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 position of phylum Nemertini, inferred from 18S rRNA sequences: molecular data as a test of morphological character homology.

Partial 18S rRNA sequence of the nemertine Cerebratulus lacteus was obtained and compared with those of coelomate metazoans and acoelomate platyhelminths to test whether nemertines share a most recent common ancestor with the platyhelminths, as traditionally has been implied, or whether nemertines lie within a protostome coelomate clade, as suggested by more recent morphological analyses. Maximum-parsimony analysis supports the inclusion of the nemertine within a protostome-coelomate clade that falls within a more inclusive coelomate clade. Bootstrap analysis indicates strong support for a monophyletic Coelomata composed of a deuterostome and protostome-coelomate clade. Support for a monophyletic protostome Coelomata is weak. Inference by distance analysis is consistent with that of maximum parsimony. Analysis of down-weighted paired sites by maximum parsimony reveals variation in topology only within the protostome-coelomate clade. The relationships among the protostome coelomates cannot be reliably inferred from the partial sequences, suggesting that coelomate protostomes diversified rapidly. Results with evolutionary parsimony are consistent with the inclusion of the nemertine in a coelomate clade. The molecular inference corroborates recent morphological character analyses that reveal no synapomorphies of nemertines and flatworms but instead suggest that the circulatory system and rhynchocoel of nemertines are homologous to coelomic cavities of protostome coelomates, thus supporting the corresponding hypothesis that nemertines belong within a protostome-coelomate clade. The sequence data provide an independent test of morphological character homology.     

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.     

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).      

Metazoan Relationships on the Basis of 18S rRNA Sequences: A Few Years Later...

SYNOPSIS. The 18S rRNA database is continuously growing andnew tree construction methods are being developed. The presentstudy aims at assessing what effect the addition of recentlydetermined animal 18S rRNA sequences and the use of a recentlydeveloped distance matrix calculation method have on the resultsof some previously published case studies on metazoan phytogeny.When re-assessing three case studies, part of their conclusionswas confirmed on the basis of the present 18S rRNA data set:1) the monophyly of Arthropoda; 2) the monophyly of the Vestimentifera-Pogonophoraand their protostome character; 3) the doubt about the monophyleticorigin of Echiura-Sipuncula and 4) the coelomate characterofNemertea. Yet, it is also demonstrated that some of the previousresults are no longer warranted when updating the analyses:1) the monophyly of both the Annelida and the Eutrochozoa; 2)the sister-group relationship of Echiura to Vestimentifera-Pogonophoraand 3) the polyphyly of the Mesozoa and their close relationshipto Myxozoa and Nematodes. In addition, some new very preliminaryevidence is provided for: 1) a common ancestry of Platyhelminthesand Mesozoa and the monophyly of the latter group and 2) themonophyly of Clitellata, Hirudinida and Oligochaeta. Finally,doubt is casted on the monophyly of the Polychaeta and the polychaeteorders Spionida, Phyllodocida, and Sabellidae. Of course, thesehypotheses also need further testing.     

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.     

REVIEW Evolution and systematics of the Chelicerata

After approximately 40 years of discussion about the question of whether the Arthropoda are a monophyletic or a paraphyletic group or even a polyphyletic assemblage of unrelated taxa, most morphologists, palaeontologists and molecular taxonomists agree that the Arthropoda are a monophylum. The Euarthropoda are composed of the Arachnomorpha and Mandibulata. Myriapods are usually considered to be mandibulates; however, new molecular data as well as some morphological characters show similarities which the Myriapoda share with the Chelicerata, suggesting that there is no taxon Antennata or Atelocerata. Chelicerata are usually considered to be the sister group of Trilobita or, more correctly, Trilobita branch off from the chelicerate stem line. The first adaptive radiation of the Chelicerata took place in the Cambrian. All extant and some extinct orders were present during the Carboniferous. Two systems are compared. It is suggested that the Chelicerata contain the Pantopoda and Euchelicerata. The Euchelicerata are divided into Xiphosura and terrestrial Arachnida. Scorpiones are considered to be the sister group of all other arachnids, the Lipoctena and these are further divided into the Megoperculata (Uropygi, Amblypygi, and Araneae) and Apulmonata (all other groups). The Acari are tentatively considered to be a monophylum and the sister group of the Ricinulei. However, the Actinotrichida and Anactinotrichida diverged early and therefore have had a long history of independent evolution.     

Phylogenetic relationships of annelids,molluscs, and arthropods evidenced from molecules and morphology

Annelids and arthropods have long been considered each other's closest relatives, as evidenced by similarities in their segmented body plans. An alternative view, more recently advocated by investigators who have examined partial 18S ribosomal RNA data, proposes that annelids, molluscs, and certain other minor phyla with trochophore larva stages share a more recent common ancestor with one another than any do with arthropods. The two hypotheses are mutually exclusive in explaining spiralian relationships. Cladistic analysis of morphological data does not reveal phylogentic relationships among major spiralian taxa but does suggest monophyly for both the annelids and molluscs. Distance and maximum-likelihood analyses of 18S rRNA gene sequences from major spiralian taxa suggest a sister relationship between annelids and molluscs and provide a clear resolution within the major groups of the spiralians. The parsimonious tree based on molecular data, however, indicates a sister relationship of the Annelida and Bivalvia, and an earlier divergence of the Gastropoda than the Annelida–Bivalvia clade. To test further hypotheses on the phylogenetic relationships among annelids, molluscs, and arthropods, and the ingroup relationships within the major spiralian taxa, we combine the molecular and morphological data sets and subject the combined data matrix to parsimony analysis. The resulting tree suggests that the molluscs and annelids form a monophyletic lineage and unites the molluscan taxa to a monophyletic group. Therefore, the result supports the Eutrochozoa hypothesis and the monophyly of molluscs, and indicates early acquisition of segmented body plans in arthropods. Received: 25 September 1995 / Accepted: 15 March 1996     

Phylogeny of protostome worms derived from 18S rRNA sequences

The phylogenetic relationships of protostome worms were studied by comparing new complete 18S rRNA sequences of Vestimentifera, Pogonophora, Sipuncula, Echiura, Nemertea, and Annelida with existing 18S rRNA sequences of Mollusca, Arthropoda, Chordata, and Platyhelminthes. Phylogenetic trees were inferred via neighbor-joining and maximum parsimony analyses. These suggest that (1) Sipuncula and Echiura are not sister groups; (2) Nemertea are protostomes; (3) Vestimentifera and Pogonophora are protostomes that have a common ancestor with Echiura; and (4) Vestimentifera and Pogonophora are a monophyletic clade.      

Diplopod hemocyanin sequence and the phylogenetic position of the Myriapoda

Hemocyanins are copper-containing respiratory proteins of the Arthropoda that have so far been thoroughly investigated only in the Chelicerata and the Crustacea but have remained unstudied until now in the Myriapoda. Here we report the first sequence of a myriapod hemocyanin. The hemocyanin of Spirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two distinct subunits that are arranged in a 6 x 6 native molecule. The cloned hemocyanin subunit cDNA codes of for a polypeptide of 653 amino acids (75.5 kDa) that includes a signal peptide of 18 amino acids. The sequence closely resembles that of the chelicerate hemocyanins. Molecular phylogenetic analyses reject with high statistical confidence the integrity of the Tracheata (i.e., Myriapoda + Insecta) but give conflicting results on the position of the myriapod hemocyanin. While distance matrix and maximum-likelihood methods support a basal position of the Spirostreptus hemocyanin with respect to the other hemocyanins, parsimony analysis suggests a sister group relationship with the chelicerate hemocyanins. The latter topology is also supported by a unique shared deletion of an alpha-helix. A common ancestry of Myriapoda and Chelicerata should be seriously considered.     

Phylogenetic analysis based on 18S ribosomal RNA gene sequences supports the existence of class polyacanthocephala (acanthocephala)

Members of phylum Acanthocephala are parasites of vertebrates and arthropods and are distributed worldwide. The phylum has traditionally been divided into three classes, Archiacanthocephala, Palaeacanthocephala, and Eoacanthocephala; a fourth class, Polyacanthocephala, has been recently proposed. However, erection of this new class, based on morphological characters, has been controversial. We sequenced the near complete 18S rRNA gene of Polyacanthorhynchus caballeroi (Polyacanthocephala) and Rhadinorhynchus sp. (Palaeacanthocephala); these sequences were aligned with another 21 sequences of acanthocephalans representing the three widely recognized classes of the phylum and with 16 sequences from outgroup taxa. Phylogenetic relationships inferred by maximum-likelihood and maximum-parsimony analyses showed Archiacanthocephala as the most basal group within the phylum, whereas classes Polyacanthocephala + Eoacanthocephala formed a monophyletic clade, with Palaeacanthocephala as its sister group. These results are consistent with the view of Polyacanthocephala representing an independent class within Acanthocephala.     

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.     

The partial mitochondrial genome of the Cephalothrix rufifrons (Nemertea, Palaeonemertea): characterization and implications for the phylogenetic position of Nemertea

A continuous 10.1kb fragment of the Cephalothrix rufifrons (Nemertea, Palaeonemertea) mitochondrial genome was sequenced and characterized to further assess organization of protostome mitochondrial genomes and evaluate the phylogenetic potential of gene arrangement and amino acid characters. The genome is A-T rich (72%), and this biased base composition is partly reflected in codon usage. Inferred tRNA secondary structures are typical of those reported for other metazoan mitochondrial DNAs. The arrangement of the 26 genes contained in the fragment exhibits marked similarity to those of many protostome taxa, most notably molluscs with highly conserved arrangements and a phoronid. Separate and simultaneous phylogenetic analyses of inferred amino acid sequences and gene adjacencies place the nemertean within the protostomes among coelomate lophotrochozoan taxa, but do not find a well-supported sister taxon link.     

Two ancient bacterial endosymbionts have coevolved with the planthoppers (Insecta: Hemiptera: Fulgoroidea)

Background

Pseudoscorpions are chelicerates and have historically been viewed as being most closely related to solifuges, harvestmen, and scorpions. No mitochondrial genomes of pseudoscorpions have been published, but the mitochondrial genomes of some lineages of Chelicerata possess unusual features, including short rRNA genes and tRNA genes that lack sequence to encode arms of the canonical cloverleaf-shaped tRNA. Additionally, some chelicerates possess an atypical guanine-thymine nucleotide bias on the major coding strand of their mitochondrial genomes.

Results

We sequenced the mitochondrial genomes of two divergent taxa from the chelicerate order Pseudoscorpiones. We find that these genomes possess unusually short tRNA genes that do not encode cloverleaf-shaped tRNA structures. Indeed, in one genome, all 22 tRNA genes lack sequence to encode canonical cloverleaf structures. We also find that the large ribosomal RNA genes are substantially shorter than those of most arthropods. We inferred secondary structures of the LSU rRNAs from both pseudoscorpions, and find that they have lost multiple helices. Based on comparisons with the crystal structure of the bacterial ribosome, two of these helices were likely contact points with tRNA T-arms or D-arms as they pass through the ribosome during protein synthesis. The mitochondrial gene arrangements of both pseudoscorpions differ from the ancestral chelicerate gene arrangement. One genome is rearranged with respect to the location of protein-coding genes, the small rRNA gene, and at least 8 tRNA genes. The other genome contains 6 tRNA genes in novel locations. Most chelicerates with rearranged mitochondrial genes show a genome-wide reversal of the CA nucleotide bias typical for arthropods on their major coding strand, and instead possess a GT bias. Yet despite their extensive rearrangement, these pseudoscorpion mitochondrial genomes possess a CA bias on the major coding strand. Phylogenetic analyses of all 13 mitochondrial protein-coding gene sequences consistently yield trees that place pseudoscorpions as sister to acariform mites.

Conclusion

The well-supported phylogenetic placement of pseudoscorpions as sister to Acariformes differs from some previous analyses based on morphology. However, these two lineages share multiple molecular evolutionary traits, including substantial mitochondrial genome rearrangements, extensive nucleotide substitution, and loss of helices in their inferred tRNA and rRNA structures.     

The effect of model choice on phylogenetic inference using mitochondrial sequence data: lessons from the scorpions

Chelicerates are a diverse group of arthropods, with around 65,000 described species occupying a wide range of habitats. Many phylogenies describing the relationships between the various chelicerate orders have been proposed. While some relationships are widely accepted, others remain contentious. To increase the taxonomic sampling of species available for phylogenetic study based on mitochondrial genomes we produced the nearly complete sequence of the mitochondrial genome of the scorpion Mesobuthus gibbosus. Mitochondrial gene order in M. gibbosus largely mirrors that in Limulus polyphemus but tRNA secondary structures are truncated. A recent analysis argued that independent reversal of mitochondrial genome strand-bias in several groups of arthropods, including spiders and scorpions, could compromise phylogenetic reconstruction and proposed an evolutionary model that excludes mutational events caused by strand-bias (Neutral Transitions Excluded, NTE). An arthropod dataset of six mitochondrial genes, when analyzed under NTE, yields strong support for scorpions as sister taxon to the rest of Chelicerata. We investigated the robustness of this result by exploring the effect of adding additional chelicerate genes and taxa and comparing the phylogenies obtained under different models. We find evidence that (1) placement of scorpions arising at the base of the Chelicerata is an artifact of model mis-specification and scorpions are strongly supported as basal arachnids and (2) an expanded chelicerate dataset finds support for several proposed interordinal relationships (ticks plus mites [Acari] and spiders plus whip spiders plus whip scorpions [Araneae+Pedipalpi]). Mitochondrial sequence data are subject to systematic bias that is positively misleading for evolutionary inference and thus extreme methodological care must be taken when using them to infer phylogenies.     

First molecular data on the phylum Loricifera: an investigation into the phylogeny of ecdysozoa with emphasis on the positions of Loricifera and Priapulida

Recent progress in molecular techniques has generated a wealth of information for phylogenetic analysis. Among metazoans all but a single phylum have been incorporated into some sort of molecular analysis. However, the minute and rare species of the phylum Loricifera have remained elusive to molecular systematists. Here we report the first molecular sequence data (nearly complete 18S rRNA) for a member of the phylum Loricifera, Pliciloricus sp. from Korea. The new sequence data were analyzed together with 52 other ecdysozoan sequences, with all other phyla represented by three or more sequences. The data set was analyzed using parsimony as an optimality criterion under direct optimization as well as using a Bayesian approach. The parsimony analysis was also accompanied by a sensitivity analysis. The results of both analyses are largely congruent, finding monophyly of each ecdysozoan phylum, except for Priapulida, in which the coelomate Meiopriapulus is separate from a clade of pseudocoelomate priapulids. The data also suggest a relationship of the pseudocoelomate priapulids to kinorhynchs, and a relationship of nematodes to tardigrades. The Bayesian analysis placed the arthropods as the sister group to a clade that includes tardigrades and nematodes. However, these results were shown to be parameter dependent in the sensitivity analysis. The position of Loricifera was extremely unstable to parameter variation, and support for a relationship of loriciferans to any particular ecdysozoan phylum was not found in the data.     

Progress in nemertean biology: development and phylogeny

This paper reviews progress in developmental biology and phylogenyof the Nemertea, a common but poorly studied spiralian taxonof considerable ecological and evolutionary significance. Analysesof reproductive biology (including calcium dynamics during fertilizationand oocyte maturation), larval morphology and development anddevelopmental genetics have significantly extended our knowledgeof spiralian developmental biology. Developmental genetics studieshave in addition provided characters useful for reconstructingmetazoan phylogeny. Reinvestigation of the cell lineage of Cerebratuluslacteus using fluorescent tracers revealed that endomesodermforms from the 4d cell as in other spiralians and that ectomesodermis derived from the 3a and 3b cells as in annelids, echiuransand molluscs. Studies examining blastomere specification showthat cell fates are established precociously in direct developersand later in indirect developers. Morphological characters usedto estimate the phylogenetic position of nemerteans are criticallyre-evaluated, and cladistic analyses of morphology reveal thatconflicting hypotheses of nemertean relationships result becauseof different provisional homology statements. Analyses thatinclude disputed homology statements (1, gliointerstitial cellsystem 2, coelomic circulatory system) suggest that nemerteansform the sister taxon to the coelomate spiralian taxa ratherthan the sister taxon to Platyhelminthes. Analyses of smallsubunit rRNA (18S rDNA) sequences alone or in combination withmorphological characters support the inclusion of the nemerteansin a spiralian coelomate clade nested within a more inclusivelophotrochozoan clade. Ongoing evaluation of nemertean relationshipswith mitochondrial gene rearrangements and other molecular charactersis discussed.     

Phylogenetic position of the Tardigrada based on the 18S ribosomal RNA gene sequences

The phylogenetic position of the Tardigrada remains uncertain. This is due to the limited information available, and the uncertainty of whether some characters are homologous or analogous with other taxa. Based on some morphological characters, current discussion centres on whether the taxon branches from the annelid-arthropod lineage, or lies within the arthropod complex. The molecular data presented here from an analysis of the 18S rRNA gene sequences are used to test the validity of these two hypotheses. Phylogenetic inference by the maximum parsimony and distance (neighbour-joining) methods suggests that the Tardigrada is a sister group of the major protostome eucoelomate assemblage that emerged before the arthropods, annelids, molluscs, and sipunculids evolved. The tardigrade clade also appears as an independent lineage separate from the nematode clade, thus supporting the current idea that tardigrades do not have a close aschelminth relationship. The molecular data also imply that several morphological features, considered significant in determining the phylogenetic relationships of tardigrades, are not synapomorphic characters.