Systematic searches for molecular synapomorphies in model metazoan genomes give some support for Ecdysozoa after accounting for the idiosyncrasies of Caenorhabditis elegans

There has been broad acceptance among evolutionary biologists of the Ecdysozoa hypothesis that, based principally on molecular phylogenetic studies of small and large subunit ribosomal RNA sequences, postulates a close relationship between molting taxa such as arthropods and nematodes. On the other hand, recent studies of as many as 100 additional genes do not support the Ecdysozoa hypothesis and instead favor the older Coelomata hypothesis that groups the coelomate arthropods with the coelomate vertebrates to the exclusion of the nematodes. Here, exploiting completely sequenced genomes, we examined this question using cladistic analyses of the phylogenetic distribution of 1712 orthologous genes and 2906 protein domain combinations; we found stronger support for the Coelomata hypothesis than for the Ecdysozoa hypothesis. However, although arrived at by considering very large data sets, we show that this conclusion is unreliable, biased toward grouping arthropods with chordates by systematic high rate of character loss in the nematode. When we addressed this problem, we found slightly more support for Ecdysozoa than for Coelomata. Our identification of this systematic bias even when using entire genomes has important implications for future phylogenetic studies. We conclude that the results from the intensively sampled ribosomal RNA genes supporting the Ecdysozoa hypothesis provide the most credible current estimates of metazoan phylogeny.     

Palaeontological and Molecular Evidence Linking Arthropods, Onychophorans, and other Ecdysozoa

Membership of Arthropoda in a clade of molting animals, the Ecdysozoa, has received a growing body of support over the past 10 years from analyses of DNA sequences from many genes together with morphological characters involving the cuticle and its molting. Recent analyses based on broad phylogenomic sampling strengthen the grouping of cycloneuralian worms and arthropods as Ecdysozoa, identify the velvet worms (Phylum Onychophora) as the closest living relatives of arthropods, and interpret segmentation as having separate evolutionary origins in arthropods and annelid worms. Determining whether the water bears (Phylum Tardigrada) are more closely related to onychophorans and arthropods or to unsegmented cycloneuralians such as roundworms (Nematoda) is an open question. Fossil taxa such as the Cambrian anomalocaridids provide a combination of arthropod and cycloneuralian characters that is not observed in any living ecdysozoan. Fossils break up long branches and help to resolve the sequence of character acquisition at several critical nodes in the arthropod tree, notably in a suite of Cambrian lobopodians that may include the stem groups of each of the major panarthropod lineages.     

Phylogenomics and the number of characters required for obtaining an accurate phylogeny of eukaryote model species

MOTIVATION: Through the most extensive phylogenomic analysis carried out to date, complete genomes of 11 eukaryotic species have been examined in order to find the homologous of more than 25,000 amino acid sequences. These sequences correspond to the exons of more than 3000 genes and were used as presence/absence characters to test one of the most controversial hypotheses concerning animal evolution, namely the Ecdysozoa hypothesis. Distance, maximum parsimony and Bayesian methods of phylogenetic reconstruction were used to test the hypothesis. RESULTS: The reliability of the ecdysozoa, grouping arthropods and nematodes in a single clade was unequivocally rejected in all the consensus trees. The Coelomata clade, grouping arthropods and chordates, was supported by the highest statistical confidence in all the reconstructions. The study of the dependence of the genomes' tree accuracy on the number of exons used, demonstrated that an unexpectedly larger number of characters are necessary to obtain robust phylogenies. Previous studies supporting ecdysozoa, could not guarantee an accurate phylogeny because the number of characters used was clearly below the minimum required.     

Multigene analyses of bilaterian animals corroborate the monophyly of Ecdysozoa, Lophotrochozoa, and Protostomia

Almost a decade ago, a new phylogeny of bilaterian animals was inferred from small-subunit ribosomal RNA (rRNA) that claimed the monophyly of two major groups of protostome animals: Ecdysozoa (e.g., arthropods, nematodes, onychophorans, and tardigrades) and Lophotrochozoa (e.g., annelids, molluscs, platyhelminths, brachiopods, and rotifers). However, it received little additional support. In fact, several multigene analyses strongly argued against this new phylogeny. These latter studies were based on a large amount of sequence data and therefore showed an apparently strong statistical support. Yet, they covered only a few taxa (those for which complete genomes were available), making systematic artifacts of tree reconstruction more probable. Here we expand this sparse taxonomic sampling and analyze a large data set (146 genes, 35,371 positions) from a diverse sample of animals (35 species). Our study demonstrates that the incongruences observed between rRNA and multigene analyses were indeed due to long-branch attraction artifacts, illustrating the enormous impact of systematic biases on phylogenomic studies. A refined analysis of our data set excluding the most biased genes provides strong support in favor of the new animal phylogeny and in addition suggests that urochordates are more closely related to vertebrates than are cephalochordates. These findings have important implications for the interpretation of morphological and genomic data.     

Support for the Coelomata clade of animals from a rigorous analysis of the pattern of intron conservation

Many intron positions are conserved in varying subsets of eukaryotic genomes and, consequently, comprise a potentially informative class of phylogenetic characters. Roy and Gilbert developed a method of phylogenetic reconstruction using the patterns of intron presence-absence in eukaryotic genes and, applying this method to the analysis of animal phylogeny, obtained support for an Ecdysozoa clade (Roy SW, Gilbert W. 2005. Resolution of a deep animal divergence by the pattern of intron conservation. Proc Natl Acad Sci USA. 102:4403-4408). The critical assumption in the method was the independence of intron loss in different branches of the phylogenetic tree. Here, this assumption is refuted by showing that the branch-specific intron loss rates are strongly correlated. We show that different tree topologies are obtained, in each case with a significant statistical support, when different subsets of intron positions are analyzed. The analysis of the conserved intron positions supports the Coelomata topology, that is, a clade comprised of arthropods and chordates, whereas the analysis of more variable intron positions favors the Ecdysozoa topology, that is, a clade of arthropods and nematodes. We show, however, that the support for Ecdysozoa is fully explained by parallel loss of introns in nematodes and arthropods, a factor that does not contribute to the analysis of the conserved introns. The developed procedure for the identification and analysis of conserved introns and other characters with minimal or no homoplasy is expected to be useful for resolving many hard phylogenetic problems.     

The multimeric beta-thymosin found in nematodes and arthropods is not a synapomorphy of the Ecdysozoa

The Ecdysozoa hypothesis proposes a clade of animals including arthropods and nematodes that share the characteristic of periodic molting or ecdysis. The original evidence supporting this hypothesis came from molecular phylogenies based on ribosomal RNA gene sequences. Contrary evidence has come from studies of multiple protein coding genes. One of the most convincing bits of supporting evidence for this theory has been the observation of an unusual multimeric form of the beta-thymosin gene in the genomes of Drosophila melanogaster and Caenorhabditis elegans where, in other metazoans that had been studied, a monomeric form has been found. Here I show that recently deposited sequence data reveal that the multimeric form is in fact a characteristic of all major subdivisions of the Metazoa. The multimeric form is present in a deuterostome, Ciona intestinalis, a lophotrochozoan, Hermissenda crassicornis, and in the ecdysozoans and also exists outside the Metazoa in a fungus. The presence of the multimeric form in nematodes and arthropods, therefore, although not contradicting the Ecdysozoa hypothesis, gives it no support. The absence of the monomeric form in the completely sequenced flies and nematodes may suggest they are linked but, lacking the complete genomes of other ecdysozoans, proving its total absence from the Ecdysozoa is not possible. Furthermore, the absence of the monomeric form from the genome of the deuterostome Ciona suggests that the absence of this character is an unreliable indicator of relationships.     

The phylogenetic status of arthropods, as inferred from 18S rRNA sequences

Partial 18S rRNA sequences of five chelicerate arthropods plus a crustacean, myriapod, insect, chordate, echinoderm, annelid, and platyhelminth were compared. The sequence data were used to infer phylogeny by using a maximum-parsimony method, an evolutionary-distance method, and the evolutionary-parsimony method. The phylogenetic inferences generated by maximum-parsimony and distance methods support both monophyly of the Arthropoda and monophyly of the Chelicerata within the Arthropoda. These results are congruent with phylogenies based on rigorous cladistic analyses of morphological characters. Results support the inclusion of the Arthropoda within a spiralian or protostome coelomate clade that is the sister group of a deuterostome clade, refuting the hypothesis that the arthropods represent the "primitive" sister group of a protostome coelomate clade. Bootstrap analyses and consideration of all trees within 1% of the length of the most parsimonious tree suggest that relationships between the nonchelicerate arthropods and relationships within the chelicerate clade cannot be reliably inferred with the partial 18S rRNA sequence data. With the evolutionary-parsimony method, support for monophyly of the Arthropoda is found in the majority of the combinations analyzed if the coelomates are used as "outgroups." Monophyly of the Chelicerata is supported in most combinations assessed. Our analyses also indicate that the evolutionary-parsimony method, like distance and parsimony, may be biased by taxa with long branches. We suggest that a previous study's inference of the Arthropoda as paraphyletic may be the result of (a) having two few arthropod taxa available for analysis and (b) including long-branched taxa.      

The complete mitochondrial genome of the onychophoran Epiperipatus biolleyi reveals a unique transfer RNA set and provides further support for the ecdysozoa hypothesis

Onychophora (velvet worms) play a crucial role in current discussions on position of arthropods. The ongoing Articulata/Ecdysozoa debate is in need of additional ground pattern characters for Panarthropoda (Arthropoda, Tardigrada, and Onychophora). Hence, Onychophora is an important outgroup taxon in resolving the relationships among arthropods, irrespective of whether morphological or molecular data are used. To date, there has been a noticeable lack of mitochondrial genome data from onychophorans. Here, we present the first complete mitochondrial genome sequence of an onychophoran, Epiperipatus biolleyi (Peripatidae), which shows several characteristic features. Specifically, the gene order is considerably different from that in other arthropods and other bilaterians. In addition, there is a lack of 9 tRNA genes usually present in bilaterian mitochondrial genomes. All these missing tRNAs have anticodon sequences corresponding to 4-fold degenerate codons, whereas the persisting 13 tRNAs all have anticodons pairing with 2-fold degenerate codons. Sequence-based phylogenetic analysis of the mitochondrial protein-coding genes provides a robust support for a clade consisting of Onychophora, Priapulida, and Arthropoda, which confirms the Ecdysozoa hypothesis. However, resolution of the internal ecdysozoan relationships suffers from a cluster of long-branching taxa (including Nematoda and Platyhelminthes) and a lack of data from Tardigrada and further nemathelminth taxa in addition to nematodes and priapulids.     

Evaluating hypotheses of deuterostome phylogeny and chordate evolution with new LSU and SSU ribosomal DNA data

We investigated evolutionary relationships among deuterostome subgroups by obtaining nearly complete large-subunit ribosomal RNA (LSU rRNA)-gene sequences for 14 deuterostomes and 3 protostomes and complete small-subunit (SSU) rRNA-gene sequences for five of these animals. With the addition of previously published sequences, we compared 28 taxa using three different data sets (LSU only, SSU only, and combined LSU + SSU) under minimum evolution (with LogDet distances), maximum likelihood, and maximum parsimony optimality criteria. Additionally, we analyzed the combined LSU + SSU sequences with spectral analysis of LogDet distances, a technique that measures the amount of support and conflict within the data for every possible grouping of taxa. Overall, we found that (1) the LSU genes produced a tree very similar to the SSU gene tree, (2) adding LSU to SSU sequences strengthened the bootstrap support for many groups above the SSU-only values (e.g., hemichordates plus echinoderms as Ambulacraria; lancelets as the sister group to vertebrates), (3) LSU sequences did not support SSU-based hypotheses of pterobranchs evolving from enteropneusts and thaliaceans evolving from ascidians, and (4) the combined LSU + SSU data are ambiguous about the monophyly of chordates. No tree-building algorithm united urochordates conclusively with other chordates, although spectral analysis did so, providing our only evidence for chordate monophyly. With spectral analysis, we also evaluated several major hypotheses of deuterostome phylogeny that were constructed from morphological, embryological, and paleontological evidence. Our rRNA-gene analysis refutes most of these hypotheses and thus advocates a rethinking of chordate and vertebrate origins.     

A congruent solution to arthropod phylogeny: phylogenomics,microRNAs and morphology support monophyletic Mandibulata

While a unique origin of the euarthropods is well established, relationships between the four euarthropod classes—chelicerates, myriapods, crustaceans and hexapods—are less clear. Unsolved questions include the position of myriapods, the monophyletic origin of chelicerates, and the validity of the close relationship of euarthropods to tardigrades and onychophorans. Morphology predicts that myriapods, insects and crustaceans form a monophyletic group, the Mandibulata, which has been contradicted by many molecular studies that support an alternative Myriochelata hypothesis (Myriapoda plus Chelicerata). Because of the conflicting insights from published molecular datasets, evidence from nuclear-coding genes needs corroboration from independent data to define the relationships among major nodes in the euarthropod tree. Here, we address this issue by analysing two independent molecular datasets: a phylogenomic dataset of 198 protein-coding genes including new sequences for myriapods, and novel microRNA complements sampled from all major arthropod lineages. Our phylogenomic analyses strongly support Mandibulata, and show that Myriochelata is a tree-reconstruction artefact caused by saturation and long-branch attraction. The analysis of the microRNA dataset corroborates the Mandibulata, showing that the microRNAs miR-965 and miR-282 are present and expressed in all mandibulate species sampled, but not in the chelicerates. Mandibulata is further supported by the phylogenetic analysis of a comprehensive morphological dataset covering living and fossil arthropods, and including recently proposed, putative apomorphies of Myriochelata. Our phylogenomic analyses also provide strong support for the inclusion of pycnogonids in a monophyletic Chelicerata, a paraphyletic Cycloneuralia, and a common origin of Arthropoda (tardigrades, onychophorans and arthropods), suggesting that previous phylogenies grouping tardigrades and nematodes may also have been subject to tree-reconstruction artefacts.     

Phylogeny of Wolbachia in filarial nematodes.

Intracellular bacteria have been observed in various species of filarial nematodes (family Onchocercidae). The intracellular bacterium of the canine filaria Dirofilaria immitis has been shown to be closely related to Wolbachia, a rickettsia-like micro-organism that is widespread among arthropods. However, the relationships between endosymbionts of different filariae, and between these and the arthropod wolbachiae, appear not to have been studied. To address these issues we have examined ten species of filarial nematodes for the presence of Wolbachia. For nine species, all samples examined were PCR positive using primers specific for the ftsZ gene of Wolbachia. For one species, the examined samples were PCR negative. Sequences of the amplified ftsZ gene fragments of filarial wolbachiae fall into two clusters (C and D), which are distinct from the A and B clusters recognized for arthropod wolbachiae. These four lineages (A-D) are related in a star-like phylogeny, with higher nucleotide divergence observed between C and D wolbachiae than that observed between A and B wolbachiae. In addition, within each of the two lineages of filarial wolbachiae, the phylogeny of the symbionts is consistent with the host phylogeny. Thus, there is no evidence for recent Wolbachia transmission between arthropods and nematodes. Endosymbiont 16S ribosomal DNA sequences from a subset of filarial species support these findings.     

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.      

Ecdysozoa: The Relationship between Cycloneuralia and Panarthropoda

The hypothesis that molting protostomes such as nematodes and arthropods form a monophyletic group known as Ecdysozoa is directly opposed to Articulata, in which some segmented protostomes such as annelids and arthropods form a monophyletic taxon. Ultrastructural and cladistic studies have led to the widely accepted hypothesis that nematodes belong among the protostomes. While early molecular studies suggested that nematodes were basal triploblasts, more recent molecular evidence suggests that this was an artifact of ‘long branch attraction’ and 18S rRNA gene, total evidence and hox gene studies all support the placement of nematodes within Ecdysozoa. The branching pattern within Ecdysozoa has been difficult to elucidate, but it now appears that priapulids and kinorhynchs form the earliest branching clade, followed by nematodes + nematomorphs, and finally the panarthropods. This suggests that Cycloneuralia is paraphyletic and that arthropods are the most derived of the ecdysozoans.     

Phylogenetic relationships of the Wolbachia of nematodes and arthropods

Wolbachia are well known as bacterial symbionts of arthropods, where they are reproductive parasites, but have also been described from nematode hosts, where the symbiotic interaction has features of mutualism. The majority of arthropod Wolbachia belong to clades A and B, while nematode Wolbachia mostly belong to clades C and D, but these relationships have been based on analysis of a small number of genes. To investigate the evolution and relationships of Wolbachia symbionts we have sequenced over 70 kb of the genome of wOvo, a Wolbachia from the human-parasitic nematode Onchocerca volvulus, and compared the genes identified to orthologues in other sequenced Wolbachia genomes. In comparisons of conserved local synteny, we find that wBm, from the nematode Brugia malayi, and wMel, from Drosophila melanogaster, are more similar to each other than either is to wOvo. Phylogenetic analysis of the protein-coding and ribosomal RNA genes on the sequenced fragments supports reciprocal monophyly of nematode and arthropod Wolbachia. The nematode Wolbachia did not arise from within the A clade of arthropod Wolbachia, and the root of the Wolbachia clade lies between the nematode and arthropod symbionts. Using the wOvo sequence, we identified a lateral transfer event whereby segments of the Wolbachia genome were inserted into the Onchocerca nuclear genome. This event predated the separation of the human parasite O. volvulus from its cattle-parasitic sister species, O. ochengi. The long association between filarial nematodes and Wolbachia symbionts may permit more frequent genetic exchange between their genomes.     

Testing the new animal phylogeny: first use of combined large-subunit and small-subunit rRNA gene sequences to classify the protostomes

Although the small-subunit ribosomal RNA (SSU rRNA) gene is widely used in the molecular systematics, few large-subunit (LSU) rRNA gene sequences are known from protostome animals, and the value of the LSU gene for invertebrate systematics has not been explored. The goal of this study is to test whether combined LSU and SSU rRNA gene sequences support the division of protostomes into Ecdysozoa (molting forms) and Lophotrochozoa, as was proposed by Aguinaldo et al. (1997) (Nature 387:489) based on SSU rRNA sequences alone. Nearly complete LSU gene sequences were obtained, and combined LSU + SSU sequences were assembled, for 15 distantly related protostome taxa plus five deuterostome outgroups. When the aligned LSU + SSU sequences were analyzed by tree-building methods (minimum evolution analysis of LogDet-transformed distances, maximum likelihood, and maximum parsimony) and by spectral analysis of LogDet distances, both Ecdysozoa and Lophotrochozoa were indeed strongly supported (e.g., bootstrap values >90%), with higher support than from the SSU sequences alone. Furthermore, with the LogDet-based methods, the LSU + SSU sequences resolved some accepted subgroups within Ecdysozoa and Lophotrochozoa (e.g., the polychaete sequence grouped with the echiuran, and the annelid sequences grouped with the mollusc and lophophorates)-subgroups that SSU-based studies do not reveal. Also, the mollusc sequence grouped with the sequences from lophophorates (brachiopod and phoronid). Like SSU sequences, our LSU + SSU sequences contradict older hypotheses that grouped annelids with arthropods as Articulata, that said flatworms and nematodes were basal bilateralians, and considered lophophorates, nemerteans, and chaetognaths to be deuterostomes. The position of chaetognaths within protostomes remains uncertain: our chaetognath sequence associated with that of an onychophoran, but this was unstable and probably artifactual. Finally, the benefits of combining LSU with SSU sequences for phylogenetic analyses are discussed: LSU adds signal, it can be used at lower taxonomic levels, and its core region is easy to align across distant taxa-but its base frequencies tend to be nonstationary across such taxa. We conclude that molecular systematists should use combined LSU + SSU rRNA genes rather than SSU alone.     

A three-phase model of arthropod segmentation

Molecular and morphological evidence (expression patterns of pair-rule genes and segmental position of the genital openings and other segmental markers) suggest that the segmental units of the arthropod body are specified, in early ontogeny, by three spatially and/or temporally distinct mechanisms and do not appear in a strict antero-posterior sequence. A first anterior set of indivisible segments (naupliar segments, possibly three in all arthropods) is followed by a set of more caudal (post-naupliar) primary units (eosegments, possibly ten in all arthropods) which then undergo a process of secondary segmentation, thus giving rise to a higher number of definitive segments (merosegments). The number of merosegments deriving from each eosegment is characteristic of the different arthropod clades and is mostly stable at the level of the traditional arthropodan classes or subclasses. All their segmentation patterns, however, including those found in the segmental organisation of highly segmented forms (such as centipedes and millipedes, notostracan, lipostracan and anostracan crustaceans, and trilobites) are reducible to the basic groundplan with three naupliar and ten postnaupliar segments. These basic units of arthropod segmentation may also have an equivalent in other Ecdysozoa, despite the lack of any segmentation (nematodes) or, at least, of an overt segmentation (kinorhynchs).     

Phylogenetic analysis of the Metastrongyloidea (Nematoda: Strongylida) inferred from ribosomal RNA gene sequences

Phylogenetic relationships among nematodes of the strongylid superfamily Metastrongyloidea were analyzed using partial sequences from the large-subunit ribosomal RNA (LSU rRNA) and small-subunit ribosomal RNA (SSU rRNA) genes. Regions of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction, directly sequenced, aligned, and phylogenies inferred using maximum parsimony. Phylogenetic hypotheses inferred from the SSU rRNA gene supported the monophyly of representative taxa from each of the 7 currently accepted metastrongyloid families. Metastrongyloid taxa formed the sister group to representative trichostrongyloid sequences based on SSU data. Sequences from either the SSU or LSU RNA regions alone provided poor resolution for relationships within the Metastrongyloidea. However, a combined analysis using sequences from all rDNA regions yielded 3 equally parsimonious trees that represented the abursate Filaroididae as polyphyletic, Parafilaroides decorus as the sister species to the monophyletic Pseudaliidae, and a sister group relationship between Oslerus osleri and Metastrongylus salmi. Relationships among 3 members of the Crenosomatidae, and 1 representative of the Skrjabingylidae (Skrjabingylus chitwoodorum) were not resolved by these combined data. However, members of both these groups were consistently resolved as the sister group to the other metastrongyloid families. These relationships are inconsistent with traditional classifications of the Metastrongyloidea and existing hypotheses for their evolution.     

The origins of the arthropod nervous system: insights from the Onychophora

A revision of evolutionary relationships of the Arthropoda has provided fresh impetus to tracing the origins of the nervous system of this group of animals: other members of the Ecdysozoa possess a markedly different type of nervous system from both the arthropods and the annelid worms, with which they were previously grouped. Given their status as favoured sister taxon of the arthropods, Onychophora (velvet worms) are a key group for understanding the evolutionary changes that have taken place in the panarthropod (Arthropoda + Onychophora + Tardigrada) lineage. This article reviews our current knowledge of the structure and development of the onychophoran nervous system. The picture that emerges from these studies is that the nervous system of the panarthropod ancestor was substantially different from that of modern arthropods: this animal probably possessed a bipartite, rather than a tripartite brain; its nerve cord displayed only a limited degree of segmentation; and neurons were more numerous but more uniform in morphology than in living arthropods. These observations suggest an evolutionary scenario, by which the arthropod nervous system evolved from a system of orthogonally crossing nerve tracts present in both a presumed protostome ancestor and many extant worm-like invertebrates, including the onychophorans.     

EST sequencing of Onychophora and phylogenomic analysis of Metazoa

Onychophora (velvet worms) represent a small animal taxon considered to be related to Euarthropoda. We have obtained 1873 5' cDNA sequences (expressed sequence tags, ESTs) from the velvet worm Epiperipatus sp., which were assembled into 833 contigs. BLAST similarity searches revealed that 51.9% of the contigs had matches in the protein databases with expectation values lower than 10(-4). Most ESTs had the best hit with proteins from either Chordata or Arthropoda (approximately 40% respectively). The ESTs included sequences of 27 ribosomal proteins. The orthologous sequences from 28 other species of a broad range of phyla were obtained from the databases, including other EST projects. A concatenated amino acid alignment comprising 5021 positions was constructed, which covers 4259 positions when problematic regions were removed. Bayesian and maximum likelihood methods place Epiperipatus within the monophyletic Ecdysozoa (Onychophora, Arthropoda, Tardigrada and Nematoda), but its exact relation to the Euarthropoda remained unresolved. The "Articulata" concept was not supported. Tardigrada and Nematoda formed a well-supported monophylum, suggesting that Tardigrada are actually Cycloneuralia. In agreement with previous studies, we have demonstrated that random sequencing of cDNAs results in sequence information suitable for phylogenomic approaches to resolve metazoan relationships.     

Chordate evolution and the three-phylum system

Traditional metazoan phylogeny classifies the Vertebrata as a subphylum of the phylum Chordata, together with two other subphyla, the Urochordata (Tunicata) and the Cephalochordata. The Chordata, together with the phyla Echinodermata and Hemichordata, comprise a major group, the Deuterostomia. Chordates invariably possess a notochord and a dorsal neural tube. Although the origin and evolution of chordates has been studied for more than a century, few authors have intimately discussed taxonomic ranking of the three chordate groups themselves. Accumulating evidence shows that echinoderms and hemichordates form a clade (the Ambulacraria), and that within the Chordata, cephalochordates diverged first, with tunicates and vertebrates forming a sister group. Chordates share tadpole-type larvae containing a notochord and hollow nerve cord, whereas ambulacrarians have dipleurula-type larvae containing a hydrocoel. We propose that an evolutionary occurrence of tadpole-type larvae is fundamental to understanding mechanisms of chordate origin. Protostomes have now been reclassified into two major taxa, the Ecdysozoa and Lophotrochozoa, whose developmental pathways are characterized by ecdysis and trochophore larvae, respectively. Consistent with this classification, the profound dipleurula versus tadpole larval differences merit a category higher than the phylum. Thus, it is recommended that the Ecdysozoa, Lophotrochozoa, Ambulacraria and Chordata be classified at the superphylum level, with the Chordata further subdivided into three phyla, on the basis of their distinctive characteristics.