Molecular phylogeny of the major arthropod groups indicates polyphyly of crustaceans and a new hypothesis for the origin of hexapods

A phylogeny of the arthropods was inferred from analyses of amino acid sequences derived from the nuclear genes encoding elongation factor-1 alpha and the largest subunit of RNA polymerase II using maximum- parsimony, neighbor-joining, and maximum-likelihood methods. Analyses of elongation factor-1 alpha from 17 arthropods and 4 outgroup taxa recovered many arthropod clades supported by previous morphological studies, including Diplopoda, Myriapoda, Insecta, Hexapoda, Branchiopoda (Crustacea), Araneae, Tetrapulmonata, Arachnida, Chelicerata, and Malacostraca (Crustacea). However, counter to previous studies, elongation factor-1 alpha placed Malacostraca as sister group to the other arthropods. Branchiopod crustaceans were found to be more closely related to hexapods and myriapods than to malacostracan crustaceans. Sequences for RNA polymerase II were obtained from 11 arthropod taxa and were analyzed separately and in combination with elongation factor-1 alpha. Results from these analyses were concordant with those derived from elongation factor-1 alpha alone and provided support for a Hexapoda/Branchiopoda clade, thus arguing against the monophyly of the traditionally defined Atelocerata (Hexapoda + Myriapoda).      

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.     

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.     

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.      

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.     

Gene expression reveals evidence for EGFR‐dependent proximal‐distal limb patterning in a myriapod

Evolution of segmented limbs is one of the key innovations of Arthropoda, allowing development of functionally specific specialized head and trunk appendages, a major factor behind their unmatched evolutionary success. Proximodistal limb patterning is controlled by two regulatory networks in the vinegar fly Drosophila melanogaster, and other insects. The first is represented by the function of the morphogens Wingless (Wg) and Decapentaplegic (Dpp); the second by the EGFR‐signaling cascade. While the role of Wg and Dpp has been studied in a wide range of arthropods representing all main branches, that is, Pancrustacea (= Hexapoda + Crustacea), Myriapoda and Chelicerata, investigation of the potential role of EGFR‐signaling is restricted to insects (Hexapoda). Gene expression analysis of Egfr, its potential ligands, and putative downstream factors in the pill millipede Glomeris marginata (Myriapoda: Diplopoda), reveals that—in at least mandibulate arthropods—EGFR‐signaling is likely a conserved regulatory mechanism in proximodistal limb patterning.     

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.     

Phylogeny of protostome moulting animals (Ecdysozoa) inferred from 18 and 28S rRNA gene sequences

Reliability of reconstruction of phylogenetic relationships within a group of protostome moulting animals was evaluated by means of comparison of 18 and 28S rRNA gene sequences sets both taken separately and combined. Reliability of reconstructions was evaluated by values of the bootstrap support of major phylogenetic tree nodes and by degree of congruence of phylogenetic trees inferred by various methods. By both criteria, phylogenetic trees reconstructed from the combined 18 and 28S rRNA gene sequences were better than those inferred from 18 and 28S sequences taken separately. Results obtained are consistent with phylogenetic hypothesis separating protostome animals into two major clades, moulting Ecdysozoa (Priapulida + Kinorhyncha, Nematoda + Nematomorpha, Onychophora + Tardigrada, Myriapoda + Chelicerata, Crustacea + Hexapoda) and unmoulting Lophotrochozoa (Plathelminthes, Nemertini, Annelida, Mollusca, Echiura, Sipuncula). Clade Cephalorhyncha does not include nematomorphs (Nematomorpha). Conclusion was taken that it is necessary to use combined 18 and 28S data in phylogenetic studies.     

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).     

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.     

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.     

Brain organization in Collembola (springtails)

Arthropoda is comprised of four major taxa: Hexapoda, Crustacea, Myriapoda and Chelicerata. Although this classification is widely accepted, there is still some debate about the internal relationships of these groups. In particular, the phylogenetic position of Collembola remains enigmatic. Some molecular studies place Collembola into a close relationship to Protura and Diplura within the monophyletic Hexapoda, but this placement is not universally accepted, as Collembola is also regarded as either the sister group to Branchiopoda (a crustacean taxon) or to Pancrustacea (crustaceans + hexapods). To contribute to the current debate on the phylogenetic position of Collembola, we examined the brains in three collembolan species: Folsomia candida, Protaphorura armata and Tetrodontophora bielanensis, using antennal backfills, series of semi-thin sections, and immunostaining technique with several antisera, in conjunction with confocal laser scanning microscopy and three-dimensional reconstructions. We identified several neuroanatomical structures in the collembolan brain, including a fan-shaped central body showing a columnar organization, a protocerebral bridge, one pair of antennal lobes with 20-30 spheroidal glomeruli each, and a structure, which we interpret as a simply organized mushroom body. The results of our neuroanatomical study are consistent with the phylogenetic position of Collembola within the Hexapoda and do not contradict the hypothesis of a close relationship of Collembola, Protura and Diplura.     

Phylogeny of Arthropoda inferred from mitochondrial sequences: strategies for limiting the misleading effects of multiple changes in pattern and rates of substitution

In this study, mitochondrial sequences were used to investigate the relationships among the major lineages of Arthropoda. The data matrix used for the analyses includes 84 taxa and 3918 nucleotides representing six mitochondrial protein-coding genes (atp6 and 8, cox1-3, and nad2). The analyses of nucleotide composition show that a reverse strand-bias, i.e., characterized by an excess of T relative to A nucleotides and of G relative to C nucleotides, was independently acquired in six different lineages of Arthropoda: (1) the honeybee mite (Varroa), (2) Opisthothelae spiders (Argiope, Habronattus, and Ornithoctonus), (3) scorpions (Euscorpius and Mesobuthus), (4) Hutchinsoniella (Cephalocarid), (5) Tigriopus (Copepod), and (6) whiteflies (Aleurodicus and Trialeurodes). Phylogenetic analyses confirm that these convergences in nucleotide composition can be particularly misleading for tree reconstruction, as unrelated taxa with reverse strand-bias tend to group together in MP, ML, and Bayesian analyses. However, the use of a specific model for minimizing effects of the bias, the "Neutral Transition Exclusion" (NTE) model, allows Bayesian analyses to rediscover most of the higher taxa of Arthropoda. Furthermore, the analyses of branch lengths suggest that three main factors explain accelerated rates of substitution: (1) genomic rearrangements, including duplication of the control region and gene translocation, (2) parasitic lifestyle, and (3) small body size. The comparisons of Bayesian Bootstrap percentages show that the support for many nodes increases when taxa with long branches are excluded from the analyses. It is therefore recommended to select taxa and genes of the mitochondrial genome for inferring phylogenetic relationships among arthropod lineages. The phylogenetic analyses support the existence of a major dichotomy within Arthropoda, separating Pancrustacea and Paradoxopoda. Basal relationships between Pancrustacean lineages are not robust, and the question of Hexapod monophyly or polyphyly cannot be answered with the available mitochondrial sequences. Within Paradoxopoda, Chelicerata and Myriapoda are each found to be monophyletic, and Endeis (Pycnogonida) is, surprisingly, associated with Acari.     

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.     

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.     

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 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.     

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.      

Phylogeny of Molting Protostomes (Ecdysozoa) as Inferred from 18S and 28S rRNA Gene Sequences

Phylogenetic relationships within the group of molting protostomes were reconstructed by comparing the sets of 18S and 28S rRNA gene sequences considered either separately or in combination. The reliability of reconstructions was estimated from the bootstrap indices for major phylogenetic tree nodes and from the degree of congruence of phylogenetic trees obtained by different methods. By either criterion, the phylogenetic trees reconstructed on the basis of both 18 and 28S rRNA gene sequences were better than those based on the 18S or 28S sequences alone. The results of reconstruction are consistent with the phylogenetic hypothesis classifying protostomes into two major clades: molting Ecdysozoa (Priapulida + Kinorhyncha, Nematoda + Nematomorpha, Onychophora + Tardigrada, Myriapoda + Chelicerata, and Crustacea + Hexapoda) and nonmolting Lophotrochozoa (Plathelminthes, Nemertini, Annelida, Mollusca, Echiura, and Sipuncula). Nematomorphs (Nematomorpha) do not belong to the clade Cephalorhyncha (Priapulida + Kinorhyncha). It is concluded that combined data on the 18S and 28S rRNA gene sequences provide a more reliable basis for phylogenetic inferences.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 4, 2005, pp. 590–601.Original Russian Text Copyright © 2005 by Petrov, Vladychenskaya.