HEADS AND TAILS: A CHORDATE PHYLOGENY

Abstract— A cladistic analysis of chordates is presented, based on some 320 nested characters. All the principal higher taxa are defined by synapomorphies, including extinct acanthodians and placoderms. The data base draws broadly from adult anatomy (including osteological data for Recent and fossil taxa), embryology, physiology, and biochemistry. A conventional sequence of chordate higher taxa is generated (hemichordates, urochordates, cephalochordates, craniates). Among the craniates, cyclostomes are considered paraphyletic. Gnathostomes are monophyletic, but two fossil "agnathan" groups (galeaspids, osteostracans) are regarded as stem gnathostomes. Chondrichthyans and osteichthyans are monophyletic. New arguments for osteichthyan affinity of acanthodians are presented. The phylogenetic position of placoderms is still problematic, but they can no longer be perceived as stem chondrichthyans or even as "elasmobranchiomorphs." Recent dipnoans and tetrapods are sister groups, but new paleontological discoveries refute many of their supposed osteological synapomorphies, thereby reopening the possibility of a closer relationship between tetrapods and osteolepiform rhipidistians.     

Soft anatomy and the affinities of conodonts

Recent claims that conodonts are members of the Craniata or Vertebrata are based in part upon soft tissue features that have been preserved in a small number of specimens. These features include what appear to be radials in the caudal fin and paired structures that have been identified as eye remnants. The evidence for radials is limited, but credible. However, the anatomy of extant cyclostomes suggests that the paired structures are more reasonably interpreted as otic capsules than the remnants of sclerotic eye capsules. Moreover, even if these structures are the remnants of eyes, conodonts might equally well be a sister group to the craniates as a member of that group. Aside from these paired structures, conodont fossils exhibit no features that are suggestive of a cartilaginous skeleton. Given that cyclostome fossils sometimes show evidence of the cartilages of the head, the apparent absence of a similar skeleton in conodont animals calls into question the claim that they are craniates. The simple single chevron shape of conodont myomeres also suggests that they lie outside of the Craniata. All living craniates have double-chevron myomeres as adults, whereas simple myomeres of the conodont type are found in the non-craniate cephalochordates. Thus the available soft tissue evidence suggests that conodonts are best regarded as the sister group of the craniates.     

Deuterostome phylogeny and the sister group of the chordates: evidence from molecules and morphology

Complete coding regions of the 18S rRNA gene of an enteropneust hemichordate and an echinoid and ophiuroid echinoderm were obtained and aligned with 18S rRNA gene sequences of all major chordate clades and four outgroups. Gene sequences were analyzed to test morphological character phylogenies and to assess the strength of the signal. Maximum- parsimony analysis of the sequences fails to support a monophyletic Chordata; the urochordates form the sister taxon to the hemichordates, and together this clade plus the echinoderms forms the sister taxon to the cephalochordates plus craniates. Decay, bootstrap, and tree-length distribution analyses suggest that the signal for inference of dueterostome phylogeny is weak in this molecule. Parsimony analysis of morphological plus molecular characters supports both monophyly of echinoderms plus enteropneust hemichordates and a sister group relationship of this clade to chordates. Evolutionary parsimony does not support chordate monophyly. Neighbor-joining, Fitch-Margoliash, and maximum-likelihood analyses support a chordate lineage that is the sister group to an echinoderm-plus-hemichordate lineage. The results illustrate both the limitations of the 18S rRNA molecule alone for high- level phylogeny inference and the importance of considering both molecular and morphological data in phylogeny reconstruction.      

Evolution and development of the chordates: collagen and pharyngeal cartilage

Chordates evolved a unique body plan within deuterostomes and are considered to share five morphological characters, a muscular postanal tail, a notochord, a dorsal neural tube, an endostyle, and pharyngeal gill slits. The phylum Chordata typically includes three subphyla, Cephalochordata, Vertebrata, and Tunicata, the last showing a chordate body plan only as a larva. Hemichordates, in contrast, have pharyngeal gill slits, an endostyle, and a postanal tail but appear to lack a notochord and dorsal neural tube. Because hemichordates are the sister group of echinoderms, the morphological features shared with the chordates must have been present in the deuterostome ancestor. No extant echinoderms share any of the chordate features, so presumably they have lost these structures evolutionarily. We review the development of chordate characters in hemichordates and present new data characterizing the pharyngeal gill slits and their cartilaginous gill bars. We show that hemichordate gill bars contain collagen and proteoglycans but are acellular. Hemichordates and cephalochordates, or lancelets, show strong similarities in their gill bars, suggesting that an acellular cartilage may have preceded cellular cartilage in deuterostomes. Our evidence suggests that the deuterostome ancestor was a benthic worm with gill slits and acellular gill cartilages.     

Conodont anatomy, chordate phylogeny and vertebrate classification

Interpretations of conodont anatomy and affinity continue to generate controversy. Fossilized soft-tissue evidence indicates that conodonts possessed eyes, extrinsic eye muscles, a notochord, myomeres, a differentiated tail with fin radiais, possible otic capsules and possible branchial structures. Indirect evidence suggests a differentiated brain and cartilaginous head skeleton. The multi-component phosphatic tissue complexes of the conodont feeding apparatus cannot be compared to the amorphous apatite of extant agnathan otoliths. By limiting cladistic analysis to a restricted selection of these characters the hypothesis that conodonts are a sister group of the clade comprising extant hagfish, lampreys and gnathostomes can be supported. However, exhaustive analysis of a more complete character-set strongly supports the hypothesis that conodonts are more derived than hagfish. From a taxonomic perspective, these two hypotheses have no effect on how conodonts should be classified. Whether they are a stem group (the former hypothesis) or part of the crown group (the latter), conodonts are clearly part of the total group Vertebrata (=Craniata).     

An aboral-dorsalization hypothesis for chordate origin

Chordates originated from a common ancestor(s) shared with two other deuterostome groups, echinoderms and hemichordates, by creating a novel type of tadpole-like larva, which was characterized by a dorsal hollow neural tube and notochord. Recent molecular phylogeny supports the notion that echinoderms and hemichordates form a clade named the Ambulacraria and that, among the chordates, cephalochordates are more basal than urochordates and vertebrates. An aboral-dorsalization hypothesis is proposed to explain how the tadpole-type larva evolved. Embryological comparison of cephalochordates with nonchordate deuterostomes suggests that, because of limited space on the oral side of the ancestral embryo, morphogenesis to form the neural tube and notochord occurred on the aboral side of the embryo. Namely, the dorsalization of the aboral side of the ancestral embryo may have been a key developmental event that led to the formation of the basic chordate body plan.     

Development and evolution of chordate cartilage

Deuterostomes are a monophyletic group of animals containing vertebrates, lancelets, tunicates, hemichordates, echinoderms, and xenoturbellids. Four out of these six extant groups-vertebrates, lancelets, tunicates, and hemichordates-have pharyngeal gill slits. All groups of deuterostome animals that have pharyngeal gill slits also have a pharyngeal skeleton supporting the pharyngeal openings, except tunicates. We previously found that pharyngeal cartilage in hemichordates and cephalochordates contains a fibrillar collagen protein similar to vertebrate type II collagen, but unlike vertebrate cartilage, the invertebrate deuterostome cartilages are acellular. We found SoxE and fibrillar collagen expression in the pharyngeal endodermal cells adjacent to where the cartilages form. These same endodermal epithelial cells also express Pax1/9, a marker of pharyngeal endoderm in vertebrates, lancelets, tunicates, and hemichordates. In situ experiments with a cephalochordate fibrillar collagen also showed expression in pharyngeal endoderm, as well as the ectoderm and the mesodermal coelomic pouches lining the gill bars. These results indicate that the pharyngeal endodermal cells are responsible for secretion of the cartilage in hemichordates, whereas in lancelets, all the pharyngeal cells surrounding the gill bars, ectodermal, endodermal, and mesodermal may be responsible for cartilage formation. We propose that endoderm secretion was primarily the ancestral mode of making pharyngeal cartilages in deuterostomes. Later the evolutionary origin of neural crest allowed co-option of the gene network for the secretion of pharyngeal cartilage matrix in the new migratory neural crest cell populations found in vertebrates.     

The serial transformation hypothesis of vertebrate origins: comment on "The new head hypothesis revisited"

In "The New Head Hypothesis Revisited," R.G. Northcutt (2005. J Exp Zool (Mol Dev Evol) 304B:274-297) evaluates the original postulates of this hypothesis (Northcutt and Gans, 1983. Quart Rev Biol 58:1-28). One of these postulates is that the brain-particularly the forebrain-evolved at essentially the same time as many neural crest and neurogenic placode derivatives-including sensory ganglia, dermal skeleton and sensory capsules of the head, and branchial arches. Northcutt's subsequent paper in 1996 concluded with the idea that transitional forms might not have occurred at the origin of vertebrates. Butler proposed a "Serial Transformation" hypothesis in 2000, which disputed the latter idea in that paired eyes and an enlarged brain (but lacking telencephalon) were envisioned to have been gained before elaboration of most neural crest and neurogenic placodal derivatives. In 2003, J. Mallatt and J.-Y. Chen analyzed fossils of the Cambrian animal Haikouella, which strongly support its affinity to craniates and aspects of several hypotheses, including Butler's transformational model, because although branchial bars are present, most other neural crest and placodal derivatives are absent, while paired eyes and an enlarged brain (but probably without telencephalon) are present. A more complete picture of vertebrate origins can be realized when the various hypotheses are constructively reconciled.     

The unusual cartilaginous tissues of jawless craniates, cephalochordates and invertebrates

A collagenous extracellular matrix was previously considered to be a requirement for classification of true cartilage. Data from the lamprey and hagfish now clearly indicate that both of these jawless craniates have extensive non-collagenous, yet cartilaginous endoskeletons. Non-collagenous cartilages are present in the cephalochordates (amphioxus) and in the invertebrates, although collagen-containing cartilages also are found in the invertebrates. This review summarizes current knowledge of the morphological, biochemical and molecular characteristics of the unusual non-collagenous cartilages in jawless craniates and the cartilaginous tissues in amphioxus and invertebrates. A least two types of non-collagenous cartilage matrix proteins are found in both the hagfishes and the lampreys, all of which are resistant to digestion by cyanogen bromide (CNBr). Although all four of these matrices show some similarities with each other, suggesting a family of non-collagenous, elastin-like proteins, it is clear that the major matrix proteins of each are different. New morphological and biochemical information on the cartilaginous tissues in squid, horseshoe crab and amphioxus reveals the presence of CNBr-insoluble, non-collagenous matrix proteins, potentially extending the jawless craniate family of cartilaginous proteins into the invertebrates. Details of the evolutionary relationships between these non-collagenous matrix proteins and the significance of the occurrence of these proteins as the major components of the cartilaginous tissues of jawless craniates, amphioxus, horseshoe crab and squid, all of which are capable of producing a variety of collagens in other tissues, remain to be investigated.     

The evolutionary origin of chordate segmentation: revisiting the enterocoel theory

One of the definitive characteristics of chordates (cephalochordates, vertebrates) is the somites, which are a series of paraxial mesodermal blocks exhibiting segmentation. The presence of somites in the basal chordate amphioxus and in vertebrates, but not in tunicates (the sister group of vertebrates), suggests that the tunicates lost the somites secondarily. Somites are patterned from anterior to posterior during embryogenesis. How such a segmental pattern evolved from deuterostome ancestors is mysterious. The classic enterocoel theory claims that chordate mesoderm evolved from the ancestral deuterostome mesoderm that organizes the trimeric body parts seen in extant hemichordates. Recent progress in molecular embryology has been tremendous, which has enabled us to test this classic theory. In this review, the history of the study on the evolution of the chordate mesoderm is summarized. This is followed by a review of the current understanding of genetic mapping on anterior/posterior (A/P) mesodermal patterning between chordates (cephalochordates, vertebrates) and a direct developing hemichordate (Saccoglossus kowalevskii). Finally, a possible scenario about the evolution of the chordate mesoderm from deuterostome ancestors is discussed.     

A cladistic analysis of the anomalocystitid mitrates

A cladistic analysis of the anomalocystitid mitrates is presented. A neutral terminology for the anomalocystitid skeleton is proposed, independent of the zoological interpretation of the fossils as echinoderms or as craniates. The anomalocystitid monophyly is supported by parsimony, although the instability of several basal taxa makes it difficult to ascertain the sequence in which characters were acquired or modified in the transition from the mi-trocystitids to the anomalocystitids. The genus Barrandeocarpus falls outside the anomalocystitids as traditionally defined in the literature, and is either polyphyletic or monophyletic. Diamphidiocystis drepanon is either a basal anomalocystitid or the sister taxon to the group ( Enoploura popei + Allanicytidiidae). Ateleocystites guttenbergensis is placed either at the base of the anomalocystitids or as the sister taxon to a group including mainly boreal forms, the only non-boreal members being the South African Bokkeveldia oosthuizeni and the Australian Victoriacystis wilkinsi.     

Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives

Deuterostomes are a monophyletic group of animals that include the vertebrates, invertebrate chordates, ambulacrarians and xenoturbellids. Fossil representatives from most major deuterostome groups, including some phylum-level crown groups, are found in the Lower Cambrian, suggesting that evolutionary divergence occurred in the Late Precambrian, in agreement with some molecular clock estimates. Molecular phylogenies, larval morphology and the adult heart/kidney complex all support echinoderms and hemichordates as a sister grouping (Ambulacraria). Xenoturbellids are a relatively newly discovered phylum of worm-like deuterostomes that lacks a fossil record, but molecular evidence suggests that these animals are a sister group to the Ambulacraria. Within the chordates, cephalochordates share large stretches of chromosomal synteny with the vertebrates, have a complete Hox complex and are sister group to the vertebrates based on ribosomal and mitochondrial gene evidence. In contrast, tunicates have a highly derived adult body plan and are sister group to the vertebrates based on the analyses of concatenated genomic sequences. Cephalochordates and hemichordates share gill slits and an acellular cartilage, suggesting that the ancestral deuterostome also shared these features. Gene network data suggest that the deuterostome ancestor had an anterior-posterior body axis specified by Hox and Wnt genes, a dorsoventral axis specified by a BMP/chordin gradient, and was bilaterally symmetrical with left-right asymmetry determined by expression of nodal.     

Phylogenetic analysis of phenotypic characters of Tunicata supports basal Appendicularia and monophyletic Ascidiacea

With approximately 3000 marine species, Tunicata represents the most disparate subtaxon of Chordata. Molecular phylogenetic studies support Tunicata as sister taxon to Craniota, rendering it pivotal to understanding craniate evolution. Although successively more molecular data have become available to resolve internal tunicate phylogenetic relationships, phenotypic data have not been utilized consistently. Herein these shortcomings are addressed by cladistically analyzing 117 phenotypic characters for 49 tunicate species comprising all higher tunicate taxa, and five craniate and cephalochordate outgroup species. In addition, a combined analysis of the phenotypic characters with 18S rDNA-sequence data is performed in 32 OTUs. The analysis of the combined data is congruent with published molecular analyses. Successively up-weighting phenotypic characters indicates that phenotypic data contribute disproportionally more to the resulting phylogenetic hypothesis. The strict consensus tree from the analysis of the phenotypic characters as well as the single most parsimonious tree found in the analysis of the combined dataset recover monophyletic Appendicularia as sister taxon to the remaining tunicate taxa. Thus, both datasets support the hypothesis that the last common ancestor of Tunicata was free-living and that ascidian sessility is a derived trait within Tunicata. “Thaliacea” is found to be paraphyletic with Pyrosomatida as sister taxon to monophyletic Ascidiacea and the relationship between Doliolida and Salpida is unresolved in the analysis of morphological characters; however, the analysis of the combined data reconstructs Thaliacea as monophyletic nested within paraphyletic “Ascidiacea”. Therefore, both datasets differ in the interpretation of the evolution of the complex holoplanktonic life history of thaliacean taxa. According to the phenotypic data, this evolution occurred in the plankton, whereas from the combined dataset a secondary transition into the plankton from a sessile ascidian is inferred. Besides these major differences, both analyses are in accord on many phylogenetic groupings, although both phylogenetic reconstructions invoke a high degree of homoplasy. In conclusion, this study represents the first serious attempt to utilize the potential phylogenetic information present in phenotypic characters to elucidate the inter-relationships of this diverse marine taxon in a consistent cladistic framework.     

Ambulacrarian prototypical Hox and ParaHox gene complements of the indirect-developing hemichordate <Emphasis Type="Italic">Balanoglossus simodensis</Emphasis>

The Hox genes and its evolutionary sister, the ParaHox genes, are widely distributed among animals. Although it has been expected that hemichordates and echinoderms have a single set of Hox genes and most likely a single set of ParaHox genes, it is not known whether the ortholog of Hox8 is absent in hemichordates, and in turn, consensus view about Hox/ParaHox gene complements in hemichordates has not been established. In this study, we isolated either complete or nearly complete coding sequences of 12 Hox genes, including the ortholog of the Hox8 that has not been reported in the previous studies, and three ParaHox genes from the recently discovered indirect-developing acorn worm, Balanoglossus simodensis. Our data suggest that the ancestral hemichordate had intact complements of ambulacrarian prototypical Hox and ParaHox genes, consisting of 12 and three members, respectively.     

A phylogenetic test of the calcichordate scenario

The calcichordate scenario of Jefferies and colleagues purports to explain the origin and early evolution of the phyla Echinodermata and Chordata. Calcichordate proponents have argued that echinoderms are the sister group of the chordates and urochordates are the sister group of the craniates. These phylogenetic hypotheses, which differ from the traditional groupings, are derived primarily from morphological interpretations of carpoids (solutes, cornutes, and mitrates), an enigmatic fossil group usually held to be primitive stem-group echinoderms. Although the scenario has received only limited support, it has yet to be falsified. The difficulty with falsifying the calcichordate scenario is proving that the morphological interpretations, for example, that carpoids possessed notochords, dorsal hollow nerve cords, and other typical chordate or craniate characters, are incorrect. Here, rather than argue over the interpretation of fossils, the phylogenetic hypotheses embedded within the scenario are tested. It is found that the calcichordate scenario fails such a test, even if both the Recent and fossils forms are coded according to the calcichordate scenario. It is argued that: (1) the erection of scenarios must follow the construction of a cladogram; and (2) fossils are unable to dictate the relationships among phyla. □ Calcichordate scenario, Carpoidea, Deuterostomia, Echinodermata, Chordata, phylogeny, cladistics.     

Towards the reconstruction of the bilaterian ancestral pre-MHC region

In this article, we describe how we reconstructed a precise, minimal proto-MHC region in the ancestor of euchordates, which was based on a comparison of the MHC-paralogy group of vertebrate with the MHC-like chromosome of cephalochordates. This deduced ancestral region was compared with the genomes of extant species, other deuterostomes and protostomes. Our analysis revealed statistically significant traces of conservation in these species, suggesting that a proto-MHC region existed at the origin of all bilaterian species. We also propose a new approach to reconstruct ancestral genomes, which combines both stringent statistical testing and phylogenetic analysis.     

Olfactory systems: common design, uncommon origins?

In both vertebrates and invertebrates, odorant molecules reach the dendrites of olfactory receptor cells through an aqueous medium, which reflects the evolutionary origin of these systems in a marine environment. Important recent advances, however, have demonstrated striking interphyletic differences between the structure of vertebrate and invertebrate olfactory receptor proteins, as well as the organization of the genes encoding them. While these disparities support independent origins for odor-processing systems in craniates and protostomes (and even between the nasal and vomeronasal systems of craniates), olfactory neuropils share close neuroanatomical and physiological characters. Whereas there is a case to be made for homology among members of the two great protostome clades (the ecdysozoans and lophotrochozoans), the position of the craniates remains ambiguous.     

Cranial anatomy and phylogenetic position of Suminia getmanovi, a basal anomodont (Amniota: Therapsida) from the Late Permian of Eastern Europe

Suminia getmanovi , a recently discovered basal anomodont from the Late Permian of Russia, is characterized by robust, 'leaf-shaped' teeth, and a masticatory architecture that is similar to that of the highly diverse and cosmopolitan group of Permo-Triassic herbivores, Dicynodontia (Anomodontia). Based on new material, the skull is reconstructed in three dimensions and described in detail. A cladistic analysis of the basal anomodonts, Patranomodon, Galeops, Otsheria, Ulemica , and Suminia , using 37 cranial characters, resulted in a single most parsimonious tree, in which Suminia is united with the Russian taxa, Ulemica and Otsheria. This clade, diagnosed by four unambiguous characters, is designated as Venyukovioidea. The South African anomodont, Galeops , appears as the sister taxon to Dicynodontia. Patranomodon is the most basal anomodont. The cladistic analysis suggests that a 'dicynodont-type' masticatory architecture, with an expanded adductor musculature and sliding jaw articulation, may have originated prior to the advent of the (Venyukovioidea + ( Galeops + Dicynodontia)) clade.     

Remarks on the inner ear of elasmobranchs and its interpretation from skeletal labyrinth morphology.

The structure and function of the craniate inner ear is reviewed, with 33 apomorphic characters of the membranous labyrinth and associated structures identified in craniates, gnathostomes, and elasmobranchs. Elasmobranchs are capable of low-frequency semi-directional phonoreception, even in the absence of any pressure-to-displacement transducer such as ear ossicles. The endolymphatic (parietal) fossa, semicircular canals, and crista (macula) neglecta are all adapted toward phonoreception. Some (but not all) of the morphological features associated with phonoreception can be inferred from the elasmobranch skeletal labyrinth. Endocranial spaces such as the skeletal labyrinth also provide suites of morphological characters that may be incorporated into phylogenetic analyses, irrespective of how closely these spaces reflect underlying soft anatomy. The skeletal labyrinths of Squalus and Notorynchus are compared using silicone endocasts and high-resolution CT-scanning. The latter procedure offers several advantages over other techniques; it is more informative, nondestructive, preserves relationships of surrounding structures, and it can be applied both to modern and fossil material.     

Structural evolution of Otx genes in craniates

Using a degenerate PCR approach, we performed an exhaustive search of Otx genes in the reedfish Erpetoichthys calabaricus, the dogfish Scyliorhinus canicula, and the hagfish Myxine glutinosa. Three novel Otx genes were identified in each of these species, and their deduced protein sequences were determined over a large C-terminal fragment located immediately downstream of the homeodomain. Like their lamprey and osteichthyan counterparts, these nine genes display a tandem duplication of a 20--25-residue C-terminal domain, which appears to be a hallmark of all craniate Otx genes identified thus far, including the highly divergent Crx gene. Phylogenetic analyses show that, together with their osteichthyan counterparts, the dogfish and reedfish genes can be classified into three gnathostome orthology classes. Two of the three genes identified in each of these species belong to the Otx1 and Otx2 orthology classes previously characterized in osteichthyans. The third one unambiguously clusters with the Otx5/Otx5b genes recently characterized in Xenopus laevis, thus defining a novel orthology class. Our results also strongly suggest that the highly divergent Crx genes identified in humans, rodents, and oxen are the mammalian representatives of this third class. The hagfish genes display no clear relationships to the three gnathostome orthology classes, but one of them appears to be closely related to the LjOtxA gene, previously identified in Lampetra japonica. Taken together, these data support the hypothesis that the Otx multigene families characterized in craniates all derive from duplications of a single ancestral gene which occurred after the splitting of cephalochordates but prior to the gnathostome radiation. Using site-by-site sequence comparisons of the gnathostome Otx proteins, we also identified structural constraints selectively acting on each of the three gnathostome orthology classes. This suggests that specialized functions for each of these orthology classes were fixed in the gnathostome lineage prior to the splitting between osteichthyans and chondrichthyans.