Phylogenetic analysis of arthropods using two nuclear protein-encoding genes supports a crustacean + hexapod clade

Recent phylogenetic analyses using molecular data suggest that hexapods are more closely related to crustaceans than to myriapods, a result that conflicts with long-held morphology-based hypotheses. Here we contribute additional information to this debate by conducting phylogenetic analyses on two nuclear protein-encoding genes, elongation factor-1 alpha (EF-1 alpha) and the largest subunit of RNA polymerase II (Pol II), from an extensive sample of arthropod taxa. Results were obtained from two data sets. One data set comprised 1092 nucleotides (364 amino acids) of EF-1 alpha and 372 nucleotides (124 amino acids) of Pol II from 30 arthropods and three lobopods. The other data set contained the same EF-1 alpha fragment and an expanded 1038-nucleotide (346-amino-acid) sample of Pol II from 17 arthropod taxa. Results from maximum-parsimony and maximum-likelihood analyses strongly supported the existence of a Crustacea + Hexapoda clade (Pancrustacea) over a Myriapoda + Hexapoda clade (Atelocerata). The apparent incompatibility between the molecule-based Pancrustacea hypothesis and morphology-based Atelocerata hypothesis is discussed.     

On the phylogenetic position of insects in the Pancrustacea clade

The current views on the phylogeny of arthropods are at odds with the traditional system, which recognizes four independent arthropod classes: Chelicerata, Crustacea, Myriapoda, and Insecta. There is compelling evidence that insects comprise a monophyletic lineage with Crustacea within a larger clade named Pancrustacea, or Tetraconata. However, which crustacean group is the closest living relative of insects is still an open question. In recent phylogenetic trees constructed on the basis of large gene sequence data insects are placed together with primitive crustaceans, the Branchiopoda. This topology is often suspected to be a result of the long branch attraction artifact. We analyzed concatenated data on 77 ribosomal proteins, elongation factor 1A (EF1A), initiation factor 5A (eIF5A), and several other nuclear and mitochondrial proteins. Analyses of nuclear genes confirm the monophyly of Hexapoda, the clade uniting entognath and ectognath insects. The hypothesis of the monophyly of Hexapoda and Branchiopoda is supported in the majority of analyses. The Maxillopoda, another clade of Entomostraca, occupies a sister position to the Hexapoda + Branchiopoda group. Higher crustaceans, the Malacostraca, in most analyses appear a more basal lineage within the Pancrustacea. We report molecular synapomorphies in low homoplastic regions, which support the clade Hexapoda + Branchiopoda + Maxillopoda and the monophyletic Malacostraca including Phyllocarida. Thus, the common origin of Hexapoda and Branchiopoda and their position within Entomostraca are suggested to represent bona fide phylogenetic relationships rather than computational artifacts.     

Mitochondrial genomes suggest that hexapods and crustaceans are mutually paraphyletic

For over a century the relationships between the four major groups of the phylum Arthropoda (Chelicerata, Crustacea, Hexapoda and Myriapoda) have been debated. Recent molecular evidence has confirmed a close relationship between the Crustacea and the Hexapoda, and has included the suggestion of a paraphyletic Hexapoda. To test this hypothesis we have sequenced the complete or near-complete mitochondrial genomes of three crustaceans (Parhyale hawaiensis, Squilla mantis and Triops longicaudatus), two collembolans (Onychiurus orientalis and Podura aquatica) and the insect Thermobia domestica. We observed rearrangement of transfer RNA genes only in O. orientalis, P. aquatica and P. hawaiensis. Of these, only the rearrangement in O. orientalis, an apparent autapomorphy for the collembolan family Onychiuridae, was phylogenetically informative.We aligned the nuclear and amino acid sequences from the mitochondrial protein-encoding genes of these taxa with their homologues from other arthropod taxa for phylogenetic analysis. Our dataset contains many more Crustacea than previous molecular phylogenetic analyses of the arthropods. Neighbour-joining, maximum-likelihood and Bayesian posterior probabilities all suggest that crustaceans and hexapods are mutually paraphyletic. A crustacean clade of Malacostraca and Branchiopoda emerges as sister to the Insecta sensu stricto and the Collembola group with the maxillopod crustaceans. Some, but not all, analyses strongly support this mutual paraphyly but statistical tests do not reject the null hypotheses of a monophyletic Hexapoda or a monophyletic Crustacea. The dual monophyly of the Hexapoda and Crustacea has rarely been questioned in recent years but the idea of both groups' paraphyly dates back to the nineteenth century. We suggest that the mutual paraphyly of both groups should seriously be considered.     

Elongation factor-2: a useful gene for arthropod phylogenetics.

Robust resolution of controversial higher-level groupings within Arthropoda requires additional sources of characters. Toward this end, elongation factor-2 sequences (1899 nucleotides) were generated from 17 arthropod taxa (5 chelicerates, 6 crustaceans, 3 hexapods, 3 myriapods) plus an onychophoran and a tardigrade as outgroups. Likelihood and parsimony analyses of nucleotide and amino acid data sets consistently recovered Myriapoda and major chelicerate groups with high bootstrap support. Crustacea + Hexapoda (= Pancrustacea) was recovered with moderate support, whereas the conflicting group Myriapoda + Hexapoda (= Atelocerata) was never recovered and bootstrap values were always <5%. With additional nonarthropod sequences included, one indel supports monophyly of Tardigrada, Onychophora, and Arthropoda relative to molluscan, annelidan, and mammalian outgroups. New and previously published sequences from RNA polymerase II (1038 nucleotides) and elongation factor-1alpha (1092 nucleotides) were analyzed for the same taxa. A comparison of bootstrap values from the three genes analyzed separately revealed widely varying values for some clades, although there was never strong support for conflicting groups. In combined analyses, there was strong bootstrap support for the generally accepted clades Arachnida, Arthropoda, Euchelicerata, Hexapoda, and Pycnogonida, and for Chelicerata, Myriapoda, and Pancrustacea, whose monophyly is more controversial. Recovery of some additional groups was fairly robust to method of analysis but bootstrap values were not high; these included Pancrustacea + Chelicerata, Hexapoda + Cephalocarida + Remipedia, Cephalocarida + Remipedia, and Malaocostraca + Cirripedia. Atelocerata (= Myriapoda + Hexapoda) was never recovered. Elongation factor-2 is now the second protein-encoding, nuclear gene (in addition to RNA polymerase II) to support Pancrustacea over Atelocerata. Atelocerata is widely cited in morphology-based analyses, and the discrepancy between results derived from molecular and morphological data deserves greater attention.     

The Tetraconata concept: hexapod-crustacean relationships and the phylogeny of Crustacea

A growing body of evidence indicates that Crustacea and Hexapoda are sister groups, rather than Hexapoda and Myriapoda. Some recent molecular data even suggest that Mandibulata is not monophyletic, with Myriapoda and Chelicerata instead being sister groups. Here, arguments for homology of the mandible throughout mandibulate arthropods and for a monophyletic Mandibulata will be presented, as well as arguments supporting the taxon Tetraconata (i.e. Crustacea + Hexapoda). The latter include molecular data (nuclear and mitochondrial ribosomal RNAs and protein coding genes), and morphological characters such as ommatidial structure, the presence of neuroblasts and a very similar axonogenesis of pioneer neurons. However, crustaceans are insufficiently sampled for the molecular data, and studies of neurogenesis are lacking for many crustacean taxa. Remipedia, Cephalocarida and Maxillopoda are particularly problematic. This is important for the entire problem, because monophyly of the Crustacea has not yet been proven beyond doubt and several molecular analyses suggest a paraphyletic Crustacea. Here, arguments for the monophyly of the Crustacea are reviewed and two alternatives for the relationships between the five higher taxa Remipedia, Cephalocarida, Maxillopoda, Branchiopoda and Malacostraca are discussed: the Entomostraca concept sensu Walossek with Malacostraca as sister group to Cephalocarida, Maxillopoda and Branchiopoda, and the Thoracopoda concept sensu Hessler with Cephalocarida, Branchiopoda and Malacostraca forming a monophylum.     

A new view of insect-crustacean relationships I. Inferences from neural cladistics and comparative neuroanatomy

Traditional hypotheses regarding the relationships of the major arthropod lineages focus on suites of comparable characters, often those that address features of the exoskeleton. However, because of the enormous morphological variety among arthropods, external characters may lead to ambiguities of interpretation and definition, particularly when species have undergone evolutionary simplification and reversal. Here we present the results of a cladistic analysis using morphological characters associated with brains and central nervous systems, based on the evidence that cerebral organization is generally robust over geological time. Well-resolved, strongly supported phylogenies were obtained from a neuromorphological character set representing a variety of discrete neuroanatomical traits. Phylogenetic hypotheses from this analysis support many accepted relationships, including monophyletic Chelicerata, Myriapoda, and Hexapoda, paraphyletic Crustacea and the union of Hexapoda and Crustacea (Tetraconata). They also support Mandibulata (Myriapoda + Tetraconata). One problematic result, which can be explained by symplesiomorphies that are likely to have evolved in deep time, is the inability to resolve Onychophora as a taxon distinct from Arthropoda. Crucially, neuronal cladistics supports the heterodox conclusion that both Hexapoda and Malacostraca are derived from a common ancestor that possessed a suite of discrete neural centers comprising an elaborate brain. Remipedes and copepods, both resolved as basal to Branchiopoda share a neural ground pattern with Malacostraca. These findings distinguish Hexapoda (Insecta) from Branchiopoda, which is the sister group of the clade Malacostraca + Hexapoda. The present study resolves branchiopod crustaceans as descendents of an ancestor with a complex brain, which means that they have evolved secondary simplification and the loss or reduction of numerous neural systems.     

ARTHROPOD PHYLOGENY: A COMBINED APPROACH

Abstract— Ribosomal and ubiquitin protein coding sequence data are generated from 20 arthropods and five close relatives. These molecular data are combined with morphological characters derived from the literature to approach arthropod phylogenetics from the perspective of total evidence. Trilobita were included in the analysis through morphological comparison alone. The overall data strongly support arthropod monophyly. Recent molecular analyses which have yield different results are shown to have been based on few characters, few taxa or both. The most parsimonious explanation of the data is (Annelida + (Onychophora + (Trilobita + Chelicerata) + (Crustacea + (Myriapoda + Hexapoda))))). The data are largely concordant both internally among data sets and externally with previous cladistic anatomical analyses.     

A new view of insect-crustacean relationships II. Inferences from expressed sequence tags and comparisons with neural cladistics

The enormous diversity of Arthropoda has complicated attempts by systematists to deduce the history of this group in terms of phylogenetic relationships and phenotypic change. Traditional hypotheses regarding the relationships of the major arthropod groups (Chelicerata, Myriapoda, Crustacea, and Hexapoda) focus on suites of morphological characters, whereas phylogenomics relies on large amounts of molecular sequence data to infer evolutionary relationships. The present discussion is based on expressed sequence tags (ESTs) that provide large numbers of short molecular sequences and so provide an abundant source of sequence data for phylogenetic inference. This study presents well-supported phylogenies of diverse arthropod and metazoan outgroup taxa obtained from publicly-available databases. An in-house bioinformatics pipeline has been used to compile and align conserved orthologs from each taxon for maximum likelihood inferences. This approach resolves many currently accepted hypotheses regarding internal relationships between the major groups of Arthropoda, including monophyletic Hexapoda, Tetraconata (Crustacea + Hexapoda), Myriapoda, and Chelicerata sensu lato (Pycnogonida + Euchelicerata). "Crustacea" is a paraphyletic group with some taxa more closely related to the monophyletic Hexapoda. These results support studies that have utilized more restricted EST data for phylogenetic inference, yet they differ in important regards from recently published phylogenies employing nuclear protein-coding sequences. The present results do not, however, depart from other phylogenies that resolve Branchiopoda as the crustacean sister group of Hexapoda. Like other molecular phylogenies, EST-derived phylogenies alone are unable to resolve morphological convergences or evolved reversals and thus omit what may be crucial events in the history of life. For example, molecular data are unable to resolve whether a Hexapod-Branchiopod sister relationship infers a branchiopod-like ancestry of the Hexapoda, or whether this assemblage originates from a malacostracan-like ancestor, with the morphologically simpler Branchiopoda being highly derived. Whereas this study supports many internal arthropod relationships obtained by other sources of molecular data, other approaches are required to resolve such evolutionary scenarios. The approach presented here turns out to be essential: integrating results of molecular phylogenetics and neural cladistics to infer that Branchiopoda evolved simplification from a more elaborate ancestor. Whereas the phenomenon of evolved simplification may be widespread, it is largely invisible to molecular techniques unless these are performed in conjunction with morphology-based strategies.     

Pancrustacean phylogeny: hexapods are terrestrial crustaceans and maxillopods are not monophyletic

Recent molecular analyses indicate that crustaceans and hexapods form a clade (Pancrustacea or Tetraconata), but relationships among its constituent lineages, including monophyly of crustaceans, are controversial. Our phylogenetic analysis of three protein-coding nuclear genes from 62 arthropods and lobopods (Onychophora and Tardigrada) demonstrates that Hexapoda is most closely related to the crustaceans Branchiopoda (fairy shrimp, water fleas, etc.) and Cephalocarida + Remipedia, thereby making hexapods terrestrial crustaceans and the traditionally defined Crustacea paraphyletic. Additional findings are that Malacostraca (crabs, isopods, etc.) unites with Cirripedia (barnacles, etc.) and they, in turn, with Copepoda, making the traditional crustacean class Maxillopoda paraphyletic. Ostracoda (seed shrimp)--either all or a subgroup--is associated with Branchiura (fish lice) and likely to be basal to all other pancrustaceans. A Bayesian statistical (non-clock) estimate of divergence times suggests a Precambrian origin for Pancrustacea (600 Myr ago or more), which precedes the first unambiguous arthropod fossils by over 60 Myr.     

Origin of the Ostracoda and their maxillopodan and hexapodan affinities

There are Cambrian fossils attributed to the Ostracoda but the extant subclasses Podocopa and Myodocopa do not appear until the Ordovician. At this time the morphologically similar, free-living ancestors of the now sedentary Thecostraca (Ascothoracida, Acrothoracica and Cirripedia) may have still been extant, and from an ecological point of view it seems likely that, by and large, ostracods replaced them. However, living ostracods have an abbreviated, direct development, and some key aspects of their morphology, such as the nature of the maxillary segment and abdomen, are conjectural. Thus the affinities between these and related taxa remain uncertain; e.g., while some contemporary carcinologists place Ostracoda as a taxon coordinate with the Branchiopoda, Remipedia, Cephalocarida, Maxillopoda, Malacostraca, others tentatively or unequivocally ally them with the Maxillopoda (generally Mystacocarida, Copepoda, Tantulocarida and Thecostraca, and sometimes Branchiura and Pentastomida). Others, largely involved with fossils, have stretched the definition of the Maxillopoda even further, to the point where it seems even less likely a monophyletic taxon. Until recently cladistic analyses utilizing genetic (largely 18S rDNA) as well traditional morphological characteristics have given confusing results regarding the affinities between these taxa, and an important one suggested the Ostracoda might even be diphyletic. Furthermore, a very recent genetic study utilizing protein encoding genes places a podocopine ostracod among the most primitive of the extant crustaceans (Branchiopoda, Cephalocarida Remipedia and Mystacocarida), and then generally at the base of a lineage leading to the Malacostraca, a lineage giving rise to copepods and cirripeds along the way. This indicates these so-called maxillopodan taxa evolved independently from a malacostracan-like ancestor, and if so they are convergent. And finally, from genetic studies it is not only becoming well documented the Crustacea rather than Myriapoda gave rise to the Hexapoda, but it appears the Hexapoda stem from among the lower rather than the higher crustaceans, possibly even from the Ostracoda. Whether there were terrestrial ostracods at the time hexapods appeared in the Lower Ordovician is unknown, but the modest diversity of terrestrial ostracods today are podocopines which also first appeared in the Lower Ordovician. Thus, if current interpretations of living ostracodan and fossil hexapodan body plans are largely correct, it can be hypothesized the Ostracoda are close to the ancestor of the Hexapoda.     

The ovary structure and oogenesis in the basal crustaceans and hexapods. Possible phylogenetic significance

Recent large-scale phylogenetic analyses of exclusively molecular or combined molecular and morphological characters support a close relationship between Crustacea and Hexapoda. The growing consensus on this phylogenetic link is reflected in uniting both taxa under the name Pancrustacea or Tetraconata. Several recent molecular phylogenies have also indicated that the monophyletic hexapods should be nested within paraphyletic crustaceans. However, it is still contentious exactly which crustacean taxon is the sister group to Hexapoda. Among the favored candidates are Branchiopoda, Malacostraca, Remipedia and Xenocarida (Remipedia + Cephalocarida). In this context, we review morphological and ultrastructural features of the ovary architecture and oogenesis in these crustacean groups in search of traits potentially suitable for phylogenetic considerations. We have identified a suite of morphological characters which may prove useful in further comparative studies.     

Arthropod phylogeny: An overview from the perspectives of morphology,molecular data and the fossil record

Monophyly of Arthropoda is emphatically supported from both morphological and molecular perspectives. Recent work finds Onychophora rather than Tardigrada to be the closest relatives of arthropods. The status of tardigrades as panarthropods (rather than cycloneuralians) is contentious from the perspective of phylogenomic data. A grade of Cambrian taxa in the arthropod stem group includes gilled lobopodians, dinocaridids (e.g., anomalocaridids), fuxianhuiids and canadaspidids that inform on character acquisition between Onychophora and the arthropod crown group. A sister group relationship between Crustacea (itself likely paraphyletic) and Hexapoda is retrieved by diverse kinds of molecular data and is well supported by neuroanatomy. This clade, Tetraconata, can be dated to the early Cambrian by crown group-type mandibles. The rival Atelocerata hypothesis (Myriapoda + Hexapoda) has no molecular support. The basal node in the arthropod crown group is embroiled in a controversy over whether myriapods unite with chelicerates (Paradoxopoda or Myriochelata) or with crustaceans and hexapods (Mandibulata). Both groups find some molecular and morphological support, though Mandibulata is presently the stronger morphological hypothesis. Either hypothesis forces an unsampled ghost lineage for Myriapoda from the Cambrian to the mid Silurian.     

The complete sequence of the mitochondrial genome of the crustacean Penaeus monodon: are malacostracan crustaceans more closely related to insects than to branchiopods?

The complete sequence of the mitochondrial genome of the giant tiger prawn, Penaeus monodon (Arthropoda, Crustacea, Malacostraca), is presented. The gene content and gene order are identical to those observed in Drosophila yakuba. The overall AT composition is lower than that observed in the known insect mitochondrial genomes, but higher than that observed in the other two crustaceans for which complete mitochondrial sequence is available. Analysis of the effect of nucleotide bias on codon composition across the Arthropoda reveals a trend with the crustaceans represented showing the lowest proportion of AT-rich codons in mitochondrial protein genes. Phylogenetic analysis among arthropods using concatenated protein-coding sequences provides further support for the possibility that Crustacea are paraphyletic. Furthermore, in contrast to data from the nuclear gene EF1alpha, the first complete sequence of a malacostracan mitochondrial genome supports the possibility that Malacostraca are more closely related to Insecta than to Branchiopoda.     

Higher-level crustacean phylogeny: Consensus and conflicting hypotheses

This paper presents an overview of current hypotheses of higher-level crustacean phylogeny in order to assist and help focus further research. It concentrates on hypotheses proposed or debated in the recent literature based on morphological, molecular and combined evidence phylogenetic analyses. It can be concluded that crustacean phylogeny remains essentially unresolved. Conflict is rife, irrespective of whether one compares different morphological studies, molecular studies, or both. Using the number of recently proposed alternative sister group hypotheses for each of the major tetraconatan taxa as a rough estimate of phylogenetic uncertainty, it can be concluded that the phylogenetic position of Malacostraca remains the most problematic, closely followed by Branchiopoda, Cephalocarida, Remipedia, Ostracoda, Branchiura, Copepoda and Hexapoda. Future progress will depend upon a broader taxon sampling in molecular analyses, and the further exploration of new molecular phylogenetic markers. However, the need for continued revision and expansion of morphological datasets remains undiminished given the conspicuous lack of agreement between molecules and morphology for positioning several taxa. In view of the unparalleled morphological diversity of Crustacea, and the likely nesting of Hexapoda somewhere within Crustacea, working out a detailed phylogeny of Tetraconata is a crucial step towards understanding arthropod body plan evolution.     

Phylogenetic relationships of basal hexapods among the mandibulate arthropods: a cladistic analysis based on comparative morphological characters

In this paper we propose a reappraisal of the relationships between the basal hexapod lineages (the former 'apterygote' insects) and the other major groups of mandibulate arthropods. It results from a cladistic analysis including 72 characters based on external morphology, internal anatomy and development. Detailed comments are provided on the various characters used and the scoring of their states. The 35 terminal taxa include 12 hexapods (9 of which are basal 'apterygote' representatives), 7 myriapods, 13 crustaceans, and 3 chelicerates taken as outgroups. The results of our analyses are discussed in detail for each of the taxonomic groupings, and compared with those recently obtained by other authors using different approaches based on morphological, palaeontological, developmental or molecular sequence data. Our results support the monophyly of the Mandibulata, Crustacea, Atelocerata (Tracheata) and Hexapoda, but the assemblage of Myriapoda appears poorly supported. A close relationship between Crustacea and Hexapoda, as hypothesized by several authors, is not found in any of our analyses. Within Hexapoda, the Protura and the Collembola appear as independent clades, whereas the two unresolved dipluran taxa are grouped with the monophyletic Ectognatha (Archaeognatha, Zygentoma and Pterygota).     

First molecular evidence for the existence of a Tardigrada + Arthropoda clade

The complete 18S rDNA gene sequence of Macrobiotus group hufelandi (Tardigrada) was obtained and aligned with 18S rDNA and rRNA gene sequences of 24 metazoans (mainly protostomes). Discrete character (maximum-parsimony) and distance (neighbor-joining) methods were used to infer their phylogeny. The evolution of bootstrap proportions with sequence length (pattern of resolved nodes, PRN) was studied to test the resolution of the nodes in neighbor-joining trees. The results show that arthropods are monophyletic. Tardigrades represent the sister group of arthropods (in parsimony analyses) or they are related with crustaceans (distance analysis and PRN). Arthropoda are divided into two main evolutionary lines, the Hexapoda + Crustacea line (weakly supported), and the Myriapoda + Chelicerata line. The Hexapoda + Crustacea line includes Pentastomida, but the internal resolution is far from clear. The Insecta (Ectognatha) are monophyletic, but no evidence for the monophyly of Hexapoda is found. The Chelicerata are a monophyletic group and the Myriapoda cluster close to Arachnida. Overall, the results obtained represent the first molecular evidence for a Tardigrada + Arthropoda clade. In addition, the congruence between molecular phylogenies of the Arthropoda from other authors and this obtained here indicates the need to review those obtained solely on morphological characters.      

The endoskeletal structures in arthropods: cytology, morphology and evolution

The paper proposes an overview of the endoskeletal structures of the head and trunk in the different arthropod groups: Chelicerata, Crustacea, Myriapoda and Hexapoda (=Insecta s.l.). Two major endoskeletal systems are reported with their cytological characteristics: those made up of connective tissue derived from muscular tendons, and those consisting of cuticular rods or plates arising from integumentary ingrowths. The morphological value of the various endoskeletal structures, their possible homologies in different groups, and their presumed evolutionary changes are discussed. This survey may be considered as a first step to use morphological characteristics of the endoskeleton in future cladistic analyses to assess the phylogeny of arthropods.     

Arthropod Cladistics: Combined Analysis of Histone H3 and U2 snRNA Sequences and Morphology

Morphological, developmental, ultrastructural, and gene order characters are catalogued for the same set of arthropod terminals as we have scored in a recent study of histone H3 and U2 snRNA sequences (D. J. Colgan et al. , 1998, Aust. J. Zool. 46, 419–437). We examine the implications of separate and simultaneous analyses of sequence and non-sequence data for arthropod relationships. The most parsimonious trees based on 211 non-sequence characters (273 apomorphic states) support traditional higher taxa as clades, including Mandibulata, Crustacea, Atelocerata, Myriapoda, and Hexapoda. Combined analysis of morphology with histone H3 and U2 sequences with equal character weights differs from the morphological results alone in supporting Progoneata + Hexapoda (= Labiophora) in favor of a monophyletic Myriapoda, resolves the entognathous hexapods as a grade, and supports pycnogonids as sister group to Euchelicerata (rather than as basal euarthropods). Monophyly of Chelicerata (including pycnogonids), Mandibulata, Crustacea, Progoneata, Chilopoda, and Hexapoda is maintained under a range of transition/transversion and third codon weights, whereas Atelocerata and Myriapoda/Labiophora do not withstand all sensitivity analyses.     

Ecdysozoan phylogeny and Bayesian inference: first use of nearly complete 28S and 18S rRNA gene sequences to classify the arthropods and their kin

Relationships among the ecdysozoans, or molting animals, have been difficult to resolve. Here, we use nearly complete 28S+18S ribosomal RNA gene sequences to estimate the relations of 35 ecdysozoan taxa, including newly obtained 28S sequences from 25 of these. The tree-building algorithms were likelihood-based Bayesian inference and minimum-evolution analysis of LogDet-transformed distances, and hypotheses were tested wth parametric bootstrapping. Better taxonomic resolution and recovery of established taxa were obtained here, especially with Bayesian inference, than in previous parsimony-based studies that used 18S rRNA sequences (or 18S plus small parts of 28S). In our gene trees, priapulan worms represent the basal ecdysozoans, followed by nematomorphs, or nematomorphs plus nematodes, followed by Panarthropoda. Panarthropoda was monophyletic with high support, although the relationships among its three phyla (arthropods, onychophorans, tardigrades) remain uncertain. The four groups of arthropods-hexapods (insects and related forms), crustaceans, chelicerates (spiders, scorpions, horseshoe crabs), and myriapods (centipedes, millipedes, and relatives)-formed two well-supported clades: Hexapoda in a paraphyletic crustacea (Pancrustacea), and 'Chelicerata+Myriapoda' (a clade that we name 'Paradoxopoda'). Pycnogonids (sea spiders) were either chelicerates or part of the 'chelicerate+myriapod' clade, but not basal arthropods. Certain clades derived from morphological taxonomy, such as Mandibulata, Atelocerata, Schizoramia, Maxillopoda and Cycloneuralia, are inconsistent with these rRNA data. The 28S gene contained more signal than the 18S gene, and contributed to the improved phylogenetic resolution. Our findings are similar to those obtained from mitochondrial and nuclear (e.g., elongation factor, RNA polymerase, Hox) protein-encoding genes, and should revive interest in using rRNA genes to study arthropod and ecdysozoan relationships.