprise this, the first part of volume 6, are classification and phylogeny as revealed by molecular biology; anatomy of the male reproductive tract and organs; anatomy and evolution of copulatory structures; development and anatomy of the female reproductive tract; endocrinology of reproduction; ovarian dynamics and follicle development; spermatogenesis and testicular cycles; avian spermatozoa: structure and phylogeny; testis size, sperm size and sperm competition and, lastly, fertilization. Many new illustrations are included. About the Series and Volume Editor Dr. Barrie Jamieson is Emeritus Professor of Zoology in the School of Integrative Biology, University of Queensland. He holds a Ph.D. from the University of Bristol, England, a D.Sc. from the University of Queensland, and is a former Visiting Fellow of, and member of the Association of Corpus Christi College, Cambridge. In 1990 he was awarded the Clarke Medal for Research in Natural Sciences, early recipient of which were Thomas Henry Huxley and Richard Owen. His chief field of research is spermatozoal ultrastructure and its relevance to phylogeny but he is also an authority on taxonomy of earthworms and has published on bioluminescence, trematode taxonomy and life cycles, and DNA-based phylogenetics. He has published nearly 200 scientific papers and is the author, coauthor or editor of fourteen books.

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CHAPTER

1

Classification and Phylogeny of Birds John Harshman

1.1

INTRODUCTION

Avian phylogenetics is currently in its infancy, and paradoxically it is also nearing maturity. By infancy I mean that we do not currently know much about the relationships among birds, and by maturity I mean that within a few years we will know most of what there is to know. The reason for this apparent paradox lies in the rapidly increasing ease of gathering and analyzing molecular data. Our ignorance is largely a matter of our not yet having gathered enough DNA sequences or other molecular data for enough species, and that condition is being remedied at an increasing rate. If I had written this chapter a year or two from now, I would have been able to cite studies incorporating perhaps twice the data so far in published form—this purely from yet-unpublished studies of whose existence I am now aware— and I would have been able to say much more about the structure of the avian tree. But the editor was not anxious to follow that schedule, and so we press on with what is currently available. During more than two thousand years of avian systematics—counting from Aristotle—researchers were able to divide birds into a number of robust groups based on distinctive morphologies: the non-passerine families. (We will get to passerines in a moment.) This process was largely completed by the beginning of the last century. For reviews see Sibley and Ahlquist (1990) and Cracraft et al. (2004). Progress since then, on relationships between and within families, has been highly incremental, so much so that at least one prominent avian systematist (Stresemann 1959) announced that no further progress was possible. The cladistic revolution did little to help, though at least it focused our attention on the right questions; apparently it’s very difficult to find reliable morphological characters that will build a robust tree of birds. And the first 30 years of the molecular revolution have illuminated only a few of 4869 Pepperwood Way, San Jose, CA 95124; E-mail: [email protected]

Reproductive Biology and Phylogeny of Birds

those questions (Sheldon and Bledsoe 1993), leading another pair of systematists, once again, to wonder if there would be no further progress (Poe and Chubb 2004). However, recent discoveries suggest that there is more reason for optimism. This chapter will concentrate on what we do know confidently about avian relationships. Necessarily, it must treat mostly relationships between larger groups, since the nearly 10,000 living species are too many to be covered in detail. It is also convenient to divide the birds into two groups, only one of them monophyletic: passerines and non-passerines. One reason for separate treatment is that it splits the species roughly in half, but the main reason is that our state of knowledge differs radically between the groups. For nonpasserines, most traditional families are clearly supported as monophyletic, and we can summarize relationships as being between these entities. (The same is not true of traditional orders, as we will see.) I have used the classification of del Hoyo et al. (1992-2002) as representative of traditional familial and ordinal assignments. For passerines, however, most traditional families that have been adequately sampled are not monophyletic, and we must either speak in extremely general terms or choose individual species as our units of discussion. In both groups, genera have not fared well, and many have proven not to be monophyletic when examined closely. Non-monophyly should come as no surprise. Traditional taxonomy did not take monophyly into account. What mattered was distinctness, and whether that distinctness arose from apomorphy or plesiomorphy was not considered important. Thus if some subset of a group possessed a prominent apomorphy, that served to characterize two subgroups: one by its presence, and another by its absence. This practice has continued even to the present, e.g. the vigorous defense by Short and Horne (2002) of separating Ramphastidae from Capitonidae. One caveat should be kept in mind when assessing the results of phylogenetic analyses: they can discern the relationships only among those species actually included in the analysis. This rule may seem obvious, but it has been violated frequently in the literature. If an analysis of gruiform phylogeny includes only gruiforms and a single outgroup, the monophyly of gruiforms cannot be tested. If an analysis of bird phylogeny includes only one representative from each order, the monophyly of orders cannot be tested. If an attempt to find the sister group of hoatzins does not include the actual sister group (whatever it may be), that relationship will not be found. It would thus seem that every phylogenetic analysis, in order to be valid, must include every species of bird (and that further assumes that birds are monophyletic, and that each individual species is also), which is clearly impossible. Fortunately, chaos is not total, and uncertainties are of limited taxonomic scope: families whose ordinal relationships are unclear have themselves so far proven to be monophyletic; all members of non-monophyletic families can be constrained within single orders, and all members of non-monophyletic genera within single families. (The latter does not apply to passerines, however.) Some

Classification and Phylogeny of Birds

!

groups do have clear morphological synapomorphies, and these can be easy to assay across species. Genetic distances offer some comfort too. If we find all species of Anas to be genetically similar, it’s unlikely that any of them is really a bustard, even if we do not include them all in an analysis with a bustard. But we must still be careful not to overinterpret any results. Because of their importance to avian systematics, I must discuss the DNA hybridization studies of Sibley and Ahlquist (1990) and the classification based on them (Sibley and Monroe 1990). Their final product, the “Tapestry”, is a comprehensive and nearly fully resolved tree of over 1100 species (Sibley and Ahlquist 1990, figs. 357-385). However important, this tree was severely flawed as a representation of their data, and the data themselves were insufficient to resolve objectively many of the relationships asserted by the Tapestry (Lanyon 1992; Harshman 1994). A later attempt to correct some of the flaws of the Tapestry by using rigorous methods of analysis on subsets of the data (Sibley and Ahlquist 1990, figs. 325-352)—since they used the FitchMargoliash (1967) method of distance analysis, we may call them “Fitch trees”—is an improvement but suffers from the absence of an evaluation of strength of support, which Harshman (1994) attempted to repair by reanalyzing the data. These latter analyses should be preferred to the Tapestry in the several cases of conflict. Sibley and Ahlquist’s studies contain some exciting, corroborated insights, e.g., the sister relationship of mockingbirds (Mimidae) and starlings (Sturnidae), but also some major mistakes, e.g., the placement of storks (Ciconiidae) and New World vultures (Cathartidae) as sister taxa, and even some decisions unjustified by any data, e.g., the placement of hoatzin (Opisthocomidae) with cuckoos (Cuculiformes). In the end, without corroboration, it is difficult to separate the Tapestry’s good estimates from the bad, even with access to their original data (courtesy of the late C. G. Sibley). Below, I will mention Sibley and Ahlquist’s findings only when they have been independently corroborated or if their conclusions are clearly supported by their data, properly analyzed.

1.2

NON-PASSERINES

Relationships among the non-passerine families are not well resolved at present. If we consider a tree of relationships among the 103 non-passerine families recognized by del Hoyo et al. (1992-2002) plus the order Passeriformes, that tree, if fully resolved, will have 103 internal nodes (counting the root position). We can be fairly confident of only 60 of those nodes (Figs. 1.1 and 1.2). In contrast to many other taxa, the most basal relationships are among the best known, but most other resolved nodes unite small numbers of closely related families, such as Dromaiidae (emu) and Casuariidae (cassowaries).

1.2.1

Basal Relationships

Modern birds—Aves or Neornithes depending on one’s taste in terminology (Gauthier et al. 2001)—are divided basally into two clades, Palaeognathae and

" Reproductive Biology and Phylogeny of Birds

Fig. 1.1 Relationships among non-passerine families, so far as they can be confidently asserted at present according to my perhaps biased estimate. This tree does not derive from a formal analysis, either of a combined supermatrix or by a rigorous supertree method. Branches in gray represent paraphyletic groups. This figure shows Paleognathae through Metaves. For Coronaves see Fig. 1.2. For reasons behind the topology chosen, see text.

Neognathae. Palaeognathae includes the ratites and tinamous. Neognathae is divided into Galloanserae, consisting of the sister orders Anseriformes and Galliformes, and Neoaves, consisting of all other birds. Recent analyses, both molecular and morphological, have been nearly unanimous on these four basal clades (Prager and Wilson 1978; Caspers et al. 1997; Groth and Barrowclough 1999; García-Moreno and Mindell 2000; van Tuinen et al. 2000; Paton et al. 2002; Cracraft and Clarke 2001; García-Moreno et al. 2003; Mayr and Clarke 2003; Cracraft et al. 2004). Some other studies lacked an outgroup root but are consistent with these clades (Sibley and Ahlquist 1990; Chubb 2004a; Fain and Houde 2004). A few studies using whole mitochondrial genomes or large fractions thereof have found a different topology, in which passerines are basal to all other


#

Fig. 1.2 Relationships among non-passerine families, continued. Coronaves only. For reasons behind the topology chosen, see text.

$ Reproductive Biology and Phylogeny of Birds birds (Mindell et al. 1997; Härlid and Arnason 1999; Mindell et al. 1999). But these have been shown to be the result of long branch attraction (Braun and Kimball 2002; Paton et al. 2002; García-Moreno et al. 2003). Also see Ericson et al. (2001), which failed to find a monophyletic Galloanserae. A note on non-monophyly: there are two forms of non-monophyly, paraphyly and polyphyly. Strictly speaking, the two cannot be distinguished on a phylogenetic tree, since the definitions require either explicit assignment of ancestral nodes to groups or optimization of diagnostic characters. In this chapter, I will define paraphyly artificially: if we code group membership as a presence/absence character, a group is paraphyletic if that state can be optimized as gained only once on the tree and lost one or more times. It is polyphyletic if group membership must be optimized as being gained at least twice. This favors paraphyly in ambiguous cases. Results may differ from those under other definitions, but at least the condition can be determined from the tree itself.

1.2.2 Palaeognathae Almost all studies using non-avian outgroups have found paleognaths to be monophyletic (García-Moreno and Mindell 2000; van Tuinen et al. 2000; Paton et al. 2002; Cracraft et al. 2004; but see Chapter 8). Under that assumption, we can add studies using neognath outgroups, and these have found the two paleognath orders, Struthioniformes (ratites) and Tinamiformes (tinamous) also to be monophyletic (Sibley and Ahlquist 1990; Lee et al. 1997; van Tuinen et al. 1998; Cooper et al. 2001; Cracraft and Clarke 2001; Haddrath and Baker 2001). Within ratites, however, there is considerable contention. It is universally agreed in all these studies that Dromaiidae (emu) and Casuariidae (cassowaries) are sister taxa. Molecular studies commonly make Apterygidae (kiwis) the sister of these two, forming an Australasian clade (Sibley and Ahlquist 1990; Lee et al. 1997; van Tuinen et al. 1998; Cooper et al. 2001; Haddrath and Baker 2001). The question of whether the Rheidae (rheas) or Struthionidae (ostrich) is the sister of all other ratites is less clear; the majority of studies have found the rhea basal (Cooper et al. 1992; Lee et al. 1997; Cooper et al. 2001; Haddrath and Baker 2001), but others have found the ostrich basal (Sibley and Ahlquist 1990; van Tuinen et al. 1998). The sole morphological study has the same unrooted topology as the molecular studies, but the root attaches to the kiwi, making the rooted topology quite different (Lee et al. 1997). Phylogenetic relations within tinamous have been little studied. There is only one published morphological phylogeny (Bertelli et al. 2002) and no molecular studies.

1.2.3 Galloanserae Monophyly of Galloanserae is affirmed by a large number of studies (Sibley and Ahlquist 1990; Caspers et al. 1997; Livezey 1997; Groth and Barrowclough 1999; García-Moreno and Mindell 2000; van Tuinen et al. 2000;


%

Zusi and Livezey 2000; Cracraft and Clarke 2001; Paton et al. 2002; GarcíaMoreno et al. 2003; Mayr and Clarke 2003; Sorenson et al. 2003; Cracraft et al. 2004). But see Ericson (1997) and Ericson et al. (2001). There is also strong support for monophyly of both Galliformes and Anseriformes (Livezey 1986; 1997a; Cracraft and Clarke 2001; Zusi and Livezey 2000; Ericson et al. 2001; Sorenson et al. 2003; Chubb 2004a; Cracraft et al. 2004), though molecular studies with large taxon samples of both orders have not been published. Sibley and Ahlquist (1990) found strong support for monophyly of Galliformes, but monophyly of Anseriformes could not be confirmed (Harshman 1994), and Chubb (2004a) also could not confirm anseriform monophyly.

1.2.3.1

Relationships within Galliformes

All families are monophyletic except Phasianidae (pheasants), which includes the two traditional families Meleagrididae (turkeys) and Tetraonidae (grouse) (Dimcheff et al. 2002). The monophyly of Numididae (guineafowl) is not supported by morphological characters (Dyke et al. 2003), but at least two of the four genera have been shown to be closely related by DNA hybridization (Sibley and Ahlquist 1990). Basal relationships are clear: Megapodiidae (megapodes) and Cracidae (curassows, guans, and chachalacas) are successive sister groups to the rest (Sibley and Ahlquist 1990—the Fitch tree, contradicting the Tapestry; Ericson et al. 2001; Dimcheff et al. 2002; Dyke et al. 2003; Sorenson et al. 2003; Mayr and Weidig 2004; Pereira and Baker 2006). The major puzzle is whether the sister group of Phasianidae is Odontophoridae (New World quail) (Armstrong et al. 2001; Dimcheff et al. 2002) or Numididae (Sibley and Ahlquist 1990; Kornegay et al. 1993; Kimball et al. 1999; Armstrong et al. 2001; Dimcheff et al. 2002; Pereira and Baker 2006). Some studies support both possibilities in different analyses. There are several studies of relationships within families: megapodes (Birks and Edwards 2002), cracids (Pereira et al. 2002), and phasianids (Kimball et al. 1999; Dimcheff et al. 2002). The phasianid genus Francolinus is polyphyletic (Bloomer and Crow 1998), and the tetraonid genera Bonasa and Falcipennis are paraphyletic (Ellsworth et al. 1996; Dimcheff et al. 2002; Drovetski 2002).

1.2.3.2

Relationships within Anseriformes

Monophyly of Anhimidae (screamers) is easily confirmed, as the three species are quite closely related (Livezey 1986; Sibley and Ahlquist 1990). Sibley and Ahlquist’s (1990) Tapestry makes Anseranas (magpie goose, sometimes given its own monotypic family Anseranatidae) the sister group of Anhimidae, thus making Anatidae (ducks) paraphyletic, but other analyses of the same data (Sibley and Ahlquist 1990’s Fitch tree; Harshman 1994) restore duck monophyly. In all other analyses, Anatidae is monophyletic (Livezey 1986; 1997a; Ericson 1997; Ericson et al. 2001; Cracraft et al. 2004). Relationships within Anatidae are contentious. Though the basal relationships are well established (Anseranas and Dendrocygninae as successive

& Reproductive Biology and Phylogeny of Birds sister groups to the remaining anatids), the remaining relationships differ greatly between morphological (Livezey 1997b and references therein) and molecular (Madsen et al. 1988; Sraml et al. 1996; Donne-Goussé et al. 2002; Callaghan and Harshman 2005 and references therein) analyses. Only one genus has been shown to be paraphyletic. As might be expected, this is the largest, Anas, which includes the steamer ducks (Tachyeres) as well as three monotypic genera that are sometimes merged with Anas (Amazonetta, Speculanas, Lophonetta) (Johnson and Sorenson 1999).

1.2.4

Neoaves

Monophyly of Neoaves, also called Plethornithes by Groth and Barrowclough (1999), has been confirmed in all recent studies (Groth and Barrowclough 1999; Sorenson et al. 2003; Cracraft et al. 2004; Fain and Houde 2004). It encompasses the great majority of all birds, including Passeriformes.

1.2.4.1

Monophyly of neoavian orders

There are 23 non-passerine orders within Neoaves. Of these, 9 consist of just a single family, and their monophyly is trivial. Two more orders, Psittaciformes (parrots) and Strigiformes (owls) each consist of just two closely related families, the psittaciform pair often merged into a single family, Psittacidae. Thus there are 12 orders for which monophyly is a serious question. Of these, we have good evidence of monophyly for four, good evidence against monophyly for five, and no strong evidence either way for three. The single-family orders are Sphenisciformes (penguins), Gaviiformes (loons/divers), Podicipediformes (grebes), Phoenicopteriformes (flamingos), Opisthocomiformes (hoatzin), Pterocliformes (sandgrouse), Columbiformes (pigeons), Coliiformes (colies), and Trogoniformes (trogons). The clearly monophyletic orders (other than those with only one family) are Procellariiformes (tubenoses), Apodiformes (swifts and hummingbirds), Galbuliformes (puffbirds and jacamars), and Piciformes (woodpeckers, barbets, and honeyguides). The monophyly of both Pelecaniformes and Ciconiiformes is falsified by the close relationship among the pelecaniform family Pelecanidae (pelicans) and the two ciconiiform families Scopidae (hamerkop) and Balaenicipitidae (shoebill) (Hedges and Sibley 1994; Siegel-Causey 1997; van Tuinen et al. 2001; Cracraft et al. 2004; Fain and Houde 2004; but see Mayr 2003). There remains a group that we might call “core pelecaniforms” except that it fails to include pelicans, consisting of Sulidae (boobies), Phalacrocoracidae (cormorants), Anhingidae (darters), and Fregatidae (frigatebirds) (Sibley and Ahlquist 1990; Harshman 1994; Cracraft et al. 2004). Relationships of the remaining traditional pelecaniform family, Phaethontidae (tropicbirds), are unclear, as are relationships of the remaining traditional ciconiiforms. Caprimulgiformes is at least paraphyletic, since one caprimulgiform family, Aegothelidae (owlet-nightjars) is the sister of Apodiformes (Mayr 2002a; Cracraft et al. 2004); whether the order would be monophyletic if apodiforms were included is unclear.


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The monophyly of both Gruiformes and Charadriiformes is falsified by the discovery that one gruiform family, Turnicidae (buttonquails) is deeply nested within charadriiforms (Paton et al. 2003). However, charadriiforms are monophyletic with the addition of turnicids (Paton et al. 2003). But there is no evidence for monophyly of gruiforms minus turnicids, and some evidence against it (Fain and Houde 2004). There is a group we might call “core gruiforms”, consisting of Gruidae (cranes), Aramidae (limpkin), Psophiidae (trumpeters), Rallidae (rails), and Heliornithidae (finfoots) (Houde et al. 1997; Livezey 1998; Cracraft et al. 2004; Fain and Houde 2004). For another view, see Livezey (1998), in which both Turnicidae and Pedionomidae (plains wanderer) are included in a monophyletic Gruiformes. There is some morphological evidence for monophyly of Falconiformes (Griffiths 1994), but no molecular analyses have so far succeeded in putting all families together. Nor, however, is there strong evidence for any falconiform family’s relationship to any non-falconiform family. The hypothesized relationship between Cathartidae (New World vultures) and Ciconiidae (storks) (e.g., Sibley and Ahlquist 1990) has not survived rigorous analysis. There is no evidence for monophyly of Coraciiformes, though there is some support for a “core coraciiforms” including Alcedinidae (kingfishers), Todidae (todies), Momotidae (motmots), Meropidae (bee-eaters), Coraciidae (rollers), and Brachypteracidae (ground-rollers) (Johansson and Ericson 2003; Cracraft et al. 2004). There are so far only two studies that have included Leptosomidae (cuckoo-roller), and there was no support for its inclusion in or exclusion from Coraciiformes (Kirchman et al. 2001; Mayr et al. 2003). The position shown on Sibley and Ahlquist’s (1990) Tapestry is not based on valid data (C. G. Sibley, unpublished data). The remaining three coraciiform families, Upupidae (hoopoe), Phoeniculidae (woodhoopoes), and Bucerotidae (hornbills) also form a clade, but their relationship to core coraciiforms is unsupported (Cracraft et al. 2004). There is no strong evidence for monophyly of Cuculiformes, consisting of Cuculidae (cuckoos) and Musophagidae (turacos) (Sorenson et al. 2003).

1.2.4.2

Relationships between neoavian orders (or their monophyletic fragments)

Confirmed relationships between orders are few. Perhaps the most interesting published hypothesis is that of Fain and Houde (2004), who divided Neoaves into two basal clades: Metaves, consisting of Caprimulgiformes, Apodiformes, Podicipedidae, Phaethontidae, Phoenicopteridae, Opisthocomidae, Mesitornithidae, Rhynochetidae, Eurypygidae, Pteroclidae, and Columbidae; and Coronaves, consisting of the remaining Neoaves. So far, these clades are supported entirely by analyses of beta-fibrinogen, intron 7, but it is encouraging that both sequence characters and indel characters, which evolve largely by independent processes, support the same clades. In addition, no rigorous studies contradict this hypothesis. After some internal debate, I have

Reproductive Biology and Phylogeny of Birds added these groups to Figs. 1.1 and 1.2. Note that, if accepted, these two clades make Pelecaniformes polyphyletic (through the inclusion of Phaethontidae in Metaves and the remaining families in Coronaves) and Gruiformes highly polyphyletic. One amply confirmed though surprising relationship is that between Phoenicopteridae (flamingos) and Podicipedidae (grebes). First discovered by van Tuinen et al. (2001), it has since been confirmed by many authors (Chubb 2004a; Cracraft et al. 2004; Mayr 2004). Another well confirmed relationship is that between Aegothelidae (owletnightjars) and Apodiformes (Mayr 2002a; Mayr et al. 2003; Cracraft et al. 2004). The traditional relationship of Galbuliformes and Piciformes has recently been questioned strongly enough that the two orders, generally united as Piciformes, were separated in the classification used by del Hoyo et al. (19922002). However, that relationship has subsequently been conclusively confirmed (Johansson and Ericson 2003; Mayr et al. 2003; Cracraft et al. 2004). The long-standing hypothesis that Procellariiformes and Sphenisciformes are sister groups has received some support in molecular analyses (van Tuinen et al. 2001); but see Mayr (2005) for a placement of penguins within “core pelecaniforms”. One more possible grouping, though it has ambiguous support and its boundaries are not clearly defined, is sometimes called “water birds”. It includes most of the traditional members of Pelecaniformes (except Phaethontidae), Ciconiiformes, and Procellariiformes, plus Spheniscidae (penguins) and Gaviidae (loons) (van Tuinen et al. 2001; Cracraft et al. 2004). Support in any individual analysis is not strong, however, and some analyses include additional groups within the clade (e.g., Cuculiformes, “core gruiforms”), while other analyses do not sample all groups. Because of the ambiguity surrounding this potential group, I have omitted it from the tree (Fig. 1.2). One prominent hypothesis that has no support is a clade made up of any combination of Opisthocomidae, Musophagidae, and Cuculidae. Groups uniting different pairs or all three have been proposed several times (Sibley and Ahlquist 1990; Hedges et al. 1995; Hughes 1996, 2000; Hughes and Baker 1999), but they have been plagued by data problems (Sorenson et al. 2003), and as of now no resolution is possible. Note that in the analysis by Fain and Houde (2004), the hoatzin belongs to Metaves, while turacos and cuckoos are Coronaves. There are a few other hypotheses with some support from morphological data that either contradict or are unconfirmed by molecular data. Mayr (2004b) proposed a sister group relationship between Mesitornithidae (mesites) and Cuculidae (cuckoos). Mayr (2003b) proposed a sister group relationship between Trogonidae (trogons) and Steatornithidae (oilbirds). Both relationships contradict Fain and Houde’s (2004) Metaves/Coronaves split, though they do not contradict any strongly supported clades in any other analyses.


1.2.4.3

Relationships within neoavian orders (or their monophyletic fragments)

Procellariiformes. Pelecanoididae (diving-petrels) are probably nested within Procellariidae (petrels and shearwaters) (Cracraft et al. 2004), so I show them as sister taxa in Fig. 1.2. This is the only resolved node in my reanalysis (Harshman 1994) of Sibley and Ahlquist’s (1990) data. Hydrobatidae may be polyphyletic (see below) and this prevents any further resolution among families. Pelecaniformes. This order is divided into three pieces. Phaethontidae (tropicbirds) is apparently not closely related to any of the other families. The relationships of Pelecanidae (pelicans) have been noted above. This leaves a clade I will call “core pelecaniforms” despite the absence of pelicans from the group, composed of Fregatidae (frigatebirds), Sulidae (boobies), Phalacrocoracidae (cormorants), and Anhingidae (darters). The only resolved node in my reanalysis (Harshman 1994) of Sibley and Ahlquist’s (1990) data unites these four families. Siegel-Causey (1997) and Cracraft et al. (2004, fig. 27.4) also find this grouping. In other studies (van Tuinen et al. 2001; Cracraft et al. 2004, fig. 27.7) only the last three are united or Fregatidae is only weakly attached to the others (Cracraft et al. 2004, fig. 27.6). Relationships among Sulidae, Phalacrocoracidae, and Anhingidae are ambiguous, with either phalacrocoracids (van Tuinen et al. 2001; Fain and Houde 2004) or sulids (Siegel-Causey 1997; Cracraft et al. 2004) sister to the other two. Falconiformes. Though the order cannot be confidently asserted as monophyletic, relationships are well established among families of the suborder Accipitres, with Sagittariidae (secretary bird) the sister of Pandionidae (osprey) and Accipitridae (hawks) (Cracraft et al. 2004; Fain and Houde 2004); but see Griffiths (1994). Gruiformes. The relationships of Otididae (bustards), Cariamidae (seriemas), and Mesitornithidae (mesites) to any other families (gruiform or otherwise) cannot be determined at present. Turnicidae (buttonquail) belongs to Charadriiformes (see above). Eurypygidae (sunbittern) and Rhynochetidae (kagu) are sister taxa (Houde et al. 1997; Livezey 1998; Cracraft et al. 2004; Fain and Houde 2004), but their relationships to other families cannot be determined. This leaves a “core gruiforms” consisting of Gruidae (cranes), Aramidae (limpkin), Psophiidae (trumpeters), Rallidae (rails), and Heliornithidae (finfoots), for which there is strong support (Houde et al. 1997; Livezey 1998; Cracraft et al. 2004; Fain and Houde 2004). There is strong support for a sister group relationship between Gruidae and Aramidae and between Rallidae and Heliornithidae (Houde 1994; Houde et al. 1997; Livezey 1998; Cracraft et al. 2004; Fain and Houde 2004). The placement of Psophiidae is disputed, but the only strong support is for a position as sister of Gruidae plus Aramidae (Livezey 1998; Fain and Houde 2004). Charadriiformes. All molecular analyses (Sibley and Ahlquist 1990; Ericson et al. 2003; Paton et al. 2003; Fain and Houde 2004) are agreed on the


relationships of families within Charadriiformes. Paton et al. (2003) discuss incongruence between their tree and that of Sibley and Ahlquist (1990), but they are referring there to the Tapestry, while I refer here to the more rigorous Fitch tree (Sibley and Ahlquist 1990; Harshman 1994). The order is traditionally divided into three suborders, Charadrii, Lari, and Alcae (del Hoyo et al. 1996), but Alcae is nested within Lari, and the traditional Charadrii is polyphyletic. The three basal clades do consist of plover-like birds, gull-like birds, and sandpiper-like birds, which might be given the names Charadrii, Lari, and Scolopaci, respectively, with the first being sister to the other two. Ibidorhynchidae (ibisbill) has never been sampled and cannot be placed. Lacking any better notion, I have placed it at the basal node of Charadriiformes. Caprimulgiformes. There is no study strongly supporting the relationship of any caprimulgiform family to any other (Harshman 1994; Mariaux and Braun 1996; Fidler et al. 2004). The only supported relationship is that of Aegothelidae to Apodiformes, as discussed above. Apodiformes. Remarkably few studies have included representatives of all three families of apodiforms, but those that did have unsurprisingly found Apodidae (swifts) and Hemiprocnidae (treeswifts) to be sister taxa (Johansson et al. 2001; Mayr 2002a; Mayr et al. 2003; Chubb 2004a). Coraciiformes. Relationships within “core coraciiforms” are partially resolved. One clade is composed of Alcedinidae, Todidae, and Momotidae, though relationships among these families are contradictory (Sibley and Ahlquist 1990; Harshman 1994; Espinosa de los Monteros 2000; Johansson et al. 2001; Johansson and Ericson 2003), and another clade of Coraciidae and Brachypteracidae (Johansson et al. 2001; Kirchman et al. 2001; Johansson and Ericson 2003). The position of Meropidae is unresolved, though there is some evidence that it is sister to the first clade (Johansson and Ericson 2003). There is another clade of traditional coraciiforms whose relationships to core coraciiforms are unresolved; it consists of Bucerotidae, Upupidae, and Phoeniculidae, in which the last two are sister taxa (Sibley and Ahlquist 1990; Harshman 1994; Johansson et al. 2001; Cracraft et al. 2004). Leptosomidae certainly does not belong to the roller clade and probably does not belong to the core coraciiforms (Kirchman et al. 2001). Piciformes. There is a basal split between Capitonidae (which includes Ramphastidae) and the other two families, Indicatoridae and Picidae (Sibley and Ahlquist 1990; Harshman 1994; Johansson and Ericson 2003; Cracraft et al. 2004).

1.2.4.4

Monophyly of neoavian families

Most non-passerine, neoavian families have clear morphological synapomorphies, and the monophyly of many has been supported by molecular analyses. But there are a few exceptions. Charadriidae (plovers) is both paraphyletic, since it includes the two families Recurvirostridae (stilts


!

and avocets) and Haematopodidae (oystercatchers) (Ericson et al. 2003; Fain and Houde 2004) and polyphyletic, since Pluvianellus (Magellanic plover) is not a close relative of the other plovers, but is instead sister to Chionidae (sheathbills) (Paton et al. 2003). Glareolidae (coursers and pratincoles) is polyphyletic, since Pluvianus (Egyptian plover), as its common name might suggest, is not a member of the family; its actual position is not clear (Fain and Houde 2004). In parrots, Psittacidae is paraphyletic to Cacatuidae (de Kloet and de Kloet 2005). There is some evidence to suggest that the procellariiform family Hydrobatidae (storm petrels) is paraphyletic (Nunn and Stanley 1998) to the rest of the order, or even polyphyletic (Cracraft et al. 2004), but topologies are contradictory. I have represented this in Fig. 1.2 by making Hydrobatidae paraphyletic and its relationships to other families unresolved. There is also strong evidence that Procellariidae (petrels and shearwaters) is paraphyletic to Pelecanoididae (diving-petrels) (Nunn and Stanley 1998, Cracraft et al. 2004). And Capitonidae (barbets) is paraphyletic to Ramphastidae (toucans) (Sibley and Ahlquist 1990; Lanyon and Hall 1994; Barker and Lanyon 2000; Johansson and Ericson 2003; Cracraft et al. 2004; Moyle 2004). There may be other exceptions that we are unaware of and that will be exposed by increased taxon sampling, but I do not expect many.

1.2.4.5

Relationships within neoavian families

Space doesn’t permit a lengthy discussion of intrafamilial relationships. Instead I have provided a list of references (Table 1.1) that investigate such relationships with a large enough taxon sample to be useful. Table 1.1

References to studies of phylogeny within individual neoavian families

Family

Reference

Spheniscidae Diomedeidae Procellariidae Hydrobatidae Phaethontidae Fregatidae Ardeidae

Bertelli and Giannini 2005 Nunn and Stanley 1998 Nunn and Stanley 1998 Nunn and Stanley 1998 Kennedy and Spencer 2004 Kennedy and Spencer 2004 Sheldon 1987 Sheldon et al. 2000 Slikas 1997 Griffiths 1999 Griffiths 1999 Griffiths et al. 2004 Wink 1995 Krajewski and King 1996 Livezey 1998 Pitra et al. 2002 Broders et al. 2003

Ciconiidae Accipitridae Falconidae Cathartidae Gruidae Rallidae Otididae

Table 1.1 Contd. ...

" Reproductive Biology and Phylogeny of Birds Table 1.1 Contd. ...

Family

Reference

Jacanidae Scolopacidae

Whittingham et al. 2000 Ericson et al. 2003 Paton et al. 2003 Thomas et al. 2004 Braun and Brumfield 1998 Thomas et al. 2004 Crochet et al. 2000 Thomas et al. 2004 Pons et al. 2005 Friesen et al. 1996 Thomas et al. 2004 Johnson and Clayton 2000 Miyaki et al. 1998 Brown and Toft 1999 de Kloet and de Kloet 2005 Hughes and Baker 1999 Veron and Winney 2000 Aragón et al. 1999 Hughes and Baker 1999 Hughes 1996, 2000 Johnson et al. 2000 Sorenson et al. 2003 Mariaux and Braun 1996 Bleiweiss et al. 1997 Chubb 2004b Espinosa de los Monteros 2000 Johansson and Ericson 2004 Moyle 2005 Sibley and Ahlquist 1990 Johansson and Ericson 2003 Kirchman et al. 2001 Barker and Lanyon 2000 Johansson and Ericson 2003 Nahum et al. 2003 Moyle 2004 Prychitko and Moore 1997 Webb and Moore 2005

Stercorariidae Laridae

Alcidae Columbidae Psittacidae/ Cacatuidae

Musophagidae Cuculidae

Nyctibiidae Trochilidae Trogonidae

Alcedinidae Brachypteracidae Capitonidae/ Ramphastidae

Picidae

1.2.4.6

Monophyly of neoavian genera

There are a limited number of families in which taxon sampling has been dense enough to determine the monophyly of genera. But in those families, paraphyly of genera has been shown to be frequent. The sparse data so far suggest that the larger a genus, and the more genera within the family, the more likely it is that non-monophyly will be found. Most often, as with families, paraphyly results from aberrant species being assigned their own genera. But there are additional cases without such clear causes.


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The greatest prevalence so far is in the most heavily sampled family with large genera, Picidae, in which at least six genera are non-monophyletic: Colaptes, Piculus, Picoides, Dendropicos, Veniliornis, and Picus (Weibel and Moore 2002; Webb and Moore 2005). Other examples include Eupodotis and Ardeotis in Otididae (Pitra et al. 2002; Broders et al. 2003), Columba and Streptopelia in Columbidae (Johnson and Clayton 1999; Johnson et al. 2001), Larus in Laridae (Pons et al. 2005), Tauraco in Musophagidae (Veron and Winney 2000), and Stercorarius in Stercorariidae (Braun and Brumfield 1998). We can expect many more non-monophyletic genera to come to light as species sampling increases.

1.2.5

Classification of Non-passerines

It would be premature to offer a new classification of the non-passerines, given our great current uncertainty and the hope that it will soon be repaired, but I can at least make a few recommendations for modification of the classification I have been using, from del Hoyo et al. (1992-2002). Paraphyletic and polyphyletic groups should be reconciled so as to make them monophyletic, and though Linnean ranks are arbitrary, we may as well avoid leaving traditional family-rank names nested within other family-rank names. Therefore sevaral families should be merged into the families that enclose them: Tetraonidae and Meleagrididae into Phasianidae, Pelecanoididae into Procellariidae, Haematopodidae and Recurvirostridae into Charadriidae, Cacatuidae into Psittacidae, and Ramphastidae into Capitonidae. It may be that Hydrobatidae will need to be split into two families, but more study is needed. Turnicidae should be moved from Gruiformes to Charadriiformes. Coraciiformes should be limited to the “core coraciiforms”, and a new order established for Bucerotidae, Upupidae, and Phoeniculidae, for which Bucerotiformes is a reasonable name. Likewise, Gruiformes should be limited to the “core gruiforms”. We should probably wait a while before redefining Pelecaniformes; perhaps a clade can be found that includes both pelicans and “core pelecaniforms”, though it will surely include other families too. Galbuliformes should be returned to its traditional state as a suborder of Piciformes.

1.3

PASSERINES

The order Passeriformes contains more than half the species of birds (Sibley and Monroe 1990). In contrast to the difficulties with non-passerines, molecular techniques have proven to be highly successful in determining relationships at all levels. The major difficulties have been the sheer number of species and the unreliability of previous taxonomies as guides to phylogeny. Thus, single species cannot be used as handy proxies for entire families, or even genera. The rules of thumb used for non-passerines—in which genera did not commonly change families, and non-monophyletic

$ Reproductive Biology and Phylogeny of Birds genera at least had their separate parts all within one family, etc.—cannot be counted on here. What that means for this review is that any summary of relationships will be difficult, as the species assigned to families and superfamilies are in flux, and new surprises are expected as more species are sampled. Further, it becomes even more important to consider the exact nature of the taxon sampling in any given study and its comparability to sampling in other studies. I will attempt only an impressionistic summary tree of higher taxa (Figs. 1.3 and 1.4), for whose suggested species membership the reader must consult the references provided. One monotypic traditional family, Hypocoliidae, has never been included in any molecular analysis or any rigorous, cladistic analysis of morphology, and it has been omitted from further discussion here. Many of the analyses used in assembling the passerine tree have used Bayesian methods (Huelsenbeck and Ronquist 2001). Recently it has become apparent that Bayesian support values can be highly inflated (Suzuki et al. 2002; Yang and Rannala 2005). Very small differences in data or choice of evolutionary model can produce contradictory but strongly supported clades (pers.obs.), and there are many contradictory clades in the sources used in this review that have high Bayesian support. Consequently, I have not accepted any relationships based solely upon Bayesian analysis of any single data set. Either there must be some other measure of support used (e.g., parsimony or likelihood bootstrap), or different data sets (including indel data if any) must agree on the topology. Neither has any contradiction in relationships supported by Bayesian analyses alone caused me to remove resolution from any clade.

1.3.1

Monophyly of Passeriformes

Passeriform monophyly is well supported by morphological apomorphies (Raikow 1982). There have been several molecular analyses with moderately large taxon samples of passerines and other orders, and passerine monophyly has always been found (Johansson et al. 2001; Johansson and Ericson 2003; Sorenson et al. 2003; Cracraft et al. 2004; Fain and Houde 2004).

1.3.2

Monophyly of Passerine Families and Genera

One reason that it’s difficult to summarize passerine relationships in the same way I did for non-passerines is that there are no convenient, easily recognized subtaxa to use in constructing a summary tree. Sibley and Ahlquist (1990) showed that many of the traditional passerine families were not monophyletic, and others since then have found that many of the groups with which Sibley and Ahlquist (1990) and Sibley and Monroe (1990) replaced those traditional groups are themselves not monophyletic (see below). Further, misplaced taxa are not limited to small movements, but may span almost the range of the passerine tree—“almost” because so far no putative suboscine has been shown to be oscine, or vice versa. However, species have moved between superfamilies. Pseudopodoces (Hume’s ground jay), supposed to be a


%

member of Corvidae and thus of Corvoidea, is instead a member of Paridae (in fact within the genus Parus, which thereby becomes paraphyletic) and thus of Sylvioidea (James et al. 2003; Gill et al. 2005). Yuhina (Erpornis) zantholeuca (white-bellied yuhina) is not a yuhina (Sylvioidea) but a vireo (Corvoidea) (Barker et al. 2004). Macgregoria (Macgregor’s bird of paradise) is not a member of Paradiseidae (Corvoidea) but of Meliphagidae (Meliphagoidea) (Cracraft and Feinstein 2000). Sapayoa (broadbilled sapayoa) is not a New World suboscine but an Old World suboscine, in terms of phylogeny if not geography (Fjeldså et al. 2003; Chesser 2004). There is no reason to suppose that these examples exhaust the list. Traditional or recently defined (Sibley and Ahlquist 1990; Sibley and Monroe 1990) families that have proven non-monophyletic include Eurylaimidae (e.g., Prum 1993), Formicariidae (traditional and Sibley and Ahlquist versions), Rhinocryptidae, Furnariidae, Tyrannidae, Cotingidae, and Pipridae, in fact the large majority of suboscine families (Sibley and Ahlquist 1990; Irestedt et al. 2002; Chesser 2004). Oscines are similar in their proportion of non-monophyletic families, though I will not list them here (e.g. Sibley and Ahlquist 1990; Cibois and Cracraft 2004; Voelker and Spellman 2004; Beresford et al. 2005; Alström et al. 2006). Genera of passerines are also not reliably monophyletic. While this is true also of non-passerines, there are certainly many more visible examples within passerines, and genera are more likely to be split into more widely separated pieces. In an informal poll of bird systematists I conducted at a recent meeting, I asked them to guess what percentage of passerine genera were nonmonophyletic (excluding monotypic genera). The modal answer was “about half”. Of course the majority of genera have not been rigorously investigated so far, which requires sampling most or all the species in the genus plus many species outside it. A few examples should suffice. In addition to finding New World warblers polyphyletic, Lovette and Bermingham (2002) found six out of seven investigated genera with two or more sampled species to be nonmonophyletic; Driskell and Christidis (2003) found three non-monophyletic genera within Meliphagidae out of sixteen examined; Irestedt et al. (2004a) found three of four sampled genera of Thamnophilidae non-monophyletic, and monophyly of the fourth was not confirmed; and Irestedt et al. (2004b) found two of six sampled genera of Dendrocolaptidae non-monophyletic.

1.3.3

Basal Relationships within Passerines

Basal relationships are clear (Fig. 1.3). The family Acanthisittidae (New Zealand wrens), with only two genera and three extant species, is the sister group of all other passerines (Lovette and Bermingham 2000; Barker et al. 2002, 2004; Ericson et al. 2002a; Chesser 2004; Beresford et al. 2005). The remaining passerines are split into two clades, Suboscines and Oscines (Sibley and Ahlquist 1990; Lovette and Bermingham 2000; Barker et al. 2002, 2004; Ericson et al. 2002a; Chesser 2004; Beresford et al. 2005).

& Reproductive Biology and Phylogeny of Birds

Fig. 1.3 Relationships among passerine families and other groups, so far as they can be confidently asserted at present according to my perhaps biased estimate. This tree does not derive from a formal analysis, either of a combined supermatrix or by a rigorous supertree method. Branches in gray represent paraphyletic groups. For Passerida see Fig. 1.4. For reasons behind the topology chosen, see text.


'

Within the suboscines basal relationships are also easy to describe, as there are New World and Old World clades, Tyrannides and Eurylaimides (Sibley and Ahlquist 1990; Irestedt et al. 2001; Barker et al. 2002, 2004; Chesser 2004; Beresford et al. 2005). (Recall that Eurylaimides has a single New World representative, Sapayoa, as mentioned above.) Old World suboscines are also simple: Pittidae (pittas) is either sister to or paraphyletic to Sapayoa, with a larger taxon sample necessary to determine which (Fjeldså et al. 2003; Chesser 2004), and Eurylaimidae (broadbills) is paraphyletic to Philepittidae (asities) (Prum 1993; Irestedt et al. 2001; Barker et al. 2002, 2004; Beresford et al. 2005). New World suboscines can be divided into two clades, Tyranni and Furnarii (Lovette and Bermingham 2000; Irestedt et al. 2001; Barker et al. 2002, 2004; Chesser 2004; note that Sibley and Ahlquist 1990 gave the name Tyranni to a different group), In the oscines, Sibley and Ahlquist (1990) erected two groups Corvida and Passerida. Corvida is however paraphyletic to a (mostly) monophyletic Passerida (Barker et al. 2002). Within the corvidan grade, there are three superfamilies: Menuroidea, Meliphagoidea, and Corvoidea, each very roughly corresponding to Sibley and Ahlquist’s (1990) groupings, plus a number of additional taxa unincluded in any superfamily (Barker et al. 2002, 2004; Beresford et al. 2005). Within Passerida, three superfamilies, Sylvioidea, Muscicapoidea, and Passeroidea, are also roughly similar to Sibley and Ahlquist’s (1990) groups (Sheldon and Gill 1996; Barker et al. 2002, 2004; Ericson and Johansson 2003; Beresford et al. 2005). It has however been necessary to add a fourth, Certhioidea (Barker et al. 2004), and there are also a number of groups that do not fit securely into any superfamily (Sheldon and Gill 1996; Barker et al. 2002, 2004; Ericson and Johansson 2003; Beresford et al. 2005; Fuchs et al. 2006). Ignoring for simplicity groups that do not fit into a superfamily, the relationships among those superfamilies can be resolved: Menuroidea, Meliphagoidea, and Corvoidea are successive sister groups to all other oscines (Barker et al. 2002, 2004; Beresford et al. 2005). Sylvioidea is sister to all other passeridans, and Muscicapoidea is sister to Certhioidea (Sheldon and Gill 1996; Barker et al. 2002, 2004; Beresford et al. 2005). Relationships are described in more detail below.

1.3.4 Relationships within New World Suboscines 1.3.4.1 Furnarii There is a basal polytomy among three clades: Melanopareia (crescent-chests), a genus traditionally assigned to Rhinocryptidae (Irestedt et al. 2002; Chesser 2004), Thamnophilidae (typical antbirds) plus Conopophagidae (gnateaters), and remaining Furnarii (Irestedt et al. 2001, 2002; Chesser 2004; but see Barker et al. 2002, 2004 for a different placement of conopophagids). Formicariidae (ground antbirds) is divided into two parts; one of them, ant-pittas, is the sister group of Rhinocryptidae (tapaculos) while the other, ant-thrushes, is sister to Furnariidae (ovenbirds) and Dendrocolaptidae (woodcreepers). Furnariidae is paraphyletic to Dendrocolaptidae (Irestedt et al. 2002; Chesser 2004).


1.3.4.2

Tyranni

Several clades within Tyranni are clearly established: Pipridae (manakins), Tityridae (tityras), Cotingidae (cotingas), Oxyruncus (sharpbill), and Tyrannidae (tyrant flycatchers). But relationships among them are contradictory in different analyses, and no resolution is strongly supported in any analysis (Barker et al. 2002, 2004; Johansson et al. 2002; Chesser 2004). Relationships within Tyrannidae are likewise contentious, and no molecular study has so far accumulated a large taxon sample.

1.3.5 Relationships within the Corvidan Grade 1.3.5.1 Menuroidea and other near-basal groups Sibley and Ahlquist’s (1990) Menuroidea included Menuridae (lyrebirds), Atrichornithidae (scrub-birds), Climacteridae (Australasian treecreepers), and Ptilonorhynchidae (bowerbirds). More recent studies show that the latter two are sister taxa and are more closely related to other oscines than to Menuridae (Barker et al. 2002, 2004; Ericson et al. 2002b; Beresford et al. 2005; but see Ericson et al. 2002a). There is so far no sequence data available for Atrichornithidae, and I have retained it within a reduced Menuroidea (Sibley and Ahlquist 1990). The unnamed group including Climacteridae and Ptilonorhynchidae is sister to all oscines other than Menuroidea (Barker et al. 2002, 2004; Ericson et al. 2002b; Beresford et al. 2005; but see Ericson et al. 2002a).

1.3.5.2

Meliphagoidea and other groups outside Corvoidea

Meliphagoidea consists of Maluridae (fairy wrens), Meliphagidae (Honeyeaters), Pardalotidae (pardalotes), Acanthizidae (scrubwrens, thornbills), and Dasyornis (bristleheads) (Cracraft and Feinstein 2000; Barker et al. 2002, 2004) and is sister to the remaining oscines (Barker et al. 2002, 2004; Beresford et al. 2005). Relationships within Meliphagidae and Acanthizidae have been investigated by Driskell and Christidis (2004). Two more families, Pomatostomidae (Australian babblers) and Orthonychidae (logrunners) are successively more closely related to Corvoidea and Passerida (Barker et al. 2002, 2004). Three more clades form a polytomy with Corvoidea and Passerida: Callaeatidae (New Zealand wattlebirds), Cnemophilinae (traditionally supposed to be birds of paradise, belonging to the corvoid Paradiseidae), and Melanocharitidae (most berrypeckers—one genus, Paramythia, is corvoid) (Cracraft and Feinstein 2000; Barker et al. 2002, 2004; Beresford et al. 2005).

1.3.5.3

Corvoidea

This large clade contains most of the member of Sibley and Ahlquist’s (1990) Corvoidea (Barker et al. 2004, 2004; Beresford et al. 2005). It consists of two large, resolved subclades, a few pairs of taxa, and a large, basal polytomy. One large subclade is a collection of mostly shrike-like birds including Cracticidae (butcherbirds), Gymnorhina (Australian magpie), Strepera (currawongs), Artamidae (wood-swallows), Aegithinidae (ioras), Malaconotinae (bush-


shrikes), Dryoscopus (puffbacks), Batis (batises), Lanioturdus (chatshrike), Vangidae (vangas), and Prionopidae (helmet shrikes) (Barker et al. 2002, 2004; Beresford et al. 2005; Fuchs et al. 2006). The other group consists of taxa close to Corvidae, including Laniidae (shrikes), Paradiseidae (birds of paradise), Monarchidae (monarch flycatchers), Struthidea (apostlebird), Corcorax (whitewinged chough), Grallina (mud-nest builders), Melampitta (melampittas), Dicruridae (drongos), and Rhipiduridae (fantails) (Barker 2002, 2004; Beresford et al. 2005).

1.3.6

Relationships within Passerida

These are shown in Fig. 1.4.

1.3.6.1

Groups not within any superfamily

Picathartidae (rockfowl) and Petroicidae (Australian robins) are basal members of Passerida (Barker et al. 2002, 2004; Beresford et al. 2005). Regulidae (kinglets) and Hyliota are closer to other Passerida than the previous two families, but cannot so far be linked to any of the superfamilies (Barker et al. 2002, 2004; Ericson and Johansson 2003; Beresford et al. 2005; Fuchs et al. 2006).

1.3.6.2

Sylvioidea

The most difficult problem in establishing sylvioid monophyly has been the inclusion of Paridae (titmice) and its relatives within the superfamily. Though no study has supported that conclusion strongly, several have supported it and none have strongly contradicted it (Sheldon and Gill 1996; Barker et al. 2002, 2004: Beresford et al. 2005; Alström et al. 2006). In addition to its traditional allies, Remizidae (penduline tits), the clade around Paridae has recently been shown to include a clade of refugees from other families, termed Stenostiridae by Beresford et al. (2005). This clade includes Stenostira (fairy warbler, previously considered a sylviid), Culicicapa (canary-flycatchers, usually considered muscicapid, but placed among petroicids by Sibley and Ahlquist 1990), and Elminia (blue-flycatchers, previously considered monarchids) (Barker et al. 2002, 2004; Beresford et al. 2005; Fuchs et al. 2006). Monophyly of the remainder of Sylvioidea is strongly supported, though this group differs from that of Sibley and Ahlquist (1990) by inclusion of Alaudidae (larks) (Sheldon and Gill 1996; Barker et al. 2002, 2004; Ericson and Johansson 2003; Alström et al. 2006; Fuchs et al. 2006). Alaudidae, with its unexpected sister taxon Panurus (bearded tit) (Ericson and Johansson 2003; Alström et al. 2006; Fuchs et al. 2006) is strongly supported as the sister group of all remaining sylvioids (Sheldon and Gill 1996, Barker et al. 2002, 2004; Ericson and Johansson 2003; Beresford et al. 2005; Alström et al. 2006; Fuchs et al. 2006). In the rest of Sylvioidea there is a large polytomy consisting of several genera, a few families, and one large group of families. There are still a great many sylvioid (or supposed sylvioid) genera and species yet to be sampled;


Fig. 1.4 Relationships among passerine families, continued. Passerida only. For reasons behind the topology chosen, see text.


!

more sampling of taxa and genes may resolve this polytomy and illuminate unknown clades. Beresford (2005) proposed a “Sphenoeacus group” consisting of Sphenoeacus, Sylvietta, Achaetops, Bradypterus victorini (the genus being polyphyletic), and Macrosphenus; but this was supported solely by Bayesian analysis. Other studies, with only partly overlapping taxon samples, have produced contradictory results (Alström et al. 2006; Fuchs et al. 2006). Alström et al. (2006) found Macrosphenus to belong to Pycnonotidae (bulbuls) with strong support; however, the two studies sequenced different species, and it’s quite possible that both are right and the genus is polyphyletic. The sylvioid polytomy includes four large clades (Alström et al. 2006). Two are the families Cisticolidae (cisticolas) and Timaliidae (babblers). Note that Timaliidae as used here includes the genera Sylvia and Zosterops; there are no families Sylviidae or Zosteropidae, following the terminology of Alström et al. (in press). A third clade includes the families Megaluridae (grassbirds) and Acrocephalidae (acrocephaline warblers) plus the monotypic Donacobius, once considered a troglodytid (Barker 2004; Alström et al. 2006). The final clade consists of a number of families: Hirundinidae (swallows), Pycnonotidae (bulbuls), Phylloscopidae (leaf warblers), Cettiidae (bush warblers), and Aegithalidae (long-tailed tits), plus the genus Hylia (Alström et al. 2006), and the last three form a subclade (Beresford et al. 2005).

1.3.6.3

Certhioidea

This superfamily was erected by Cracraft et al. (2004) to cover a clade of four families removed from Sibley and Ahlquist’s (1990) Sylvioidea. It includes Troglodytidae (wrens), Polioptilidae (gnatcatchers), Certhiidae (treecreepers), and Sittidae (nuthatches) (Barker et al. 2002, 2004; Ericson and Johansson 2003; Beresford et al. 2005). Within this clade, Sittidae is basal and Troglodytidae and Polioptilidae are sister taxa (Sibley and Ahlquist 1990; Harshman 1994; Sheldon and Gill 1996; Ericson and Johansson 2003; Barker 2004; Alström et al. 2006; but see Barker et al. 2002, 2004).

1.3.6.4

Muscicapoidea

The most difficult question is whether Bombycillidae (waxwings, silky flycatchers, palmchat) belongs to this superfamily. No analysis has been conclusive, but several have given weak support (Sibley and Ahlquist 1990; Barker et al. 2002, 2004; Cibois and Cracraft 2004) and I consider the combination to offer strong support. Monophyly of the rest of Muscicapoidea is clear (Sibley and Ahlquist 1990; Harshman 1994; Barker et al. 2002, 2004; Ericson and Johansson 2003; Cibois and Cracraft 2004; Voelker and Spellman 2004; Alström et al. 2006). Relationships among the five families Sturnidae (starlings), Mimidae (mimic thrushes) Cinclidae (dippers), Turdidae (thrushes), and Muscicapidae (sensu stricto—Old World flycatchers) are also contentious, but a strong case can be made for the topology I have chosen, in which Sturnidae and Mimidae are sisters, Turdidae and Muscicapidae are sisters, and Cinclidae is sister to

" Reproductive Biology and Phylogeny of Birds the turdid-muscicapid clade (Sibley and Ahlquist 1990; Barker et al. 2002, 2004; Cibois and Cracraft 2004; Beresford et al. 2005; Fuchs et al. 2006). An alternative arrangement in which Cinclidae is sister to the sturnid-mimid clade is supported only by Bayesian analysis, though the relationship between Turdidae and Muscicapidae is also supported by parsimony jackknifing (Ericson and Johansson 2003). Voelker and Spellman (2004) show a substantially different topology, in which Muscicapidae is basal, but contradictory nodes again have weak support. Two additional genera form a polytomy with Sturnidae and Mimidae (Cibois and Cracraft 2004). Buphagus (oxpeckers) is traditionally considered part of Sturnidae. Rhabdornis is a genus of previously uncertain relationships, sometimes placed in Certhiidae or Timaliidae (Sibley and Monroe 1990).

1.3.6.5

Passeroidea

It may be that Promeropidae (sugarbirds) is the basal family in Passeroidea, but that conclusion is supported by Bayesian analyses alone, and only by different versions of one data set (Barker et al. 2002, 2004; Beresford et al. 2005). What may be partial confirmation is offered by Fuchs et al. (2006). The two genera Modulatrix (spot-throat) and Arcanator (dapple-throat, sometimes merged into Modulatrix) are strongly supported as the sister group of Promerops (Barker et al. 2002, 2004; Beresford et al. 2005). Fuchs et al. (2006), again supported only by Bayesian analysis, show Modulatrix and Arcanator as the sister group of Passeroidea, but Promerops appears in a polytomy at the base of Passerida, and in light of the ambiguity of this information, that is where I have left the entire family. However, there does seem to be good evidence for inclusion of those two species, whose relationships have previously been highly uncertain (Sibley and Monroe 1990), as sister taxa within Promeropidae. The remainder of Passeroidea is clearly monophyletic (Barker et al. 2002, 2004; Ericson and Johansson 2003; Beresford et al. 2005). Nectariniidae (sunbirds) and Dicaeidae (flowerpeckers) are sisters (Barker et al. 2002, 2004; Ericson and Johansson 2003; Beresford et al. 2005), and form a trichotomy with Irenidae (fairy bluebirds) and remaining passeroids (Barker et al. 2002, 2004; Beresford et al. 2005). Peucedramus (olive warbler), previously thought to be either a parulid or basal within 9-primaried oscines (Sibley and Monroe 1990) is instead sister to Prunellidae (accentors) (Ericson and Johansson 2003), and these are sister to the remaining passeroids (Barker et al. 2002, 2004; Ericson and Johansson 2003; Beresford et al. 2005). Passeridae (Old World sparrows) and Motacillidae (pipits and wagtails) are successive sister groups to the nine-primaried oscines (Barker et al. 2002, 2004; Ericson and Johansson 2003). Though this is weakly supported in each analysis, agreement between analyses and a three-codon insertion in the c-myc sequences (Ericson et al. 2000) are conclusive. The nine-primaried oscines form a strongly supported group (Klicka et al. 2000; Ericson and Johansson 2003; Barker et al. 2004), including the families


#

Fringillidae (finches), Emberizidae (buntings and New World sparrows), Icteridae (New World blackbirds), Parulidae (New World warblers), Thraupidae (tanagers), and Cardinalidae (grosbeaks). Fringillidae is the sister of the others (Klicka et al. 2000; Yuri and Mindell 2002; Ericson and Johansson 2003; Barker 2004). Two genera previously considered emberizids, Calcarius (longspurs) and Plectrophenax (snow buntings) are sister taxa of each other and together the sister of the remaining nine-primaried oscines (Klicka et al. 2000; Yuri and Mindell 2002; Ericson and Johansson 2003). Relationships among the remaining five families are contentious. Of analyses with representatives of all five families, most unite Cardinalidae and Thraupidae (Bledsoe 1988; Barker et al. 2002, 2004; Yuri and Mindell 2002), though two (Sibley and Ahlquist 1990; Harshman 1994; Klicka et al. 2000) do not. Again, most unite Icteridae and Parulidae (Bledsoe 1988; Barker et al. 2002, 2004), though two (Klicka et al. 2000; Yuri and Mindell 2002) do not. But support for the minority arrangements are weak. The position of Emberizidae is unclear; Barker et al. (2002, 2004) have strong support for relationship to the cardinalidthraupid clade; Bledsoe (1988) found a relationship to the parulid-icterid clade; Yuri and Mindell (2002) and Klicka et al. (2000), though failing to find the parulid-icterid clade, did find a clade consisting of Emberizidae, Icteridae, and Parulidae. For the present, relationships of Emberizidae are best treated as unresolved.

1.4

CONCLUSION

Progress in avian systematics during the past few years has finally, after a long period of frustration, become worthy of optimism. As more and more homologous DNA sequences and other genetic characters become available for more and more species, We will gradually chip away at the remaining uncertain nodes of the tree, and this trend will only accelerate as DNA sequencing becomes both cheaper and more reliable. These are heady times in avian systematics.

1.5

ACKNOWLEDGMENTS

I would like to thank Keith Barker and Fred Sheldon for their comments on the manuscript, and the other members of the Early Bird project, particularly Kathy Miglia, for sending me copies of many of the referenced articles.

1.6

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CHAPTER

2

Anatomy of the Testis and Male Reproductive Tract Tom A. Aire

2.1

INTRODUCTION

The testes of birds are intra-abdominal, and, unlike in most mammals, they do not migrate from their site of embryological origin. They are, thus, closely related, topographically, to the kidneys. As in mammals, birds have two testes, one on either side of the midline, bordering the aorta and caudal vena cava, laterally. They are attached from their dorso-medial borders to the dorsal abdominal wall by a short mesorchium, and as the kidneys with which they are related embryologically, they are largely retroperitoneal, and ventral to the vertebral column. Topographically, the cranial poles (extremitates craniales) of the testes lie close to the ventral border of the lungs, while their caudal poles (extremitates caudales) lie cranio-ventral to the cranial divisions of the kidneys (Nickel et al. 1977). The dorsomedial aspect of the testis attaches to the relatively small epididymis. The ductus deferens runs distally from the caudal border of the epididymis, toward the cloaca, into which it opens. There are no known accessory sex organs or glands in birds that are either homologous or analogous to those found in mammals. Mammalian terminologies have often been erroneously applied to certain structural modifications in birds, such as seminal vesicle in passerine birds, and the ampulla of the ductus deferens. These have to be understood for what they are, structurally and functionally, as segmental convolutions or enlargements, respectively, of the ductus deferens. The testis is surrounded by the cranial and caudal thoracic as well as abdominal air sacs, but contrary to the opinion of Cowles and Nordstrom (1946) that this relationship helps to cool the testes, as the mammalian scrotum does, Williams (1958) has observed no differences in the temperature Department of Anatomy and Physiology, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, Republic of South Africa. E-mail: [email protected]

!& Reproductive Biology and Phylogeny of Birds in the area of the viscera and at the testicular surface in the domestic fowl. Herrin et al. (1960) have also disproved the assertion by Cowles and Nordstrom (1946), that adjacent air sacs cool the testis. Testicular color is nearly white in sexually mature and active birds, but it may be grey or black, or a mixture of black and white patches or may be entirely greyish black due to the presence of melanin pigments in melanoblasts, in the testicular connective tissues. The testes of seasonally resting birds are very small, being functionally atrophic. Organs of sexually immature or resting birds have yellowish white to black or grey colour, being quite black in regressed testes that already contain melanoblasts, as a result of the concentration of these pigment cells in the connective tissues of much smaller, regressed organs.

2.1.1

Testis Shape, Asymmetry and Size

The avian testes are usually bean- or oval-shaped (Fig. 2.1), but they are normally vermiform in Cypseloides spp., swifts (Marshall 1961). Although both left and right testes are symmetrically situated on either side of the median plane, they are, however, often dissimilar in size. The left testis is often larger than the right one in most species of birds. Riddle (1918, 1925) observed that the left testes were larger than the right in pigeons (Columba species), but in 36 of 39 in the dove (Streptopelia risorsia), the right was larger than the left. In rooster (Gallus domesticus), the left testis is larger than the right in 57% (Mimura 1928) and 65.3% (Marvan 1969) of the birds studied. The right was larger in only 26.5% while both testes were equal in 8.2% of the birds (Marvan 1969). In 169 genera of birds he studied, Friedmann (1927) concluded that the right testis was not larger than the left in any genus, but in 104 genera, both testes were of the same size and the left was larger than the right in 60 genera. Five genera were inconstant, varying between the first two positions. The left organ is, also, larger than the right organ in the ratite, Emu (Dromaius novaehollandiae), although each gram of testis tissue contributes equally to the production of androgen and, possibly, also, spermatozoa (Malecki et al., 1998). It seems that the right testis diminishes in size with age, as the left testis was larger than the right in 90.9% of day-old Turkey toms (Meleagris gallopavo), 94.3% in toms 53-55 wk old and 100% in toms 63-67 wk of age. Care is needed in removing and identifying each testis especially when the carcass of the bird is placed on dorsal recumbency, with the left side of the specimen being on the right of the operator. That is probably why Law and Kosin (1958) found that all Turkey toms they investigated had larger right testes than the left, whereas Burke (1973) reported that the left testis surpassed the right testis in size, with increasing age in their own Turkey toms. The reason for the generally observed difference in testis size, in favor of the left, is not clearly understood, especially given the variations that have been reported by different authors. However, the embryological study by Witschi (1935) shows that the cortex of the right indifferent gonad loses its chemotactic attraction for primordial germ cells, in favor of the cortex of the left gonad, from the beginning of the 3rd day of embryogenesis. Nevertheless, there are no

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Anatomy of the Testis and Male Reproductive Tract

Fig. 2.1 The topography of the reproductive organs of the quail (Coturnix japonica), from a ventral view. T, testis; E, epididymis; D, ductus deferens; P, pars recta ductus deferentis; R, receptacle of the ductus deferens; L, lung; K, kidney. Original.

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" Reproductive Biology and Phylogeny of Birds differences in sizes between the left and right testes in the Marsh hawk (Circus cyaneus) (Witschi 1935; Stanley and Witschi 1940). The size of the avian testis, except that of several breeds of Domestic Fowl and Turkey developed for agricultural production, varies considerably between phases of the reproductive cycle. Thus, the testis reaches maximum size for the species or breed during the peak of the breeding season, and becomes considerably smaller, following regression, during the sexually inactive or resting period of the reproductive cycle, in most wild birds. This variation in size may be as much as 400- to 500-fold in seasonally breeding species (Lofts and Murton 1973). During the active breeding season, testis size is positively related to sperm production rate (Møller 1991), as has been observed in mammals (Møller 1989). Testis sizes and body weights of 247 species of birds, from 152 genera, 37 families, and 16 orders, have been compiled by Møller (1991). Testis size in birds is further discussed in Chapter 9.

2.1.2

Body Temperature and Testis Function in Birds

Whereas in most mammals the testes migrate from the site of their embryogenesis distally and ventrally into the scrotum, those of birds retain their position of origin, close to the kidneys. Mammalian male germ cells degenerate at the body’s internal temperature of 37∞C, and even slight and temporary elevations of scrotal temperature may cause male infertility (Plöen 1972, 1973a,b). On the contrary, spermatogenesis occurs normally at core body temperature (of 40-41∞C) in birds (Béaupre et al. 1997). Mezquita et al. (1998) recently demonstrated a number of apparently genetically controlled biochemical effects that may contribute to ensure the synthesis of essential proteins which achieve thermotolerance during avian, but not mammalian, spermatogenesis.

2.1.3

Consistency and Structure of the Testis

The testis of sexually active birds is soft to touch and quite fragile compared to the mammalian testis. The main exception is the Ostrich (Struthio camelus), whose testicular substance is enclosed in a firm testicular capsule which is relatively very thick (vide infra); in other investigated birds it is thin. An incision through the testicular capsule into the testis causes a mild protrusion of the testicular substance from which there is a dripping of a relatively copious amount of milky fluid, even in the Emu whose testis is very dark in color. Sexually active avian testes have a considerable amount of fluid content (Lake 1957; Aire 1979a; Clulow and Jones 1988).

2.1.4

The Excurrent Ducts of the Testis

The avian epididymis is a small spindle-shaped structure that is attached intimately to the medial border of the testis (Fig. 2.1). A thin cranial extension of this structure is incorporated into the adrenal gland capsule, as the appendix paradidymidis (Gray 1937; Budras and Sauer 1975a; Budras and Meier 1981).


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The caudal end of the epididymis also thins out, and is continued by the ductus deferens. The epididymis of the rooster is 3-5 mm thick at its base, by which it is attached to the testis (Hodges 1974; Pers. obs.). The epididymis is relatively large in the ostrich, being between 21 and 31 g in weight, 12 and 15.4 cm in length and 3-7 cm in height, at its highest point at the caudal onethird of the organ (Pers. obs.). It is about 2.4 cm wide (Soley 1992). The epididymis, as the testis, is also firm to touch in the Ostrich. In all birds, the ductus deferens leaves the caudal end of the epididymis in a slightly wavy manner, but becomes considerably convoluted, increases in diameter, cranio-caudally, and is situated lateral to the relatively firm and regular ureter. A short segment of the duct straightens out to form the pars recta ductus deferentis, just before it enters the cloaca. The ductus deferens terminates thereafter, in a spindle- or barrel-shaped enlargement, the receptaculum ductus deferentis, which is embedded in the cloacal wall, but opens into the urodeum through its protruding and pointed distal end, the papilla ductus deferentis. Birds do not have accessory sex organs that are to be found in mammals.

2.1.5

Blood Supply to the Reproductive Organs

The blood supply to the male reproductive organs of birds has been studied fully only in the rooster (Nishida 1964) and Pigeon (Bhaduri et al.1957), and partly by Siller and Hindle (1969) and Kurihara and Yasuda (1973) in their reports of the vasculature of the kidney in the rooster. Blood supply to the male organs in the ostrich has also been studied grossly to complement these reports (Elias M, Aire, T. A. and Soley, J. T. unpublished results), and the pattern of arterial supply as well as venous drainage of the male organs is generally similar in these three species of birds. The following account is based on these reports. The avian male reproductive organs are intra-abdominal and retain their embryological positions, and therefore their blood supply is relatively simpler than in mammals. A short testicular artery (Arteria testicularis) arises from the A. cranialis renalis, and runs into the testis, along its dorso-medial border, supplying the testis, and sending thin branches to the epididymis (Figs. 2.2, 2.3 and 2.4). One or two short accessory testicular arteries (Aa. testiculares accessoria) may originate directly from the aorta, unilaterally or bilaterally, and similarly run into the hilus of the testis. The testicular arteries run short courses into the hilus of the testis from where they divide into intratesticular loops (Fig. 2.3). Branches ramify in the interstitial tissue, from these loops. The cranial part of the ductus deferens is supplied by the R. ureterodeferentialis cranialis that arises from the A. cranialis renalis. The A. renalis communis originates from A. sciatica in the Ostrich (Elias et al., unpublished observations) and immediately divides into the A. renalis medius and A. renalis caudalis. The former sends one or two twigs to the middle part of the ductus deferens. The A. pudenda sends several branches (Rr. ureterodeferentiales caudales) to the caudal part of the ductus deferens, and several other branches (Rr. cloacales) into the cloaca and root of the phallus. The general disposition of the blood

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Fig. 2.2 The arterial supply to the reproductive organs of male Gallus domesticus. Ara, A. renalis cranialis; At, A. testicularis; Ae, Aa. epididymicae; Auda, Aa. ureterodeferentiales craniales; Audm, Aa. uretero-deferentiales mediae; Audp, Aa. uretero-deferentiales caudales; Apc, A. pudenda communis. From Nishida, T. 1964 Japanese Journal of Veterinary Science 26: 211-221. Figure 1. Reproduced with the permission of Japanese Society of Veterinary Medical Science.


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Fig. 2.3

Fig. 2.4 Figs. 2.3 and 2.4 A semi-diagrammatic illustration of blood vascular supply in the right testis of Gallus domesticus. Aoa, aorta; At, A. testicularis; Ata, A. testicularis accesorius; Vic, V. iliaca communis; Vcp, V. cava caudalis. From Nishida, T. (1964) Japanese Journal of Veterinary Science 26: 211-221. Figure 4. Reproduced with the permission of Japanese Society of Veterinary Medical Science.

"" Reproductive Biology and Phylogeny of Birds vessels in the rooster, pigeon and ostrich are similar, with only minor intraand inter-species variations. The veins draining the testicular substance are tributaries of larger veins which run peripherally into the testicular capsule (Figs. 2.3 and 2.4). The latter are large, wide and clearly visible as they run radially toward the hilus from which they emerge and open into the caudal vena cava as numerous short veins (Nishida 1964). The veins draining the ductus deferens are mostly satellites of the arteries that supply the duct (Nishida 1964).

2.2 2.2.1

THE TESTIS The Testicular Capsule

The avian testicular capsule is generally very thin (Lake 1971). In a preliminary study, figures for testicular capsule thickness in certain species of birds are, on the average, 578.1 mm in the ostrich, 81.5 mm in the rooster, 91.7 mm in the Japanese quail (Coturnix japonica) and 91.8 mm in the drake (Anas platyrhynchos) (Pers. obs.). However, at the interface between the testis and epididymis, the capsule is much thicker, and has been found to be, on the average, 1215.9 mm thick in the ostrich, 515.0 mm in the rooster, 255.4 mm in the Japanese quail, and 233.7 mm in the drake. There are no readily available data on testicular thickness in mammals, but Davis et al. (1970) indicate that the thickness of the testicular capsule varies from one testicular region to another. The tunica albuginea in man is approximately 1000 mm thick, at 75 years of age (Yoshimura and Fukunishi 1965, cited by Davis et al. 1970). The testicular capsule in the bird divides in the epididymal region, and sends a thick branch around the epididymis. The other branch, devoid of the tunica serosa, runs on the orchido-epididymal border, and is pierced at a number of points, in most birds, by the intracapsular portion of the rete testis (RT) ductules, to enter the epididymis. Histologically, the testicular capsule is composed of three main tissue layers: an outer, thin tunica serosa, a thick tunica albuginea and the innermost, very thin tunica vasculosa (Fig. 2.5). A basement membrane separates the thin mesothelial cells of the t. serosa from the bulky connective tissue of the t. albuginea. The latter forms the bulk of the capsule, and is composed of collagen, elastic fibers and abundant fibroblasts (Hodges 1974). Blood vessels, especially veins of varying sizes run within these bundles of fibroblasts and collagen, and are easily observed running radially on the surface of the testis, grossly. Smooth muscle actin intermediate filaments have been demonstrated, immunohistochemically, in the testicular capsule of the Japanese quail, Drake, Turkey, rooster and Ostrich (Aire, T.A. unpublished observations). This indicates that there are smooth muscles in the testicular capsule of birds, as has been demonstrated in some mammals (Davis et al. 1970; Hargrove et al. 1977). Smooth muscle cells in the testis capsule are probably responsible for the spontaneous and drug-induced contractions of the capsule of mammals. These contractions may assist in moving immobile testicular spermatozoa


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Fig. 2.5 Histological section of testicular capsule of the ostrich (A, C) and drake (B). A. The capsule is thick and shows the tunica serosa (arrowheads), tunica adventitia (T) and tunica vasculosa (arrow). V, large intracapsular blood vessels; S, seminiferous epithelium. B. A thinner capsule, displaying the three tissue layers, as in A. Nu, an elongated nucleus of a myofibroblasts. C. testicular capsule (T) of a juvenile ostrich. There are no obvious septa originating from the testicular capsule, rather, connective tissue containing oval, euchromatic nuclei aggregate and pass between the seminiferous cords (Sc) of the testis. Bars: All figures = 100 µm. Original.

"$ Reproductive Biology and Phylogeny of Birds toward the epididymis (Davis et al. 1970). A similar function may be expected to occur in birds. There are no septa running from the testicular capsule into the testicular substance, as occurs in most mammals (Fig. 2.5A, C). Loose connective tissue, which appears better formed in the ostrich than in other birds, may be seen conducting blood vessels from the subcapsular region into the testis substance, and sending slivers of connective tissue between the seminiferous tubules (Fig. 2.5A, C).

2.2.2 Seminiferous Tubules The seminiferous tubules of birds are dissimilar to those of mammals by forming highly and complexly anastomotic, non-blind-ending network of tubules (Huber 1916; Bailey 1953; Lake 1957; Marvan 1969). Evidence of this structure is commonly found in histological sections (Fig. 2.6), and is probably responsible for the lack of connective tissue septa, as well as the nonlobulation of the avian testis. The seminiferous epithelium is several cells thick, and contains two cell types: fixed, somatic cells, represented by the Sertoli cells, and temporary and mobile germ cells, comprising a series of differentiating cells (spermatogonia, primary and secondary spermatocytes and spermatids) (Fig. 2.6 B, inset). The basal stem cell is the spermatogonium that divides repeatedly to form larger, upward-mobile, cells in successive stages of meiotic division and maturity. Spermatogenesis in birds, as in mammals, involves a series of divisions of spermatogonia, resulting in primary spermatocytes and secondary spermatocytes, both of which undergo meiotic divisions, culminating in the evolution of spermatids. The latter differentiate to form motile, itinerant spermatozoa. Spermatogenesis is discussed fully in Chapter 7, but the Sertoli cell is discussed below.

2.2.3

Sertoli Cells

The Sertoli (sustentacular) cell is the fixed or permanent and non-germinal cell in the epithelium of the seminiferous tubule. It is known as a “nurse” cell because it supports and provides nutrition for the developing germ cells. Avian Sertoli cells are generally similar to those described for the mammal (Cooksey and Rothwell 1973). Sertoli cells are tall, columnar and extend from the basal lamina to the luminal border of the seminiferous epithelium (Fig. 2.7A, B). Sertoli cells in the rooster have fewer broad base contacts with the basement membrane than in mammals (Osman et al. 1980). A similar observation has been made in the Japanese quail (Fig. 2.7A, B). The cytoplasm of this cell is generally more electron-dense than that of attaching or adjacent germ cells (Aire, T. A. personal observations), and arborizes between the germ cells that attach to, or embed in, it in an irregular manner. The nucleus lies at the level immediately above the spermatogonia, and is relatively large and euchromatic. It may therefore be relatively of high activity. It is usually irregular, has a prominent nucleolus, and is situated close to the basal lamina in most birds except in the ostrich where it lies in the middle part of the cell


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Fig. 2.6 Coturnix japonica. A. A survey photomicrograph of the seminiferous tubules showing complex branching of the seminiferous tubules. B. Higher power view of seminiferous tubules displaying various germ cells in the epithelium. Inset: shows Spermatogonia (arrows); Sertoli cell nucleus (arrowhead); round spermatids (Sr); elongated spermatids (Sel). Bars: A = 100 µm, B, including inset = 200 µm. Original.

"& Reproductive Biology and Phylogeny of Birds

Fig. 2.7 Coturnix japonica. A. A resin section of seminiferous tubules stained with toluidine blue, showing positions of concentrated Sertoli cell cytoplasm (arrowheads). Dark particles and elongated profiles in the Sertoli cell cytoplasm are sections of embedded elongated spermatids. B. A TEM micrograph of the basal half of the seminiferous epithelium showing a Sertoli cell (S) with its characteristic elongated and irregular nucleus (N), electron-dense cytoplasm (stars), spermatogonium (Sg) and spermatocyte (Sp). T, peritubular and interstitial tissue. Bar: A = 200 µm, B = 2 µm. Original.


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and hence epithelium (Aire, T. A. personal observations). The cytoplasm contains abundant, small, smooth endoplasmic reticulum (SER), numerous free ribosomes and polyribosomes, a well-developed Golgi complex and micropinocytotic vesicles, both basally and between adjacent cells (Fig. 2.8). Microtubules are conspicuous in the cell body and processes that surround the germ cells. Lipid droplets are dense and small in size, and are few in the rooster (Cooksey and Rothwell 1973) unlike in the Budgerigar (Melopsittacus undulatus) (Humphreys 1975), where they are numerous. Sertoli cells are absolutely necessary for the development of germ cells, from spermatogonia to spermatozoa, not only because they provide physical support and nutrition for the germ cells, but also because they provide the avenue for substances to pass between the blood and germ cells. In mammals, there is a close temporal relationship between the development of the secretion of many Sertoli cell products and the appearance and increase in number of primary spermatocytes and early spermatids (Jégou 1991). The same is probably true in birds because Jégou (1991) considers that the interaction between late spermatids and the Sertoli cell has survived throughout evolution and therefore constitutes a major aspect of the paracrine regulation of spermatogenesis. The passage of substances from Sertoli cells to germ cells is regulated by a peculiar structural feature of Sertoli cells (Mann and LutwakMann 1981), which involves cell-cell contacts in the seminiferous epithelium. These contacts are necessary because there is an intimate interdependence of germ cells and Sertoli cells. Elongating and elongated spermatids are difficult to isolate from Sertoli cells without their being considerably damaged. Besides, early spermatids have not survived well in cell culture (Jutte et al. 1981; Le Magueresse and Jégou 1988). Indeed, Jégou (1991) emphasises that spermatids are major regulators of Sertoli cell function. Details of the cellular contacts between the Sertoli and germ cells have been reported in mammals (Fawcett 1973; Dym 1973; Dym and Fawcett 1970; Fawcett 1975) and birds (Cooksey and Rothwell 1973; Osman et al. 1980; Bergmann and Schindelmeiser 1987; Sprando and Russel 1987; Pelletier 1990; Pfeiffer and Vogl 1993). The main cell contacts in the seminiferous epithelium of the rooster (Osman et al. 1980; Bergmann and Schindelmeiser 1987; Pelletier 1990) are similar to those described in mammals, and are classified as Sertoligerm cell junctions and Sertoli-Sertoli cell junctions (Fig. 2.9). Sertoli-germ cell contacts consist of gap and adhering junctions that have subsurface condensations of 7 nm filaments that are lined internally by cisternae of endoplasmic reticulum (Pelletier 1990). Where two germ cells are apposed, contacts are made by means of gap junctions that are similar to those of Sertoli-germ cell adhesions (Fig. 2.9). The Sertoli-Sertoli cell junctions comprise occluding, gap and adhering junctions, with the occluding junction being most basal, but situated above spermatogonia as well as early primary spermatocytes (Pelletier 1990). The gap junction separates the occluding junction from the adhering junction. The inter-Sertoli cell-occluding junction is the morphological basis of the intra-testicular and extremely effective blood-

# Reproductive Biology and Phylogeny of Birds

Fig. 2.8 Coturnix japonica. High power view of the cytoplasm of the Sertoli cell of the quail exhibiting an abundance of smooth endoplasmic reticulum (SER), a few lipid droplets (L), some mitochondria (M), Golgi complex (G), a few microtubules (upper arrowheads), and a lysosome (Y). Sp, spermatogonium; RER (lower arrowheads) B, basal lamina. Bar = 1 µm. Original.


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testis barrier. The avian occluding junction is different from that of mammals in only a few details, such as the absence, in birds, of subsurface filaments, disposed of as bundles, that lie between the adjacent tight junctions and the subsurface cisternae (Fig. 2.9). This structural barrier, as in mammals, separates the seminiferous epithelium into the basal and adluminal compartments, and is able to prevent tracer compounds in the basal compartment from entering the adluminal compartment of the seminiferous epithelium in the rooster (Osman et al. 1980; Bergmann and Schindelmeiser 1987; Pelletier 1990). The occluding Sertoli-Sertoli cell junctions are present and relatively constant in position, above the spermatogonia, in both regressed testes (Pelletier 1990), and active testes (Osman et al. 1980; Bergmann and Schindelmeiser 1987; Pelletier 1990) of birds. Thus, in the birds investigated, the Sertoli cell junction is constantly present and active, irrespective of the phase in the reproductive cycle.

2.2.3.1

Functions of the Sertoli cell

Since avian Sertoli cells are similar to those of mammals, structurally, it is tempting to assume that the main functions in both classes of animals are generally similar. The abundance of SER in the mammalian Sertoli cell indicates a hormone-secreting cell (Fawcett 1975). Sertoli cells are unable to significantly synthesise steroids de novo, per se, but promote interconversion of steroids e.g. progesterone and androstenedione to testosterone and reduced 5 a-androgens (Mann and Lutwak-Mann 1981). Whereas the presence of steroids was demonstrated in extracts of the fowl testis (Delrio et al. 1967), it was Tingari’s (1973) histochemistry study that demonstrated steroidogenic activities in the seminiferous epithelium, and particularly in those parts that are consistent with Sertoli cell locations, in the rooster. Androgen binding protein (ABP), inhibin and activin are endocrine regulators of follicularstimulating hormone (FSH) production and of testicular steroidogenesis and spermatogenesis, not only in mammals but also in birds (Johnson and Brooks 1996; Lovell et al. 2000; Onagbesan et al. 2004). It is likely that the avian Sertoli cell functions as its mammalian counterpart, in secreting ABP, inhibin, activin, and a part of the testicular fluid (Hagenäs et al. 1975) that have the same types of functions, as in mammals. The phagocytic ability of the Sertoli cell is well known in both mammals and birds. These cells have been observed to remove residual bodies in the rooster and Japanese quail (Cooksey and Rothwell 1973; Sprando and Russel 1987; Lin and Jones 1992) and India ink particles (Fig. 2.10) that were introduced into the seminiferous tubular lumen in the rooster (Pers. obs.). Perhaps one of the most important functions of the Sertoli cell is the establishment of the most durable component of the blood-testis barrier, which creates two fluid compartments within the seminiferous epithelium. This barrier, already described above, is similar to the mammalian barrier, in major particulars. It is present and functional in both sexually mature and active as well as regressed testes in birds, by preventing tracers from entering the adluminal compartment, from the basal compartment (Osman et al. 1980;

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Fig. 2.9. A. A diagrammatic representation of Sertoli-germ cell and Sertoli-Sertoli cell ectoplasmic specializations and junctions in an active testis. Sert, Sertoli cell; Fig. 2.9 Contd. ...


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Pelletier 1990). It has an important protective role in safeguarding the germ line from noxious influences originating both from within and external to the individual, “…including both isolation of sperm-specific autoantibodies and autoimmunocytes from the tubule in the event that sensitization does occur” (Neaves 1977). In addition to these functions, there are peritubular-Sertoli cell interactions. Peritubular cells constitute an important extra-tubular part of the blood-testis barrier, and they also have important regulatory interactions with Sertoli cells in the seminiferous tubule, via the production of paracrine factors that enhance Sertoli cell functions, including the production of ABP and transferrin (see Skinner et al. 1991). Sertoli cell-Leydig cell interactions also exist, in which both cells influence each other with regard to steroidogenic activities, as well as inhibin and activin secretions (see Skinner et al. 1991).

2.2.4

Interstitial Tissue of the Testis

The interstitial tissue of the testis consists of two main components. The first is that compact layer of myofibroblasts and connective tissue which closely surrounds the seminiferous tubule, and known as the boundary tissue. The other is the loose connective tissue, the interstitium, which lies between seminiferous tubules, with full expression in the angular areas or wedges between 3 or more adjacent seminiferous tubules (Fig. 2.11). The boundary or peritubular tissue consists of subepithelial tissue and layers of alternating elongated cells and their interconnecting amorphous tissue. The interstitium, on the other hand, consists of single, or groups of a few, Leydig cells (Cellulae interstitiales), blood vessels, fibroblasts and cells of the macrophage system, such as macrophages, lymphocytes and monocytes. The interstitial tissue of Fig. 2.9 Contd. ...

Sg, spermatogonium; Sp, spermatocyte; Sd, elongating spermatids; B, basal lamina; L, lumen of seminiferous tubule. Arrowheads indicate a junctional complex between adjacent Sertoli cells (Sertoli-Sertoli junctional complex) situated above the spermatogonium, and consisting of a varying number of tight junctions and parallel-running subsurface cisternae of endoplasmic reticulum (highlighted in Figure B). Other less frequently encountered inter-Sertoli cell junctions, lying apical to the tight junctions, are adhering junctions (Ad) (comprising intracytoplasmic condensation of material, on both sides of the Sertoli cell plasma membranes, and a line of dense material between the two cell membranes, and gap junctions (Gp). The former is also found between adjoining Sertoli and germ cells. A special type of Sertoli cell-elongating spermatid cell junction (arrows) displays a subsurface condensed material in the Sertoli cell cytoplasm, but none in the spermatid cytoplasm (hence also known as hemi-ectoplasmic specialization). B. A higher power representation of tight junctions between adjacent Sertoli cells. Arrowheads, focal tight junctions; Sert, Sertoli cell; er, cisternae of endoplasmic reticulum. Unlike in mammals, there are no layers of filaments between the cisternae and the cell membranes, on either side. Original.

#" Reproductive Biology and Phylogeny of Birds

Fig. 2.10 Coturnix japonica. India ink particles (arrowheads) occur in the apical half of the Sertoli cytoplasm after 15 m of retrograde infusion of India ink through the rete testis into the seminiferous tubules. Bar = 50 µm. Original.

the testis has been described in a number of mammals (Christensen 1965; Fawcett et al. 1969, 1970; Dym 1973; Fawcett et al. 1973; Weaker 1977; Skinner et al. 1991), but reports on this tissue in birds are rather scanty, and are, in the main, on domestic species of birds, such as the rooster (Rothwell and Tingari 1973, 1974; Rothwell 1975; Aire 1997), Duck (Marchand 1973; Aire 1997), Guineafowl (Numida meleagris) and Japanese quail (Aire 1997). Although a very important component of the interstitium, Leydig cells have been described in only a few species of birds (Connel 1972; Garnier et al. 1973; Marchand 1973; Rothwell 1973; Scheib 1973; Aire 1997). The following review of the testicular interstitial tissue is based largely on these reports, and a number of unpublished observations from our laboratory.

2.2.4.1

The boundary (peritubular) tissue

Figures 2.11A and B show interstitial tissue located between three seminiferous tubules in the drake. Figure 2.11C illustrates the relationship between the seminiferous tubules, the boundary tissue, and the interstitium containing blood vessels and Leydig cells. The boundary tissue in the rooster (Rothwell 1975) and Ostrich (Soley, J. T., van Wilpe, E. and Aire, T. A. unpublished observations) consists of an inner fibrous layer, subjacent to the seminiferous epithelium, and an outer cellular layer of myofibroblasts. The inner fibrous layer or lamella exhibits a homogeneous, moderately dense basal lamina, and an adjacent layer of multi-directional collagen fibrils. The latter layer, of collagen fibrils, is absent in the birds studied by Aire (1997) (see Fig. 2.12A). It is not known why this structural difference occurs between the bird


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Fig. 2.11 Anas platyrhynchos (A, B), Coturnix japonica (C). A. Wedges of interstitial tissue between three seminiferous tubules shows a peripheral lymphatic vessel (arrows). V, blood vessels; L, Leydig cells. B. The lymphatic vessel (arrow) in this histological section appears to be centrally located in the interstitium. L, Leydig cell. C. An electron micrograph displays a lymphatic vessel (star) situated between the boundary tissue (B) of a seminiferous tubule (S), a blood vessel (V) and a Leydig cell (L). The blood vessel and Leydig cell are within the interstitium. Bars: A = 60 µm; B = 60 µm; C = 2 µm. Original.

#$ Reproductive Biology and Phylogeny of Birds tissues that were fixed by immersion fixation (Rothwell and Tingari 1973; Rothwell 1975; Soley, J. T., van Wilpe, E. and Aire, T. A. unpublished observations) and those fixed by vascular perfusion (Aire 1997). The basal lamina rests on a thin layer of microfibrils and amorphous, moderately electron-dense material in perfused birds (Aire 1997). However, Humphreys (1975) reports that the basement membrane of the budgerigar testis exhibits “irregular collagen content, and that many birds do not show collagen”, at all. Microfilament rearrangement and re-configuration in the Leydig cells of the dog has been described in tissues that were re-fixed by immersion in glutaraldehyde after a previous perfusion fixation (Connel and Christensen 1975). Collagen fibrils have been observed, recently, in unpublished micrographs, below the basal lamina in a patchy manner, in some, but not all, of the seminiferous tubules, in intravascularly perfused adult sexually active Japanese quail, in our laboratory (Fig. 2.12B). This is in accord with the findings by Humphreys, referred to above. Further studies are necessary, using various fixatives and buffers, as well as fixation methods in several species of birds in order to clarify this concern. The mesenchymal layer of myofibroblast cells varies in thickness, based upon the number of concentric, alternating or overlapping cells. There are up to 5, and occasionally, more, cellular lamellae (Aire 1997; Soley, J. T., van Wilpe, E and Aire, T. A. unpublished observations). The myofibroblasts contain highly elongated, uniformly granular nuclei displaying a few foci of marginated chromatin and eccentric nucleoli. Short profiles of moderately distended RER, a few oval mitochondria, a well-developed Golgi complex and a number of micropinocytotic vesicles are found in the cytoplasm, which, also, abounds in typical 5 nm-thick intermediate filaments and associated focal intracytoplasmic densities. In the rooster and ostrich tissues fixed by immersion, bundles of collagen fibres lie in the intercellular spaces between myofibroblast cells (Fig. 2.13). Myofibroblasts that exhibit features which are found in either fibroblasts or smooth muscle cells are not uncommonly seen. In the quail and ostrich, the peritubular cells are invested by what appears to be an incomplete layer of basal lamina (Fig. 3.12B). The endothelium of blood or lymphatic capillary forms the peripheral limit between the boundary tissue and the interstitium (Fig. 2.11C). Birds are similar to the rat, chinchilla, guinea pig and mouse, in possessing a relatively small volume of Leydig cells and in the location of the lymphatic vessels, but differ from these mammals in lacking an extensive peritubular lymphatic system (Fawcett et al. 1973). However, the arrangement of the myofibroblasts is similar to that in human and cat (Burgos et al. 1970), in being multilayered. The peritubular tissue of birds therefore varies in certain particulars between species (Rothwell 1975; Aire 1997; Soley, J. T., van Wilpe, E. and Aire, T. A. unpublished observations), and combines structural features of the interstitium found variably in mammals.

2.2.4.2

The interstitium

The interstitium is relatively compact in birds (Aire 1997) except in the ostrich in which it forms a loose and ‘oedematous’ connective tissue (Soley, J. T., van


#%

Fig. 2.12 Anas platyrhynchos (A) and Coturnix japonica (B). A. The basal lamina (B) of the seminiferous tubule does not rest on an internal lamella of collagen fibers, but directly on an amorphous material adjacent to myofibroblast cells (M) of the boundary tissue. Extensions of the basal lamina (arrows) into the seminiferous epithelium occurs frequently. B. The internal (fibroreticular) lamella of the boundary tissue displays a patchy presence of collagen fibers (C) below the basal lamina (B); Arrow indicates a part of the internal lamella devoid of collagen fibers. Arrowheads, dense material, similar to the lamina densa of the basal lamina, interposed between myofibroblasts (M); S, seminiferous epithelium. Bars: A = 2 µm; B = 1 µm. Figure A is from Aire, T. A. 1997 Onderstepoort Journal of Veterinary Research 64: 291-299, with permission of the Editor. Figure B is original.

#& Reproductive Biology and Phylogeny of Birds

Fig. 2.13 Struthio camelus. A. The boundary tissue at the base of the germinal epithelium (GE) displays a regular basal lamina (arrowheads), the inner fibrous lamellum (F) and the outer peritubular layer consisting of alternating cellular (1-4) and acellular lamellae. Collagen fibrils (cf) are the most conspicuous element of the acellular lamellae. B, Dilated profiles of rough endoplasmic reticulum (RER), bundles of microfilaments (arrow), focal densities (squat arrow) and coated pit (arrowhead) are present in the myofibroblasts. Cross-sections of collagen fibers occur in the acellular lamellae. Basal lamina (double arrowhead). Bars for both figures = 1 µm. (From Soley, J. T., van Wilpe, E. and Aire, T. A. unpublished micrographs).


#'

Wilpe, E. and Aire, T. A. unpublished observations). The component tissue and cell types include a number of centrally situated blood vessels, varying in size from capillaries to large vessels (Fig. 2.11). Lymphatic vessels are sparse and usually peripherally, but occasionally, centrally, located in the interstitium. When peripheral, they meander between the boundary tissue and the central components of the interstitium (Aire 1997). They are commonly located centrally in the Ostrich (Soley, J. T., van Wilpe, E. and Aire, T. A. unpublished observations). Other component cells include very few macrophages that are closely associated with a few, single or groups of a small number of Leydig cells. It has not been determined in birds if there are structural and functional relationships between these two cell types, as in mammals in which they are morphologically and functionally coupled (Bergh 1985, 1987; Gayton et al. 1994). Other blood-derived cells, such as lymphocytes, plasma and mast cells may occur in the interstitium. The central role of the Leydig cell in the secretion of the primary male sex hormone, and possibly also testicular estrogens in man, the stallion and boar (Akingbemi et al. 1999) sets this cell apart for special treatment in this review.

2.2.4.3

The Leydig cell

Leydig cells of birds (Connel 1972; Nicholls and Graham 1972; Garnier et al. 1973; Marchand 1973; Rothwell 1975; Aire 1997; Soley, J. T., van Wilpe, E. and Aire, T. A. unpublished observations) are generally similar, structurally, to those of mammals (Neaves 1975; Russel 1996; Akingbemi et al. 1999). The number of Leydig cells that are present in the interstitial wedges between multiple seminiferous tubules is small (Fig. 2.11C). Leydig cells seem to form columns of cells in the interstices in the turkey (personal observations). References to information on the ostrich are taken from unpublished results of studies done on the interstitial tissue of this bird by Soley, J. T., van Wilpe, E. and Aire, T. A. Ultrastructurally, Leydig cells of gonadally active birds are large, relatively electron-dense, and contain euchromatic, oval, polygonal or elongated nuclei, depending on location in the interstitial tissue (Fig. 2.14). The Golgi complex is generally moderately developed in birds (Aire 1997) except in the ostrich, in which it is well developed and displays numerous Golgi fields (Fig. 2.15B). Prominent, relatively numerous, oval or elongate mitochondria contain tubular cristae that are embedded in an electron-dense matrix (Fig.2.14). The rough endoplasmic reticulum (RER) is poorly developed, relative to the smooth endoplasmic reticulum (SER) which is more developed (Fig. 2.14), but not nearly as well as in mammals (Neaves 1975; Aire 1997). The SER profiles are short, moderately dilated, or, in the ostrich, may form branched or anastomotic strands. Whorls of SER are present only in the guineafowl (Aire 1997), and, in the ostrich, there are concentric collections of thickened membranes which develop within existing cisternae of SER, resulting in arrays of parallel-oriented, long, rod-shaped structures of greater electron density than normal strands of SER with which they are continuous (Fig. 2.15). Their functions are not known. Scattered ribosomes, polyribosomes,

$ Reproductive Biology and Phylogeny of Birds

Fig. 2.14 Coturnix japonica. Part of an electron micrograph of a Leydig cell showing mitochondria (M) with tubular cristae within a dense matrix. Abundant profiles of smooth endoplasmic reticulum (SER), several lipid droplets (D) and only a few, short profiles of rough endoplasmic reticulum (arrowhead) occur in the cytoplasm. The nucleus (N) is oval in shape. F, microfilaments in peritubular myofibroblasts (P); S, seminiferous tubule. Bar = 1 µm. Original.


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dense bodies and intermediate filaments of variable prominence and location within the cell are present in the ostrich. Lipid droplets, often partially extracted, are fewer and larger in the drake, guineafowl and rooster, than in the Japanese quail (Fig. 2.14) and ostrich. In several cells, a solitary cilium, of undetermined axoneme structure, originates deeply in the cytoplasm and, therefore, traverses an intracytoplasmic canal to reach the cell surface, in the ostrich. Non-myelinated nerve cell processes, as well as occasional naked axons are to be found throughout the interstitium. These nerves are transmitted into the substance of the testis by septa-like strands of connective tissue that surround the seminiferous tubules. The boundary tissue, however, does not display any neural elements, but some nerve fibers seem to lie close to the basal lamina of the seminiferous tubules in the rooster (Tingari and Lake 1972a). Adrenergic nerves appear to innervate interstitial cells in Mute swan (Cygnus olor) (Baumgarten and Holstein 1968).

2.2.4.4

Functions of the intertubular tissue

Peritubular cells are involved not only in environmental interactions, that is structural interactions, but also regulatory interactions, in which they produce a paracrine or an autocrine agent to elicit a signal transduction event that influences cellular functions on a molecular level (Skinner 1987). Considerable and varying interactions occur between the cells of the testis (Skinner et al. 1991). These include peritubular cell-Sertoli interactions, all of which are vital to the proper structural and functional roles of the various cells in the testis. For example, the peritubular cells secrete PModS which influences Sertoli cell functions that are vital for the maintenance and control of spermatogenesis (Skinner 1987).

2.3

THE EXCURRENT DUCTS OF THE TESTIS

The excurrent ducts of the testis which comprise various ducts that transport spermatozoa and fluid produced by the seminiferous epithelium, in birds, have, surprisingly, been described in only a few species: the rooster (Gray 1937; Lake 1957; Marvan 1969; Tingari 1971, 1972; Budras and Sauer 1975a,b), Turkey (Hess and Thurston 1977; Hess et al. 1976; Aire 2000a; Aire and Josling 2000), Guineafowl (Aire et al. 1979; Aire 1980, 1982a), Japanese quail (Aire 1979a, 1980, 1982b, 2000a; Clulow and Jones 1982, 1988), Ostrich (Budras and Meier 1981; Aire and Soley 2000, 2003), passerine birds (Bailey 1953; Traciuc 1967, 1969; Middleton 1972; Barker and Kendall 1984), Pigeon (Stefanini et al. 1999), Mute Swan (Mehrotra 1962, 1964) and drake (Marchand and Gomot 1973; Aire 1982a,b; 2000a). The excurrent duct system of birds, as in mammals, comprises (a) the rete testis (RT) unit, (b) the efferent duct unit and (c) the epididymal duct unit. The epididymal duct unit, in turn, comprises the ductus conjugens or connecting duct, the ductus epididymidis or epididymal duct and the ductus deferens or deferent duct. The epididymis of birds is not divided into the

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Fig. 2.15 Struthio camelus. A. A Leydig cell displays an array of rod-shaped structures (R) in longitudinal profiles. These structures, some of which appear branched (arrows), are more electron dense than the smooth endoplasmic reticulum (SER) (arrowheads). Only a few profiles of rough endoplasmic reticulum or sparsely granulated endoplasmic reticulum (squat arrows) occur in the Fig. 2.15 Contd. ...


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customary gross segments of caput, corpus and cauda epididymides, seen in mammals, but it is a complex structure (Fig. 2.16) consisting of several duct units (Lake 1957; Tingari 1971; Budras and Sauer 1975; Hess et al. 1976; Aire 1979a; Aire et al. 1979; Budras and Meier 1981). This organ incorporates the extratesticular portion of the RT, the efferent ducts, the connecting and epididymal ducts (Fig. 2.17). A description of the histological and ultrastructural features of the various duct units of the epididymis will be based, in the main, on the various reports referred to, above.

2.3.1

The Rete Testis

The rete testis (RT) of birds is connected to the seminiferous tubules whose products it transports to the succeeding segment of the excurrent duct system. The occurrence of divisions, based on their location, of the RT has been a controversial subject. Tingari (1971) considers that the rete is located entirely outside the testis but it is generally agreed that there are intracapsular (intratunical) and extratesticular portions of this duct unit. In addition, Bailey (1953), Budras and Meier (1981), Aire (1982a) and Stefanini et al. (1999) have described an intratesticular portion which is made up of rete channels that are similar in epithelial lining to those in the intracapsular and extratesticular segments, or appear in the form of tubuli recti in various species of birds (Gray, 1937; Mehrotra 1964; Aire 1979a; Aire et al. 1979; Budras and Meier 1981; Barker and Kendall 1984). Osman (1980), however, reports that the seminiferous tubules of the testis of the domestic fowl are joined to the RT in three ways: by way of a terminal segment and a tubulus rectus, a terminal segment only, or by direct opening into the rete lacunae and Lake (1957) describes a transitional tubule, lined by modified Sertoli cells, at the junction between the seminiferous tubule and RT. The intratesticular portion of the RT is rarely observed in birds, but in both birds and mammals it constitutes a minor part of the duct unit (Amann et al. 1977; Roosen-Runge and Holstein 1978; Budras and Meier 1981). They are more distinctly evident in regressed testes (Fig. 2.17B). However, in birds, most of the seminiferous tubules terminate by opening into the RT directly (Aire 1982b). The main part of the RT of birds is extratesticular, forming an integral part of the epididymis. Both the intracapsular and extratesticular parts of the RT of the Ostrich (Budras and Meier 1981) are even more complex in organisation than in most of the other birds studied. They consist of a series of connected Fig. 2.15 Contd. ...

cytoplasm. The perimeter of the cell is demarcated by a basal lamina-like material (double arrowheads). B. Rod-shaped structures, as in Fig. 2.15A, are sectioned transversely. Strands of SER and the rods seem to be continuous or closely associated (arrowheads). Some SER profiles display thickened membranes in the lumen (arrows), probably indicating the formation of rod-shaped structures from normal elements of the SER. An extensive Golgi complex (G), a dense body (D) and a vesicular nucleus (Nu) are present in the cell. Bars: A = 1 µm; B = 2 µm. (From Soley J. T., van Wilpe, E. and Aire, T. A. unpublished micrographs)

$" Reproductive Biology and Phylogeny of Birds

Fig. 2.16 Gallus domesticus. A diagrammatic representation of the epididymis and its duct units, based partly on casts. T, testis; RT, rete testis; P, proximal efferent ductule; D, distal efferent ductule; CD, connecting ductule; DE, ductus epididymidis; DD, ductus deferens. From Nasu, T., Nakai, M., Murakami, T., Saito, I., and Takahara, H. 1985 Japanese Journal of Zootechnical Science 56: 81-85. Fig. 2.1-7. Reproduced with the kind permission of Japanese Society of Animal Science.

longitudinal cisterns and ducts that open ultimately into the efferent duct unit (Budras and Meier 1981). The distribution of the RT in mammals also varies between species, being septal and mediastinal in the rat, Rattus norvegicus (Roosen-Runge 1961; Dym 1976; Hermo and Dworkin 1988), Guinea pig, Cavia porcellus (Fawcett and Dym 1974), Goat, Capra hircus (Ezeasor 1986; Goyal et al. 1992), bull, Bos taurus (Hees et al. 1987) and man, Homo sapiens (Roosen-Runge and Holstein 1978). An extratesticular portion appears to be found in only a few mammalian species, e.g. in man (Roosen-Runge and Holstein 1978) and goat (Goyal et al. 1992). In birds, the RT is the smallest duct unit, by volume, in both the epididymis (Table 2.1), and the entire excurrent duct system, constituting only 2.3% of the extra-testicular ducts in the Japanese quail (Clulow and Jones 1988). But, the ratio of surface area of luminal border:luminal volume for this duct unit (43.4:1) is quite large in the Japanese quail (Clulow and Jones 1988).

2.3.1.1

Surface features of the rete testis tubules/lacunae

Scanning electron microscopical features of the RT of birds, have been reported in the drake (Aire 1982a), Ostrich (Aire and Soley 2000) and certain domestic galliform birds—Turkey, rooster, Guineafowl and Japanese quail (Bakst 1980; Aire and Josling 2000). The RT spaces are, not infrequently, broken up into interconnecting cavernous spaces by lamellae or sheets of connective tissue which are, themselves, lined by the rete epithelium. Narrow cylindrical struts of connective tissue or chordae retis, lined by rete epithelium,


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Fig. 2.17 Coturnix japonica. A. Low power histological view of the epididymis showing profiles of the rete testis (RT) opening into ductuli efferentes proximales (PED). DED, ductuli efferentes distales; DC, ductus conjugens and DE, ductus epididymidis. B. A histological section of the para-epididymal region of an involuting testis showing exaggerated profiles of the intratesticular duct system (asterisks), which are portions of the rete testis. TC, testicular capsule; IR, capsular portion of the rete testis. Bar: A = 200 µm, B = 100 µm. Original.

$$ Reproductive Biology and Phylogeny of Birds Table 2.1

Species variation in volumetric proportions (%) of epididymal ducts and structures Species and number of birds

Structures

Chicken (4) Japanese quail (5) Guinea-fowl (3) Turkey (3) Ostrich (4)

Rete testis Proximal efferent duct Distal efferent duct Connecting duct Ductus epididymidis Connective tissue Blood vessels Aberrant ducts

13.3 ± 1.5* 27.6 ± 3.9

9.9 ± 0.7 40.8 ± 3.5

10.7 ± 1.8 45.7 ± 4.2

7.7 ± 1.3 2.3 ± 0.4

15.2 ± 2.5 1.7 ± 0.4

16.2 ± 2.1 0.7 ± 0.0

7.6 ± 0.4 38.7 ± 3.7 2.5 ± 0.4 0.3 ± 0.0

2.4 ± 0.6 27.3 ± 5.4 2.7 ± 0.5 –

1.8 ± 0.2 22.6 ± 1.5 2.3 ± 0.3 –

14.0 ± 6.8 28.1 ± 3.9

2.4 ± 1.8

38.8 ± 4.1 4.4 ±1.6 –

58.2 ± 4.9 1.8 ± 1.4 –

78 11.8 ± 1.8 6.2 ± 2.8 9 5.2 ± 2.2 7 8 26.1 ± 4.1 3.3 ± 1.6 9

* ± Standard error. Adapted from Aire, T.A. 1979. Journal of Anatomy 129: 703 – 706, Table 1, with permission of Blackwell Publishing Ltd.

may be seen to traverse the lacuna, linking opposite walls. Chordae retis, named by Roosen-Runge and Holstein (1978), may be a common feature of the RT in many species, and probably acts as transluminal coupling device for the contractile system of myofibroblasts (Hees et al. 1989), as do the trabeculae septomarginales of the heart. The surface of the epithelial lining of the RT ductules, except for a few minor details, is generally regular, but may bear a few shallow grooves in the ostrich (Aire and Soley 2000). The apical cell outlines are elongated or polygonal in shape. The cell surfaces extend into short, stubby regular microvilli which vary from very few and sparsely distributed to very numerous and evenly distributed (Fig. 2.18). A single, central cilium projects from most cells into the duct lumen in all birds studied (Aire 1982a,b; Aire and Soley 2000; Aire and Josling 2000). The solitary cilium, that exhibits the 9+2 axonemal structure in the bull (Hees et al. 1989), and is probably sensory in function, appears to be a common feature of the rete cells in animals, as it has also been reported in other mammals, including man (Dym 1976; RoosenRunge and Holstein 1978; Goto 1981; Hees et al. 1989). The solitary cilia of the non-ciliated cells of the human oviduct, which are similar to those described in the male reproductive organs, have been shown, using phase- or videomicroscopy, to have vortical or funnel-like movement (Odor and Blandau 1985; Nonaka et al. 1998), contrary to Ghadially’s (1997) assertion that they are immotile. In most birds studied, especially in the rooster, macrophages are seen in the RT lumen, resting on the epithelium (Aire and Malmqvist 1979a; Osman 1980; Budras and Meier 1981; Aire 1982b; Aire and Josling 2000). Only a few spermatozoa and earlier germ cell series occur in the lumen, and each spermatozoon in the RT of the ostrich bears a single, spindle-shaped, distal cytoplasmic droplet (Aire and Soley 2000), a phenomenon that appears to be unique to this bird, and perhaps other ratites. The motility and fertilizing ability of the rete spermatozoa, and, indeed, of spermatozoa in other segments of the excurrent ducts in the ostrich need to be investigated, as has been done


%$Fig. 2.18 Coturnix japonica (A), Gallus domesticus (B, C). Surface morphology of the rete testis epithelium. A. Short, stubby microvilli are concentrated centrally and around the edge of the cell in the quail, or B. are evenly and sparsely distributed throughout the surface. In C. the microvilli are restricted to the edges of the cell surfaces. In B. and C., solitary cilia project from most cells. Bars: A = 10 µm; B and C = 2 µm. Original.

$& Reproductive Biology and Phylogeny of Birds for some other birds (Munro 1938; Bedford 1979; Howarth 1983, 1995), for purposes of comparability.

2.3.1.2

The histology and ultrastructure of the rete testis cells

The rete epithelium varies from simple, low cuboidal to squamous. Because the apical portions of some of the rete cells overlap sections of adjacent cells, the histological sections of the epithelium often display a pseudostratified appearance (Fig. 2.19). This appears to be a common feature in all birds except the ostrich in which the epithelium is almost always simple (Aire and Soley 2003). In mammals, the rete epithelium is simple squamous to low columnar (Leeson 1962; Dym 1976; Bustos-Obregon and Holstein 1976; Osman 1978; Hees et al. 1989). The rete epithelium contains only one, non-ciliated cell type. Other cell types that may be found in the epithelium are intraepithelial lymphocytes (Fig. 2.19A) and an occasional ciliated cell. Ciliated cells have been described by Barker and Kendall (1984) as being a usual component of the epithelium in some wild birds, but Aire (2002a) is of the opinion that a few scattered ciliated cells occur in the rete epithelium of gonadally-resting birds. Most of these cells, obviously, are lost during recrudescence, preparatory to resumption of active gonadal function. The surfaces of the rete cells display short, straight and regular microvilli, in well-fixed tissue. Adjacent lateral cell membranes, or parts thereof, are either straight and regular (Tingari 1972) or form complex interdigitations (Aire 1982b; Aire and Soley 2003) that may extend to the basal part of the cell. Extensive, intricate cell membrane interdigitation is probably associated with active transport of substances (Morales et al. 1984) but there is little net fluid reabsorption in the RT of the Japanese quail (Clulow and Jones 1982). The apical tight junctional complexes between the rete cells contain only one or two punctate fusions which, however, do not permit tracer compounds to reach the duct lumen via the paracellular space (Nakai and Nasu 1991). This type of junctional complex has also been reported in the mammalian rete epithelium (Dym 1976) but Claude and Goodenough (1973) regard it as a ‘leaky’ junctional complex. Desmosomes are also present, both in the apical and basal zones of the plasma membrane (Barker and Kendall 1984). In the ostrich, a unique lateral cell membrane modification, similar, in some respects, Fig. 2.19 Coturnix japonica (A) and Anas platyrhynchos (B). Transmission electron micrographs of rete testis epithelial cells. A. The epithelium often appears pseudostratified. The nuclei (N) are deeply indented, euchromatic and irregular in shape. The cytoplasm contains sparse organelles. L, an intraepithelial lymphocyte; P, periductal tissue. B. Part of the rete cell exhibiting short, stubby microvilli (arrowheads), a number of multivesicular bodies (arrows), a small Golgi complex (G), a few subapical vesicles (V) and round/oval profiles of mitochondria (M). Bars: A = 20 µm; B = 1 µm. A is adapted from Aire, T. A. 2002 Anatomy Histology Embryology 31: 113-118, Fig. 2, with permission of Blackwell Publishing Ltd., and B is adapted from Aire, T. A. 1982 Journal of Anatomy 135: 97-110, Fig. 15, with permission of Blackwell Publishing Ltd.


Fig. 2.19

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% Reproductive Biology and Phylogeny of Birds to a hemi-desmosome, occurs frequently along the length of this membrane. Its function is unknown (Aire and Soley 2003). Rete cell nuclei are typically irregular in outline, being often deeply indented and exhibiting marginated chromatin as well as a single, central nucleolus (Fig. 2.19A). However, in the ostrich, the nuclei have regular, vertically elongated profiles, and are more euchromatic than in other birds. Oval to elongate mitochondria occur above and below the nucleus in moderate numbers, and are more numerous in the ostrich rete cells (Aire and Soley 2003) than has been reported for other birds (Tingari 1972; Aire 1982b; Barker and Kendall 1984). The Golgi complex is moderately large and usually lies in the perinuclear region (Fig. 2.19B). Other organelles worthy of note are multivesicular bodies and strands of rough endoplasmic reticulum, both of which may be numerous in the drake (Aire 1982b). A few, small, lipid droplets are a constant feature of this cell. However, in the ostrich, a solitary, large heterogeneous lipid droplet is a consistent feature in the immediate supranuclear region (Aire and Soley 2003). Intermediate filaments commonly surround the nuclei and support the basal part of the cell, especially in the ostrich (Aire 1982b; Aire and Soley 2003). The chordae retis is lined by an epithelium that is similar to that of the rest of the RT, but its core substance is composed of loose spongy connective tissue, as has also been observed in the bull (Hees et al. 1989). Phagocytised sperm fragments are found occasionally in the rete cell of the rooster (Tingari 1972), and in certain mammalian species (Sinowatz et al. 1979; Holstein 1978). Luminal macrophages contain ingested fragments of spermatozoa only occasionally (Aire and Malmqvist 1979a; Aire and Josling 2000), apparently, removing by ingestion, only damaged or abnormal spermatozoa, but they become avidly spermiophagic in vasectomized or vasoligated birds (Tingari and Lake 1972b; Aire and Heath 1977; Nakai et al. 1989b; Aire 2002a). The subepithelial tissue is composed of dense connective tissue and myofibroblasts or smooth muscle cells (Tingari 1972; Aire 1982b; Aire and Soley 2003). This tissue is richly innervated (Tingari 1972), but poorly vascularized (Barker and Kendall 1984; Nakai et al. 1988) by a non-fenestrated capillary network that is irregularly arranged (Fig. 2.20) (Nakai et al. 1988). Aire (1982b) has observed a rich lymphatic drainage system in the subepithelial tissue in the vicinity of the RT in the drake. Intraepithelial lymphocytes, exhibiting irregular outlines, light-staining cytoplasm and considerably heterochromatic nuclei (Fig. 2.19A), are not uncommon components of the rete epithelium in birds (Aire and Malmqvist 1979b; Aire 1982b; Osman 1980; Stefanini et al. 1999) and mammals (Dym and Romrell 1975; Hees et al. 1989). They have sparse organelles, as in mammals, and there are no junctional complexes between them and epithelial cells. They occur at all levels in the epithelium. These cells probably monitor the epithelium with a view to sequestering germ cell antigens and preventing their escape into the blood or periductal connective tissue (Dym and Romrell 1975; Osman and Plöen 1978).


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Fig. 2.20 Gallus domesticus. Three types of microvasculature of the rete testis ductule (a), efferent ductule (b) and epididymal duct and ductus deferens (c) are shown diagrammatically. The rete testis has a sparse and irregular capillary network, while both the efferent ductule and epididymal duct unit have a dense capillary network. The meshwork of capillaries is polygonal in (b) and elongated in (c). From Nakai, M., Hashimoto, Y., Kitagawa, H., Kon, Y. and Kudo, N. 1988 Japanese Journal of Veterinary Science, with the kind permission of Japanese Society of Veterinary Medical Science.

2.3.1.3

Functions of the rete testis

The functions of the avian RT are still quite conjectural. The RT constitutes only 2.4% in a preliminary study in the ostrich, and between 10% and 13% of the entire volume of the epididymis in galliform birds (see Table 2.1). This does not take into account the small intratesticular and intracapsular portions of this duct unit. The RT is, understandably, the smallest component (2.3%) of the units of the extratesticular genital ducts of the Japanese quail (Clulow and Jones 1988). The histology and ultrastructure of the RT epithelium in birds (Tingari 1971 1972; Budras and Sauer 1975; Hess and Thurston 1977; Hess et al.1976; Aire 1979a, 1982b; Budras and Meier 1981; Barker and Kendall 1984; Aire and Soley 2003) is similar, in most respects, to those of mammals (Dym 1976; Roosen-Runge and Holstein 1978; Goto 1981; Hees et al. 1989), and because of the ‘leaky type’ apicolateral junctional complex between the epithelial cells (Claude and Goodenough 1973; Lopez et al. 1997), this segment of the excurrent duct system may be a particularly weak one, with regard to the blood-epididymal barrier. In mammals, the RT epithelium secretes about 65% of total testicular fluid (Tuck et al. 1970), but the fluid entering the rete testis is the primary secretion of the seminiferous tubules in the quail (Clulow and Jones 2004). However, even though there is a net reabsorption of testicular fluid by all of the epididymal ducts in the Japanese quail, there is little net fluid transport across the RT epithelium (Clulow and Jones 1988).

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Spermatozoa spend only 25 sec in traversing the RT in the Japanese quail (Clulow and Jones 1988), and therefore the through-flow that enters the proximal efferent duct is highly diluted fluid which contains only a few germ cells (Aire 1979a, 1982b). The RT cells are capable of endocytic activity by both fluid-(bulk)-phase and adsorptive endocytosis in the rat (Morales et al. 1984). Tracers interiorised from the lumen by rete cells are disposed of by the lysosomal system in a similar fashion, irrespective of mode of endocytosis. Morales et al. (1984) have concluded that the RT cells may play a role in determining the composition of the RT fluid. These cells in the Japanese quail are also capable of interiorizing intraluminally-introduced India ink particles which are then conveyed to the lysosomal system of the cells (Aire T.A. unpublished observations). However, RT cells fail to phagocytize intraluminally-introduced avirulent strain of Salmonella gallinarium organisms in the Japanese quail (Aire et al. 2004). The myofibroblast layer surrounding the RT lacunae is well developed, and probably acts to move testicular fluid forward into the efferent ducts. The relaxation of the contractile layer, including the chordae retis, could suck fluid from the seminiferous tubules, by means of vis-a-fronte forces, into the cavernous spaces of the RT. Actin, desmin and vimentin filament staining are strongly positive in the myofibroblast layer in the Japanese quail and drake but only slightly positive in the ostrich (Pers. obs.).

2.3.2

The Efferent Ducts (Ductuli Efferentes)

In birds, the RT lacunae are continued by the efferent duct unit (Bailey 1953; Lake 1957; Traciuc 1969; Tingari 1971; Budras and Sauer 1975a; Hess et al. 1976; Aire 1979a, 1980; Aire et al. 1979; Budras and Meier 1981; Bellamy and Kendall 1985), as in mammals (Reid and Cleland 1957; Ladman and Young 1958; Ilio and Hess 1994). The volume proportion of the efferent ducts in the epididymis varies between 35% and 62% in domestic Galliform species of birds and 12% in the ostrich (Table 2.1), and about 19% of the total volume of the genital ducts in the Japanese quail (Clulow and Jones 1988). Two different but serially arranged segments of the efferent duct occur in birds, viz., proximal efferent duct [ductuli efferentes proximalis] (PED) and the distal efferent duct [ductuli efferentes distalis] (DED). The latter is the distal continuation of the PED, and opens into the connecting duct [ductulus conjugens] (Budras and Sauer 1975a; Aire 1979a). The ductuli efferentes, according to Ilio and Hess (1994), are unique in being the only duct unit of the male reproductive tract that is lined by a ciliated epithelium. Whereas in birds there is a distinct histological division of the efferent ducts into the two segments, PED and DED, such a division is not clearly marked in mammals, among which are species-based differences in the non-ciliated cell types present along the length of the duct. Thus, in the laboratory animals, there is a single type of non-ciliated (NC) cell (Hamilton 1975; Robaire and Hermo 1988) and three types in the cane rat, Threonomys swinderianus (Aire and van der Merwe 2003), man, goat and bull (Goyal et al.1992; Morita 1966; Goyal and Hrudka 1981; Gray et al. 1983; Goyal and Williams 1988). Ciliated (C) cells in


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birds, as in mammals, do not vary structurally along the efferent duct, but the non-ciliated (NC) cell types (types I and II, in the PED and DED, respectively) exhibit different and characteristic cytological features (Aire et al. 1979; Aire 1980). Tingari (1971,1972), Marchand and Gomot (1973), Hess et al. (1976), Hess and Thurston (1977) regarded the distal efferent ductule to be the connecting duct, in error, and, consequently, described only a single nonciliated cell type in the efferent duct system.

2.3.2.1

Surface morphology of the efferent ducts

The surface features of the efferent ducts have been described, using scanning electron microscopy, in the rooster and Turkey (Bakst 1980; Aire and Josling 2000), drake (Aire 1982a), rooster, drake and Japanese quail (Aire and Josling 2000) and Ostrich (Aire and Soley 2000). The following account is derived from these reports. Proximal Efferent Ducts (PED). The epithelial lining, and, in some instances, the mucosa of the PED is highly folded, presenting an irregular, festoon appearance (Fig. 2.21A,B). The folds project prominently into the ductal lumen and are lined by cells whose apical surfaces extend into a lush brush border of microvilli (non-ciliated cells) or numerous cilia (ciliated cells). In all investigated birds, the non-ciliated cells (NC) are more numerous than the ciliated (C) cells, and their microvilli are closely packed, long and regularly cylindrical in shape. The microvilli of the C cells are fewer, shorter and thinner than those of the NC cells, and are scattered between the cilia which usually overshadow adjacent NC cells. The microvilli of the NC type I cell of the PED are shorter in the Turkey and very much so in the ostrich than in the rooster and Japanese quail (Aire and Josling, 2000; Aire and Soley 2000). A single cilium projects into the duct lumen from the central region of most NC cells (Aire 1982b; Aire and Soley 2000; Aire and Josling 2000). In vascularly perfused birds (Aire 1982a; Aire and Soley 2000; Aire and Josling 2000), the NC cells do not exhibit apical blebbing, as reported by Bakst (1980), whose specimens were fixed by immersion fixation. Aire (1980; 2000a) regards apical blebbing in this cell type to be a fixation artifact. Distal Efferent Ducts (DED). The epithelium of this round or oval duct is regular and exhibits no folds that are very prominent in the PED. The cilia of the predominant cell type, C cell, overhang the fewer NC type II cells whose apical surface features are similar to those of the NC type I cell of the PED (Fig. 2.21C).

2.3.2.2

Histology and ultrastructure of the efferent duct epithelia

The histology of the efferent ducts in birds has been reported in only a small number of species: in the rooster (Lake 1957; Tingari 1971; Budras and Sauer 1975a, b), turkey (Hess et al. 1976), Japanese quail (Aire 1979a), Guineafowl (Aire et al. 1979), duck (Marchand and Gomot 1973), Pigeon (Stefanini et al. 1999) and the Common Starling, Sturnus vulgaris (Bellamy and Kendall 1985). Histology of Efferent Ducts. The epithelial lining of the ductuli efferentes proximalis (proximal efferent duct) (PED) or occasionally, the mucosa, projects

%" Reproductive Biology and Phylogeny of Birds

Fig. 2.21 Gallus domesticus. Scanning electron microscopy of the surface features of the efferent ducts. A. The duct wall of the proximal efferent duct (PED) displays variably-sized epithelial and mucosal folds. Note that the epithelial surface is devoid of apical cytoplasmic blebs. B. A higher power view of the epithelial surface of PED. C, cilia of ciliated cells; V, microvilli of non-ciliated cells. Cilia of ciliated cells frequently over-shadow the non-ciliated cells which are more numerous that the former cells. C shows an overwhelming preponderance of ciliabearing ciliated cells over the non-ciliated type II cell of the DED. Bars: A = 50 µm; B and C = 5 µm. Original.


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into the duct lumen as folds of varying length and thickness, thus conferring a ‘festoon’ appearance on transverse profiles of the PED (Fig. 3.17A). The epithelium is columnar and pseudostratified because the C cells usually appear truncated between NC cells, and their nuclei are usually situated in the apical half of the cells, while those of the NC type I cells are located in the basal half (Fig. 2.22A). Both cell types make contact with the basement membrane. The NC cells are predominant over the C cells, and, in the common starling in the ratio of 5:1 (Bellamy and Kendall 1985). The luminal content is mainly proteinaceous fluid, in which there is a suspension of sparse spermatozoa and earlier germ cell series. The apical surface of the NC cell is extended by long, closely bunched, regularly cylindrical microvilli that project into the duct lumen. In plastic sections, the subapical region of the cell displays a few vacuoles of varying sizes, below which are rows of round dense bodies extending to the level of the nucleus. The nucleus is round or, more commonly, oval in shape, and contains one or two nucleoli. The C cell is generally of a lighter stain than the NC cell. The nuclei of the C cells are also round or oval, but may be irregular in shape. Even in plastic sections, profiles of organelles in the C cell, other than the nuclei, are hardly discernible. The epithelium rests on a distinct basement membrane that is supported by periductal tissue of fibroblasts, collagen fibers and myofibroblasts. The ductuli efferentis distalis (DED) has a regular profile, both externally and internally (Fig. 2.22B). It has a columnar or high cuboidal, pseudostratified epithelium consisting of NC cells, and a preponderance of C cells. The apical morphological features of these cells are similar to those of the PED. In plastic sections, there are no obvious subapical vacuoles or dense bodies in the NC type II cells of this duct. The C cell is similar, structurally, to that in the PED. The microvasculature of the avian epididymis is derived from branches of testicular artery, including the cranial ureterodeferential ramus, that supply blood to the epididymis in the rooster (Nishida 1964; Nakai et al. 1988) and Ostrich (Elias M, Aire T.A. and Soley J.T. unpublished observations). Smaller branches of these arteries run along the length of the efferent duct and produce a rich periductal network of fenestrated capillaries (Fig. 2.20B). Nerves that supply the epididymis of the rooster are derived from the testicular plexus, and reach the organ by accompanying the testicular arteries (Nishida 1964; Tingari 1971). Both cholinergic and adrenergic components, which have a similar distribution, have been demonstrated in the efferent ducts. These nerve fibers run around the ducts and are associated with the walls of blood vessels and muscle fibers (Tingari and Lake 1972a). Ultrastructure of the Efferent Ducts. Detailed reports of the fine structure of the epithelium in both segments of the efferent ducts (PED and DED) have been published mainly in Galliformes (Tingari 1972; Budras and Sauer 1975a; Hess and Thurston 1977; Aire 1980), drake (Aire 1982b, 2002b) and Pigeon (Stefanini et al. 1999).

%$ Reproductive Biology and Phylogeny of Birds

Fig. 2.22 Gallus domesticus. Histological sections of the PED (A) and DED (B). In A., the NC type I cell (N) contains numerous supranuclear, dense bodies; C, ciliated cell. In B., the NC type II cell (N) lacks the dense bodies found in the corresponding cell type of the PED; C, ciliated cell, and S, spermatozoa in the DED lumen. Bars: A = 200 µm; B = 10 µm. Original.


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The classification of the NC cell into types I and II is based upon important characteristic organelle differences and disposition between the two nonciliated cells lining the PED and DED, respectively (Aire 1980). In mammals, NC cells have also been known to vary structurally along the length of the efferent ducts: thus there is only one type in rodents (Hoffer and Greenberg 1978; Ilio and Hess 1994), but there are three types in the Great cane rat, Threonomys swinderianus (Aire and van der Merwe 2003), and 3 in man, bull, Goat and Dog, Canis canis (Morita 1966; Goyal and Hrudka 1980, 1981; Gray et al. 1983; Goyal and Williams 1988). Variations in the disposition and number of vacuoles and/or granules have been used as criteria for classifying the non-ciliated cells in the efferent ducts of animals. It is not clearly understood whether or not these organelle differences influence the functions of these cells, individually in mammals, but there appears to be an obvious dichotomy in function(s) between the NC type I and NC type II, in birds (Aire 1979a, 1980, 2000b, 2002b; Aire et al. 2004; Clulow and Jones 1988). The NC Type I Cell. The apical surface extends into a microvillous brush border of closely bunched, long, and uniformly cylindrical microvilli (Fig. 2.23). The apical surface may also invaginate as fuzzy-lined, tubular coated pits into the subapical cytoplasm which is remarkably endowed with an elaborate endocytic or tubulovacuolar system. The tubular coated pits are continued by straight or coiled apical tubules, which together with endosomes made up of large dilated membranous vacuoles, as well as a few multivesicular bodies (MVBs) occupy the apical one-fourth of the cell (Fig. 2.24). The apical tubules frequently contain an amorphous inspissated material, probably protein, taken in from the duct lumen. Distal to the endocytic system there occurs a large number of round, variably-sized homogeneous or heterogeneous dense bodies. The dense bodies may become heterogeneous as a result of endocytosis and lysosomal activity by the cell. This type of dense body is probably a telolysosome. Numerous, elongated or oval mitochondria occur in both the supranuclear, and to a greater extent, in the subnuclear regions of the cell. They may measure up to 0.6 mm in breadth (Aire 2002b). The Golgi complex is moderately developed in the supranuclear region of the cell. The oval nucleus is situated in the basal half of the cell, contains a central or eccentric nucleolus, and is generally euchromatic. Lipid droplets are uncommonly encountered in the cytoplasm. Strands of RER and short, small profiles of SER are scattered in the cytoplasm. The apical junctional complexes between the NC cells of the efferent ducts in G. domesticus show, apico-basally, a series of punctate fusions (zonula occludentes), adhering or intermediate junctions (zonula adherens), and desmosomes (macula adherens), in that order, along the complex. A continuous line of fusion between the outer leaflets of adjacent cell membranes in the apical junctional complex is also evident (Nakai and Nasu 1991). But Claude and Goodenough (1973) and Suzuki and Nagano (1978) regard these junctional complexes as being of the ‘leaky type’, in the rat. However, Nakai and Nasu (1991) have shown that the tight junctions in both the rete and

%& Reproductive Biology and Phylogeny of Birds

Fig. 2.23 Gallus domesticus. Transmission electron micrograph of epithelial cells of the PED. N, non-ciliated type I cell; C, ciliated cell. Note the large number of dense bodies (D) and long mitochondria (arrowheads) in the supranuclear region of the NC cells. The mitochondria of the C cells (arrow) are thinner than those of the NC cells. The lush microvilli (m) of the NC cells are regularly cylindrical and longer than the inter-cilial microvilli of C cells. Bar: 1 µm. Adapted from Aire, T. A. and Josling, D. 2000 Onderstepoort Journal of Veterinary Research 67: 191-199, Figure 12, with permission of the Editor.


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Fig. 2.24 Anas platyrhynchos (A), Gallus domesticus (B, C, D). Non-ciliated type I cells of the PED. A. Supranuclear region of NC type I cells showing an elaborate subapical tubulovacuolar or endocytic system, comprising coated pits (arrowheads), coated apical tubules (arrows) and vacuole (V). D, dense bodies; N, nucleus. B. A coated pit (arrowhead) leads into a non-coiled apical tubule (T); M, microvilli; J, junctional complex. C. Transverse sections of apical tubules contain inspissated material (arrowhead). D. Heterogeneous dense bodies (H) and mitochondria in a cell that has experienced phagocytosis. Bars: A = 10 µm; B = 2 µm; C and D = 1 µm. Adapted from Aire, T. A. 2002 Journal of Morphology 253: 64-75, Fig. 4., with permission of Wiley-Liss, Inc.

& Reproductive Biology and Phylogeny of Birds efferent duct epithelia are able to exclude lanthanum nitrate from the duct lumen in the rooster. It is not known if this complex can exclude compounds of smaller molecular weights from passing into or out of the lumen. Large and extensive intercellular spaces occur between adjacent NC cells in the PED in the Ostrich. These spaces usually occur in the basal two-thirds of the epithelium, and are frequently seen to extend to the basal lamina in electron micrographs. Dilated intercellular spaces are usually associated with enhanced fluid absorption by epithelia (Pudney and Fawcett 1984), and in this case, paracellular fluid movement, apparently by active solute transport (Suzuki and Nagano 1978). The NC Type II cell. The microvillous brush border is similar to that of the NC type I cell. The type II cell lacks the characteristic, elaborate, subapical endocytic apparatus and numerous dense bodies in the supranuclear zone of the type I cell (Fig. 2.25). Instead, only a few, sparsely-distributed, subapical coated pits and apical tubules, containing inspissated material, are scattered between the bundles of microfilaments which project from the microvilli into the subapical cytoplasm. Vacuoles are usually few, small and scattered in the apical half of the cytoplasm. Mitochondria are fewer than in the NC type I, and are scattered within the supranuclear, and to a lesser extent, in the subnuclear cytoplasm. The nuclei are similar to those of NC type I cells in location, size, shape and configuration. Strands of RER and a few quite small dense bodies and lysosome-like bodies may be seen in the cytoplasm. Ciliated Cells. Readily discernible differences are not to be found in the ultrastructure of the C cells in both segments of the efferent ducts, but the number of C cells relative to NC cells increases markedly in the DED, with a ratio of 4:5, respectively, in the Common starling (Bellamy and Kendall 1985) and 9:1 in the ostrich (Budras and Meier 1981) than in the PED that has a respective ratio of C to NC cells of 1:5 in the common starling (Bellamy and Kendall 1985) and 3:7 in the Ostrich (Budras and Meier 1981). Uniformly-spaced cilia, interspersed with a few, short and thinner microvilli than in the NC cells, project into the lumen (Figs. 2.23 and 2.25). A few coated apical pits are also present. Mitochondria are mainly in the supranuclear region of the cell; they are only about 30% as broad as those of the NC cells (Aire 2002b). The Golgi complex is of moderate size. Numerous bundles of microfibrils, possibly assisting in stabilizing the cell whose role includes movement of the luminal through-flow, may be seen running in different directions, in the supranuclear and perinuclear zones of the cell (Aire 1980). A euchromatic nucleus, which is generally oval in profile, but is often indented or invaginated, is situated in the apical half of the cell. Profiles of RER, polyribosomes, a few small dense bodies and lysosomes may be seen in the cytoplasm. The C cell is thought to assist in moving the seminal content of the efferent ducts, in addition to a limited endocytic activity, and, thus, also influencing the composition of the luminal content. Intraepithelial lymphocytes are not uncommonly present, in varying numbers and at various levels, in the epithelium of both segments of the


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Fig. 2.25 Gallus domesticus. A. A survey transmission electron micrograph of the DED epithelium. C, ciliated cell; N, non-ciliated type II cell showing one or two subapical vacuoles; L, intraepithelial lymphocyte. B. High power of the supranuclear region of ciliated (C) and non-ciliated type II (N) cells. The N cell lacks the numerous dense bodies found in the supranuclear region of the corresponding cell in the PED, and only a few apical tubules (arrowheads) are present in the DED. Arrow, microfibrillar bundles in the supranuclear region of a C cell. Bars: A = 200 µm, B = 2 µm. Original.

efferent ducts (Fig. 2.25A), as in the RT epithelium (Aire and Malmqvist 1979a). Function of the Efferent Ducts. The structure of the efferent ducts is generally similar in mammals and birds (Burgos 1960; Ladman 1967; Tingari 1972; Ramos and Dym 1977; Jones et al. 1979; Aire 1980; Hermo et al. 1988; Robaire and Hermo 1988). It is therefore very tempting to ascribe similar functions to the ducts in most, if not all, animals. In birds, however, unlike in mammals, the proximal segment (PED) of the duct possesses a highly folded epithelium

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which increases considerably the surface area that is available and exposed to the luminal content. Besides, the PED constitutes a greater proportion of the epididymal volume (about 300% more) than the distal segment (DED), but, together, both of them constitute between 35% and 62% of the entire epididymal volume in various species of birds (Aire 1979b). This is significant because the avian testis has a high fluid content and sperm production, the latter being also very rapid. The structure of the PED epithelium is consistent with active uptake of luminal macromolecules and considerable luminal fluid reabsorption, and transport across the epithelium. The PED must therefore play a major role in modifying luminal content, and, in general, the functioning of the avian excurrent duct system, as in mammals. However, little is known about endocrine regulation of the avian male reproductive tract and its role in production of fertile spermatozoa (Janssen et al. 1998). The efferent ducts are probable sites of steroidogenesis in the rooster (Tingari 1973), but androgens do not prolong sperm viability in the ductus deferens of the rooster (Munro 1938). Estrogen receptors are strongly expressed in its efferent ducts (Kwon et al. 1997) and in several mammals (Goyal et al. 1997, 1998; Fisher et al. 1997). Estrogens seem to have a profound effect on the ability of the efferent ducts in Mouse (Mus musculus) to reabsorb luminal fluid (Hess et al. 1997). In mammals, micropuncture studies indicate that the efferent ducts reabsorb most of the testicular fluid entering the excurrent ducts of the testis (Crabo 1965; Jones 1980; Jones and Clulow 1987; Clulow and Jones 1988; Clulow et al. 1994; Man et al. 1997). In their excellent studies in the Japanese quail, Clulow and Jones (1988) show that even though spermatozoa (in their fluid medium) spend only 3 min traversing the PED, yet about 86% of the fluid leaving the testis is reabsorbed there, and another 6.5% in the DED. Important ion transporters, such as sodium-potassium ATPase [Na+, K+-ATPase] carbonic anhydrase II (CA II) and sodium hydrogen exchanger isoform 3(NHE3) have been immunolocalized in the efferent ducts of the rooster (Bahr et al. 2006). Transmembrane water channel proteins (aquaporins –2, –3, and –9) that are responsible for water flow, have similarly been localized in efferent ducts of the large white turkey (Zamboni et al. 2004). The arrangement of the efferent duct unit into a large number of narrow ducts in a parallel array provides a large ratio of luminal surface area:luminal volume in the Japanese quail (Clulow and Jones 1988). Spermatozoa traverse the entire efferent duct unit in 8 min in the Japanese quail (Clulow and Jones 1988) in contrast with about 45 min in the rat (English and Dym 1981). It is clear that the rate of fluid reabsorption of testicular fluid by the efferent ducts of the testis is much higher in birds than in mammals (Jones 1998). The inspissated material in the lumen of apical tubules of the NC type I cell in birds (Fig. 2.24C) is probably proteinous, and, if so, it indicates that this type of cell, and therefore the entire efferent duct unit, is capable of absorption of testicular proteins secreted into the testicular fluid, as has been established for mammals (Koskimies and Kormano 1975; Olson and Hinton 1985; Jones and Jurd 1987; Veeramachaneni and Amann 1991; Clulow et al. 1994).


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Morales and Hermo (1983) and Hermo and Morales (1984) have demonstrated that the non-ciliated cells of efferent ducts are capable of internalizing specific substances from the duct lumen by both adsorptive and fluid-phase endocytosis in the rat. Nakai et al. (1989a) and Aire (2000a) have also demonstrated the respective ability of NC cells in the PED of birds to interiorize testicular proteins and India ink particles (Fig. 2.26). The absorbed materials are destined for the lysosomal system of the cell. The NC cells in the PED are able to recognise, by an unknown mechanism, and remove cationic ferritin, even in bicameral chambers (Janssen et al. 1998), as well as ‘designated’ spermatozoa (Hess et al. 1982; Aire 2000b, 2002a) and intraluminally introduced avirulent strain of Salmonella gallinarium (Aire et al. 2004) from the luminal through-flow in the rooster and Japanese quail. In vasectomized birds, desquamated germ cells that are transported from the seminiferous tubules to the excurrent ducts are sequestered in the PED where they degenerate and their fragments are removed by phagocytic acitivities of the NC type I cells (Tingari and Lake 1972b; Aire and Heath 1977; Nakai et al. 1989b; Aire 2002a). The DED seems to be screened, by an unknown mechanism, from such germ cell debris by the PED. Remarkably, the NC type I cells are also capable of proliferating and forming new adluminal sheets of cells which are highly spermiophagic on both their free surfaces, in the process of removing an overwhelming accumulation of sperm debris, following vasectomy in the Japanese quail and rooster (Aire 2000b, 2002a) or carbendazim exposure in Coturnix (Aire 2004). These new sheets of cells subsequently sequester small ducts from the original duct lumen, during the process of microrecanalization in the efferent ducts (specifically the PED segment) in birds (Aire 2004). Microrecanalization, specifically of the transected end of the vas deferens, has been described previously only in vasectomized men, and was probably responsible for unexpected fathering of babies by such men (Cruickshank et al. 1987; Freund et al. 1989).

2.3.3 The Epididymal Duct Unit 2.3.3.1 General organization and features From ontogenetical, structural and functional perspectives, the connecting duct (ductus conjugens), epididymal duct (ductus epididymidis) and deferent duct (ductus deferens) are essentially similar and constitute a functional unit (Gray 1937; Lake 1957; Tingari 1971, 1972; Budras and Sauer 1975a; Budras and Meier 1981; Aire 1979a; Aire et al. 1979; Aire 2000a). This is also in accord with the observations made by Tingari (1971, 1972) and Aire et al. (1979), that the NC type III cell is identical in the epithelia of these ducts, which shows that the avian ductus epididymidis and ductus deferens are merely different segments of the same organ, an organ equivalent to, but not grossly structured as, the epididymis in mammals. This functional unit may therefore be referred to, for convenience, as the “epididymal duct unit”. This duct unit forms the greater proportion (about 74%) of the entire excurrent duct volume than all other duct units put together, in the Japanese quail (Clulow and Jones 1988).

&" Reproductive Biology and Phylogeny of Birds

Fig. 2.26 Coturnix japonica. A. Rete testis-infused India ink particles are present in the non-ciliated, but not the ciliated cells of the PED epithelium, and are absent in the epithelial cells of the DED and ductus epididymidis (DE). B. India ink particlefilled lysosomes are present in transverse sections of non-ciliated (N) cells but not ciliated (C) cells of the PED. Bars: A = 200 µm; B = 1 µm. Original.


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Similarly, the epididymis and vas deferens in mammals, together, constitute, a priori, the greatest proportion by volume of the excurrent duct system. The efferent ducts are continued distally by the epididymal duct unit that is derived from the Wolffian duct (Budras and Sauer 1975a). This duct is lined by a non-ciliated epithelium. The first portion of this unit is the short connecting duct (CD) that joins the efferent duct, specifically, the DED segment of the efferent duct unit, to the epididymal duct (ED). The CD opens into the ED which is a slightly wavy duct (Fig. 2.16), but may be complexly tangled in the Common Tern (Sterna hirundo) and Jackdaw (Corvus monedula) (Traciuc 1967, 1969). It is situated longitudinally on the dorsomedial border of the epididymis, and runs caudally into the ductus deferens (DD). Neither the ED nor DD is therefore similar to those of mammals in length, gross features or configuration.

2.3.3.2

The ductus deferens and its modifications

In non-passerine birds, the ductus deferens is highly wavy and increases in diameter cranio-caudally, in sexually active birds (Fig. 2.1). Close to the cloaca, the ductus deferens straightens out for a variable length, depending on the species and size of bird, to form the pars recta ductus deferentis (Marvan 1969; Lake 1971; Marchand and Gomot 1973). This part subsequently enlarges into a thick-walled, barrel-or spindle-shaped structure, the receptaculum ductus deferentis that pierces the wall of the cloaca to open into the urodeum of the cloaca by an opening in its pointed end, the papilla ductus deferentis. In passerine birds, the ductus deferens is complexly thrown into coils that form a compact ball, the seminal glomus or sac, caudally (Bailey 1953) (Fig. 2.27). The seminal glomus is a tubular and highly coiled structure that is encapsulated in loose connective tissue (Bailey 1953; Salt 1954 and Wolfson 1954). It lies beneath the skin, dorso-lateral to the cloaca (Salt 1954), where its coiled ducts may be visible through the skin of the cloacal protuberance (Mulder and Cockburn 1993). The latter, akin to the scrotum in scrotal mammals, is a swelling of the skin, that accommodates the seminal glomus. The seminal glomus is a characteristic feature of passerine birds (though also reported for the psittacid Melopsittacus undulatus, see Chapter 8), and is regarded as an anatomical adaptation that ensures a storage and probable maturation site for spermatozoa (Lake 1981) that are necessary for sperm competition (Birkhead et al. 1993). It is tempting to regard the lower temperature in the seminal glomus as beneficial to spermatozoa in passerine birds, as it is in scrotal mammals. Wolfson (1954) believes that the lower temperature is necessary for sperm maturation. But, avian sperm do not require maturation and capacitation in order to be fertile (Munro 1938; Bedford 1979; Howarth 1983). The seminal glomus may therefore serve as a daily sperm store since, it appears, sperm production in the testis as well as sperm transport to the glomus seminalis occurs more rapidly at night, while its sperm content declines significantly during the day (Birkhead et al. 1994). The seminal glomus may, in addition to acting as a storage area, provide a

&$ Reproductive Biology and Phylogeny of Birds

Fig. 2.27 A diagrammatic representation of a ventral view of the reproductive organs and tract of a sexually mature and active passerine bird, Passer domesticus (the House sparrow). T, testis; E, epididymis; D, ductus deferens; SG, seminal glomus; CL, cloaca; R, rectum; V, vent; Arrowhead, skin of the cloacal protuberance. Not drawn to scale. Original.

lower temperature for spermatozoa, in order to conserve their energy or perhaps permit a biochemical or physiological reaction or adjustment in the spermatozoa, before insemination into the female tract. The precise function of the seminal glomus remains speculative. This structure is neither homologous nor analogous to the seminal vesicle of mammals. The cloacal protuberance is a very useful structure for sexing passerine birds, and in determining the prevailing phase of their reproductive cycle. The size of the protuberance is strongly and positively correlated both with the mass of the seminal glomus and its sperm content (Birkhead et al. 1993)


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and, therefore, a guide to the sperm production capacity of the bird under examination. In the Superb Fairy-Wren (Malurus cyaneus), a pointed anterior ‘tip’ of the cloacal protuberance, not yet described in other passerine birds, probably facilitates effective sperm transfer, during short periods of copulation (Mulder and Cockburn 1993).

2.3.3.3

Surface features of the epididymal duct unit

The epididymal epithelium, at low power magnification, appears smooth and presents no folds except at sharp angulations and at the entry of the CD into the ED where several longitudinal ridges and grooves occur (Aire and Soley 2000). A number of irregularly distributed invaginations or ‘craters’ (Fig. 2.28) occur on the epithelial surface in the drake (Aire 1982b). In all birds studied (the ostrich by Aire and Soley 2000; Turkey, rooster and Japanese quail by Aire and Josling 2000), except the drake (Aire 1982b), the luminal surfaces of the CD, ED and DD appear cobbled, with distinct intercellular grooves. Close-up views show numerous, evenly distributed, regular microvillar extensions of the apical surfaces of the principal epithelial cells (Fig. 2.28B). A single, central cilium projects from several cell surfaces into the duct lumen.

2.3.3.4

Histology of the epididymal duct unit

In non-passerine birds, the histological features of the epididymal duct unit have been reported in several species. Discrepancies or errors in the nomenclature of the ducts, as well as the interpretation of normal structure, are to be found in the literature (Gray 1937; Lake 1957; Tingari 1971, 1972; Budras and Sauer 1975a; Hess et al. 1976; Aire 1979a, 2000a). These will be highlighted in this review. The CD, ED and DD are lined by cuboidal to columnar, non-ciliated epithelium, simple or pseudostratified, depending on the angle of section (Fig. 2.28). The epithelium consists of the non-ciliated type III cell (Aire et al. 1979) which is distinctly different, ultrastructurally, from the non-ciliated types I and II cells of the PED and DED, respectively. Basal cells, not present in the more proximal duct units, are wedged between the NC type III cells, and their long axes typically lie parallel to the basal membrane. Basal cells increase in number, cranio-caudally, i.e., they are least numerous in the CD and ED, but quite numerous and virtually form a distinct layer of cells in the caudal part of the DD. The ductal lumen is regular in outline and oval in cross-section, save at the entry of the CD into the ED where epithelial ridges and grooves occur (Aire and Soley 2000). A short microvillous brush border, varying in height from 0.7 mm in the drake to 1.6 mm in the Japanese quail (Aire 2000a) projects from the NC type III cells into the lumen. Nuclei of the NC type II cells are round or oval in shape, and display single, central nucleoli. Intraepithelial lymphocytes are also present in the epithelium (Aire and Malmqvist 1979b). The epithelium rests on a compact, richly vascular peritubular boundary tissue composed of fibroblasts, collagen fibers and several layers of smooth muscle cells (Aire 2000a). In passerine birds, not much has been reported on the normal structure of the excurrent ducts of the testis, even though passerines constitute a

&& Reproductive Biology and Phylogeny of Birds

Fig. 2.28 Anas platyrhynchos (A), Meleagris gallopavo (B) and Struthio camelus (C). In A., A SEM view of the surface epithelium of the epididymal duct, showing a regular surface interspersed with crater-like depressions. Several solitary cilia Fig. 2.28 Contd. ...


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significantly large proportion of all birds. In fringillids, the epithelium of the epididymal duct is reported to be columnar and ciliated (Bailey 1953) but that of the ductus deferens is columnar or pseudostratified and non-ciliated. Bedford (1979) considers that the DD has a ciliated epithelium. The apical cytoplasm of NC cells blebs into the duct lumen (Bailey 1953), in a manner that is reminiscent of apocrine secretion. Care must be taken in regarding these blebs as representing apocrine secretion, because histological sections of this tissue, fixed by immersion, but not by vacular perfusion, in non-passerine birds, display such apical blebs that are regarded as artifacts of fixation (Aire 1980, 1982b, 1997, 2000a). Basal cells that lie on the basement membrane are present between non-ciliated cells. The epithelial lining of the seminal glomus is cuboidal to low columnar, and is, also, composed by non-ciliated and basal cells (Bailey 1953; Salt 1954; Wolfson 1954; Middleton 1972). However, Bullough (1942) and Bhat and Maiti (2000) have described a ciliated columnar epithelium. Middleton (1972) considers that the epithelium contains secretory cells whose product is PASpositive, and probably glycogen. The duct is filled with spermatozoa the heads of which are pointed toward, and make contact with, the epithelium, which in some cases is penetrated and eroded by the spermatozoa, thus creating the impression of holocrine secretion by this epithelium. The epithelium is invested by a well-defined fibromuscular layer of boundary tissue. There is a great deal of discrepancy in the nomenclature of the various ducts of the epididymis in these reports. Further and more detailed studies of the structure of the epithelial lining of the various excurrent ducts of the testis of passerine birds is necessary in order to minimise the confusion that exists in the literature. The Receptaculum Ductus Deferentis. This structure displays a large number of epithelial and mucosal folds that are not obliterated, even when full of spermatozoa. The folds are longer and more complex toward the papilla of the receptacle. The epithelium is columnar, may be pseudostratified and comprises non-ciliated cells and numerous basal cells (Fig. 2.29). Several deep grooves or crypts, and their cross-sections occur in histological sections. In the ostrich, the cross-sections of the crypts, especially toward the papilla, Fig. 2.28 Contd. ...

(arrowheads) project from most cells. S, spermatozoon. B. Higher power view of the surface epithelium of the epididymal duct, showing uniformly distributed, regular microvilli and solitary cilia projecting from several cells. C. A histological section of the epididymal duct of the ostrich, exhibiting a columnar profile; basal cells (arrowheads) are present, and increase considerably in number craniocaudally. Bars: A = 200µm; B = 2 µm; C = 200 µm. Figure A is adapted from Aire, T. A. 2000 Anatomy Histology Embryology 29: 179-191, Figure 2. Reproduced with permission; Figure B is from Aire, T. A. and Josling, D. 2000 Onderstepoort Journal of Veterinary Research 69: 191-199, reproduced by permission of the Editor. Figure C is original.

' Reproductive Biology and Phylogeny of Birds

Fig. 2.29 Struthio camelus. Histological sections of the receptacle of the ductus deferens. A. The epithelium projects finger-like processes into the duct lumen. Eosinophilic and PAS-positive, but not glycogen, secretory material (arrowheads) occurs within the epithelium, particularly in the region of the crypts between epithelial folds. B. Basal cells (arrows) are extremely numerous, and line the basal part of the epithelium. Bar: A = 100 µm, B = 200 µm. Original.

often display spermatozoa and/or an eosinophilic content that is PASpositive, non-glycogen polysaccharide material (Fig. 2.29), of which the mode of secretion and function are unknown (pers. obs.). The epithelium of the receptaculum ductus deferentis therefore appears to be secretory and capable of influencing or modulating sperm viablility or nutrition. Its sperm content is highly concentrated, and therefore can be rapidly regulated (Clulow and Jones 1988). In the drake, the epithelial grooves contain only spermatozoa, if any (Aire et al. 1979). The epithelium is enclosed by a very thick coat of fibromuscular tissue. In the rooster, this tissue contains subepithelial sinuses and tortuous arteries, akin to the situation in the mammalian penis (Lake 1957), but the function and mechanism of action of this structural peculiarity, with regard to ejaculation in birds, is unknown. Nevertheless, the receptacle appears to be a temporary storage compartment for a relatively large number of spermatozoa, close the point of their emission.

2.3.3.5

Ultrastructure of the epididymal duct unit

Reports of ultrastructural studies on the avian epididymal duct unit are scarce, and are mainly on the rooster (Tingari 1972; Aire 2000a), turkey (Hess and Thurston 1977; Aire 2000a), drake and Japanese quail (Aire 2000a). Non-ciliated Type III Cells. The apical surface of the type III cells extend into a microvillous brush border (Figs. 2.30 and 2.31), which is only about 65% as


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Fig. 2.30 Anas platyrhynchos. A survey electron micrograph of epithelial cells lining the epididymal duct unit, taken from the ductus epididymidis. Short microvilli, a well developed Golgi complex (G), complexly folded lateral plasmalemma (open arrows); nucleus (N) and an investing layer of intermediate filaments (IF). Numerous mitochondria occupy the supranuclear region of the cell. Bar = 1 µm. Original.

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Fig. 2.31 Anas platyrhynchos. Magnified portions of the non-ciliated type III cell of the epididymal duct unit. A. The microvilli may branch (small arrowhead), the Golgi Fig. 2.31 Contd. ...


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long as those of NC types I and II cells in efferent ducts of the Japanese quail (Aire 2000b). The microvilli are regularly cylindrical in profile, and blebbing of the apical cytoplasm is rarely seen in tissues that are well fixed, either by very good immersion fixation or, more importantly, by good intravascular perfusion fixation. Apical blebbing in NC types I and III cells has been described by various authors (Budras and Sauer 1975a; Hess and Thurston 1977; Bakst 1980; Stefanini et al. 1999). However, Aire (1979a, 1980, 1982b), Aire and Josling (2000) and Aire et al. (1979) have found that these blebs are absent in very well fixed tissues. Nicander (1970) and Hamilton (1975) have also regarded these structures, in mammalian epididymal tissues, as artifacts of fixation. Ericsson (1964) has made similar observations in poorly fixed homologous cells of the kidney. Adjacent lateral plasma membranes are intricately folded (Fig. 2.30), especially in the basal two-thirds of the cell length. These foldings may serve a mechanical purpose of stabilizing intercellular attachments, or in water transport (Pease 1956). Apicolateral junctional complexes are well formed (Friend and Gilula 1972) and both the tight junction (zonula occludens) and adhering intermediate junction (zonula adherens) are composed of multiple punctate fusions which exclude tracer compounds from the duct lumen, in the rooster (Nakai and Nasu 1991). Desmosomes on the lateral membranes and hemi-desmosomes in the basal plasma membrane are also present. The nucleus of the NC type III cell is round or oval in shape in the drake (Aire 2000a) and Ostrich (pers. obs.) or vertically elongated in the rooster, Turkey and Japanese quail (Tingari 1972; Hess and Thurston 1977; Aire 2000a). It is moderately heterochromatic, contains a usually eccentric nucleolus, and is basally situated in the cell. Bundles of intermediate filaments (IFs), up to 640 nm wide, may surround the nucleus, partially or wholly (Fig. 2.30), particularly in the drake (Aire 2000a) and may be seen to attach to other organelles as well as the plasma membrane. The function of this distinct assemblage of IFs in the drake is not clearly understood, but IFs are known to form the cytoskeleton component that connects different parts of the cell into an organic network, act as organizers that control the distribution of different subcellular structures, and as integrators of the cellular space (Lazarides 1980; Geiger 1987 and Zhu et al. 1997). A moderately abundant endoplasmic reticulum occurs in the cell, and are mainly of the sparsely granulated (SGER) type in the drake, in particular, while in other birds, it is represented mainly Fig. 2.31 Contd. ...

complex (G) is well developed but consists of only a few saccules; the cytoplasm contains abundant, distended profiles of SER or sparsely granulated endoplasmic reticulum (SGER) (large arrowheads); the subapical region shows numerous secretory vesicles with a dense content (arrows). B. Apical half of part of the NC type III cell displaying profiles of microtubules (arrowheads) extending to the apical membrane of the cell. Arrows, clathrin-coated vesicles. Bars for both A and B = 2 µm. Original.

'" Reproductive Biology and Phylogeny of Birds by profiles of SER and RER. In Turkey, a whorl of RER occurs invariably in the immediate supranuclear zone (Aire 2000a). Abundant free ribosomes or rossettes of ribosomes occur in the cells. Lipid droplets are uncommon in all birds (Tingari 1972; Aire 2000a) except the turkey in which they are quite abundant, as lipid aggregates, in the supranuclear, and to a lesser extent, infranuclear regions of the NC type III cell (Hess and Thurston 1977; Aire 2000a). The lipid content in the cells, particularly in the Turkey, may be related to some form of steroidogenesis, as Tingari (1973) has shown that 3b-and 17b-hydroxysteroid dehydrogenases are moderately localized in the epididymal duct of the rooster. Mitochondria are dispersed evenly in the cytoplasm, and are each usually surrounded by a strand of RER. A well developed, supranuclear Golgi apparatus occurs in the drake and Japanese quail. Numerous smooth-surfaced vesicles and clathrincoated vesicles are observed in the region of the Golgi complex. The Golgi complex, in this duct unit, is very well developed (Fig. 2.31), but is not as elaborate as in the epididymal duct of mammals (Nicander and Glover 1973; Ramos and Dym 1977; Hermo et al. 1991; Stoffel and Friess 1994). Its function in birds appears quite significant (Aire 2000a). Noteworthy features include numerous secretory vesicles, with increasing condensation of their content, as they move from the peri- and supra-nuclear zone toward the apical plasmalemma (Fig. 2.31B), in the rooster (Tingari 1972) and drake (Aire 2000a). Microtubules extend from the region of the Golgi complex to the apical zone in the Japanese quail (Aire 2000a) and drake. Movement of secretions by means of vesicles and/or microtubules is well established in, possibly, all eukaryotic cells (Palade 1975; Cooper et al. 1990; Darnell et al. 1990). The secretory vesicles contain dense, amorphous material, which is probably discharged in a merocrine manner of secretion into the duct lumen (Tingari 1972; Aire 2000a). The nature of this secretion is unknown. However, about four Wolffian duct proteins bind to spermatozoa as they pass through the epididymis and the ductus deferens in the rooster (Esponda and Bedford 1985; Morris et al. 1987). Only one androgen-dependent protein of 17kDa has been identified in the Japanese quail (Kidd 1982, cited by Jones 1998). These proteins seem to have a specificity that is confined to the same order of birds, in this case, Galliformes (Esponda and Bedford 1985; Morris et al. 1987). Coated pits invaginate into the subapical cytoplasm, and lead to only a few, scattered subapical coated vesicles. These indicate that the NC type III cell is capable of micropinocytosis, probably of luminal proteins, along with a very small quantity of fluid, because Clulow and Jones (1988) have shown that the epididymal duct unit (CD, ED and DD) reabsorbs only about 0.8% of the total testicular plasma output in the Japanese quail. The NC type III cells appear to be incapable of endocytosis of India ink particles infused into the excurrent duct system through the RT (Fig. 2.26A), whereas numerous rete cells and almost all of efferent duct NC type I cells, but not NC type II or ciliated cells, avidly take up the ink particles (Aire 2000a, and unpublished observations). Similarly, the epididymal duct unit fails to interiorize


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horseradish peroxidase, while both the RT cells and NC type I contain large amounts of the substance (Nakai et al. 1989b). On the whole, the NC type III cell, in birds, appears to be similar, functionally, to the principal cell of the rat epididymis, in being involved primarily, but not exclusively, in secretion, while the clear cells, not described in birds, are primarily involved in absorption (Hamilton 1975; Robaire and Hermo 1988; Hermo et al. 1988). Basal Cells. Basal cells are found between the bases of NC type III cells only in the epididymal duct unit, and they increase in frequency, cranio-caudally (Figs. 2.26, 2.28 and 2.29C). They are cuboidal or pyramidal in shape, containing nuclei of varying shape, from elongated to triangular or irregular (Fig. 2.32). The nucleus contains a central nucleolus and heterochromatin aggregations attached to the inner nuclear membrane. The organelle content of the cell is sparse, and includes a small Golgi apparatus, a few mitochondria and strands of RER (Fig. 2.32). The nucleus is encircled by bundles of fibrillar material that are best developed in the rooster and Japanese quail, but are also present, to a lesser extent, in the drake and Turkey (Aire 2000a). Tingari (1972) therefore speculates that basal cells subserve a similar function as myoepithelial cells, in assisting the contraction of the muscular coat, during ejaculation. Croisille et al. (1978) suspect that basal cells serve as stem cells for the regeneration of the periodically exfoliating epithelial lining of the epididymal duct unit. Intraepithelial lymphocytes are also seen in the epithelium of the epididymal duct unit, as in other duct units, described above. Periductal Tissue. One or two layers of fibroblasts and up to ten concentric layers of smooth muscle cells constitute the periductal tissue which progressively thickens, cranio-caudally. The smooth muscle cells contain highly elongated, euchromatic, regular nuclei, surrounded by longitudinally orientated filaments, and organelles that are typical of smooth muscle cells (Fig. 2.33). This tissue, in the epididymal duct unit, is richly vascularized, being penetrated by a dense peritubular blood capillary network (Nakai et al. 1988; Aire 2000a), contrary to Tingari’s (1971) observation. The blood capillaries are fenestrated and close to the epithelium in the domestic fowl (Nakai et al. 1988) but not as close to the epithelium as in the mammalian epididymis (Abe et al. 1984). Encapsulated nerve endings are found between the blood capillaries and the ductal epithelium (Nakai et al. 1988), and both cholinergic and adrenergic nerve components have been demonstrated (Tingari, and Lake 1972a). The adrenergic nerve component is particularly closely associated with the epithelium, and ramifies abundantly in the walls of the ducts as well as the receptacle and papilla of the ductus deferens. The abundance of fine intrinsic nerves in the epididymal duct unit may be related to the higher level of development of smooth muscle cells in the boundary tissue of this duct unit than in the more cranial duct units, particularly the RT and efferent ducts. Therefore this unit may have a greater contractile force for onward movement of spermatozoa toward the cloaca than the more cranial duct units (Tingari and Lake 1972a).

'$ Reproductive Biology and Phylogeny of Birds

Fig. 2.32 Gallus domesticus. A basal cell rests on the basal lamina of the epithelium of the epididymal duct, and its nucleus (N) is oval in shape and surrounded by bundles of microfilaments (arrows) which may attach to the cell membrane (arrowheads). Organelles are sparse. Bar: 1 µm. Adapted from Aire, T. A. 2000 Anatomy Histology Embryology 29: 179-191, Figure 15. Reproduced with permission of Blackwell Publishing Ltd.

2.4

APPENDIX EPIDIDYMIDIS

The appendix epididymidis is made up of blind-ending tubules in the rooster (Budras and Sauer 1975a) and lies close to the adrenal gland, into which its cranial portion may be incorporated, for which reason, the appendix epididymidis is usually separated from the epididymis, on the removal of the testis from the body cavity. In the ostrich, the appendix epididymidis is very large and forms the cranial 40% of the entire length of the epididymis in the


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Fig. 2.33 Gallus domesticus. The epithelium of the ductus epididymidis rests on a relatively thick periductal layer of smooth muscle cells (S), displaying typical organelle features. Bar: 2 µm. Adapted from Aire, T. A. 2000 Anatomy Histology Embryology 29: 179-191, Figure 6. Reproduced with the permission of Blackwell Publishing Ltd.

sexually mature and active bird (Budras and Meir 1981). It is attached to the dorsal body wall by the mesepididymis, to the testis by the epiorchium and to the adrenal gland, cranially, by connective tissue as well as by some of its ductules and duct, whose free ends embed in the adrenal gland. The appendix epididymidis contains two vestigial duct components: (i) the ductus aberrans, that represents the straight, cranial blind end of the ductus epididymidis (Wolffian duct), and into which open (ii) the ductuli aberrantes. The latter are vestiges of the nephrons of the mesonephros that are farther away than others from the testis, and, instead, lie close to the adrenal gland. Ontogenetically, the ductuli aberrantes fail to make contact with the RT ducts because of the distance between them. They have lost their Bowman’s capsules, and become blind-ended (Budras and Sauer 1975b). The ductulus aberrans therefore constitutes the distal end of the nephron that normally gives rise to the DED and CD, developmentally (Budras and Sauer 1975b; Croissile et al. 1978). Other categories of ductules, are ductuli aberrantes which are connected to the RT but not to the epididymal duct, as well as tubuli paradidymidis, which are blind at both ends, and are seldom seen in histological sections along the length of the epididymis (Budras and Sauer 1975b).

'& Reproductive Biology and Phylogeny of Birds Transverse sections of these ducts exhibit regular outlines, simple cuboidal or low columnar epithelium, and a homogeneous, non-cellular luminal content (Tingari 1971), or small clumps of cellular debris in the lumen (Fig. 2.34). Budras and Sauer (1975b) have demonstrated, by histochemical and ultrastructural methods, moderate steroid hormone synthesis in the ductuli efferentes, ductus conjugens, and ductus epididymidis, and, in particular, high synthesis in the ductuli efferentes proximales segment of the efferent ductule, ductus aberrans and ductuli aberrantes in the rooster. The noduli epididymidis, in the Ostrich, is situated within the substance of the appendix epididymidis rostrally, and is derived from the swollen ends of the ductuli aberrantes. It is involved in steroidogenesis (Budras and Meier 1981).

2.5

THE PHALLUS

In most birds there is no organ that is homologous to the mammalian penis structurally. Closely related folds of tissue, derived from the ventral wall of the proctodeum of male birds, collectively form the phallus. The phallus is analogous to the penis in mammals because it is the organ of transfer of ejaculated spermatozoa from the male into the female reproductive tract. The phallus in most birds, for example, the domestic fowl, turkey and quail, is small and not intromittent. An engorgement of the vascular folds by lymph in the phallus causes an ‘erection’ that permits contact between this spermbearing organ and the slightly everted vagina of the female, during copulation.

Fig. 2.34 Coturnix japonica. A histological section of the rostral part of the epididymis, containing profiles of proximal efferent ductules (PED), ductus epididymidis (ED) and ductuli aberrantes (DA) which are round/oval and display cuboidal, ciliated epithelium and relatively empty lumina. Bar: 100 µm. Original.


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On the other hand, in Anseriforms and Ratites, the phallus is long, may be coiled, and intromittent (see King 1981 for an excellent review of the structure of avian phallus). This better developed phallus consists of paired fibrous bodies that constitute the bulk of the organ, a phallic sulcus that bears and transports the ejaculated semen, and an elastic vascular body which probably inclines the phallus cranioventrally, during erection. The avian phallus is in the subject of Chapter 3.

2.6

HISTOCHEMISTRY OF THE MALE REPRODUCTIVE ORGANS

A number of enzymes and other chemical compounds have been studied in specific regions of the male genital tract in mammals (Nicander 1954, 1957; Dawson and Rowlands 1959; Allen and Slater 1961; White et al. 1961; Mann 1964; Risley and Skrepetos 1964a,b; Stallcup and Roussel 1965; Blackshaw and Samisoni 1967). In order to ascertain homologies between parts of the male reproductive tract in mammals and birds, and to relate structure to function, it is necessary to evaluate a number of relevant enzymes and compounds in the male reproductive tract of birds. Glycoproteins, but not glycogen, are present in small amounts in the epithelium of the reproductive tract of the rooster (Tingari 1972), and Esponda and Bedford (1985) have shown that most of the sperm-binding secretions in the excurrent ducts of the rooster, save for one glycoprotein, are proteins. Four Wolffian duct proteins have thus been identified in the ducts of the rooster (Morris et al. 1987). Efferent duct non-ciliated cells seem capable of many metabolic functions, including the probable production of glutamic acid by mitochondrial-rich NC type I cells. Most of the amino acid content of the semen of the rooster is glutamate (Lake and McIndoe 1959). Lactic dehydrogenase (LDH) exhibits strong activity in the seminiferous tubules, but lipid reactivity and GDH, SDH and G-6-DH activities are very weakly expressed in the seminiferous tubules and RT (Tingari and Lake 1972c). The more distal excurrent ducts, however, have shown the presence of lipids (phopholipids, neutral lipids and free fatty acids) in both the epithelial cells and lumen (Tingari and Lake 1972c). Of all of the excurrent ducts, the PED exhibits the highest level of lipid content specifically in the NC type I cells. The function of the lipids may be related to estrogen production and/or utilization by efferent ducts of the rooster (Hess et al. 1997). Tingari (1973) concludes rightly that there exists a mechanism for the metabolism of steroids in the male fowl tract. The presence of both 3b- and 17b-hydroxysteroid dehydrogenases, and 3b-ol-steroid dehydrogenase (Budras and Sauer 1975b) in the various epithelia, and the presence of estrogen receptors (ER) in the germ cells and efferent ducts of the rooster (Kwon et al. 1995, 1997) confirm this assumption. Similar findings have been reported in the mouse (Janulis et al. 1996). It has been established that the absence of estrogen receptors in the efferent ducts of mice compromises considerably the indispensable fluid-resorptive ability of these ducts (Hess et al. 1997). The non-ciliated cells in efferent ducts appear to have a common function

Reproductive Biology and Phylogeny of Birds in all species studied (Hess 2000). Thus, it is assumed that estrogen receptors in the homologous avian ducts have a similar function, perhaps, among others, of regulating fluid reabsorption in the efferent ducts of these animals, as in mammals. Strong cholinesterase activity is expressed in the epididymal duct, and this may be related to ionic movements, as found in homologous mammalian ducts, but it is absent in the seminiferous tubules, RT and efferent ducts (Tingari 1972). Recent reports show that the efferent ductule epithelium immunohistochemically expressed sodium-potassium ATPase (Na+, K+ ATPase), carbonic anhydrase II (CAII) and sodium hydrogen exchanger isoform 3 (NHE3), and that the connecting ductule and epididymal duct epithelia immunoexpressed Na+, K+ -ATPase and CA II (Bahr et al. 2006). Similarly, Zamboni et al. (2004) have shown that transmembrane water channel proteins (aquaporins –2, –3, and –9), that are responsible for water flow, are present in the epithelia of efferent ducts, collecting ducts and ductus epididymidis. Acid phosphatase activity is present only in the luminal macrophages in the RT, as well as in the dense bodies in NC type I cells of the PED, indicating that these bodies are lysosomal (Nakai et al. 1989a). The microvillous border, as also the lateral plasma membrane of the epididymal duct unit, shows intense acid phosphatase activity. Alkaline phosphatase activity is present only on the outer covering of the microvilli of the efferent ducts, and is completely absent in the epididymal duct unit. The functions of these enzymes in the male tract are only speculative. However, according to Aitken (1971), a significant concentration of acid phosphatase is characteristic of all spermstorage areas although its exact significance in storage is not clearly understood. Sperm are stored in the ductus deferens in birds, albeit for only short periods of time.

2.7

ACKNOWLEDGMENTS

The University of Pretoria kindly provided library support for this review, as well as research grants for new material contained in the text. I also acknowledge the assistance of Mrs. Wilma Olivier who made all the line diagrams. The assistance of both Dr. Peter Ozegbe and Dr. Wahab Kimaro in computerization and composition of the text and figures is gratefully acknowledged.

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Reproductive Biology and Phylogeny of Birds Mulder, R. A. and Cockburn, A. 1993. Sperm competition and the reproductive anatomy of male Superb Fairy-Wrens. The Auk 110: 588-593. Munro, S. S. 1938. Functional changes in fowl sperm during their passage through the excurrent ducts of the male. Journal of Experimental Zoology 79: 71-92. Nakai, M., Hashimoto, Y., Kitagawa, H., Kon, Y. and Kudo, N. 1988. Microvasculature of the epididymis and ductus deferens of domestic fowls. Japanese Journal of Veterinary Science 50: 371-381. Nakai, M., Hashimoto, Y., Kitagawa, H., Kon, Y. and Kudo, N. 1989a. Histological study on seminal plasma absorption and spermiophagy in the epididymal region of domestic fowl. Poultry Science 68: 582-589. Nakai, M., Hashimoto, Y., Kitagawa, H., Kon, Y. and Sugimura, M. 1989b. Effects of ligation of the ductus deferens on the fowl epididymal region. Japanese Journal of Veterinary Science 51: 521-529. Nakai, M. and Nasu, T. 1991. Ultrastructural study on junctional complexes of the excurrent duct epithelia in the epididymal region in the fowl. Journal of Veterinary Medical Science 53: 677-681. Neaves, W. B. 1975. Leydig cells. Contraception 11: 571-606. Neaves, W. B. 1977. The blood-testis barrier. Pp. 125-162. In A. D. Johnson and Gomes, W. R. (eds.), The Testis. Volume IV. Academic Press, New York. Nicander, L. 1954. Glycogen secretion in the epididymis. Nature 174: 700-701. Nicander, L. 1957. On the regional histology and cytochemistry of the ductus epididymidis in rabbits. Acta morphologica neerlando-scandinavica 1: 99-118. Nicander, L. 1970. On the morphological evidence of secretion and absorption in the epididymis. Morphological Aspects of Andrology 1: 12-124. Nicander, L. and Glover, T. D. 1973. Regional histology and fine structure of the epididymal duct in the golden hamster (Mesocricetus auratus). Journal of Anatomy 114: 347-358. Nicholls, T. J. and Graham, G. P. 1972. Observation on the ultrastructure and differentiation of Leydig cells in the testis of the Japanese quail (Coturnix coturnix japonica). Biology of Reproduction 6: 179-192. Nickel, R., Schummer, A. and Seiferle, E. 1977. Urogenital system. Pp. 70-84. In Anatomy of the Domestic Birds. Verlag Paul Parey, Berlin and Hamburg. Nishida, T. 1964. Comparative and topographical anatomy of the fowl. XLII. Blood vascular system of the male reproductive organs. Japanese Journal of Veterinary Science 26: 211-221. Nonaka, S., Tanaka, Y., Okada, Y., Takeda, S., Harada, A., Kanai, Y., Kido, M. and Hirokawa, N. 1998. Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor. Cell 95: 829-837. Odor, D. L. and Blandau, R. J. 1985. Observations on the solitary cilium of rabbit oviductal epithelium: its motility and ultrastructure. American Journal of Anatomy 174: 437-453. Olson, G. E. and Hinton, B. T. 1985. Regional differences in luminal fluid polypeptides of the rat testis and epididymis revealed by two-dimensional gel electrophoresis. Journal of Andrology 6: 20-34. Onagbesan, O. M., Safi, M., Decuypere, E. and Bruggeman, V. 2004. Development changes in inhibin a and inhibin/activin bA and bB mRNA levels in the gonads during post-hatch prepuberal development of male and female chickens. Moelcular Reproduction and Development 68: 319-326.


Osman, D. I. 1978. On the ultrastructure of modified Sertoli cells in the terminal segment of seminiferous tubules in the boar. Journal of Anatomy 127: 603-613. Osman, D. I. 1980. The connection between the seminiferous tubules and the rete testis in the domestic fowl (Gallus domesticus)—morphological study. International Journal of Andrology 3: 177-187. Osman, D. I. and Plöen, L. 1978. The terminal segment of the seminiferous tubules and the blood-testis barrier before and after efferent ductule ligation in the rat. International Journal of Andrology 1: 235-249. Osman, D. I., Ekwall, H. and Plöen, L. 1980. Specialized cell contacts and the bloodtestis barrier in the seminiferous tubules of the domestic fowl (Gallus domesticus). International Journal of Andrology 3: 553-562. Palade, G. 1975. Intracellular aspects of the process of protein synthesis. Science 189: 347-358. Pease, D. C. 1956. Infolded basal plasma membranes found in epithelia noted for their water transport. Journal of Biophysical and Biochemical Cytology, Supplement 2: 203-208. Pellietier, R. M. 1990. A novel perspective: the occluding zonule encircles the apex of the Sertoli cell as observed in birds. American Journal of Anatomy 188: 87-108. Pfeiffer, D.C. and Vogl, A. W. 1993. Ectoplasmic (“Junctional”) specializations in Sertoli cells of the rooster and turtle: evolutionary implications. The Anatomical Record 235: 33-50. Plöen, L 1972. An electron microscope study of the immediate effects on spermateliosis of a short-term experimental cryptorchidism in the rabbit. Virchows Archives Abt B Zellpatologie 10: 293-309. Plöen, L. 1973a. An electron microscope study of the delayed effects on rabbit spermateliosis following experimental cryptorchidism for twenty-four hours. Virchows Archives Abt B Zellpatologie 14: 159-184. Plöen, L. 1973b. A light microscope study of the immediate and delayed effects on rabbit spermiogenesis following cryptorchidism for twenty-four hours. Virchows Archives Abt B Zellpatologie 14: 185-196. Pudney, J. and Fawcett, D. W. 1984. Seasonal changes in fine structure of the ductuli efferentes of the ground squirrel Citellus lateralis (Say). The Anatomical Record 188: 453-476. Ramos Jr., A. S. and Dym, M. 1977. Ultrastructure of the ductuli efferentes in monkeys. Biology of Reproduction 17: 339-349. Reid, B. L. and Cleland, K. W. 1957. The structure and function of the epididymis. I. The histology of the rat epididymis. Australian Journal of Zoology 5: 223-246. Riddle, O. 1918. Further observation on relative size and form of the right and left testes of pigeons in health and disease and as influenced by hybridity. The Anatomical Record 14: 283-334. Riddle, O. 1925. On the sexuality of the right ovary of birds. The Anatomical Record 30: 365-383. Risley, P. L. and Skrepetos, C. N. 1964a. Histochemistry of distribution of cholinesterases in the testis, epididymis and vas deferens of the rat. The Anatomical Record 148: 231-249. Risley, P. L. and Skrepetos, C. N. 1964b. Cholinesterase distribution in the rat epididymis and vas deferens after castration and sex hormone treatments. The Anatomical Record 150: 195-208. Robaire, B. and Hermo, L. 1988. Efferent ducts, epididymis, and vas deferens: Structure, functions, and their regulation. Pp. 999-1080. In E. Knobil and J. Neill (eds.), The Physiology of Reproduction. Raven Press, New York.


Roosen-Runge, E. C. 1961. The rete testis in the albino rat: Its structure, development and morphological significance. Acta Anatomica 45: 1-30. Roosen-Runge, E. C. and Holstein, A. F. 1978. The human rete testis. Cell and Tissue Research 189: 409-433. Rothwell, B. 1973. The ultrastructure of Leydig cells in the testis of the domestic fowl. Journal of Anatomy 116: 245-253. Rothwell, B. 1975. Designation of the cellular component of the peritubular boundary tissue of the seminiferous tubule in the testis of the fowl (Gallus domesticus). British Poultry Science 16: 527-529. Rothwell, B. and Tingari, M. D. 1973. The ultrastructure of the boundary tissue of the seminiferous tubule in the testis of the domestic fowl (Gallus domesticus). Journal of Anatomy 114: 321-328. Rothwell, B. and Tingari, M. D. 1974. The ultrastructural differentiation of the boundary tissue of the seminiferous tubule in the testis of the domestic fowl. British Veterinary Journal 130: 587-592. Russel, L. D. 1996. Mammalian Leydig cell structure. Pp. 43-96. In A. H. Payne, M. P. Hardy and L. D. Russel (eds.), The Leydig Cell. Cache River Press, Vienna, Illinois. Salt, W. R. 1954. The structure of the cloacal protuberance. Auk 71: 64-73. Scheib, D. 1973. Les cellules secretices testiculaires du poussin de la caille japonaise: differenciation de leur ultrastructure et rapports avec leurs potentialities steroidogenes. Development, Growth and Differentiation 15: 315-328. Siller, W. G. and Hindle, R. M. 1969. The arterial blood supply to the kidney of the fowl. Journal of Anatomy 104: 117-135. Sinowatz, F., Wrobel, K.-H., Sinowatz, S. and Kugler, P. 1979. Ultrastructural evidence for phagocytosis of spermatozoa in the bovine rete testis and testicular straight tubules. Journal of Reproduction and Fertility 57: 1-4. Skinner, M. K. 1987. Cell-cell interactions in the testis. Annals of New York Academy of Sciences 513: 158-171. Skinner, M. K., Norton, J. N., Mullaney, B. P., Rosselli, M., Whaley, P. D. and Anthony, C. T. 1991. Cell-cell interactions and the regulation of testis function. Annals of New York Academy of Sciences 637: 354-363. Soley, J. T. 1992. A histological study of spermatogenesis in the ostrich (Struthio camelus). Ph.D. thesis, University of Pretoria, Pretoria, 187 pp. Sprando, R. L. and Russel, L. D. 1987. A comparative study of Sertoli cell cytoplasmic specializations in selected non-mammalian vertebrates. Tissue and Cell 19: 479493. Stallcup. O. T. and Roussel, J. D. 1965. Development of lactic acid dehydrogenase enzyme system in the testis and epididymis of young dairy bulls. Journal of Dairy Science 48: 1511-1516. Stanley, A. J. and Witschi, E 1940. Germ cell migration in relation to asymmetry in the sex glands of hawks. The Anatomical Record 76: 329-342. Stefanini, M. A., Orsi, A. M., Grégorio, E. A., Viotto, M. J. S. and Baraldi-Antoni, S. M. 1999. Morphologic study of the efferent ductules of the pigeon (Columba livia). Journal of Morphology 242: 247-255. Stoffel, M. H. and Friess, A. E. 1994. Morphological characteristics of boar efferent ductules and epididymal duct. Microscopy Research and Techniques 29: 411-431. Suzuki, F. and Nagano, T. 1978. Regional differentiation of cell junctions in the excurrent duct epithelium of the rat testis as revealed by freeze-fracture. The Anatomical Record 191: 503-520.


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Tingari, M. D. 1971. On the structure of the epididymal region and ductus deferens of the domestic fowl (Gallus domesticus). Journal of Anatomy 109: 423-435. Tingari, M. D. 1972. The fine structure of the epithelial lining of the excurrent duct system of the testis of the domestic fowl (Gallus domesticus). Quarterly Journal of Experimental Physiology 57: 271-295. Tingari, M. D. 1973. Histochemical localization of 3b- and 17b-hydroxysteroid dehydrogenases in the male reproductive tract of the domestic fowl (Gallus domesticus). Histochemical Journal 5: 57-65. Tingari, M. D. and Lake, P. E. 1972a. The intrinsic innervation of the reproductive tract of the male fowl (Gallus domesticus). A histochemical and fine structural study. Journal of Anatomy 112: 257-271. Tingari, M. D. and Lake, P. E. 1972b. Ultrastructural evidence for resorption of spermatozoa and testicular fluid in the excurrent ducts of the testis of the domestic fowl, Gallus domesticus. Journal of Reproduction and Fertility 31: 373381. Tingari, M. D. and Lake, P. E. 1972c. Histochemical localization of glycogen, mucopolysaccharides, lipids, some oxidative enzymes and cholinesterases in the reproductive tract of the male fowl (Gallus domesticus). Journal of Anatomy 112: 273-287. Traciuc, P. E. 1967. L’anatomie microscopique de l’épididyme chez Sterna hirundo L. Anatomischer Anzeiger 121: 382-386. Traciuc, E. 1969. La structurede l’epididyme de Coloeus monodula (aves, Corvidae). Anatomische Anzeiger 125: 49-67. Tuck, R. R., Setchell, B. P., Waites, G. M. H. and Young, J. A. 1970. The composition of fluid collected by micropuncture and catheterization from the seminiferous tubules and rete testis of rats. European Journal of Physiology 318: 225-243. Veeramachaneni, D. N. R. and Amann, R. P. 1991. Endocytosis of androgen-binding protein, clusterin, and transferrin in the efferent ducts and epididmis of the ram. Journal of Andrology 12: 288-294. Weaker, F. J. 1977. The fine structure of the interstitial tissue of the testis of the ninebanded armadillo. The Anatomical Record 187: 11-27. White, I. G., Wallace, J. C., Wales, R. G. and Scott, T. W. 1961. The occurrence and metabolism of glycerylphosphorycholine in semen and the genital tract. Proceedings of the IVth International Congress of Animal Reproduction and Artificial Insemination, The Hague, 266-269. Williams, D. D. 1958. A histological study of the effects of subnormal temperature of the testis of the fowl. The Anatomical Record 130: 225-242. Witschi, E. 1935. The origin of asymmetry in the reproductive system of birds. American Journal of Anatomy 56: 119-141. Wolfson, A. 1954. Sperm storage at lower than body temperature outside the body cavity in some passerine birds. Science 120: 68-71. Zamboni, L., Akuffo, V. and Bakst, M.R. 2004. Aquaporins are observed in the duct epithelia of the epididymal region of the Large White turkey. Poultry Science 83: 1917-1920. Zhu, L. J., Zong, S. D., Phillips, D. M., Moo-Young, A. J. and Bardin, C. W. 1997. Changes in the distribution of intermediate filaments in the rat Sertoli cells during the seminiferous epithelium cycle and postnatal development. The Anatomical Record 248: 391-405.

n n

CHAPTER

3

Anatomy and Evolution of Copulatory Structures Robert Montgomerie1 and James Briskie2

3.1

INTRODUCTION

Unlike most other animal Classes, Aves (birds) is a taxon in which males of some species possess an intromittent organ (IO), whereas males of other species do not (King 1981a). Indeed, birds are virtually unique among internal fertilizers that most species lack an IO. Thus, in birds at least, the IO is not necessary for internal fertilization. This raises the question, then, whether the avian IO has evolved as a primary sexual trait simply for the delivery of sperm, as is sometimes assumed for other taxa, or as a secondary sexual trait (Eberhard 1990; Briskie and Montgomerie 1997). Most of the scant literature on avian IOs has focused on the seemingly odd presence of IOs in a few orders, but it is in fact their absence in so many species that is the evolutionary puzzle. Despite this interesting question about the absence of IOs in birds, relatively little is known about the anatomy, physiology, and evolution of structures that facilitate copulation in this taxon. There was quite a bit of interest in the phallus of ratites in the 19th century with J. Müller’s (1836) anatomical study being the most comprehensive—King (1981a) provides a useful review of this early work. Later, Eckhard (1876), R. Müller (1908) and Liebe (1914) showed that the Mallard (Anas platyrhynchos) phallus is erected by a lymphatic, rather than blood-vascular mechanism as had previously been thought. Gerhardt (1933) and other early workers also noted that there were two types of true phalluses in birds—those with and without a blind cavity— and there was a smattering of other studies on the phalluses of wild birds in the early part of the 20th century. Beyond that early work, only the non1 2

Department of Biology, Queen’s University, Kingston, ON K7L 3N6, Canada School of Biological Sciences, University of Canterbury, Christchurch, New Zealand

$ Reproductive Biology and Phylogeny of Birds intromittent phalluses of the domestic chicken (Gallus gallus) and turkey (Meleagris gallopavo) received much anatomical or physiological study (King 1981a). However, during the past decade or so, there has been renewed interest in the avian IO, with new hypotheses to explain the evolutionary patterns of occurrence (Briskie and Montgomerie 1997; Wesolowski 1999; Briskie and Montgomerie 2001; Wesolowski 2001), comparative analyses of anatomical structure (Coker et al. 2002), detailed anatomical work using modern techniques (Oliveria and Mahecha 2000; Oliveria et al. 2003, 2004), and some excellent natural history on the most bizarre copulatory structures yet discovered in birds (Birkhead et al. 1993; Mulder and Cockburn 1993; Birkhead and Hoi 1994; Wilkinson and Birkhead 1995; Winterbottom et al. 1999, 2001; McCracken 2000; McCracken et al. 2001). In this chapter we present an overview on what is known about the anatomy, histology, and physiology of the avian phallus—both intromittent (phallus protrudens) and non-intromittent (phallus nonprotrudens) forms—as well as other copulatory structures involved directly in the transfer of sperm from male to female. King’s (1981a) comprehensive review is our point of departure for this material and should be referred to by the reader needing more detail on work done up to 1980. We also discuss the evolutionary history of IOs in birds in relation both to phylogeny and to the various selective pressures that may have favored the evolutionary loss of the IO and the appearance of phallus-like copulatory structures in some lineages. Our goal in this chapter is to provide a framework for understanding the evolution of all copulatory structures in birds in the context of both the evolutionary history of birds and the influence of both natural and sexual selection on the modification and loss of phalluses in some lineages.

3.2

EVOLUTIONARY HISTORY OF COPULATORY STRUCTURES

The various kinds of copulatory structures found in modern birds (Neornithes) can be mapped onto the most recent avian phylogenies (e.g., Figs. 3.1-3.3) to illustrate their probable evolutionary history. In birds, a true phallus (both intromittent and non-intromittent) is found only in the orders Struthioniformes (ostrich, rheas, emu, kiwis and cassowaries), Tinamiformes (tinamous), Anseriformes (waterfowl and screamers), and Galliformes (pheasants, grouse, megapodes and currasows). The true phallus is clearly homologous to the phallus of crocodiles, turtles and tortoises (King 1981a), their closest living relatives (Fig. 3.1). A phallus-like structure (or pseudophallus) used in copulation is known from only three other bird genera (see below); in only one of these is it intromittent, and in none is it synapomorphic with the crocodile phallus. Male birds in all remaining avian taxa apparently have no phallus, but instead copulate solely by cloacal apposition, usually for only a few seconds (see Volume 6B, Chapter 6). Problems in rooting the avian tree with respect to the Crocodylia has made placement of the sister taxa Galliformes and Anseriformes controversial with

Anatomy and Evolution of Copulatory Structures

%

Fig. 3.1 Phylogenetic relationship of birds to crocodiles, turtles, lizards, snakes, and some extinct dinosaurs. Modified after Padian, K. and Horner, J. R. 2002. Trends in Ecology and Evolution 17: 120-124, Fig. 3.

respect to the Palaeognathae and Neognathae (Cracraft et al. 2004). Thus to provide some context for the discussion of phallus evolution in birds, we have mapped the avian phallus and other copulatory organs onto two different phylogenies that have recent support (Figs. 3.2 and 3.3). These two phylogenetic hypotheses are based on both molecular and morphological data and are different in many respects from earlier phylogenies based on morphology alone. The comprehensive phylogeny (‘Tapestry’; see Chapter 1) of bird evolution proposed by Sibley and Ahlquist (1990), based on DNA-DNA hybridization, grouped the ratites (ostrich, rheas, and tinamous) into one clade (Palaeognathae), and the Galliformes and Anseriformes into a separate clade

& Reproductive Biology and Phylogeny of Birds

Fig. 3.2 Summary of the Sibley-Ahlquist ‘Tapestry’ of bird phylogeny, based on DNA hybridization, illustrating the evolution of the avian phallus. The Megapodiidae are marked as equivocal because IOs have been recorded in some species but not others, based on very sketchy evidence (see text). Modified after Cracraft, J., et al. 2004. Pp 468-489. In Cracraft, J. and Donoghue, M. J. (eds), Assembling the Tree of Life. Oxford University Press, New York, Fig. 27.2.


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Fig. 3.3 Summary of a recent consensus phylogeny of birds, based on both molecular and morphological evidence, illustrating the evolution of the avian phallus. The Megapodiidae are marked as equivocal because IOs have been recorded in some species but not others, based on very sketchy evidence (see text). Modified after Cracraft, J., et al. 2004. Pp 468-489. In Cracraft, J. and Donoghue, M. J. (eds), Assembling the Tree of Life. Oxford University Press, New York, Fig. 27.10.

Reproductive Biology and Phylogeny of Birds (Galloanserae) that is the sister clade to the Palaeognathae (Fig. 3.2). According to this phylogenetic hypothesis, the clade comprising the Palaeognathae and the Galloanserae is separate from the clade comprising all other birds (Neoaves). Though the Sibley-Ahlquist Tapestry was radically different from accepted phylogenies at the time, it received widespread support because it was based on DNA analyses, and made sense in many respects. Problems with both the use of DNA-DNA hybridization and the interpretation of data, however, encouraged other researchers to employ a large number of other kinds of molecular and phylogenetic analyses that continue to refine our understanding of the relationships among and within the clades that Sibley and Ahlquist (1990) proposed. Probably the best consensus phylogeny currently available, based on nuclear genes as well as other traits, agrees with the Sibley-Ahlquist Tapestry in many respects, but does not isolate the Galloanserae and Palaeognathae in a separate clade (Fig. 3.3). The evolution of avian phalluses maps onto both of these general molecular phylogenies in a similar fashion (Figs. 3.2 and 3.3), with the true phallus being monophyletic in both cases. The intromittent true phallus is clearly the ancestral type and is shared by all species in the Palaeognathae and several families of the Galloanserae; the non-intromittent true phallus, on the other hand, has probably arisen only once, in the ancestor to the clade comprising the Phasianidae, Odontophoridae, and Numididae, though possibly also in the Megapodiidae (see below). Several other copulatory structures that have so far been discovered in birds are all in the Neoaves, but only in the Psittaciformes (genus Coracopsis) has a copulatory structure evolved with an intromittent function. In all other species in which the genitalia of males appear to have been modified for copulation, none is known to have a structure that is inserted into the female’s cloaca.

3.3 3.3.1

FORM AND FUNCTION Ancestral Phalluses

The true phalluses of birds all have traits similar to the phalluses of their closest living relatives—the crocodiles, turtles, and tortoises (Fig. 3.4)—and appear to be monophyletic (Figs. 3.1-3.3). Given our current understanding of the evolutionary relationships among birds and dinosaurs (Fig. 3.1), it seems quite likely that theropod dinosaurs also possessed an intromittent phallus (Larson and Frey 1992) not unlike that of crocodiles, chelonians, and the intromittent true phalluses of birds. The phalluses of male chelonians (turtles and tortoises) comprise a thickening along the midline of the ventral wall of the proctodeum (Wood Jones 1915). In most species, the caudal portion of the phallus is separated from the wall of the proctodeum so that most of the phallus is free (Fig. 3.4A), and is inserted into the female during copulation. The phallus is made up of two fibroelastic bodies (fibrous tissue with large vascular spaces), which in


Fig. 3.4 Typical intromittent true phalluses of (A, B) turtles and (C) crocodiles showing the location of the ejaculatory groove (phallic sulcus). B. Cross section of A. Not drawn to scale, nor were scale bars shown on the original drawings. Modified after King, A.S. 1981a. Pp 107-147. In King, A. S. and McLelland, J. (eds), Form and Function in Birds. Academic Press, London, Fig. 3.2.

most species are fused into a single structure of erectile tissue (Fig. 3.4). The fibroelastic bodies are separated by a median ejaculatory groove, called the phallic sulcus, that extends from the opening of the urogenital sinus nearly to the tip of the phallus. As the phallus becomes erect, the fibroelastic bodies swell such that this groove becomes a closed channel along which the semen travels (Wood Jones 1915). The phalluses of crocodilians are clearly homologous to those of chelonians, but the free part is longer and projects more prominently from the proctodeal wall when it is not erect, the shape tends to be more cylindrical (Fig. 3.4C), and the glans may be more complex (Gerhardt 1933). In addition, the crocodilian phallus is distinctly curved, particularly when it is erect, compared to the relatively straight phallus of turtles and tortoises (Fig. 3.4). In chelonians and crocodilians, the mechanism of erection appears to be blood vascular, though King (1981a) hints that this needs to be better documented. In both taxa, females also have a phallus (i.e., clitoris) that is very similar to that of the male, but is much smaller and cannot be extruded from the cloaca.

3.3.2

Bird Phalluses

King (1981a) and earlier workers classified true avian phalluses into intromittent and non-intromittent types based on their gross anatomy, and further divided the intromittent phallus into two types based on the presence (Type B) or absence (Type A) of a blind cavity. Type B phalluses are similar in


shape to Type A but have a blind tubular cavity, like the invaginated finger of a glove, when flaccid, and tend to be twisted in a spiral from the base to the tip when erect. This categorization, based on the presence/absence of a blind cavity, appeared to make some evolutionary (phylogenetic) sense at one time, but more recent work on the anatomy and histology of tinamou phalluses (Oliveira and Mahecha 2000), as well as the best current phylogenies of birds (Fig. 3.3), suggest that the blind cavity may be an adaptive trait, possibly with some erectile function. Thus, the distinction between Type A and B phalluses does not appear to be particularly useful from a phylogenetic perspective. Like the phalluses of crocodilians and chelonians (Fig. 3.4), all intromittent true phalluses in birds are composed of fibrous, erectile tissue that arises from the ventral wall of the proctodeum and have a ventral sulcus (ejaculatory groove) along which the semen travels during ejaculation (e.g., Figs. 3.5-3.10). [In birds, the cloaca has three compartments separated from each other by folds: (1) the proctodeum, nearest the vent, (2) the urodeum, a narrow zone that the urogenital ducts empty into (except in tinamous and rheas; Oliveira et al. 2003), and (3) the coprodeum, into which the rectum empties (King 1981b).] When the phallus is erect, the lips of the ejaculatory groove also become engorged, sealing the groove and thus preventing the spillage of semen. Intromittent phalluses all have a fixed base of fibrous tissue and a free conical, or tubular portion (composed of two fused fibrolymphatic bodies) that is everted during erection and copulation. Such phalluses usually curve towards the male’s left, and thus during copulation will most likely deposit sperm into the female’s left oviduct, which in birds is the functional one (King 1981a). The fixed base of the phallus has lymphatic spaces that fill and become dilated during erection (Oliveira and Mahecha 2000); vascular bodies in the floor of the urodeum provide the lymph that engorges the phallus,

Fig. 3.5 Intromittent true phallus of the male Spotted tinamou in its erect state: A. Caudal end. B. Left side view. Modified from Oliveira, C. A. and Mahecha, G. A. B. 2000. Annals of Anatomy 182: 161-169, Figs. 18 and 19.


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possibly through the action of the cloacal sphincter muscle. The base of the phallus also has glandular tissue that secretes mucous that lubricates the phallus and facilitates both eversion and, presumably, copulation (Komárek and Marvan 1969). Below we review the taxonomic distribution of phalluses among extant birds; classification and species numbers are taken from Dickinson (2003).

3.3.2.1

Palaeognathae

Males of all species of Palaeognathae studied so far have an IO that is similar to the crocodilian phallus (Fig. 3.4C), characterized a conical base of fibrous tissue, and a bend to the bird’s left when erect due to the two fused fibrolymphatic bodies being laterally asymmetrical (e.g., Figs. 3.5-3.8). In all species studied so far, the female also has a phallus that is much smaller than that of the male and does not extrude from the cloaca (e.g., Fig. 3.6B). Tinamidae (tinamous, 47 species). Tinamous were classified by King (1981a) as having a Type A phallus (i.e., lacking a blind cavity), based on work by Müller (1836) and Gerhardt (1933) on Crypturellus cinnamomeus. However, a recent anatomical and histological study of the Spotted tinamou (Nothura maculosa) clearly documents the presence of a blind cavity (Fig. 3.5A; Oliveira and Mahecha 2000) like that in the Anseriformes, rheas, emus, and cassowaries. It is quite possible that other species of tinamou possess such a cavity but there is too little information available (even in Gerhardt 1933) to be certain. The phallus of the Spotted tinamou is the best studied in this family, based on a sample of 26 males captured at different times of the year (Oliveira and

Fig. 3.6 North Island kiwi (Apteryx australis mantelli): A. Intromittent true phallus of the male. B. Vestigial phallus (clitoris) of the female. Modified after Caithness, T. A. 1971. International Zoo Yearbook 11: 206-208, Figs. 2 and 3.

" Reproductive Biology and Phylogeny of Birds Mahecha 2000). To describe the morphology of the phallus, Oliveira and Mahecha (2000) conducted modern histological and anatomical studies of birds with the phallus both flaccid and erect (the latter induced by cloacal massage). In this species, the phallus is composed of a fixed, fibrous, conical base attached to the floor of the proctodeum and a tubular portion with a blind-ended tube at the tip (Fig. 3.5A). When the phallus is not erect, it lies entirely within a phallic pouch in the floor of the proctodeum. The erect phallus is distinctly coiled in a spiral (Fig. 3.5B) and extends about 3 cm out of the male’s vent during copulation, directed towards the male’s left. The fixed base of the phallus of both Spotted and Red-winged tinamous (Rhynchotus rufescens) also has intraepithelial plasma cells that increase at least 8-fold in number during the breeding season (Oliveira et al. 2003). These cells contain granular material indicative of immunoglobulin accumulation that might be important for an immune response to protect the phallus tissue from infection resulting from sexually transmitted diseases. Alternatively, secretions from these cells may be added to the seminal fluid and thus protect sperm within the female’s reproductive tract (Oliveira et al. 2003). Apterygidae (kiwis, 3 species). Except that it is asymmetrical and curves to the male’s left, the phallus of male kiwis (Fig. 3.6A) is very similar to that of crocodilians and chelonians (Fig. 3.4). The phallus of the female (i.e., clitoris) is very small and cannot be everted from the cloaca (Fig. 3.6B), making this a relatively easy trait for sexing these externally monomorphic birds (Caithness 1971). Casuariidae (cassowaries, 3 species). Phalluses have been observed in the Southern (Casuarius casuarius) and Dwarf Cassowaries (C. bennetti), and in the latter were described as being indistinguishable from the phalluses of rheas and emus (Gerhardt 1933). Dromaiidae (emus, 3 species). Emus are reported to have the same phallus structure as rheas (Müller 1836, Gerhardt 1933). Struthionidae (ostrich, 1 species). The large, bright red phallus of the male Ostrich (Struthio camelus) is up to 20 cm long when flaccid and extrudes 40 cm or more out of the male’s cloaca when erect (Fig. 3.7A; Gerhardt 1933). Even in its flaccid state, it has to be partly protruded from the cloaca to allow defecation as it takes up so much space in the cloacal lumen that it blocks the opening of the ureter. At rest, it lies within a wide pocket in the ventral wall of the proctodeum. As in other paleognaths, the walls of the ejaculatory groove (Fig. 3.7B) in the Ostrich are composed of erectile tissue that fills with lymph during erection, and presumably seals the lip of the groove to form a tube down which the semen passes. During erection a pair of retractor muscles (m. lecator phalli) extrude the flaccid phallus from its pocket (Fig. 3.7A), followed by the vascular bodies filling the phallus with lymph. King (1981a), however, notes that the literature on this lymphatic mechanism (and the tissues involved) is old, equivocal, and biased. It is unlikely that the mechanisms of erection in


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Fig. 3.7 Intromittent true phallus of the male Ostrich: A. Erect phallus extruded from vent, with dissection showing musculature. B. Cross section of A (at about the dotted line) showing asymmetrical sizes of the left and right fibrolymphatic bodies. Not drawn to scale (especially since the erect phallus should be about 40 cm long, or about 10 times the width of the vent), nor were scale bars shown on the original drawings. Modified after King, A. S. 1981a. Pp 107-147. In King, A. S. and McLelland, J. (eds), Form and Function in Birds, vol. 2. Academic Press, London, Figs. 3.3b and 3.4d.

ostriches is different from that in the other paleognaths but this deserves some study using modern techniques Rheidae (rheas, 2 species). Müller’s (1836) description of the phallus of the Greater rhea (Rhea americana) is still the best and most detailed (Fig. 3.8). The other species of rhea has not been studied but its phallus should have essentially the same structure. In the Greater rhea, the resting phallus has an orifice at the tip that leads to a blind tube or cavity, as in both the Spotted tinamou and the Anseriformes. When the phallus is erect, about half of this blind cavity is evaginated. The proximal end of this cavity is continuous with the phallic sulcus, and when the phallus is at rest (i.e., flaccid) part of the phallic sulcus is actually pulled into the cavity. Unlike the other paleognaths studied so far, the fibrous bodies at the base of the rhea’s phallus are spirally intertwined, and the free tubular portion of the phallus continues in a slight left-turning spiral throughout its length (Fig. 3.8). In addition a band of elastic fibers (lig. elasticum phalli) runs along the entire length of the everted phallus and probably retracts the phallus during detumescence (Müller 1836; Gerhardt 1933).

$ Reproductive Biology and Phylogeny of Birds

Fig. 3.8 Intromittent true phallus of the male Rhea. Not drawn to scale, nor was there a scale bar shown on the original drawing. Modified after Briskie, J. V. and Montgomerie, R. 1997. Journal of Avian Biology 28: 73-86, Fig. 1a, who redrew this from King, A. S. 1981a. Pp 107-147. In King, A. S. and McLelland, J. (eds), Form and Function in Birds, vol. 2. Academic Press, London, Fig. 3.3d.

3.3.2.2

Neognathae

Galloanserae (452 species) The Galliformes and Anseriformes comprise the Galloanserae (Sibley and Ahlquist 1990), recognizing that these two avian orders are sister taxa (Fig. 3.3). Like the paleognaths, both of these orders possess a true phallus but only in the Anseriformes, and probably in the Cracidae and Megapodidae, is it intromittent. Thus the common ancestor of these two orders most likely had an IO but the size of this phallus became reduced and no longer involved in intromission in the galliform lineage leading to the Phasianide, Numididae, and Odontophoridae, and possibly in the Megapodiidae (Fig. 3.3). Anseriformes (162 species) Anatidae (ducks, geese, swans; 158 species). The family Anatidae is distinguished by having (i) the species with the longest (relative to body size) IO of any bird (McCracken 2000), (ii) the species with the best-studied IO, and (iii) by far the largest number of species for which there are quantitative data on IO size and morphology (Coker et al. 2002). Not only is the Anatidae the most speciose family of birds with IOs, but it displays a wide range of mating systems and male reproductive tactics and thus provides a useful model for studying the evolution of IOs in relation to sexual selection. Coker et al. (2002) have made an excellent start at this but there is still much to be done. The Anatidae are also relatively easy to keep and study in captivity, even while breeding (Johnsgard 1978), and so would lend themselves well to both critical


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experiments about IO function in relation to structure and detailed anatomical/physiological study during the critical period when males copulate. The phallus of the male Mallard is probably better studied than that of any other bird, save possibly the non-intromittent phallus of the domestic chicken. At rest, the Mallard’s phallus lies coiled within a thin peritoneal sac in the ventral wall of the proctodeum; when erect, the base of the phallus fills the male’s vent and the phallus extrudes 4 cm out of the cloaca in wild birds (but 8 cm in domesticated stock; Rautenfeld et al. 1974). The right fibrolymphatic body is much larger that the left and the phallus twists 3-4 turns in a left spiral from the base to the tip (Fig. 3.9). These two bodies are fused and thus continuous with each other but are separated at the surface by a deep ejaculatory groove (phallic sulcus) along which semen travels during ejaculation. The surface of the phallus is relatively smooth at the base but within a half turn of the spiral the surface of the fibrolymphatic bodies becomes cornified with rough transverse ridges about 2 mm apart. The tip of the IO has a small opening into a blind cavity, typical of Type B avian phalluses (Fig. 3.9). The Mallard phallus becomes erect by filling with lymph from two small (1 ¥ 4 cm) vascular bodies in the cloacal wall near the base of the phallus. Peristaltic contractions of the cloacal sphincter increase the flow of lymph to the fibrolymphatic bodies (Guzsal 1974). The swelling of these bodies seals the lips of the phallic sulcus, converting it into a closed tube such that semen can leave the phallus only at the tip. The phallus detumesces when the cloacal sphincter relaxes and lymph flows out of the phallus into general circulation.

Fig. 3.9 Intromittent true phallus of the male Mallard as viewed from the left side when fully erect. No scale bar shown on the original drawing. Note that the right fibrolymphatic body is much smaller than the left, and that the ejaculatory groove (phallic sulcus) is between them. Modified after Briskie, J. V. and Montgomerie, R. 1997. Journal of Avian Biology 28: 73-86, Fig. 1a, who redrew this from King, A. S. 1981a. Pp 107-147. In King, A. S. and McLelland, J. (eds), Form and Function in Birds, vol. 2. Academic Press, London, Fig. 3.6.

& Reproductive Biology and Phylogeny of Birds This process is assisted by contractions of two pairs of muscles as well as by elastic fibers in the phallus that were stretched during erection. Detumescence begins with the immediate invagination of the base of the phallus, then over about 30 s the entire phallus is retracted into the cloaca by the action of the retractor muscles and the elastic fibers. Finally, over a 2-4 min period, the whole flaccid phallus is folded back inside the peritoneal sac, aided by the antiperistaltic action of the cloacal sphincter as well as by mucous that is secreted from the glandular base of the phallus. Coker et al. (2002) studied the IOs of 54 species of Anatidae for which they had detailed, scaled drawings of IOs (one per species) made from formalinpreserved museum specimens of males taken during the breeding season. As they did not work with fresh or live specimens, it is unknown whether the species that they studied have phalluses that are similar in structure to that of the Mallard. From each drawing, they measured the flaccid length of the IO, estimated its circumference, and quantified the numbers and heights of knobs and ridges (Fig. 3.10, Plate 3.1) on its surface. In the species studied, flaccid IO length varied from 1.25 cm in the Red-breasted goose (Branta ruficollis) to an incredible 28.5 cm in the Australian blue-billed duck (Oxyura australis). IO length was not related to body length (r = –0.11, P = 0.36) in this sample of species, suggesting that variation in size might be adaptive. Indeed, the largest geese and swans had among the smallest IOs (e.g., 2.4 cm in the Canada goose, Branta canadensis, and 2.3 cm in the Tundra swan, Cygnus columbianus), whereas the relatively small stiff-tailed ducks (subfamily Oxyurinae) had among the largest (e.g., 23.6 cm in the Ruddy duck, O. jamaicensis). Even between closely-related species of similar size, the IO can be quite different in

Fig. 3.10 Intromittent true phallus of the North American ruddy duck, showing knobs and ridges (not to scale). The base of the phallus is to the left and the view is from the right side of the phallus. Modified after Coker, C. R. et al. 2002. Auk 119: 403-413, Fig. 1.


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size (e.g., 9.2 cm in the Common eider, Somateria mollissima, versus 15.0 cm in the King eider, S. spectabilis; 9.2 cm in the White-faced whistling duck, Dendrocygnus viduata, versus 18.8 cm in the Black-bellied whistling duck, D. autumnalis). There was also considerable variation in both the number (density) and size of knobs and ridges on the surface of the IOs, with the density of ridges strongly negatively correlated with IO circumference (r = – 0.86, P

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