Harvestmen in the first twenty years: a scientometric analysis of Zootaxa's contribution to opilionology (Arthropoda, Arachnida, Opiliones).

In its first twenty years of existence Zootaxa has been widely utilized among researchers of Opiliones, mainly those coming from Latin American countries, principally Brazil. During 20032020, a total of 141 papers on Opiliones were published in Zootaxa (no papers were published on Opiliones in the first two years, 20012002). The journal has greatly facilitated the dissemination of knowledge on Opiliones, especially with respect to the taxonomy and systematics of harvestmen from the Neotropical Realm. Those 141 papers in Zootaxa include almost a quarter (191) of the new species of Opiliones described between 2001 and 2020, as well as 112 new synonymies. Additionally, 27 of those papers proposed 182 new or restored combinations. A total of 108 authors working in 25 countries have contributed papers to Zootaxa focusing on Opiliones. There is clearly a predominance for collaborative contributions with more than twice as many papers authored by two or more authors compared to single-author publications. In general, the majority of papers deal within the local biogeographic realm (where the lead author resides). Studies of harvestmen from seven of the eight biogeographic realms were published in Zootaxa during 20032020. The largest portion of these contributions were by far focused on the Neotropical opiliofauna, but Australasian, Nearctic, Indomalayan, Palearctic, Afrotropical and Antarctic opiliofauna are covered as well. No papers on Opiliones have been published in Zootaxa by authors representing countries in Africa. We also recognize a strong gender bias in authorship and the current composition of Opiliones subject editors. We will strive to create an inclusive environment and aim to promote diversity of scientists who study Opiliones.

On the problematic placement of the fossil arthropod Devonopilio hutchinsoni in Opiliones (Arachnida).

Harvestmen (Arachnida: Opiliones) were among the earliest terrestrial arthropods but their unmineralized exoskeletons are scarce and often fragmentary as fossils (Palencia et al. 2019). Consequently, the discovery and interpretations of fossil harvestmen from the early Palaeozoic can have disproportionate effects on the understanding of evolution in Opiliones. Recently, Devonopilio hutchinsoni Tihelka, Tian Cai, 2020, was described as a new fossil harvestman from the well-known Rhynie chert deposits of Scotland, an important source of information on freshwater and terrestrial ecosystems of the early Devonian (Tihelka et al. 2020). This species would be one of the earliest records of harvestmen. The description was based on a single slide showing fragments of arthropod cuticle, which Tihelka et al. interpreted as a harvestmen penis and unspecified associated body parts. As specialists on harvestman morphology and systematics, we were intrigued by these conclusions. However, based on the material presented by Tihelka et al. we find no compelling evidence supporting the proposal that the specimen is a harvestman.

Opiliones of Canada.

The taxonomic diversity of the Opiliones fauna of Canada is reviewed and summarised. At present, 36 native and seven non-native species have been documented in Canada using traditional morphological taxonomy, although more than 20 species may remain undiscovered based on species diversity in the adjacent United States and evidence from DNA barcoding. Consequently, the native fauna is yet to be fully explored and the number, distribution and ecological effects of non-native species remain unclear. Until the 1960s, work on the Canadian Opiliones fauna was largely conducted by researchers based outside the country. From that time on, several Canadian workers became active. However, these taxonomists have now retired and no one has assumed their role. Thus, there is a need to invigorate taxonomic research on the harvestmen of Canada and for the production of easy-to-use identification tools for use by non-taxonomists.

A guide to the identification of the terrestrial Isopoda of Maryland, U.S.A. (Crustacea).

The terrestrial isopod fauna of Maryland is inferred using the taxonomic literature, internet-based citizen science projects, and original collecting. Twenty-two species are either known or are likely to occur in the state. This includes 17 mostly-European adventive species that comprise the vast majority of records. Of the five expected native species, three occur in or near marine or estuarine littoral habitats and each has yet to be recorded or recorded from only a single locality. This situation likely reflects the long history of systematic work on the European fauna and the availability of keys for the identification of these taxa, which contrasts with the limited taxonomic work on native species. A taxonomic key, illustrations, and brief descriptions of species known or expected to occur in Maryland are provided.

A new species of Leiobunum from Arizona, U. S. A. highlights the limits of typological classification in harvestmen (Opiliones: Sclerosomatidae: Leiobuninae).

A new species of leiobunine harvestman from the Chiricahua Mountains of Arizona is described. The species lacks pro- and retrolateral submarginal rows of coxal denticles, a feature often considered diagnostic for the polyphyletic Nelima, and has greatly reduced ventral dentition on the palpal claw, as in the monotypic Leuronychus. In most other respects, the species is uniquely similar to members of a clade from central and western Mexico currently in the poly- and/or paraphyletic Leiobunum. These traits include a supracheliceral lamina with a wide transverse plate and a canaliculate ocularium, with an anterior surface that slopes dorsoposteriorly and a posterior surface that bulges rearward and is constricted at its base.  There is thus a conflict between classification using traditional diagnostic characters and classification using unique similarity of non-traditional characters. The problem is exacerbated by the problematic status of each candidate genus. Here the species is placed in Leiobunum as L. silum sp. nov., a decision that gives weight to probable phylogenetic affinity with species currently placed in that genus. Leiobunum silum provides an excellent example of the limits of traditional typological classification and the need for a broad-scale morphological and molecular revision of sclerosomatid harvestmen.

Biomechanical Diversity of Mating Structures among Harvestmen Species Is Consistent with a Spectrum of Precopulatory Strategies.

Diversity in reproductive structures is frequently explained by selection acting at individual to generational timescales, but interspecific differences predicted by such models (e.g., female choice or sexual conflict) are often untestable in a phylogenetic framework. An alternative approach focuses on clade- or function-specific hypotheses that predict evolutionary patterns in terms neutral to specific modes of sexual selection. Here we test a hypothesis that diversity of reproductive structures in leiobunine harvestmen (daddy longlegs) of eastern North America reflects two sexually coevolved but non-overlapping precopulatory strategies, a primitive solicitous strategy (females enticed by penis-associated nuptial gifts), and a multiply derived antagonistic strategy (penis exerts mechanical force against armature of the female pregenital opening). Predictions of sexual coevolution and fidelity to precopulatory categories were tested using 10 continuously varying functional traits from 28 species. Multivariate analyses corroborated sexual coevolution but failed to partition species by precopulatory strategy, with multiple methods placing species along a spectrum of mechanical antagonistic potential. These findings suggest that precopulatory features within species reflect different co-occurring levels of solicitation and antagonism, and that gradualistic evolutionary pathways exist between extreme strategies. The ability to quantify antagonistic potential of precopulatory structures invites comparison with ecological variables that may promote evolutionary shifts in precopulatory strategies.

Comparative analyses of reproductive structures in harvestmen (opiliones) reveal multiple transitions from courtship to precopulatory antagonism.

Explaining the rapid, species-specific diversification of reproductive structures and behaviors is a long-standing goal of evolutionary biology, with recent research tending to attribute reproductive phenotypes to the evolutionary mechanisms of female mate choice or intersexual conflict. Progress in understanding these and other possible mechanisms depends, in part, on reconstructing the direction, frequency and relative timing of phenotypic evolution of male and female structures in species-rich clades. Here we examine evolution of reproductive structures in the leiobunine harvestmen or "daddy long-legs" of eastern North America, a monophyletic group that includes species in which males court females using nuptial gifts and other species that are equipped for apparent precopulatory antagonism (i.e., males with long, hardened penes and females with sclerotized pregenital barriers). We used parsimony- and Bayesian likelihood-based analyses to reconstruct character evolution in categorical reproductive traits and found that losses of ancestral gift-bearing penile sacs are strongly associated with gains of female pregenital barriers. In most cases, both events occur on the same internal branch of the phylogeny. These coevolutionary changes occurred at least four times, resulting in clade-specific designs in the penis and pregenital barrier. The discovery of convergent origins and/or enhancements of apparent precopulatory antagonism among closely related species offers an unusual opportunity to investigate how major changes in reproductive morphology have occurred. We propose new hypotheses that attribute these enhancements to changes in ecology or life history that reduce the duration of breeding seasons, an association that is consistent with female choice, sexual conflict, and/or an alternative evolutionary mechanism.

Phylogenomic resolution of paleozoic divergences in harvestmen (Arachnida, Opiliones) via analysis of next-generation transcriptome data.

Next-generation sequencing technologies are rapidly transforming molecular systematic studies of non-model animal taxa. The arachnid order Opiliones (commonly known as "harvestmen") includes more than 6,400 described species placed into four well-supported lineages (suborders). Fossil plus molecular clock evidence indicates that these lineages were diverging in the late Silurian to mid-Carboniferous, with some fossil harvestmen representing the earliest known land animals. Perhaps because of this ancient divergence, phylogenetic resolution of subordinal interrelationships within Opiliones has been difficult. We present the first phylogenomics analysis for harvestmen, derived from comparative RNA-Seq data for eight species representing all suborders. Over 30 gigabases of original Illumina short-read data were used in de novo assemblies, resulting in 50-80,000 transcripts per taxon. Transcripts were compared to published scorpion and tick genomics data, and a stringent filtering process was used to identify over 350 putatively single-copy, orthologous protein-coding genes shared among taxa. Phylogenetic analyses using various partitioning strategies, data coding schemes, and analytical methods overwhelmingly support the "classical" hypothesis of Opiliones relationships, including the higher-level clades Palpatores and Phalangida. Relaxed molecular clock analyses using multiple alternative fossil calibration strategies corroborate ancient divergences within Opiliones that are possibly deeper than the recorded fossil record indicates. The assembled data matrices, comprising genes that are conserved, highly expressed, and varying in length and phylogenetic informativeness, represent an important resource for future molecular systematic studies of Opiliones and other arachnid groups.

Molecular phylogeny of the leiobunine harvestmen of eastern North America (Opiliones: Sclerosomatidae: Leiobuninae).

Phylogenetic relationships among the leiobunine harvestmen or "daddy-longlegs" of eastern North America (Leiobunum, Hadrobunus, Eumesosoma) are poorly known, and systematic knowledge of the group has been limited largely to species descriptions and proposed species groups. Here we obtained mitochondrial (NADH1, 16S and 12S rDNA) and nuclear (28S rDNA, EF-1α introns and exons) DNA sequences from representatives of each genus, virtually all Leiobunum species from the USA and Canada, four western North American outgroup species and the distantly related Phalangium opilio. We applied bayesian, maximum-likelihood and parsimony methods under various data-partition treatments to reconstruct phylogeny and to test taxonomy-based phylogenetic hypotheses. Results were largely congruent among methods and treatments and well supported by bootstrap and posterior probability values. We recovered Leiobunum as paraphyletic with respect to Eumesosoma and Hadrobunus. Most species were encompassed by five well-supported clades that broadly correspond to groups based on male reproductive morphology (Hadrobunus group, an early-season Leiobunum group, L. vittatum group, L. politum group and L. calcar group). Relationships within species groups were often ambiguous or inconsistent with morphology, suggesting the presence of gene introgression or deep coalescence and/or the need for taxonomic revision.

Molecular systematics of sclerosomatid harvestmen (Opiliones, Phalangioidea, Sclerosomatidae): geography is better than taxonomy in predicting phylogeny.

Phylogenetic relationships within the Sclerosomatidae, the largest family of harvestmen, are explored using molecular data from four nuclear genes (28S and 18S rRNA, Histone 3 and Elongation factor-1α) and two mitochondrial gene regions (COI-COII, 16S and 12S rRNA). The taxon sample includes representative species from all families in Phalangioidea and all subfamilies of Sclerosomatidae (Gagrellinae, Gyinae, Leiobuninae, Sclerosomatinae). Our results solve several major taxonomic problems, including placement of Gyinae sensu stricto in Phalangiidae, the monophyly of the Metopilio group and its exclusion from Sclerosomatidae, and reaffirmation of the familial rank of Protolophidae. However, most major groups of sclerosomatids (Leiobuninae, Gagrellinae, Leiobunum, Nelima) are recovered as polyphyletic, although with a phylogenetic structure suggesting a strong association between geography and monophyly as well as notable morphological convergence in traditional diagnostic characters. Phylogenetic affinities between biotas of the New World and Asian tropics, as well as between temperate North American and East Asia, suggest that sclerosomatid historical biogeography may conform with the Boreotropic Concept. Finally, we discuss how the many problems that remain in sclerosomatid systematics might be addressed.

Surprising flexibility in a conserved Hox transcription factor over 550 million years of evolution.

Although metazoan body plans are remarkably diverse, the structure and function of many embryonic regulatory genes are conserved because large changes would be detrimental to development. However, the fushi tarazu (ftz) gene has changed dramatically during arthropod evolution from Hox-like to a pair-rule segmentation gene in Drosophila. Changes in both expression and protein sequence contributed to this new function: ftz expression switched from Hox-like to stripes and changes in Ftz cofactor interaction motifs led to loss of homeotic and gain of segmentation potential. Here, we reconstructed ftz changes in a rigorous phylogenetic context. We found that ftz did not simply switch from Hox-like to segmentation function; rather, ftz is remarkably labile, having undergone multiple changes in sequence and expression. The segmentation LXXLL motif was stably acquired in holometabolous insects after the appearance of striped expression in early insect lineages. The homeotic YPWM motif independently degenerated multiple times. These "degen-YPWMs" showed varying degrees of homeotic potential when expressed in Drosophila, suggesting variable loss of Hox function in different arthropods. Finally, the intensity of ftz Hox-like expression decreased to marginal levels in some crustaceans. We propose that decreased expression levels permitted ftz variants to arise and persist in populations without disadvantaging organismal development. This process, in turn, allowed evolutionary transitions in protein function, as weakly expressed "hopeful gene variants" were coopted into alternative developmental pathways. Our findings show that variation of a pleiotropic transcription factor is more extensive than previously imagined, suggesting that evolutionary plasticity may be widespread among regulatory genes.

Hydrodynamic pumping by serial gill arrays in the mayfly nymph Centroptilum triangulifer.

Aquatic nymphs of the mayfly Centroptilum triangulifer produce ventilatory flow using a serial array of seven abdominal gill pairs that operates across a Reynolds numbers (Re) range from 2 to 22 during ontogeny. Net flow in small animals is directed ventrally and essentially parallel to the stroke plane (i.e. rowing), but net flow in large animals is directed dorsally and essentially transverse to the stroke plane (i.e. flapping). Detailed flow measurements based on Particle Image Velocimetry (PIV) ensemble-correlation analysis revealed that the phasing of the gills produces a time-dependent array of vortices associated with a net ventilatory current, a fluid kinematic pattern, here termed a 'phased vortex pump'. Absolute size of vortices does not change with increasing animal size or Re, and thus the vortex radius (R(v)) decreases relative to inter-gill distance (L(is)) during mayfly growth. Given that effective flapping in appendage-array animals requires organized flow between adjacent appendages, we hypothesize that rowing should be favored when L(is)/R(v)<1 and flapping should be favored when L(is)/R(v)>1. Significantly, the rowing-to-flapping transition in Centroptilum occurs at Re∼5, when the mean dynamic inter-gill distance equals the vortex radius. This result suggests that the Re-based rowing-flapping demarcation observed in appendage-array aquatic organisms may be determined by the relative size of the propulsive mechanism and its self-generated vortices.

Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences.

The remarkable antiquity, diversity and ecological significance of arthropods have inspired numerous attempts to resolve their deep phylogenetic history, but the results of two decades of intensive molecular phylogenetics have been mixed. The discovery that terrestrial insects (Hexapoda) are more closely related to aquatic Crustacea than to the terrestrial centipedes and millipedes (Myriapoda) was an early, if exceptional, success. More typically, analyses based on limited samples of taxa and genes have generated results that are inconsistent, weakly supported and highly sensitive to analytical conditions. Here we present strongly supported results from likelihood, Bayesian and parsimony analyses of over 41 kilobases of aligned DNA sequence from 62 single-copy nuclear protein-coding genes from 75 arthropod species. These species represent every major arthropod lineage, plus five species of tardigrades and onychophorans as outgroups. Our results strongly support Pancrustacea (Hexapoda plus Crustacea) but also strongly favour the traditional morphology-based Mandibulata (Myriapoda plus Pancrustacea) over the molecule-based Paradoxopoda (Myriapoda plus Chelicerata). In addition to Hexapoda, Pancrustacea includes three major extant lineages of 'crustaceans', each spanning a significant range of morphological disparity. These are Oligostraca (ostracods, mystacocarids, branchiurans and pentastomids), Vericrustacea (malacostracans, thecostracans, copepods and branchiopods) and Xenocarida (cephalocarids and remipedes). Finally, within Pancrustacea we identify Xenocarida as the long-sought sister group to the Hexapoda, a result confirming that 'crustaceans' are not monophyletic. These results provide a statistically well-supported phylogenetic framework for the largest animal phylum and represent a step towards ending the often-heated, century-long debate on arthropod relationships.

Resolving arthropod phylogeny: exploring phylogenetic signal within 41 kb of protein-coding nuclear gene sequence.

This study attempts to resolve relationships among and within the four basal arthropod lineages (Pancrustacea, Myriapoda, Euchelicerata, Pycnogonida) and to assess the widespread expectation that remaining phylogenetic problems will yield to increasing amounts of sequence data. Sixty-eight regions of 62 protein-coding nuclear genes (approximately 41 kilobases (kb)/taxon) were sequenced for 12 taxonomically diverse arthropod taxa and a tardigrade outgroup. Parsimony, likelihood, and Bayesian analyses of total nucleotide data generally strongly supported the monophyly of each of the basal lineages represented by more than one species. Other relationships within the Arthropoda were also supported, with support levels depending on method of analysis and inclusion/exclusion of synonymous changes. Removing third codon positions, where the assumption of base compositional homogeneity was rejected, altered the results. Removing the final class of synonymous mutations--first codon positions encoding leucine and arginine, which were also compositionally heterogeneous--yielded a data set that was consistent with a hypothesis of base compositional homogeneity. Furthermore, under such a data-exclusion regime, all 68 gene regions individually were consistent with base compositional homogeneity. Restricting likelihood analyses to nonsynonymous change recovered trees with strong support for the basal lineages but not for other groups that were variably supported with more inclusive data sets. In a further effort to increase phylogenetic signal, three types of data exploration were undertaken. (1) Individual genes were ranked by their average rate of nonsynonymous change, and three rate categories were assigned--fast, intermediate, and slow. Then, bootstrap analysis of each gene was performed separately to see which taxonomic groups received strong support. Five taxonomic groups were strongly supported independently by two or more genes, and these genes mostly belonged to the slow or intermediate categories, whereas groups supported only by a single gene region tended to be from genes of the fast category, arguing that fast genes provide a less consistent signal. (2) A sensitivity analysis was performed in which increasing numbers of genes were excluded, beginning with the fastest. The number of strongly supported nodes increased up to a point and then decreased slightly. Recovery of Hexapoda required removal of fast genes. Support for Mandibulata (Pancrustacea + Myriapoda) also increased, at times to "strong" levels, with removal of the fastest genes. (3) Concordance selection was evaluated by clustering genes according to their ability to recover Pancrustacea, Euchelicerata, or Myriapoda and analyzing the three clusters separately. All clusters of genes recovered the three concordance clades but were at times inconsistent in the relationships recovered among and within these clades, a result that indicates that the a priori concordance criteria may bias phylogenetic signal in unexpected ways. In a further attempt to increase support of taxonomic relationships, sequence data from 49 additional taxa for three slow genes (i.e., EF-1 alpha, EF-2, and Pol II) were combined with the various 13-taxon data sets. The 62-taxon analyses supported the results of the 13-taxon analyses and provided increased support for additional pancrustacean clades found in an earlier analysis including only EF-1 alpha, EF-2, and Pol II.

Morphology of the prosomal endoskeleton of Scorpiones (Arachnida) and a new hypothesis for the evolution of cuticular cephalic endoskeletons in arthropods.

Skeletomuscular anatomy of the scorpion prosoma is examined in an attempt to explain the evolution of two endoskeletal features, a muscular diaphragm dividing the prosoma and opisthosoma and cuticular epistomal entapophyses with a uniquely complex arrangement of muscles, tendons and ligaments. Both structures appear to be derived from modifications of the mesodermal intersegmental endoskeleton that is primitive for all major arthropod groups. The scorpion diaphragm is a compound structure comprising axial muscles and pericardial ligaments of segments VI to VIII and extrinsic muscles of leg 4 brought into contact by longitudinal reduction of segment VII and integrated into a continuous subvertical sheet. This finding reconciles a long-standing conflict between one interpretation of opisthosomal segmentation based on scorpion embryology and another derived from comparative skeletomuscular anatomy. A new evolutionary-developmental mechanism is proposed to account for the complex morphology of the epistomal entapophyses. Each entapophysis receives 14 muscles and tendons that in other taxa would attach to the anterior connective endoskeleton in the same relative positions. This observation suggests that the embryological precursor to the connective endoskeleton can initiate and guide ectodermal invagination and thereby serve as a spatial template for the development of cuticular apodemes. This mesoderm-template model of ectodermal invagination is potentially applicable to all arthropods and may explain structural diversity and convergence in cephalic apodemes throughout the group. The model is used to interpret the cephalic endoskeletons of two non-chelicerate arthropods, Archaeognatha (Hexapoda) and Symphyla (Myriapoda), to demonstrate the generality of the model.

Mineralization of soft-part anatomy and invading microbes in the horseshoe crab Mesolimulus from the Upper Jurassic Lagerstätte of Nusplingen, Germany.

A remarkable specimen of Mesolimulus from the Upper Jurassic (Kimmeridgian) of Nusplingen, Germany, preserves the musculature of the prosoma and associated microbes in three dimensions in calcium phosphate (apatite). The musculature of Mesolimulus conforms closely to that of modern horseshoe crabs. Associated with the muscles are patches of mineralized biofilm with spiral and coccoid forms. This discovery emphasizes the potential of soft-bodied fossils as a source for increasing our knowledge of the diversity of fossil microbes in particular settings.

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.

Phylogenetic analysis of Myriapoda using three nuclear protein-coding genes.

We assessed the ability of three nuclear protein-encoding genes-elongation factor-1alpha (EF-1alpha), RNA polymerase II (Pol II), and elongation factor-2 (EF-2)-from 59 myriapod and 12 non-myriapod species to resolve phylogenetic relationships among myriapod classes and orders. In a previous study using EF-1alpha and Pol II (2134 nt combined) from 34 myriapod taxa, Regier and Shultz recovered widely accepted classes, orders, and families but failed to resolve interclass and interordinal relationships. The result was attributed to heterogenous rates of cladogenesis (specifically, the inability of the slowly evolving sequences to capture phylogenetic signal during rapid phylogenetic diversification) but the possibility of inadequate taxon sampling or limited sequence information could not be excluded. In the present study, the myriapod taxon sample was increased by 25 taxa (73%) and sequence length per taxon was effectively doubled through addition of EF-2 (4318 nt combined). Parsimony and Bayesian analyses of the expanded data set recovered a monophyletic Myriapoda, all four myriapod classes and all multiply sampled orders, often with high node support. However, except for three diplopod clades (Colobognatha, Helminothomorpha, and a subgroup of Pentazonia), few interordinal relationships and no interclass relationships were well supported. These results are similar to those of the earlier study by Regier and Shultz, which indicates that taxon sample and sequence length alone do not readily explain the weakly supported resolution in the earlier study. We review recent paleontological evidence to further develop our proposal that heterogeneity in phylogenetic signal provided by our slowly evolving sequences is due to heterogeneity in the temporal structure of myriapod diversification.

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.