N. B. Petrov

PHYLOGENY OF NEMATODA AND CEPHALORHYNCHA DERIVED FROM 18S RDNA

Abstract. 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.

Molecular Phylogeny of Gastrotricha on the Basis of a Comparison of the 18S rRNA Genes: Rejection of the Hypothesis of a Relationship between Gastrotricha and Nematoda

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.

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

Phylogenetic relationships within the group of molting protostomes were reconstructed by comparing the sets of 18S and 28S rRNA gene sequences considered either separately or in combination. The reliability of reconstructions was estimated from the bootstrap indices for major phylogenetic tree nodes and from the degree of congruence of phylogenetic trees obtained by different methods. By either criterion, the phylogenetic trees reconstructed on the basis of both 18 and 28S rRNA gene sequences were better than those based on the 18S or 28S sequences alone. The results of reconstruction are consistent with the phylogenetic hypothesis classifying protostomes into two major clades: molting Ecdysozoa (Priapulida + Kinorhyncha, Nematoda + Nematomorpha, Onychophora + Tardigrada, Myriapoda + Chelicerata, and Crustacea + Hexapoda) and nonmolting Lophotrochozoa (Plathelminthes, Nemertini, Annelida, Mollusca, Echiura, and Sipuncula). Nematomorphs (Nematomorpha) do not belong to the clade Cephalorhyncha (Priapulida + Kinorhyncha). It is concluded that combined data on the 18S and 28S rRNA gene sequences provide a more reliable basis for phylogenetic inferences.

Analysis of 18S rRNA gene sequences suggests significant molecular differences between Macrodasyida and Chaetonotida (Gastrotricha).

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 "Linh Duong" Pseudonovibos spiralis (Mammalia, Artiodactyla) is a new buffalo

Abstract. 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.

Secondary structure of some elements of 18S rRNA suggests that strongylid and a part of rhabditid nematodes are monophyletic

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.

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.

Trefusiidae Are a Subtaxon of Marine Enoplida (Nematoda): Evidence from Primary Structure of Hairpin 35 and 48 Loops of SSU rRNA Gene

A rare nucleotide substitution was found in the evolutionarily conserved loop of hairpin 35 of the 18S rRNA gene of marine free-living nematode, Trefusia zostericola(Nematoda: Enoplida). The same substitution was found in all the marine Enoplida studied but not in other nematodes. Such a molecular synapomorphy indicates that marine enoplids are more closely related to T. zostericolathan to freshwater Triplonchida. Maximum parsimony, neighbor-joining, and maximum likelihood analyses of complete nucleotide sequences of the gene, with the heterogeneity of nucleotide sites in evolution rates taken into account, support this conclusion. Hence, the hypothesis of particular primitiveness of Trefusiidae among nematodes should be rejected. Phylogenies based on molecular data support the morphological reduction of metanemes in Trefusiidae. Alongside with the unique change in hairpin 35 loop among marine Enoplida (including T. zostericola), hairpin 48 is also modified by a rare transversion which could be found among Mesorhabditoidea nematodes, in related genera Pelodera, Mesorhabditis, Teratorhabditis, Parasitorhabditis, Crustorhabditis, and Distolabrellus, and in 11 orders of Rhodophyta. Rare mutations in hairpins 35 and 48 tend to be fixed correlatively in evolution and could be found in all the Acanthocephala species. X-Ray data show that these regions (H31 and H43, in alternative nomenclature) are spatially brought together in native ribosomes. The nature and distribution of molecular autoapomorphies in phylogenetic trees of high-rank taxa are discussed.

Do we need many genes for phylogenetic inference

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.

Testing of Microsatellite Primers with Different Populations of Eurasian Spruces Picea abies (L.) Karst. and Picea obovata Ledeb.

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 Fst 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.