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Parasitic life-strategies in the phylum Nematoda (roundworms) are remarkably diverse and intricate in terms of evolution and taxonomy. By analysing novel rDNA data obtained on rare host-associated groups with unusual biology, we reveal paraphyly of the last major taxon with uncertain higher-rank classification that united solely parasitic nematodes (Marimermithida) to show that primarily marine parasitism only emerged independently and repeatedly in a few free-living lineages. We report secondary seaward ingression of land-based parasites (Mermithida) via invading hosts in the subtidal zone to illustrate the host-borne scenario of oceanic fish and mammal colonization by primarily terrestrial parasites (Spiruria). We also present the first molecular data on marine nematodes from unicellular hosts (foraminiferan protozoans) to demonstrate the independent origins of exploitative nematode associations at a microscopic scale. We argue that, in contrast with primarily intestinal associations arising from saprotrophy and commensalism, non-intestinal host capture (colonization of host body cavity or internal organs) is likely to be a primary route of transition to truly exploitative parasitism in roundworms. Predispositions to host capture in nematode morphology, ecology and life cycles imply its evolution as part of innate pre-adaptations to crossing environmental boundaries to enable multiple successful transitions to parasitism in the phylum history.

Nematodes (roundworms) are ubiquitous animals commonly dominating in ecological communities and networks, with many parasites and pathogen vectors of great economic and medical significance. Nematode parasites are remarkably diverse in life strategies and adaptations at a great range of hosts and dimension scales, from whales to protozoan cells. Their life history is intricate and requires understanding to study the genomic, structural and ecological bases of successful transitions to parasitism. Based on analyses of rDNA for a representative sampling of host-associated and free-living groups, we dismiss the last higher-rank nematode taxon uniting solely parasitic forms (Marimermithida) and show that primarily marine parasitism emerged independently and repeatedly within only few free-living lineages. We re-evaluate the significance of some traditionally important phenotypic characters and report the phenomenon of dramatic adaptation to parasitism on very short evolutionary timescales. A cross-phylum character interpretation vindicates that non-intestinal (in-tissue or cavitary) host capture was likely a primary route of transition to truly exploitive parasitism (vs. intestinal commensalism) in roundworms, and extant nematode parasitoids (larval parasites) infesting the host body cavity or internal organs realise this primary lifestyle. Parasitism may have evolved in nematodes as part of innate pre-adaptations to crossing environmental boarders, and such transitions have been accomplished multiple times successfully in the phylum history.

Morphological–functional analysis suggests that the adaptations of sea stars of the family Porcellanasteridae to burrowing and deposit feeding are related to morphological simplification. This simplification led to reduction of the morphological structures responsible for respiration (papulae and paxillae), decrease in the number of cribriform organs, reduction of body size, and retention of juvenile characters by adults. The forced-circulation water system inside the burrow also changes. The water generated by the cribriform organs becomes upwardly directed. The adaptive strategy in the family is characterized by intensification of nonselective deposit feeding by means of active moving within the muddy sediment and removal of fecal materials from the burrow.

The phylogenetic relationships and taxonomic position of sea stars of the family Porcellanasteridae have been a subject of debate for over a hundred years. In this paper, the methods of molecular phylogenetic analysis were used to solve these issues. The nucleotide sequences of fragments of the mitochondrial gene of subunit 1 of cytochrome oxidase (COI) and those of the 16S rRNA gene were obtained for 9 and 6 species of porcellanasterids for the first time, respectively. The phylogenetic trees including these sequences indicate the nearest relationships of the family Porcellanasteridae with the families Ctenodiscidae and Goniopectinidae, thereby supporting the validity of the allocation of these three families in the suborder Cribellina.

A fragment of the mitochondrial COI gene from isolates of several echinoderm species was sequenced. The isolates were from three species of starfish from the Asteriidae family (Asterias amurensis and Aphelasterias japonica collected in the Sea of Japan and Asterias rubens collected in the White Sea) and from the sea urchin Echinocardium cordatum (family Loveniidae) collected in the Sea of Japan. Additionally, regions including internal transcribed spacers and 5.8S rRNA (ITS1 - 5.8S rDNA - ITS2) were sequenced for the three studied starfish species. Phylogenetic analysis of the obtained COI sequences together with earlier determined homologous COI sequences from Ast. forbesii, Ast. rubens, and Echinocardium laevigaster from the North Atlantic and E. cordatum from the Yellow and North Seas (GenBank) placed them into strictly conspecific clusters with high bootstrap support (99% in all cases). Only two exceptions - Ast. rubens DQ077915 sequence placed with the Ast. forbesii cluster and Aph. japonica DQ992560 sequence placed with the Ast. amurensis cluster - were likely results of species misidentification. The intraspecific polymorphism for the COI gene within the Asteriidae family varied within a range of 0.2-0.9% as estimated from the genetic distances. The corresponding intrageneric and intergeneric values were 10.4-12.1 and 21.8-29.8%, respectively. The interspecific divergence for the COI gene in the sea urchin of Echinocardium genus (family Loveniidae) was significantly higher (17.1-17.7%) than in the starfish, while intergeneric divergence (14.6-25.7%) was similar to that in asteroids. The interspecific genetic distances for the nuclear transcribed sequences (ITS1 - 5.8S rDNA - ITS2) within the Asteriidae family were lower (3.1-4.5%), and the intergeneric distances were significantly higher (32.8-35.0%), compared to the corresponding distances for the COI gene. These results suggest that the investigated molecular-genetic markers could be used for segregation and identification of echinoderm species.

The position of brachiopods in the animal system remains controversial, with morphological data often being inconsistent with the results of molecular phylogenetic analysis. In this study, we investigated the structure of the spermatozoa and determined the combined sequences of the 18S and 28S rRNA genes of the articulate brachiopod Coptothyris adamsi (Davidson, 1871). The spermatozoa of C. adamsi are similar to those of other articulate brachiopods. Two types of sperm structure can be distinguished within Brachiopoda: the first type is characteristic of the articulate brachiopods (Rhynchonelliformea), the second type, the inarticulate brachiopods (Linguliformea and Craniiformea). Rhynchonelliformea spermatozoa are similar to those of the deuterostome animals, in particular to the sperm of the Echinodermata, whereas Linguliformea and Craniiformea spermatozoa are similar to the typical sperm of the Trochozoa, such as annelids and mollusks. Molecular phylogenetic analysis has shown that the brachiopods and phoronids form two monophyletic groups within the group Brachiozoa, where phoronids are united in a separate basal clade, while brachiopods are divided into two clades.

Representatives of the subfamily Vesicomyinae (Bivalvia, Vesicomyidae) are tiny deep-sea molluscs distributed worldwide and reaching huge abundances of hundreds and thousands of specimens in trawl catches. During the German–Russian deep-sea expedition KuramBio (R/V Sonne, 2012) for the first time two vesicomyin species were collected from the abyssal plain adjacent to the Kuril–Kamchatka Trench from the depths of 4861–5787 m, Vesicomya pacifica ( Smith, 1885 ) and “Vesicomya” filatovae sp.n. Two species of vesicomyins, V. sergeevi Filatova, 1971 and V. profundi Filatova, 1971 , which were previously reported from the hadal of the Kuril–Kamchatka Trench, were not collected at the abyssal depth despite of the close geographical proximity of the sampling area to their distribution ranges. Altogether nine species of vesicomyins are recorded now from the West and Indo-West Pacific; data on distribution and morpho-anatomical characters of these species are provided. Taxonomic description of V. pacifica is revised including information on its soft part anatomy, new localities and COI sequences. For the first time for a vesicomyin bivalve molecular data is given for a species with an explicit morphological description and unambiguous taxonomic affiliation. Molecular analysis of 160 published COI sequences of vesicomyids and newly obtained molecular data on V. pacifica showed that V. pacifica and two undescribed vesicomyin species forming a monophyletic clade which exhibits sister relationships with the Pliocardiinae, the group of chemosymbiotic vesicomyids. “Vesicomya” filatovae sp.n. is provisionally assigned to the genus Vesicomya (s.l.) until additional morphological and molecular data are obtained. It differs from Vesicomya s.s. by a broader hinge margin with more radiating teeth and the presence of only one pair of demibranchs.

Many features of mitochondrial genomes of animals, such as patterns of gene arrangement, nucleotide content and substitution rate variation are extensively used in evolutionary and phylogenetic studies. Nearly 6,000 mitochondrial genomes of animals have already been sequenced, covering the majority of animal phyla. One of the groups that escaped mitogenome sequencing is phylum Kinorhyncha-an isolated taxon of microscopic worm-like ecdysozoans. The kinorhynchs are thought to be one of the early-branching lineages of Ecdysozoa, and their mitochondrial genomes may be important for resolving evolutionary relations between major animal taxa. Here we present the results of sequencing and analysis of mitochondrial genomes from two members of Kinorhyncha, Echinoderes svetlanae (Cyclorhagida) and Pycnophyes kielensis (Allomalorhagida). Their mitochondrial genomes are circular molecules approximately 15 Kbp in size. The kinorhynch mitochondrial gene sequences are highly divergent, which precludes accurate phylogenetic inference. The mitogenomes of both species encode a typical metazoan complement of 37 genes, which are all positioned on the major strand, but the gene order is distinct and unique among Ecdysozoa or animals as a whole. We predict four types of start codons for protein-coding genes in E. svetlanae and five in P. kielensis with a consensus DTD in single letter code. The mitochondrial genomes of E. svetlanae and P. kielensis encode duplicated methionine tRNA genes that display compensatory nucleotide substitutions. Two distant species of Kinorhyncha demonstrate similar patterns of gene arrangements in their mitogenomes. Both genomes have duplicated methionine tRNA genes; the duplication predates the divergence of two species. The kinorhynchs share a few features pertaining to gene order that align them with Priapulida. Gene order analysis reveals that gene arrangement specific of Priapulida may be ancestral for Scalidophora, Ecdysozoa, and even Protostomia.

Using AP-PCR, the genome of Kamchatka mykiss (Parasalmo (O.) mykiss) was examined. Polymorphic fragments, implying geographic differences among the samples, were selected, cloned, and sequenced. Based on these sequences, longer, specific SCAR primers were selected and constructed. Using the BLAST software program, the sequences were analyzed for analogy to those from the GenBank database. It seemed likely that all sequences obtained belonged to earlier unexamined repeated sequences, variable in the populations of the species of interest. A total of seven SCAR markers, characterized by population-significant variability of the DNA products in Kamchatka geographic group of rainbow trout were constructed. These markers can be used for further investigation of the species Parasalmo (O.) mykiss. The SCAR marker sequences were deposited in GenBank under the accession numbers EU805500 to EU805506.

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.

Fifty-six nuclear protein coding genes from Taxonomically Broad EST Database and other databases were selected for phylogenomic-based examination of alternative phylogenetic hypotheses concerning intergroup relationship between multicellular animals (Metazoa) and other representatives of Opisthokonta. The results of this work support sister group relationship between Metazoa and Choanoflagellata. Both of these groups form the taxon Holozoa along with the monophyletic Ichthyosporea or Mesomycetozoea (a group that includes Amoebidium parasiticum, Sphaeroforma arctica, and Capsaspora owczarzaki). These phylogenetic hypotheses receive high statistical support both when utilizing whole alignment and when only 5000 randomly selected alignment positions are used. The presented results suggest subdivision of Fungi into Eumycota and lower fungi, Chytridiomycota. The latter form a monophyletic group that comprises Chytridiales + Spizellomycetales + Blastocladiales (Batrachochytrium, Spizellomyces, Allomyces, Blastocladiella), contrary to the earlier reports based on the analysis of 18S rRNA and a limited set of protein coding genes. The phylogenetic distribution of genes coding for a ubiquitin-fused ribosomal protein S30 implies at least three independent cases of gene fusion: in the ancestors of Holozoa, in heterotrophic Heterokonta (Oomycetes and Blastocystis), and in the ancestors of Cryptophyta and Glaucophyta. Ubiquitin-like sequences fused with ribosomal protein S30 outside of Holozoa are not FUBI orthologs. Two independent events of FUBI replacement by the ubiquitin sequence were detected in the lineage of C. owczarzaki and in the monophyletic group of nematode worms Tylenchomorpha + Cephalobidae. Bursaphelenchus xylophilus (Aphelenchoidoidea) retains a state typical of the rest of the Metazoa. The data emphasize the fact that the reliability of phylogenetic reconstructions depends on the number of analyzed genes to a lesser extent than on our ability to recognize reconstruction artifacts.

Gastrotricha are the small meiobenthic acoelomate worms whose phylogenetic relationships between themselves and other invertebrates remain unclear, despite all attempts to clarify them on the basis of both morphological and molecular analyses. The complete sequences of the 18S rRNA genes (8 new and 7 known) were analyzed in 15 Gastrotricha species to test different hypotheses on the phylogeny of this taxon and to determine the reasons for the contradictions in earlier results. The data were analyzed using both maximum likelihood and Bayesian methods. Based on the results, it was assumed that gastrotrichs form a monophyletic group within the Spiralia clade, which also includes Gnathostomulida, Plathelminthes, Syndermata (Rotifera + Acanthocephala), Nemertea, and Lophotrochozoa. Statistical tests rejected a phylogenetic hypotheses considering Gastrotricha to be closely related to Nematoda and other Ecdysozoa or placing them at the base of the Bilateria tree, close to Acoela or Nemertodermatida. Among gastrotrichs, species belonging to the orders Chaetonotida and Macrodasyida form two well-supported clades. The analysis confirmed monophyly of the families Chaetonotidae and Xenotrichulidae from the order Chaetonida, as well as the families Turbanellidae and Thaumastodermatidae from the order Macrodasyida. Lepidodasyidae is a polyphyletic family, because the genus Mesodasys forms a sister group for Turbanellidae; genus Cephalodasys forms a separate branch at the base of Macrodasyida; and Lepidodasys groups with Neodasys between Thaumastodermatidae and Turbanellidae. To confirm these conclusions and to get an authentic view of the phylogeny of Gastrotricha, it is necessary to study more Gastrotricha species and to analyze some other genes.

Recent molecular phylogenetic studies have led to the erection of the phylum Amoebozoa, uniting naked and testate lobose amoebae, the mycetozoan slime moulds and amitochondriate amoeboid protists (Archamoebae). Molecular data together with ultrastructural evidence have suggested a close relationship between Mycetozoa and Archamoebae, classified together in the Conosea, which was named after the cone of microtubules that, when present, is characteristic of their kinetids. However, the relationships of conoseans to other amoebozoans remain unclear. Here, we obtained the complete small-subunit (SSU) rRNA gene sequence (2746 bp) of the enigmatic, multiflagellated protist Multicilia marina, which has formerly been classified either in a distinct phylum, Multiflagellata, or among lobose amoebae. Our study clearly shows that Multicilia marina belongs to the Amoebozoa. Phylogenetic analyses including 60 amoebozoan SSU rRNA gene sequences revealed that Multicilia marina branches at the base of the Conosea, together with another flagellated amoebozoan, Phalansterium solitarium, as well as with Gephyramoeba sp., Filamoeba nolandi and two unidentified amoebae. This is the first report showing strong support for a clade containing all flagellated amoebozoans and we discuss the position of the root of the phylum Amoebozoa in the light of this result.

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.

Molecular sampling of the taxonomic diversity of the living world is nowadays a task of paramount importance. Heliozoa represents one of the major eukaryotic taxa, which remain significantly underrepresented in molecular databases. The term Heliozoa was coined to embrace organisms with a rounded body and stiff pseudopodia. Despite evidences from ultrastructural studies, which conclusively show the polyphyly of selected heliozoan groups, contemporary morphological systems retain Heliozoa as a monophyletic taxon. From the perspective of reconstructing the true phylogeny of Eukaryota, molecular approaches to analyse relationships within this large protist group are evidently necessary. Phylogenetic analysis of our data shows that the four heliozoan taxa branch either independently or within different eukaryotic phyla. The actinophryids (Actinosphaerium, Actinophrys) appear as a lineage of stramenopiles, while the desmothoracids (Clathrulina, Hedriocystis) branch within “core Cercozoa”. The position of both groups is strongly supported in all analyses and is congruent with ultrastructure-based taxonomic revisions. The centrohelids (Chlamydaster, Heterophrys, Pterocystis, and Raphidiophrys) do not seem to be related to any particular eukaryotic phylum, in agreement with a recent molecular study. The taxopodid Sticholonche was found to branch between Polycystinea and Acantharea, two classes of radiolarians. Results obtained in this study suggest that the heliozoan body form cannot be used as a diagnostic argument to unite Heliozoa. Instead, we discriminate between the three heliomorphic taxa of independent origin, Actinophryida, Desmothoracida and Sticholonche, and propose the novel higher rank taxon Centrohelida. The term Heliozoa should thus be used solely in historical context.

The current phase of molecular phylogenetics can be named the 18S rRNA gene era, which is now approaching the end. To date, almost all phyla of metazoans and many taxa of protists are represented in databases of 18S rRNA gene sequences. The elements of the phylogenetic tree of Metazoa inferred from 18S rRNA genes are characterized by unequal validity: some of them seem to be well grounded; others are not adequately supported, and probably will be revised later. The validity of phylogenetic reconstruction is influenced by two main factors: (1) erroneous grouping of long branches that occur because of abnormally high evolution rate; (2) deficit of phylogenetically informative characters. A method for overcoming these difficulties is suggested in addition to known tools: using phylogenetic markers that are stable within individual taxa and evolve by punctuated equilibrium. These markers are least influenced by the convergence caused by a high evolution rate of the entire gene. The nature of these markers of ancient taxa, paradoxical from the perspective of neutral evolution, is discussed, as well as their importance for establishing monophyly of both new large-scale taxonomic groups of invertebrates (Bilateria + Rhombozoa + Orthonectida + Myxozoa + Cnidaria + Placozoa and Echinodermata + Hemichordata) and some major taxa of Nematoda.

The phylogenetic relationships of two Mesozoa groups were studied by the comparative analysis of complete sequences of 18S and 28S rRNA. Two groups of Mesozoa were found to form a statistically supported clade in phylogenetic trees. The results of the analysis placed Mesozoa in the Spiralia group of Lophotrochozoa and showed the tendency of Mesozoa to converge with one of the Annelida groups.

Phylogenetic relationships of nematodes, nematomorphs, kinorhynchs, priapulids, and some other major groups of invertebrates were studied by 18S rRNA gene sequencing. Kinorhynchs and priapulids form the monophyletic Cephalorhyncha clade that is the closest to the coelomate animals. When phylogenetic trees were generated by different methods, the position of nematomorphs appeared to be unstable. Inclusion of Enoplus brevis, a representative of a slowly evolving nematode lineage, in the set of analyzed species refutes the tree patterns, previously derived from molecular data, where the nematodes appear as a basal bilateral lineage. The nematodes seem to be closer to the coelomate animals than was speculated earlier. According to the results obtained, nematodes, nematomorphs, tardigrades, arthropods, and cephalorhynchs are a paraphyletic association of closely related taxa.

From a clone library containing microsatellite DNA fragments of Norway spruce, seven pairs of primers were selected. These primers were tested as markers in the genetic structure analysis of nine populations of Eurasian spruce species Picea abies (L.) Karst. and Picea obovata Ledeb. Five pairs of these primers identified polymorphic loci with allele numbers ranging from 6 to 15. In the populations examined, the observed and expected heterozygosity values assessed at five loci varied from 0.1778 to 0.6556 and from 0.7800 to 0.900, respectively. In the populations examined, the values of F st index varied from 0.0691 to 0.2551 with the mean value of 0.1318. On the dendrogram based on Nei genetic distances, the populations formed three groups: Pskov-Ciscarpathya, Komi-Tatarstan-Arkhangelsk, Kazakhstan-Karelia(natural)-Karelia(plantation)-Krasnoyarsk. Five of the primer pairs tested proved to be useful for analysis of the population genetic structure in Eurasian spruce species.

The controversial phylogenetic position of the recently described South-East Asian endemic bovid, Pseudonovibos spiralis, was evaluated on the basis of phylogenetic analyses of originally obtained nearly complete 12S mitochondrial rDNA sequences for this species and Bubalus bubalis and 26 sequences of Bovidae from the Genbank using Cervus elaphus (Cervidae) as outgroup. In most of the phylogenetic analyses performed using PAUP 4.0 (maximum parsimony, maximum likelihood and neighbour-joining), Bovidae consisted of two major clades: Bovinae including the tribes Bovini, Tragelaphini and Boselaphini, and Antilopinae + Caprinae, incorporating all other bovids. In most trees P. spiralis fell within the buffalos (subtribe Bovina) between Bubalus and Syncerus. Therefore, our phylogenetic analyses of bovid mitochondrial 12S rRNA gene sequences suggest the close relationship of this enigmatic species with the buffalos and its placement within the subtribe Bovina.

Recent molecular phylogenetic studies revealed the extraordinary diversity of single-celled eukaryotes. However, the proper assessment of this diversity and accurate reconstruction of the eukaryote phylogeny are still impeded by the lack of molecular data for some major groups of easily identifiable and cultivable protists. Among them, amoeboid eukaryotes have been notably absent from molecular phylogenies, despite their diversity, complexity, and abundance. To partly fill this phylogenetic gap, we present here combined small-subunit ribosomal RNA and actin sequence data for the three main groups of “Heliozoa” (Actinophryida, Centrohelida, and Desmothoracida), the heliozoan-like Sticholonche , and the radiolarian group Polycystinea. Phylogenetic analyses of our sequences demonstrate the polyphyly of heliozoans, which branch either as an independent eukaryotic lineage (Centrohelida), within stramenopiles (Actinophryida), or among cercozoans (Desmothoracida), in broad agreement with previous ultrastructure-based studies. Our data also provide solid evidence for the existence of the Rhizaria, an emerging supergroup of mainly amoeboid eukaryotes that includes desmothoracid heliozoans, all radiolarians, Sticholonche , and foraminiferans, as well as various filose and reticulose amoebae and some flagellates.

Partial 18S rRNA gene sequences of four macrodasyid and one chaetonotid gastrotrichs were obtained and compared with the available sequences of other gastrotrich species and representatives of various metazoan phyla. Contrary to the earlier molecular data, the gastrotrich sequences did not comprise a monophyletic group but formed two distinct clades, corresponding to the Macrodasyida and Chaetonotida, with the basal position occupied by the sequences of Tetranchyroderma sp. and Xenotrichula sp., respectively. Depending on the taxon sampling and methods of analysis, the two clades were separated by various combinations of clades Rotifera, Gnathostomulida, and Platyhelminthes, and never formed a clade with Nematoda. Thus, monophyly of the Gastrotricha is not confirmed by analysis of the presently available molecular data.

The phylogenetic position and taxonomic status of the recently described Southeast Asian endemic bovid Pseudonovibos spiralis were deduced from nearly complete 12S mitochondrial rDNA sequences of this species and Bubalus bubalis alongside 26 sequences of Bovidae from GenBank using Cervus elaphus (Cervidae) as outgroup. Maximum-likelihood analyses performed by PUZZLE and fastDNAml nested P. spiralis at the base of the subtribe buffalo Bovini, suggesting the close relationship of this enigmatic species with buffalo and enabling its distinction into the separate subtribe Pseudonovibovina.

In order to ascertain a phylogenetic position of the freshwater amitochondriate amoeboflagellate Pelomyxa palustris its small subunit (SSU) rRNA gene was amplified and sequenced. It was shown to be 3502 bp long. The predicted secondary structure of its rRNA includes at least 16 separate expansion zones located in all the variable regions (V1–V9), as well as in some conservative gene regions. Most insertions are represented by sequences of low complexity that have presumably arisen by a slippage mechanism. Relatively conservative, uniformly positioned motifs contained in regions V4 and V7, as well as in some others, made it possible to perform folding. In maximum likelihood, maximum parsimony, and neighbor-joining trees, P. palustris tends to cluster with amitochondriate and secondary lost mitochondria amoebae and amoeboflagellates Entamoeba , Endolimax nana , and Phreatamoeba balamuthi , comprising together with them and aerobic lobose amoebae Vannella , Acanthamoeba , Balamuthia , and Hartmannella a monophyletic cluster. Another pelobiont, Mastigamoeba invertens , does not belong to this cluster. No specific similarity was discovered between the SSU rRNA of P. palustris and amitochondriate taxa of ‘Archezoa’: Diplomonada, Parabasalia, Microsporidia. Pelomyxa palustris SSU rRNA does not occupy a basal position in the phylogenetic trees and could be ascribed to the so-called eukaryotic ‘crown’ group if the composition of the latter were not so sensitive to the methods of tree building. Thus, molecular and morphological data suggest that P. palustris represents a secondarily modified eukaryotic lineage.

Analysis of the secondary structure of 18S rRNA molecules in nematodes revealed some new traits in the secondary structure peculiar to their hairpin 17. Some of them are characteristic of all the nematodes, whereas others are characteristic exclusively of the order Rhabditida. The loss of a nucleotide pair in the highly conservative region of hairpin 17 distinguishes 18S rRNA of the Strongylida and some species of the Rhabditida from other nematodes and, moreover, from all other organisms. Hence, it is possible to regard the Strongylida and a part of the Rhabditida including Caenorhabditis elegans as a new monophyletic taxon.