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A PHYLOGENETIC ANALYSIS OF THE METAZOA WITH SPECIAL CONSIDERATION OF THE ACANTHOCEPHALA
Scott Monks
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Zoology University of Toronto
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A phylogenetic analysis of the Metazoa with special consideration of the Acanthocephala. Doctor of Philosophy, 1998. Scott Monks, Department of Zoology, University of Toronto.
The purpose of this study was to examine phylogenetic relationships among the Metazoa, identify appropriate outgroups to the Acanthocephala, and make a phylogenetic analysis of the Acanthocephala. A database of 144 characters for the metazoan phyla was obtained by examination of primary and secondary literature. The Choanoflagellata was used as outgroup for 33 multicellular phyla. Phylogenetic analysis yielded 17 equally parsimonious trees. Using successive weighting, these were reduced to two trees (Length=361; CI=0.46; HI=0.58) differing only in placement of the Platyhelminthes. Monophyly of the Mesozoa was not supported but mesozoans were placed basal to the Porifera +(Cnidaria + (Ctenophora + remaining Metazoans)). The remaining Metazoans formed two major clades. The Acanthocephala was the basal member of a clade, the Ecdysozoa, containing Rotifera + (Gastrohicha + ((Nematoda + Nematomorpha) + (Pentastomida + (Kinorhynchida + (Tardigrada + (Onychophora + (Chelicerata + (Uniramia + Crustacea)))))))). The second clade consisted of two sub-clades; Chaetognatha basal to Chordata + (Echinodermata + (Phornida + Brachiopoda)) and Priapulida basal to Gnathostomulida + (Platyhelminthes + Nemertea + Mollusca + (Sipuncula + (Echiura + (Pogonophora + (Clitellata + Polychaeta))))). A novel approach to identification of early cleavage patterns involved coding of individual cleavage events as independent characters. Reinterpretation of the dassic assignment of 'spiral' cleavage to arthropods supported placement within the Ecdysozoa. Deuterostomes were only monophyletic by including protostomes, thus 'spiral' cleavage is derived from 'radial' cleavage. The position of the Ecdysozoa as sister-group to the deuterostomes + protostomes supports the conclusion that 'modified-spiral' cleavage is not derived from 'spiral' deavage. Priapulida and Rotifera were supported as outgroups for the analysis of relationships among the Acanthocephala. Direct examination of specimens identified 138 characters for 21 well-defined genera. Phylogenetic analysis yielded a single tree (Length=404; CI=0.545;HI=0.455) which provided support for the monophyly of the Palaeacanthocephala and Eoacanthocephala but not the Archiacanthocephala, which were placed basal to the two other classes. To rectify taxonomic problems encountered while characters were being evaluated, Koronacantha, was described with a single species, k mexicana. Teeorhynchus brevis was redescribed and Z pectinarius transferred to Koronacantha. Results supported placement of 1.pectinarius in Koronacantha and retention of both Tegorh nchus and Illiosentis as distinct genera. Acknowledgments
The person whose name is listed as author of a manuscript usually receives the credit, or blame, for the piece of work. However, it is obvious to all except the most egotistical that little that is worthwhile is accomplished alone. This dissertation is no different. One could argue that this project could not have been accomplished in exactly the fashion it was without m~!having done it, but I would have to argue even more strongly that it might not even have been accomplished at all without the assistance, guidance, and friendship of those around me. For the inevitable errors, I take full blame, but recognition of any value in this work I gratefully share with aLl who contributed. Acknowledgments in publications often go at the end of manuscripts, and I suspect that they are not often noticed by the majority of readers who look only for 'hard data'. I believe that it is more appropriate to acknowledge others here at the beginning, because in this manner their contributions may receive a little more attention. 1 would like to thank the following people, in temporal order rather than by rank or importance, for their contributions, both personal and professional. First, I would like to thank Brent Nick01 for providing my first instruction in the systematics of the Acanthocephala. I regret that I was no able to 'convince' him of my interest in this area, but the fault is mine for being too bashful to approach the subject rather than his. I do not expect him, or anyone else, to agree with all of my work, but it is one of my strongest hopes that he will never feel ashamed to claim me as a former student-I will always be proud of my association with him! I am indebted to Dan Brooks for his interest and encouragement of my desire to work with the Acanthocephala. Since our first meeting in 1986, long before I came to the University of Toronto, he has continued to convince me that I was capable of accomplishing this project. The 'stories' of Dan's accomplishments during his time at the University of Nebraska that I had heard from his former fellow-students, as well as his vast publication record, had not given me any hope of ever being able to work with him. His encouragement of my coming to the University of Toronto to work in his lab was a big surprise to me, and it made me feel that what I wanted to accomplish there was also worthwhile to others. Dan also gave me the opportunity to work in Central America, where I met Griselda, and the many other friends I now have there. I have long way to go before I am able to 'absorb' and put into practice all that Dan has hied to teach me about Phylogenetics. I hope I have not forgotten more than 1 remember! I cannot even begin to thank Fernando Marques for his support during the past four years- there is nothing more than my continued friendship that deserves be offered in return for this. He has been beside me during my darkest hours as well as my brightest. Sometimes nothing else could keep me going except his 'kicking' me in the butt when I needed it, and he was, and still is, a source of encouragement. The figures of Koronacantha and Tegorhynchus included herein were originally drawn by Femando, and are an example of artistic, yet scientifically accurate, drawings that all should aspire to make. Femando also was my introduction to jazz, although some other Zoology Department 'inhabitants' might not have appreciated that as much as I did! Now that my graduate 'career' is over, the one thing I will miss is the hours he and I spent working together in the lab together. tn many ways, those were the happiest and most fulfilling times of my life. I will miss the inspiration of our discussion while we processed specimens, 'arguments' over methodology, and his constant borrowing of my dissection tools-Fernando is the brother that I never had. I consider it a great honor, and a privilege, to be able to continue collaborating with him. I especially thank him for arranging my first 'date' with the woman that is now my wife. You can always be sure, Fernando, that "nuestra casa es tu casa". I am particularly fortunate to have been able to spent time with Malcolm Telford. In contrast to some others in the Department, Malcolm is very quiet and unassuming, so many students may not even be aware of the loss the University has suffered because of his retirement. The focus of science has turned away from organismal biology to more reductionist pursuits, and I do not believe that a student today can receive the broad appreciation of the diversity of life that someone with Malcolm's background can offer. As well, Malcolm is the kind of professor I would like to bealways encouraging, treating me as an equal (even though I am far from it!), and never laughing or belittling me even when my ideas were naive. I never felt hesitant to discuss ideas with him because I knew he would not make me feel stupid even if it turned out that I didn't have a 'clue' to what was going on. I greatly appreciate the times we spent together discussing the various invertebrate groups. Lastly, I owe Malcolm special thanks for his causing me to examine classical 'ideas' more rigorously, as well as his example of always being eager to give more credit to those working with him than he took himself. He is a worthy example for all graduate students, as well as many professors! I am also very fortunate to have been able to work with Gerardo Perez-Ponce de Leon. He has been generous with his time, lab space, and specimens. I hope my future students will respect me in the manner his students respect him-it is one of my goals to learn from Gerardo how to be that kind of professor! What was at first a professional relationship between us has become a friendship, and it was Gerardo that accompanied me as my Papa when I went to ask Griselda's parents for permission to marry her, and later he was my best man at the wedding. "Gracias por todo, Papi!". This is not the end of the list of people that provided help and support to me, but I cannot thank all of the rest individually. among these are Fernando Alvarez, Jefe del Departamento de Zoologia, Instituto de Biologia, Universidad Nacional Aut6noma de Mkico; Fernando Garcia; Martin Garcia; Luis Garcia Prieto; Agustin Jimenez; Greg Klassen; Virginia Leh-Regagnon; Alejandro Masis and the staff of the hstituto Nacional de Biodiversidad (INBIO), Costa Rica; Berenit Mendoza; Felipe Noguera, Chief of the Estacih de Biologia Chamela, Mexico; Coral Rosas; and Ulises Razo. AU of those at the Laboratorio de Helmintologia deserve special thanks for their hospitality and patience with my poor Spanish. I have also greatly benefited from the amiable assistance of the various support staff of the Department of Zoology. As well, Sherwin Desser, Chairman, Department of Zoology, has also been a source of encouragement. I am sure there are those whom I have left out here, but it is because of my poor memory for names and not for my lack of appreciation for their contribution. I also want to thank those who loaned specimens that were used in this study. My work was partially supported by operating grants IN201593 from the Programa de Apoyo a Proyectos de Investigacibn e Inovacih Tecnolbgica-UNAM to Gerardo Perez-Ponce de Leon and A7696 from the Natural Sciences and Engineering Council (NSERC) of Canada to Daniel R. Brooks. Lastly, I want to thank my wife, Griselda Pulido-Flores de Monks, for being almost continually at my side during the past year. She has been my companion in the field, my Spanish teacher, and best of all, she is a friend with whom I can share all things. She also has labored to help me complete my dissertation-entering vast numbers of publications into the computer and helping keep me from burying myself in the piles of paper that I could never find time to file! As those who nicknamed me "Oso roncador" can attest, she also deserves recognition for putting up with my snoring!
vii Table of contents . . Abstract...... 11
Acknowledgements...... iv ... Table of Contents ...... vlll
...... List of Tables...... xi .. List of Figures...... xu
List of Appendices...... xv
Chapter 1: Introduction ...... 1
Chapter 2: Historical review of the relationships of the Metazoa...... 3 The need for classification systems ...... 3 Pre-Darwinian 'essentialist' views ...... 4 Aristotle ...... 4 Georges Cuvier ...... 5 Charles Lyell ...... 6 Jean Baptiste Lamarck ...... 7 Louis Agassiz ...... 11 K . E . von Baer ...... 11 Darwin's views on classification and metazoan evolution ...... 12 Post-Darwinian views on metazoan evolution...... 14 Grastrea theories ...... 17 Planula theories ...... 20 Acoel theories ...... 22 Phylogenetic theories ...... 24 Non- theories ...... 29 Conclusions...... 29 Figures ...... 31 Chapter 3: Phylogeny of the Met- based on morphological characters ...... 50 Methodology ...... 54 Taxa ...... 54 Character and character state coding...... 57 Analytical procedures ...... -61 Results of the analyses...... 63 Discussion and comparison with previous studies ...... -64 Status of taxa ...... 64 Rela tionships among taxa ...... -65 'Lower' phyla ...... 65 'Higher' phyla ...... 67 Protostomes and deuterostomes ...... 67 Ecdysozoa ...... 76 Cleavage pattern ...... 82 Classification of multicellular animals...... -86 Conclusion ...... 88 Figures ...... 90
Chapter 4: Historical review of the relationships of the Acanthocephala ...... 105 + Historical background ...... 105 Sister group affinity of the Acanthocephala ...... 105 ...... Hyman...... 106 Kholodkovsku...... 107 Meyer ...... 107 Van Cleave...... 107 Petrochenko ...... 108 Conway-Morris and Crompton ...... 109 Cladistic analyses ...... 109 Historical views of relationships within the phylum ...... 111 Evolutionary hypotheses ...... -111 Meyer ...... 112 Van Cleave ...... 112 Petrochenko ...... 112 Golvan ...... 112 Yamaguti...... 112 ...... Bullock ...... 112
Phylogenetic hypo theses ...... 113 Amin ...... 113 Brooks and McLennan...... 113 Figures ...... -...... 114 Chapter 5: Phylogeny of the Acanthocephala based on morphological characters ...... 120 Materials and Methods ...... 123 Results ...... 127 ...... Discussion . . 130 Taxonomic revismn ...... 137 Description of Koronacantha mexicana n . gen.,
n . SD1 ...... *...... -...... 138 Materials and methods ...... 138 Description ...... 139 Taxonomic summary...... 144 Discussion ...... 144 Redescription of Tngorhynchus bris Van Cleave, 1921 and transfer of 1. pectinarius Van Cleave. 1940 to Koronarantha ...... 147 Materials and methods ...... 148 Redescription and reassignment of chua pectinariua ...... 148 Taxonomic summary...... 153 Remarks ...... 153 Redescription of &gorhynchus brevis Van Cleave, 1921 ...... 156 'J&grhynchus Van Cleave. 1921 ...... 156 borhvnchus brevis Van Cleave. 1921 ...... 157 . Taxonomic summary ...... 162 Remarks ...... 162 Review of past taxonomic problems with the genus &qprhvnchus ...... 164 Conclusions...... 167 Figures ...... 169
Chapter 6 Conclusion ...... 184
Literature cited ...... 188
Appendices ...... 218 List of Tables
Table I. Coding of the first three cleavages of the zygote ...... 60
Table 11. Embryological and developmental features of protostomes and deuterostomes...... - ...... - ...... -...69
Table 111. Branch support for the nodes of the phylogenetic hypothesis of acanthocephalan relationships...... 128
Table IV. Total support indices for the phylogenetic hypothesis of acan thocep halan rela tionships...... 129
Table V. Hook lengths of male and female Koronacantha mexicana...... - ...... -....- ...... 141
Table VI. Hook lengths of male and female Koronacantha pectinaria...... 150
Table VII. Comparison of measurements of Worhynchus pectinariu~and Koronacantha ~ectinaria...... 155
Table VII. Hook lengths of male and female brevis ...... 159 List of Figures
Figure 1. Lamarck's classification and tree of the Metazoa ...... 31
Figure 2. Haeckel's evolutionary stages ...... -32
Figure 3. Tree of Life according to Haeckel, 1866...... 33
Figure 4 . Tree of the Metazoa according to Jagersten, 1955...... 34
Figure 5. Tree of the Metazoa according to Siewing. 1976...... -35
Figure 6 . Tree of the Metazoa according to Hyman. 1940...... 36
Figure 7. Tree of the Metazoa according to Salvini.Plawen. 1978...... 37
Figure 8. Tree of the Metazoa according to Hadzi. 1963...... 38
Figure 9 . Tree of the Metazoa according to Steinbiick. 1963...... 39
Figure 10. Tree of the Metazoa according to Jefferies. 1988...... 40
Figure 11. Tree of the Metazoa according to Schram. 1991...... Al
Figure 12. Tree of the Metazoa according to Meglitsch and *am. 1991..... 42
Figure 13. Tree of the Metazoa according to Backeljau. 1993...... 43
Figure 14. Tree of the Metazoa according to Schram and Ellis. 1994...... 44
Figure 15. Tree (1st consensus) of the Metazoa according to Nielsen et al.. 1996...... 45
Figure 16. Tree (2nd consensus) of the Metazoa according to Nielsen et al.. 1996...... 46
Figure 17. Tree (preferred) of the Metazoa according to Nielsen et al.. 1996...... 47
xii Figure 18. Tree of the Metazoa according to Nielsen. 1995 ...... 48
Figure 19. Tree of the Metazoa according to Wilmer. 1990 ...... 49
Figure 20 . Tree of the Metazoa according to Eernisse et al.. 1992...... 90
Figure 21 . Comparison of the tree of Nielsen. 1995 with that of Nielsen et al.. 1996 ...... 91
Figure 22 . Trees resulting from the primary analysis of unweighted charactars and strict consensus of those 17 equally parsimonious trees ...... 92
Figure 23. Classic view of the evolution of cleavage types ...... 98
Figure 24 . Four types of cleavage planes exhibited in metazoans ...... 99
Figure 25 . Two equally parsimonious trees for multicellular animals produced by the analysis of weighted characters...... 100
Figure 26 . Consensus tree for multicellular animals and unambiguous character changes...... 101
Figure 27. Single tree produced by the secondary analysis...... 103
Figure 28 . Two scenarios of the evolution of cleavage patterns ...... 104
Figure 29 . Tree of the 'aschelminths' according to Conway-Morris and Crompton. 1982...... -114
Figure 30 . Tree of the Acanthocephala according to Meyer. 1931 ...... 115
Figure 31 . Tree of the Acanthocephala according to Van Cleave. 1948 ...... 116
Figure 32 . Tree of the Acanthocephala according to Petrochenko. 1956...... 117
Figure 33 . Tree of the Acanthocephala according to Yamaguti. 1963 ...... 118
Figure 34 . Three trees of the higher relationships of the Acanthocephala according to Brooks and McLennan. 1993 ...... 119
Figure 35 . Rela tionships of the Acanthocephala according to this study...... 169 Figure 36 . Morphological details of Koronacantha mexicana (1.)...... 171
Figure 37. Morphological details of Koronacantha mexicana (2.)...... 173
Figure 38 . Morphological details of Koronacantha pectinaria (male)...... 175
Figure 39 . Morphological details of Koronacantha pectinaria (female)...... A77
Figure 40 . Morphological details of Wrhy"chus brevk (male)...... 179
Figure 41 . Morphological details of Wrhynchus brevb (female)...... 182
xiv List of Appendices
Appendix I ...... 218
Appendix 11 Appendix 11. A ...... 219 Appendix 11, B ...... ZO Appendix n0C ...... 221 Appendix D-...... 222 Appendix III ...... 231
Appendix N...... -253
Appendix V Appendix V. A ...... 277 Appendix V0B ...... 283 Appendix V, C ...... 2M Appendix VI ...... 285
Appendix W ...... -291
Appendix VIII Appendix VIII. A ...... 316 Appendix VIII, B ...... 336
Appendix IX ...... 337 Chapter 1: Introduction
The purpose of this study is to examine the phylogenetic relationships among the Metazoa, identify appropriate outgroups to the Acanthocephala, and then make a phylogenetic analysis of the Acanthocephala. The study involved the following steps: 1.)build a database of characters for the metazoan phyla that were phrased in a format useful for phylogenetic analysis; 2.) perform a methodologically rigorous phylogenetic analysis that would identify outgroups and characters that could be used to polarize character transformations for the analysis of the Acanthocephala; 3.) build a database of characters for acanthocephalans that were phrased in a format useful for phylogenetic analysis; and, 4.) perform a rigorous analysis of relationships among acanthocephalans using this database. The approach to this project seems straightforward, but each analysis was hampered by a lack of suitably defined characters. First, it will be demonstrated that very little of the information about the morphology and development of metazoan phyla is suitable for use in phylogenetic analysis in the format presented in previous studies. Character descriptions and assignment are theory-laden as a result of
-a priori assumptions of evolutionary mode, relationships, and character evolution. Even in recent studies using a cladistic approach, most researchers have uncritically accepted characters without evaluation of primary homology or presentation in a testable format. In addition, in all previous studies, the character sets have been woefully inadequate to resolve relationships within the group of interest. Second, although it has been claimed that phylogenetic studies are not appropriate at the phylum level because the methodology is inadequate (Willmer, 1990) and not possible among the Acanthocephala because sufficient characters are not present (VanCleave, 1941), it will be demonstrated that neither assertion is correct. It will be shown that studies of metazoans have been hampered by (i.) the lack of properly phrased hypotheses in a testable format and (ii.) attempts to postdate evolutionary scenarios without basing them upon rigorous objective methodology. It will also be shown that acanthocephalans possess a rich store of morphological characters that have been overlooked for phylogenetic analyses. As well, it will be demonstrated that attempts to clanfy character assignment for the Acanthocephala cannot be accomplished without direct examination of specimens because of the taxonomic problems inherent in the current arrangement of groups within the phylum. Finally, the results of the analyses will be used to test hypotheses of character evolution for two examples of character 'suites' that are considered to be important as support for various theories of evolution within each group. In the Metazoa, early embryonic cleavage pattern will be examined and in the Acanthocephala, the structure of the cement glands. Both characters will be shown to have levels of inference both among metazoan phyla and among acanthocephalans. Chapter 2: Historical review of the relationships of the Metazoa
The need for classification systems
Part of the human psyche is a tendency to categorize things. This tendency has probably existed since our ancestors became capable of recognizing and remembering the different objects they saw. The first classification may have been no more than 'I can eat this' and 'this can eat me'. As long as the groups were no more complicated or numerous than this, only a simple system of classification was necessary. As experience increased, the need for a more organized classification became important. Food organisms had to be identified as 'things that are edible, things that are dangerous to eat, things I will eat only if mom makes me,' and 'things I like to eat'. Sirnpson (1961) carries this idea one step farther, and suggested that the necessity for recognizing categories of things is a general attribute of life. He further noted that Amoeba, like humans, cannot survive without the ability to differentiate 'food' and 'non-food' items. Later, the need for more groups, and more sub-divisiow of groups, resulted from the increased ability to recognize objects on an increasingly finer scale. Once more than even a few categories of objects were included, the need for a hierarchical system became essential (Raven, et al., 1971). As pointed out by Brooks and McLennan (1993),our compulsion to class@ objects arose "...out of our seemingly inherent need to organize the world into familiar units" (Brooks and McLeman, 1993b; 22). At this basic level, categorization is merely a way of sorting out and organizing various kinds of items into a manageable system. Pre-Darwinian 'essentialist' views
Details of the earliest attempts at dassification of metazoans have been lost in history. Greek scholars are usually credited with formulating the first ideas on classification systems. Hippocrates (460-377 B. C.) and Democritus (465-370 B. C.) included animals in their studies. Aristotle was the first to provide justification for formal descriptions of animals on the basis of "...their way of living, their actions, their habits, and their body parts ..." (Mayr, et al., 1953; 5) in an attempt to discover and define the 'essence' of a taxonomic group. Aristotle's classification was built upon logic, thus, the essence of a taxon was expressed in axioms that give rise to the properties of the taxon as an inevitable consequence. For this reason, pre-Darwinian definitions of species have often been called 'essentialist' because the definition depended upon the perceived essence of the organism. Hull (1988) suggested that recent non-phylogenetic classifications and nomenclature continue to embody essentialism. He prefered the term "idealist" to broadly characterize all explanations based on "...timeless general patterns or 'archetypes" (Hull, 1988; 41: see also Hull, 1970,1988). Sneath and Sokal(1973)showed that Aristotelian logic works well with mathematical constructs for which all of an object's properties are known. For example, the 'essence' of a plane triangle is expressed by its definition as a figure bounded by three straight sides. The inevitable consequence of this definition is that the sum of the lengths of any two sides is greater than the length of the third. Early systematists supposed that biological classifications could be similar because they believed that there was a finite set of immutable species that, once identified, could be fully investigated just as geometric figures could be analyzed. Yet, as understood by more recent systematists, this system of logic is not appropriate for biological classification, which is composed of unanalyzed entities, whose properties cannot be inferred from their definition (Ghiselin, 1966). 5
Hull (1988) characterized p re-Darwinian species concepts as falling into one or the other of two categories: supernatural special creation or "reverent silence" (Hull, 1988; 40). Special creationists hold the belief that each type of organism was created by a supreme being who originally brought into existence some finite number of species. Through time, these species might die out as a result of some catastrophe, such as the Biblical flood, or become degraded due to the effects of sin in the world. Any changes in a species, then termed 'mutation', was the direct result of divine intervention such as when God removed the legs of all snakes as punishment for a "serpent" aiding in the fall of mankind (Genesis 3:l-14). Although many of these early concepts were a product of Western religious culture, similar beliefs in the origin and divine intervention in the formation of organisms exist in many other cultures. Georges Cuvier was the foremost comparative anatomist of his time, and was the epitome of those who combined religion and science. His belief in the correlation of structural parts brought him fame for his ability to reconstruct extinct species from partial fossil remains. According to Cuvier, organisms are functionally constrained by their environment, and form a complete organized system, so delicately balanced that if any sigruficant variation occurred in its structural parts it would perish. Because he believed God would create no new species, he thought that through time the earth was being depleted of its original stock of species. Catastrophic events, or major changes in an environment, resulted in the extinction of the species therein. To Cuvier, the divine origin of each species made them distinct and welated and there was no connection between the various groups. Because they were the product of a single creative act, species must be immutable and could not have changed through time since the passage of time could not mar God's perfect creations. Although he gained wider acceptance than many for his ideas on classification of metazoans, Cuvier's "catastrophism" was in direct opposition to that of Lamarck, and most others of the time. These beliefs cawed him to view species not as a series of entities characterized by progressive changes, but as examples of God's divine organization. Accordingly, Cuvier's classifications were constructed to reflect this organization, and thus display the handiwork of God. He broke up the 'tree of life' into what he termed four 'embranchements': Vertebrata, Molluscs, Articulata, and Radiata. Cuvier's classification was built upon thorough examination of specimens, but because he believed each structure had a unique origin, he made no attempt to link any of the embranchements together. It was his carehl work, rather than his religious beliefs, that made him a leading figure in the scientific world of his time (Appel,. 1987). Those who had less strong religious beliefs were not eager to challenge the church-supported version of how species arose. Their strategy was to present scientific works from which they removed any reference to the possibility, or impossibility, of God's direct intervention in human existence. This approach of reverent silence was maintained in many areas of scientific study. For example, the physicist JohnHerschel made no reference to God's influence in the existence or function of the solar system, and geologists such as Charles Lyell simply avoided any discussion of a "miraculous"formation of the earth (Hull, 1988). Those who assumed the position of reverent silence tended to view species in a similar essentialist perspective. For example, Lyell recognized the existence of extinct organisms, but viewed each as the expression of a particular archetype that exactly fit an earlier role in nature. Thus, he believed that if those exact conditions were to reappear, those extinct organisms might reappear: "Then might those genera of animals return, of which the memorials are preserved in the ancient rocks of our continents. The huge iguanadon might reappear in the woods, and the ichthyosaur in the sea, while the pterodactyle [sic] might fit again through umbrageous groves of ferns." (Lyell, 1830; v.l,123 in Hull, 1988; 36). Although Lyell, as with others, did not offer any explanation of how this might come about, the idea was generally based on the belief in a progressive 'force' that propelled 'lower' organisms to 'higher' roles (now called niches) in nature . According to this perspective, species were thought to be natural 'kinds', and any species was capable of becoming any other species without regard to heredity or genealogy. This view of species as natural kinds was consistent with the beliefs of Aristotle and his successors, and remained popular until the time of Darwin. The distinction between species as classes, or kinds, of similar organisms, regardless of descent, and the idea that they are spatiotemporally continuous lineages has been debated much in recent literature (for a hli discussion, see Hull, 1976,1979,1988 and references therein). Reference to them is made here only to contrast early views with post-Darwinian perspectives. Pre-Danvinian systematists perceived the organisms themselves as having the ability to move horn one species to another and to acquire new characteristics, much as one element might be changed in identity and properties by a change in atomic components (similarly termed 'transmutation'). Today, Jean Baptiste Lamarck is often given credit for advancing the theory that there was an innate drive to perfection in all organisms which would cause each species to advance in complexity through successive generations. Some have termed this 'Lamarckian evolution', yet Larnarck was not alone in his belief in the inheritance of acquired characteristics, a view which was widely held during that period. Of the leading zoologists of the time, only Cuvier (as discussed above) believed strictly in the immutability of both organisms and species. Likewise, Larnarck's theories were not based on a conception of evolution, as we know it, and it is better termed "transformism" (for contrasting views, see Mayr, 1982 and Padcard, 1901). In Larnarck's theory of transformism, it is not the species, but organisms, that move up the 'tree of life'. As in more modem phylogenetics, he represented such transformations as branching patterns, in which it would not be equally possible for an organism to become gny other species. Hence, once an organism began moving up a particular branch, the options of its descendants were constrained to that branch, and could not jump from branch to branch. Organisms, such as parasites, could occasionally degenerate, but the drive was toward overall perfection and greater complexity. In addition, he also viewed the environment as having inherent trend.^, or channeling forces, that moved organisms upward toward and then along certain paths, or branches. Lamarck's 'tree of life' (Fig. 1B) was not a continuous route, as if organisms were figuratively climbing up the trunk of tree, but it was as if the tree were composed of discrete, stacked cubicles, and organisms moved upward from one cubicle to another. Lamarck's cubicles were themselves the actual species and, as natural kinds, were immutable. Individual organisms changed, often in a single generation, to fill the particular requirements for 'residence' within a given species, just as if they were residing within a real cubicle. Thus, within the bounds of the branches of his tree of life, any organism could be transformed into any other form, or species. In a manner he did not fully explain, Lamarck considered local environments and the organisms therein to be inseparably linked. Organisms responded to the forces of the environment, which stimulated the changes necessary for them to become different species, exactly fitted to the requirements of that environment. If an environment disappeared locally, the organisms in that environment would disappear, and those species would become extinct- As did LyeU (1830),Lamarck believed that if a former environment reappeared, in that or another location, organisms from 'below' would rise to fill the then vacant 'species' positions. As discussed by Hull (1988), it is difficult to evaluate the work of early systematists because it is necessary to remember that they lacked most of the basic biological information that we take for granted. For example, Larnarck is often faulted for not explaining how acquired characteristics were passed from parent to offspring, yet to criticize him for this shows a lack of recognition that all scientists of his day were unable to resolve this problem. Lamarck's theories also have been severely criticized for the gaps in the character qualities that separated each species, and, more obviously, his higher taxa. When discussing perceived gaps, Lamarck insisted that "...races may, nay must, exist near the boundaries, halfway between two classes..." (Lamarck, 1809; 66),yet he offered no suggestion concerning the status of these between-species "races". Both Hull (1988) and Packard (1901) suggest that much of our perceived ambiguity may be more a result of Lamarck's writing style and lack of concrete examples to illustrate points rather than shortcomings in his theories. Although Lamarck's view of species and evolution is somewhat different from modem views, his concept of order, or arrangement, in nature was more similar to current views. He suggested that arrangements should be a combination of the more traditional linear classification system and tree-like diagrams. His classification of organisms into two major groups, Invertebrata and Vertebrata (Fig. IA), is more linear than current classifications and contains as little information about phylogenetic relationships as most present classifications. His tree is more similar to a phylogenetic tree than an evolutionary tree in that it presents a branching pattern and does not attempt to provide information about degree of differentiation or absolute time. As well, Larnarck gave no indication of how to translate his trees into a classification or of an objective method for deriving the trees. From Lamarck's use of branching trees, and his claim that a knowledge of fossil organisms is essential to systematists, Mayr (1982) suggests that Lamarck was "...rather close to the concept of common descent, but he never developed it." Larnarck's work is generally viewed with little respect, but it did have a lasting effect on scientific thinking. Darwin stated that it was "...veritable rubbish..." and that he "...got not a fact or idea from it ..." (Rousseau, 1969 in Mayr, 1982), but Lamarck's essentialist views had a stronger influence on the work of many Germans, including Meckel and Haeckel (Mayr, 1982). Although his life and work has been well studied (Locy, 1908), no one has made a thorough examination of how persistent his ideas have been. In addition, it is unclear how much of our own system of classification is based upon his essentialist ideas of species identity. The theories of Lamarck and Cuvier have been presented here as typifymg pre-Darwinian thinking because their ideas concerning the relationships of metazoan taxa illustrate the pinnacle of pre-Darwinian systematics. Numerous other systematists, such as Belon, Rondelet, Ray, Geoffroy, Madeay, and Linnaeus also proposed classifications for the Metazoa, but although their contributions in other areas of zoo1ogy were substantial (see Laaon, 1971, Stresemann, 1975, and Mayr 1982 for further discussion) their efforts in metazoan systematics did not persist past Darwin. Even Linnaeus was only influential in affecting nomenclatural format. The underlying basis for Cuvier's and Lamarck's arrangements of metazoans may have been far different, but, as discussed above, both men believed that species could be viewed as having an ideal, or archetypal, quality. As pointed out by Hull (1988; 40-41), many of the examples and arguments of Darwin were directed toward the beliefs of those such as Cuvier who invoked spiritual forces to explain the diversity of organisms. It is interesting, as Hull (1988) further notes, that Darwin was less willing to directly attack the ideal morphologists of his time than he was to confront creationists. In the single paragraph concerning the essential nature of species, Darwin only attributes reliance on archetypal species to the "...illustrious Cuvier ..." (Darwin, 1859; 158). Curiously, adherents to idealist philosophy may have caused systematists to stray farther from Darwin's goal of classifymg by descent than the creationists would have. The philosophic school of thought, the German Naturphilosovhie originated from eighteenth- and early nineteenth-century philosophers (Hull, 1988; Mayr, 2982; Mayr, et al., 1953). The German school was primarily concerned with identdying archetypes, or hypothetical forms, that were thought to embody the essence of each specific animal kind. Comprehensive early studies of parallelism between embryos and adults were made by Meckel(1821)as well as others outside Germany (Geoffroy Saint-Hilaire, 1830; Owen, 1848; Serrh, 1860). This essentialist idea that ontogeny paralleled the scala natura later came to be called the Mskel-Serrgs Law and formed the basis of subsequent recapitulationist theories (Mayr, 1982). Louis Agassiz (1857), an opponent of Darwin, expanded the Meckel-Semi% Law to include a progressionist interpretation of the fossil record and suggested that ontogeny also paralleled succession of fossil organisms. Darwin was not in disagreement with this as a general principle, but he doubted that it would be "...proved true" (Darwin, 1859; 377). These conclusions were disputed by K. E. von Baer (1828,1876), who soundly rejected the concepts of a scala naturae, a single archetype for all organisms, Cuvier's four embranchements, and as well, evolution in any form. von Baer described ontogeny in terms of changes from general characters in embryos to more specific characters in adults, and it is thought that Darwin learned much of his embryology from reading von Baer's work (Mayr, 1982). As mentioned above, Darwin generally avoided confrontation with idealists, but earlier, von Baer had been quick and caustic in challenging their ideas as well as recapitulationism and parallelism (Mayr, 1982; 472). Probably because of his anti- evolutionist views, von Baer's work was rejected after Darwin published the of Species (Darwin, 1859). Mayr (1982) suggests that the decline in popularity- of Naturphilosophie in response to the rise in acceptance to the theory of evolution also contributed to von Baer's laws being forgotten until de Beer (1940) attempted to resurrect them in response to the fading popularity of Haeckelian recapitulation. Regardless of whether they were creationist or idealist, pre-Darwinian systematists believed in the immutability of species. Those who believed in what some have termed 'evolution' thought only that organisms could be transformed into different forms,fitting into one of the archetypal species. The arrangements of Cuvier and Lamarck represent the state of thought concerning metazoan evolution and classification before Darwin.
Darwin's views on classification and metazoan evolution
As Mayr (1982) recognized, Darwin "...was the founder of the whole field of evolutionary taxonomy..." (Mayr, 1982). Darwin devoted a sigruficant portion of the Origin (Darwin, 1859) to the theoretical bases and methodology of a classification consistent with descent (Darwin,1859: Chapter XIV; 345-384). Darwin stated that a "...true classification [is] genealogical" (Darwin, 1859; 346) and thus reflects the genealogical relationships of extant organisms. Even though a particular taxon might "...depart from its allies in several characters..." (Darwin, 1859; 349), it must be grouped with those to which it was most closely related rather than with those taxa with which it shared only an overall similarity. It should be the "...community of descent... which naturalists [should be] seeking, and not some unknown plan of creation, or the enunciation of general propositions and the mere putting together and separating of objects more or less alike..." (Darwin, 1872: 346). Darwin further stated that a natural classification is not "...an artificial method of enunciating, as briefly as possible, general propositions-that is, by one sentence to give the characters common, for instance to all mammals, by another those common to all carnivora [sic], by another those common to the dog-genus, and then, by adding a single sentence, a full description is given of each kind of dog" (Darwin, 1859; 347). Though Darwin's theory of evolution revolutionized systematic thinking in other areas, the importance of relying solely on descent, with the complete exclusion of archetypally defined taxa, was soon lost to most of the world (Ghiselin, 1969; Ghiselin and Jaffe, 1973; Hull, 1976,1988; Mayr, 1982; Mayr, et al., 1953). Systematists had long viewed the sharing of characteristics as an indication that species should be grouped together, but Darwin took this one step farther. He recognized that character distributions are composites of both similarities and dissimilarities, and the ratio between the two is not a particularly good indicator of rela tionships (Darwin, 1859; 351). He further recognized that overall similarity was made up of true similarities, or homologies, and deceptive similarities, or homoplasies, which were only analogous features. Homologous features, Darwin declared, should form the basis of a natural classification. The task of the systematist was to distinguish between the two types of similarity and then to construct a phy logenetic classification based only on homologies (also see Ma yr, 1982; 209-213). The sticking point was that Darwin could not put forward a method for distinguishing between homology and homoplasy or a way to use these features in reconstructing phylogeny. Furthermore, he did not have a method to convert phylogenies into classification (Hull, 1985).
Lest this seem disparaging of Danvin, it should be clearly recognized that Darwin's intent was to provide a complete, well supported theory for the process of speciation and evolution of species. His focus was on developing systematic methodology. His development of the theory of evolution was brilliant, and he also had the insight to recognize, almost in a prophetic sense, the impact of the theory on systematic methodology. His work, therefore, should not be denigrated simply because he did not see how to bring his innovative proposal to fruition. It is well to remember that no one else until the time of Hennig (Hennig, 1950,1966) was able devise a method to recover phylogenetic history. The plethora of publications since Hennig's work, even until present time, demonstrates that the solutions to this dilemma are far from simple. Darwin's concept of reflecting phylogeny in classifications, was revolutionary (Hull, 1988). Although his ideas on dassification created some interest during his lifetime, they left few traces later. Hull (1988) suggested that failure to accept these concepts was principally the result of Darwin's inability to provide answers, or methodology, to the questions he proposed. Although he recognized that species were evolving individuals, he could provide no sound manner for recognizing, or delimiting them. Consequently, the sigruficance of this novei idea was largely overshadowed by discussion of his excitingly contentious theory of evolution. Likewise, his declaration that phylogeny must somehow be incorporated into classification was ignored because he could suggest no method for deriving phylogeny. The purpose behind much of the discussion presented above is to demonstrate that one's philosophic view has an important effect on perceptions of the origin of the Metazoa and subsequent arrangements of the phyla. While this idea might seem ridiculously obvious at first glance, no one would suggest that one's philosophy influences one's views on relationships between the elements or acceptance of the periodic table. A major difference between these two is that the methodology for deriving the periodic table has long been accepted, but, although there is an almost universal acceptance of the role of evolution in the past production of the various phyla, the methodology and most appropriate characters for accurately elucidating evolutionary patterns at the phylum level is still in flux.
Post-Darwinian views on metazoan evolution
During the post-Darwinian period, classification and evolution of the Metazoa has been viewed from either an evolutionary or a cladistic perspective. Examination of recent systematic literature would suggest that adherents to each philosophy consider themselves to be vastly different than those holding the opposite view. However, it is the philosophies behind the proffered arrangements that are the most strikingly different rather than the final classifications. Undoubtedly, there exist other perspectives on metazoan relationships, such as the still existent creationist belief, but these have only a minor effect on current scientific thinking, so only evolutionary and phylogenetic perspectives, which are characteristic of the vast majority of post-Darwinian systematists, will be discussed herein. Evolutionary systematists begin their speculations by describing the way they believe evolution has progressed. These scenarios are based upon their current understanding of the particular taxa, characteristics possessed by the taxa, and the processes of evolution. Second, they create an evolutionary tree depicting the relationships between the taxa. This tree commonly embodies elements of time and relative divergence. 'Ancestral' taxa in the form of archetypes are often postulated and placed on the tree to illustrate proposed patterns of evolutionary change. Thud, the systematist elucidates characters, usually only a few of which they consider to be pivotal, and use them as evidence that their scenario correctly depicts the evolutionary history of the group. The important consideration here is that one either accepts or rejects the scenario on the basis of confidence in the systematist's experience and in their ability to recognize and correctly interpret the characters supporting the scenario. If one adheres to this philosophy, no part of the process is testable in an objective manner. To put forward a competing hypothesis one merely postulates a new scenario, builds a tree based on it, and argues that the newly selected characters are more 'fundamental' than those used in the previous hypothesis. Again, the choice between the two hypotheses is made according to the perceived abilities or arguments of their authors. Phylogenetic systematists begin the process in almost exactly the reverse of this procedure. First, the systematist examines the taxa, gathering as many characters as practical. In a rigorous study, these characters are polarized by comparison with conditions found in the putative sister-group, and often additional outgroups, without regard to the systematists personal understanding of the evolutionary process. Second, the character information is analyzed in a manner designed to produce a tree that best represents congruence of homologous characters rather than overall similarity. The third step is to interpret the relationships supported by the tree in light of our understanding of evolutionary processes, but only within the scope of the actual data used in the analysis. Unlike the evolutionary method, each step in this process, except the last, which is conjectural, is independently testable, and can be refuted based on additional evidence. The original analysis can even be tested by re-analysis of the same data set in a more appropriate manner, although the use of additional characters provides the strongest test and/or support. This process, designed by Hennig (Hennig, 1950,1965,1966), and further developed by subsequent workers (Brooks and McLennan, 1991; Wiley, 1981; Wiley, et al., 1991), allows the posing of clear, falsifiable hypotheses, independent of assumptions of character importance. In addition, it provides a means for testing homology of characters and transformation series that is lacking in the evolutionary method. It is interesting to note that the two methodologies are, in appearance, almost mirror images of each other-the steps of the phylogenetic method are the reverse order of those in the evolutionary method. Additionally, the scenario is all important in the evolutionary method, with confidence placed in the scenario because of the systematists experience, and the characters, or lack of them, are of little consequence. In the phylogenetic method, all confidence is in the characters and the method of analysis, and the experience of the one making the analysis, or lack thereof, is unimportant except in relation to having the training sufficient to recognize character states. In the next sections, the three major evolutionary theories, the gastrea, planula, and acoel theories, will be discussed. It will be demonstrated that each is consistent with evolutionary methodology. First, each is built upon preconceived archetypal ancestors, and each scenario is dependent upon the existence of these archetypes. Second, using this method, no objective manner exists with which to postulate relationships among taxa, resulting in taxa that are either grouped arbitrarily or on the basis of perceived similarity. Thus, hypothesized relationships are untestable and confidence in arrangements rests solely on the reputation of the systematist. Lastly, except for differences in origins of multicellular groups, it will be shown that each of the theories produce similar arrangements for the higher taxa because they all rely on a common conception of evolutionary mechanisms. In contrast, phylogenetic theories will be shown to have no dependence on hypotheses of archetypal ancestors, relying only on a sufficiently large number of characters to resolve relationships. The hypothesized relationships deduced from analysis of these characters is testable independently by re-analysis or by additional characters. Additionally, hypotheses of character evolution and assumptions of character homology are tested by reference to the resultant phylogenetic hypothesis. Phylogenetic hypotheses of relationships then provide the basis on which to develop scenarios of possible mechanisms and modes of evolution. Although the scenarios themselves are still subjective, because they are based upon objective evidence they can be tested by the addition of new information. Lastly, it will be shown that only the phylogenetic method will provide an objective basis for a classification that, as Darwin suggested, reflects genealogy rather than overall similarity.
Grastrea theories
Haeckel was one of the most productive of the early embryologists. An early supporter of Darwin, he originated several ideas that became the central focus for phylogenetic speculation about metazoan evolution. He is remembered most for his transformation of the Meckel-SerrLs ideas of parallelism into an evolutionary law called the "biogenetic law", or theory of recapitulation (Haeckel, 1866a, 1866b). His theory, often summarized by his own overly simplistic phrase "ontogeny recapitulates phylogeny", characterized the developmental pattern as a step-by-step collection of the adult forms of ancestors manifest in the embryology of each extant taxa. Each embryonic stage demonstrated characters found in adult ancestors, 18 appearing in the developmental sequence in the order in which they evolved, and thus provided a fundamental tool for determination of ancestral forms and character states. The most influential of Haeckel's conhibutions were the blastula and gastrea theories of metazoan origin, both of which were founded on his observations of comparative embryology (Haeckel, 1874,1875). The gastrea theory postdated a metazoan origin involving stages of complexity resembling blastula-like and gastrula-like organisms (Fig. 2) and was heavily dependent on his earlier recapitulation theories. He suggested that the blastula stage seen in embryos of extant organisms recapitulates their ancestry as a blastea organism. Thus, the primitive metazoan was a pelagic, radially symmetrical aggregation of flagellated cells, thought to resemble Volvox, which has some degree of specialization among cells with separation of somatic and gametic cells and coordination of flagellar movements. Just as in embryology, the blastea stage of complexity was succeeded by the gastrea form. It was a simple two-layered, hollow sac-like organism, that could readily be envisioned to have given rise to cnidarians, from which all other metazoans subsequently developed. The gastrea theory was simple, yet logically attractive and had a strong appeal in its orderliness of explanation. Its strongest supporters came from those who had supported the Nahrmhiloso~hie.Haedcel's work stimulated the field of embryology and established embryological studies as paramount in understanding phylogenetic relationships as well as a means of discovering archetypal forms. The earlier work of von Baer (1828,1876) and Miiller (1864) was invoked in support of Haeckel's theory and provided information for him to extend his theory to include chordate embryology. Haeckel(1866b) used these ideas to suggest a tree-like classification dividing living organisms into three kingdoms; Plantae, Protista, and Animalia (Fig. 3). Despite its popularity, Haeckel's theories were not long without attack (Garstang, 1922; Lankester, 1873,1877, and several objections, apart from the growth of disenchantment with recapitdationist laws and theories, caused it to lose favor. First, there are many colonial flagellate protists, but all of the blastula-like types are unequivocally plants (Kerkut, 1960), and all have incomplete separation of constituent cells, with fine cytoplasmic bridges connecting the colony. Hardy (1953) and Faure-Fremiet (1958) both strongly criticized all systems with protist origins and independently suggested a phyto flagellate origin, but, other than this change, neither offered a different classification for metazoans. Second, the colonial volvocines are primarily haploid. To counter this, colonial zooflagellates, which show prolonged diploidy, have been suggested as ancestral types for metazoans, but the idea has not gained popularity. To avoid the problem of a lack of blastula-like animal protists mentioned above, several modifications of Haeckel's original theory have been suggested. Principally, these have come from Europeans where belief in "...Neo-Hadelian ..." (Hanson, 1977; 587) recapitulation is the strongest. Each theorist accepts a blastea origin, and then focuses more on the stages following the blastea. They depend heavily on patterns of embryological development of the coelom, hence these theories are more generally termed 'enterocoelic'. Jagersten (l955,1959), followed by Siewing (1969,1976,1980), proposed bilaterogastraea theories in which a bilateral, benthic organism evolved gastrulation and thus, coelomic pouches. These forms then become ctenophores, anthoroam, and archecoelomate organisms (Fig. 4-5). Remane (1963a, 1963b, 1963c) suggested the slightly different cyclomerism theory, in which the gastric pouches of cnidarians were transformed into a trirneric coelomate condition. All three theories rely heavily on a pmassumptions of archetypal forms. Jiigersten (1955), for example, postulated 15 archetypes in-between the ancestral blas tea and the extant Cnidaria, a view Kohn called "...science fiction. .." (Kohn, 1973; 790). 20
Even with these modifications to Haeckel's theory, the authors did not solve earlier criticisms, and they added what some consider to be new complications. First, while some of the 'lower' invertebrates do undergo gastrulation by invagination, poriferans and cnidarians do not. Second, their theories are inextricably linked to the enterocoel theory of early coelom acquisition, according to which they postulate that in early archemetazoans the fully-formed gastric pouches of the cnidarian-Like ancestors underwent rapid transformation into enclosed coelomic body cavities. By implication, all acoelomate and pseudocoeIomate organisms must have 'regressed' from a coelomate condition. In addition, none of the gastrea theories can account for the existence of the high diversity of non-coelomates. Third, because they are tied to enterocoelic formation of the coelom, they have difficulty accounting for the existence of the various other diverse patterns seen in coelomate embryology. Of these problems, the most objectionable to many is the idea that acoelomate and pseudocoelomate organisms regressed from coelomate ancestors. However, this objection seems to be based on the a priori assumption that organisms possessing coeloms are more 'advanced' than non-coelomate taxa. As mentioned above, any theory with recapitulationist overtones has tended to be viewed with suspicion.
Planula theories
One attempt to circumvent the problems of the gastrea theory mentioned above, was retention of the blastula as the first stage in metazoan evolution (Jagersten, 1972; Remane, 1963b; Siewing, 1976). A second modification was the proposal of a solid blastea, as the second stage in evolution of the archemetazoans. This second stage has been termed the 'planaea', or 'planula' (referring to the planula larva of cnidarians), for which the theory is named. This idea was first suggested by Lankester (1873) and later by Metschnikoff (1882). Lankester claimed that his theory did not originate from Haeckel's becaw the "...substance of [his theory] had been previously given in [his]lectures, and was in no way influenced by the closely similar doctrine enunciated by Haeckel." (Lankester, 1877; 401-402). Hyman (1940) is usually given credit for the theory, although she herself cited both of Lankester's (1873,1877) and Metschdcoff s (1882,1886) major contributiow to the theory. Regardless, Hyman dearly developed the theory to its fullest extent, and produced a full classification based on the theory (Fig. 6). Hand (1963), Ivanov (1968), Beklemishev (1963,1969a, 1969b), and Reisinger (1970,1972) offered modifications of earlier forms of the theory, but none have received as much acceptance as Hyman (1940). Salvini-Plawen (1978) provided a review of all previous versions of the planula theory, along with his own modifications. He included a summary of the archetypal stages in metazoan evolution based primarily on Ivanov (1968). Salvini-Plawen's classification only extends to the major groups of the Metazoa, and is one of the more linear systems because he does not resolve archetypal groups. In this system, Salvini-Plawen, adopted the new terminology for ancestral forms and higher taxa that is popular with many of the modem Germanic authors (Willmer and Holland, 1991), especially Siew ing (1980) and Reisinger (1970,1972). In Salvini-Pla wen's scheme, he suggests that acoelomates gave rise to the 'spiral' groups, which then gave rise to non-spiral groups (Fig. 7). There are more points that favor the planula theory than the gastrea theory. First, it embraces both pelagic and benthic organisms, and radial and bilateral forms, which permits the Metazoa to be monophyletic, arising from flagellate ancestors. Second, it provides good correlation with ow knowledge of the 'lower' Metazoa because planuloid larvae occur in many cnidarians, a few poriferans, and at least one ctenophore (Greenberg, 1959). It also accommodates the Placozoa, which are planula-like as adults (Ivanov, 1973). Third, it seems to fit our ecological ideas that various forms of planulae could diversify through time according to a particular mode of life as a planktonic or a benthic, crawling form. Fourth, flatworms, a problematic group for many theories, are placed near the root of the tree, and are considered not to have been modified greatly from the primitive planulae. The primary objections are, as mentioned earlier, the difficulties in having a colonial flagellate as the first stage and problems with the occurrence of cilia and flagella. Monociliated cells, as derived from a flagellate ancestor, occur in cnidarians, placozoans, gnathostomulids, but multiciliated cells occur in ctenophores and platyhelminths, a condition which cannot be accounted for in any of the current versions of the theory. As well, Greenberg (1959), Grimstone (1959), Dillon (1962), Whi ttaker (1957,1959,1977), and more recently Valentine (1992), have suggested that the planula form is almost obligate for simple organisms with a benthic existence. As such, they argue that the form has likely evolved several times in response to ecological needs and should not be used as indicative of a monophyletic origin of life.
Acoel theories
The acoel theory of Hadzi (1953,1963) is considered to be the most heretical, and has often been dismissed outright. Hadzi envisioned an early evolution of multicellular animals as arising from multinucleate ciliate protozoa, the ancestors of which were flagellate protozoa. In his arrangement the Metazoa, as it is currently defined, is not monophyletic; poriferans and cnidarians each are derived independently from flagellate stock, and primitive ciliates, also derived independently, gave rise to both extant ciliates and the remaining metazoans (Fig. 8). As mentioned earlier, poriferans and cnidarians are monociliate, suggesting an independent origin from the uni-flagellate protozoans. Multinudeate ciliate protozoans, functionally multiciliate with a large number of independent organelles, are thought to share many similarities with the Acoela, although it has been suggested that the similarity is due to convergence. Hadzi drew on this resemblance as evidence that primitive acoels have arisen from the ancestor of extant ciliate protozoa. Acoel ancestral forms then gave rise to the remaining metazoan lineage; thus, extant ciliates are the sister group to the acoeis and 'higher' metazoans. Hadzi's theory has been attacked on several fronts. First, he has been strongly criticized by the German school for his postulated homologies between the ciliates and acoels. Even Willmer (1990), who generally presented theories without negative commentary, suggested that Hadzi was "...somewhat rash ..." (Willrner, 1990; 173) in his advocacy of some homologies (i.e., trichocysts with rhabdites and cytosome with pharynx). Others (Carter, 1954) did not even want to discuss parts of Hadzi's work because of a ''...doubt whether a definite decision [on Hadzi's proposal] is possible ..." (Carter, 1954; 164). Much of the extensive criticism of Hadzi was because he did not follow Remane's (1952) criteria for homologies, the 'standard' among those of the German school. Additionally, Remane (1963a, 1963b) and Ivanov (1968) attacked the plausibility of his ideas on celIularization in ancestral ciliates. Hadzi's arrangement generally has been ridiculed and has not received acceptance (Willmer, 1990), which he attributed largely to previous theories being held as scientific "...dogmas...". He further stated, "It is well known how difficult it is to fight against dogmas, particularly when they have been accepted for so long, when they have been supported by renowned scholars, and when they appear to be well founded." (Hadzi, 1963; xii). Two notable exceptions to those criticizing Hadzi's work are SteinbCick (1963) and Hanson (1958,1963,1977). Steinbijck was not able to publish his work because of the outbreak of the Second World War, but Hadzi gave Steinbock ,who he claimed as a friend, credit for coming to similar conclusions as he (Hadzi, 1963; viii). Steinbock (1963) later published his theory, the main difference in which was the movement of the Turbellaria to a position on his tree above the acoels and into the stem of the 'higher' metazoans (Fig. 9). Hanson also has been a strong supporter of the acoel theory, and provided solutions for most of its problem areas, while rejecting the few extreme points of Hadzi's original arrangement (Hanson, 1958,1963,1975,1977). In a large volume, Hawon (1977) presented selectionist arguments for the ciliate ancestry of turbellarians and extensive answers to criticisms of the cellularization process. The most obvious difference in his arrangement (Hanson, 1977) is the independent derivation of cnidarians from flagellate protists. Despite this support, the acoel theory has not become popular. However, considering the complete dependence upon a ~rioriassumptions of all previously discussed theories, it is difficult to see how this one is any worse than any oher. In conclusion, it should be noted that Hanson's (1977) treatment of the Metazoa was the first published analysis of relationships at this level. Hanson provided an explicit and detailed explanation of his methodology, even including the algorithms used in the analyses. He included a complete data matrix, with information for each taxon, including protozoa. However, because he used the then popular phenetic distance method for his analyses, his efforts are almost entirely ignored. Sadly, it is possible that his work has also been dismissed because he was a supporter of Hadzi.
Phylogenetic theories
One common feature in the approaches of all previous studies except those of Hanson (1958,1963,1975,1977) is a lack of the building of one study upon another. It is as if each believes that only they have the 'true vision' and that all others are so misguided that no part of their theories has the slightest validity or usefulness. Only in the work of Willmer (1990), which, in the spirit of Hyman (1940-1967), is more a compendium of previously published information, is each theory evaluated in a fair and largely unbiased manner. Those using phylogenetic methodology are no different in that some believe they have come closest to revealing the 'true' phylogeny, but they differ in that each builds somewhat on the data used by previous authors. Thus, there is a continuity in procedure not seen in other work, yet this is likely due to a common methodology rather than a spirit of collegiality. The five studies discussed here (Schram,1991; Megiitxh and Sduam, 1991b; Eemisse, et al., 1992; Backeljau, et al., 1993; Nielsen, et al., 1996; Schram and Ellis, 1994) do not actually fit in a common group except that they employ phylogenetic methods (Hennig, 1950,1966) not used in any previous studies of evolutionary relationships of metazoans. The intent of each, as expressed by Sham (1991), was to first carry out the analysis and then propose an evolutionary narrative about the Metazoa-the opposite procedure used in all previous works. The data sets of Schram (1991), Backeljau, et al. 1993), and Schrarn and Ellis (1994) are identical (except for typographical errors), so their differences in results are primarily due to differences in procedure. The study by Nielsen, et al., (1996), a phylogenetic analysis and test of a previous arrangement of Nielsen (1995), involved a different data set, so only comparison of hypothesized relationships of higher taxa will be made here; detailed comparison of results will be made elsewhere (pg. 28). The first cladograrn, sensu strictu, for the Metazoa at the phylum level is that of Jefferies (1986). This was the first study to provide an explicit data set and precise information on character states. The focus of Jefferies (1986) was on vertebrate origin, so not all phyla were included as individual groups, and several of the characters that he used were overly 'generalized' for application at this level of analysis. For example, he used the presence of an anus to characterize all groups above flatworms, a character that has been demonstrated with some conviction to have been developed independently several times. Yet, interestingly, his simplified tree (Fig. 10) is not that different from some of those hypothesized earlier. Whether this is due to a ~rioriassumptions favoring this arrangement or some other factor cannot be examined here because he did not include explicit details concerning derivation of his tree. Jefferies' (1986) tree has largely been ignored by those studying invertebrate systematics, but as the first of its kind it deserves mention here. This study also signaled a return to Darwin's (1859) ideal of a classification based on descent. The first published attempt to make a rigorous study of metazoans was that of Schram (1991). The work was published simultaneously in virtually the same form in a volume discussing paleontology (Schram, 1991) and as a chapter in an invertebrate text (Meglitsch and Schram, 1991b). Schram (1991) included 32 of 37 phyla and provided an explicit data matrix containing 77 characters. The greatest shortcoming of this study is that in neither publication did Schram provide full details of his analytical methods, nor information on the number of trees resulting from the analysis. He also presented only a single tree (Fig. 11-12), presumably chosen arbitrarily from a larger group of equally parsimonious trees. He has been extensively criticized for this (Backeljau, et al., 1993), but the criticism is unjustified given that the analysis was likely performed a year or more previous to the publication date, before methodology was as rigorous as at the time of Backeljau, et al. (1993). Also, as stated by the author (Meglitsch and Schram, 1991b), the intent of the original analysis was to present students with a "...first approximation toward an understanding of invertebrate relationships, a hypothesis, if you will, that can and should be tested with further observations and data." (Meglitsch and Schram, 1991b; 598). In their critique, Backeljau, et al. (1993) claimed to have found "...at least seven characters with debatable or erroneous character states..." (Backeljau, et al., 1993; l7O), which they corrected and then reanalyzed the data set. Their analysis produced 2444 equally parsimonious trees, of which they presented a strict consensus tree (Fig. 13). The authors made much of their results, stating that "...many of Sduam's (1991) conclusions about metazoan phylogeny are not warranted" (Backeljau, et al., 1993; 173), but they offered nothing better in its place. Furthermore, it is clear that they had no interest in the use of morphological data, stating that their "...adapted data matrix still contains several doubtful homologies and debatable states..." (Backeljau, et al., 1993; 174). It appears that they only used Meglitsch and Muam, (1991b) as a 'straw man' to knock down as an introduction to future studies because they went on to state "...further testing [with] independent data sets is needed. Small-subunit ribosomal RNA sequences, for example, are such data." (Backeljau, et al., 1993; 175),a direction which the majority of those authors have pursued in all subsequent research (for example, see Winnepenninckx and Backeljau, 1996; Winnepenninckx, et al., 1995). Scluam's (Meglitxh and Schram, 1991b; Schram, 1991) data set was also re-analyzed by Eernisse, et al. (1992) as part of an attempt to resolve questions concerning the ancestry of the Arthropods. Eemisse, et al. (1992) reported no errors in the original data set. They further extended the data set to include all 37 phyla, and their subsequent analyses produced 1422 equally parsimonious trees. In addition, they approximately doubled the size of the data matrix by adding 64 characters, but only included 26 taxa in the expanded data set in order to narrow the focus of the study. Schram and Ellis (1994) later corrected a single mistake in the original data matrix, which they attributed to a typographical error by the printer, and reaffirmed all remaining characters questioned by Backeljau, et al. (1993). They then re-analyzed the data, and produced one of the best-resolved hypotheses of relationships among multicellular animals published to date (Fig. 14). They conceded that the tree in the original studies was arbitrarily chosen from several equally parsimonious trees, but that it was only intended to serve "...as a pedagogical device to show students how this kind of analysis [could] be done ..." (Schram and Ellis, 1994; 331) The character matrix, made up of 77 characters, was not extensive enough to resolve all relationships, but several of the unresolved groups are likely a product of a coding bias related to the pseudocoelomate vs. coelomate controversy and a misinterpretation of cleavage patterns, both of which will be discussed below (Chapter 3). The most recent phylogenetic analysis is that of Nielsen, et al. (1996), which included 32 of the 37 then-known phyla. The data set was drawn from an earlier work by Nielsen (1995) which consisted of 61 characters. The analysis produced 306 equally parsimonious trees. The consensus of these trees, not given in the original study, is shown in Fig. 15. Of these 306 trees (a consensus shown in Fig. 16), they arbitrarily chose 116, which they subjected to successive weighting (Goloboff, 1993, 1994a, 1994b). By reweighting the characters according to maximum homoplasy they were able to reduce the number to four equally parsimonious trees, of which they arbitrarily chose one (Fig. 17). This tree differed from the 'intuitive' tree of Nielsen (1995) (Fig. 18) produced by what Nielsen, et al. (1996) called a "...'manual1 cladistic analysis..." (quotes in original: Nielsen, et al., 1996; 385), although most of the differences were the result of a lack of resolution in the more recent study. The results of each phylogenetic analysis discussed here, except for that of Eernisse, et al. (1992), are largely in agreement with previous non-phylogenetic hypotheses. This does not come as a surprise if one examines the data sets closely. All include many characters that are interpreted in exactly the same manner as in previous non-phylogenetic studies. For example, the character 'pseudocoelom: absent/presentWis used in each data set, even though it is well known that the pseudocoelom is produced in several different ways, suggesting that a broad definition is not appropriate (Bmsca and Bmsca, 1990; Meglitsch and Schram, 1991a). A similar coding is used for the coelom, not only known to be homoplasious in a general sense, but also known to be derived in several different ways. These, as well as many similarly vague characters, contribute a high degree of homoplasy to each study, and produce a large number of equally parsimonious trees. The consensus trees of each study cannot be directly compared with those of previous evolutionary studies because it cannot be determined if they are similar to earlier hypotheses because the results are actually the same, or if the lack of difference is due to the lack of resolution in the consensus trees making them only $ern consistent. Detailed comparisons of the hypotheses from each study will be made later in relation to the results of the current phylogenetic study.
Theories that might form a fifth 'null'category will not be discussed here. These all share a common view that metazoans are polyphyletic, but Little else. Some of the authors prefer one or the other of the hypothesized evolutionary arrangements, but believe that the characteristics of higher taxa, such as cleavage pattern (Greenberg, 1959), reflect origins from different ancestral groups. Several (Grimstone, 1959; Nursall, 1959; Nursall, 1962) object to a monophyletic interpretation of metazoan evolution because of a lack of confidence in putative homology of characters. For others, such as Willrner (1990), polyphyly of the Metazoa is, in the absence of a single compelling hypothesis, the result of a consensus of all previous theories (Fig. 19). Because each, based on a perceived lack of compelling evidence, opts for the null hypothesis of 'no known relationships1, they will not be discussed further here.
Conclusions
From the previous discussion, it is apparent that none of the hypotheses presented here have much to recommend them. All depend upon an a posteriori fitting of characters to phylogenies that were based on theories of evolutionary processes rather than the use of characters to build phylogenies, which in turn provide information about evolutionary processes. This brief discussion of the implications of the various perspectives is suffiaent to demonstrate the importance of 3 priori assumptions of character value and character evolution in 'steering' the final hypotheses (for a more extensive review see Dougherty, 1963, Willmer, 1990, and Willmer and Holland, 1991). This observation is especially true if one allows the possibility of a polyphyletic origin of multicellular animals. Although questions of homology at the phylum level cannot be resolved merely by using phylogenetic methods, there must at least be a frame of reference in which primary homology statements are testable (Brooks and McLennan, 1991; de Pinna, 1991; Hawkins, et al., 1997; Hennig, 1966; Wiley, 1981). Recent studies using phylogenetic methodology are a step forward in the right direction. Invertebrate Animals Vertebrate Animals I. Infusorians XI. Fishes n. polyps W. Reptiles III. Radiarians XIII. Birds IV. Worms XIV. Mammals V. Insects VI. Arachnids VII. Crustaceans VUI. Annelids IX. Cirrhipedes X. Molluscs A
Unguiculate Mammals Ungulate Mammals
Cetacean Mammals
Mono tremes
I Amphibian Mammals
Fishes Reptiles
Annelids Cirrhipedes Insects Molluscs Arachnids
Polyps Radiarians
Figure 1. Classification and phylogeny of the Metazoa according to Lamarck, 1809. A. Arrangement of metazoans that Lamarck considered to be most in conformity with the order of nature. B. Lamarck's 'tree of life' depicting the origin of various metazoans (after Lamarck, 1809; 179). Ancestoral stages hollow, flagellated, hypothetical hypothetical green algal colony blastaea gastraea
blastula invaginated gastrula Developmental stages
Figure 2. Evolutionary stages suggested by Haeckel(1866) to correspond with developmental stages. Figure 3. Tree depicting relationships among the Metazoa as suggested by Haeckel, 1866 (Modified from Plate 1 horn Volume 2, Generelle Mor~hol~). nCnidaria Coelomata
Figure 4. Tree depicting relationships among the Metazoa as suggested by Jagersten, 1955. Articulates
Notoneuralia (Chordata)
Spiralian Coelomates Lophophorata
Deuteros/tomia
I Blas tea
Figure 5. Tree depicting relationships among the Metazoa as suggested Siewing, 1976. Arthropoda Annel ida Mollusca Hernichordata Other Echiuroidea Schizocoela
Nemertina Trochophore Platyhelmintt Diplerula - Chaetognatha Aschelminthe: -. Primitive acoel flatworms
Ctenophora Cnidaria
Primitive medusae
Mesozoa
Planula ? Other Pori f era
\ I /rOtOzOa Flagellata
Figure 6. Tree depicting relationships among the Metazoa as suggested by Hyman, 1940. No toneuralia (Chordates)
Heteroneuralia (Trimeric groups)
Coeloma te Gastroneura
Acoeloma te Gastroneura
Trip loblas tica
Diploblastica
Figure 7. Tree depicting relationships among the Metazoa as suggested by Salvini-Plawen, 1978. Chordata Urochordata
Pterobrancha 1 / 1 Echinoderrnata
I Ectoprocta Brachiopoda Phoronida Ameiida
Echiura
I Mollusca
Asche lminthes I Turbellaria
I Cnidaria Acoeloid Ciliata
II/ ,Other Protozoa Porifera '~la~ellata
Figure 8. Tree depicting relationships among the Metazoa as suggested by Hadzi, 1963. Higher Metazoa
Higher Turbellaria
Acoela
Acoeloid . Prociliata Pats \ ,other protoma \ Flagellata
Figure 9. Tree depicting relationships among the Metazoa as suggested by Steinbock, 1963. Deu teros tomes
Nemerteans *. radial cleavage, trimerism PseudocoeIomates
Platyhelminthes
Coelenterates
** spiral cleavage, mesoderm, bilaterality ** mouth, nerves, diploblasty
multicellularity
Figure 10. Tree depicting relationships among the Metazoa as suggested by Jefferies, 1986. 1 Placozoa
Porifera Cnidaria Ctenophora Onychophora Uniramia Chelicerata Tardigrada Crustacea Pentastomida Pogonophora Annelida Sipuncula Echiura Mollusca Nemertea Entoprocta Ectoprocta Phoronida Brachiopoda Pterobranchia Echinodermata En teropneusta Urochordata Cephalochordata Gnathostomulida Platyhelminthes Chaetognatha Nematoda Nematomorpha Acanthocephala Rotifera Gastrotricha Priapulida Loricifera Kinorhyncha
Figu .re 11.. Tree depicting relationships among the Metazoa irs suggested by Schram, 1,991. Cnidaria Nematoda Priapulida Kinorhyncha Acoelomorp ha Rhabditophora Gnathostomulida Chordata Echinodennata Brachiopoda Phoronida Nemertea Sipuncula Echiura Caudofoveata Solenogastres Polyplacophora
Conchifera Polychaeta Clitellata Pogonophora Crustacea Tardigrada Chelicerata Onychophora Uniramia
Figure 12. Tree depicting relationships among the Metazoa as suggested by Meglitsch and Schram, 1991a, b. ata
na
Figure 13 Tree depicting relationsMps among the Metazoa as suggested Backeljau et al., 1993. Placozoa Porifera Cnidaria Ctenophora Chaetognatha Nematoda Nematomorpha Acanthocephala Rotifera Gas trotricha Priapulida Loricifera Kinorhyncha Gnathos tomulida Pla tyhelminthes Onychophora Uniramia Chelicerata Tardigrada Crustacea Sipuncula Echiura Mollusca Amelida Pogonophora Nemertea Entoprocta Ectoprocta Phoronida Brachiopoda Pterobranchia Echinoderrnata Enteropheusta Urochordata Cephalachordata
Figure 14. Tree depicting relationships among the Metazoa as suggested by Schrarn and Ellis, 1994. Choanoflagellata Porifera Placozoa Cnidaria Ctenophora Sipunda Mollusca Annelid a Onychophora Arthropoda Tardigrada Platyhelminthes Nemertini Entoprocta Ectoprocta Gastrotricha Nematoda Nema tomorpha Priapula Kinorhyncha Loricifera Chaetognatha Rotifera
Acanthocephala ' Phoronida Brachiopoda Pterobranchia Echinoderms ta En teropneus ta Urochordata Cephalochordata Vertebrata gure 15. Tree depicting relationships among the Metazoa as suggested by Nielsen al., 1996. Frist consensus tree. Choanoflagellata Porifera Placozoa Cnidaria Ctenophora Sipuncula Mollusca Annelida Onychophora Arthropoda Tardigrada Platyhelminthes Nemertini Entoprocta Ectoprocta Gastrotricha Nematoda Nematomorpha Priapula Kinorhyncha Loricifera Chaetognatha Rotifera Acan thocephala Phoronida Brachiopoda Pterobranchia Echinoderms ta Enteropneusta Urochordata Cephalochordata Vertebrata
Figure 16. Tree depicting relationships among the Metazoa as suggested by Nielsen et al., 1996. Second consensus tree.
I Choanoflagellata
I Porifera Placozoa Cnidaria Sipuncula Mollusca Annelida Onychophora Arthropoda Tardigrada En t op roc ta Ectoprocta Platyhelminthes Nernertini Rotifera Acan thocep hala Chaetognatha Gastrotricha Nematoda Nematomorpha Priapula Kinorhyncha Loricifera C tenophora Phoronida Brachiopoda Pterobranchia Echinodermata Enteropneusta Urochordata Cephalochordata Vertebrata
Figure 18. Tree depicting relationships among the Metazoa as suggested by Nielsen, 1995. Chordata
Echinodermata
Cnidaria Gastrotricha
Figure 19. Tree depicting relationships among the Metazoa as suggested by Willmer, 1990. Chapter 3: Phylogeny of the Metazoa based on morphological characters
In the previous chapter, it was shown that various systematists had produced phylogenetic taxonomic groupings with more of less predictive power depending upon their a ~rioriassumptions of the origin of the Metazoa and character evolution. No method was then known with which to recover phylogenetic information, so early workers did not have the 'tools' to produce formally-testable hypotheses in the manner used today. Despite this problem, these systematists made great advances in the knowledge of morphology of invertebrate groups. Now that a phylogenetic method is available, the extensive knowledge of morphology which they amassed is available for use in cladistic analyses of metazoan relationships. Yet, comparatively few investigators are studying relationships at the major-taxon level. Of those, only a small number (Backeljau, et al., 1993; Meglitsch and Schram, 1991b; Nielsen, et al., 1996; Schram, 1991; !%hamand Ellis, 1994) have attempted to include all metazoan phyla. Each of the cladistic studies discussed in Chapter 2 used data sets that were insufficient to resolve all relationships among the phyla included in their study. Of studies at the phylum level, the most inclusive in relation to number of characters was that of Eernisse, et al. (1992). The focus of their study was to test the hypothesized sister-group relationship of annelids and arthropods. This view was largely based on the presence of segmentation in each phylum and a perceived similarity in the body 'plans' of the two groups (see Brusca and Brusca (1990), Meglitsch and Sdvam 1991a, and Eemisse, et al., 1992 for a full discussion). The study by Eemisse, et al. (1992) included 26 taxa, 19 of which were considered to be phyla. Their characters (141) were drawn from published data sets including larval characters from the early work of Nielsen (Nielsen, 1985; Nielsen, 1987). Their treatment of characters is problematic because all were accepted uncritically as given in the previous studies, and many were phrased in an evolutionary rather than phylogenetic context. Some of these, such as the characters coding for cleavage pattern and body cavities comprise character 'suites' made up of several independent features rather than a single character (see Table I). These character 'suites' had been assigned to higher taxa without determining whether all features of the 'suite' were possessed by all members of the taxon. Despite this, analysis of these characters by Eernisse, et al. (1992) produced six equally parsimonious hypotheses for the relationships of included taxa. A strict consensus of these resolved all but three clades, one of which contained taxa considered to make up the Arthropods (Fig. 20). Much of the phylogeny of Nielsen, et al. (1996) is built upon Nielsen's (1979, 1985,1987; Nielsen and Nerrevang, 1985) earlier works on land development and an earlier treatise (Nielsen, 1995) on metazoan relationships viewed from an evolutionary perspective. The purpose of Nielsen, et al. (1996) was to "...present a quantitative cladistic analysis of the data in Nielsen (1995)" (Nielsen, et al., 1996; 386). Nielsen, et al. (1996) used only 61 characters in their analyses, and did not include many of the characters discussed earlier by Nielsen (1995). Their (Nielsen, et al., 1996) justification was that inclusion of those additional characters would have "...posed significant problems of interpretation, introducing either homoplasy...or biased interpretations..." (Nielsen, et al., 1996; 393). The (Nielsen, et al., 1996) analyses produced 306 equally parsimonious trees, which they reduced to 116 trees by removing all those which contained polytomies that were resolved in other trees (a method not tested by empirical studies). Using successive weighting of the 116 trees, they further reduced the number to four compatible trees, of which they discarded three because they contained less resolution than the fourth (Fig. 17). Nielser,, et al. (1996) concluded that the differences between their tree and Nielsen's 1995 tree (Nielsen, 1995) were "...not great but do point towards the need for greater knowledge in certain key areas" (Nielsen, et al., 1996; 396). The two most obvious differences between the trees of the two studies (Fig. 21) were that (1.)the 1996 tree 52
(Nielsen, et al., 1996) fully resolved all relationships and that (2.) it failed to support some of the higher taxa of the earlier study. Of all previous phylogenetic studies, only Nielsen, et al. (1996) have suggested a classification system for the Metazoa based on the results (Appendix I). Finally, as discussed in the historical review of the Metazoa, evolutionary hypotheses of metazoan relationships have some unique details, but regardless of these details, they share several common features. First, although many include all phyla in their discussion, each hypothesis focuses on only a few of the many available characters. Although they often recognized the priori assumptions of competing hypotheses, instead of attempting to remove these biases to make characters from previous studies more useful, many past investigators have dismissed other's work outright and selected their own characters, with concomitant personal biases. One aim of the work presented here was to use data from as many previous studies as possible, in a manner consistent with the homology criteria mentioned in the introductory chapter (pg. 1) and the historical review (pg. 16,29), and discussed more fully below (pg. 52-53,57-58). To accomplish this, available characters were drawn from original descriptions, phylogenetic studies of more restricted groups, and secondary sources, as well as examination of specimens of selected taxa. Once these data were gathered, as many taxa as practical (given our comparative lack of knowledge of some phyla) were included in the analysis in order to provide a broad base for examination of character evolution within the Metazoa. Most investigators have approached metazoan phylogeny with a ~riori assumptions about hypothetical ancestors and how the genealogy must have been based on an assumed developmental sequence pattern. A second aim of this study was therefore to minimize biases from g priori assumptions of character evolution by viewing primary homology assumptions from a strict non-phylogenetic perspective (Brooks and McLennan, 1991; 24-26). The recognition of similarity, as 53
noted by de Pinna (1991), is "...an essentially imprecise and subjective process" (de Pinna, 1991; 377), so if features appeared 'similar' they were assumed to be homologous (i.e. assumed to have primary homology sensu de Pinna, 1991). When available, information on comparative development and embryonic source of structures was used, but, when necessary, character transformations were recast without regard for 'primitive' or 'advanced' features (- Mayr, 1969), or the commonly assumed importance of particular developmental characters. In this manner, the weaknesses of previous studies because of their acceptance of biased character interpretations could be avoided. The third goal of this study was to produce a hypothesis of phylogenetic relationships among multicellular animals using morphological characters. The analyses were carried out using rigorous phylogenetic methods to provide hypotheses of relationships based on the information provided by the data set rather than using the characters to support a preconceived genealogy. Because these hypotheses are independent of known pre-analysis biases, they provide a sound foundation upon which to establish hypotheses about the evolution of the Metazoa. Reference to the results of the phylogenetic analyses provided a test of assumptions of primary homology4aracters that were not congruent with the phylogenetic pattern were deemed homoplasies. By evaluating characters independently of ideas of origin and evolution of metazoan taxa, the current analysis provides a preliminary phylogenetic test of primary character homology on which to base future studies. It also provides an objective viewpoint for a posteriori interpretation of character evolution. A primary need for a phylogenetic hypothesis of relationships of the Metazoa was to provide information on which to select outgroups for a phylogenetic analysis of the Acanthocephala. The relationships supported by this analysis are used to support the choice of priapulids and rotiferans as outgroups for the analysis of relationships among acanthocephalans. It is also another step in providing a phylogenetic basis for a classification scheme for multicellular animals based on descent, in accord with Darwin's (1859) ideas.
Methodology Taxa
The Choanoflagellata is commonly held to be the sister-group to multicellular animals (Nielsen, 1995), although altemative groups, such as the ciliate protists discussed in the chapter above, have been suggested. Comparative data is most comprehensive for this group, and, since no generally accepted morphology-based p hylogenetic hypo thesis for unicellular organisms exists, the Choanoflagellata was used as the single outgroup for the present analysis. Use of the choanoflagellates alone as an outgroup probably provided polarization of most characters consistent with that from any altemative outgroup (Willrner, 1990; Willmer and Holland, 1991). However, the use of a single outgroup did not allow testing of hypotheses concerning monophyly of multicellular animals. An attempt was made to use ciliate protozoa as a second outgroup, but because they did not share any of the five characters which were used to test relationships at the above-phylum level (i.e., MC1, MC2, MC3, MC4, and MCS), their inclusion did not provide any benefit in character polarization. In addition, a complete phylogenetic hypothesis for the ciliate assemblage based on morphological characters does not exist, so there was not way to choose additional characters to include in the analyses nor to determine the piesiomorphic condition of any character found in the group. Future analyses should include additional outgroups, especially the ciliate protozoa, which Hadzi, at least, thought were more closely related to the Porifera, Cnidaria, and Ctenophora (Hadzi, 1953,1963; Hanson, 1963, 1977). However, appropriate protist groups cannot be identified until relationships among protozoan groups have been clarified. The actual number of phyla depends entirely upon one's interpretation of what each phylum comprises. Brusca and Bmxa (1990) recognize 36 animal phyla (Appendix I1 A), four mesozoan and 32 metazoan phyla, but various other authors divide the Molluua into four phyla, the Arthropoda. into three, the Annelids, into two, and the Chordata, into three phyla. The number of recognized phyla is also dependent upon acceptance of a particular evolutionary scenario and resultant classification. Only Nielsen's (1995) scheme (Appendix I) has a cladistic basis (according to his claim that he performed a 'manual'analysis), but he was arbitrary in designation and inclusion of tnxa, although he did restrict his definitions to rnonophyletic groups. Although Meglitsch and Schram (1991a) discussed consideration of particular taxa as phyla, neither they, nor any of the others using cladistic methodology (Backeljau, et al., 1993; Eernisse, et al., 1992; Nielsen, et al., 1996; Schram, 1991; Schram and Ellis, 1994) have proposed a classification based on their results. Nielsen, et al. (1996) did not propose modification of the classification arrangement or designation of phyla by Nielsen (1995), even though their results were not in complete agreement with his classification. Systematists have always been concerned with designating taxa of equal rank as members of categories of equal taxonomic rank (Agassiz, 1962), yet the intermingling of partial classifications by systematists using different methodology has completely confounded rank and category positions in current classifications. It may be arguable whether, for example, the Arthropoda (as defined by Nielsen, 1995) is a phylum, or the component groups (the Uniramia, Crustacea, Chelicerata, and Pentas tomida) are phyla. Because categories and ranks of taxa in current systems are largely based on incongruent evolutionary scenarios, no defense of appropriateness of rank of the taxa included in this study is offered here, and should not be until each group has been tested for monophyly. Whether terminal taxa are considered to be phyla is not important to this analysis, but for convenience, all will be referred to as such. In initial phases of the analysis, 40 phyla were included, but there was sufficient information to include only 33 multicellular groups (Appendix II B) in the final analyses. One objective was to clarify the relationship between the Acanthocephala and potential sister groups for use in an ongoing study of the phylogenetic relationships of acanthocephalans. Exclusion of the seven taxa did not compromise this aspect of the analyses. An posteriori secondary analysis was performed to further investigate the position of the Priapulida in relation to the ecdysozoan (Aguinaldo, et al., 1997) dade because in two of the 17 original trees (Fig. 22) the Priapulida was placed intermediately within the clade. For the secondary analysis, taxa were arbitrarily deleted leaving only 20 phyla (Appendix 11 C). The data for the reduced number of taxa were analyzed in the same manner as that of the primary analysis. For ease of reference to particular groups of taxa the following non- phylogenetic conventions were observed for common names without specdying the exact taxa within each group. Deuterostomes are those taxa commonly grouped with the Chordata and protostomes are taxa commonly grouped with the Platyhelminthes. The Ecdysozoa (Aguinaldo, et al., 1997) are taxa typically referred to as the 'pseudocoelomatest,all of which molt during their life cycle. These terms are used for convenience and their use does not indicate an acceptance of classical evolutionary designations of membership within or status of groups, or a formal name for groups. In a similar manner, the terms 'higher', suggesting greater complexity (Bell and Mooers, 1997), and 'lower', suggesting lesser complexity, is used in quotes, and does not imply either an advanced or primitive condition (sensu Mayr, 1969), although 'higher' often is correlated with a more terminal (vs. basal) position on a tree. Character and character state coding
Characters used in the analysis were extracted from primary and secondary literature on morphology and development of multicelldar animals. This material yielded 144 characters that could be phrased in a testable phylogenetic format. Character assignment for the Choanoflagellata was taken from Brusca and Brusca (1990), Meglitsch and Schram (1991a), Willmer (1990), and Nielsen (1995). The source of initial ideas for some characters was Eernisse, et al. (1992). The characters used by Eemisse, et al. (1992) had been taken from earlier studies without critical assessment of either primary homology or character assignment. Because characters had been accepted uncritically by these authors, were applied to only a few phyla, and some were not phrased in a phylogenetic context, each character was re-examined and cross-checked with the original source. Characters assignment was re-evaluated for each phylum and characters were broken down into single features when practical. Characters and citations for the major sources for character information is provided, when appropriate, in the character list given in Appendix III. The morphological categories of Eernisse, et al. (1992) were used to group characters for descriptive purpose. Similarly, character numbers of Eemisse, et al. (1992) were retained as part of the character names for ease of reference to that source (see Appendix HI). The complete data matrix is given in Appendix IV. Characters of Eernisse, et al. (1992) that were excluded from the analyses because they not phrased in a Hennigian format were retained in the matrix for reference to the original work. Characters for which more than one state was reported in different representatives of terminal taxa were scored as polymorphic. Character information was screened for primary homology (sensu de Pinna, 1991) based on phenotypic similarity. Primary homology of some structures is difficult to assess at the phylum level (see Willmer 1990 and Willrner and Holland Willmer and Holland, 1991), so primary homology of characters based on general similarity was accepted unless there were overriding reasons to do otherwise. Conflicting opinions on suitability of some features for phylogenetic analysis have been expressed in previous studies. Too liberal acceptance of these characters has likely increased the number of homoplasies, which will be discwed later. However, this was deemed more appropriate than outright rejection of these characters. In some cases, it was necessary to accept previous declarations of homology because reference to original works was not provided by authors. This, again, probably increased the number of homoplasies, but was considered more advantageous than rejection. The original concept of characters from Eemisse, et al. (1992) was used to maintain continuity with the previous work, but each transformation series was evaluated for independence from other characters. Whenever possible, characters were coded in binary form. Character suites, a 'suite' of features considered to have evolved as a single trait, are problematic because the entire set of features is not always found in all members of higher taxa. As well, assignment to a higher taxon is often based solely on the supposed possession of a given suite, often without every member of a phylum being assessed for all features of the character suite. Until now, none of these character suites have been tested in a phylogenetic context, and none have been broken down into their component features to evaluate the their status as co-inherited units. One such character suite is early cleavage pattern, commonly referred to as 'longitudinal', 'radial', 'spiral', and 'modified spiral', which has been hypothesized to have evolved in the form depicted in Figure 23. Each cleavage type is established by the first three cell cleavages. The suitability of combining the first three cleavages into a single character suite was specifically tested. To break each pattern down into its component parts, cleavage steps were coded individually according to the plane of cleavage (Fig. 24). To avoid confusion with previous designation, resultant cleavage patterns are termed meridional, equatorial, oblique, and multi-plane, respectively. In this entirely new approach, the steps in the cleavage sequence were coded as unordered transformation series, as shown in Table I. TABLEI. Coding of the first three cleavages of the zygote.
Characters Separation of First cell Second cell Third cell daughter cells division division division (CLEI) (CLE2) (CLW (unordered) (unordered) (unordered) (unordered)
0 = Complete 0 = Meridional 0 = Meridional 0 = Meridional separation of cleavage cleavage cleavage daughter cells
1 = Daughter cells 1 = Cleavage 1 = Multi-plane 1 = Equatorial not spearated plane from cleavage cleavage animal to vegetal poles
2 = Oblique cleavage
cleavage Analytical procedures
Phylogenetic analysis was performed using the computer program Paup 3.1.1 (Swofford, 1993) and Hennig86 (Farris, 1988). Initial heuristic search procedures were carried out using Paup 3.1.1 (Swofford, 1993) under the options "addition sequence simple" with 'TBR branch-swapping, MULPARS" option in effect, and by collapsing zero-length branches. The branch-and-bound search algorithm was used for all searches, but it did not find any trees not found by a heuristic search algorithm. Because of the large sue of the data matrix, initial Branch and Bound searches took 40-48 hours of computer (Macintosh LC475) time to complete. Subsequent Branch and Bound searches were made using Hennig86 with the commands kc-; mh bb;" as an independent check the results of the Paup 3.1.1 analysis. Characters were initially run unordered and equally weighted (weight = 1). One purpose of the analysis was to identify possible sister groups to the Acanthocephala for use in a phylogenetic analysis of members of that phylum. Because, as discussed above, a large proportion of the characters were expected to be homoplasious, and the main goal was resolution of ecdysozoan taxa, successive weighting (Farris, 1969) of characters was used to enhance the phylogenetic 'signal' of homologous characters. The use of successive weighting has been discussed extensively in the literature (Carpenter, 1994,1993; Simon, et al., 1994; Swofford, et al., 1996). When a single tree is necessary for use in a subsequent analysis, successive weighting schemes result in trees that best explain the data and produce hypotheses of sister-group relationships that have the most predictive power (Farris, 1969,1979, 1982). Willmer (1990) and Nielsen (1995), discussed current information on the characters of metazoans which contain a large amount of phylogenetic information. However, they suggested that this information is 'masked' by the many homoplasious characters that result from improper understanding of homologous structures, development, and morphology. This is especially true in littlestudied enigmatic groups such as the Acanthocephala, Kinorhyncha, and Gastrotricha. Since homologous characters co-vary and homoplasious ones are not expected to co-vary (Hennig, 1966), successive weighting based on homology was deemed appropriate for these analyses. A scheme using parsimony methods such as that of Goloboff (1993) was preferred over one that was strictly probabilistic, such as that of Sharkey (1989) or Wilkinson (1994). The first step in the analysis was performed with all characters assigned a weight of one, and in the second step of the analysis, the "reweight characters" option in Paup was used to reassign weights to characters based on their maximum homology in the group of 17 equally parsimonious trees (Fig. 22) produced by the anaiysis. Using this algorithm, weights were assigned according to the retention index and based on the weight of 10 for maximum homology. Characters were down-weighted from the initial weight, based on their lack of fit to the 17 trees produced in the initial analysis. The characters were then re-analyzed using the assigned weights given in Appendix V, A. The selection of the initial weight (10) was made arbitrarily because it was the largest number allowed by the computer program Hennig86 (Farris, 1988), which was used to verify the results produced by Paup 3.1.1 (Swofford, 1993). Alternative weighting schemes and values have been advanced (Carpenter, 1994; Sharkey, 1989), but that of Goloboff (1993) was chosen before the analysis based on his and Carpenter's (1994) arguments that it was the most parsimonious. Goloboff (1993) and Carpenter (1994) argued that, by evaluating characters according to homoplasy, rather than perceived importance, successive weighting implied characters that were maximally reliable. Although, as mentioned above, the initial weight of 10 was chosen because the value was appropriate for use in another program, Goloboff suggested that this value provided coarse weight differences that insured "...onIy relatively important differences in homoplasy will cause a difference in weight" (Goloboff, 1993; 90). Selection of weighting method and character weight
was chosen before the analysis, and not changed 9 posteriori.
Results of the analyses
The primary analysis of the relationships of 34 taxa using 144 morphological and biochemical characters yielded 17 equally parsimonious trees (Fig. 22), each 361 steps long; consistency index (CI)= 0.460; consistency index, excluding uninformative characters (CIX) = 0.405; homoplasy index (HI) = 0.582; homoplasy index, excluding uninformative characters (HR)= 0.621; retention index (N)= 0.689, and rescaled consistency index (RC) = 0.317 (see Appendix V, B). After re-weighting the characters, only two of the 17 trees were retained, differing in the placement of the Platyhelminthes (Fig. 25). On one tree the Platyhelminthes are placed basal to the Nemertea (Fig. 25A), and on the other, they are the sister group to the Mollusca (Fig. 25B). The 10 characters responsible (Chars. 7,17,39,40,44,92, 104,105,112, and 116) for this difference in placement are shown in Appendix V, C. In both trees, the Acanthocephala was the most basal member of a clade, the Ecdysozoa (Aguinaldo, et al., 1997), composed of the (Rotifera, (Gastrotricha, (Nematoda, Nematomorpha), (Pentastomida, (Kinorhynchida, (Tardigrada, (Onychophora, (Chelicerata, (Unirarnia, Crustacea))))))))). The strict consensus of the two final trees is depicted in Figure 26. The secondary analysis was performed to clarify the position of the Priapulida in relation to the ecdysozoan clade. This was necessary because in two of the 17 original trees (Fig. 22K,P) the Priapulida was placed above the Acanthocephala, Rotifera, and Gastrotricha and basal to the (Nematoda + Nematomorpha) + (Pentastomida + (Kinorhynchida + (Tardigrada + (Onycho p ho ra + (Chelicerata, (Unirarnia + Crustacea)) ))))) . The secondary analysis yielded a single tree that was 199 steps long, CI = 0.618, CIX = 0.549, HI = 0-410, HIX = 0.465, RI = 0.756, and RC = 0.467. In this tree (Fig. 27), the Priapulida was placed basal to the Ecdysozoa, and the (Rotifera + Gastrotricha) was moved basal to the Acanthocephala. Relationships among all other taxa remained the same as in the primary analyses (Fig. 26).
Discussion and comparison with previous studies Status of taxa
One goal of this study was to provide a phylogenetic hypothesis for all multicellular animals. Sufficient information could be gathered to include only 33 multicellular groups; three mesozoan, and 30 metazoan phyla (Appendix II, A). Information was not available for several recognized phyla, so they were excluded from the analysis. Lndusion of these taxa, (see Appendix II, C for excluded taxa) especially the Ectoprocta, Entoprocta, and the Loricifera, could have yielded interesting hypotheses, but the amount of information concerning their morphology is hopelessly inadequate. Although not included in the formal results of this study, analysis of a greatly reduced number of taxa did suggest probable placement of the Ectoprocta, Enteropneusta, Urochordata, Cephalochordata and Pterobranchia within the chordate clade, Entoprocta within the protostome clade, and the Loricifera in the ecdysozoan clade somewhere 'above' the Nematoda + Nematomorpha branch. Exact placement, of course, will not be possible until additional information is available for these enigmatic taxa. Relationships among taxa 'Lower' phyla
That multicelldar animals are monophyletic is very widely accepted. Under this assumption, and that extant choanoflagellates have not secondarily lost any of the characters used in the analysis, monophyly of muiticellular animals is supported by 16 unambiguous characters (0~11,0G21,0~3~,0~41,0~7~,0~111,0~121,
CEL~~,CEW~, cIL,ll, 301, 311,321,361, 391,and 100~)( see Fig. 26). This deserves further testing by inclusion of at least the ciliates as an outgroup, but, from the results of this analysis, no other conclusion is supported by the available data. Contrary to current arrangements, Dicverna. Trirho~lax,and Rho~hra, representing the Mesozoa, do not form a monophyletic group. It has been suggested that the orthonectids (Rho~lura)arose from the Echiura (Lameere, 1922) or from the Platyhelminthes (Stunkard, 1954,1972,1982), but neither relationship is supported by this analysis. The three phyla are placed basal to the Metazoa indicating that they are not degenerate forms of one of the other phyla. The relationship Choanoflagellata + (Dicyemida + (Rhoplura + (Trichoplax + (Metazoa))))is supported in all 17 original trees (Fig. 22). Rho~lurahas not been included in any previous analysis, but Dicvemida and Trichoplax were included by Schram (1991) and Schram and Ellis (1994). In both studies, Dicyemida and Trichoplax were sister-groups, and in Schram and Ellis (1994), the Porifera was placed basal, as sister-group to the two mesozoan phyla. As noted above, neither of the three mesozoans cluster together in the results of the current analyses and all are placed basal to the Porifera. Diploblastic organisms are considered to have a body wall composed of two tissue layers, ectoderm and endoderm, separated by an acellular jelly-like mesoglea. The so-called 'Diploblasta', has been considered either to consist of the Porifera, Cnidaria, and Ctenophora (Saivini-Plawen, 1978,1985) or only the Cnidaria and Ctenophora (Hadzi, 1963; HFan, 1940; Jagersten, 1955; Steinbikk, 1963). Few stiU hold this view, and there is a growing recognition that the 'aceUularl mesoglea always has a cellular component, and only appears acellular in some groups because the ratio of cell volume to 'jelly1volume is lower than observed in other organisms (Meglitsch and Schrarn, 1991a). Recent interpretation of Mebchnikoffs (1885) study of development in the ctenophore Callianira bilata suggests that ctenophores have a more extensive mesoderm that also contains muscle cells (Nielsen, 1995). The results of the current study thus support the conclusion that there is no basis for the concept of the 'Diploblasta'. In most of the evolutionary scenarios discussed earlier, poriferans have been placed basal to all other multicellular animals (excluding mesozoans), and in many schemes, are said to have an independent protist origin. Pre-Darwinian systematists often organized animals based on the number of tissue layers (Brusca and Brusca, 1990). Thus, the most simple organisms consist of a single cell or single-layered group of cells. At the next level of complexity, organisms have two-layered bodies, such as those attributed to poriferans, cnidarians, and ctenophorans. These two-layered organisms were envisioned as giving rise to the more complex three-layered organisms. In addition, Cnidaria and Ctenophora have often been united, primarily because of the aforementioned diploblastic body form, but grouping of the Cnidaria and Ctenophora as sister taxa is not supported by the current analysis. The results of this analysis support the basal position of the Porifera, Cnidaria, and Ctenophora, but an origin independent from other multicellular animals is not supported. Although, as discussed above, characterization of organisms by number of tissue layers is problematic, and Likely artificial, a general trend in complexity is inherent in the arrangement presented here. 'Higher' phyla Protostomes and deuterostomes
The remaining metazoans are divided into two major clades (Fig. 26). The larger clade consisting of 15 taxa, often identified as the Bilateria, contains phyla usually referred to as protostomes and deuterostomes. Protostomes are characterized as possessing determinate oblique (= 'spiral') cleavage, mouth formed from the blastopore, mesoderm formed from proliferation of the 4d mesentoblast cell, coelom (if present) from schizocoelous splits within the mesodermal bands, and a trochophore larva (Willmer, 1990). In contrast, deuterostomes are characterized as having indeterminate radial cleavage, anus formed from the blastopore, mesoderm formed from out-folding of the gut wall, coelom formed by entercxoelic pouches off of the gut wall, and a dipleurula larva (Wiher, 1990). Each group is generally thought to be monophyletic, although this has only been tested by Nielsen, et al. (1996). The results of this analysis support the monophyly of the protostomes, but not of the deuterostomes; deuterostomes are only monophyletic if they include the protostomes (Fig. 26). Of the characters usually used to support each group (Table 11), only the secondary mouth formation in deuterostomes (Char. 93, and oblique cleavage (CLE3) and entomesoblast origin of mesoderm (Char. 5) are supported by the results of this analysis. Thus, classical definitions of protostomes and deuterostomes are not consistent with the current analyses and previous scenarios must be re-examined in light of these results. One sub-clade of this larger clade contains 5 taxa usually considered to be deuterostomes, the Chaetognatha + (Chordata + (Echinodermata + (Phoronida + Brachiopoda))). This arrangement is somewhat the reverse order suggested by Nielsen (1995) (Fig. 18),who considered the Chordata to be the most derived phylum, with the Phoronida and Brachiopoda basal. Nielsen (1995) also placed the Ctenophora as the most basal member of this dade because he considered them to share a similar mode of mesoderm formation. However, he then went on to say that all mesoderm may not be homologous (Nielsen, 1995; 65), thus weakening his argument. He further stated that cnidarians and ctenophores share a common "...gastrea-typeorganization ..." (Nielsen, 1995; 307) and a unique first cleavage which begins at the blastoporal pole. TABLEn. Embryological and larval features of protostomes and deuterostomes. Items marked with "" were not evaluate in the cumtanalyses. All other previously hypothesized synapomorphies are equivical at this time.
Classical view Phylogenetic analysis
Protostomes Deu terostomes Protostomes Deuteros tomes
Cleavage Transverse Equatorial Transverse Equatorial
Determinate tndetenninate rC 4
Blastopore Forms mouth Forms anus * IC
Mesoderm From 4d cell From gut wall From 4d cell From gut wall
By proliferation By infolding By proliferation By infolding
Coelom Schizocoelic Enterocoelic No No Somewhat: Larval type Trochophore Dipleurula (Cliteilata- No; No Phoronida- Yes)
New features Monocilia ted epidermis Yes No (Char. 32)
Median cerebral Yes ganglion and (but some taxa statocyst not evaluated) organ. (Char. 1101 Nielsen (Nielsen, 1995; Fig. 421,308) presented two scenarios, one based upon his trochaea theory (Nielsen, 1985) and the other on an alternative hypothesis. According to the trochaea theory, he postulated that Cnidaria was basal to (protostomes + (Ctenophores + deuterostomes)),and alternatively that the Cnidaria was basal to (Ctenophora + (protostomes + deuterostomes)). Nielsen's larval characters (Chars. 38,39, and 44) were induded in the present analysis, and his altemative theory, with the addition of the ecdysozoan clade, is supported rather than the trochaea theory (Fig. 26). Nielsen, et al. (1996) analyzed a subset of the characters from Nielsen's (1985) earlier work, and the results of their analyses (Fig- 17) also favor the altemative hypothesis with placement of the Ctenophora as sister-group to the 'higher' Metazoa. Such a position was also supported by all other cladistic analyses discussed here which induded these taxa, and confirmed by the current analyses. The other bilaterian sub-clade contains nine taxa regarded as protostomes (Fig. 26). The clade is well supported (14 character changes) although full resolution of relationships was not achieved. The Gnathostomulida are most basal, with the remaining taxa, three phyla and a smaller clade, forming a polytomy supported by four character changes. The polytomy in the consensus tree is the result of the Platyhelrninthes being placed as sister to the Molluscs in one arrangement, and basal to the Nemertea + (Molluscs + (Sipuncula + (Echiura + (Pogonophora + (Clitellata + Polychaeta)))))in the other. The optimization of 10 characters (Appendix V, C) changed depending upon the placement of the Platyhelrninthes. Two of these characters, Char. 7, Entomesoblast proliferation contributing to mesoderm (Anderson, 1973; Wilson, 1898) and Char. 112, Paired olfactory fossae of pre-oral lobes (Beklemishev, 1969a, 1969b), could not be scored for the Gnathostomulida because of insufficient developmental information for members of this phylum. If the condition of these two characters could be determined, it might resolve the placement of the Platyhelminthes. The Arthropods and arthropod-like taxa, i.e. Tardigrada, Onychophora, etc. are conspicuously absent kom the protostome dade in the current analyses. These taxa are placed in the ecdysozoan dade, which will be discussed below. Arthropods and annelids have long been regarded as sister taxa, primarily because Cuvier (1817) and Haeckel (Haeckel, 1866a, 1866b) considered the presence of segmentation as evidence of a common origin. The near universal acceptance of this relationship (Barnes, 1987; Bmsca and Brusca, 1990; Meglitsch and Schram, 1991a) has only been challenged by the study of Eernisse et al. (1992). Although the number of taxa examined by Eernisse et al. (1992) was less than in the present study, both are in agreement that arthropods are not closely related to annelids, and are not members of the protostome clade. Acoel platyhelminths have often been suggested as archetype metazoans (Hadzi, 1953,1963; Hanson, 1958,1963,1977; Steinbijck, 1963), or more recently, either as the sister-group to protostomes + deuterostomes (Meglitsch and *am, 1991a; -ram, 1991; Schram and Ellis, 1994) or to the protostomes alone (Bmsca and Brusca, 1990; Eernisse, et al., 1992). Neither of the three hypotheses are supported by the current study, although the Platyhelminthes is basal to seven of the eight other taxa included in the clade in one of the two final trees (Fig. 25A). In the tree of Nielsen, et al. (1906) and Nielsen (1995), the Platyhelminthes and Nemertea form a clade, which is then sister to the remaining protostomes. That relationship is not supported here, either. It is also interesting to note that Nielsen, et al. (1996) and Nielsen (1995) combined the Gnathostomulida with the Annelids, even though Nielsen (1995) suggested that his a ~riorichoice was "...based on few characters, and none of them can be taken as definitive" (Nielsen, 1995; 141). He went on to state that he was "...inclined to regard the gnathostornulids as another highly specialized polychaete group" (Nielsen, 1995; 141). Nielsen's (1995) concern for deficiency in his chosen characters is clearly supported by the results of the current study in which gnathostomulids are basal to all other protostomes, rather than as sister-group to either platyhelminths or polychaetes. Similarly, his (Nielsen, 1995; 141) statement that they are not closely related to gastrotrichs is supported. The position of the gnathostomulids as basal to other protostomes, and not part of the Annelids, is consistent with the results of all phylogenetic studies other than Nielsen's (1995) and Nielsen, et al. (1996) discussed herein. The Priapulida have long been an enigmatic group of uncertain placement (Bmsca and Brusca, 1990; Lang, 1963; *am, 1991; Willmer, 1990). Some of this incertitude is related to lack of knowledge concerning the exad nature of the body cavity in priapulids. Shapeero (1961) held that the body cavity was a true coelom, lined with tissue of mesodermal origin. Others (Calloway, 1982; MacLean, 1984) have suggested that the lining is a simple non-cellular membrane secreted by muscle cells in the inner wall of the body cavity, which implies that it is a pseudocoelom. These conflicting opinions have been problematic because the presence and type of body cavity, i-e. acoel, coelom, pseudocoelom, etc., has long been used as dear evidence of common origin of taxa. Nielsen (1995) and Nielsen, et al. (1996) support a placement of priapulids as sister-group to the (Kinorhyncha + Loricifera), a position somewhat consistent with Schram (Schram, 1991), Meglitsch and *am (1991a), and Schram and Ellis (Schram and Ellis, 1994) who posit the Kinorhyncha basal to the (Priapulida + Loricifera). Eernisse et al. (1992) were not able to resolve this question, and in their consensus tree (Fig. 20) the Priapulids, protostomes, and ecdysozoans form a polytomy. However, they did state that priapulids were placed basally in the ecdysozoan clade in two of the six equally parsimonious trees. A similar uncertainty exists in that in two of the 17 equally parsimonious trees produced in the unweighted analysis shown herein, the Priapulida was included in the ecdysozoan clade (Fig. 22K,P). In both trees produced from the analysis of weighted characters, the Priapulida was the sister-group to the protostome clade. This placement suggests many questions for future studies. First, although cleavage in priapulids is generally considered to be equatorial (Bmsca and Brusca, 1990; Nielsen, 1995), Nielsen et al.
(1996; 406, Appendix 1) codes the priapulids as "?", Schram (1991; 40, Table 1) as equatorial twice (Chars. 18 and 24), and Eernisse et al. (1992; 315; Fig. 3) as neither oblique, multi-plane, nor equatorial. If deavage in priapulids is truly equatorial, as stated above and coded herein, then oblique cleavage is derived from equatorial cleavage. This provides some support for the hypothesis of Salvini-Plawen (1978) that acoelomate ancestors gave rise to the protostome groups. Although cleavage in priapulids is generally considered to be equatorial, given past uncertainties in other features of the phylum, verification of this in a modem study of early development is necessary. None the less, the placement of the Priapulida strongly refutes their candidacy as the sister-group to the Acanthocephala. However, placement basal to rotiferans in the secondary analysis does support priapulids as a second outgroup for use in the analysis of relationships among acanthocephalans presented in Chapter 5. Second, regardless of whether the body cavity in priapulids is a coelom or not, its presence, along with a body cavity in the ecdysozoan clade ('pseudocoelom' and coelom), and deuterostomes (coelom), suggests that body cavities evolved before the acoelomate condition in Platyhekninthes. Thus, the condition in the acoels is secondarily derived from an ancestor with some type of body cavity. If priapulids are coelomate, then the acoel condition is a secondary loss of body cavity from the coelomate condition. This, of course contradicts the hypothesized scenario of Salvini-Plawen (1978) mentioned in the preceding paragraph. This arrangement does provide some support for the bilaterogastraea theories of Jagersten (Jagersten, 1955,1959), Siewing (1969,1976,1980),Remane (1963a, 1963b, 1963c), as well as Haeckel(l866a, 1866b), who postulated the evolution of coelomate organisms from cnidarian-like ancestors, which then gave rise to the remaining taxa. However, the placement of the ecdysozoan clade as sister to the protostomes and deuterostomes suggests that it was 'pseudocoelomates' and coelomates that were derived from these cnidarian-like ancestors, rather than a coelomate first and 'pseudocoelomate' second. The acoel theory of Hadzi (1953,1963), Steinbijdc (1963), and Hanson (1958, 1963,1977) in which coelomates and 'pseudocoelomates' arose from acoel ancestors is likewise not supported. Lastly, the cwlom in arthropods (derived from a 'pseudocoelom') is not homologous with that of members of the protostome + deuterostome clade. Depending upon the optimization of the bodycavity characters, it is possible to formulate two competing hypotheses. Either the coelom evolved first, in the ancestor to the three clades, and the acoelomate and 'pseudocwlomate' condition was independently derived or, the 'pseudocoelomate' condition evolved first, giving rise to the coelomate condition, which then gave rise to the acoelomate condition in platyhelrninths. Either way, the acoelomate condition must be regarded as derived. Each hypothesis is equally supported by this analysis. In future studies, alternate coding of body cavity characters in accordance with each interpretation may provide support for one or the other of the two. Third, Conway-Morris (1981,1985), Conway-Moms and Crompton (1982), and van der Land and Nerrevang (1985), whose claims are based upon those of Conway-Morris, suggested that the Priapulida was the sister group of the Acanthocephala. The main support for this idea was the similarity in general body- shape of fossil priapulids and extant Acanthocephala. Conway-Morris and Crompton (1982) further speculated that since priapulids Lived in marine sediment, the ancestors of acanthocephalans, which they suggested was a fossil priapulid, made the ecological switch to parasitism after being eaten by a fish (Conway-Morris and Crompton, 1982). In two of the 17 original trees produced in my first unweighted analysis, the Priapulida emerged in the middle of the ecdysozoan dade. Analysis of the weighted characters (see p. 60-61) strongly refuted this placement. None the less, a further test the placement of the Priapulida because of its sigruiicance for the acanthocephalan analysis. Therefore, the hypotheses of Conway-Morris (1985) were tested in an a posteriori secondary analysis by arbitrarily pruning most of the taxa located 'distantly' from the Acanthocephala, and then re-analyzing the reduced data set with a11 characters unweighted. This analysis yielded a single tree (Fig. 27) in which the Priapulida was sister to the ecdysozoan dade, but the order of taxa within the clade was slightly changed from that of the more comprehensive analysis. In this dade (Fig. 27), Rotifera and Gastrotricha were sister groups, and they were basal to the Acanthocephala. This a ~osteriorianalysis was not part of the original plan, so the implications of the differences in order of taxa were not extensively explored. Although the data are not included here, several similar analyses were made by randomly including and excluding different non-ecdysozoan taxa. In most of the analyses with reduced data sets, relationships of all taxa the Priapulida remained in the same relative positions within the cladogram. In contrast, the Priapulida 'jumped' around to various places in a manner that seemed to have no obvious correlation with the taxa included or excluded. Because previous studies of priapulids have not included a phylogenetic perspective, various authors have stressed features that reflect their preferential placement of the phylum. Because this has likely produced a data set that is a composite of conflicting characters, each subset was chosen to support the position of the taxa favored by the particular author. Thus, it is not surprising that the position of this group is highly dependent upon the balance of contrasts of polarity in character transformations that is suggested by the inclusion or exclusion of particular taxa. An attempt was made to clarify potential conflicting character assignments by direct examination of specimens, but only whole adult worms could be borrowed from reference collections, and permission to section this material could not be obtained. Solution of this problem must await future studies that include collection and examination of priapulids or additional material from another source that can be sectioned. Despite this, the results of the analysis do not support the priapulids as sister group to acanthocephalans and, accordingly, they were rejected as sister-group to the Acanthocephala for the following analysis (Chapter 5). Although the results of the weighted analyses support the sister-group relationship of the Priapulida and protostomes, the less conclusive results of the unweighted analysis should lead us to concur with Barnes (1985), and conclude that "The phylogenetic position of the priapulids continues to be puzzling ..." (Barnes, 1985; 365). A full understanding of the relationship of this phylum to other metazoans cannot be achieved until the phylum itself has been examined more thoroughly using phylogenetic methodology.
Ecdysozoa
The most sigruhcant new result from this analysis is the placement of the Arthropoda in a strongly supported clade, the Ecdysozoa. This is especially interesting because the Acanthocephala (the target group of this research program) emerge as the basal taxon in this newly defined clade. Placement of the taxa within the Ecdysozoa has been the most problematic of all phyla, even though some, such as the Nematoda, are relatively well-studied. The name for this clade, Ecdysozoa, was suggested by Aguinaldo, et al. (1997) to encompass phyla of animals that molt. The results of the current study support this idea, and indude the arthropods and arthropod-like phyla (Fig. 26) within the concept of the group. In this clade, here called the Ecdysozoa because ecdysis is common within the group, the Acanthocephala is basal to the Rotifera + (Gastrotricha + (Nematoda + Nematomorpha) + (Pentastornida + (Kinorhyncha + (Tardigrada + (Onychophora + (Chelicerata + (Uniramia + Crustacea)))))))). The results of the current analysis strongly support the Ecdysozoa as sister-clade to the protostomes + deuterostomes. Prior hypotheses that the taxa included in the Ecdysozoa were derived from protostome ancestors is refuted. The Ecdysozoa clade is well supported by a common multi-plane cleavage pattern (Chars. CLEI, CLE2, and CLE3), absence of mitosis in epidermal cells (Char. 28), the presence of ecdysone (Char. Sl), as inferred by molting (Char.82) or empirical studies (Willmer, 1990). Additionally, five of the 12 members of the clade possess cement glands of one type or another, which provides further support for the Ecdysozoa. Based on the results of the analyses, it can be further hypothesized that the seven taxa coded as unknown for cement glands may possess structures that are modified forms of cement glands. For example, the spinnerets of chelicerates may have been derived from cement glands, but homology of these structures must await further examination. Arthropods and annelids have been regarded as sister taxa, primarily because of Cuvier's (1817) long untested claim that the presence of segmentation in both phyla was evidence of a common origin. That this hypothesis has gone untested, and is so widely held (Brusca and Brusca, 1990), is amazing, especially because Cuvier was a creationist (see the historical review, p. 5). Evidence of a common origin had gone wtudied until the work of Eernisse et al. (1992). Using a larger number of characters than any others, these authors found that arthropods were placed in a dade with the nematodes, tardigrades, and kinorhynchs; Nematoda + (Kinorhyncha + (Tardigrada + (Onychophora + (Crustacea + Uniramia + Chelicerata)))).However, many of their characters were uncritically accepted from previous non-phylogenetic studies and thus, were not phrased in a format that allowed independent tests of primary homology (see discussion of primary homology, p. 52-53; 57-58), and they only included 19 phyla in their study. However, despite the use of a larger data set, Eernisse et al. (1992) were not able to resolve relationships among these ecdysozoan phyla. More recently, Moura and Christoffersen (1996) focused on relationships within uniramians and, although their number of taxa is much reduced from that of Eemisse et al. (1992), they also agreed that arthropods were not closely related to annelids, and were not members of the protostome clade. The results of an unpublished study by Christoffersen et al. (D. S. Amorim, pers. comm. Christoffersen, et al., 1998), also support inclusion of arthropods within the ecdysozoan clade, although they suggest a more basal placement for arthropod taxa. In the Ecdysozoa, taxa commonly referred to as the Arthropoda form a monophyletic group, although if pentastomes are the basal-most member of the clade, the extent of the group is greater than previous arrangements. The Onychophora is often postulated as sister to the taxa within the classic Arthropoda (e.g., chelicerates, uniramians, and crustaceans) (Brusca and Brusca, 1990; Meglitsch and Schram, 1991a) and this is supported by the current analyses. Questions of division of arthropods into various subgroups have been argued by several authors (Brusca and Brusca, 1990; Eemisse, et al., 1992; Meglitsch and Schrarn, 1991a; Moura and Christoffersen, 1996; Nielsen, 1995) without resolution, but, as in the current study, all support the monophyly of arthropods and arthropod-like groups. Schram (1991), Meglitsch and Sduam, (1991a), Moura and Christoffersen (1996), and Nielsen (1995) were able to provide full resolution of relationships among arthropod taxa. However, Schram and Ellis (1994) and Eemisse et al. (1992) could not resolve relationships among these taxa, although each placed the classic Arthropoda terminal among the arthropod-like taxa. This brings into question the earlier hypothesized homology of the coelom in Arthropoda and Annelids-a topic for future studies. The results of the current study support placement of arthropods as terminal within ecdysozoans, and the placement of tardigrades, kinorhynchs, and pen tas tomes, which possess segmentation, musculature, and muscle attachment similar to other arthropods, basal to the classic Arthropoda. The placement of the Kinorhyncha within this group has not been suggested previously. Although Eemisse et al. (1992) did place the phylum basal to their dade of arthropod-like phyla, they did not suggest placement of kinorhynchs within the group. The Kinorhyncha are usually placed with the 'pseudocoelomates', rather than the protostornes, even though they are considered to have true metameric segments (Brusca and Brusca, 1990) similar to the classic Arthropoda. Segmentation in each is demonstrated to be homologous in the current study. Pentastomids are now considered to be either parasitic crustaceans (Abele, et al., 1989; Wingstrand, 1972) or chelicerates (Walossek and Miiller, 1994), but are basal to all other arthropod-like taxa. Thus, according to the results of these analyses, the Arthropoda must be redefined to include the pentastomids, kinorhynchs, tardigrades, and onychophorans, plus the classic arthropods, to be monophyletic. This differs from previous hypotheses by the inclusion of kinorhynchs as an arthropod-like taxon. Nematoda and Nematomorpha from a sub-clade within the Ecdysozoa, a relationship not unexpected, but it is based primarily on shared secondary loss of features. One exception, a synapomorphy for this clade, is the presence of dorsal and ventral nerve chords (Char. 108). Several features of musculature and cuticle structure were evaluated in preliminary stages of this study, but they could not be included in the analysis because of insufficient information. Homology of the muscle layer in the body wall of nematomorphs with other taxa is not known, in part because lack of developmental studies for most members of the phyla. At least in Nectonema (Class Nectonematoida), the body wall muscles appear similar to those of nematodes, although enervation has not been described (Swanson, 1982; Carvalho, 1942). Sections of an unidentified species of the class Gordioida were examined, but the muscles appeared different than those described for Nectonrma, and enervation was not evident. Although sectioned specimens of Nectonema could not be examined, these muscles, if similar, would likely provide an additional synapomorphy for the clade. Consideration of this character, as with others, must await further examination of specimens and a phylogenetic hypothesis for nematomorphs in order to idenbfy homologies between that phylum and nematodes. Regardless, the sister-group relationship of the two taxa is well supported by these analyses. A close relationship between rotifers and gastrotrichs has frequently been hypothesized (Brusca and Bmsca, 1990; Meglitsch and !3chram, 1991a), but this is likely as much a function of a common relative lack of phylogenetically signihcant information as shared features. Several recent studies of cuticle morphology (Ahlrichs, 1997; Rieger and Tyler, 1995) have suggested gastrotrichs as sister-taxa to the Rotifera + Acanthocephala, an earlier hypothesis of Storch (1979) and Lorenzen (1985). Although recent studies (Ahlrichs, 1997; Rieger and Tyler, 1995) have used phylogenetic methods, they have included very few characters and a priori assumptions of character evolution not based on more comprehensive studies. They are more similar to earlier evolutionary views in which a few characters were selected to support g priori ideas of relationships than they are of the studies by Schram (1991), Schram and Ellis (1994), Nielsen, et al. (1996), or this study. Despite this, all are in agreement that rotifers and gastrotrichs are closely related, a conclusion supported by the current analyses, which place rotifers just basal to gastrohichs. This relationship must be tested further by additional information concerning gastrotrich development, which is not well known, and reproductive mode (i.e., hermaphrodism vs. gonochorism) within the phylum. The Acanthocephala has been allied to almost every group of invertebrate that contains parasitic members. The phylogenetic relationship of the Acanthocephala to other parasitic groups was explored by Nicholas (Nicholas, 1967, 1971; ) and Nicholas and Hynes (1963), but, other than the discussion by Conway-Morris and Crompton (1982), the topic has not received attention in any comprehensive cladistic study- Lorenzen (1985) suggested several characters to tie together the Rotifera and Acanthocephala. His primary reasons for this relationship were a similar embryology Uoffe, 1979) and similarity in integument (Storch, 1979), which he further suggested were traits "...which can be derived from Turbellaria..." (Lorenzen, 1985; 241). His suggested similarities have been supported by the studies of Rieger and Tyler (1995) and Ahlrichs (1997), although, as discussed above, neither included a large number of taxa or characters. The results of the current analyses suggest a dose relationship between the two phyla, but they strongly suggest that they are not sister groups. The Acanthocephala is the most basal ecdysozoan, situated just below the Rotifera. The remaining taxa are separated from the Acanthocephala by presence of pedal retractor muscle bundles (Char. 48), chitin in the cuticle (Char. 76), terminal alimentary zones of cuticle (Char. 94) (although this character is confounded by absence of gut in acanthocephalans), an anus (Char. 96), and epidermal sensory setae (Char-116). The basal position of acanthocephalans within the Ecdysozoa provides an interesting contrast with what is seen in other parasitic groups. Parasitic organisms are generally thought to be degenerate forms of their free-living relatives (Noble and Noble, 1971; Schmidt and Roberts, 1985; Smith, 1949). For example, in the Platyhelminthes, the parasitic monogeneans, trematodes, and cestodes were postulated by Ehlers (1985,1986) and Rohde (1990,1994) to be derived from free-living platyhehinth ancestors subsequent to the origin of extant free-living taxa. The assumption of degeneracy has been refuted by Brooks and McLennan (1993a, 1993b), who found that parasitic flatworms are characterized by having more highly-derived features rather than a loss of features possessed by their free-living ancestors. Derivation of parasitic forms from free-living forms is intuitive given that parasitism is a feature of ecology that has arisen independently numerous times, as is evidenced by parasitic groups such as trematodes, nematodes, acanthocephalans, etc. not forming a monophyletic group. However, the basal placement of the Acanthocephala within the Ecdysozoa provides no dues to its free-living ancestor, other than a hypothetical ancestor based upon plesiomorphic characters of the protostome + deuterostome clade and the ecdysozoans.
Cleavage pattern
Great emphasis has been placed on a few character suites which have been used to characterize groups of taxa and support particular evolutionary scenarios. Such characters suites include early embryonic development (Hadel, 1866b; Lankester, 1873,1877), types of larvae (Nielsen, 1979,1985; Nielsen and Nsrrevang, 1985), and mode of coelom formation (Clark, 1967,1979). One of the most touted of these character suites is the pattern of early cleavage. Early cleavage is typically broken down into three categories: 'longitudinal'/'meridional','radial', and 'spiral'. Meridional cleavage planes are parallel to the animal-vegetal poles. 'Radial' cleavage planes are initially meridional, but then switch to equatorial. In 'spiral' cleavage, initial cleavage planes meridional, and then they switch to a oblique pattern such that the new cells form in a spiral fashion. A fourth type of cleavage, termed 'modified spiral', has been held to be a derivation of 'spiral' cleavage, just as the name implies. In this pattern cleavage occurs first in an equatorial plane, and then such that micromeres cleave in one plane and macromeres in a different plane (Fig. 24) (see (Brusca and Brusca, 1990; 97-106 for a more extensive discussion of cleavage patterns). Evolutionary systematists have long used character suites, with their inherent implications for evolution, as support for particular scenarios without benefit of initial phylogenetic testing. Just as with other character suites, early cleavage patterns have been used to just@ hypotheses of relationships. For example, because it is 'known'that 'modified spiral' is derived from 'spiral' cleavage, then organisms 83 exhibiting 'modified spiral deavage' must have arisen from 'spiral' ancestors. Even in the previous studies discussed above that have used phylogenetic methods, none has tested this hypothesis, even though some, such as Eemisse et al. (1992), have avoided the coelom/pseudocoelom problem. One goal of the current study was to test hypothesized patterns of early cleavage without bias related to a ~rioriassumptions of evolution of this character. The approach used here is to break cleavage stages into individual characters. In this form, each cleavage stage is independent of other stages to allow the homology of each stage to be tested by the analyses. Thus, cleavage pattern is separated from a priori assumptions of character evolution as well as presumptions of metazoan evolution. As discussed above, assignment of deavage characters (Table I) was made strictly from descriptions of cleavage planes without reference to perceived 'type' of cleavage. To avoid confusion with previous designation of deavage types and traditional association of types with particular taxa, resultant deavage patterns are termed meridional, equatorial, oblique, and multi-plane, as discussed above. One of the most interesting features of the phylogenetic hypothesis produced by this study is the placement of the Arthropoda within the Ecdysozoa. As discussed previously, Eemisse, et al. (1992) suggested that arthropods were not closely related to annelids. This relationship was based primarily on 'segmentation' in both groups. In a preliminary step in the analysis, before all questions in character assignment had been finalized, the Arthropoda were coded as having a oblique cleavage pattem (Chars. CLE1, CLE2, and CLE3), the same as the Gna thostomulida-clade. This coding was based upon information from secondary sources (Bmsca and Bruxa, 1990; Meglitsch and Scham, 1991a; Nielsen, 1995; Willmer, 1990). Initial placement of the arthropods within the Ecdysozoa in all 17 trees produced by the analysis of unweighted characters led me to re-examine primary descriptions of arthropod embryology. Nielsen (1995), along with classical embryologists (see (Brusca and Brusca, 1990) for a full discussion), considered the cleavage pattem of arthropods to be of the same type as the platyhelrninths, molluscs, and other protostomes. The differences in pattem among the arthropods compared to that of the other taxa has always been attributed to the high proportion of yolk in arthropod eggs (Anderson, 19 79). Although much is known about final cell fate, discussions of the first three cleavages in arthropods is typically vague, and such statements as "...cleavage may be more-or-less radial or may show traces of spiral pattern... However, [this] appears to be somewhat different from the typical spiralian pattem seen in [other protos tomes]..." (Bmsca and Brusca, 1990; 484-485) are common. Curiously, in all of the fate maps examined to resolve this ambiguity, none identifies mesoderm as originating from a 4d cell, although this is supposed to be characteristic of 'spiral' cleavage. The only study that fully described the first three stages of cleavage in an arthropod is that of Balanus balanoides. a arriped crustacean with relatively little yolk in its eggs, by Delsman (1917). Nielsen re-drew the original figures (Nielsen, 1995; 169, Fig. 20.2) of Delsman (1917), and revised Delsman's cleavage notation into the form used today (Nielsen, 1995; 16, Table 20.2). A closer examination of the cleavage pattem of Balanus balanoides figured in Delsman's (1917) original work, and Nielsen's (1995) revised figures and notation, showed that the steps in early cleavage were almost identical to multi-plane cleavage that is seen in other ecdysozoans. Re-coding of characters related to early cleavage and cell fate did not affect placement of taxa within the 17 trees produced by the unweighted analysis, or the two trees of the weighted analysis. However, this change provided stronger support for the assignment of arthropods to the Ecdysozoa, as seen in the results of Eemisse et al. (1992) and the current study. To further test reliance of the analysis upon assignment of cleavage pattem, all characters relating to cleavage were removed from the analysis. Re-analysis of the reduced data set (excluding Chars. 85
CLE1, CLEZ, and CLE3) did not alter placement of any taxa, and trees identical to those produced by analysis of the complete data set were obtained. Identical topology in these trees with those produced from the data set including cleavage characters confirms the validity of assigning multi-plane rather than oblique cleavage to the arthropods and arthropod-like taxa. The Porifera, Cnidaria, and Ctenophora have the plesiomorphic meridional cleavage pattem, although the patterns in Cnidaria and Ctenophora are each somewhat modified (Brusca and Brusca, 1990). Members of the protostome dade all show oblique cleavage, and the Priapulida and all deuterostomes have equatorial cleavage. The Ecdysozoa exhibit what is termed here multi-plane cleavage. Thus, the results of the current study suggest that the form of deavage pattern is a synapomorphy for the deuterostomes + protostomes, protostome clade, and the Ecdysozoa. To test hypotheses of treating cleavage pattem as a 'suite' that combined the first three cleavages into a single character, each cleavage was coded independently. This coding allowed either of the two interpretations of evolution of cleavage pattem shown in Figure 28. Complete independence of the third cleavage in non-ecdysozoans (Fig. 28A) was not supported. Instead, the pattem of optimization of the character on the tree suggests that meridional cleavage with complete separation of daughter cells is a synapomorphy for multicellular animals (Fig. 28B), with cleavage patterns in mesozoans independently derived. Equatorial cleavage is derived from meridional (CLE3: 0->I), and oblique, from equatorial (CLE3: 1->2). Multi-plane cleavage is also derived from meridional, but from an earlier stage of cleavage (CLEI: 0->I), with difference in the second and third cleavages not independent of the change in the first cleavage. This further suggests that an ordered transformation is more appropriate for these three characters than an unordered one. The classical view of the importance of early cleavage in the evolutionary history of multicellular animals, expressed in a novel and entirely modem form, has been supported by the results of the analysis. It must be emphasized that placement
of taxa did not depend upon a pried assignment based on perceptions of an evolutionary scenario, but on a rigorous phylogenetic analysis. Characterization of groups was possible only after monophyletic groups and their relationships were identified, and primary homology of character transformations were tested in a phylogenetic context. Hypothesized concordant patterns of early origins and cleavage has not been tested using phylogenetic methods in any previous study.
Classification of multicellular animais
The widely-accepted monophyly of multicellular animals is well supported by 16 unambiguous character changes (Chars. OG1,OG2,OG3,OG4,OG7,OG11, OG12, CEL1, CEW, CILl, 30,31,32,36,39, and 100). Strong support for the choanoflagellates as the sister-group does not preclude a different group, such as the ciliates, from consideration as an additional outgroup. Hanson (Hanson, 1963,1977) and Hadzi (Hadzi, 1953,1963) provided some information that could be used for a future test of this hypothesis, but it was phrased as an evolutionary scenario that is not suitable for phylogenetic analysis. At this time, the only morphological data set useful for comparison of protist groups with multicellular animals is that of Nielsen (1995). This study provides a great increase in our knowledge of relationships among multicellular animals by clearly defining the placement of the 'aschelminth' phyla and refuting the hypothesized 'Aschelminthes' clade. Identification of the Ecdysozoa, and inclusion of the Arthropods and arthropod-like groups within that clade, is a large step forward in our understanding of relationships among these enigmatic groups. One taxon that is obvious in its exclusion from this analysis is the Loricifera. Although it has been suggested as a member of the 'pseudocoelomates', very little information on internal structures exists, and its embryology is unknown. Once the Loricifera is better studied, inclusion in the analysis will probably strengthen the concept of the Ecdysozoa. The results of the current study challenge the monophyly, and therefore, validity of the Protostornia and Deuterostomia. The two together appear to be a monophyletic assemblage united by the presence of a cuticle formed from non-living external layers containing protein (Char. 74) and the presence of collagen sequestered in the cuticle (Char. 75). However, it is premature to name this group until the excluded taxa are placed within the two sub-clades. The Ectoprocta and Entoprocta in particular are missing from the analysis. Although a great deal more is known about these two groups, the secondary Literature is not helpful in providing information about many of the characters used in the current analysis. With these, as with many other more obscure taxa, because particular structures have not traditionally been thought to have phylogenetic 'importance', they have not been well-described. Only direct examination will provide dues to particular features. As well, a new phylum, the Cydiophora (Funch and Kristensen, 1995), has recently been discovered that is thought to be dosely related to the Ectoprocta or Entoprocta. Inclusion of this taxon, more well-described than many of the other enigmatic groups, will Likely yield further clues concerning relationships of Ectoprocts to its nearest relatives. A classification based upon descent as determined from the results of this analysis is presented in Appendix 11, D. However, no new names for these groups, other than the aforementioned Ecdysozoa, are offered, and should not be until a more an analysis including all phyla is performed. In addition, taxonomic decisions based upon the results of analyses which provide full resolution of relationships of the groups of inference is preferred. For example, resolution of the protostome clade is seen in all 17 trees, so that dade could be identified in a phylogenetic context and named. However, the polytomy within the dade precludes decisions on those taxa. Because additional data, or re-interpretation of existing character assignments will 88 be necessary to resolve the ambiguities seen herein, it is better to be conservative at this time, at least until all taxa can be included in the analysis. Thus, only fully resolved assemblages are named in this classification.
Conclusion
A number of authors (Nursall, 1959,1962; Whittaker, 1957,1959,1969; Willmer, 1990) have concluded that it may be impossible to determine relationships among phyla, regardless of the technique used. For example Willmer declared that "... [phylogenetics may] not work at the phyletic level..." (Willmer, 1990; 1). The results of this study do not answer all questions, but they dearly demonstrate that this challenge can be met. Using an extensive character data set and phylogenetic methodology, a robust phylogenetic hypothesis for multicellular animals can be produced. Three goals for this study were initially identified: 1.) use of an adequate data set which included as many putative phyla and as large a number of characters identified by previous investigators as practical, 2.) to avoid inherent biases and a priori assumptions of character evolution or perceived importance by viewing primary homology from a strict Hennigian perspective, and 3.) to produce a phylogenetic hypothesis for multicellular animals that would provide a basis for p hy logenetic interpretation of evolutionary patterns, rela tionships among metazoan phyla, and suggest a classification by descent based on the results of the analysis. Each of those goals was met to a greater or lesser extent. First, a large data set was gathered from characters identified by previous investigators and primary sources. Second, each of those characters and character coding was evaluated for primary homology. Third, a strict phylogenetic analysis was performed to identify relationships among included taxa. As well, the analysis provides a strong test of hypothesized primary homology of all characters used in the analysis. Although some taxa were excluded from the analysis, and not all relationships were resolved, sufficient resolution was achieved to identdy taxa useful for polarization of characters in a phylogenetic analysis of the Acanthocephala. Additionally, hypothesized patterns of evolution of the stages of early cleavage were tested in a phylogenetic context for the first time. The results suggest that a similar approach would provide clarification concerning the evolution of body cavities and reduce the confounding effect of a priori assumptions about this feature. Lastly, strong support for the assignment of the arthropod-like phyla to the Ecdysozoa was found, resolving previous confusion concerning segmentation and early cleavage of arthropods. Cnidaria Rhabditophora Acwlomorpha Gnathostomulida Nemertea Conchifera Polyplacophora Solenogastres Caudofoveata Clitellata PoIychae ta Pogonophora Echiura Sipuncula Nematoda Uniramia Crus tacea Chelicerata Onychophora Tardigrada Kinorh yncha Priapulida Echinodermata Chordata Phoronida Brachiopoda
Figure 20. Tree depicting relationships among the Metazoa as suggested by Eernisse et al., 1992. Figure 21. Comparison of the tree of Nielsen (1995) (A.) with that of Nielsen, et al. (1996) (B.). Taxa not supported in the tree of Nielsen et al. (1996): 1. Aschelrninthes; 2. Protomaezoa; 3. Neorenalia. Figure 22. Trees resulting from the primary analysis of unweighted characters of 33 multicellular phyla. AQ. Equally parsimonious trees (17trees). R. Consensus tree of these 17 trees (A-Q). Choanoflagellata Dicyemida Tnchoplax ~icyemida- Rho alura Tnchoplax porikra Rho alura Cnidaria pori Pera Cteno hora Cnidaria ~canttoce~hala Cteno hora Rotifera ~canttoce~hala Gasuotricha Rotifera Nernatoda Gastrotricha Nematomo ha Nematoda ~cntastomiTa Ncmatomo ha Kinorhyncha ~cntastomiTa Tdgrada Kinorhyncha Onychophora Tardlgrada Chelicerata Onychophora Uniramia Chelicerata Cmstacea Uniramia Chaetognatha Cmstacea Chordata Chaetognatha Echinodennata Chordara Phoronida Echinodemata Brachio oda Phoronida priaPuliSa Brachio oda Gnathostomulida priapuda Nemertea Gnathostomulida Platy helminthes Nemenea Mollusca Platyhelminthes Sipuncula Mollusca Echiura Sipuncula Pogonophora Echiura Clitellata Pogonophora Polychaeta Clitellata \ Polychaeta
Choanoflagellata Choanoflagellata Dicyemida Dicyemida Tricho lax Tricho lax ~ho~aha ~ho~aira Porifera Pori fen Cnidaria Cnidaria Ctenophora Ctenophora Chaetognatha Acanthocephala Chordata Rotifera Ec hinodermata Gastrotricha Phoronida Nernatoda Brachiopoda Nematomorpha Priapulida Pentastomida Gnathostomulida Kinorhyncha Nemertea Tardi da Platyhelminthes 0nycGhora Mollusca Chelicerata S&u'faula Uniramia Cmstacea Pogocophora Chaetognatha Clrtellata Chordata Polychaeta Echinodennata Rotifera Phoronida Gastrotricha B rach iopoda Acanthocephala Priapulida Nematoda Gnathostornulida Nematomo ha Nernertea ~entastomi7a Platyhelminthes Kinorhyncha Mollusca Tardi da S$um&a ~nycg~hora Chelicerata Pogonophora Uniramia Clltellata Crustacea Polychaeta ChoanoflagelIata A Choanoflaeellata Dicyemida ~ic~ernida- Trichoplax Tnchoplax Rho alum Rho dura ~oriFen poriPera Cnidaria Cnidaria Ctenophora Cteno hora Rotifera ~crntRoce~hala Gastrotricha Rotifera Acanthocephala Gastrotricha Nematoda Nematoda Nematorno ha Nematomo ha pen tastomiz ~entastorniTa Kinorhyncha Kinorhyncha Tardcgrada Tardlgrada Onychophora Onychophora Chelicerata Chelicerata Uniramia Uniramia Crustacea Crustacea Chaetognatha Chaetognatha Chordata Chordata Echinodermata Echinodermata Phoronida Phoronida Srachio oda Bnchio oda priapuliSa ~riaou~iia Gnathostomulida ~naihostomulida Nemertea Platyhelminthes Platyhelrninthes ~emertea Mollusca Mollusca Sipuncula Sipuncula Echiura Echiura Pogonophora Pogonophora Clitellata Clitellata Pol yc haeta \ Polychaeta
Choanoflagellata I Choanoflagellata Dic yemida Dicyemida- Tricho lax Tricho lax ~ho~ain ~ho~afira Pori fera Porifera Cnidaria Cnidaria Ctenophora Ctenophora Chaetognatha Acanthocephala Chordata Roti fera Echinodermata Gastrotricha Phoronida Nematoda Brachiopoda Nematomorpha Priapulida Pentastomida Gnathostomulida Kinorhyncha Nemertea Platyhelminthes TardipOnyc ophora Mollusca Chelicenta sipd" Unirarnia Cmstacea Pogonophora Chaetognatha ClltelIata Chordata Polychacta Echinodermata Rotifera Phoronida Gastrotricha Brachiopoda Acanthocephala PriapuIida Nematoda Gnathostomulida Nematomor ha Nemertea ~entastomiBa Platyhelminthes Kinorhync ha Mollusca S&m,"la OnycTardip ophora Chelicerata Pogonop hora Uniramia Clrtellata Crustacea Polychaeta I Choanoflanellata Choanoflagellata ~ic~emida- Dicyemida Tnchoplax Trichoplax Rho alura Rho alura ~oriFera ~oriPsn Cnidaria Cnidaria Ctenophora Cteno hora Rotifera ~cantioce~hala Gastrotricha Rotifera Acanthocephala Gastrotricha Nematoda Nematoda Nematomo ha Nematomorpha ~entastomiTa Kinorhyncha Kinorhyncha Pentastomida Tardigrada Tardi grada Onychophora Onychophora Chelicerata Chelicerata Uniramia Uniramia Cmstacea Crustacea Chaetognatha Chaetognatha Chordata C hordata Echinodermata Echinodermata Phoronida Phoronida Brachio oda Brachio oda Priapuli!a ~ria~uliSa Gnathostomulida Gnathostomulida Nemenea Nemertea Platyhelminthes Platyhelminthes Mollusca Mollusca Sipuncula Sipuncula Echiura Echiura Pogonophora Pogonophora Clitellata Clitellata Polychaeta Polychaeta
Choanoflagellata Choanoflagellata Dicyemida Dicyemida Tricho lax Tricho lax ~ho~a&ra ~ho~a&n Pon fer? Pori fera Cnidana Cnidaria Ctenophora Ctenophora Acanthocephala Chaetognatha Rotifera Chordata Gastrotricha Echinodermata Priapulida Phoronida Nematoda Brachiopoda Nematomo ha Priapulida ~cntastomiTa Gnathostornulida Kinorhyncha Platyhelminthes Tard~grada Nemenea Onychophora Mollusca Chelicerata S&u;da Uniramia Crus tacea Pogonophora Chaetognatha Clitellata Chordata Polychaeta Echinodermata Rotifera Phoronida Gastrotricha Brachiopoda NcrnatodaAcanthocephala Gnathostomulida Platyhclminthes Nemalorno ha Nemcnca ~entastomiXi Mollusca Kinorhyncha Sp~Ia Tardi da 0nycEphora Pogonophora Chelicerata Chtellata Uniramia Polychaeta Cmstacea Choanoflagellata Dicyemida- ~ic~crnida~ Trichoplax Tnchoplax Rho dura Rho aiura PO ri Pe ra ~oriFen Cnidaria Cnidaria Cteno hora Ctenophora ~cantiocc~hala Rotifera Rotifera Gastrotricha Gastrotricha Acanthocephala Nernatoda Nematoda Nematomo ha Nematomo ha ~entastorniTa ~entmtomi% Kinorhyncha Kinorhyncha Tamwja Tard~grada Onychophora Onychophora Chelicerata Cheliccrata Uniramia Uniramia Crustacea Cmstacea Chaetognatha Chaetognatha Chordata Chordata Echinodermata Ec hinoderrnata Phoronida Phoronida Brachio oda Brachio oda ~ria~uIi%a priapuIiSa Gnathostomulida Gnathostornulida Platyhelminthes Platyhelrninthes Nemertea Nemenea Mollusca Mollusca Sipuncula Sipuncula Echiura Echiura Pogonophora Pogonophora Clitcllata Clitellata Polychaeta Polychacta
Choanoflagcl lata Choanoflagellata Dicyemida Dicyemida Tricho lax Tricho lax ~ho~afira ~ho~aira Pori fera Pori fera Cnidaria Cnidaria Ctenophora Ctenophora Acant hocephala Acanthocephala Rotifera Rorifera Gastrotricha Gastrotricha Nernatoda Priapulida Nernatornorpha Nematoda Kinorhyncha Nematornorpha Pentastomida Kinorhyncha Tardi da Pentastomida ~n~c'~hora Tardigrada Cheiicerata Onychophora Uniramia Cheliccrata Cmstacea Unirarnia Chaetognatha Crustacea Chordata Chaetognatha Echinodermata Chordata Phoronida Echinodermata Brachio poda Phoronida Priapulida Brachiopoda Gnathostomulida Gnathostornulida Nernenea Platyhelrninthes Platyhelminthes Nemenea Mollusca Mollusca S&mda !S&u;h Pogonophora Pogonophora Chtellata Cl~tellata Polychaeta Polychaeta ~ic~cmida~ Trichoplax Rho aha pori Pe ra Cnidaria Cteno hora ~cantke~hala Rotifeta Gastrotricha Nematoda Nematomorpha Kinorhyncha Pentastomida Tardigfada Onychophora Chelicerata Uninmia Crustacea Chaetognatha Chordata Echi noderrnata Phoronida
Gnathostomulida Platyhelminthes Nemertea lMollusca Sipuncula Echiura Pogonophora Clitellata \ Polychaeta Modified-spiral Spiral Radial
00 Longtudinal
Ancestor: Complete separation of daughter cells
Figure 23. Classic view of the evolution of cleavage types. Meridiaonal @qpa-
Equatorial a 00 a m
plane
Figure 24. Cleavage planes exhibited by metazoans. The third cleavage is not depicted for multi-plane cleavage. Figure 25. Two equally parsimonius trees produced from the analysis of weighted characters. Figure 26. Consensus tree resulting from a phylogenetic analysis of 33 multicellular animals. Letters accompanying slash marks on the tree indicate putative synapomorphies based on unambiguous character state changes; character state changes (autapomorphies) for individual taxa and ambiguous changes are not shown.
X-841, 861,871,88l, 1131,1291; and, 8-83', 1111,1141,1271,1281. Choanoflagella ta
Trichoplax RhoDalura Porifera Cnidaria Ctenophora Priapulida Gnathostomulida PIatyhelminthes Nemertea Mollusca Sipuncula Echiura Pogonophora Clitellata Polychae ta Chaetogna tha Chordata Echinodennata Phoronida Brachiopoda Acanthocephala Rotifera Gastrotricha Nematoda Nematomorpha Pentastomida Kinorhyncha Tardigrada Onychophora Chelicera ta Uniramia Crustacea Choanoflagellata
Picyemida
Porifera
Cnidaria
Gnathostomulida
Platyhelminthes
Echinodermata
Chordata
Priapulida.
Rotifeta
Gastrotricha Acanthocephala
Nematoda
Nema tomorpha
Pentastomida
Kinorhyncha
Tardigrada
Onychophora
Chelicerata
Uniramia
Crus tacea
Figure 27. Single tree produced by the secondary analysis of a reduced number of taxa. The box indicates members of the Ecdysozoa and arrows indicate Priapulida, Rotifera, and Acanthocephala. Meridional Meridional + Meridional I Complete I separation of daughter cells I I (Choanoflagelata) I v Oblique Centrioles a; animal and vegetal poles + Multi-plane
Meridional l) Meridional Meridional Meridional +- + Complete I separation of 8 daughter cells I + Equatorial (Choanoflagelata) I I + Oblique Centrioles at animal Multi-plane and vegetal poles +
Figure 28. Cleavage evolution scenario. Two scenarios of cleavage evolution that were explored in the current analyses. A. Coding of states as "unordered". B. Equatorial and oblique patterns are derived from meridional cleavage and multi- plane cleavage is a & novo pattern as suggested by results of the analyses. Chapter 4: Historical review of the relationships of the Acanthocephala Historical background
The study of the Acanthocephala has had an erratic history, going from almost unnoticed, then making important contributions to systematics and development (during the era of Anton Meyer), back to being ignored, back to importance again (during the era of Harley J. Van Cleave), and then another return to obscurity. Now, with a renewed interest in the relationships of the 'pseudocoelomates' and their positions within the Metazoa, the phylum is again receiving attention. Parasitic organisms are generally thought to be relatively uninformative for the understanding of metazoan relationships because they are said to be degenerate forms of their free-living relatives (Noble and Noble, 1971; Schmidt and Roberts, 1985; Smith, 1949). Despite challenges to this assumption (Brooks and McLennan, 1993b), this view of parasites, and the Acanthocephala in particular, has not changed much with the growth in understanding of parasite evolution. The increase in comparative and co-evolutionary studies (Brooksand McLeman, 1991,1993b) underscores the necessity of reexamining all hypothesized relationships within a phylogenetic context, including the Acanthocephala and other neglected phyla.
Sister group affinity of the Acanthocephala
Since the first descriptions of acanthocephalans, the placement of the group in relation to other taxa has been problematical (Amin, 1985; Van Cleave, 1948). Because of the group's worm-like characteristics, it was often considered to be closely related to the Nematoda. In many early classification systems (e.g. Southwell 106
and MacFie, 1925), it was placed with the Nematoda and other roundworms in the Phylum 'Nemathelminthes' . Possible placement with the roundworms was supported mainly by such features as body shape, presence of a pseudocoelomic body cavity, cell constancy with a small relative number of cells, dioecism, and other characters reflecting outward similarity. A survey of invertebrates shows that these characters, such as dioecism, are not limited to acanthocephalans, or even to 'nemathelminths'. A more thorough examination of their characters suggests that acanthocephalans have only a superficial resemblance to the other 'Nemathelminthes'. Even Hyman (1951) could not decide where to place the Acanthocephala,
declaring that their morphology was more similar to the 'Aschelminthes' but embryology was more similar to that of Platyhelminthes. In addition, in acanthocephalan gastrulation, a solid stereo-gastrula, is formed rather than the hollow coeloblastula common to many pseudocoelomates. Yet the pattern of early cleavage in acanthocephalans, commonly called 'modified spiral', is similar to that of other so-called 'pseudocoelomates' instead of the more regular 'spiral' cleavage found in platyhelminths. Hyrnan (1951) also based her conclusion, in part, on the pseudocoelomic body cavity of acanthocephalans, yet it bears only a general resemblance to the pseudocoelom of nematodes. During development, many internal cell membranes of acanthocephalans disappear and the body becomes a syncytium, and remains so throughout subsequent development and adult life. The central syncytial body mass then becomes divided into cortical and medullary regions. Along the junction of these two regions a split occurs that forms the body cavity (Sdunidt, 1985). The pseudocoelom of nematodes is a persistent blastocoel (Hyman,1951). A careful comparison of each process with that seen in other supposed 'pseudocoelomate' taxa is necessary before homology of the body cavity in each group can be established. Similarly, cell constancy, first described in acanthocephalans by Van Cleave (1913), 107
is often attributed to all 'pseudocoelomates', but a comparative examination of all taxa must be made before its existence can be determined with certainty. Conversely, because some of the earliest known species are pseudo-segmented and flattened in unfixed material, the phylum has also been thought to have a dose relationship with the Platyhelminthes. Kholodkovskii (or "Cholodkovsky" of Van Cleave, 1941) examined available data a study of Acanthocephala and compared them with other groups of worm-like organisms. In a little noticed article (Kholodkovskii, 1897), he suggested that acanthocephalans had few features in common with roundworms and that they were more similar to Platyhelminthes. He further suggested that characters such as structure of the cuticle, body walls, musculature, excretory system, and nervous system; external adaptations; and features of developmental life-cycle stages indicated acanthocephalans may share a common ancestor with the Cestoda. Meyer (1933) departed from these early views and suggested that the Acanthocephala was closely related to the Priapulida and proposed an arrangement for the Aschelrninthes composed of the following evolutionary 'series' of taxa: Rotifera, Kinorhyncha, Priapulida, Acanthocephala, Nematomorpha, Nematoda. He considered division of the body into presoma and metasoma, the presence of armature, cuticle, type of body cavity, and the absence of metameres to be most important in determining affinity of the Acanthocephala with other groups. As discussed above, none of these characters are Limited to this group of taxa. Later, Van Cleave (1941) disagreed with Meyer and came to a conclusion, similar to that of Kholodkovskii (1897), that acanthocephalans were the sister group to the Turbellaria. However, Van Cleave's scheme was primarily based on characters of general similarity such as: lack of digestive system; cuticle; chitinized hooks; lacunar/vascular system; protonephridia; and introvert (proboscis) structure. He stated that, because of their degenerate nature, this type of character was the only sort available and the "...usual avenues for seeking evidences of phylogenetic 108 relationships [were] seriously obstructed..." (VanCleave, 1941; 36). He went on to say that paleontological, morphological, and ontogenetic evidence was insufficient to provide strong support for any hypothesis of origin of the Acanthocephala. He reasoned that this was primarily because of the ancient origin of the Acanthocephala as revealed in parallel co-evolution of European and Japanese species of Acanthocephalus from amphibians (Van Cleave, 1925). Some characters used by early systematists, such as body shape, are actually artifacts of methods of examination rather than actual characteristics. In live Acanthocephala, the body is mostly flat and only assumes a cylindrical shape during fixation. Because some of the earliest known species were pseudo-segmented and flattened in unfixed material, the phylum also was thought to have a dose relationship with the Platyhelminthes. Van Cleave (1941) suggested acanthocephalans had many similarities to the Cestoda; the absence of an alimentary canal in all lifecycle stages, a proboscis with hooks (which he equated with the cestode rostellum as well as tentacles in trypanorhynchs), presence of both circular and longitudinal muscles, and similarities between the hexacanth embryo of cestodes and the acanthor of acanthocephalans. However, others (Nicholas, 1971; Nicholas and Hynes, 1963) have questioned the hamology of these characteristics, preferring placement within the 'Aschelminthes'. Petrochenko (1956a, b) agreed with the conclusions of Kholodkovskii (1897) and Van Cleave (1941) but suggested that their evidence was weak because their chosen characters were the product of convergence related to similarity in habitat. He concluded that features of the nervous system, ultrastructura1 characteristics of the body wall, excretory system, and reproductive system, and early-embryonic development would give better clues to the sister group of the Acanthocephala than outward features of similarity. He proposed the Turbellaria as the sister-group of the Acanthocephala. 109
A more recent treatment by Conway-Morris and Crompton (1982) suggests a return to placement of the Acanthocephala with the Aschelminthes, and a reappraisal of Meyer's (1933) suggestion of the Priapulida as sister group. As would be expected, Conway-Morris and Crompton approached this problem from a more modem phylogenetic systematics viewpoint than has been used in the past. They argued that recently discovered Cambrian fossil evidence supports earlier suggestions by Nicholas (1971) and Whitfield (1971) that free-living ancestors of acanthocephalans were part of the burrowing marine meiofauna. They hypothesized that ancestors of the Acanthocephala + Priapulida were the sister group to ancestral forms of the extant Kinorhyncha (Fig. 29). This hypothesis is reminiscent of Meyer's (1933) 'series'. The most recent attempts at clarification of the relationship of the Acanthocephala to other taxa have used more rigorous cladistic techniques to understand metazoan evolution. As discussed in the historical review of the Metazoa, Sham (1991), Eemisse et al. (1992), Badceljau et al. (1993), and Sdvam and Ellis (1994) postulated the Rotifera as sister group to the Acanthocephala based on p hy logene tic analyses of anatomical and larval features. Studies using molecular techniques also have suggested a close relatiowhip between the Acanthocephala and Rotifera (Garey, et al., 1996; Winnepenninckx, et al., 1995), although they are not in complete agreement on relationships among all included taxa. Based largely on similarity of the syncytial ectoderm found in both acanthocephalans and rotifers, several recent workers have regarded acanthocephalans as parasitic rotifers (Lorenzen, 1985; Storch, 1979; Storch and Welsch, 1970) closely related to the bdelloids. This position is supported by one molecular study (Garey, et al., 1996). Others consider the common structure of the ectoderm and shared embryology to be synapomorphies for the two phyla, and the differences in embryological origin of the retrocerebral organ and the acanthocephalan proboscis to be apomorphic for each group, thus supporting the monophyly of each phylum (Nielsen, 1995). As discussed earlier, one's philosophic view has an important effect on perceptions of the origin of the Metazoa and subsequent arrangements of the phyla. This has a doubly-confounding effect on arrangements when combined with preconceived notions of parasites as degenerate organisms. Many previous hypotheses of metazoan relationships, such as that of Nielsen (1995), have been constrained by their a priori assumption that any lack of characters in parasitic groups is the product of secondary losses of previously evolved structures. These structures are considered to have been lost as a result of adaptation to a parasitic existence (see Brooks and McLennan, 1993b; 121-126 for a discussion and test of this hypothesis). This line of reasoning appears intuitive given the perceived lack of selection for these characters. However, for most parasitic groups, the assumption has not been tested in a phylogenetic context- One of the goals of the current study was to perform an analysis of the Acanthocephala that is as free of a priori assumptions concerning character evolution as possible, in which all decisions of character polarity were based on outgroups chosen by a methodologically rigorous analysis of the Metazoa. Because no previous studies of metazoans were found that met these criteria for rigor, it was necessary to perform the analysis of the Metazoa discussed earlier in this work (see Chapter 3). The results of that analysis place the Priapulida in the clade that is the sister group to the Acanthocephala + (Rotifera + Gastrohicha) + (remaining 'aschelrninths' and arthropods)) (Fig. 26). Based on their placement in the analysis above, the Priapulida and Rotifera were each used as outgroups in the analysis of the Acanthocephala presented below. At this time, the condition of most characters used in the following analysis is unknown for several of the classes of rotifers. Therefore, evaluation of the hypotheses of Storch (1979; Storch and Welsch, 1970), Lorenzen (1985), Garey et al. (1996) and Ahlrichs (1995,1997) that the Acanthocephala are a member of the Ro tifera, and thus represent parasitic rotifers, cannot be accomplished at this time.
Historical views of relationships within the phylum Evolutionary hypotheses
Koelreuther (1771) provided the first description of an acanthocephalan and proposed the name Acanthocephalus for species known from fishes. Miiller (1776) described another species horn fishes, naming it Echinorhynchus. The first classification of the group using distinct genera and species appeared in the thirteenth edition of Systema Naturae, edited by Gmelin, published between 1788 and 1793 (Amin, 1985). Several prominent 18th and 19fh century parasitologists were fascinated with Acanthocephala (e-g., (Goeze,1782; Schrank, 1782; Tilesius, 1810; Zeder, 1800; Zeder, 1803). Rudolphi (1808-1809) correlated existing information on acanthocephalans and established the first modem taxonomy of the group. He placed all acanthocephalans in the order Acanthocephala. The single genus Echinorhynchus was divided into two groups according to the presence or absence of body armature, or spines. Each of these groups was then subdivided on the basis of a different set of morphological characters. Other arrangements were produced by Westrumb (1821), Diesing (1851), and Vogt (1851). Hamam (1892) recognized the diversity of structure in the genus Echinorhynchus and further divided acanthocephalans into Echinorhynchidae, Gigantorhynchidae, and Neorhynchidae, based on differences in the structure of the proboscis, proboscis receptacle, and body wall. An increasing number of recognized species necessitated yet more revisions of Hamam's taxonomic system by Southwell and MacFie (1925),Travassos (1926), and Thapar (1927). This classification later formed the conceptual basis for the orders recognized by Meyer (1931) and Van Cleave (1936). Meyer (1931,1932,1933) proposed a comprehensive scheme based on characteristics of the lacunar system, protonephridia, and arrangement of proboscis hooks (Fig. 30). His system divided acanthocephalans into 12 families and 58 genera, and formed the foundation for the most widely accepted current classification. Van Cleave (1936,1941,1948) revised Meyefs system, elevated Acanthocephala to phylum level, and divided it into two classes and four orders (Fig. 31). Petrochenko (1956a, 1956b) devised a different taxonomic arrangement relying heavily on acanthor spination (Fig. 32). His attempt was largely ignored after attention was drawn back to the Meyer-Van Cleave system by Golvan (1959, 1960-1961,1962,1969; Golvan and Houin, 1964). Yamaguti (1963), who suggested that Golvan's system placed too much importance on the number of cement glands, proposed a system also based on the Meyer-Van Cleave arrangement, but focused more on external characters such as trunk spination (Fig. 33). Bullock (1969) expanded the Meyer-Van Cleave system to include additional morphological features such as distribution of main longitudinal vessels of the lacunar system, number of cement glands and ligament sacs, nature of subcuticular nuclei, embryonic membranes, and characteristics of the proboscis receptacle. He accommodated some features of Petrochenko's system as well as information on the nature of definitive and intermediate hosts, but did not propose formal changes to the higher taxa of Van Cleave's (1948) scheme. Other than additions of new species and subfamilial taxa, and the rearrangements of arc.hiacanthocephalans by Sdunidt (1972) and Schmidt and Kuntz (1977), little new insight concerning relationships among acanthocephalans has occurred. This arrangement has remained substantially intact, except for a few minor changes and the addition a new class, the Polyacanthocephala by Amin (1982,1985,1987). Phylogenetic hypo theses
It is important to note that none of the previous hypotheses of relatiowhips among acanthocephalans have been tested using rigorous phylogenetic methodology. To date, only two studies of acanthocephalans have included phylogenetic analyses in their procedures. Amin (1986) examined the relationships between three species of Acanthocevhalus found in North American fishes and ). Brooks and McLennan (1993b) examined proposed sister-groups of the Acanthocephala and phylogenetic hypotheses of relationships among the classes of acanthocephalans (Fig. 34). Brooks and McLennan (1993b), using the characters discussed by Conway-Morris and Crompton (1982) to indicate the plesiomorphic condition for acanthocephalans, explored higher level relationships within the phylum. They found the Eoacanthocephala and Palaeacanthocephala to be monophyletic, with the Archiacanthocephala paraphyletic. None of their three equally parsimonious trees completely resolved the relationships of these taxa. Except for these two studies, no previous authors have tested hypothesized rela tionships among acanthocephalans in a rigorous phylogenetic context.
Order Family Subfamily
Acanthogyridae Quadrigyridae - Palaeacanthocephala Rhadinorhynchidae Polymorphinae Polymorphidae Centrorhynchinae Fessisentidae --E Plagiorhynchinae Echinorhynchidae Echinorhynchinae Acanthocephala Cavisominae Pomphorhynchinae Neoechinorhynchidae Apororhynchindae - Archiacanthocephal Gigantorhynchidae Oligacan thorhynchidae Moniliformidae Pachysent idae
Figure 30. Tree depicting relationships among the Acanthocephala as suggested by Meyer, 1931. Palaeacanthocephala
Metacant hocephala
Archiacanthocephala
Acanthocephala Pallisentidae Cyracanthocephala Quadrigyridae Eoacan thocephala Neoechinorhynchidae L Neoacanthocephala 4 L Hebesomidae
Figure 31. Tree depicting relationships among the Acanthocephala as suggested by Van Cleave, 1948. Class Subclass Order Family Subfamily
Neochinorhynchinae Neochinorhynchidac! Gracilisentinae Neocchinorhynchida Hebcsomidae Eocollinae Atactorhynchinae ' Neoecl\inorhynchinea I Tenuisentidae Tenuisentinae Acanthogyridae Acanthog~rids Quadrigyrudae Hypoechinorhynchidae Echinorhynchidae Echinorh ynchinae Fessisen t idae Heteracanthocephalinae Cavisomatidae Leptorhynchoidinae
Acanthocephala - E~hinorh~nchinea-1 Pomporhynchidae Arhythmacanthidae Polyacanthorhynchinae Palymorphida -7 Rhadinorhy nchidadRhadinorhynehinae Telosentidae L Serrasentinae t Poly morphidae Poly morphinae Corynosominae tPlagiorhynchinae Pseudoacanthocephalidae Giganthorhynchida Filicollidae - Gigantorhynchinea Prosthorhynchidae Gigantorhynchidae Centrorhynchinae Apororhynchidae -€ Gigantorhynchinae
Moniliformidae Oligacanthorhynchida Oligaranthorynchidae Pachisentidae
Figure 32. Tree depicting relationships among the Acanthocephala as suggested by Petrochenko, 1956. Class Order Family Subfamily
' Apororhynchidca -Apororhynchidac Eocollinae Neoechinorhynchidae Neoechinorhynchinae . Neoechinorhynchide Hebesoma tidac Gracilisentinae Acanthogyridae Atactorhynchinae Quadrigyridae Tenuisentinae Pessisen tidae Hypoechinorhynchinae Filicollidae Cav isomatinae Diplosen tidae Leptorhynchidinae Echinorhynchidae Heteracanthocephalinae Acanthocephala . Echinorhynchide Pomphorhynchidae L Echinorhynchinae Plagiorhynchidae Sphaerechinorhynchinae +Plagiorh ynchinae Micracanthorhynchinidae serra,,nt inae Rhadinorhynchidae Polyacanthorhynchinae Illiosentinae Arhythmacanthidae--€ Rhadinorhynchinae Aspersentidae Corynosomatinae POlYmOr Phidae- Polymorphinae Centrorhynchidae Macracanthorhynchinae - Giganthorhynchidea Oligacanthorhynchidae Prosthenorchiinae Pseudacanthocephalidaef Oligacanthorhynchinae Prosthorhynchidae Giganthorhy nchidae Moniliformidae Figure 33. Tree depicting relationships among the Acanthocephala as suggested by Yamaguti, 1963. Figure 34. Relationships among the major groups of the Acanthocephala as interpreted by Brooks and McLennan, 1993 (after Brooks and McLennan, 1993). Chapter 5: Phylogeny of the Acanthocephala based on morphological characters
The Acanthocephala is a fascinating, enigmatic, and largely neglected phylum of parasitic helminths. Like most groups of parasites, they are potentially excellent model systems for studying general evolutionary principles and for examining the evolution of complex inter-specific ecological associations (Brooks and McLennan, 1991,1993b; Price, 1980). However, to date, only the two studies of acanthocephalans mentioned in the previous chapter have induded phylogenetic analyses in their procedures. Arnin (1986) examined the relationships between three species of Acanthoce~hahsfound in North American fish and Brooks and McLennan (1993b) examined proposed sister groups of the Acanthocephala and phylogenetic hypotheses of relationships among the classes of acanthocephalans. Amin's (1986) study was the first to address problems of acanthocephalan taxonomy using phylogenetic methods. He used 11 characters to evaluate the taxonomic status of six species of the Acanthoce~halus.Most of his characters were coded from continuous data, and some of the ranges for character states had values that overlapped, a method recognized today as problematical (Archie, 1985; Goldman, 1989; Scotland, 1992). Arnin (1986) proposed plesiomorphic states for the genus and did not use outgroups in his analysis. In addition, Amin (1986) was not explicit in details of his analyses (as was common for the time) and reported only a single tree. Reanalysis of his original coding for the data set produces multiple equally parsimonious trees (EPT), none of which have the same topology as his tree. The analysis of relationships among several major groups of Acanthocephala by Brooks and McLennan (1993b) was more rigorous than that of Amin (1986), but it also did not involve sufficient characters to resolve relationships within the Acanthocephala. Brooks and McLennan used 19 characters hypothesized by Conway-Morris and Crompton (1982) and the conditions they suggested were plesiomorphic. The results of their analyses produced three EPT (Fig. 34), a consensus of which was a polytomy with the only resolved dade the (Moniliformidae + Palaeacanthocephala). In addition, in one of their trees the archiacanthocephalans are basal in a second, eoacanthocephalans are basal, and in the third, all taxa occur in an unresolved polytomy. This does not detract from the value of their study, because it was only "...presented to stimulate interest in studying this enigmatic group" (Brooks and McLennan, 1993b; 370) and not intended as an empirical study. To date, these two studies (Amin, 1986; Brooks and McLennan, 1993b) are the only cladistic studies of relationships among acanthocephalans. Neither provides a strong basis for any decisions about relationships- Brooks and McLennan remarked that their results were "Based on the data [available] at present ..." (Brooks and McLennan, 1993b; 370), and that 'Clearly, more characters are needed to provide a robust starting point for elucidating the phylogenetic relationships of this fascinating group of parasites" (Brooks and McLe~an,1993b; 373). One of the aims of this study of the Acanthocephala was to identify a larger database of characters useful for a cladistic analysis of relationships among acanthocephalans. Since the first descriptions of acanthocephalans, the placement of the group in relation to other taxa has been problematical (Amin, 1985; Van Cleave, 1948). The most recent attempts at clarification of the relationships of the Acanthocephala to other taxa have used more rigorous cladistic techniques to understand metazoan evolution, but they have not provided more generally accepted hypotheses. The most prominent studies were discussed in the chapter on metazoan phylogeny. Schram (1991), Backeljau, et al. (1993), Eernisse, et al. (1992), and Schram and Ellis (1994) all postulated the Rotifera as sister group to the Acanthocephala, but Nielsen (Nielsen, 1995) and Nielsen et al. (Nielsen, et al., 1996) supported the Priapuiida. Although not in complete agreement, studies using molecular techniques also have suggested a close relationship between the Acanthocephala and Rotifera 122 (Garey, et al., 1996; Winnepenniflckx, et al., 1995). Based largely on similarity of the syncytial ectoderm found in both taxa, several recent workers have regarded acanthocephalans as parasitic rotifers (Storch and Welsch, 1970; Storch, 1979; Lorenzen, 1985; Ahlrichs, 1997), closely related to the bdelloids. This position is in accordance with molecular data (Garey, et al., 1996). Others consider the common structure of the ectoderm and shared embryology to be synapomorphies for the two phyla, and the differences in embryological origin of the rotiferan retrocerebral organ and the acanthocephalan proboscis to be apomorphic for each group, thus supporting the monophyly of each phylum (Nielsen, 1995; Nielsen, et al., 1996). In the present analysis, the Priapulida and Rotifera were each used as outgroup taxa. The Cestoda and Turbellaria (discussed in Chapter 4) have been suggested as sister-groups to the Acanthocephala, but, based on the results of the analyses of the Metazoa presented above (Chap. 4), the Platyhelminthes is not closely related to the Acanthocephala. Additionally, the Platyhelminthes was excluded from this analysis because it did not provide any clues to character polarity not seen by using the Priapulida, which was sister to the clade containing the Platyhelminthes (Fig. 26). The results of those analyses (Chapter 3) support the Acanthocephala as the most basal member of the Ecdysozoa, the clade containing Rotifera and the remaining ecdysozoans as the sister-group. Both the Priapulida and Rotifera were included in the analysis rather than producing hypothetical ancestors based on their position in their respective clades primarily because of past hypotheses of metazoan relationships (Conway-Morris and Crompton, 1982; Whitfield, 1971). In this way, primary homology of characters of each taxa that had been proposed as evidence of relationship to the Acanthocephala could be evaluated independently as a test of previous hypotheses. Despite the suggestions by Lorenzen (1985) and Garey, et al. (1996) that, without including the Acanthocephala as a highly derived rotifer the Rotifera may be paraphyletic, insufficient attempts have been made to evaluate the monophyly of the Rotifera in a broadscale phylogenetic context that included more than a few select characters. For the current analyses, it is sufficient that both taxa are diagnosably distinct from the ingroup and that their relationship is close enough to provide maximal polarization of character states within the ingroup. In the phylogenetic analysis of the Metazoa (Chap. 3), the relationship
Priapulida + (Rotifera i-Acanthocephala)) was consistent the results of all analyses. Excluding features of the cement glands, discussed below, the Priapulida provided polarization for 30 characters, and the Rotifera for 40 characters, 18 of which were also scored for priapulids (Appendices W; VIII). These numbers represent the approximate number (42) of homologous structures that could be identified in both priapulids and rotiferans. The remaining characters pertain to features of those structures which are derived within the Acanthocephala. Lack of coding of these characters does not represent missing data.
Materials and Methods
Phylogenetic systematic analyses were performed using methodology outlined by Hennig (Hennig, 1966), Wiley (1981), and Wiley, et al. (1991). Heuristic search procedures were carried out using Paup 3.1.1 (Swofford, 1993) under the options "addition sequence simple" with "TBR branch-swapping, h4ULPARSWoption in effect, and by collapsing zero-length branches. Because of the large size of the data matrix, Branch and Bound searches were made using Hennig86 (Farris, 1988) with the commands "cc-; rnh bb;". Assessment of branch support for individual branches (Bremer index; Bremer, 1988) was calculated using TreeRot (Sorenson, 1996). Total support (Kallersjo et al., 1992) and total support indices (Bremer, 1994) were calculated according to Bremer (1994). Information concerning outgroup taxa was largely taken from published descriptions of morphology and the works of Hyman (1951), Willmer (1990), and Nielsen (1995). In addition specimens of adults of both taxa were also examined. Among other characters (see Appendix W;Vm), the Rotifera and Acanthocephala share a common embryological pattern (Chars. 136,137, and 138; CLE1, CLE2, CLE3 in the analysis of the Metazoa), gonochorism (Char. 133), and absence of mitosis in epidermal cells (Char. 28 in the analysis of the Metazoa). These characters are not found in the Priapulida; relationships of the three phyla are discussed further in Chapter 3. Thus, all trees were rooted with the Priapulida and Rotifera as paraphyletic. The study included over 1340 specimens comprising 73 species of Acanthocephala (Appendix VI). Of the 32 genera represented by these specimens, 21 genera were found to be sufficiently well-described and represented by a great enough number of specimens such that identification of the concept of the genus was not ambiguous. Fifteen of these genera were represented by more than one species, so specimens from multiple species from those genera were evaluated for each character. This insured that the species used in the analysis were an accurate representative of the concept of the genus as it is currently defined. Thus, these 15 species represent the current concept of the genus of which they are members, and serve as exemplars of that genus. Each of the 22 species included in the analysis possesses the character states discussed in the character argumentation (Appendix VII) and coded in the data matrix (Appendix VIII, A). Those genera that were represented by too few specimens, or encompassed a concept that required major revision, were excluded from the analysis pending revision of all currently assigned species and the concept of the genus. An example of this sort of taxonomic problem is discussed below (pg. 137). Except for taxa described since 1985, nomenclatural designations and authorities for taxa are those of Amin (1985), and are given in Appendix VI. Some specimens (e.g., Pach sentis sp., Neoechinorh us sp., and Phadinorh represent undescribed species which were collected under the auspices of the Colecci6n Nacional de Helminthosf [Ntituto de Biologia, Universidad Nacional Authoma de M&cico,M6xico City, Mexico. The type material for each species will be placed in their collections after the descriptions are completed. Other specimens (see Appendix VI) are retained in the author's personal collection. Each specimen was evaluated for 138 morphological and developmental characters, 134 of which were recognized by direct examination of specimens. The remaining four characters were taken from published studies. The three early cleavage characters (Chars. 136,137,138 = CLEI, CLEZ CLE3 in Chap. 3) were taken from the analysis of the Metazoa in Chapter 3. Reproductive mode was assessed by one character (Char. 133 = Chars 117 and 119 in Chap. 3), also taken from the analysis of the Metazoa. Embryo type, anechinate, herniechinate, and holoechinate were assigned based on published descriptions. All other characters were assessed only by direct examination of specimens. Whenever possible, characters were polarized using outgroup comparison (Lorenzen, 1993; Maddison, et al., 1984). Characters found only in the ingroup, or with uncertain homology of character states between the ingroup and outgroup, were coded as "unknown" in the outgroup (see Appendix W). The lack of understanding of homology between many of structures that could have a common origin in the Rotifera and Acanthocephala prohibits making a large number of hypotheses of character evolution. Even among structures that appear to be putative homologues, the potentially ancient origin of the phyla has obscured much of the information necessary to directly relate fine structural components between taxa. For others, primary homologies between gross structures are more clearly indicated, but either fine structural features common to acanthocephalans are missing in outgroups or there are several transformation states which occur in the outgroups for which no phylogenetic hypothesis exists. One example is the cement glands, a feature common to several invertebrate groups. 126
Cement glands occur in two general forms, as individual glands (or follicles, sensu Van Cleave, 1949) or as a syncytium. Both conditions occur in the Rotifera and in the Acanthocephala, but it is unknown whether syncytial or individual glands is plesiomorphic. Various other syncytial structures occur in both of these taxa, as well as other invertebrate groups, suggesting that the presence of syncytial structures could have been independently derived more than once (Willmer, 1990). Brooks and McLeman (1993b) showed that the polarization of the cement gland strongly affected the outcome of phylogenetic analysis based on a small number of characters presented by Conway-Morris and Crompton (1982). Consequently, three assumptions of character evolution of cement gland structure within the Acan thocephala were explored. Assumption i-The plesiomorphic type of cement glands in the Rotifera is undetermined. This hypothesis provides the least amount of information concerning characters related to the structure of the cement glands. The outgroup state for only three of the 16 characters (chars. 83,87, and 88; Appendix Vm, 8) could be coded under this assumption. Assumption ii-Non-syncytial cement glands are plesiomorphic. This hypothesis provides the most information concerning cement glands (10 chars.). The outgroup state for seven additional characters were added to the three coded as plesiomorphic under Assumption i. ass urn^ tion iii-Syncytial cement glands are plesiomorphic. Characters 74, 78,80, and 84 (Appendix VIII, B) relating to syncytial features coded as "absent" or "unknown" under Assumption fi were changed to "present" and the coding of four characters was changed to "unknown". Results
Phylogenetic analysis of the data set produced a single most parsimonious tree topology. Using Assumptions i and fi for the cement glands, the analyses produced the same single most parsimonious tree (length = 404 steps, CI = 0.545, CIX = 0.517, HI = 0.455, HIX = 0.483, RI = 0.670, RC = 0.365). Using Assumption the tree was four steps longer (length = 408 steps, CI = 0.539, CIX = 0.512, HI = 0.461, HIX = 0.488, RI = 0.665, RC = 0.359). Bremer support indices for individual branches under the three assumptions of cement gland evolution are given in Table EI and total support indices are given in Table IV. Total support was higher under
Assumptions i and 11 (-t = 56) than Assumption iii (1 = 44) as was the total support index (G = 0.14 vs. = 0.11). The tree (Fig. 35) indicates that the Palaeacanthocephala and Eoacanthocephda both form monophyletic groups and are sister taxa. The members of the Archiacanthocephala are basal to the other two clades, but do not themselves form a clade. TABLE111. Branch support (h) (Bremer, 1988) calculated for each node of the tree depicting relationships among the Acanthocephala (Fig. 35) under the three assumptions of cement gland structure. TABLEW. Total support indices for the tree depicting relationships among the Acanthocephala (Fig. 35) under the three assumptions of cement gland structure. Note: & = total support, s = length of the tree, and a = total support index; ti = (1/S ) (Bremer, 1994). Discussion
Bullock (1969) suggested that acanthocephalans possess a rich store of morphological characters for phylogenetic analyses. This study supports that contention. Examination of over 1340 specimens comprising 73 species of Acanthocephala yielded 138 morphological and developmental characters. Polarization of these characters was possible using the Priapulida and Rotifera as outgroups, as indicated by the analyses of metazoan relationships in Chapter 3. Analysis of these characters produced a single tree (Fig. 35) in which relationships among 21 genera were fully resolved and well supported. Brooks and McLennan (1993b). in the study discussed above, used 19 characters to explore higher level relationships within the phylum, but none of their three EPT completely resolved the relationships of these taxa. In addition, in one of their trees the archiacanthocephalans are basal in a second, eoacanthocephalans are basal, and in the third, all taxa occur in an unresolved potytomy. These different trees were produced by changing only the plesiomorphic condition of type of cement gland from non-syncytial to syncytial. Analysis of the much larger data set in my study yielded a single tree under each of the three assumptions of cement gland evolution. Using Assumptions i or k the resultant tree was the same as under Assumption a but it was four steps shorter. If Assumption ~ that syncytial cement glands were plesiomorphic, was used, and the Eoacanthocephala were forced to be basal, 14 extra steps were required, producing a much less parsimonious result. As well, values for total support and total support indices are higher under Assumption (f = 56; ri = 0.14) than Assumption iii (1 = 44; fi = 0.11). These suggest that individual cement glands are plesiomorphic for acanthocephalans, and syncytial glands arose independently in the Rotifera and Eoacanthocephala (Assumption u). This hypothesis can be tested further with additional information from a robust phylogeny of the Rotifera. Brerner (1988) suggested that tree stability could be assessed by determining the number of character changes needed in the data matrix to yield a different arrangement. Thus, those clade with strong support provide more bold hypotheses on which to base character evaluations. Kuersjo et a1 (1992) later proposed that the number of steps required to collapse a particular branch be termed "Bremer support" @). Clearly, the most robust hypotheses depend upon higher values of although Bremer support values can only be compared within a given data set (Bremer, 1992). Bremer (1994) later proposed a tree stability index (dcalculated by dividing the total support for the tree (t = sum of Bremer indices for all nodes of a tree) by the length of the tree (2). The total support index provides a measure of tree stability in terms of supported resolution rather than in terms of synapomorphy and homoplasy (Bremer, 1992); information for the latter two are provided by the consistency and retention indices (Farris, 1989). In the absence of homoplasy, with a single completely resolved tree, f = s and = 1, and in a completely unresolved tree, & = 0 and ti = 0. No confidence interval has been calculated for but, as with the Bremer support index, larger values indicate stronger support. In a study of 12 phylogenetic analyses of morphological and molecular data sets for plant taxa, Bremer (1992) calculated values ranging from 0.07 to 0.40. The overall average value was ti = 0.23, as were the individual total support values for both molecular and morphological data sets; six data sets had values below the average and six above. The value of a = 0.14 for the data under Assumptions i and fi is below the average in Bremer's (1992) study, but 12 of 21 nodes (Table III) had Bremer support values greater than one. As discussed later in relation to support for the monophyly of the Archiacanthocephala (pg. 134435). it is likely that a greater understanding of characters within the outgroups resolve some questions concerning homoplasy among the classes. Only weaker support (h = 1) is seen for 132
the basal nodes within the Palaeacanthocephala. This may be a result of the limited representation of members of this very diverse group. Despite the greater number of characters used in the current study than in the previous analyses cited above, there is no support for the monophyly of the Archiacanthocephala as a monophyletic group. None of the species, which included three from the class Oligacanthorhynchidae, were grouped together. Many of the characters used taxonomically to diagnose the class (Amin, 1982,1987; Van Cleave, 1923a) are plesiomorphic. The class may not be a natural group, but examination of more taxa currently assigned to the class is necessary to determine if there are any synapomorphies for it. It is also possible that character states coded herein as "unknown" in the outgroups are unduly constraining the analysis by not providing information necessary to idenbfy putative synapomorphies. Each of these features must be more closely examined in the outgroups to evaluate actual status and primary homology. Petrochenko (1956a) considered the Archiacanthocephala to be the most recently-derived of the acanthocepalan classes and postulated Eoacanthocephalan ancestors for the Acanthocephala. In contrast, Meyer (1932,1933) characterized the Archiacanthocephala, as evidenced by their name, as being the most primitive among acanthocephalans, although it should be noted that he originally placed eoacanthocephalans within this group, and his hypothesis of primitiveness was based on their inclusion. Since the Eoacanthocephala is not part of the Archiacanthocephala, neither of their hyotheses are supported by the results of this analysis. If the Archiacanthocephala are most like the ancestors of the phylum, as their basal placement suggests, they would be expected to possess a high proportion of plesiomorphic characters. Archiacanthocephalans do not form a group, but all are basal to the the clade Eoacanthocephala + Palaeacanthocephala (Fig. 35), and all five species, as well as the other archiacanthocephalan species examined but not included in the analysis (Appendix VI), possess many plesiomorphic characters. 133
Brooks and Bandoni (1988) and Brooks and O'Grady (1989) have suggested that very old parasite groups may be "relicts" ($emuSimpson, 1944 and Brooks and Bandoni, 1988) of a formerly more spedose group that has been reduced because of extinction of host species. This hypothesis cannot be applied to the Archiacanthocephala at this time because the phylogenetic hypothesis presented here does not include all members of the dass, but several factors suggest that archiacanthocephalaw might fit this pattern. First, the results of this study support the species of the archiacanthocephala as representing the oldest members of the phylum, and thus, possibly are phylogenetic relicts. Petrochenko (1956a) considered use of only mammals and birds as definitive hosts a result of ecological 'advancement', but current restriction of hosts to only these two groups could also be viewed as resulting from extinction of now extinct early fishes or reptiles. Second, the Archiacanthocephala is numerically less than the other two groups, and may also be a numerical relict. If the number of genera is used to represent diversity in general type within the Acanthocephala, the ratio of genera of Archiacanthocephala: Eoacanthocephala: Palaeacanthocephala is 1.0: 2.2: 6.4. Admitedly, the number of genera is an artifact of taxonomy, but the ratio of species in each group somewhat supports a similar conclusion (1: 1.1: 3.7, respectively) and the Archiacanthocephala only comprises about 21% of the known species of the Acanthocephala. Inclusion of a greater number of species in future studies will allow status as relicts to be evaluated and possibly determination of whether the class is a phylogenetic relict, a numerical relict, or both. This will also permit evaluation of ecological diversification among the classes, and more extensive comparisons between host and parasite phylogenies. In contrast, the phylogenetic relationships among the Palaeacanthocephala and Eoacanthocephala are fully resolved, and did not change under any of the three assumptions. Within each class, however, at least one currently-defined family was paraphyletic. Not all families are included in this analysis, but it does indicate genera that should be reexamined for indusion within other groups. The Eoacanthocephala is monophyletic based on five unambiguous character state changes (71,202,532,741, and 841). The Brerner support index for the node is h = 4. Among the Neoechinorhynchidae, the Neoechhorhynchinae is paraphyletic unless Floridosentis is included (Fig. 35). The palaeacanthocephalans included in this study also form a monophyletic group based on 11 unambiguous character state changes (23,302,552, 662, 691, $81,
962,982,1071,1164, and 1301). The Bremer support index for the node is = 2. Within the class are two subgroups. Le~torhvnchoidesand Koronacantha form a clade supported by six synapomorphies (511,870,880,921, 1011, and 1162). Four genera currently assigned to the Uiosentidae form a monophyletic group based on five unambiguous character state changes (182,332,980,1023, and 1092). The Rhadinorhynchidae is paraphyletic, with Rhadinorhvnchus the sister group of the clade containing Leotorhvnchoides + Koronacantha and the Illiosentidae. Telosentis, currently considered a member of Illiosentidae, requires 13 extra steps to include it with the other Illiosentidae. Neither Van Cleave (1923b), in his original description of Telosentis, nor Kostylew (1926). in his study of 1.exiguus (Linstow 1901) Kostylev 1926 (erroneously attributed to Van Cleave, 1923b in Golvan, 1969 and Amin, 1985) made reference to the number of cement glands in Telosentis. Yet, in their review of the Rhadinorhynchidae, Van Cleave and Lincicome (1940) listed Telosentis with those genera possessing eight cement glands. The co-types of -T. molini and specimens of 1.exiguus examined for this study possess only four cement glands. This clearly indicates the need for an examination of all current material and a revision of the genus. The Eoacanthocephala and Palaeacanthocephala form a monophyletic group based on seven unambiguous character state changes (51,241,250,593,701,1001, and 1311). The Bremer support index for the node is h = 2. Of these, only the absence of an apical sense organ (240) has been used in the past as diagnostic of these classes. Although various anterior sensory organs in the outgroups have been identified (WiIlmer, 1990), homology of these structures with those of the Acanthocephala is unknown. Despite that, this analysis supports the interpretation that the Eoacanthocephala and Palaeacanthocephala have secondarily lost the apical sense organ that is present in the Archiacanthocephala. Petrochenko (1956a) and Van Cleave (1941) (and Meyer, 1931, in with respect to the neoechinorhynchids) suggested that the Eoacanthocephala was the most primitive of the three dasses included in this study. This idea was based on the lack of variability among members of the class, especially in relation to the proboscis and its armature, presence of syncytial cement glands, single-walled receptacle, delicate egg membranes, giant nuclei, and absence of body armature, embryonic armature (i.e., anechina te) and low host diversity (primarily fishes). Petrochenko (1956a) further suggested that the Echinorhynchidae (whichhe considered a "...branch of the Neoechhorhynchida ..." (Petrochenko ,1956a; 150) were nearly as primitive as the neoechinorhynchids. The Echinorhynchidae (represented by Acanthocephalaus and Echinorhynchus in this study), are currently assigned to the Palaeacanthocephala. This assignment is supported by the results of this analysis, but they are mentioned here because they are the most basal members of that clade. Petrochenko (1956a) also suggested that Leptorhynchoides and Filisoma both showed a plesiomorphic lack of body spines. The analysis supports this hypothesis for Filisoma, given the included taxa, but not for Leptorhynchoides. He further supported placement of Telosentis in a separate family, the Telosentidae Petrochenko, 1956, based on a more anterior location of the cerebral ganglion. He postulated that Telosen tis would be placed "...intermediate.. ." (Petrochenko, l956a; 152) between the Rhadinorhynchidae and the Arhythmacanthidae. His inclusion of the Arhythmacanthidae was based on shared davate-shaped cement glands, but he considered the Telosentidae distinct beause of its genital spines. The Polyacanthorhynchidae, which Amin (1987) considered to be a separate class, were omitted because insufficient specimens were available at the initial stages of the study. As discussed by Amin (1987), Golvan (1956,1962), and Petrochenko (1956b), the characters used to diagnose the family are common to more than one class, so it is expected that the class will not be supported and the family will be placed within one of the other classes. The status of this group must be tested by future analyses. One goal of this study was to test the placement of '&gorhynchus pectinarius in Koronacantha and the proposal to consider &orhvnchus and Illiosentis as distinct taxa by Monks and Perez-Ponce de L&n (1996) and Monks, et al. (1997). Koronacantha mexicang and K. pectinaria form a dade based on nine unambiguous character state changes (83,162,183,& 352,383,811,1154, and 1170) and a Bremer support index of h = 6. Dollfusmtis is the sister group to Jlliosentis and Tegorhynchus, and the three form a clade supported by five unambiguous character state changes (542,1022,1172~121~,1172,and 12621, although the Bremer suppoa index for the node is h = 1. The recognition of the independent status of Tegorhynchus and Illiosenti~is well supported; -orhynchus, on the basis of five apomorphies (181,191,222, 442, and 1155) and nliosentis, nine apomorphies (142, 15~,363,475,595,642,710,913, and 1052). As with many of the characters used in this analysis, eight of these changes are homoplasious at the class level (I@, 191, Z2 and 152,363,595,710,933, respectively), but only three are homoplasious within the Illiosentidae (152,363, and 710). The terminal node of the Illiosentidae, containing Tegorhynchus and Illiosentis, has a Bremer support index of h = 4. Thus, the results of this study provide strong support for the recognition of both genera as distinct taxa, as well as the transfer of 1.pectinarius to Koronacantha as suggested by Monks and Perez-Ponce de Le6n (1996) and Monks, et al. (1997). However, evaluation of placement of Koronacanthq within the Illiosentidae must await further inclusion of taxa. Taxonomic revision
One of the prerequisites of phylogenetic studies are that terminal taxa are clearly defined and monophyletic, or natural groups @mu Wiley, 1981; 72-73) if species are the terminal taxa. As discussed above in the historical review of the Acanthocephala, both higher taxa as well as species have often been described on the basis of only a few characters. Given the known species of the particular era, these each group could easily be distinguished using only these few characters. However, as additional species were discovered, the perceived unique quality of many of these characters was diminished. One example is the presence of body spines, genital spines, and parasitism of marine fishes, a suite of characters once thought to distinguish Telosentis. Later it was necessary to expand these application of these characters to include Illiosentis, however, species of Illiosentis were subsequently found that did not possess genital spines. In a phylogenetic context, these three characters are homoplasious because each is found in several additional taxa not included within the Illiosentidae. The necessary first step in producing the set of characters used in the current phylogenetic analysis was to determine if specimens assigned to particular species formed natural groups. In some cases, it was necessary to examine a large number of specimens, over 100 for some species, to ensure that the presence of characters used in the analysis was consistent within the group. Nine genera
(Caballerorhvnchus. Centrorhvnchu~ 8 Corvnosorna. Fessise no& m~na~ug, Neoacanthoce~haIoides.Oncicola, Polymorphus. and Porn~horhynchudwere excluded from the analysis because either insufficient specimens were available at that time (three genera) or because the concept of the more speciose genera was defined so broadly that no single species could be chosen to represent the genus (six genera). One of the most problematic areas of this study was clarification of previous taxonomic assignments of species. Questionable placement of some taxa, such as Telosentis, was not obvious until after the analysis was performed. For others, initial examination of specimens clearly indicated a need for taxonomic revision prior to the actual analysis and before questions concerning the concept of particular genera could be assessed. One such genus is wrhvnchus, currently assigned to the Illiosentidae. A review of taxonomy of the genus is given in the next section. In addition, as an example of the taxonomic problems that hamper those attempting to study phylogenetic relationships among acanthocephalans, descriptions of a new genus and a new speaes, redescription and transfer of a species to the new genus, and re-establishment of two genera are presented below.
Description of Koronacantha mexicana n. gen., n. sp.
During a study of helminth parasites of marine fishes of Chamela Bay, Jalisco StateI Mexico, numerous acanthocephalans were collected from the intestinal tracts of six species of fish. Among the parasites collected were specimens of an undescribed acanthocephalan genus and species which is described below.
Materials and methods
Commercial fishermen captured specimens of Anisotremus intern 1862), Haemulon sexfasciaturn Gill, 1863, H. sudden Gill, 1863, Pomadasvs leuciscus (Giinther, 1864), and Eugerres sp. Jordan and Evermann, 1927, with gill nets and long lines 1-2 km from the shore of Chamela Bay, Jalisco State, Mexico (19O31' N, 105O04' E) during March 1995. Acanthocephalans were removed from the cecae and intestine, allowed to die in refrigerated tap water, transferred to AFA for 24-48 hr, and then stored in 70% ethanol. Specimens were stained with either Mayer's carmalum or Ehrlich's hematoxylin and mounted in Canada balsam for examination as whole mounts. Figures were drawn with the aid of a camera lucida. Measurements were taken from sexually mature specimens as indicated by the presence of sperm in the seminal vesicle of males and shelled eggs in the body cavity of females. Measurements are in micrometers unless otherwise stated; for some traits, ranges are given, followed in parentheses by mean values f1 standard deviation (x = mean; n = sample size). CNHE refers to the Colecci6n Nacional de Helmintos, Instituto de Biologia, Universidad Nacional Autonoma de M6xic0, Ciudad de Mexico, Mexico; FIOCRUZ refers to the Colg3o Helmintologica, Ins ti tu to Oswaido Cruz, Rio de Janeiro, Brasil; HWML refers to the University of Nebraska State Museum, Harold W. Manter Laboratory, Division of Parasitology, Lincoln, Nebraska, U.S.A.; INBIO refers to the Instituto Nacional de Biodiversidad, San Dorningo, Heredia, Costa Rica; NHR refers to the Naturhistoriska Riksmuseet (Swedish Museum of Natural History), Stockholm, Sweden; USNPC refers to the US. National Parasite Collection, BeltsviUe, Maryland, U.S.A., VC refers to Van Cleave's original numbering system.
Description Koronacantha n. gen.
TNnk cylindrical, greatest width approximately at anterior one-third. Proboscis cylindrical, elongate, bearing numerous longitudinal rows of hooks, with heavy cuticle Posterior-most hooks in each row small, thin, rootless, forming closely set, comb-like series of thorns. Heavy, strongly recurved hooks shaped like inverted apostrophe immediately anterior to comb-like hook rings; hook blades small, roots simple but exaggerated in size. Remaining hooks with simple roots. Armature similar in both sexes with slight dorso-ventral dimorphism of size; dorsal hooks slightly larger. Trunk spines present, extending over most of trunk length. Main canals of lacunar system lateral. Proboscis receptacle sheath double-walled, attached at anterior end of neck; cerebral ganglion immediately anterior to mid-receptacle. Two lemnisci, long, not bound to body wall at distal ends, reaching midbody. Body wall thin, containing highly branched subcuticular nuclei. Single ligament sac, persistent, ruptured in adult- Testes oval, tandem, contiguous. Eight claviform cement glands. Genital pores terminal. Females with band-like vestibular muscle; posterior lacking muscular protuberances. Type and only species: Koronacantha mexicana n. sp.
Koronacantha mexicana n. sp.
Description (based on 20 male and 14 female specimens): Trunk cylindrical, greatest width approximately at anterior one-third. Proboscis (Fig. 36A, B) cylindrical, elongate, bearing 12 (n=32) longitudinal rows of hooks, with heavy cuticular coating; each row with 20 to 26 hooks (2M, n=22). Posterior four or five hooks in each row small, thin, rootless, forming a closely set, comb-like series of thorns. Heavy, strongly recurved hooks shaped like inverted apostrophe immediately anterior to comb-like hook rings; hook blades small, roots simple but exaggerated in size. Remaining hooks with simple roots showing little regional modification. Armature (Table V) similar in both sexes with slight dorso-ventral dimorphism of size; dorsal hooks slightly larger (Fig. 36E, F). Single lateral sensory papilla (sensu Nick01 and Holloway, 1968) present on each side of neck at approximately mid-neck (Fig. 378). Trunk spines (Fig. 37F), most with small rootlets, extending from posterior end of receptade to posterior end of trunk. Genital spines on both female and male identical to trunk spines; four genital spines distributed equidistantly from each other in circle around genital pore. Main canals of the lacunar system lateral; secondary branches simple. TABLEV. Range of hook lengths of 10 mature male and female Koronacantha mexicana. 'Apostrophe'refers to the apostropheshaped hooks and 'comb'refers to the comb-like posterior hooks.
Male Female Hook Dorsal Ventral Hook Dorsal Ventral no. no. -
1-6 32-41(37S) 31-39(363+) 1-6 41-47 (W) 31-39 (363+)
7-9 38-41 (3%) 38-43 (41s) 7-9 41-47 (4W) 38-43 (4M)
11-13 38-44 (41s) 35-42 (3833) 11-13 33-50 (4W5) 35-42 (38s)
14-15 35-38 (36Q) 28-30 (29i1) 14-15 32-38 (3W) 28-30 (2%1)
16-17 28-35 (3M) 16-19 (17s) 16-17 23-28 (2W) 16-19 (17s)
'apostrophe' 13-19 (16s) 17-20 (19f1) 'apostrophe' 15-19 (17f2) 17-20 (19fl)
'comb' 13-17(15E) 16-19 (17s) 'comb' 16-17 (1&1) 16-19 (17s) Proboscis receptade sheath double-walled, attached at level immediately posterior to last row of hooks at anterior end of neck; length of outer wall less than inner wall (Fig. 37B). Cerebral ganglion just anterior to mid-receptacle, retinacula emerging from receptade at approximately or slightly posterior to mid-receptacle. Two lemnisci, long, subequal, often coiled or twisted, not bound to body wall at distal ends, reaching to midbody; one lemnixus typically approximately 80% length of the other. Nuclei in lemnisci elongate, partially fragmented in mature specimens. Body wall thin, containing highly branched subcuticular nuclei not fragmented in mature specimens (Fig. 37C). Genital pores terminal. Male: (Fig. 36A-F, 378, D): 4121 to 7282 (52e761, n=19) long, 477 to 706 (6O3f 75, n=19) wide at greatest width. Proboxis 687 to 1,024 (838k106, n=17) long, 95 to 145 (11210, n=19) wide. Neck 35 to 69 (49f 10, n=18) long, 88 to 126 (107k10, n=16) wide. Proboscis receptade 1,021 to 1,528 (124Ok127, n=19) long, 123 to 198 (150+23, n=19) wide. Cerebral ganglion 183 to 284 (225-0, n=17) long, 32 to 57 (M8,n=16) wide. Short lemniscus 1,254 to 2,630 (2,050+403, n=18) in length, long lernniscus 1,717 to 4,171 (2,581k617, n=lS) long; width of lemnisci equal, 44 to 88 (59+11,n=19) wide. Male reproductive system 2,104 to 4,347 (2,7=55, n=20) long, occupying approximately 53% of trunk length. Testes oval, tandem, contiguous with slight overlap. Anterior testis 432 to 857 (631+120, n=19) long and 164 to 271 (202328, n=19) wide; generally longer and less wide than posterior testis. Posterior testis 347 to 797 (%Sf 111, n=19) long and 183 to 258 (216+23, n=19) wide. Vas efferens from each testis joining seminal vesicle separately, 432 to 1,101 (697k155, n=20) long; 643 to 1,101 (79W135, n=7) long in specimens with bursa extended, 432 to 989 (646k146, n=13) long in specimens without bursa extended. Vas efferentia slightly widened to provide two to four individual seminal reservoirs arranged tandemly (Fig 36D); reservoirs with terminal sphincter. Vas deferens not present. Seminal vesicle elongate oval, 205 to 359 (275k49, n=20) long and 63 to 164 (120m, n=20) wide. Ejaculatory duct 126 to 318 (l77i56, n=18) long and 35 to 63 (46k9,n=18) wide. Eight elongate, slightly daviform cement glands, each with two granular unfragmented nuclei (Fig. 36C); individual glands 101 to 227 (178k34, n=20) long and 57 to 117 (9lf 16, n=20) wide (Fig. 36C). Cement duct from each gland slightly widened anteriorly to provide individual cement reservoirs. Saefftigen's pouch 410 to 977 (614+125, n=19) long, 132 to 227 (171=, n=18) wide, located immediately posterior to the duster of cement glands. Female: (Fig. 37A, C, E, F): 4,388 to 10,449 (6,8888211,542, n=14) long, 490 to 731 (607k86, n=14) wide at greatest width. Proboscis 756 to 1,159 (1,002132, n=14) long, 101 to 158 (133k14, n=14) wide. Neck 35 to 63 (51k9, n=14) long, 101 to 158 (125t18, n=14) wide. Proboscis receptacle 1,024 to 1,821 (1,407k231, n=13) long, 134 to 224 (186k27, n=14) wide. Cerebral ganglion 197 to 258 (W7,n=9) long, 32 to 50 (40+7, n=9) wide. Shorter lemniscus 2,007 to 2,835 (2,448+379, n=5) in length, longer lemniscus 2,400 to 3,900 (2,905+546, n=7) long; width of lemnisci equal, 47 to 63 (52+5, n=7) wide. Female reproductive system 665 to 951 (763k73, n=14) long, occupying approximately 11% of trunk length. Single ligament sac, persistent, but ruptured in adult, connected inside uterine bell to egg sorting apparatus. Uterine bell 189 to 268 (240-6, n=13) long, 38 to 110 (78k19, n=14) wide at anterior opening (Fig. WE), slightly tapering to junction with sorting apparatus. Uterus 135 to 208 (161e0, n=14) long, 38 to 63 (5127, n=14) wide. Vagina elongate, 126 to 167 (l49f 13, n=13) long, 28 to 47 (38k6, n=13) wide. Mature eggs elongate fusiform, prolongation of inner membranes slightly pinched at the poles (Fig. 37G); size measured through body wall 66 to 72 (69E3, n=7) long, 11 to 19 (169, n=7) wide. Females lacking posterior muscular protuberance; genital vestibule not found. A pair of moderately developed bands of vestibular muscle with fan-shaped posterior attachment extending from dorsal body wall at level of uterine bell posteriorly to ventral body wall adjacent to genital pore (Fig. 37E). Taxonomic summary Host: Anisotremus intemmtus (Gill, 1862). Other hosts: Haemulon sexfasciatwn Gill, 1863, maGill, 1863, Pomadasys leuciscus (Giinther, 1864), and Wrressp. Jordan and Everrnann, 1927. Site of infection: cecae and intestine. Locality: Charnela Bay, Jalisco State, MWco (19'31' N, 105'04' E). Holotype: CNHE 2740. Paratypes: CNHE 2741-2747; HWML 39119; USNPC 8675886759. Voucher specimens: CNHE 2748 Etymology: The genus is named for the apostrophe-shaped hooks on the proboscis. The name is a combination of the Greek words koronis, an apostrophe-shaped flourish, and &antha, a thorn-Like hook. The species is named for M6xic0, where it was first discovered.
Discussion
The elongate proboscis with a heavy cuticle, cuticular body spines, and eight cement glands indicate that Koronacantha should be assigned to the family Illiosentidae Golvan, 1960. The new species differs from species of Telosentis Van Cleave, 1923, the only other illiosentid reported to have genital spines in both sexes, by possessing (1.)genital spines identical with trunk spines, (2.) genital spines spaced equidistantly around genital pore, (3.) lacking trunk spines on the anterior body, (4.) band-like vestibular muscle with fanshaped posterior attachment, (5.) lateral sensory papilla on the neck, and (6.) an elongate vagina. Telosentis possesses (1.)genital spines smaller and finer than trunk spines, (2.) genital spines not spaced equidistantly around genital pore, (3.) hunk spines on the anterior body, (4.) vestibular muscle absent, (5.) lateral sensory papilla absent, and (6.) a short, rounded vagina. In these characters, Koronacantha mexicana most resembles Dollfusentis Golvan, 1969, Tegorhynchus Van Cleave, 1921 and Illiosentia Van Cleave and Lincicome, 1939. The new species possesses a single pair of band-like vestibular muscles with fan-shaped posterior attachment whereas in Dollfusentis the vestibular muscles are double pairs of band-like muscle with narrow attachment and in Illiosentis and &gQrhynchusthey are pad-like. It further differs from Illiosentis and Doll fusentis by lacking long proboscis hooks in the posteriormost ring, lacking trunk spines on the anterior body, having trunk spines extending posteriorly past mid-body, and possessing apostrophe-shaped hooks. Therefore, we propose the new genus to accommodate the new species. Koronacantha mexicana can be further distinguished from Illiosentis by the following characters: (1.)the cerebral ganglion in K. mexicana is located just anterior to mid-receptacle and in Illiosentis it is located at the attachment point of the receptacle and the proboscis, (2). the posterior end of female K. mexicana is rounded and simple because it lacks the large, thick pad-like vestibular muscle attached to the posterior body wall which forms a transverse cleft at the posterior end of female Illiosentis, and (3.) the lateral sensory papilla is located in the neck in K. mexicana and in the proboscis in Illiosentis. The new genus can be further distinguished from Dollfusentis, in which (1.) the cerebral ganglion is located in the neck, and (2.) the lateral sensory papilla is located in the proboscis. Koronacantha mexicana has two nuclei in each cement gland whereas flliosentis. Dollfusentis, and mrhvnchus all have more than 20 nuclei in each gland. The new species further differs from Tegorhynchus in the following characters: (1.) IS. mexicana lacks the blunt lateral papillae described in worh chus, and (2.) in the new species the cerebral ganglion is located just anterior to mid-receptacle, but in wrhynchus, it is at the anterior extremity of the receptacle. Bullock and Mateo, in an abstract of a paper given at the Second Intemational Congress of Parasitology (Bullock and Mateo, 1970, Proceedings of the Second International Congress of Parasitology, Part 1, Journal of Parasitology 56: 41-42) suggested that Tegorhychus vectinarius Van Cleave, 1940 should not be retained in the genus, but did not formally place the species in a new genus. The two original specimens of 1.pectinarius have been lost to science so a complete evaluation of this species could not be made until additional material was collected, but comparison with the data and figures presented in the original description (Van Cleave, 1940) suggested that the two species were similar. Koronacantha rnexicana has normal hooks in the dorsal rows and lacks the mid-dorsal space devoid of hooks described for I.pectinarius. A full comparison of these two species and evaluation of the taxonomic placement of 1.pectinarius is given below. Bullock and Mateo (np.. &.) further suggested that since the genera Tegorhynchus and Illiosentis both have a "fan-shaped muscular organ" (called "pad-like" in the present study) they considered the two genera to be "congeneric" (q.a.). Examination of type material of both genera prior to the phylogenetic analysis presented above, supports their interpretation of similarity in vestibular musculature, but differences between the two genera in other characters (i.e. lack of long proboscis hooks in the posteriormost ring in wrhvnchus but present in Illiosentis and additional characters discussed above) indicated that a more comprehensive study the two genera was necessary. Van Cleave (Van Cleave, 1913) first described a pair of band-like vestibular muscles with fan-shaped posterior attachment in Gracilisentis gracilisentis (Van Cleave, 1913) Van Cleave, 1919, an eoacanthocephalan, that is similar to that of
-K. mexicana, but attached more dorsally. Van Cleave (Van Cleave, 1945a; Van Cleave, 1947) later discwed the importance of the genital vestibule and its associated muscles as a taxonomic characters. Oetinger and Budcner (Oetinger and Buckner, 1993) reviewed the various types of vestibular muscle, which they termed "genital vestibule retractor muscles" (Oetinger and Buckner, 1993), previously described from acanthocephalans and suggested several possible functions. A more broad study of vestibular muscles, involving a wide range of taxa, is necessary to evaluate the homology and evolution of the different types of vestibular muscle, but the current study reinforces Van Cleave's assertion of the importance of this character as an indicator of phylogenetic relationships.
Redescription of Wrhynchus brevis Van Cleave, 1921 and transfer of -T. pectin arius Van Cleave, 1940 to Koronacantha
The genus Tegorhynchus Van Cleave, 1921 was originally designated to accommodate 1-brevis Van Cleave, 1921 found in Malapterns reticulatus Valenciennes in Cuvier and Valenciennes, 1839 collected near the Juan Femandez Islands, Chile, by the Swedish Expedition in 1916 (Van Cleave, 1921). Van Cleave originally reported the host of 1.brevis as "Malacopterus reticulatus (C. V.)" (Van Cleave, 1921), but this name was based on an unjustified emendation by (Giinther, 1862). Later, Van Cleave (Van Cleave, 1940) expanded the diagnosis of Tegorhynchus and described 1.pectinarius Van Cleave, 1940 . The original specimens of 1. pectinarius consisted of two females removed from a fish tentatively identified as "Medialuna (?) taken from stomach of Sriola sp-,..." (italics not in original) from Puerto Culebra, Bahia Culebra, Costa Rica (VanCleave, 1940). Since the original material was collected, no additional reports of either species have been published. During the course of an investigation of the parasites of marine fish from Chamela Bay, Jalisco, Mexico, numerous male and female specimens later identified chus pectinarius were collected from the intestine of Microlevidom brevipinnis Steindachner, 1869. The original specimens of 1.pectinarius are currently unavailable and may have been lost, so additional specimens were collected from the type locality, Bahia Culebra, Costa Rica, to confirm the diagnosis.
-T. pectinarius was then redesaibed based on both male and female specimens from Costa Rica and M6xico and transferred to Koronacantha. In determining the taxonomic position of 1.pectinarius, the original description and specimens of T.- brevis were reexamined. The descriptions of the genus and type species, Z brevis, was emended to include characters not discussed by Van Cleave (1921).
Materials and methods
Commercial fishermen captured Microle~idotusbrevi innis with gill nets and long lines 1-2 km from the shore of Chamela Bay, Jalisco State, Mkico (19'31' N, 105'06' W; February, May 1995) and Playa Cuajiniquil(10'57' N, 85-48' W; February 1996). Fishes were collected using spear guns at 10-151x1 depth from Bahia Culebra, Playas Panaml and Hermosa (10'38' N, 85'39' W) (Feb. 1996). No difference in measurements of traits of specimens from Mkico and Costa Rica was found, so measurements of specimens horn both localities were combined.
Redescription and reassignment of aorh chus pectinarius Koronacantha ~ectinaria(Van Cleave, 1940 ) Monks et. al., 1997
Redescriotion (based on 16 male and 32 female specimens): Trunk cylindrical, 3,358 to 8,000 (5,498+1,187, n=42) long, 382 to 1,070 (623k152,n=39) wide with greatest width approximately 1/3 total body length posterior to neck (Fig. 38B,39A). Proboscis cylindrical, elongate, 1,005 to 1,707 (1,329f197, n=31) long, 104 to 230 (169-2, n=44) wide, bearing 12 to 13 (Imf n=38) longitudinal rows of hooks, with heavy cuticular coating (Fig. 38A). Ventral and medial longitudinal hook rows with 21 to 28 (2W,n=32) hooks, dorsal rows with fewer due to hooks lacking from mid- dorsal space located immediately anterior to recurved hooks. Posterior five to seven hooks in each row small, thin, rootless, forming closely set, comblike series of thorns. Heavy, strongly recurved hooks shaped like inverted apostrophe immediately anterior to comblike hook rings; hook blades small, roots simple but exaggerated in size. Mid-dorsal space occurring anterior to recurved hooks either entirely devoid of hooks or with poorly developed vestiges of hooks. Remaining hooks with simple roots. Armature (Table IV) similar in both sexes with slight dorso-ventral dimorphism of size; dorsal hooks slightly larger than ventral (Fig. 39D). Single lateral sensory papilla ($emu Nick01 and Holloway, 1968) present on each side of neck at approximately mid-neck (Fig. 38A, B; 39A). Apical organ 47 to 95 (69&13, n=20) long, 13 to 28 (21k4, n=20) wide at anterior end. Neck 70 to 145 (98k18, n=37) long, 107 to 235 (157+28, n=38) wide. Proboscis receptacle double-walled (Fig. 38A, 39D), 155 to 350 (232k46, n=45) wide at midpoint. Inner wall 1,335 to 2,525 (1,798319, n=42) long, 13 to 40 (27k7, n=42) thick, attached at level immediately posterior to last row of hooks at anterior end of neck. Length of outer wall less than inner wall, 1,260 to 2,405 (1,6Sm20, n=40) long, 13 to 48 (2839, n=42) thick, beginning 58 to 163 (10gf26, n=40) below anterior end of inner wall, not attached to trunk (Fig. 38A). Cerebral ganglion located at midpoint of anterior 1/2 of receptacle, 175 to 347 (238k43, n=28) long, 30 to 65 (45k9,n=28) wide. Retinacula emerging from receptacle slightly anterior to mid-receptacle. Two lemnisci, long, reaching to mid-body, often coiled or twisted, not attached to body wall at distal ends. One lemniscus typically shorter, approximately 88% the length of the other in females, approximately 94% the length of the other in males. Nuclei in lemnisci elongate, partially fragmented in mature specimens. Single ligament sac, persistent, but ruptured in adult females, connected inside of uterine bell to egg-sorting apparatus. Uterine bell variable in shape from slightly vase-shaped (wide at anterior opening, with slightly narrower portion, middle widened then tapering to posterior end) to slightly funnel-shaped (widest at opening, tapering to posterior end) (Fig. 398,ZC). Vagina elongate, widest at midpoint. TABLEVI. Hook lengths of 5 female and 5 male mature Koronacantha pectinaria. Hooks of similar shape were arbitrarily grouped as indicated in the table.
Male Female Hook Dorsal Ventral Hook Dorsal Ventral no. no.
apical 38-70 (57k14) apical 63-74 (69i4)
2-3 48-53 (49+2) 2-3 53-83 (6213)
4-6 50-63 (54s) 4-6 53-73 (63s)
7-8 53-68 (59-7)+ 7-8 56-85 (e12)
9-11 45-58 (536) 9-11 53-88 (W15)
12-IS* 23-38 (28%) 12-15* 30-54 (44i10)
16-18 "patch" 16-18 "patch"
"apostrophe"t 20-28 (2323) 88apostrophe"t 23-28 (25s)
anterior* 23-28 (2M) anterior* 23-30 (27s)
terminal* 20-28 (2M) 16-25 (23k4) terminal* 23-30 (263) 23-28 (25s) 'Depending upon the number of hooks per row, proboscis hook 12 on the ventral surface approximately corresponds to the hook just anterior to the dorsal patch devoid of hooks (Fig. 38A D). t~e~endin~upon the number of hooks per row, proboscis hooks 15-19 on the ventral surface approximately correspond to the hook opposite the apostropheshaped dorsal hook (Fig. 38A D). *Anteriormost and terminal hooks of comb. Females with pair of moderately developed bands of vestibular muscle with fanshaped posterior attachment extending from dorsal body wall at level of uterine bell posteriorly to posteroventral body wall adjacent to genital pore (Fig. 39B,2C). Testes oval, tandem, contiguous with slight overlap (Fig. 38C); anterior testis generally longer and wider than posterior testis. Vas efferens from each testis joining seminal vesicle separately; vas deferens not present. Each duct of vas efferentia slightly widened to provide two to four individual seminal reservoirs arranged tandemly (Fig 38C); reservoirs with terminal sphincter. Seminal vesicle elongate oval; ejaculatory duct present. Eight elongate slightly claviform cement glands, each with two granular unfragmented nuclei. Cement duct from each gland slightly widened anteriorly to provide individual cement reservoirs. Saefftigen's pouch located immediately posterior to duster of cement glands. Body wall thin, containing highly branched subcuticular nuclei not fragmented in mature specimens (Fig. 39F, 2G). Main canals of lacunar system lateral. Trunk spines, most with small rootlets, extending from the necktrunk junction posteriorly over about 85% of length of W; spines lacking on dorsal trunk approximately at level of receptacle (Fig. 388,39A). Trunk spines more numerous on females than males. Genital pores terminal in relaxed specimens; genital spines lacking. Mature eggs elongate fusiform, prolongation of inner membranes slightly pinched at the poles (Fig. 39E). Male (Fig. 38): 3,358 to 7,980 (4,54343f1186, n=13) long, 382 to 1,070 (62743f193, n=13) wide at greatest width (Fig. 388). Proboscis 1,005 to 1,340 (1,177k120, n=ll) long, 104 to 185 (1-3, n=15) wide, bearing 12 to 13 (12W-, n=15) longitudinal rows of hooks. Ventral and medial longitudinal hook rows with 21 to 25 (24_tl,n=13) hooks, dorsal rows with fewer hooks (Fig. 38A). Apical organ 55 to 95 (66f13, n=7) long, 15 to 25 (21M, n=7) wide at anterior end. Neck 75 to 110 (95+11, n=13) long, 107 to 175 (145e1, n=14) wide. Proboscis receptacle 155 to 287 (208k34,n=14) wide. Inner wall 1,370 to 2,070 (1,699+219, n=13) long, 13 to 28 (21&5, n=12) thick. Outer wall, 1,270 to 1,948 (1,574+216, n=ll) long, extending to within 58 to 163 (105fl7, n=ll) of anterior end of inner wall, 13 to 38 (21k7, n=12) thick, outer wall approximately as thick as inner wall. Cerebral ganglion 175 to 347 (234k47, n=12) long, 30 to 65 (43+10, n=12) wide. Shorter lemniscus 1,950 to 3,015 (2,441k437, n=7) in length, longer lemniscus 2,055 to 3,020 (2,601+364, n=7) long; width of lemnisci equal, 44 to 103 (68337, n=22) wide at level of posterior end of receptacle. Male reproductive system 1,339 to 2,945 (2,096k479, n=12) long, occupying approximately 46% of trunk length (Fig. 38C). Anterior testis 384 to 895 (565k146, n=ll) long and 192 to 416 (267L-74, n=ll) wide. Posterior testis 347 to 715 (534k129, n=ll)long and 180 to 362 (266k55, n=ll) wide. Vas efferens from posterior testis 325 to 725 (456k119, n=9) long. Seminal vesicle 143 to 410 (236k63, n=14) long and 63 to 211 (125E37, n=14) wide. Ejaculatory duct 128 to 275 (203k46, n=13) long and 30 to 58 (41+10, n=13) wide. Cement glands 113 to 277 (170f42, n-45) long and 47 to 140 (88k22, n=45) wide. Saefftigen's pouch 288 to 677 (453f 108, n=ll) long and 142 to 274 (174k37, n=ll) wide; pouch wall nine to 23 (13k5, n=ll) thick. Female (Fig. 39): 4,450 to 8,000 (5,926B21, n=29) long, 400 to 930 (62M131, n=26) wide at greatest width (Fig. 39A). Proboscis 1,055 to 1,707 (1,412H81, n=20) long, 135 to 230 (183k28, n=29) wide, bearing 12 (n=23) longitudinal rows of hooks. Ventral and medial longitudinal hook rows with 22 to 28 (ZS, n=19) hooks, dorsal rows with fewer hooks. Apical organ 47 to 95 (71+13, n=13) long, 13 to 28 (234, n=13) wide at anterior end. Neck 70 to 145 (9ge1, n=24) long, 120 to 235 (164k29, n=24) wide. Proboscis receptade 173 to 350 (24248, n=31) wide. Inner wall 1,335 to 2,525 (1,842+349, n=29) long, 13 to 40 (2%, n=30) thick. Outer wall, 1,260 to 2,405 (1,732f345, n=29) Long, beginning 63 to 163 (llOE27, n=29) below anterior end of inner wall, 18 to 48 (31k7, n=30) thick; outer wall slightly thicker than inner wall. Cerebral ganglion 180 to 340 (241+41, n=16) long, 30 to 63 (47k8, n=16) wide. Shorter lemniscus 1,960 to 4,051 (2,975k573, n=12) long, longer lemniscus 2,130 to 4,820 (3,366k766, n=12) long; lemnisci equal width, 35 to 101 (71k20, n=41) wide at level of posterior end of receptacle. Female reproductive system 693 to 904 (805k92, n=4) long, occupying approximately 14% of trunk length (Fig. 39B, 2C). Uterine bell 213 to 356 (266t36, n=18) long, 38 to 110 (71X21, n=19) wide at anterior opening, 43 to 126 (76fl0,n=19) wide at middle. Uterus 135 to 250 (177525, n=26) long, 38 to 63 (50k7, n=26) wide. Vagina 100 to 189 (124320, n=29) long, 35 to 73 (48k10, n=30) wide at middle. Size of mature eggs measured through body wall 55 to 98 (73+11, n=36) long, 15 to 25 (18+3,n=36) wide.
Taxonomic summary Hosts: "Medialuna (?) taken from stomach of Seriola sp." (sic); Microlepidotus brevi~innisSteindachner, 1869. Site of infection: Intestine and cecae. Localities: Bahia Culebra, Playa Panama, and Playa Hermosa, Costa Rica (10°38' N, 85"39' W); Golfo de Santa Elena, Playa Cuajiniquil, Costa Rica (10'57' N, 85'48' W); Chamela Bay, Jalisco State, M6xico (19'31' N, 105'06' W). Specimens deposited: CNHE 3016-3021,3022-3023; FIOCRUZ three specimens; HWML 39287-39288; INBIO five specimens; USNPC 87006-87007.
Remarks
Van Cleave's two original specimens of Tegorh chus pectinarius are unavailable and may have been lost, so some ambiguity in the characteristics of the species exists as well as his reasons for assigning it to mrhynchus. One specimen, the holotype (VC3124.2), was deposited in the collection of the AUan Hancodc Foundation of The University of Southern California and the other, a paratype, was retained in Van Cleave's personal collection (Van Cleave, 1921). The Foundation collection has since been broken up and disbursed to various institutional collections and no record of the disposition of the holotype could be found. Van Cleave's personal collection was subsequently transferred to the USNPC collection. The paratype was catalogued, but has been Listed as missing for an undetermined number of years. The large number of specimens collected permitted a more complete description of this species; for example, this study presents the first report of males. At this time, few descriptions of illiosentids encompass the sample size included in this material, but the general differences between these specimens and the values given in Van Cleave's (1940) description are consistent with those exhibited by members of the family IUiosentidae. The material collected as part of the current study is somewhat smaller in overall body size than the original material (Table V), but in all other traits it approximately corresponds with Van Cleave's (1940) description of the two females. Therefore, it was determined that the specimens represent a single species and that the species was 1.pectinarius. Examination of these specimens corroborated the observations of Bullock and Mateo (w.&t) who suggested that L pectinariu should be removed from Tegorhynchus. The regional specialization of the proboscis hooks typical of the species suggesed that 1.pectinarius should be transferred to Koronacantha Monks and Pkrez-Ponce de Leh, 1996. Koronacantha mexicana Monks and Perez-Ponce de Leon, 1996 and 1.pectinarius both have posterior hook rows consisting of small, thin, rootless hooks, forming a dosely set, comb-like, series of thorns. Immediately anterior to these hooks, both species have heavy, strongly recurved hooks in the shape of an inverted apostrophe such that the hook blade is small, with a simple root that is exaggerated in size. In addition to the above, the two species have sensory pores at mid-neck, two nuclei in each cement gland in males, and band-like vestibular muscles that are widened posteriorly in females. aorhpchus pectinarius, therefore was transferred (Monks, et al., 1997) to Koronacantha as -K. pectinaria (Van Cleave, 1940) Monks et al., 1997. TABLEW. Comparison of original measurements of mrhynchus pectinanus from the original description (Van Cleave, 1940) to those of Koronacantha ~edinaria included in the current study. All measurements are in p.
Original specimens* Present specimens+
Female Both sexes
Body length 8,400-8,800 4,450-8,OOO 4,450-8,000 Proboscis length 1,400 1,055-1,707 1,005-1,707 Rows of hooks 12 12 12-13 Hooks per row 28-30 21-28 21-28 Hooks in "comb" 5-7 5-7 5-7 Recurved hook 27 23 24
ERE width 21-24 15-25 Taken from Van Cleave (1940). tMean, standard deviation, and sample size for each value is given in the description. Koronaantha pectinaria differs from L -an% the only other known member of the genus, by having recurved hooks only on the mid-dorsal section of the dorsal hook rows with normal hooks on the lateral and ventral rows and a mid- dorsal space immediately anterior to the recurved hooks that is either entirely devoid of hooks or the hooks corresponding to these positions represented by poorly developed vestiges of hooks, and in genital spines lacking in both sexes (Fig. 38C; 398, C). It further differs by having spines extending from the necktrunk junction posteriorly over about 85% length of trunk, and genital spines lacking in both sexes. In K. mexicana, the mid-dorsal space in the dorsal hook rows is lacking, trunk spines extend posteriorly from about the level of the posterior end of the receptacle over the remainder of the trunk, and genital spines are present in both sexes.
Redescription of Tesorhvnchus brevis Van Cleave, 1921 &ggrhvnchus Van Cleave, 1921
Trunk cylindrical, greatest width approximately 1/3 total body length posterior to neck. Proboscis cylindrical, elongate to slightly clavate, with heavy cuticular coating, bearing numerous longitudinal rows of hooks. Posterior-most basal hooks in each row small, thin, rootless, forming comblike series. Remaining hooks with simple roots and little regional modification with slight dorso-ventral dimorphism of size; hooks slightly larger. Armature similar in both sexes. Proboscis receptacle double-walled; inner wall attached at anterior end of neck and neck-trunk junction, outer wall beginning posterior to neck-trunk junction. Cerebral ganglion in neck. Two lemnisci, long, not bound to body wall at distal ends, reaching mid-body. Dorsal and ventral neck inverter muscles present. Single ligament sac, persistent, ruptured in adult; in females, ligament sac attached inside uterine bell to egg-sorting apparatus. Females with narrow, moderately thick pad-like vestibular muscle on inner posteroventral wall of trunk. When contracted, vestibular muscle pulls mid- posterior ventral surface of trunk inward, forming trough-like genital vestibule (sensu (Van Cleave, 1945a). Testes oval, tandem to slightly diagonal, contiguous. Each duct of vas efferentia slightly widened to form individual seminal reservoirs arranged tandernly; vas deferens simple. Seminal vesicle elongate oval; ejaculatory duct present. Eight cement glands, each with more than 20 small nuclei dispersed throughout gland. Cement ducts consisting of two reservoirs separated by narrow section; anterior portion of duct enlarged to form tapering resentoir, posterior portion enlarged to form elongate reservoir. Duct from Saefftigen's pouch to genital canal expanded anteriorly to form additional reservoir, tapering toward posterior end; remainder of duct leading to genital canal short, narrow. Main canals of lacunar system lateral. Body wall thin, containing highly branched subcuticular nuclei. Trunk spines present on anterior trunk;genital spines absent in both sexes. Genital pore terminal in male, slightly dorsal in female. Mature eggs elongate fusiform with prolongation of inner membranes at poles. Type and only species: Tngorhvnchus brevis Van Cleave, 1921
Teporhvnchus brevis Van CIeave, 1921
Emended descri~tion(based on 17 specimens-Eight male and seven female whole mounts and one male and one female sectioned): Trunk cylindrical, 2,413 to 6,899 (3,796k1052, n=15) long, 525 to 1,139 (762k187, n=15) wide with greatest width approximately 1/3 total body length posterior to neck (Fig. 40A, 41A). Proboscis cylindrical, elongate, 728 to 1,085 (941+116, n=10) long, 156 to 223 (188+23, n=10) wide, bearing 13 to 15 (1&1, n=9) longitudinal rows of 16 to 19 (18+1, n=10) hooks, with heavy cuticular coating. Each row containing 16 to 19 (18+1, n-10) hooks per row. Posterior five or six hooks in each row small, thin, rootless, forming closely set, comblike series of thorns. Remaining hooks have simple roots, anterior hooks largest, decreasing in size toward posterior probosds, but with little regional modification. Armature (Table VI) similar in both sexes with slight dorso-ventral dimorphism; dorsal hooks slightly larger than ventral (Fig. 41F). Single lateral sensory papilla (~ensuNick01 and Holloway, 1968) present on each side of proboscis approximately at level of 10th or 11th hook; hooks of adjacent rows slightly displaced to accommodate papilla (Fig. 40B). Apical organ 32 to 51 (45k7, n=8) long, 27 to 48 (36+8, n=8) wide. Neck 207 to 255 (23W16, n=10) long, 128 to 207 (17M7, n=10) wide. Proboscis receptacle double-walled (Fig, 41A, E), 121 to 354 (261+62, n=14) wide at midpoint. Inner wall 1,162 to 2,042 (1,330t315, n=13) long, 19 to 48 (2828, n=14) thick, attached at level immediately posterior to last row of hooks at anterior end of neck and at neck-t~nkjunction. Length of outer wall less than inner wall, 932 to 1,771 (1,124+219, n=13) long, 16 to 40 (269,n=14) thick, beginning 137 to 271 (205-4, n=13) below anterior end of inner wall, 48 to 96 (72k19,n=9) posterior to neck-trunk junction, not attached to trunk (Fig. 408). Cerebral ganglion located approximately in anterior 1/4 of receptacle, 160 to 220 (l8Sf 19, n=9) long, 32 to 51 (4lf6, n=9) wide. Retinacula emerging from receptacle slightly anterior to rnid- receptacle. Two lemnisci, long, reaching mid-body, wider anteriorly to mid- lemniscus, tapering distally, often coiled or twisted, not attached to body wall at distal ends. Narrow dorsal and ventral neck inverter muscles present; anterior attachment at neck-trunk junction, posterior attachment to body wall at mid-body. Single ligament sac, persistent, ruptured in adult females. Uterine bell vaseshaped, wider at midpoint than at anterior opening, tapering slightly from midpoint to junction with sorting apparatus (Fig. 41B,4C). Vagina elongate, widest at base; base slightly rounded. Females with narrow, moderately thick pad-like vestibular muscle on inner posteroventral wall of trunk (Fig. 41B, 4C). In mature females, contraction of vestibular muscle forming genital vestibule with two lateral folds that resemble pseudo-papillae (sensu (Van Cleave, 1921) and pulling the genital pore to a relatively more ventral position; pseudo-papillae not obvious in less mature females. TABLEwI. Hook lengths of three female and three male mature mrhynchus brevis. Hooks of similar shape were arbitrarily grouped as indicated in the table.
Male Hook Dorsai Ventral no.
terminal* 43-50 (463) 52-63 (58k4)
Female Hook Dorsal Ventral no.
terminal* 44-45 (44,+0) 55-58 (56e) 'Anteriormost and terminal hooks of comb. Testes oval, tandem to slightly diagonal, contiguous to slightly overlapping (Fig. 40% F); anterior testis generally longer and narrower than posterior testis. Each duct of vas efferentia slightly widened to provide two to four individual seminal reservoirs arranged tandemly (Fig. 40F). Vas deferens simple, approximately as long as vas efferens from posterior testis. Seminal vesicle elongate oval; ejaculatory duct present. Eight cement glands; individual glands broadly oval. Each cement gland with more than 20 small nuclei dispersed throughout cement gland. Cement ducts divided into two reservoirs. Anterior portion of cement duct enlarged forming reservoir tapering posteriorly, ending in short, narrow portion; posterior portion of cement duct enlarged forming elongate cement reservoir (Fig. 40E). Saefftigen's pouch located immediately posterior to cluster of cement glands, overlapping anterior portion of cement ducts. Body waLl thin, containing highly branched nuclei not fragmented in mature worms; nuclei not assessed for oldest specimens. Main canals of Iacunar system lateral. Trunk spines, most with small rootlets, extending approximately anterior 1/4 length of hunk (Fig. 40A; 41A). Genital pore terminal in male, slightly dorsal in female; genital spines lacking. Mature eggs elongate fusiform, prolongation of inner membranes slightly pinched at the poles. Male (Fig. 40A-F; 41E): 2,413 to 4,320 (3,473612, n=8) long, 576 to 813 (717k91,n=8) wide. Proboscis 728 to 1,034 (906f124, n=6) long, 156 to 204 (175+18, n=6) wide (Fig. 40B), bearing 13 to 15 (14k1, n=6) longitudinal rows of 16 to 18 (1721, n=6) hooks. Apical organ 32 to 51 (4W,n=6) long, 27 to 48 (3327, n=6) wide at anterior end. Neck 214 to 255 (229k17,n=6) long, 150 to 207 (18W19, n=6) wide. Proboscis receptacle 207 to 351 (26950, n=8) wide. Inner wall 1,181 to 1,516 (l,28l+ll3, n=8) long, 19 to 37 (26M, n=8) thick. Outer wall 986 to 1,305 (1,OWf 109, n=8) long, beginning 137 to 230 (19Dt-30, n=8) below anterior end of inner wall, anterior end 48 to 96 (8Ok17, n=6) below neck-trunk junction (Fig. 40A, B), 16 to 32 (24k6, n=8) thick; outer wall slightly thinner than inner wall. Cerebral ganglion 163 to 191 (179k12, n=5j long, 32 to 51 (41f7, n=5) wide. Lemnisci equal in size, 1,532 to 2,042 (1,767k199, n=6) in length, 64 to 153 (113B1, n=8) wide from anterior end to mid-lemniscus, tapering to 22 to 51 (3%12, n=7) at posterior end. Male reproductive system 2,087 to 3,015 (2,463G97, n=8) long, occupying approximately 71% of trunk length (Fig. 40A, E, F). Anterior testis 284 to 622 (48lf 103, n=7) long and 204 to 332 (267fi1, n=7) wide. Posterior testis 284 to 485 (414k70, n=8) long and 236 to 380 (280+53, n=8) wide. Vas efferens from posterior testis 262 to 635 (433k121, n=7) long. Presence of terminal sphincter of reservoirs could not be assessed. Vas deferens simple, 495 to 734 (659f89, n=7) long. Seminal vesicle 185 to 348 (27M1, n=8) long and 108 to 223 (174k40, n=8) wide. Eight cement glands; individual glands oval, wider, 89 to 160 (118k23, n=8), than long, 70 to 128 (90k20, n=8) (Fig. 40D, E). Saefftigen's pouch 332 to 542 (437k68, n=8) long, 176 to 319 (243k49, n=8) wide; pouch wall 11 to 19 (15k3, n=8) thick (Fig. 40A, F). Anterior portion of duct from pouch to bursa 112 to 160 (136214, n=8) wide at anterior end, tapering to 22 to 41 (36+6, n=8) wide. Female (Fig. 41A-E): 2,970 to 6,899 (4,166&1,359,n=7) long, 525 to 1,139 (813S!37, n=7) wide (Fig. 41A). Proboscis 887 to 1,085 (995392, n=4) long, 191 to 223 (207k15, n=4) wide, bearing 13 to 14 (14&1, n=3) longitudinal rows of 16 to 19 (1852, n=4) hooks. Apical organ 48 to 51 (49k2, n=2) long, 41 to 48 (45k5, n=2) wide at anterior end. Neck 207 to 243 (23lf 16, n=4) long, 128 to 195 (160+Blf n=4) wide. Proboscis receptacle 121 to 354 (249k79, n=6) wide. Inner wall 1,162 to 2,042 (1,4072363, n=5) long, 21 to 48 (31+10, n=6) thick. Outer wall 932 to 1,771 (1,178S43, n=5) long, beginning 191 to 271 (229k28,n=5) below anterior end of inner wall, 48 to 64 (56+8, n=3) below neck-trunk junction, 19 to 40 (27k7, n=6) thick; outer wall slightly thinner than inner wall. Cerebral ganglion 160 to 220 (192+25, n=4) long, 38 to 48 (424, n=4) wide. Lemnisci equal in size, 1,229 to 2,361 (1,688+439, n=5) long, 70 to 112 (93215, n=6) wide from anterior end to mid-lexnniscus, 45 to 64 (52k8, n=6) in width at posterior end. Female reproductive system 574 to 769 (687k85, n=4) long, occupying approximately 17% of trunk length (Fig. 41B, C). Uterine bell 182 to 303 (233S7, n=4) long, 70 to 112 (93+21, n=4) wide at anterior opening, 182 to 303 (233fi7, n=4) wide at midpoint. Uterus 121 to 160 (139k17, n=4) long, 43 to 54 (5W5, n=4) wide. Vagina 96 to 137 (113219, n=4) long, 22 to 32 (27k7, n=4) wide at midpoint. Mature eggs 51 to 55 (53+2,n=7) long, 11 to 14 (13k1, n=7) wide measured through body wall (Fig. 41D).
Taxonomic summary Hosts: Malapterus reticulatus Valenciennes in Cuvier and Valenciennes, 1839 Site of infection: Intestine. Locality: Masatierra, Juan Femandez Islands, Chile, by the Swedish Expedition in 1916 (VanCleave, 1921). Specimens examined: NHR 4768, kctotype; NHR 4766, paralectotype female, NHR 4767, paralectotype; USNPC 37535, paralectotypes; USNPC 81405, voucher (sectioned material). Van Cleave deposited three specimens, NHR 4766-4768, at NHR and retained all other specimens in his personal collection. He labeled all specimens except USNPC 81405 as cotypes, but his deposit of three specimens in a recognized institution (NHR) suggests that he considered those as the name-bearing types. Thus, lectotypes and paralectotypes were designated from this material as listed above.
Remarks
Teeorhynchus brevis is presently assigned to the Illiosentidae (Amin, 1985), because it has thick cuticular trunk spines in a single undivided region, long proboscis receptacle, cerebral ganglion at a level anterior to the middle of the receptacle, and eight elongate, clavate cement glands, all characters of the Illiosentidae as it is currently defined (Amin, 1987). The species also shares several characters with members of Illiosent~in particular. Members of both genera have cuticular spines on the anterior trunk, sensory pores located on the proboscis, posterior five or six hooks on proboscis comblike, a heavy cuticle on the proboscis. Females of both genera have a pad-like vestibular musde, posterodorsal placement of the genital pore and ligament sac attached inside uterine bell to eggsorting apparatus; males have eight davate to pynform cement glands with about 20 small nuclei, cement ducts of each gland divided into tubular anterior and posterior reservoirs, simple vas deferens, and vas efferentia with tandemly arranged seminal reservoirs. Members of both genera have some median basal hooks that are slightly recurved, but they do not have the greatly enlarged roots seen in members of Koronacantha. aorhynchus brevis differs fkom members of Illiosentis by having a more short, robust trunk and hooks in the basal circle of proboscis hooks only slightly enlarged. In addition, females have a genital vestibule with two lateral folds that resemble pseudo-papillae formed by the contraction of the vestibular muscle and males have vas efferentia consisting of two to four reservoirs. Members of Illiosentis have elongate, cylindrical trunks and ventral hooks in the basal cirde greatly enlarged. Females have genital vestibules formed by contraction of the vestibular muscle, but the vestibule lacks lateral pseudo-papillae and; in males, the vas efferentia are simpler than those of 1.brevis with one reservoir in the duct leading from the posterior testis and two reservoirs in the duct from the anterior testis. The position of the cerebral ganglion is variable in members of each genus, but in morhvnchus the cerebral ganglion is located approximately in the anterior 1/4 of the receptacle, never extending anteriorly past the limit of the receptacle; in Illiosen tis, the cerebral ganglion is located in the anterior neck at the base of the proboscis, often anterior to the limit of the receptacle and rarely extending posteriorly to mid-neck. Review of past taxonomic problems of mrhvnchus
In his discussion on the taxonomic placement of &ggrhynrhus, Van Cleave (1921) did not associate the new genus with any then-described family. He suggested that the presence of body spines alone should not be the basis for a determination of the phylogenetic relationships of this genus and, furthermore, that Tegorhynchus should remain "unplaced" until the species was better known and determination of relationships was possible (Van Cleave, 1921; 77). Van Cleave (1940), in expanding the generic diagnosis to include Z pectinarius, cited Van Cleave and Lincicome (1940), then in press, as authority for placing the genus in the Gorgorhynchidae Van Cleave and Linucome, 1940. In both descriptions, Van Cleave diagnosed &gorhynchus as possessing four cement glands, a characteristic of the yet-to-be-described Gorgorhynchidae, but in his emendation of the genus stated that it was "...closely related to the genus Illiosentis Van Cleave and Lincicome." (italics not in original: Van Cleave, 1940; 505) which possessed eight cement glands and was a member of the family Rhadinorhynchidae. When the review and emendation of the Rhadinorhynchidae was published, Van Cleave and Lincicome (1940), without further explanation, categorized morh chus as possessing eight cement glands and grouped it with the five other genera that they recognized as belonging to the Rhadinorhynchidae. The Gorgorhynchidae they described as having, along with other characters, four cement glands. All male specimens of both 1.bra and L pectinaria that we examined have eight cement glands. Golvan (1960) retained Norh chus in the Rhadinorhynchidae but, within the family, he established the IUiosentinae Golvan, 1960 for Jlliosentis and Telosentis. In addition to the characters of the family, the members of the sub-family were characterized as having genital spines. In his discussion of morhvnchu~, Golvan (1960) commented on the disagreement between Van Cleave's accounts of numbers of cement glands, but without examining any specimens, accepted eight as the correct number of cement glands for ~orhynchusbased on the diagnosis of the genus as having of eight glands in Van Cleave and Linacome (1940). His stated reason was that since Van Cleave and Lincicome (1940) was the most recent paper, the value given in that publication should be used (Golvan, 1960; 163). Yamaguti (1963) accepted the IUiosentinae, but disregarded Golvan's (1960) suggestions on number of cement glands. With some changes, Yamaguti redescribed the Rhadinorhynchidae and included in it the genera of the Gorgorhynchinae Golvan, 1960. In his description, family members possessed 2-8 cement glands. Golvan (1969), accepted little of Yamaguti's (1963) taxonomy, and later elevated the Illiosentinae to family status, and transferred &prhynchus and various other genera to it. Although no longer closely assodated with the Illiosentidae, he also retained Gorgorhynchinae, attributed to Van Cleave and Lincicome (1940), but placed it within Rhadinorhynchidae. Bullock and Mateo, in an abstract of a paper given at the Second International Congress of Parasitology (op. cit.), suggested that 1.pectinarius should not be retained in morhynchus, but did not formally place the species in a new genus. They further suggested that since Tegarhvnchus and Illiosentis both have a "fan- shaped muscular organ" they considered the two genera to be "congeneric" (=. &.). Van Cleave (194%) had previously noted the resemblance of the vestibular muscles of the two genera, but he considered them to be only "...somewhat similar..." (VanCleave, 1945b; 57). Until the current study, questions concerning the relationship between the two genera has received little attention, and, with the exception of Buckner, et al. (1978), who commented that it was "... a matter still requiring attention." (Buckner, et al., 1978; 196), had not been resolved. This lack of attention was likely due, in part, to a need for further collection of material, originally suggested as a prerequisite for a better understanding of these groups by Van Cleave (1921). Unfortunately, the uncertainty in the status of Jlliosentis has been reflected in the work of authors since the presentation by Bullock and Mateo (wa). Buckner et al. (1978), in a study of the intermediate host of 1. furcatus Van Cleave and Lincicome, 1939, considered the species to be a member of mthynchus and cited Bullock and Mateo's abstract (gg.&.) as authority for the placement Despite this, as mentioned above, they recognized that the matter was not fully resolved and further commented that it was particularly troublesome because Bullock and Mateo (w.&.) had mentioned only I furcatus and were not specific about the status of other members of the genus (Buckner, et al., 1978). The confusion was further propagated by Amin (1985) in a discussion of the classification of the Acanthocephala, who, without formal justification, placed all members of Illiosentis in Tegorhynchus with "...Bullock and Mateo, 1970" (Amin, 1985; 47) (the same work) as authority for the transfer. This was a change from his listing of Illiosentis as one of the eight genera in the Illiosentidae in a previous discussion of classification (Amin, 1982). The discovery of Koronacantha mexicana helped to resolve some of the uncertainty surrounding the relationships between '&gorhynchus and Illiosentis As noted in the section on I(. mexicana above (pg. 143), the species is dearly distinct &us and Illiosentis by having cerebral ganglia at about mid-receptacle rather than at the level of the neck, sensory pore at mid-neck rather than on the proboscis, recurved hooks with greatly enlarged roots rather than without enlarged roots, cement glands with two nudei per gland rather than more than 20 nuclei per gland, and females with band-like vestibular muscles rather than pad-like vestibular muscles. Evidence has been provided here demonstrating that the species originally described as 1.peainanus possesses these attributes as well. By placing 1.pectinanus in Koronacantha, aorhynchusand nliosenh*S are now diagnosably distinct from each other and from Koronacantha. At this time, a formal investigation of relationships of all taxa assigned to Illiosentidae still has not been possible because the specimens described from marine fishes of the Indian Ocean have been lost. However, with the present re-examination and redescription of a step toward this goal has been made. The phylogenetic analysis presented in this chapter supports the contentions of Monks and Perez-Ponce de Le6n (1996) and Monks et al. (1997) the synonymy of Illiosentis with &orh chus by Amin (1985) and others is unjustified. Although the shared characters of Tegorh chus and llliosentis indicate, as suggested by Bullock and Mateo (np &.), that the two are sister-groups, the differences discussed above require that the two genera be retained as separate taxa.
Conclusions
Acanthocephalans have a rich spectrum of morphological characters that provide information usable for phylogenetic studies. Although, in some ways, acanthocephalans still stand "...apart in a condition of isolation..." (VanCleave, 1941; 31), the current study of metazoan relationships provided a sound basis for selection of the Priapulida and Rotifera as outgroups. These two groups allowed many structural features to be polarized for phylogenetic analysis. As the understanding of the outgroups grows, the ability to interpret the evolution of characters within the phylum will increase, making possible a more robust know ledge of rela tionships among acan thocephalans. This study supports many of the classic views concerning acanthocephalan taxonomy; for example, both the Eoacanthocephala and Palaeacanthocephala were supported as monophyletic. Yet, the study also suggests that, if the Archiacanthocephala is actually a natural group, a search for more evidence is necessary to demonstrate its monophyly. The number of taxa included is not sufficient to serve as an exhaustive test of the entire class, but it does point out a potential taxonomic problem. In addition to the Archiacanthocephala, strong support is provided for particular clades, in particular, the Eoacanthocephala and Palaeacanthocephala. For other taxa, such as Telosentis, higher level relationships cannot be resolved without an analysis of each member of the genus, as was done for Tegorhynchus. This analysis is one step of many toward our understanding of the phylogenetic relationships of the Acanthocephala. As discussed above (p. 125,130), while many structures of the outgroups could be identified as homologous with those of acanthocephalans, many individual features of those structures could not. Examination of as great a number of specimens of the Rotifera as the number of acanthocephalans evaluated in this study would undoubtedly yield much new information on homology of those features. Thus, polarity could be established for those characters coded herein as "unknown" for the Rotifera. Establishment of an extensive character database for the Rotifera would also allow the monophyly of the Rotifera to be investigated. If the Acanthocephala is contained within the Rotifera, as suggested by Ahlrichs (1997) and Garey et al. (1996) the group to which the Acanthocephala are most closely related must be identified. Likewise, the plesiomorphic state of characters in rotifers that are also found in the Acanthocephala must be determined to allow polarization of a greater number of characters. It is important that the phylogenetic database provided here be extended to include more species. As species are examined, additional characters must be added that are appropriate to distinguish all taxonomic levels. Then, similar procedures to those used here can provide a comprehensive understanding of the evolutionary history of this fascinating phylum. Figure 35. Single most parsimonious tree resulting from a phylogenetic analysis of 22 members of the Acanthocephala. Letters accompanying slash marks on the tree indicate putative synapomorphies based on unambiguous character state changes; character state changes (autapomorphies) for individual taxa and ambiguous changes are not shown.
X-162, 441,1113; and, Y-543, 1022,1172,1211,1262.
FIGURE 36. Koronacanthq mna.A. Holotype, male (details of vas efferentia not shown). B. Proboscis of holotype. C. Cement glands of a paratype. D. Posterior end of a male paratype, showing vas efferentia. E. Ventral proboscis hooks of the holotype. F. Dorsal proboscis hooks of holotype. (Hooksdepicted correspond to types given in Table III; anteriormost and posteriormost hooks of "comb" are shown. (Scale bars in pm: A = 500; B = 200; C = 100; D = 300; E, F = 50)
FIGURE 37. Koronacantha mexicana. A. Paratype, female, external body showing spine distribution. B. Neck, anterior body, and sensory papilla of holotype; arrows indicate anteriormost extent of outer wall of receptade. C. Single subcuticular nucleus of a paratype, female. D. Holotype, posterior end of the receptacle. E. Posterior end of a female, paratype, showing one of pair of vestibular musde bands. F. Details of trunk spines from Holotype. G.Mature egg, drawn through body wall. (Scale bars in pm:A = 500; 8 = 100; C = 200; D = 100; E = 200; F, G = 25)
FIGURE 38. Koronacanthq ~ectinaria~male. A. Proboscis, neck, anterior body, sensory papilla, and receptacle. B. External body showing spine distribution. C. Posterior end showing vas efferentia, seminal receptade, and ejaculatory duct. D. Proboscis hooks (Hooks depicted correspond to Table N; hooks 2-6 are similar in shape so are combined in this illustration (Arrow points toward anterior end of proboscis.). (Scale bars in pm: A-D = 500)
FIGURE39. Koronacantha ~ectinaria,female. A. External body showing spine distribution. B. Posterior end, lateral view showing vestibular muscle (Dorsal surface on right side of image-). C. Posterior end, dorsal view showing lateral position of vestibular muscles. D. Posterior end of receptacle. E. Mature egg, drawn through body wall. F. Subcuticular nucleus from anterolateral trunk (Arrow points toward anterior end of W).G. Subcuticular nudeus from posterolaterd trunk (Arrow points toward anterior end of trunk.) (Scale bars in pm: A = 500; B-D= 200; E = 50; F, G = 200)
FIGURE 40. aorh chus bcevis, male. A. Body (details of cement glands and ducts not shown). B. Proboscis. C. Trunk spines. D. Cement gland, showing nuclei. E. Cement glands and cement ducts F. Posterior end showing vas efferentia, vas deferens, seminal receptacle, and ejaculatory duct. (Scale bars in p:A = 500; B = 250; C,D = 50; E= 200; F = 500)
FIGURE 41. ~orhvnchusbrevis, female. A. External body showing spine distribution, genital vestibule (Outline of reproductive organs to indicate placement and relative size.). B. Posterior end, showing vestibular musde fully contracted (Dorsal surface on right side of image.). C. Posterior end, showing vestibular musde slightly contracted forming pseudo-papillae (Dorsal surface on left side of image.). D. Mature egg, drawn through body wall. E. Posterior end of receptacle. F. Proboscis hooks; hooks depicted correspond to types given in Table VI (Arrow points toward anteroir end of proboscis.). (Scale baa in pm: A = 500; B = 50; C, D = 300; E, F = 200)
Chapter 6: Conclusion
The purpose of this of study was to examine the phylogenetic relationships among the Metazoa, identify appropriate outgroups to the Acanthocephala, and then make a phylogenetic analysis of the Acanthocephala. Very little of the information about the morphology and development of metazoan phyla is suitable for use in phylogenetic analysis in the format presented in previous studies. Character descriptions and assignment, even in recent studies using a dadistic approach, are theory-laden as a result of a prioxj assumptions of evolutionary mode, relationships, and character evolution. In ail previous studies of metazoan and acanthocephalan relationships, the character sets have been woefully inadequate to resolve relationships within the group of interest. Because characters have been uncritically accepted without evaluation of primary homology or presentation in a testable format, it was the necessary first step of both analyses to gather a large data set of binary and mu1tis ta te characters derived from comparative morphological and ontogene tic studies. Character of the Choanoflagellata, Mesozoa, and Metazoa were extracted from primary and secondary Literature on morphology and development of muIticellular animals, as well as examination of selected specimens. This material yielded 144 characters that could be phrased in a testable phylogenetic format. An historical review of studies of relationships among metazoans demonstrated that all have been hampered by (i.) the lack of properly phrased hypotheses in a testable format and (ii.) attempts to postulate evolutionary scenarios without basing them upon rigorous objective methodology. Second, although it had been claimed that phylogenetic studies were not appropriate at the phylum level because the methodology was inadequate (Wiher, 1990) and that character information was excessively conflictive, it was demonstrated that neither assertion was correct. The primary analysis of the relationships of 34 taxa using 144 morphoIogica1 and biochemical characters yielded 17 equally parsimonious trees, each 361 steps long (CI = 0.460, CIX = 0.405, HI = 0.582, HIX = 0.621, RI = 0.689, and RC = 0.31 7). After re-weighting the characters, only two of the 17 trees were retained, differing in the placement of the Platyhelminthes). On one tree the Platyhelminthes are the sister group to the Molluscs, and on the other, they are placed basal to the Nemertea. In both trees, the Acanthocephala was the basal member of a dade (the Ecdysozoa) composed of the (Rotifera, (Gastrotricha, (Nematoda, Nematomorpha), (Pentastomida, (Kinorhynchida, (Tardigrada, (Onychophora, (Chelicerata, (Uniramia, Crustacea))))))))). Although not all relationships were resolved, sufficient resolution was achieved to idenbfy the Priapulida and Rotifera as being useful for polarization of characters in the phylogenetic analysis of the Acanthocephala. The concepts of Deuterostomia and Protostornia were found to be inadequate to explain the relationships supported by the analyses. The Deuterostomia is only monophyletic if the Protostomia, sister-group to the Priapulida, is included. Additionally, strong support for the assignment of the arthropod-like phyla to the Ecdysozoa was found, resolving previous confusion concerning segmentation and early cleavage pattern of arthropods. The results provide support for an evolutionary scenario similar to that of Remane (1963a, 1963b, 1963c), in which the gastric pouches of cnidarians were transformed into body cavities and the acoelomate condition is secondarily derived. Lastly, hypothesized patterns of evolution of the stages of early cleavage were tested in a phylogenetic context for the first time. Cleavage was viewed a in a novel and entirely modem form that used individual cleavage stages as characters rather than as a 'suite' of features. The results of the analysis provided the first objective support for the classical conception of the importance of early cleavage in the evolutionary history of multicellular animals. The Porifera, Cnidaria, and Ctenophora showed the plesiomorphic meridional cleavage pattern, although the patterns in Cnidaria and Ctenophora were
each somewhat modified from that of poriferans. Members of the protostome clade all showed oblique cleavage, and the Priapulida and deuterostomes all possess equatorial cleavage. The Ecdysozoa all exhibit multi-plane cleavage. Thus, the results of the current study suggest that the form of cleavage pattern is a synapomorphy for the deuterostomes + protostomes, protostome clade, and the Ecdysozoa. Van Cleave (1941) suggested that relationships among the Acanthocephala were unclear because suffiaent characters were not present because of their parasitic lifestyle. Direct examination of specimens yielded 138 morphological and developmental characters and demonstrated that acanthocephalans possess a rich store of morphological characters that have been overlooked for phylogenetic analyses. Attempts to danfy character assignment for the Acanthocephala showed that taxonomic problems inherent in the current arrangement of groups within the phylum hamper phylogenetic studies unless taxonomic revisions are first undertaken to clarify the concepts of higher tam. One example, the revision of Tegorhynchu%erection of a new genus JCoronacanthpfor the new species K. mexicana, and reassignment of 1.pectinarius to Koronacantha, was used to demonstrate current taxonomic problems in many of the little-studied taxa. In addition, evolutionary patterns of the structure of the cement glands were tested using three hypotheses: (i.)that the plesiomorphic cement gland structure in the sister-group, the Rotifera, is unknown, (d.)that non-syncytial cement glands are plesiomorphic for both taxa, and (u.)that syncytial cements glands are p lesiomorphic. Phylogenetic analysis of the data set produced a single most parsimonious tree topology. Using Assumptions i and ii for the cement glands, the analyses produced the same single most parsimonious tree (length = 404 steps, CI = 0.545, CIX = 0.517, HI = 0.455, HIX = 0.483, RI = 0.670, RC = 0.365). Using Assumption the tree was four steps longer (length = 408 steps, CI = 0.539, CIX = 0.512, HI = 0.461, HIX = 0.488, RI = 0.665, RC = 0.359). This results of the study upheld many of the classic views concerning acanthocephalan taxonomy. For example, both the Eoacanthocephala and Palaeacanthocephaia were strongly supported as monophy le tic (based on five and 11 unambiguous character changes, respectively). The two classes form a monophyletic group based on seven unambiguous character state changes. However, monophyly of the more basal Archiacanthocephala was not supported. The results of the study provided strong support for the recognition of both Illiosentis and &gprhvnchus as distinct taxa, as well as the transfer of
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Note: indentation of names using " : " indicates one step in hierarchical level. : : : : Chaetognatha Porifera : : : : Cycloneuralia Placozoa : : : : : Gastrotricha Eumetazoa .... : Introverts : Cnidaria : : : : : : Nematoda : Bilateria : : : : : : Nematomorpha : : Protostomia ..... : Cephalorhyncha : : : Spiralia : : : : : : : Priapula : : : : Teloblastica : : : : : : : Kinorhyncha : : : : : Sipuncula : : : : : : : Loricifera : : : : : Articulata : : Protornaeozoa : : : : : : Mollusca : : : Ctenophora : : : : : : Euarticulata : : : Deuterostomia : : : : : : : Annelida : : : : Phoronida : : : : : : : Panarthropoda : : : : Brachiopoda : : : : : : : : Onychophora : : : : Neorenalia : : : : : : : : Arthropods ..... Pterobranchia : : : : : : : : Tardigrada : : : : : Echinodennata : : : : Bryozoa : : : : : Cyrtotreta : : : : : Entoprocta : : : : : : Enteropneusta : : : : : Ectoprocta ..... : Chordata : : : : Parenchyrnia : : : : : : : Urochordata : : : : : Platyhelminthes ...... : Notochordata : : : : : Nemertini : : : : : : : :Cephalochordata : : : Aschelminthes : : : : : : : : Vertebrata : : : : Rotifera : : : : Acanthocephala Appendix II A. Phyla of multicellular animals recognized by Brusca and Brusca, 1990. Acanthocephala Monoblastozoa Annelida Nematoda Arthlopoda Nema tomorpha Brachiopoda Nemertea Cephalochordata Onychophora Chae tognatha Orthonectida Chordata. Pentastomida Cnidaria Phoronida Ctenophora Placozoa Echinodermata Platyhelminthes Echiura Pogonophora Ectoprocta Porifera Entoprocta Priapulida Gastrotricha Rhombozoa Gna thostomulida Rotif era Hemichordata Sipuncula Kinorhyncha Tardigrada Loricifera Vestimentif era Mollusca Appendix II (cont.) 8. Taxa included in primary analysis. Acanthocephala Nematomorpha Brachiopoda Nemertea Chaetognatha Onychophora Chelicerata Pentastornida Choanoflagellata (outgroup) Phoronida Chordata PLa tyhelminthes ClitelIata Pogonophora Cnidaria Polychaeta Cmstacea Pori fera C tenophora Priap ulida Dicvemida (Rhombozoa) Rhovalura (Orthonectida) Echinoderrnata Rotifera Echiura Sipuncda Gastrotricha Tardigrada Gna thostomulida Tricoplax (Placozoa) Kinorhyncha Uniramia Mollusca Nematoda Appendix II (cont.) C. Taxa excluded from all analyses and reduced number of taxa included in the -a posterior secondary analysis. Taxa included in secondary analysis. Taxa exciuded from the analyses. Acanthocephala Cyclophora Choanoflagellata (outgroup) Cephalochordata Chordata Ectoprocta Cnidaria Enteropneusta Dicvemida (Rhombozoa) Entoprocta Echinodermata Loricifera Gastrotricha Pterobranchia Gnathostomulida Urochordata Kinorhyncha Nema toda Nematomorpha Onychophora Pentas tomida Pla tyhelminthes Pogonophora Porifera Priapulida Ro tifera Sipuncula Tardigrada Appendix II (cont.) D. Classification scheme for the Animalia based on the arrangement presented in the current study. Some names (marked by "?") are retained from those suggested by Nielsen, 1995. Note: indentation of names using " : " indicates one step in hierarchical level. KingdornAnimalia (Multicellular animals) Dicvemida ...... : Un-named Trichoplax : : : : : : : : : : : : Pogonophora Rho~lura : : : : : : : : : : : : : Un-named Porifera ...... : : : :Clitellata* Eurnetazoa : : : : : : : : : : : : : : : Polychaeta* : Cnidaria : : : : : Ecdysozoa : : Un-named ..... : Acanthocephala : : : Ctenophora : : : : : : : Un-named : : : : Un-named : : : : : : : : Rotifera : : : : : Bilateria : : : : : : : : : Cycloneuralia : : : : : : Deuterostornia : : : : : : : : : : Gastrotricha ..... : : Protornaeozoa (?) : : : : : : : : : : : Introverts : : : : : : : : Chaetognatha : : : : : : : : : : : : Un-named ...... Un-named ...... : : : : Nematoda : : : : : : : :: : Chordata : : : : : : : : : : : : : Nematomorpha ...... : : Un-named ..... : : : : : : : : : Panarthropoda (?) ...... : : : : : : : : : : : : Echinodermata ...... : : : : : : : : : Pentastomida ...... ---: : : : : : : : Un-named ...... : : : : : : : Phoronida ...... : : : : : : : Brachiopoda : : : : : : Un-named ...... : : : : : Tardigrada : : : : : : : Priapulida ::::::::::::::::::::Onychophora : : : : : : : : Protostomia ...... :::::::::::::::Arthropods : : : : : : : : Platyhelrninthes ...... :: : : :: : : ::: :Chelicerata : : : : : : : : Nemertea ...... Mollusca ...... : : : Teloblastica (?) : : : : : : : : : : Sipuncula Appendix III Characters by "system" (non-acronym characters are similar to those of Eernisse, et al., 1992) Characters that support the Choanoflagellata as the sister group to all multicellular organisms [MC] MCI. Choanocytes or choanocyte-derived cells (i.e.collar cells) (For characteristics of choanocytes and collar cells, see (Nielsen, 1995; Fig. 1): 0 = absent [Ancestor]; 1 = present [Choanoflagellata, Metazoa]; ? = [Trichoplax, Dicyemida, Rhopalura]. MC2. Accessory centriole of choanocyte perpendicular to flagellum: 0 = absent [Ancestor, Choanoflagellata]; I = present [Choanoflagellata, Trichoplax, Dicyemida, Rhopalura, Metazoa]. MC3. Actin as contractile structure (Data on presence of actin in choanoflagellates from Leadbeater, 1983): 0 = absent [Ancestor]; 1 = present [Choanoflagellata, Trichoplax, Dicyemida, Rhopalura, Metazoa]. MC4. Chloroplast (Hibberd, 1986): 0 = present [Ancestor]; I = absent [Choanoflagellata, Trichoplax, Dicyemida, Rhopalura, Metazoa]. MC5. Mitochondria1 cristae (Nielsen, 1995): 0 = tubular cristae [Ancestor]; 1 = flattened cristae (Leadbeater and Manton, 1974) [Choanoflagellata, Trichoplax, Dicyemida, Rhopalura, Metazoa]. Ingroup characters for "Animalia","Mesozoa", and "Metazoa"[OG] OG 1. Multicellularity: 0 = unicel~ular/colonial[Choanoflagellata]; 1 = multicellular [Trichoplax, Dicyemida, Rhopalura, Metazoa]. OG2. Complete separation of daughter cells during reproduction: 0 = division with complete separation of daughter cek [Choanoflagellata]; 1 = division without complete separation of daughter cells [Trichoplax, Dicyemida, Rhopalura, Metazoa] . 0G3. Inter-membranous rings of particles in choanocyte cilia (various metazoans have 3-4 rings, vertebrates have 45rings (Nielsen, 1991): 0 = various patterns, but not as in multicellular organisms [ChoanoflagelIata]; 1 = three to five rings of particles [Metazoa]; ? = [Trichoplax, Dicyemida, Rhopalura]. OG4. All somatic cells diploid (only egg and sperm haploid) (Choanoflagellata assumed to not be diploid, but no actual studies of chromosome number are known for choanoflagellates (Nielsen, 1995)): 0 = absent [Choanoflagellata]; 1 = present [Trichoplax, Dicyemida, Rhopalura, Metazoa]. OG5. Re-absorption and reformation of flagella: 0 = present [Choanoflagellata, Porifera]; 1 = absent [Metazoa excluding Porifera]; ? = [Trichoplax, Dicyemida, Rhopalura]. 0G6. General cell specialization: 0 = Somatic and reproductive cells only [Choanoflagellata, Dicyemida]; 1 = Additional specialized cell types [Trichoplax, Rhopalura, Metazoa]. OG7. Somatic cell specialization: 0 = Single type of somatic cells [Choanoflagellata]; 1 = Additional specialized somatic cell types [Dicyemida,Trichoplax, Rhopalura, Metazoa]. 0G8. Position of reproductive elements: 0 = Endogenous (intra-cellular) [Choanoflagellata, Dicyemida]; 1 = Exogenous (extra-cellular) [Trichopiax, Rhopalura, Me tazoa]. 0G9. Digestive process: 0 = retention by surface cells of inhacellular digestion [Choanoflagellata, Trichop lax, Dicyemida, Rhopalur a]; 1 = presence of specialized digestive cells [Metazoa]. OGlO. Specialized sensory cells: 0 = absent [Choanoflagellata, Trichoplax, Dicyemida, Rhopalura, Porifera]; 1 = present [Metazoa excluding Porifera]. OG11. Spermatozoa (Nielsen, 1995): 0 = absent [Choanoflagellata]; 1 = present [Trichoplax, Dicyemida, Rhopalura, Metazoa]. OG12. Occluding (septate, etc.) cell junctions (Madcie, 1984; Nielsen, 1995): 0 = absent [Choanoflagellata]; I = present [Trichoplax, Dicyemida, Rhopalura, Metazoa]. A. Early cleavage pattern [CLE] (Pogonophoran ontogeny poorly understood (Bakke, 1980; Ivanov, 1963; Ivanov, 1988); pogonophorans represented by Perviata;Meglitsch and Schram (1991) for all characters except Char. 14; Gnathostomulida, by (Riedl, 1969) and 'acoel' and 'polydad' flatworm deavage by (Boyer, 1971) and (Boyer, 1989). respectively; Solenogastces cleavage and ontogeny reviewed by Hadfield, 1979 and Salvini-Plawen, 1985; Caudofoveata ontogeny undescribed; 'Turbellaria' interpreted as polydads (Salvini-Plawen, 1988); additional information for cleavage patterns from Anderson, 1973; Brusca and Bwca, 1990; Cather, 1971; Costello and Henley, 1976; Freeman and Lundelius, 1992; Schleip, 1929; Wilson, 1898). CLE1. Cleavage type of first cell division of zygote (Centriole position in relation to animal and vegetal poles located in the vertical plane; division asynchronous in Dicyemida, but pattern similar to state 1.): 0 = meridional placement of centrioles [Ancestor, Choanoflagellata, Cnidaria, Chordata, Echinodermata, Phoronida, Brachiopoda, Platyhelminthes, Gnathostomulida, Nemertea, Priapulida, Sipuncula, Molluscs, Echiura, Pogonophora, Polychaeta, Clitellata, Porifera, Ctenophora]; 1 = polar placement of centrioles [Dicyemida, Nematoda, Acanthocephala, Rotifera, Gastrotricha, Nematomorpha, Pentastornida, Onychophora, Crustacea, Uniramia, Chelicerata, Tardigrada, Kinorhyncha, Loricifera]; ? = [Trichoplax, Rhopalura, Kinorhynchida, Loricifera]. CLEI, CLE2, and CLE3 replace Chars. 1,2and 3 of Eemisse, et al. (1992). Character 1. Spiral quartet cleavage; cells B and D with ventral transverse contact, cells A and C with dorsal saggital contact: 0 = absent; 1 = present. Character 2. Spiral cleavage with nuclear migration: 0 = absent; 1 = present. Character 3. Radial holoblastic cleavage: 0 = present; 1 = absent. From Eernisse, et al. (1992); excluded because most cells fates are known to be fixed to some extent in all tam, and the time of fixation varies even within phyla (Anderson, 1973; Brusca and Bruxa, 1990; Meglitsch and Schram, 1991a). Character 4. Cell fates of primary germ layers fixed by end of fifth cleavage: 0 = absent; 1 = present. CLEZ. Cleavage type of second cell division of zygote : 0 = cleavage planes meridional [Porifera, Cnidaria, Ctenophora, Platyhelminthes, Nemertea, Priapulida, Entoprocta, Gnathostomulida, Clitellata, Polychaeta, Sipuncula, Echiura, Pogonophora, Chelicerata, Unirarnia, Crustacea, MoUusca, Phoronida, Ectoprocts, Brachiopoda, Echinodermata, Chaetognatha, Enteropneusta, Pterobranchia, Urochordata, Cephalochordata, Chordata]; 1 = cleavage planes at right angles to each other [Dicyemida, Nematoda, Acanthocephala, Rotifera, Gastrotricha, Nematomorpha, Pentastomida, Onychophora, Crustacea, Uniramia, Chelicerata, Tardigrada]; ? = [Ancestor, Choanoflagellata, Trichoplax, Rhopalura, Kinorhynchida, LoriciferaJ. CLE3. Cleavage type of third cell division of zygote (Lack of clearly defined third division in Dicvemida coded as "unknown".): 0 = meridional cleavage plane [Porifera, Ctenophora]; 1 = transverse cleavage plane [Cnidaria, Chordata, Echinodermata, Phoronida, Brachiopoda, Priapulida]; 2 = oblique cleavage plane [Pla tyhelminthes, Gnathostomulida, Nemertea, Sipuncula, Molluscs, Echiura, Pogonophora, Polychaeta, Clitellata]; 3 = multiple cleavage planes [Nematoda, Acanthocephala, Rotifera, Gastrotricha, Nematomorpha, Pentastomida, Onychophora, Crustacea, Uniramia, Cheiicerata, Tardigrada]; ? = [Trichoplax, Dicyemida, Rhopalura, Kinorhynchida, Loricifera]. 5. Entomesoblast cell (blastomere 4d) (Beklemishev, 1969a, 1969b; Boudreaux, 1979; Verdonk and van den Biglelaar, 1983): 0 = absent; 1 = present. From Eernisse, et al. (1992); excluded because confounded with determination of the features of the 'coe1om'-a subject for future investigation. Character 6. Entomesoblast proliferation into paired anterior coelornic sacs (Brusca and Brusca, 1990): 0 = absent (non-cavitated mesenchyme); 1= present. 7. Entomesoblast proliferation contributing to mesoderm (Anderson, 1973; Wilson, 1898): 0 = absent (mesoderm absent or forming from ectoderm or archenteron); 1 = present. 8. Entomesoblast proliferation into paired dorsoposterior mesodermal tissue bands (Brusca and Brusca, 1990; Salvini-Plawen, 1985,1988): 0 = absent; 1 = present. 9. Teloblastic segmentation of mesodermal sacs with pygidial growth zone (Salvini- Plawen, 1985,1988): 0 = absent; 1 = present. 10. Epidermal mitosis by parenchymal kinetosome-containing cells (Ehlers, 1986; Smith, et al., 1986): 0 = absent; 1 = present. 11. Apical and intermediate micromere quartet form cross (Meglitsch, 1972): 0 = absent; 1 = present. 12. Cross pattern (Brusca and Bmsca, 1990; Meglitsch, 1972): 0 = radiate; 1 = inter-radiate. From Eernisse, et al. (1992); excluded because of confusion concerning 'mesoglea' in 'lower' metazoam-condition is probably an artifact (Bmsca and Brusca, 1990; Hanson, 1977; Meglitsch and Schram, 1991a; Nelson and Weisblat, 1991). Character 13. Triploblastic tissue organization : 0 = absent; 1 = present. B. Coelom (Characters of coelom and mesoderm ontogeny: mollusc references reviewed in (Salvini-Plawen, 1985); monoplacophoran "dorsal coelorns" not coded as present (Wingstrand, 1985); coelornic metamerism in Uniramia from B msca and Brusca (1990); pogonophorans represented by Vestimentifera (Bmsca and Brusca, 1990). 14. Bilaterally paired coelomic anlagen (Brusca and Brusca, 1990; Meglitsch and Schram, 1991a): 0 = absent; 1 = present. 15. Longitudinally metameric coelomic cavities with mesodermal contribution to mesenteric partitions (Bmsca and Bmsca, 1990; Meglitsch and Schram, 1991a): 0 = absent; 1 = present. 16. Tripartite coelom (Brusca and Brusca, 1990): 0 = absent; 1 = present. 17. Schizocoelous formation of body cavity lined with mesodermal peritoneum (characterization of nemertean rhynchocoel by Turberville, 1991; Turberville and Ruppert, 1985) (Brusca and Brusca, 1990; Meglitsch and Schram, 1991a): 0 = absent; 1 = present. 18. Enterocoelous formation of body cavity Lined with mesodermal peritoneum (Tardigrades in Nelson, 1982); priapulids (Lang, 1848, in Meglitsch and Schram, 1991) (Bmsca and Brusca, 1990; Meglitsch and Schram, 1991a): 0 = absent; 1 = present. 19. Hemocoel; main body cavity unlined with lymph-filled vacuities (Brusca and Brusca, 1990; Meglitsch and Schram, 1991a): 0 = absent; 1 = present. 20. Gonocoel; coelom reduced to perigonadal region (Brusca and Brusca, 1990; Meglitsch and %am, 1991a): 0 = absent; 1 = present. From Eemisse, et al. (1992); excluded because it is confounded with the 'coelom' (Willmer, 1990). The pressure component cannot be objectively quantified from current knowledge. Character 21. Hydrostatic skeleton; coelomic compartment under relatively high pressure (B~scaand Bmsca, 1990; Meglitsch and *am, 1991a): 0 = absent; 1 = present- 22. Pericardial excretory complex of coelomoducts connected to cloaca (Salvini- Plawen, 1985): 0 = absent; 1 = present. From Eemisse, et al. (1992); excluded because pseudocoelom is known to arise in several different forms (Willmer, 1990), and is primarily identified by being a true coelom. Character 23. Pseudocoelom; blastocoel persisting as unlined cavity(ies) between endodenn and mesoderm (Meglitsch and Sduam, 1991a): 0 = absent; 1= present. C.Cellular [CEL) CELL Belt desmosomes (Mackie, 1984; Nielsen, 1995): 0 = absent [Choanoflagellata]; 1 = present [Trichoplax, Porifera, Cnidaria, Ctenophora, Platyhelminthes, Nemertea, Rotifera, Gastrotricha, Kinorhyncha, Nematoda, Nema tomorp ha, Priapulida, Acanthocephala, Entoprocta, Gnathostomulida, Loricifera, Clitellata, Polychaeta, Sipuncula, Echiura, Pogonophora, Chelicerata, Uniramia, C~stacea, Onychop hora, Tardigrada, Pentastomida, Mollusca, Phoronida, Ectoprocts, Brachiopoda, Echinoderrnata, Chaetognatha, Enteropneusta, Pterobranchia, Urochordata, Cephalochordata, Chordata]; ? = [Dicyemida, Rhopalura]. CELZ. Spot desmosomes (Madcie, 1984; Nielsen, 1995): 0 = absent [Choanoflagellata, Trichoplax, Cnidaria, Ctenophora, Matyhelminthes, Nemertea, Rotifera, Gastrotricha, Kinorhyncha, Nematoda, Nematomorpha, Priapulida, Acanthocephala, Entoprocta, Gnathostomulida, Loricifera, Clitellata, Polychaeta, Sipuncula, Echiura, Pogonophora, Chelicerata, Uniramia, Cmstacea, Onychophora, Tardigrada, Pentastomida, Mollusca, Phoronida, Ectoprocts, Brachiopoda, Echinodermata, Chaetognatha, Enteropneusta, Pterobranchia, Urochordata, Cephalochordata, Chordata]; 1 = present [Porifera]; ? = [Dicyemida, Rhopalura]. CEL3. Hemidesmosomes (Mackie, 1984; Nielsen, 1995): 0 = absent [Choanoflagellata, Trichoplax, Dicyemida, Rhopalura, Porifera]; 1 = present [Cnidaria, Ctenophora, Platyhelminthes, Nemertea, Rotifera, Gastrotricha, Kinorhyncha, Nematoda, Nematornorpha, Priapulida, Acanthocephala, Entoprocta, Gnathostomulida, Loricifera, Clitellata, Polychaeta, Sipunda, Echiura, Pogonophora, Chelicerata, Uniramia, Crustacea, Onychophora, Tardigrada, Pentastornida, Mollusca, Phoronida, Ectoprods, Brachiopoda, Echinodermata, Chaetognatha, Enteropneusta, Pterobranchia, Urochordata, Cephalochordata, Chordata]. 24. Multicellular with population of gamete cells (Hanson, 1977; Nielsen, 1985): 0 = absent; 1 = present. 25. Special sense cells (Hanson, 1977; Nielsen, 1985): 0 = absent; 1 = present. 26. Gap junctions (Nielsen, 1985) ): 0 = absent; 1 = present. 27. Basal lamina (Nielsen, 1985): 0 = absent; 1 = present. 28. Mitosis lacking in epidermal cells (Ehlers, 1986; Smith, et al., 1986): 0 = absent; I = present. D. Ciliary [Cn] (Much of the information on Ciliary and flagellar Chars. 30-39 from Tyler, 1979, Nielsen, 1979,1985,1987, and Nielsen and Nerrevang, 1985). 29. CILl. Collared ciliary units (Willmer, 1990) (approximately equal to Character 29 of Eernisse, et al., 1992): 0 = absent; 1 = present. 30. Ciliated somatic cells (Nielsen, 1987): 0 = absent; 1 = present. 31. Multiciliary epidermis with ciliated rootlets (Nielsen, 1987; Smith, et al., 1986): 0 = absent; 1 = present. 32. Ciliated ventral surface in adult (Nielsen, 1987): 0 = absent; 1 = present. From Eemisse, et al. (1992); features combined with MC2 and CLI. Character 33. Monociliated cells with accessory centriole (Nielsen, 1987): 0 = present; 1 = absent (multiciliated without corresponding accessory centrioles) From Eemisse, et al. (1992); features combined with MC2 and CIL1. Character 34. Cilium with one basal body; without accessory centriole (Ehlers, 1986; Nielsen, 1987): 0 = absent; 1= present. 35. Coordinated cilia with ciliated necklace (Nielsen, 1987): 0 = absent; 1 = present. 36. Motile somatic cilia or flagella (Nielsen, 1987): 0 = absent; 1 = present. From Eemisse, et al. (1992); excluded because discocilia's paddle shape itself is a preservation artifact (Nielsen, 1987; Nielsen 1995). Character 37. Chemoreceptor cells with paddle-shaped discocilia (Haszprunar, 1985b): 0 = absent; 1 = present. E. Larval (Characters of larval morphology: taxa with exclusive direct development coded "absent" if larval character state is not known to be part of the developmental pattern.) 38. Upstream collecting bands of cilia in larvae with separate cilia on monociliate cells (Nielsen, 1987): 0 = absent; 1 = present. 39. Swimming/feeding band(s) of cilia in larvae with compound cilia (Lack of compound cilia in oweniids (Polychaeta) considered a derived exceptional case by Nielsen, 1987) (Nielsen, 1987): 0 = absent; 1 = present. 40. Protohoch; locomotory equatorial ciliary band(s) with two or four rows of broad cilia formed before gastrulation (Brusca and Brusca, 1990; Strathmann, 1987): 0 = absent; 1 = present. 41. Pelagic larvae with apical ciliary tuft and plate (Nielsen, 1987): 0 = absent; I = present. 42. Nutritive metatroch with opposed bands; post-oral (segmentally added) paired ciliary bands beating in opposite directions and sewing in food capture Sipundans and nemerteans without opposed band mechanism(Strathmann, 1978; Salvini-Plawen, 1988): 0 = absent; 1 = present- From Eernisse, et al. (1992); exduded because ocelli and pigment cups did not appear to be similar in structure even though Rosen, et al., 1979 considered them to be homologous. Character 43. Cerebral rhabdomeric larval ocelli or integumentary pigment cups (Uniramia coded "present", although myriopods possess direct development. Absence of larval oceIli viewed as secondarily derived because of loss of larval stage (Brusca and Brusca, 1990). (Bmaand Bmsca, 1990; Meglitsch and Schrarn, 1991a; Rosen, et al., 1979): 0 = absent; 1= present. 44. Telotroch; pelagic larvae with para- or circumanal ciliary tuft (Brusca and Brusca, 1990; Meglitsch and Schrarn, 1991a; Nielsen, 1987): 0 = absent; 1 = present. 45. Pre-oral fold covering larval hyposphere (Brusca and Bmsca, 1990; Megiitsch and Schram, 1991a): 0 = absent; 1 = present. 46. Pericalymna; unpaired, mineralized, epispheral test enveloping larvae (Sipunculans show a "serosa larva" and polychaetes an "endolarva" (Salvini-Plawen, 1988). (Brusca and Brusca, 1990; Salvini-Plawen, 1988): 0 = absent; 1 = present. F. Bilateral symmetry (Characters based on paired lateral structures sharing similar relative topology and ultrastructure.) 47. Paired ventral nerve bundles (Beklemishev, 1969a, 1969b; Brusca and Brusca, 1990; Kozloff, 1990): 0 = absent; 1 = present. 48. Paired ventral lateral pedal retractor musde bundles (Solenogastres in Salvini- Plawen, 1985) (Wingstrand, 1985): 0 = absent; 1 = present. From Eernisse, et al. (1992); excluded because excretory organs are dealt with in other characters and nephridiopores are not defined for most taxa (Brusca and Brusca, 1990). Character 49. Paired excretory organs and ducts open externally (nephridiopores): 0 = absent; I = present. 50. Paired gills; ectodermal filamentous or lamellar respiratory surfaces (Brusca and Brusca, 1990): 0 = absent; 1 = present. 51. Paired gonads and gonoducts (or nephridiopores used as gonoducts) (Uniramia coded "present" although Diplopoda present, Chilopoda variable and Pauropoda females have single ovary, all with paired gonoducts.): 0 = absent; 1 = present. 52. Paired endotheliurn-lined pericardial divertidae (auricle) (Wingstrand, 1985): 0 = absent; 1 = present. G. Serial repetition (See Eemisse et al, 1992 and Brusca and Bmsca, 1990 for full discussion of difference between serial repetition and metamerism.) 53. Serially repeated nerve collaterals; ladder-like nervous system with ventrolateral nerve cords and lateral connectives (Bekkmishev, 1969a, 1969b; Wingstrand, 1985): 0 = absent; 1 = present. From Eemisse, et al. (1992); excluded because Eernisse, et al. (1992) duplicated this character as Char. 56. Character 54. Serially repeated transverse discrete muscle bundles (Beklemishev, 1969a, 1969b; Wingstrand, 1985): 0 = absent; I = present 55. Serially repeated nerve ganglia (Pogonophora coded polymorphic based on Meglitsch and Sduam, 1991) (Beklemishev, 1969a, 1969b; Wingstrand, 1985): 0 = absent; 1 = present. 56. Serially repeated transverse discrete muscle bundles (Beklemishev, 1969a, 1969b; Wingstrand, 1985): 0 = absent; 1 = present. 57. Serially arranged series of excretory ducts; nephridiopores (Bmsca and Bmsca, 1990): 0 = absent; 1 = present. 58. Serially arranged ectodermal filamentous or lamellar respiratory surfaces (Chordata from Eemisse et al., 1992) (Brusca and Brusca, 1990): 0 = absent; 1 = present. 59. Serially arranged series of gonads (Bmsca and Bmsca, 1990): 0 = absent; 1 = present. 60. Atria; serially arranged muxularized regions of a dorsal blood vessel (Salvini- Plawen, 1985; Wingstrand, 1985): 0 = absent; I = present- 61. Schizocoelous metamerism between pre-oral prostomium and non-metameric pygidium: 0 = absent; 1 = present. 62. One or more transverse coelomic septa (Brusca and Brusca, 1990): 0 = absent; 1 = present. 63. Serially repeated ventricles; branchioauricular sinuses with ctenedial pores (Bmsca and Bmsca, 1990; Ruppert and Carle, 1983; Wingstrand, 1985): 0 = absent; 1 = present. 64. Metamerism in associated cuticular, muscular, and nervous tissues (Bmsca and Brusca, 1990; Meglitsch and Sduam, 1991a): 0 = absent; 1 = present. H. Circdatory (Characters of the blood vaxular system fiom Ruppert and Carle, 1983). 65. Heart(s); dorsal blood vessel with contractile epithelium formed around a vascularized longitudinal lumen by hsion of coelomic walls and lined by a basal lamina (Brusca and Brusca, 1990; Ruppert and Carle, 1983): 0 = absent; 1 = present. 66. Closed posterior circulation (Brusca and Brusca, 1990; Ruppert and Carle, 1983; Salvini-Plawen, 1985): 0 = absent; 1 = present. 67. Atrial ostia; muscularized opening(s) in dorsal blood vessel (Ruppert and Carle, 1983; Salvini-Plawen, 1985,1988): 0 = absent; 1 = present. 68. Atrial dtra-filtration (Salvini-Plawen, 1985,1988): 0 = absent; 1 = present. 69. Hemerythrin or myohemerythrin as a respiratory pigment molecule (Demuynck, et al., 1991; Mangum, et al., 1985; Richardson, et al., 1987; Takagi and Cox, 1991; Volbeda and Hol, 1989; Yano, et al., 1991): 0 = absent; 1 = present. From Eernisse, et al. (1992); combined with Char. 71. Character 70. Hemocyanin as a respiratory pigment molecule hypothesized to be homologous based on spectroscopic and sequence similarities (Lang, 1988; Lang and Holde, 1991; Mangum, et al., 1987; Mangum, et al., 1985; Voit and Feldmaier, 1990; Volbeda and Hol, 1989): 0 = absent; 1 = present. 71. Hemocyanin structure (Linzen, et al., 1985; Mangum, et al., 1987): 0 = hexamer or multihexamer "boxcar" molecules with subunits of about 75,000 molecular weight, together combining to up to 3-5 million, each containing one dinuclear copper site; 1 = cylindrical molecules made up of about 10-20 "stacked petri-plate" subunits, each of about 350,000 molecular weight, containing seven or eight domains with one oxygen-binding dinudear copper site per domain. 72. Mesodermal origin of pericardioducts (Salvini-Plawen, 1985,1988): 0 = absent; 1 = present. I. htegumentary [INTI (Characters of the ectoderrnal integument: arthropods, Manton and Anderson, 1979; rnoUuscs, Runnegar, 1983, Runnegar and Pojeta, 1985); Salvini-Plawen, 1985,1988; Wingshand, 1985). INTI. Collagen in the integument (Nielsen, 1995): 0 = absent; 1 = present. INTZ. Collagen as fibers in the integument (Nielsen, 1995): 0 = absent; I = present. 73. Cellular production of collagenous proteins (Brusca and Brusca, 1990; Meglitsch and Schrarn, 1991a): 0 = absent; 1 = present. 74. Cuticle; continuously secreted, non-living external layer(s) containing protein (Bmsca and Brusca, 1990; Meglitsch and Sduam, 1991a): 0 = absent; 1 = present. 75. Collagenous proteins sequestered in cuticle (Collagenous exoskeletal perisarc of gorgonians (Cnidaria) considered similar to the cuticle of insects (Goldberg, (Goldberg, 1976).) (Bereiter, et al., 1984; Brown, 1975; Brusca and Brusca, 1990; Meglitsch and Schram, 1991a): 0 = absent; 1 = present. 76. Chitinous proteins invested in cuticle (Chitin found in tube of Phomida, but not in the cuticle (Hyman, 1958).(Bereiterfet al., 1984; Brown, 1975; Bmsca and Brusca, 1990; Meglitsch and Sduam, 1991a): 0 = absent; 1 = present. 77. Cellular secretion of chitinous proteins (Report of chitin in Chordata (Sannasi and Hermann, 1970) rejected by Azariah (1973).) (Bereiter, et al., 1984; Brown, 1975; Bmsca and Bruxa, 1990; Meglitxh and Schram, 1991a; Willmer, 1990): 0 = absent; 1 = present. ~ticularcovering of entire external bod] lrface (Brusca and Brusca, 1990; Meglitsch and Schram, 1991a): 0 = absent; 1 = present. 79. Sclerotinization of cutide with tannin proteins (Brusca and Brusca, 1990; Meglitsch and Sduam, 1991a): 0 = absent; 1 = present. 80. Protrusible and retractable chitinous setae (Brwa and Bwca, 1990; Meglitsch and Schrarn, 1991a): 0 = absent; 1 = present. 81. Alpha and/or beta ecdysone (Willmer, 1990): 0 = absent; 1 = present. 82. Periodic ecdysis under hormonal control (Bmsca and Brusca, 1990; Meglitsch and Schram, 1991a): 0 = absent; 1 = present. 83. Anterior ecdysome-producing gland (Brusca and Brusca, 1990; Meglitsch and Schram, 1991a): 0 = absent; 1 = present. 84. Aciculae; tonofibrillae penetrating epidermis with muscle attachment (Brusca and Brusca, 1990; Meglitxh and Schram, 1991a): 0 = absent; 1 = present. 85. Mantle; thick epidermal cuticular sheet with band(s) of glands capable of secreting a hard calcareous skeleton (Brusca and Brusca, 1990; Meglitsch and Sduam, 1991a): 0 = absent; 1 = present. 86. Calcified skeletal covering secreted by epidermis (According to Fautin and Mariscal(1991),the calcification mechanisms of anthotoan and hydrocoral (Cnidaria)skeletons, which occurs within specialized regions of the epidermis, are not completely understood.) (Brown, 1975; Brusca and Brusca, 1990; Carter, 1990; Salvini-Plawen, 1988; Tidball, 1984): 0 = absent; 1 = present. 87. Lateral tergal folds or paranotal lobes (Ghiseiin, 1988; Salvini-Plawen, 1988): 0 = absent; 1 = present. 88. Anterior cephalic tagma formed from metameres and the primary sensory acron (Brusca and Brusca, 1990; Meglitsch and Sduam, 1991a): 0 = absent; 1 = present. 89. Serially arranged mineralized ectodermal plates (Brusca and Brusca, 1990; Meglitsch and Schram, 1991a): 0 = absent; 1 = present. J. Alimentary system 90. U-shaped alimentary canal (Brusca and Bwca, 1990): 0 = absent; 1 = present. 91. Crystalline stylus and associated ciliated midgut digestive organs (Bmsca and Bma,1990): 0 = absent; 1 = present. 92. Pharyngeal diverticulae (Salvini-Plawen, 1988): 0 = absent; 1 = present. 93. Esophageal pouches (Salvini-Plawen, 1988): 0 = absent; 1 = present. 94. Terminal alimentary zones of cuticle (Boudreaux, 1979): 0 = absent; I = present. 95. Secondary mouth formation (Meglitsch and Sduam, 1991a): 0 = absent; 1 = present. 96. Anus with proctodeum; complete unidirectional alimentary canal (Vestimentifera have transitory alimentary canal in early ontogeny; character coded as polymorphic for Pogonophora according to Meglitsch and Schram, 199la).(Meglitsch and *am, 1991a): 0 = absent; 1 = present. K. Excretory system [EXC] (Types of protonephridial structures from (Rohde, 1990,1993; Watson, et al., 1992) EXCI . Selenocytes (Nielsen, 1995): 0 = absent; 1 = present. EXC2. Protonephridia (Rohde, 1990): 0 = absent; 1 = present. From Eernisse et al., 1992-replaced to restrict only to structural features of protonephridia without a priori judgment as to what constitutes protonephridia. Character 99. Protonephridia; arnpullary (blind) vessels bearing multiciliated cells serving excretory /osmoregulatory function (Bmaand Brusca, 1990; Meglitsch and Schrarn, 1991a): 0 = absent; 1 = present. EXC3. An tennal gland: 0 = absent; 1 = present. From Eemisse et al., 1992-replaced to restrict only to antenna1 gland and not metanephridia. Character 97. Antenna1 gland excretory ducts; mandibular (first) pair metanephridia: 0 = absent; 1= present. EXC4. Metanephridia (Nielsen, 1995): 0 = absent; 1 = present. From Eernisse et al., 1992-replaced to restrict only to presence of metanephridia. Character 98. Metanephridia; paired mesodermal excretory ducts with ciliated funnel draining coelomic cavity(ies) (Brusca and Brusca, 1990): 0 = absent; 1 = present. EXC5. Rennet cells: 0 = absent; 1 = present. EXC6. Invertebrate 'kidney'(Willmer, 1990; Nielsen, 1995): 0 = absent; 1 = present. L. Nervous/Sensory [NER](Additional features of the nervous system from Beklemishev, 1969). NER1. Nervous system complexity: 0 = nerves absent [Ancestor, Choanoflagellata, Trichoplax, Dicyemida, Rhopalura, Porifera] 1 = nerves present, non-polar net [Cnidaria, Ctenophora] 2 = nerves present with polarized synapses [Platyhelminthes, Nemertea, Rotifera, Gastrotricha, Kinorhyncha, Nematoda, Nematomorpha, Priapulida, Acanthocephala, Entoprocta, Gnathostomulida, Loricifera, Clitellata, Polychaeta, Sipuncula, Echiura, Pogonophora, Chelicerata, Uniramia, Crustacea, Onychophora, Tardigrada, Pentastomida, Molluscs, Phoronida, Ectoprocts, Brachiopoda, Echinodermata, Chaetognatha, Enteropneusta, Pterobranchia, Urochordata, Cephalochordata, Chordata]. NER2. Nervous system central organization: 0 = nerves absent [Ancestor, Choanoflagellata, Trichoplax, Dicyemida, Rhopalura, Porifera] 1 = nerves present, non-polar net without nerve plexus [Cnidaria] 2 = nerves present, non-polar net with nerve plexus [Ctenophora] 3 = nerves present, polarized synapses, with ganglion [PlatyheLminthes, Nemertea, Rotifera, Gastrotricha, Kinorhyncha, Nematoda, Nematomorpha, Priapulida, Acanthocephala, Entoproda, Gnathostomulida, Loricifera, Clitellata, Polychaeta, Sipuncula, Echiura, Pogonophora, Chelicerata, Unirarnia, Cmstacea, Onychophora, Tardigrada, Pentastomida, Molluscs, Phoronida, Ectoprocts, Brachiopoda, Echinodermata, Chaetognatha, Enteropneusta, Pterobranchia, Urochordata, Cephalochordata, Chordata J. 100. Acetocholine (Willmer, 1990): 0 = absent; 1 = present. 101. Creatine phosphotase (Willmer, 1990): 0 = absent; I = present. From Eernisse et al., 1992-replaced because the character is so general in definition that it cannot be scored objectively and because it is covered by NERl and NEIU. Character 102. Population of specialized polar neurons with neurites and synaptic terminals (Bmsca and Brusca, 1990; Nielsen, 1985): 0 = absent; 1 = present. 103. Orthogon; dewe diffuse neural plexus with short peripheral connections and very long inter-ganglionic connections (Beklemishev, 1969a, 1969b; Brusca and Bmsca, 1990; Meglitsch and Schrarn, 1991a): 0 = absent; I = present. 104. Subcutaneous neural plexus; subepithelial location of (at least some) epiderrnally derived neurons (Beklernishev, 1969a, 1969b; Bmsca and Brusca, 1990; Meglitsch and Schram, 1991a): 0 = absent; I = present. 105. Circumpharyngeal chain of ganglia (buccal, pharyngeal, or subenteric) attached to longitudinal ventral nerve cord(s) (Beklemishev, 1969a, 1969b; Kozloff, 1990): 0 = absent; 1 = present. 106. Adnate ventral nerve cords (Kozloff, 1990): 0 = absent; 1 = present. 107. Lateral nerve cords; paired longitudinal cutaneous or subepidermal axon bundles descending from an anterior commissure or ganglion (Beklernishev, 1969a, 1969b; Brusca and Brusca, 1990; Meglitxh and Schrarn, 1991a): 0 = absent; 1 = present. 108. Dorsal nerve cord; median longitudinal cutaneous or subepidermal axon bundle descending horn an anterior commissure or ganglion (Beklemishev, 1969a; Bmsca and Bmsca, 1990; Meglitsch and Schrarn, 1991a): 0 = absent; 1 = present- 109. Ospluadia; chemosensory epithelial surfaces located on or near the gill(s); mechanoreceptive collar cells with eight or nine stereo microvilli (Brusca and Bmsca, 1990; Haszprunar, 1985a; Haszprunar, 1985b; Haszprunar, 1987): 0 = absent; 1 = present. 110. Endon; median cerebral ganglion and adjacent aboral statocyst organ (Beklemishev, 1969a, 1969b): 0 = absent; 1 = present. 111. Three pairs of cerebral ganglia; an anterior one receiving ocular input, a second receiving palpar or antenna1 input, and a third contributing to circumenteric connectives (Beklemishev, 1969a, 1969b; Brusca and Brusca, 1990): 0 = absent; 1 = present. 112. Paired olfactory fossae of pre-oral lobes (Beklemishev, 1969a, 1969b): 0 = absent; 1 = present. 113. Compound eyes with ommatidia (Bmsca and Brusca, 1990; Meglitsch and Schram, 1991a; Paulus, 1979): 0 = absent; 1 = present. 114. Ornmatidium consisting of a cornea with two comeagen cells, a tetrapartite eucone crystalline cone, and a retinula of eight cells (B~scaand Bmsca, 1990): 0 = absent; 1 = present. 115. Prostomial sensory antennae with basal ocelli with or without lens structure (Bmsca and Brusca, 1990): 0 = absent; 1 = present. 116. Unicellular sensory (tactile)setae in epidermis (Brusca and Brusca, 1990): 0 = absent; I = present. M. Reproductive: These features are known to have appeared independently many times (this conclusion is supported by assignment of low weights (Appendix V), but were left in for interest and future use. A more careful examination of these characters is becoming possible as knowledge concerning reproduction, and especially, sperm morphology continues to grow. 117. Hermaphroditic sexual system (Brusca and Bma, 1990): 0 = gonochoric; 1 = present in more than isolated species. 118. Filiform morphology of sperm (Ax, 1985; Franzen, 1987; Smith, et al., 1986; Wirth, 1984): 0 = absent; 1 = present. 119. Direct internal fertilization (Ax, 1985): 0 = external; 1 = internal. N. Respiratory 120. Gill with counter-current 02exchange (Brusca and Brusca, 1990): 0 = absent; 1 = present. 121. Cu ticie-lined tracheal tubes (Brusca and Brusca, 1990): 0 = absent; 1 = present. 122. Semi-internal lateroventral respiratory chamber (Bmsca and Bmsca, 1990): 0 = absent; 1 = present. 0.Oral 123. Subradular organ (Wingstrand, 1985): 0 = absent; 1 = present. 124. Radda; ribbon or plates or recurved chitinous teeth stretched over a supportive cartilaginous (or hemocoelic) basal expansion of the foregut epithelium (Eernisse and Kerth, 1988; Salvini-Plawen, 1988; Wingstrand, 1985): 0 = absent; 1 = present. From Eernisse et al. (1992)--excluded because "introverts" are known not be homologous developmentally (Nielsen, 1995) or morphologically (Wiher, 1990 and personal observation). In addition, as defined in Eernisse et al. (1992),the character is confounded with the presence of a digestive tract and armature. Character 125. Introvert (proboscis) at anterior end of digestive tract with barbs and hooks (Brusca and Bmxa, 1990; Meglitxh and Schrarn, 1991a; Nielsen, 1987): 0 = absent; 1 = present. 126. Lophophore; anterior ring of hollow ciliated tentacles formed by coelomic evaginations (Brusca and Bma, 1990): 0 = absent; 1 = present. 127. Mandibles; appendages of the thud postacronal head somite (Bmsca and Brusca, 1990): 0 = absent; I = present. 128. Two pairs of maxillae; appendages of postacronal head sornites four and five (Brusca and B~sca,1990): 0 = absent; 1 = present. P. MUSCU~Xsystem (Additional features of the somatic musculature and locomotor apparatus from: arthropods, Manton and Anderson, 1979; Emerson and Schram, 1990, molluxs Runnegar, 1983; Runnegar and Pojeta, 1985; Salvini-Plawen, 1980; Salvini-Plawen, 1985,1988; Wingstrand, 1985). 129. Oblique striated muscle fibers (Brusca and Brusca, 1990; Willmer, 1990): 0 = absent; 1 = present. 130. Cross striated muscle fibers (Brusca and Brusca, 1990; Willmer, 1990): 0 = absent; 1 = present. From Eemisse et al. (1992)-excluded because it is not possible to define this character in an objective format. The terms "dermal" and "external" are not adequately defined by the original author (Salvini- Plawen, 1978,1985) to spec@ the position in the body wall. Character 131. Dermal circular (or "external transverse") muscular fibers (Brusca and Brusca, 1990; Salvini-Plawen, 1978; Salvini-Plawen, 1985): 0 = absent; 1 = present. From Eemisse et al. (1992)-duded because it is not possible to define this character in an objective format. The terms "sheet" and "band" are not cleady defined by the original author (Salvini-Plawen, 1978,1985). Character 132. Longitudinal muscle sheet(s) or band@) (Brusca and Brusca, 1990; Salvini-Plawen, 1978; Salvini-Plawen, 1985): 0 = absent; 1 = present. 133. Inter-segmental tendon system (Brusca and Bmsca, 1990): 0 = absent; 1 = present. 134. Locomotor coxae with extrinsic and intrinsic muscles (Brusca and Brusca, 1990): 0 = absent; 1 = present. 135. Myofilaments (Hanson, 1977; Nielsen, 1985): 0 = absent; 1 = present. 136. Smooth muscle fibers (Brusca and Brusca, 1990; Hanson, 1977; Nielsen, 1985): 0 = absent; 1 = present. 137. Scleratized terminal structure on coxae (Brusca and Brusca, 1990): 0 = absent; 1 = present. 138. Broad creeping sole or narrow hydrostatic foot in ventral furrow (="pedal groove") (Wingstrand, 1985): 0 = absent; 1 = present. 139. Segmented serially arranged locomotor appendages with basal coxite and distal telopide (Bmsca and Brusca, 1990): 0 = absent; 1 = present. 110. Pedal glands; ventral and large with mucous secretion (Brusca and Brusca, 1990): 0 = absent; 1 = present. 141. Biramous appendages (Emerson and Schram, 1990; Grosberg, 1990; Meglitsch and Schram, 1991a): 0 = absent; 1 = present. Appendix N. Data Matrix for the Metazoa. Vertical lines indicate divisions between 'systems' (see Appendix m), 'E."indicates chars. from Eernisse et al., 1992. Character MMMMM100 0 0 0 0 0 0 CCCCCIGGGGGGGG 12345112345678 I ChoanoflageIlata Dicyernida Rhopalura Trichoplax Porifera Cnidaria C tenophora Pla tyhelminthes Nemertea Rotifera Gastrotricha Kinorhyncha Nematoda Nema tomorpha Priapulida Acan thocep hala Gna thostomulida Clitellata Appendix IV. (cont.) Character MMMMM100 0 0 0 0 0 0 CCCCCIGGGGGGGG 12345112345678 I Polychaeta Sipuncula Echiura Pogonophora Chelicerata Uniramia Crus tacea Onychophora Tardigrada Pentastomida Mollusca Phoronida Brachiopoda Echinoderrnata Chaetognatha Chordata 1 Appendix IV. (cont.) Character OOOOICCC5 78 91011 G G G G 1 L L L (E.) (E.) (E.) (E.) (E.) (E-) 9 11 1IE E E 0 1211 2 3 Choanoflagellata 00 0 0 000000000 Dicyemida 0011 113000000 Rhopalura 0011 ???OOOOOO Trichoplax 0011 ???OOOOOO Porifera 1011 000000000 Cnidaria 1111 001000000 Ctenophora 1111 0040?0??? Platyheiminthes 1 1 1 1 00210?011 Nemertea 1111 00211000? Ro tifera 1111 113000??? Gastrotricha 1111 113000??? Kinorhyncha 1111 ???OOO??? Nema toda 1111 11300000? Nematomorpha 1 1 1 1 11300000? Priapulida 1111 OOlOOO??O Acanthocephala 1 1 1 1 1130000?0 Gnathostomulida 1 1 1 1 OO21??OO? Clitella ta 1111 OO21111?? Appendix IV. (cont.) Character Character -- OOOOfCCC5 7 8 91011 G G G G I L L L (E.) (E.) (E-) (E.) (E.) (E.) 9 11 1IE E E 0 1211 2 3 Pol ychaeta Sipuncula Echiura Pogonophora Chelicerata Uniramia Crustacea Onychophora Tardigrada Pentastomida Mollusca Phoronida Brachiopoda Echinodermata Chae togna tha Chordata Appendix IV. (cont.) Character Polychaeta 1 11010000 Sipuncula 0 00010000 Echiura 1 00010000 Pogonophora ? (01)l 0 100 0 0 Chelicera ta ? O?OOOl?O Uniramia ? 1?0001?0 Crustacea ? 0?000110 Onychophora ? 010??110 Tardigrada ? 010001Z0 Pentastomida ? ???????? Mollusca 0 00010111 Phoronida ? 01101000 Brachiopoda ? 1 1 0(01) (01) 0 0 0 Echinodennata ? 11101000 Chaetognatha 0 I??????? Chordata ? 11130000 Appendix IV. (cont.) 25 26 27 28 l C 30 31 32 35 36 1 38 39 40 (E.) (E.) (E.) (E.) I 1 (E.) (E.) (E.) (E.) (E.) I (E-)(E.) (E.) I L I I1 I Choanoflagellata 00 0 0 000000 000 Dicyemida OOO? 1111?1 010 Rhopalura OOO? 1111?1 010 Trichoplax OOO? 1111?1 010 Porifeia O?OO 010OIO 000 Cnidaria 1100 110001 000 Ctenophora Ill? 1110?1 ??? Platyhelminthes 1 1 1 1 111I11 000 Nemertea 1110 111111 010 Rotifera ll?? 1110?1 ?OO Gastrotricha Il?? 110111 ?OO Kinorhyncha 1111 100001 000 Nematoda 1111 100000 000 Nematomorpha 1 1 1 1 100000 000 Priapulida Ill? 110001 000 Acanthocephala 1 1 ? 1 100000 000 Gnathostomulida 1 1 1 0 110101 000 CIitellata 1110 111011 010 Appendix IV. (cont.) Pol ychaeta 1110 111011 011 Sipuncula 110111001 001 Pogonophora 1110 111001 Ol? Chelicerata I110 100000 000 Uniramia 1110 100000 000 Crustacea 1110 100000 000 Onychophora 1110 100001 000 Tardigrada 1111 100000 000 Pentastomida 11?? 100000 000 Phoronida 1110 11OOOI 100 Brachiopoda 1110 110001 100 Echinodermata 1 1 1 0 110001 10 0 Chaetognatha Ill? 111000 000 Chordata 1110 110001 000 Appendix IV. (cont.) Character - - Choanoflagellata 0 0 0 0 0 000000 0 0 Dicyemida OO?OO 000000 0 0 Rhopalura OO?OO 000000 0 0 Trichoplax OO?OO 000000 0 0 Pori fera 00000 000000 0 0 Cnidaria 00000 000000 0 0 Ctenophora ????? ?OOOOO 0 0 Platyhelminthes 1 0 1 1 0 101010 10 Nemertea 10010 100010 11 Rotifera 00000 010010 0 0 Gastrotricha 00000 111010 0 0 Kinorhyncha 00000 111010 11 Nema toda 00000 000000 0 1 Nematomorpha 0 0 0 0 0 OO?OOO O? Priapulida 00000 000010 0 0 Acanthocephala 0 0 0 0 0 000010 0 0 Gnathostomulida 0 0 0 0 0 000000 0 0 Clitellata 00001 101010 11 Appendix IV. (cont.) Character Polychaeta Sipuncula Echiura Pogonophora Chelicerata Uniramia Crus tacea Onychophora Tardigrada Pentas tomida Mollusca Phoronida Brachiopoda Echinoderrnata Chaetognatha Chordata Appendix IV. (cont.) Character Choanoflagellata 0 00 0 0 0 00 0 0000 Dicyemida 000000000 0000 Rhopalura 000000000 0000 Trichoplax 000000000 0000 Porifera 000000000 0000 Cnidaria 000000000 0000 C tenophora 000000000 0000 Platyhelminthes 0 00 0 0 0 0 00 0000 Nemertea 010100000(01)100 Rotifera 000000000 0000 Gastro tricha 000000000 0000 Kinorhyncha 1OOOOOOOZ 0000 Nematoda 000000000 0000 Nematomorpha 0 00 0 0 0 0 00 0000 Priapulida 000000000 0000 Acanthocephala 1 00 0 0 0 0 00 0000 Gnathostomulida 0 00 0 0 0 0 00 0000 Cli tella ta 111101111 1100 Appendix IV. (cont.) Character Pol ychaeta Sipuncula Echiura Pogonophora Chelicerata Uniramia Crustacea Onychophora Tardigrada Pentas tomida Mollusca Phoronida Brachiopoda Echinodermata Chae tognatha Chordata Appendix IV. (cont.) Character ------69 71 72 I I I 73 74 75 76 77 78 79 80 (E.) (E.) (E.) I N N (E.) (E.) (E.) (E.) (E.) (E.) (E.) (E.) IT T I1 2 Cnoanoflagellata 0 0 0 0000000000 Dicyemida 000 0000000000 Rhopalura 000 0000000000 Trichoplax 000 0000000000 Porifera 000 1000000000 Cnidaria 000 1OI1101000 Ctenophora 000 10?1?00000 Platyhelminthes 0 00 1010000000 Nemertea 000 1010001000 Rotifera 000 tO1111OOOO Gastrotricha 000 1011110000 Kinorhyncha O?O 1011011100 Nematoda 000 1111101110 Nematomorpha 0 0 0 111??0?1?0 Priapulida 100 10???11000 Acanthocephala 0 00 1011?00000 Gnathostomulida 0 0 0 1010001000 CIitellata 000 1011101001 Appendix IV. (cont.) Character Pol ychae ta Sipuncuia Echiura Pogonophora Chelicerata Unirarnia Crustacea Onychophora Tardigrada Pentastornida Molhsca Phoronida Brachiopoda Echinodermata Chaetognatha Chordata Appendix IV. (cont.) Character Choanoflagellata Dicyemida Rhopalura Trichoplax Porifera Cnidaria C tenophora PIa tyhelrninthes Nemertea Rotifera Gastrotricha Kinorhyncha Nematoda Nematomorpha Priapulida Acanthocephala Gna thostomulida Cli tella ta 000000000 0000 Appendix N.(cont.) Character -- Pol ychaeta 000101000 0 0 0 0 Sipuncula 000000001 1000 Echiura 000000000 0000 Pogonophora 000000000 ?OOO Chelicerata 110101110 0 0 0 0 Uniramia 111101110 0000 Crustacea 111101110 0000 Onychophora 010000000 0000 Tardigrada 110000000 0000 Pentastomida llO?OO??O 0000 Mollusca 00001100? 0111 Phoronida 000000000 1000 Brachiopoda 000011000 1000 Echinodermata 00 0 00 1 00 0 1000 Chaetognatha OOO?OOOOO 0000 Chordata 000001000(01)000 Appendix IV. (cont.) Character 94 95 96 I E E E E E E I N N 100 103 (E.) (E.) (E.) I X X X X X X l E E (E.) (E.) IC C C C C CIR R 11 2 34 5 611 2 ------ Choanoflagellata 0 0 0 000000 0000 Dicyemida 0 0 0 000000 OO?O Rhopalura 000 000000 OO?O Trichoplax 000 000000 OO?O Porifera 000 000000 0010 Cnidaria 000 000000 1110 Ctenophora 000 000000 1210 Platyhelminthes 00 0 0(13) 0 00 0 2311 Nemertea 001 020000 2311 Rotifera ?01 020000 2311 Gastrotricha ?01 010000 2311 Kinorhyncha ?01 010000 2310 Nematoda ?01 000010 2311 Nematomorpha ? 0 1 000000 2311 Priapulida 10 1 100100 2310 Acanthocephala 0 0 0 030000 2310 Gnathostomulida 0 0 0 020000 2310 Clitellata ?01 000100 2310 Appendix IV. (cont.) Character ) (E.) (E) I X X X X X X l E E (E.) (E*) IC C C C C CIR R Polychae ta 10 I 100100 2310 Sipuncula 101 000100 2311 Echiura 101 I00100 2311 Pogonophora 001 O?OOOO 2310 Chelicerata 101 001100 2310 Uniramia 101 001100 2310 Crustacea 101 001100 2310 Onychophora 101 001100 2310 Tardigrada 101 000000 2310 Pentastornida ?01 000000 2310 Mollusca 001 000001 2311 Phoronida 001 100100 2310 Brachiopoda 0 11 000100 2310 Echinodennata 0 1 1 000100 2311 Chae togna tha 0 11 000000 2311 Chordata 011 000000 2310 Appendix N.(cont.) Character Choanoflagellata Dicyemida Rhopalura TrichopIax Porifera Cnidaria C tenophora PIa tyhelminthes Nemertea Rotifera Gastrotricha Kinor hyncha Nematoda Nematomorpha Priapulida Acanthocephala Gna thostomulida Clitella ta Appendix IV. (cont.) -- Character PoIychaeta Sipunda Echiura Pogonophora Chelicera ta Crustacea Onychophora Tardigrada Pentastornida Mollusca Phoronida Brachiopoda Echinoderms ta Chaetognatha Chordata 1000000010000 Appendix IV. (cont.) - Character Choanoflagellata 00 ? 000 00000 0 Dicyemida 001 000 00000 0 Rhopalura 001 000 00000 0 Trichoplax 001 000 00000 0 Porifera 10000 00000 0 Cnidaria 100 000 00000 0 Ctenophora 1?0 000 00000 0 Platyhelminthes 1 1 1 000 00000 1 Nemertea 000 000 00000 0 Rotifera 0?1 ?OO 00000 0 Gastrotricha (01) ? ? 300 00000 0 Kinorhyncha 001 000 00000 ? Nema toda 001 000 00000 1 Nematomorpha 0 ? ? ??? ????? 1 Priapulida 000 000 00000 I Acanthocephala 0 1 1 000 00000 ? Gnathostomulida 1 1 I 000 00000 0 Cli tella ta 101 000 00000 0 Appendix IV. (cont.) Character Pol ychaeta 000 100 00000 0 Si puncula 000 000 00000 0 Echiura 000 000 00000 0 Pogonophora 000 100 00000 0 Chelicerata 001 111 00000 1 Uniramia 001 011 00011 1 Crustacea 001 111 00011 1 Onychophora 001 011 00000 0 Tardigrada 001 000 0 0 0 0 0 (01) Pentas tomida 001 ?OO 00000 0 Mollusca 10 1 100 11000 0 Phoronida 000 100 00100 0 Brachiopoda 00 0 100 00100 0 Echinodermsta 000 100 00100 0 Chaetognatha l?l ?O? 00000 0 Chorda ta 00 (01) 10 0 00000 0 Appendix N. (cont.) Character Polychaeta Sipuncul a Echiura Pogonophora Cheiicerata Uniramia Crustacea Onychophora Tardigrada Pentastomida Mollusca Phoronida Brachiopoda Echinoderms ta Chaetognatha Chordata Appendix V A. Secondary character weights. Excluded characters are listed in Appendix III. Character Weight States Appendix V (cont.) 13. #Tissue layers (Excluded) Appendix V (cont.) Appendix V (cont.) J. Alimenfary======90. 7 01 91. 10 01 92. 10 01 93. 10 01 94. 8 01 95. 7 01 96. 8 01 K. Excretoory system======----======EXCl 0 01 EXC2 0 0123 EXC3 10 01 EXC4 6 01 Appendix V (cont.) Appendix V (cont.) Appendix V (cont.) B. Descriptive measures for trees produced from the analysis of the Metazoa. Consistency index excluding uninformative characters = CIX, homoplasy index excluding uninformative characters = I-ID<, retention index = RI, rescaled consistency index = RC, and most parsimonious trees = MPT. 1st analysis 17 361 0.405 0.621 0.689 0.317 Weighted 2 361 0.405 0.621 0.689 0.317 analysis Secondary 1 199 0.549 0.465 0.756 0.467 analysis Appendix V (cont.) C. Character diagnostics of characters for which optimization changed in the two final trees. Consistency index = CI, homoplasy index = HI, retention index = RI, and rescaled consistency index = RC. Tree Character Steps a HI RI RC Appendix VI Specimens examined; CNHE refers to the Colecah Nacional de Helmintos, htituto de Biologia, Universidad Nacional Autonoma de Mkxico, Ciudad de M6xic0, Mexico; HWML refers to the University of Nebraska State Museum, Harold W. Manter Laboratory, Division of Parasitology, Lincoln, Nebraska, U.S.A.; INPA refers to the Instituto Nacional de Pesquisa da Arnazbnia; FIOCRUZ refers to the ColeqSo Helmintologica, Instituto Oswaldo Cntz, Rio de Janeiro, Brad; NHR refers to the Naturhistoriska Riksmuseet (Swedish Museum of Natural History), Stockholm, Sweden; USNPC refers to the US. National Parasite Collection, Beltsville, Maryland, U.S.A. Specimens currently retained in the author's personal collection are indicated by "$". Vouchers of all undescribed species will be placed in the CNHE. Acanthoce~haluswills? (Miller, 1780) Liihe, 1911: USNPC-37599, USNPC-65291, USNPC-65292, USNPC-65293, USNPC-65301; IT97-257$. Acanthoce~haluadims (Van Cleave 1931) Van Cleave and Townsend 1936: USNPC-37598, USNPC-37599, USNPC-65298, USNPC-65299. Acanthoce~halu~lucii (Miiller, 1776) Liihe, 1911: K97-261$. Acanthoce~halusranae (Schrank,1788) Liihe, 1911: USNPC-65277, USNPC-65278, USNPC-65279, USNPC-77084. Brentisentis uncinus Leotta, Schmidt, and Kuntz 1982: USNPC-76537, USNPC-76538, USNPC-76539, USNPC-76540. Caballerorhvnchus (undesuibed sp.): MX97-055$, MX97-056$, MX97-058$. Caballerorhvnchug J- Salgado-Maldonado, 19m CNHE-000648, CNHE-000675. Cathavacanthus exilis (Van Cleave, 1928) Golvan, 1969 (= Rhadinorhynchu~ exilis Van Cleave, 1928): USNPC-37537. Centrorhvnchus (undescribed sp.) Liihe, 1911: RD93-19,SM93-IS$. Centrorhvnchus microcenhalus (Bravo-Hollis, 1947) Golvan, 1956: CNHE-(unaccessioned). Corvnosoma (unidentified sp.) Liihe, 1904: CNHE (unaccessioned). Corvnosorna constricturn Van Cleave, 1918: SM87-200$, NE86-74$, NE87-131$. Dollfusentis bravoae Salgado-Maldonado, 1976: CNHE-000649. Dollfusentis chandleri Golvan 1969; , USNPC-74604, MX95-90$. Dollfusentis ctenorhynchua (Cable & Linderoth, 1963) Golvan, 1969: USN fC-60344, USNPC-82476, USNPC-84687. Dollfusentis heteracanthu~(Cable & Linderoth, 1963) Golvan, 1969: USNPC-60343, USNPC-85059. Dollfusentis longis inus (Cable & Linderoth, 1963) Golvan, 1969: USNPC-38652, USNPC-60342, USNPC-84713. Echinorhvnchus g&i Zoega in Miiller 1776: USNPC-5192, USNPC-7298, USNPC-64725, USNPC-64732, USNPC-65079. Echinorhvnchus salmonis Miiller, 1784; SM95-23. Fessisentis fessus Van Cleave, 1931: HWML-37596. Fessisentis vancleavei (Hughes & Moore, 1943) Nickol, 1972: USNPC-36872, H WML-20837,HWML-20838. Filisorna bucerium Van Cleave 1940: USNPC-36877, USNPC-84877, MX95-003$. Filisoma indicurn Van Cleave, 1928: USNPC-37543, USNPC-37544. Filisoma fidum Van Cleave and Manter, 1948: USNPC-37545, USNFC-38651. Floridosentis rn- (Machado 1951) Bullock 1962: MX95-88$, MX95-89$. Floridosentig ~acificaBravo-Hollis, 1969: MX97-043$, MX97-052$, MX97-053$, MX97-054$, MX97-055$, MX97-057$, MX97-059. Go~tocephalus elon orchis Thatcher, 1979: INPA ACANTHO-002, INPA ACANTHO-003, INPA ACANTHO-004, INPA ACANTHO-005, INPA ACANTHO-006, INPA ACANTHO-011. Illiosentis a fricanus Golvan, 1955): H WML-35206. Illiosentis cetratus Van Cleave, 1945): USNPC-37539, USNPC-81401, USNPC-81402, USNPC-81403, USNPC-81404. Illiosentis furcatus Van Cleave and Lincicome 1939: USNPC-6324, USNPC-9270, USNPC-34530, USNPC-37538, USNPC-34552, USNPC-38637, USNPC-54763, USNPC-74602. Koronacantha rnexicana Monks and Perez -Ponce de Le6n 1996: CNHE-2740, CNHE-2741, CNHE-2748, HWML-39119, USNPC-86758, USNPC-86759, MX95-030$, MX95-036$, MX95-043$. Koronacantha oectinariq (Van Cleave 1940) Monks, Marques, Leon-Regapon, and Perez-Ponce de Le6n 1997 (= uorhynchus pectinarius Van Cleave, 1940): CNHE-3016, CNHE-3017, CNHE-3021; HWML-39287, HWML-39288, USNPC-87006, USNPC-87007. Le~torhvnchoidesa~hredoderi Buckner & Buckner, 1976: USNPC-61024, USNPC-74057, USNPC-74058, USNPC-74059. (Westrumb, 1821) Kostylev, 1924: Le~torhvnchoidesthecatus (Linton 1891) Kostylev 1924: USNPC-6921, USNPC-6930; NES7-007$, NE8&014$, NE8&040$, NES&045$, NEW-046$, MN92-004, MN92-0051 NE95-009$, MN93-021$,MN94006$. Macracanthorhynchus iggMS (Linstow 1879) Meyer 1932: AR97-247$, AR97-248$. Macracanthorhvnchus hirudinaceus (Pallas, 1781) Travassos, 1917: SM97-098$. Mediorhynchus uandi~(Van Cleave 1916): TX94-104$. Mediorhvnchus edmondsi midtand Kuntz, 1977: USNPC-74356, USNPC-74357 ,USNPC-74058. Megapriapus mai(Gracia-Rodrigo, 1960) Golvan, Gracia-Rodrigo and Diaz-Ungria, 1964: BR97-002*, BR97-003$, BR97-004$, BR97-005$, BR97-006$, BR97-007$, BR97-008$, BR97-009$, BR97-010$. Moniliformis clarki (Ward, 1917) Van Cleave, 1924: NE91-12$. Moniliformis moniliformis (Bremser 1811) Travassos 1915: SM86-43$, SM86-73$, SM87-14$, SM87-28$, SM87-62$, SM87-94$. Neoechinorhvnchus (undescribed sp.) Stiles 6t Hassall, 1905: MX95-77$, MX96-129$. Neoechinorhvnchus cur~rnaiNoroiiha, 1973: FIOCRUZ -30812, FIOCRUZ-32060. Neoechinorhvnchus wlindratua (Van Cleave 1913) Van Cleave 1919: NE88-023$, MN93-018$, MN93-019$, MN93-020$, MN93-021$, NE95-009$. Neoechinorhynchus golvad Salgado-Maldonado, 1978: CNHE-000603. Neoechinorhynchus rutili (Miiller, 1780) Stiles & Hassall, 1905: CAN96-008$, CAN9&112$. Neoechinorhynchus pseudemydis Cable & Hopp, 1954: SM86-72$. Neocehinorhvnchus pterodoridis Thatcher, 1981: INPA ACANTHO-024. Neoechinorhynchus spectabilis Machado, 1959: FIOCRUZ 23993, FIOCRUZ 23994, FIOCRUZ 23995, FIOCRUZ 23996. . . s~lnrcaudatusCable & Quick, 1954: USNPC-49414. Octospiniferoides chandleri Bullock 1957: CNHE-000662, CNHE-000688. Oligoacanthorhynchua (unidentified sp.) Travassos, 1955: RD93-014$. Olieacanthorhvnchus tortuosa (Leidy 1850) Schmidt 1972: AR97-249$. Oncicola (unidentified sp.) Travassos, 1916; CR96-067$. Oncicola ranis (Kaupp,1909) Hall & Wigdor, 1918: CNHE-000343. Pachvsentis (undescribed sp.) Meyer 1931; MX95-004$. Paulisentis fractus Van Cleave and Bangham 1949: USNPC-37129, US NPC-37648. Plaeiorhynchus cm(Goeze 1782) Schmidt and Kuntz 1966: NES6-21$, NE86-75$, NE87-130$, NE87-131$, NE87-133$,NE87-134$, NE88-51$. Polvmorphus (unidentified sp.) Liihe, 1911: CAN96-110$, CAN96-ill*. Pol~morphusmarilk Van Cleave, 1939: USNPC-37551. Porn~horhvnchusbulbocolli Linkins in Van Cleave, 1919: NE88-015$, NE88-041$. Pom~horh ynchus Jaevis (Zoega in Miiller, 1776) Van Cleave, 1924; IT97-258$. Rhadinorhynchu~(undescribed sp. 1) Liihe, 1911: MX97-050$. Rhad inorhynchus (undescribed sp. 2) Liihe, 1911: MX97-202$, MX97-208$, MX97-212$, MX97-W*. Rhadinorhynchu~colabis Laurs and McCauley, 1964: USNPC-39506, USNPC-39507, USNPC-39508, USNPC-60049. Rhadinorhynchus IaPioscionis Thatcher, 1980: INPA-ACANTHO-018, INPA-AC ANTHO-019, INPA-ACANTHO-021, INPA-ACANTHO-023. Rhadinorh ynchus pristis (Rudolphi 1802) Liihe 1911 (specimens originally identified as R. selkirki Van Cleave 1921): USNPC-37534, USNPC-37537. Tenorhvnchus brevis Van Cleave 1921: NHR-4766, NHR-4767, NHR-4768, USNPC-37535, USNPC-81405. Telosentis exipus (Linstow 1901) Kostylev 1926: HWML-35203, HWML-35204. Telosentis molini Van Cleave 1923: USNPC-1416, USNPC-37533. Telosentis tenuicornis (Linton, 1892) Van Cleave 1947 (= Rhadinorhvnchu~ tenuicornis (Linton, 1892) Van Cleave, 1918): USNPC-6314, USNPC-6324, USNPC-38637, USNPC-38638, USNPC-38639, USNPC-38640, USNPC-38641, USNPC-38642, USNPC-38653. Appendix W Character argumentation-Acanthocephala Many of the characters used in this analysis could not be polarized directly using outgroup comparisons because of lack of information concerning the existence of homologous structures in the outgroups. The monophyly of the Acanthocephala is not a matter of question, so a single-process method described by Maddison, et al. (Maddison, et al., 1984) (also see Donoghue and Maddison (1986) for a critique of this method) was used for simultaneous resolution of ingroup and outgroups. This method is appropriate when the outgroup is heterogeneous and the exact relationships among outgroups are not known. The choice of outgroups was made according with the results of the analysis of the Metazoa presented in Chapter 3, but, because more than one equally parsimonious tree (17 trees) was produced, the exact relationship between the two outgroups is still uncertain. Thus, the choice of method is appropriate. As required (Swofford, 1993), characters were included in the analysis that are invariant within the ingroup but informative within the outgroup. These characters provide polarity for the remaining characters that have unknown or ambiguous status in the outgroups, and which have been coded as "unhown" in the outgroups. All characters are analyzed "unordered". Characters marked with an "*" relate to features which are not found in the outgroups and thus are coded as "unknown" in the outgroups. Terminology for morphological structures of acanthocephalans follows that of Miller and Dunagan (1985) whenever possible. 1. Proboscis: There is a lack of agreement on the homology of the "introverts" of various invertebrate taxa, but the proboscis of acanthocephalans is embryologically different from other taxa that have been studied (Nielsen, 1995). Thus, I have coded the proboscis as absent in the outgroups and present in the Acanthocephala: 0-absent; 1-present, invaginable. 2.* Proboscis shape: 1-round; 2-elliptical to oval; 3-elongate to fusiform; 4-clava te; 5-cone-like. 3.' Proboscis (divided): 1-proboscis undivided; 2-teloboscis (sensu Schmidt and Kuntz, 1977). 4.' Proboscis cuticle theca (presence): I-theca absent; 2- theca present. 5.' Proboscis cuticle (swollen around hook): 1-cuticle not swollen; 2-cuticle swollen around hook. 6. Hooks: Scalids found in Priapulida are unlike the hooks of Acanthocephala and Rotifera lack hooks, so both outgroups were coded as "absent": 0-absent; 1-present. 7.' Hooks (pattern or arrangement): I-spiral; 2-longitudinal rows. 8.' Hooks (size pattern): 1-all hooks approximately same size; 2-anterior hooks largest; 3-middle hooks largest. 9.' Hooks (anterior-size equality): 1-All anterior hooks equal; 2-some anterior hooks larger. lo.* Hooks (barbed): I-hooks without barbs; 2-hooks with barbs. 11." Hooks (posterior circle perpendicular): 1-posterior hooks normal; 2-posterior hook ring perpendicular. 12.' Hooks (bare patch): I-bare patch absent; 2-bare patch on proboscis present. 13.' Hook (post ring enlarged): 1-absent; post ring normal; 2-some hooks enlarged. 14.' Hooks (ventral hooks in posterior ring enlarged): I-absent; post ring normal; 2-ventral hooks enlarged. 15.* Hooks (dorsal-ventral hook relative length): 1-dorsal and ventral hooks equal; 2-ventral shorter than dorsal; 3-ventral longer than dorsal. 16.' Hook (basal comb (~ensuMonks and Pkrez-Ponce de Leon, 1996)): 1-absent; 2-present. 17.' Hooks ("crescent" (~ensuCable and Linderoth, 1963)): 1-absent; 2-present. 18.' Hooks (recurved (sensu Monks and Perez-Ponce de Leh, 1996)): 1-absent-all hooks 'normal'; 2-recurved, root normal; hecuwed, root enlarged. 19.' Hook roots (location of rooted hooks): 1-present, all hooks rooted; 2-present, more anterior hooks only. 20.' Hook roots (type of roots): 1-posterior process only; 2-anterior and posterior processes. 21.' Hook roots (bifurcation in root): I-roots absent; 2-present posterior root bifurcation. 22.' Hook roots (posterior ring): 1-hooks without roots; 2-posterior roots only; 3-anterior roots only; 4-anterior and posterior roots. 23.' Hook rooks (roots of hooks in "crescent" (msu Cable and Linderoth, 1963)): 1-roots absent; 2-hooks rooted. 24.' Apical organ shape: 0-oval: 1-cone-Iike; 2-hanging drop shape; 3-elongated hanging drop. 25. Apical sense organ: Numerous sense organs occur in the apical region of both rotifers and priapulids, but none are known to be homologous with the apical sense organ of acanthocephalans, thus both outgroups are coded as "absent1': 0-apical sense organ absent; 1-apical organ present. 26.' Apical sense organ (type): 1-apical sense organ single; 2-apical sense organ double. 27. Sensory pore: No homologous lateral sensory pores are known in the outgroups, thus each is coded as "absent". In the Acanthocephala, sensory pores are found either on the neck or proboscis. In the specimens of Brentisentis and mrhvnchuthat were examined for this study, the pore was not found and is coded "absent": 0-vesicle absent; 1-present on neck; 2-present on proboscis. 28.' Sensory pore (alignment (-u Gee, 1987)): 1-lateral; 2-lateral but more dorsal ; 3-both pores almost dorsal. 29. Receptacle: Similarity between the pharynx of various invertebrate groups and the receptacle has been suggested, but no studies have supported homology of the two structures. Regardless, the exact structure of the acanthocephalan receptacle is a synapomorphy for the phylum, so outgroups were coded as "absent": 0-absent; 1-present. 30.' Receptacle (number of muscle layers): Lone muscle layer; 2-two muscle layers. 31.' Receptacle inner wall (attach point): 1-attached anterior proboscis; 2-a ttached mid-proboscis; 3-base of proboscis anterior neck; 4-middle of neck; 5-attached at base of neck; 6-unattached in non-muscular sheath. 32. Receptacle outer wall (attachment point): 1-base of proboscis anterior neck; 2-middle of neck; 3-base of neck at trunk junction; 4-unattached. 33 .' Receptacle secondary attachment point (location): 1-attached only at anterior end; 2-attached secondarily at neck-trunk junction. 34.' Receptacle inner wall thickness (dorsal-ventral equality): I-dorsal and ventral walls equal; 2-dorsal wall thicker. 35.' Receptacle outer wall thickness (dorsal-ventral equality): 1-both walls equal width; 2-dorsal wall thicker. 36.' Receptacle wall thickness (inner-outer equality): 1-both walls equal thickness; 2-inside wall thicker; 3-outside wall thicker. 37.' Receptacle inner wall muscle type: 1-circular fibers; 2-spiral fine fibers. 38.' Receptacle outer wall muscle (type): 1-circular fibers; 2-spiral fine fibers; 3-spiral bundles of fibers. 39.' Receptacle tubular sheath (presence): 1-absent; 2-present. 40.* Receptacle anterior tubular sheath (extent): 1-partial tubular sheath present; 2-complete tubular sheath present. 41 .' Proboscis retractor muscle (exit point): 1-posterior of proboscis; close; Zposterior of proboscis; wide; 3-ventral posterior, dorsal slightly anterior ; 4-dorsal and ventral slightly post mid-proboscis; 5-ventral at mid-, dorsal post mid-proboscis; 6-both dorsal at mid-proboscis. 42. Central Nerve Cord: Rotifera have a primary ventral nerve cord and a smaller dorsal cord (Exact position of these is somewhat variable within the phylum and may be derived within the phylum, but is coded as "1" herein.) and the Priapulida have a ventral cord. All acanthocephalans have lateral nerve cords: 0-ventral only; 1-dorsal and ventral; 2-lateral. 43. Central nervous system: Rotifera have multiple cephalic ganglions and the Priapulida have a cephalic ring. All acanthocephalans have a single cephalic ganglion: 0-cephalic nerve ring; 1-multiple ganglions; 2-single cephalic ganglion. 44. * Cephalic ganglion (position): 1-neck; 2-anterior end of receptacle at neck-body junction; 3-mid-anterior half of receptacle; Cmiddle of receptacle; 5-mid-posterior half of receptacle; 6-base of receptacle. 45.' Cephalic ganglion (attachment): 1-side of receptacle-neck; 2-attached to retractors; 3-attached to base between retractors; 4-on cords, loose in receptacle-neck. 46. Cephalic ganglion: The position of the cephalic nerve ring of the Priapulida is not homologous with that of the other two phyla so it is coded as "unknown". In Rotifera, the ganglion is located more dorsally, although it is not in a receptacle. In the acanthocephalans examined for this study, the cephalic ganglion can be located medially in the receptacle or on the ventral wall (A position at the posterior end of the receptacle is considered 'medial' for this character.): 0-ganglion dorsal; 1-ganglion ventral; 2-ganglion medial. 47.' Retinacula exit (location): 1-anterior half mid point; 2-mid-receptacle; lateral; 3-posterior half mid point; 4-posterior end of receptacle; 5-mid-receptacle, ventral only; 6-just anterior to base of receptacle, lateral. 48.' Retinacula (connection to lemnisci): 1-retinacula connected to body wall; 2-retinacula connected anterior part of lemnisci. 49. Lemnisci: Although the homology of the "lemnisci" of Rotifera and that of the Acanthocephala is still in question (Nielsen, 1995), I have considered them homologous here pending more detailed study, so the two phyla are coded as "present". Priapulids lack lemnisci and are coded as "absent": 0-absent; 1-present. 50. Lemnisci (shape): The plesiomorphic shape for the lemnixi is digitate, as exhibited by the Rotifera. Acanthocephalaw have lemnisci in all three shapes. Priapulids lack lemnisci and are coded as "unknown" : 0-digitate or fusiform; 1-claviform; 2-widest in middle. 51. Lemnisci (length equality): The lernnisci of the Rotifera are equal in length and acanthocephalans exhibit both conditions. Priapulids lack lemnisci and are coded as "unknown": 0-lemnisci equal length; 1-lemnisci unequal length. 52.' Lemnisci nuclei (type): I-round to oval; 2-elongate fusiform; 3-irregularly shaped. 53.' Lemnisci (nuclei distribution pattern): 1-no particular pattern; 2-one nucleus in one lemnisci, two in the other. 54.' Lemnisci (central canal): 1-lemnisci solid; 2-lemnisci tubular. 55.* Lemnisci (attachment): I-a ttached along entire length; 2-only at posterior end; 3-free, hanging in body cavity. 56.' Lemnisci (sheath): 1-sheath absent; 2-entirely sheathed. 57.' Lemnisci sheath (membrane type): 1-sheath absent; 2-sheath tubular; 3-sheath non-tubular. 53. Neck (bulb on neck): This character is constant ("absent") in all taxa, but was left in the analysis because it would be appropriate for a wider group of acanthocephalans: 0-absent; 1-present. 59- Trunk (shape): Various body shapes occur in both outgroups and it is not known which form is plesiomorphic, so both are coded as "unknown": l -pyriform; 2-wider anterior not pyriform; 3-widest at anterior one third; 4-"blimp-shaped"; 5-fusiform cylindrical; 6-narrower anterior. 60. Trunk (pseudo-segmentation): Rotiferans possess true segmentation, but that condition is not pertinent to this analysis. Pseudo-segmentation within acanthocephalans differs from the condition seen in either rotiferans or priapulids, so the outgroups are cod.ed as "absent": 0-pseudo-segmentation absent; I-present. 61. Trunk spines (presence): Trunk spines are not found in Rotifera, and the structure of the spines of the Priapulida is different than that of acanthocephalans. The Rotifera is coded as "absent" and the Priapulida as "unknown". 0-absent; 1-present. 62.+ Trunk spines (distribution) (only random distributions were evaluated, although various other patterns exist in species not included in this study): I-random distribution on anterior tnuk only; 2-random distribution on anterior and mid-trunk; 3-random distribution on mid- to posterior trunk. 63.+ Trunk spines (rootlets): 1-spines absent; 2-without rootlets; 3-with rootlets. 64.* Trunk (genital spines): Genital spines are not found in the Rotifera (coded "absent"), and the condition has not been thoroughly studied in the Priapulida (coded "unknown"): 0-absent; I-present both sexes; 2-present females only; 3-present males only. 65. Lacunar system: The lacunar system as found in acanthocephalans is not seen in the outgroups, so each is coded as "absent". 0-absent; I-present. 66.' Lacunar system (location): I-dorsal and ventral; 2-lateral; 3-dorsal only; 4-ventral only. 67.' Lacunar system sec. branches (type): I -even circular tubes; 2-irregular branches. 68. Hypodermic nuclei: The Rotifera have hypodermic nudei that are similar to the 'giant' nuclei of acanthocephalans. The body wall nuclei of priapulids have not been described and were not visible in the whole specimens available at the time of the current study (coded "unknown"). Acanthocephala exhibit all types: 0-'giant'; 1-tree-like-highly branched; 2-small, round to ovoid; 3-elongated (like tree branch, but not branching); 4-irregular but not amoeboid; 5-irregular connected into chain; 6-amoeboid. 69. Hypodermic nuclei: The number hypodermic within Rotifera and Acanthocephala is variable between taxa, but numbers fall within the two ranges given here. The higher number may be a result of 'fragmentation' of the plesiomorphic lower number, but data is insufficient to address that question at this time. The Rotifera have only a small number of hypodermic nuclei and Acanthocephala have both conditions. The number of hypodermic nuclei in Priapulida is not known (coded "unknown"): 0-few (425); 1-many (>loo). 70. Testes (proximity to each other): The testes of non-parthenogenetic rotiferans are not close together, and are coded as "apart". In priapulids, each testis and an ovary is combined into a single structure, so is coded as "unknown": 0-apart; 1-overlap to touching. Testes (location): This character is particular problematical in that it is confounded with changes in body length because testes position is not independent, but is dependent or the type of change in length. I chose to include this character as given only because of its use in classical descriptions. In both outgroups the testes occur in the posterior third of the body, and all conditions are found in the Acanthocephala: 0-posterior third of body; 1-middle third of body; 2-anterior third of body. 72: Testes (position): In the Rotifera, the testes lie opposite each other. In the Priapulida, they are combined with the ovaries, so, as above, they are coded as "unknown". In all of the acanthocephalans included in this analysis, the testes are approximately tandem: 0-opposition; 1-tandem. 73: Testes (proximity to cement glands): In Rotifera, the testes are separated from the cement glands. In the Priapulida, the testes are combined with the ovaries, so, as above, they are coded as "unknown". Acanthocephalans exhibit both conditions: 0-apart; I -touching. Coding and polarization of features of the cement glands are given in the 3 assumptions discussed above, and coding of the outgroups under each assumption is given in Table XX. For characters 75-84, acanthocephalans exhibit all conditions. Characters 79 and 81 apply only to syncytial cement glands. For features of individual glands, taxa with syncytial glands are coded as unknown. 74. Cement gland (type): 0-individual glands; 1- syncytial gland. 75. Cement gland (number): 0-two glands; 1- four glands; 2-six glands; 3-eight glands. 76. Cement gland (cluster type): 1-two regular to irregular rows; 2-even pairs, overlapping, each pair longer in length; 3single long row; 4-grape-like mass. 77. Cement gland (shape): O-round to oval; 1-cylindrical to elongate. 78. Cement gland-syncytial (shape): O-round; l-oval (length two times width); 2-elongate (length three times width). 79. Cement gland (elongation): O-not elongated; l-length two to three times width; 2-length 10 times width. 80. Cement gland-syncytial (number of nuclei): @not syncytial; 1-Eight nuclei. 81. Cement gland (multiple nuclei): O-one nucleus per gland; l-more than one per gland. 82. Cement gland (number of nuclei): 0-one per gland; 1-two per gland; 2-three to thirty per gland; 3-more than fifty per gland. 83. Cement gland nuclei (fragmentation): All occurrences of multiple nuclei in cement glands could be considered as fragmented in the classical sense. I have chosen to interpret multiple nuclei as the product of continued division of "single" nuclei rather than fragmentation because each of the multiple nuclei are membrane bound and appear identical in form to the larger single nuclei. Among the taxa included in this study, fragmentation into non- membrane bound particles occurs only in Telosentis, in which no nuclei were observable in any specimens examined: 0-solid, dense; 1-fragmented. 84. Cement reservoir (single reservoir): A single reservoir associated with a syncytial cement gland is exhibited by some rotiferans and acanthocephalans. The homologous condition in priapulids is undetermined: 0-absent; I-present. 85. Cement reservoir (internal): In Telosentis, Filisoma. Rhadinorhvnchus, and Pla ~iorhy nchus an internal cement 'reservoir' that is located within the main body of the cement gland is exhibited. This condition is not found in other taxa: 0-absent; I-present. 86.' Cement duct (funnel-like reservoir): 1-absent; 2-enlarged funnel-like anterior. 87.* Cement duct (anterior reservoir): I-absent; 2-enlarged into elongate reservoir. 88.' Cement duct (posterior reservoir): 0-absent; I-enlarged into elongate reservoir. 89. Cement duct (fusion): Of the taxa examined here, fusion of the cement ducts occurs only in Pach sen ti^ and Rhadinorhvnchus. Acanthocephalans with syncytial cement glands have 2 ducts leading from the reservoir that are not fused, so they are coded as having non-fused ducts. The Rotifera and Priapulida both exhibit various types of ducts, but none examined are similar in this feature to those of the ingroup, thus are coded as "unknown". 0-not fused; I-fused anterior to posterior end. 90. Vas efferens (type): In Rotifera and Priapulida the vas efferentia are simple. Acanthocephalans included here exhibit all conditions except having the entire duct enlarged: 0-simple duct; 1-entire duct enlarged; 2-several reservoirs with anterior connections; 3-several reservoirs with middle connections. 91. Vas efferens (location): In acanthocephalans, all states are present, but in the outgroups the location of the vas efferentia does not correspond with any states given here (coded "unknown"): 1-both ducts ventral; 2-posterior ventral, anterior dorsal; 3-posterior dorsal, anterior ventral; 4-posterior crosses dorsal, anterior dorsal; 5-both ducts medial. 92. Vas deferens: In the Priapulida the testes and ovaries are combined into a 'gonad' and they exhibit external fertilization, thus the structure is different (coded "unknown")than in the Rotifera and Acanthocephala. In the Rotifera which have males, the vas deferens is present. Acanthocephalans exhibit both conditions: 0-present; l-absent. 93. Seminal vesicle: The priapulids do not have a seminal vesicle (coded "absent") and the state is unclear in the Rotifera (coded "unknown"): O-seminal vesicle absent; 1-present, round; 2-present, widened vas deferens; 3-present, inverted triangle. 94. Bursa (presence): A bursa is absent in both of the outgroups, and present in all acan thocephalans: O-absent; 1-present. 95.' Bursa (muscle type): l-inner cup not heavy muscle; 2-heavy muscular inner cup. 96." Bursa (digitate lobes on posterior lip): 1-absent; 2-bursa with digitate lobes. 97. Saefftigen's pouch (shape): Saefftigen's pouch is not found in the outgroups and is coded as "unknown". Acanthocephalans exhibit all conditions (This condition was examined in numerous specimens that exhibited extension and contraction of the bursa to remove this confounding factor from the assessment of condition.): l-duct not enlarged; 2-duct enlarged posteriorly; 3-pouch as elongate tube. 98. Genital pore position (male): The genital pore is terminal in both outgroups and acanthocephalans exhibit all conditions: 0-pore terminal; I-posteroventral; 2-posterodorsal. 99. Genital sheath (presence): A genital sheath is not exhibited in the outgroups but is present in the Acanthocephala: 0-absent; 1-present. 100.+ Genital sheath (thickness): I-thin membrane; 2-thick membrane. 101." Genital sheath (tubular): 1-non-tubular; 2-tubular. 102.* Genital sheath (anterior attachment location): 1-sheath not attached to body wall; 2-attached dorsal and ventral; 3-attached dorsal only. 103.' Genital sheath (muscular): 1-not muscular; 2-heavy muscle. 104. Urogenital duct sheath (presence): The urogenital sheath is absent in the outgroups and present in all acanthocephalans: 0-absent; 1-present. 105.' Urogenital duct sheath (heavy muscle): 1-non-muscular sheath; 2-sheath in form of a muscular ball. 106. Ligament sac (presence): Ligament sacs are absent in the outgroups and found in all acanthocephalans: 0-absent; 1-present. 107.' Ligament sac (number of sacs): 1-one ligament sac; 2-two Ligament sacs. 108.' Ligament sac (attachment of 2nd sac): 1-ventral posterior; 2-dorsal posterior. 109.' Ligament sac (attadunent to uterine bell): 1-attached to total bell Lip; 2-posterior inside bell; 3-attached to dorsal lip and posterior inside bell. 110. Uterine bell (presence): A uterine bell is absent in the outgroups and found in all acanthocephalans: 0-absent; I-present. 111.' Uterine bell (shape): 1-tubular (narrow); 2-funnel-shaped; 3-vase-shaped; 4-bent "lily". 112.' Uterine bell lips (relative length): 1-dorsal and ventral lips equal; 2-dorsal lip longer. 113.' Uterine bell (secondary attachment location): 1-una ttached; 2-dorsal; 3-ventral; $-dorsal and ventral. 114. Uterus ("lining" tissue): In Acanthocephala, the inner lining of the vagina extends anteriorly toward and into the uterus. In the Rotifera, this condition is absent and in the Priapulida the uterus does not exist (coded "unknown"): 0-'lining' absent; 1-short, not to uterus; 2-mid-u terus; 3-lining entire uterus. 115. Vagina (shape): The vagina in the Rotifera is a non-muscular tube unlike that of the Acanthocephala, and does not exist in the Priapulida, thus are coded as "unknown" for each group: 1-round; 2-short with round base; 3-elongate with round base; 4-elongate with round middle; 5-elongate without round base; 6-short but not round. 116.' Vaginal sphincter (type): 1-solid, round circle-like ring; 2-two circular tubes; 3-one circular tube; 4-one circular tube with sheath extending posteriorly; 5-circular flat ring. 117. Genital pore position (female): The genital pore is terminal in both outgroups and acanthocephalans exhibit all conditions: 0-pore terminal; 1-pore posteroventral; 2-pore posterodorsal. The outgroups lack vestibular muscles similar to those in acanthocephalans, so features of vestibular muscles (chars. 118-128)are coded as "unknown" for each outgroup. Some higher taxa included in this analysis have members with muscle types other than as given here, but this data set reflects included species as representative of the current concept of the genus as based on the type species. 118.' Vestibular muscle (cuplike): This muscle is found dorsally in Macracanthorhvnchus and Filisoma, and ventrally in Telosentis and Neoechinorhvnchus, and a full cuplike muscle in Moniliformis, Mediorhvnchus, Acanthocephalu~,and Echinorhynchus. All three conditions are combined here as a single character and coded "present": 1-absent; 2-present. 119.' Vestibular muscle (half cuplike): A half-cuplike muscle is found dorsally in Macracanthorhvnchus and Filisoma, and ventrally in Telosentis and Neoeshinorhvnchus: 1-absent; 2-dorsal; 3-ventral. 120.' Vestibular muscle (full cuplike): A full cuplike muscle is found in Moniliformis, Mediorhynchus. Acanthoce halus, and Echinorhynchus: 1-absent; 2-present. 121.* Vestibular muscle (bands): Vestibular muscle bands are found in nchoides, J3ollfusentis, Koronacantha, Brentisentis, Rhadinorhvnchus, and Floridosentis: 1-absent; 2-present. 122.' Vestibular muscle bands (number of anterior muscle "heads"): Muscle bands with one anterior connection to body wall (= "head") are found in Le~torhvnchoides,Koronacantha, Rhad- and Floridosentis. Two muscle "heads" are exhibited by Dollfusentis, and 3 by Brentisentis: bone band each side; 2-two bands each side; 3-three bands each side. 123.' Vestibular bands (posterior attachment width): Narrow posterior attachment is exhibited by Le torhvnchoides, and wide attachment by Dollfusentis, Koronacantha, Brentisentis, Rhadinorhvnchus, and Floridosentis: 0-bands absent; 1-narrow attachment; 2-wide a ttachrnent. 124.* Vestibular band; ventral (presence): A ventral muscle band is found only in Bren tisentis: 1-absent; 2-present. 125.' Vestibular band; central (presence): Muscle bands that originate posteriorly in the center of the trunk are exhibited by Macracanthorh &us. Pachvsenti~. and Oligacanthorhynchus: Labsent; 2-present. 126.' Vestibular muscle; pad (presence): Heavy ventral muscle that is pad-like is exhibited only by &orhv nchu~and Jlliosentis: 1-absent or reduced; 2-heavy ventral pad. 127.' Vestibular muscle, to genitalia (presence): A muscle band that extends from . . the posterior trunk to the genitalia is exhibited by Mon~liformis.Pac hvsentis,- Olieacanthorhynchw and Octospnlferoides:. . 1-absent; 2-p resent. 128.' Vestibular muscle (to genitalia): In Pachpenb and QJqpcanthorhynchus, the muscle band connects anteriorly to the uterus, and in Monil~formis-. and Octosoiniferoides it connects to the sorting apparatus: I-band to anterior uterus; 2-band to sorting apparatus. 129. Protonephridial organ (presence): Protonephridia are exhibited by both outgroups, Macracanthorhynchus Pach sentis, and Q&pcanthorhynchus: 0-absent; I-present. 130. Egg (polar prolongations): Prolongation of the inner membrane is exhibited by all palaeacanthocephalans (Eggs of Brentisentis are not known and not present in the type specimens, so this taxa is coded "unknown".): 0-absent; 1-present. 131. Egg (shape): Eggs that are round to oval are exhibited by the Rotifera, Mediorhynchus. Moniliformis. Macracanthorhvnchus. Pachvsentis, Olieacanthorhvnchus, Neoechinorhvnchus, and Paulisentis. All other taxa have fusiform eggs. Note: other species currently assigned to Neoechinorhynchus have fusifonn eggs, but the N. cvlindratus (included here) and &J. mtili (type species for the genus) both exhibit oval eggs: 0-round to oval (length two times width); 1-fusiform (length four times width). 132. Embryo armature (type): The outgroups and Neoerhinorhynchus have -. anechinate embryos, Monlllfonn is, Acanthoceohalus. Echinorhvnchus, Leptorhynchoides. Dollfusentis, TelosentiS, Koronacantha, &prhvnchus, . . Illiosentis, Filisor~lj~Jthadinorhvnchus, - and Octos~~niferoides have hemiechinate embryos, and Mediorh chus, Macracanthdynchus, Pachvsentis. Oligacanthorhvnchus, and Paulisentis have holoechinate larvae. Note: Assignment of states to taxa were made from published descriptions of embryos: 0-anechinate; 1-hemiechinate; 2-holoechinate. 133. Fertilization (type): External fertilization is exhibited by priapulids and internal fertilization is exhibited by Rotifera having males and all acanthocephalans: 0-external; 1 -internal. 134. Ovary (number): The outgroups exhibit two or more ovaries and all acanthocephalans exhibit only a single ovary: 0-two or more ovaries or gonads; 1-single ovary. 135. Ovary (fragmentation): Fragmentation of ovarian tissue to produce ova is a synapomorphy for acanthocephalans: 0-unfragmented; 1-ovary fragments to produce ova. 136. Cleavage type of first cell division of embryo: The Priapulida exhibit meridional placement of centrioles during the first embryonic cell division. Rotifera and Acanthocephala exhibit polar placement of centrioles: 0-meridional placement of centrioles; 1-polar placement of centrioles. 137. Cleavage type of second cell division of embryo: The Priapulida exhibit meridional placement of centrioles during the second embryonic cell division. Rotifera and Acanthocephala exhibit cleavage planes that are at right angles to each other: O-meridional placement of centrioles; l-cleavage planes at right angles to each other. 138. Cleavage type of third ceil division of embryo: The Priapulida exhibit meridional placement of centrioles during the third embryonic cell division. Rotifera and Acanthocephala exhibit multiple cleavage planes: O-meridional placement of centrioles; 1-multiple cleavage planes. Appendix Vm.A. Data Matrix for the Acanthocephala. Characters are numbered as in the text. N=not applicable; +=coding as in Table 1; ?=condition unknown. -- - -- Character Priapulida Rotifera Mediorhvnc- - Monili fonnis mo- Macracanthorh&us i- Olieacanthorhvnchus tomosa Acanthoce~halusdims Echinorhvnchus Lev torhvnchoides thm- Doll fusentis ch- Telosentis emus L 3 11 Appendix VIII (cont.) Character Koronacantha 131211231111 . * Koronacantha ~ecw Te~orhvnchusbrevis Illiosentis fur- Brentisenfis un&us Filisoma bucerim . . Rhadinorhvnrhus nnshs Plapiorhvnchus cv- 131111211121 Octos~iniferoideschandleri 111?11121111 Neoechinorhecvlindratus 1 1 1 1 1 1 1 2 2 1 1 1 . . Floridosentis ma 141~11121111 Paulisentis fracu 121111121111 Appendix VIII (cont.) Priapulida ???????????O??O Rotifera ???????????0??0 Med. ma& 1111122112N2111 Mon.monilifor@ 1 1 1 11 2 1 1 2 1N 2 1 2 1 Mac. in~eng- 1111111 12N2111 Pacvsentis SD. 1111111114N2111 0.tortuosa 1111111212N2111 A. dims E. gadi L. thecatus D. chandleri 1132222121210N2 T. eximus 1131111112N10N1 Appendix V3lI (cont.) Character Taxa* 13 15 20 25 K. mexirana 1132132111NlON1 . K. ~ecbnana 1132132111N10N1 T. brevis 1132111112NlON2 I. furcatus 1222122111NlON2 B. uncinus 2121122111N10NO F. bucerium 1111111112N10N1 R. mistis 2131111111N10N1 Pla. cvlindraceus 11111111llN1ONO Oct. chandIeri 11111112?4N10N? 1111111112N30N1 1111111211N30N1 Pau. fractus 1111111214N30N1 Appendix VIII (cont.) Character Taxa* 28 30 35 40 Priapulida ?O????????????O Ro tifera ?Of?????????? Med. 1112N12NNlN21 Mon.monilifom 1 1 15 N 1 1 N N 1 N 2 2 Mac. in- Pacvsen tis s~. 0. tortuosa A. dims 312421111?21N22 Uadi 312421212?21N22 L. thecatus 112311111121N12 D. chandld 112342111111N12 T. exiguus 112421113121N12 Appendix VIII (cont.) K. mexicana 112341223131N12 K. ~ectinaria 112341223131N12 T. brevis lZ2342111121N12 I. furcatus 112342113121N12 B. uncinus ?12332111111N12 F. bucerium 112431113?21N12 R. ~ristis 212311123221N32 Pla-cvlindraceu ? 1 2 4 3 1 1 1 1 1 11 N 1 2 Oct. chandleri ?Il?N?2NN?NlN?2 N. cvlindratus 1113N12NN2NlN22 F1. mugilis 1114NllNN2NlN22 Pau. fractus 1113NllNN?NlN22 Appendix VIII (cont.) Character Priapulida O?????O???????? Rotifera 1??0??100?????? 241121100112322 . Mon. mon&forrrll,~263241101112323 Mac. in- 24111?100112322 0. tortuosa A. dims E. nadi L. thecatus D. chandleri T. exiwus 242221111111223 Appendix Vm (cont.) Character Taxa* 43 45 50 - K. mexicana 232221101212311 K. ~ectinaria 232221101212311 T. brevis I. hrcatus B. uncinus F. bucerium 263241100111223 R. ~ristis 232221100113122 Pla. cvlindraceus 24 2 2 2 11 0 1 1 2 23 1 1 Oct. chandleri 25???10012N3NN N. cvlindram 263231100121311 Fl. mudis 263261101122311 Pau. fractus 263261100122311 Appendix VIII (cont.) Character Taxa* 58 60 I I Priapulida O??????O?????O? Ro tifera 0??0??00??00000 Med. mandis 0500NN011250011 . Mon. monil~fonry. ~0510NN011250001 Pacvsentis SD. 0. tortuosa L. thecatus 0300NN012210101 D. chandleri Appendix Vm (cont.) Character K. mexicana 030133112210111 -. K. wctinana 030123012210111 T. brevis 030111012210111 I. furcatus 050111212210101 B. uncinus 030111012210101 F. bucerium 0500NN012221111 R. pristis 050112012221121 Pla-cvlindraceus 0 3 00 N N 0 1 22 2 1 1 1 1 Oct. chandleri 0300NN011200111 N. cvlindratus 0300NN011200111 F1. mudis 0500NN011200101 Pau. fractus 0300NN011200111 Appendix VIII (cont.) Character Taxa* 73 75 Priapulida ?tttttttttttttt Ro tifera Ottttttttfttttt Med. izrandis 1031000N0000110 Mon.0031000N0000120 Mac. in- 0031000N0000110 1031000N0000110 Q. tortuosa 0031000N0000120 A. dims 1021000N2100121 E. zadi 0023000N2100120 L. thecatus 1034000N2100110 D. chandleri 1034000N2100121 T. exi-?~ 1011101N??10221 Appendix Vm (cont.) Character Taxa* 73 75 80 85 -- K. mexicana 1034000N1100120 K. ~ectinaria 1034000N1100120 T. brwis 1034OOON2100121 I. furcatus 1034OOON2100121 B. uncinus 1034OOON2100121 F. bucerium 1012102N2100211 R. pristis 1032102N3200221 Pla. cvlindraceus 1 0 1 2 1 0 2 N 2 1 0 0 2 1 1 Ckt. chandleri 11NNN1NlNN01110 11NNN2N1NN01110 F1. mudis 11NNN2NlNN01110 Pau. fractus 11NNN2N1NNO11lO Appendix Vm (cont.) Character Taxa* 88 90 95 100 Priapulida ttO??OO???OO??? Rotifera tt0?0?0???00??? Med. nrandb 012402121101221 . . Mon. moniliforrllt\012102121201221 Mac. ineens 012102121301221 Pacvsentis SD. 122102121201221 0. tortuosa 0102?012130122? A. dims 112100122221111 E. cadi 110101122221221 L. thecatus 013311121221111 D. chandieri 013201111201113 T. exieuus- 110302122221113 Appendix Vm (conk) Character Taxa* 88 90 95 100 I I I I - - - K. mexicana 012211121221111 . - K. ~ectm- 012211121221111 T. brevis 113201121201222 I. furcatus 113301121201222 8. uncinus 113501121201223 F. bucerium ll????l????l??? R. pristis 12????12131112? Pla.cvlindraceus 1 12 20 0 1 2 1 1 2 1 2 2 1 Qct. chandleri 01????1???11??? N.cvlindtatus 013503121301122 F1. mu* 01351211130111? Pau. fractus 013511121211??? Appendix VIII (cont.) Character Taxa* 103 105 110 115 Priapulida ?O?O???O??????O Rotifera ?O?O???O???O??O 111121111123130 ~~1111211111?1150 Mac. in- 111121113223222 Pawsentis SD. 111121113123222 0. tortuosa 1111211132?3222 A. dims 11111N211112140 E. eadi 21111N313212240 L. thecatus 11111N311112321 D.chandleri 11111N213112331 T. exipuus 11111N211212132 Appendix VIII (cont.) Character Taxa* 103 105 K. mexicana K. mctinaria T. brevis I. furcatus 8. uncinus F. buceriu~fl R. ~ristis Pla. cvlindraceus Oct. chandleri N. cvlindratus Fl. mudis- 1111?2112242610 Pau. fractus 1111221122??111 Appendix VIII (cont.) Character Taxa* 118 120 125 130 I I i I Rotifera ???????????0000 Med. man& 2121NN1111N1002 Mac. inpens 2211NN1211N0002 Pacvsentis SD. llllNN121210002 0. tortuosa llllNN121210002 L. thecatus 1112111111N1111 D. chan- 1112221111N1121 Appendix VIII (cont.) Character K. mexicana 1112121111Nllll K. uectinaria 1112121111Nlll1 T. brevis I. furcatus 8. uncinus F. bucerium 2211NN11llNl111 R. ~ristis 1112121111Nllll Pla-cvlindracew 1 1 1 1 NN 1 1 1 1 N 1 1 1 2 Qct. chandlei ????NN??1221011 N. cvlindratus 2311NNll11N1000 Fl. mueilis 111212?111N101? Pau. fractus ????NN??1??1002 Appendix VIII (cont.) -. -- Character Priapulida 000000 Rotifera 100111 111111 on. rnd~fom-. 1 1 1 1 1 1 ac. rnw 111111 Pacysentis sp. 111111 0.tom- 111111 LukuL 111111 u 111111 111111 111111 3. ex- 111111 Appendix Vm (cont.) Character Appendix VIII (cont.) B. Character state assignment for 16 characters related to cement gland structure under the three assumptions used in the analyses. Character i ii iii Appendix IX Descriptive statistics of the three identical-topology trees produced under assumptions of cement gland evolution. Assump tion i: Assumption ii: Assumption u: Tree length = 404 Tree length = 404 Tree length = 408 CI = 0.545 CI = 0.545 CI = 0.539 HI = 0.455 HI = 0.455 HI = 0.461 CIX = 0.517 CIX = 0.517 CIX = 0.512 HIX = 0.483 HIX = 0.483 HIX = 0.488 RI = 0.670 RI = 0.670 RI = 0.665 RC = 0.365 RC = 0.365 RC = 0.359 f value = 3538 f value = 3538 f value = 3562 f-ratio = 0.5374 f-ratio = 0.5374 f-ratio = 0.5374