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

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

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

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

Robust support for tardigrade clades and their ages from three protein-coding nuclear genes

Abstract. Coding sequences (5,334 nt total) from elongation factor-1α, elongation factor-2, and the largest subunit of RNA polymerase II were determined for 6 species of Tardigrada, 2 of Arthropoda, and 2 of Onychophora. Parsimony and likelihood analyses of nucleotides and amino acids yielded strong support for Tardigrada and all internal nodes (i.e., 100% bootstrap support for Tardigrada, Eutardigrada, Parachela, Hypsibiidae, and Macrobiotidae). Results are in agreement with morphology and an earlier molecular study based on analysis of 18S ribosomal sequences. Divergence times have been estimated from amino acid sequence data using an empirical Bayesian statistical approach, which does not assume a strict molecular clock. Divergence time estimates are pre-Vendian for Tardigrada/Arthropoda, Vendian or earlier for Eutardigrada/Heterotardigrada, Silurian to Ordovician for Parachela/Apochela, Permian to Carboniferous for Hypsibiidae and Macrobiotidae, and Mesozoic for Isohypsibius/Thulinia (both within Hypsibiidae) and Macrobiotus/Richtersius (both within Macrobiotidae).     

Genome size and chromosome number in velvet worms (Onychophora)

The Onychophora (velvet worms) represents a small group of invertebrates (~180 valid species), which is commonly united with Tardigrada and Arthropoda in a clade called Panarthropoda. As with the majority of invertebrate taxa, genome size data are very limited for the Onychophora, with only one previously published estimate. Here we use both flow cytometry and Feulgen image analysis densitometry to provide genome size estimates for seven species of velvet worms from both major subgroups, Peripatidae and Peripatopsidae, along with karyotype data for each species. Genome sizes in these species range from roughly 5–19 pg, with densitometric estimates being slightly larger than those obtained by flow cytometry for all species. Chromosome numbers range from 2n = 8 to 2n = 54. No relationship is evident between genome size, chromosome number, or reproductive mode. Various avenues for future genomic research are presented based on these results.     

Analysis of expressed sequence tags from the fungus Aspergillus oryzae cultured under different conditions.

We performed random sequencing of cDNAs from nine biologically or industrially important cultures of the industrially valuable fungus Aspergillus oryzae to obtain expressed sequence tags (ESTs). Consequently, 21 446 raw ESTs were accumulated and subsequently assembled to 7589 non-redundant consensus sequences (contigs). Among all contigs, 5491 (72.4%) were derived from only a particular culture. These included 4735 (62.4%) singletons, i.e. lone ESTs overlapping with no others. These data showed that consideration of culture grown under various conditions as cDNA sources enabled efficient collection of ESTs. BLAST searches against the public databases showed that 2953 (38.9%) of the EST contigs showed significant similarities to deposited sequences with known functions, 793 (10.5%) were similar to hypothetical proteins, and the remaining 3843 (50.6%) showed no significant similarity to sequences in the databases. Culture-specific contigs were extracted on the basis of the EST frequency normalized by the total number for each culture condition. In addition, contig sequences were compared with sequence sets in eukaryotic orthologous groups (KOGs), and classified into the KOG functional categories.     

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

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

Eimeria tenella: identification of secretory and surface proteins from expressed sequence tags

To identify new vaccine candidates, Eimeria tenella expressed sequence tags (ESTs) from public databases were analysed for secretory molecules with an especially developed automated in silico strategy termed DNAsignalP. A total of 12,187 ESTs were clustered into 2881 contigs followed by a blastx search, which resulted in a significant number of E. tenella contigs with homologies to entries in public databases. Amino acid sequences of appropriate homologous proteins were analysed for the occurrence of an N-terminal signal sequence using the algorithm signalP. The resulting list of 84 entries comprised 51 contigs whose deduced proteins showed homologies to proteins of apicomplexan parasites. Based on function or localisation, we selected candidate proteins classified as (i) secreted proteins of Apicomplexa parasites, (ii) secreted enzymes, and (iii) transport and signalling proteins. To verify our strategy experimentally, we used a functional complementation system in yeast. For five selected candidate proteins we found that these were indeed secreted. Our approach thus represents an efficient method to identify secretory and surface proteins out of EST databases.     

The clade Ecdysozoa, perplexities and questions

A relatively new clade, the Ecdysozoa [Aguinaldo et al., 1997. Nature 387, 489-493] was raised based on the 18S ribosomal DNA sequences that indicate a close relationship between the moulting phyla (Arthropoda, Tardigrada, Onychophora, Nematoda, Nematomorpha, Kinorhyncha, Lorificera and Priapula), from which the Annelida, with other phyla, are excluded.However, the authors here expressed puzzlement about this conclusion. In particular they stressed that: (a) ecdysis might not be an autapomorphy for the Ecdysozoa; (b) some Ecdysozoa phyla are unrelated from one another with regard to morphology and embryogeny; (c) the annelids have a body architecture that is more similar to arthropods than some of the Ecdysozoa; (d) the annelids are moulting animals; (e) some phyla excluded from the new clade (e.g. the gastrotrichs), probably carry out a gradual ecdysis by flaking similar to that of the polychaetes.The authors concluded that the clade Ecdysozoa appears to be phylogenetically unconvincing.     

Growth patterns in Onychophora (velvet worms): lack of a localised posterior proliferation zone

Background  

During embryonic development of segmented animals, body segments are thought to arise from the so-called "posterior growth zone" and the occurrence of this "zone" has been used to support the homology of segmentation between arthropods, annelids, and vertebrates. However, the term "posterior growth zone" is used ambiguously in the literature, mostly referring to a region of increased proliferation at the posterior end of the embryo. To determine whether such a localised posterior proliferation zone is an ancestral feature of Panarthropoda (Onychophora + Tardigrada + Arthropoda), we examined cell division patterns in embryos of Onychophora.     

Immunolocalization of serotonin in Onychophora argues against segmental ganglia being an ancestral feature of arthropods

Background  

Onychophora (velvet worms) represent the most basal arthropod group and play a pivotal role in the current discussion on the evolution of nervous systems and segmentation in arthropods. Although there is a wealth of information on the immunolocalization of serotonin (5-hydroxytryptamine, 5-HT) in various euarthropods, as yet no comparable localization data are available for Onychophora. In order to understand how the onychophoran nervous system compares to that of other arthropods, we studied the distribution of serotonin-like immunoreactive neurons and histological characteristics of ventral nerve cords in Metaperipatus blainvillei (Onychophora, Peripatopsidae) and Epiperipatus biolleyi (Onychophora, Peripatidae).     

Phylogeny of the Metazoa Based on Morphological and 18S Ribosomal DNA Evidence

Cladistic analysis of traditional (i.e. morphological, developmental, ultrastructural) and molecular (18S rDNA) data sets (276+501 informative characters) provides a hypothesis about relationships of all meta-zoan higher taxa. Monophyly of Metazoa, Epith-eliozoa (= -03non-Porifera), Triploblastica, Mesozoa, Eutriploblastica (=Rhabditophora+Catenulida+“higher triploblasts”=Neotriploblastica, including Xeno- turbellida and Gnathostomulida), Rhabditophora, Syndermata (=“Rotifera”+Acanthocephala), Neotrichozoa (=Gastrotricha+Gnathostomulida), Nematozoa (=Nematoda+Nematomorpha), Panarthropoda (=Onychophora+Tardigrada+ Arthropoda), Cephalorhyncha, Deuterostomia, Ambulacralia (=Hemichordata+Echinodermata), Chordata, Phoronozoa (=Phoronida+“Brachiopoda”), Bryozoa, Trochozoa (=Eutrochozoa+Entoprocta+ Cycliophora), Eutrochozoa, and Chaetifera (=Annelida+ Pogonophora+Echiura) is strongly supported. Cnidaria (including Myxozoa), Ecdysozoa (=Cepha- lorhyncha + Nematozoa + Chaetognatha + Panarthropoda), Eucoelomata (=Bryozoa+Phoronozoa+Deuterostomia+Trochozoa, possibly including also Xenoturbellida), and Deuterostomia+Phoronozoa probably are monophyletic. Most traditional “phyla” are monophyletic, except for Porifera, Cnidaria (excluding Myxozoa), Platyhelminthes, Brachiopoda, and Rotifera. Three “hot” regions of the tree remain quite unresolved: basal Epitheliozoa, basal Triploblastica, and basal Neotriploblastica. A new phylogenetic classification of the Metazoa including 35 formally recognized phyla (Silicispongea, Calcispongea, Placozoa, Cnidaria, Ctenophora, Acoela, Nemertodermatida, Orthonecta, Rhombozoa, Rhabditophora, Catenulida, Syndermata, Gnathostomulida, Gastrotricha, Cephalorhyncha, Chaetognatha, Nematoda, Nematomorpha, Onychophora, Tardigrada, Arthropoda, Echinodermata, Hemichordata, Chordata, Phoronozoa, Bryozoa s. str., Xenoturbellida, Entoprocta, Cycliophora, Nemertea, Mollusca, Sipuncula, Echiura, Pogonophora, and Annelida) and few i ncertae sedis g roups (e.g. Myzostomida and Lobatocerebromorpha) is proposed.     

Arginine kinase from the Tardigrade, Macrobiotus occidentalis: molecular cloning, phylogenetic analysis and enzymatic properties

Arginine kinase (AK), which catalyzes the reversible transfer of phosphate from ATP to arginine to yield phosphoarginine and ADP, is widely distributed throughout the invertebrates. We determined the cDNA sequence of AK from the tardigrade (water bear) Macrobiotus occidentalis, cloned the sequence into pET30b plasmid, and expressed it in Escherichia coli as a 6x His-tag—fused protein. The cDNA is 1377 bp, has an open reading frame of 1080 bp, and has 5′- and 3′-untranslated regions of 116 and 297 bp, respectively. The open reading frame encodes a 359-amino acid protein containing the 12 residues considered necessary for substrate binding in Limulus AK. This is the first AK sequence from a tardigrade. From fragmented and non-annotated sequences available from DNA databases, we assembled 46 complete AK sequences: 26 from arthropods (including 19 from Insecta), 11 from nematodes, 4 from mollusks, 2 from cnidarians and 2 from onychophorans. No onychophoran sequences have been reported previously. The phylogenetic trees of 104 AKs indicated clearly that Macrobiotus AK (from the phylum Tardigrada) shows close affinity with Epiperipatus and Euperipatoides AKs (from the phylum Onychophora), and therefore forms a sister group with the arthropod AKs. Recombinant 6x His-tagged Macrobiotus AK was successfully expressed as a soluble protein, and the kinetic constants (K(m), K(d), V(ma) and k(cat)) were determined for the forward reaction. Comparison of these kinetic constants with those of AKs from other sources (arthropods, mollusks and nematodes) indicated that Macrobiotus AK is unique in that it has the highest values for k(cat) and K(d)K(m) (indicative of synergistic substrate binding) of all characterized AKs.     

A Review of Arthropod Phylogeny: New Data Based on Ribosomal DNA Sequences and Direct Character Optimization

Ribosomal gene sequence data are used to explore phylogenetic relationships among higher arthropod groups. Sequences of 139 taxa (23 outgroup and 116 ingroup taxa) representing all extant arthropod "classes" except Remipedia and Cephalocarida are analyzed using direct character optimization exploring six parameter sets. Parameter choice appears to be crucial to phylogenetic inference. The high level of sequence heterogeneity in the 18S rRNA gene (sequence length from 1350 to 2700 bp) makes placement of certain taxa with "unusual" sequences difficult and underscores the necessity of combining ribosomal gene data with other sources of information. Monophyly of Pycnogonida, Chelicerata, Chilopoda, Chilognatha, Malacostraca, Branchiopoda (excluding Daphnia ), and Ectognatha are among the higher groups that are supported in most of the analyses. The positions of the Pauropoda, Symphyla, Protura, Collembola, Diplura, Onychophora, Tardigrada, and Daphnia are unstable throughout the parameter space examined.     

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.      

Conservation and diversification of Msx protein in metazoan evolution

Msx (/msh) family genes encode homeodomain (HD) proteins that control ontogeny in many animal species. We compared the structures of Msx genes from a wide range of Metazoa (Porifera, Cnidaria, Nematoda, Arthropoda, Tardigrada, Platyhelminthes, Mollusca, Brachiopoda, Annelida, Echiura, Echinodermata, Hemichordata, and Chordata) to gain an understanding of the role of these genes in phylogeny. Exon-intron boundary analysis suggested that the position of the intron located N-terminally to the HDs was widely conserved in all the genes examined, including those of cnidarians. Amino acid (aa) sequence comparison revealed 3 new evolutionarily conserved domains, as well as very strong conservation of the HDs. Two of the three domains were associated with Groucho-like protein binding in both a vertebrate and a cnidarian Msx homolog, suggesting that the interaction between Groucho-like proteins and Msx proteins was established in eumetazoan ancestors. Pairwise comparison among the collected HDs and their C-flanking aa sequences revealed that the degree of sequence conservation varied depending on the animal taxa from which the sequences were derived. Highly conserved Msx genes were identified in the Vertebrata, Cephalochordata, Hemichordata, Echinodermata, Mollusca, Brachiopoda, and Anthozoa. The wide distribution of the conserved sequences in the animal phylogenetic tree suggested that metazoan ancestors had already acquired a set of conserved domains of the current Msx family genes. Interestingly, although strongly conserved sequences were recovered from the Vertebrata, Cephalochordata, and Anthozoa, the sequences from the Urochordata and Hydrozoa showed weak conservation. Because the Vertebrata-Cephalochordata-Urochordata and Anthozoa-Hydrozoa represent sister groups in the Chordata and Cnidaria, respectively, Msx sequence diversification may have occurred differentially in the course of evolution. We speculate that selective loss of the conserved domains in Msx family proteins contributed to the diversification of animal body organization.     

The NIEHS Xenopus maternal EST project: interim analysis of the first 13,879 ESTs from unfertilized eggs

The sequencing of expressed sequence tags (ESTs) from Xenopus laevis has lagged behind efforts on many other common experimental organisms and man, partly because of the pseudotetraploid nature of the Xenopus genome. Nonetheless, large collections of Xenopus ESTs would be useful in gene discovery, oligonucleotide-based knockout studies, gene chip analyses of normal and perturbed development, mapping studies in the related diploid frog X. tropicalis, and for other reasons. We have created a normalized library of cDNAs from unfertilized Xenopus eggs. These cells contain all of the information necessary for the first several cell divisions in the early embryo, as well as much of the information needed for embryonic pattern formation and cell fate determination. To date, we have successfully sequenced 13,879 ESTs out of 16,607 attempts (83.6% success rate), with an average sequence read length of 508 bp. Using a fragment assembly program, these ESTs were assembled into 8,985 'contigs' comprised of up to 11 ESTs each. When these contigs were used to search publicly available databases, 46.2% bore no relationship to protein or DNA sequences in the database at the significance level of 1e-6. Examination of a sample of 100 of the assembled contigs revealed that most ( approximately 87%) were comprised of two apparent allelic variants. Expression profiles of 16 of the most prominent contigs showed that 12 exhibited some degree of zygotic expression. These findings have implications for sequence-specific applications for Xenopus ESTs, particularly the use of allele-specific oligonucleotides for knockout studies, differential hybridization techniques such as gene chip analysis, and the establishment of accurate nomenclature and databases for this species.     

Ultrastructure, biology, and phylogenetic relationships of kinorhyncha

The article summarizes current knowledge mainly about the (functional)morphology and ultrastructure, but also about the biology, development,and evolution of the Kinorhyncha. The Kinorhyncha are microscopic,bilaterally symmetrical, exclusively free-living, benthic, marineanimals and ecologically part of the meiofauna. They occur throughoutthe world from the intertidal to the deep sea, generally insediments but sometimes associated with plants or other animals.From adult stages 141 species are known, but 38 species havebeen described from juvenile stages. The trunk is arranged into11 segments as evidenced by cuticular plates, sensory spots,setae or spines, nervous system, musculature, and subcuticularglands. The ultrastructure of several organ systems and thepostembryonic development are known for very few species. Almostno data are available about the embryology and only a singlegene has been sequenced for a single species. The phylogeneticrelationships within Kinorhyncha are unresolved. Priapulida,Loricifera, and Kinorhyncha are grouped together as Scalidophora,but arguments are found for every possible sistergroup relationshipwithin this taxon. The recently published Ecdysozoa hypothesissuggests a closer relationship of the Scalidophora, Nematoda,Nematomorpha, Tardigrada, Onychophora, and Arthropoda.