Molecular Genetic Markers of Intra- and Interspecific Divergence within Starfish and Sea Urchins (Echinodermata).

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.

[Repetitive DNA sequences as an indicator of the level of genetic isolation in fish].

Although the functional role is still unknown for most types of nuclear noncoding repetitive sequences, some of them proved to provide adequate phylogenetic and taxonomic markers for studying the genetic relationships of organisms at the species and within-species levels. Several markers were used in this work. First, microsatellite markers were used to examine populations varying in the extent of genetic subdivision in marine and anadromous fish, including the Chilean jack mackerel Trachurus murphyi, anadromous brown trout Salmo trutta, and isolated and anadromous char populations. Locus polymorphism was proportional to the gene flow between populations in all cases. Second, satellite DNA was used to study the phylogenetic relationships within the genera Salmo, Oncorhynchus, Salvelinus, and Coregonus. Genetic distances agreed well with the taxonomic relationships based on morphological traits and various biochemical markers and correlated with the evolutionary ages estimated for the groups by other markers. Third, RAPD PCR with a set of 20-mer primers was performed to study the genus Coregonus and anadromous and isolated populations and species of the genus Salvelinus. The resulting phylogenetic trees may help to resolve some disputable taxonomic issues for the groups. A comparison showed that several RAPD-detected sequences contain conserved fragments of coding sequences and polymorphic repeats (minisatellites) from intergenic regions or introns. The finding point to a nonrandom nature of repetitive DNA divergence and may reflect the evolution of the fish groups examined. Heterochromatic satellite repeats were assumed to contribute to generating a reproductive barrier.

[On the phylogenetic position of Hexapoda within the Pancrustacea].

Contemporary 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 in fact comprise a monophyletic lineage with Crustacea within a larger clade of Pancrustacea (=Tetraconata). Which crustacean group is the closest living relative of insects remains an open question. Recent phylogenetic analyses based on multiple genes suggest their sistership with "lower" crustaceans, the Branchiopoda. This relationship was often impeached to be caused by the long branch attraction artifact. We analyzed concatenated data on 77 ribosomal proteins, elongation factor 1 alpha (EF1A), initiation factor 5 alpha (alF5A) and other selected nuclear and mitochondrial proteins. Nuclear protein data supports the monophyly of Hexapoda, the clade uniting entognath and ectognath insects. Hexapoda and Branchiopoda comprise a monophyletic lineage in most analyses. Maxillopoda occupies the sister position to the Hexapoda + Branchiopoda. "Higher" crustaceans, the Malacostraca, in most reconstructions comprise a more basal lineage withinthe Pancrustacea. Molecular synapomorphies in low homoplastic regions are found for the clades Hexapoda Branchiopoda + Maxillopoda and the monophyletic Malacostraca containing the Phyllocarida. Therefore, the sistership of Hexapoda and Branchiopoda and their position within Entomostraca may in fact represent bona fide phylogenetic relationships.

[Primary structure of hairpin 35 and 48 small rRNA is proof that Trefusiidae is a subtaxon of marine Enoplida (Nemotoda)]. / Pervichnaia struktura petel' 35 i 48 maloi rRNK svidetel'stvuet, chto Trefusiidae iavliaiutsia subtaksonom morskikh Enoplida (Nematoda).

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. zostericola than 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.

The unusually long small subunit ribosomal RNA gene found in amitochondriate amoeboflagellate Pelomyxa palustris: its rRNA predicted secondary structure and phylogenetic implication.

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.

Phylogeny of Nematoda and Cephalorhyncha derived from 18S rDNA.

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.

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.

[Phylogeny of amphibia based on comparison of conserved segments of the 28S rRNA sequence]. / Filogeniia amfibii na osnovanii sravneniia konservativnykh uchastkov posledovatelnosti 28S rRNK.

The sequences of two regions flanking the 5'D1 domain of 28S rRNA of 13 vertebrate species were determined by direct rRNA sequencing through reverse transcriptase extension of DNA primers. Comparative treatment of these new data and previously reported rRNA sequences was undertaken with special reference to phylogenetic affinity of Amphibia by using some programs of Felsenstein's PHYLIP 3.3 package. The results obtained suggest that Amphibia is rather a biphyletic than a monophyletic group, and that birds and mammals are the closest relevant. These data were compared with those obtained on Vertebrata by using an analogous comparative analysis of 18S rRNA sequences.

[Repeating DNA sequences of closely related species of mosquitoes]. / Povtvoriaiushchiesiia posledovatel'nosti DNK blizkorodstvennykh vidov komarov.

Repeated DNA sequences of mosquitoes were studied by using of reassociation kinetics, molecular hybridization, restriction analysis and Southern blot-hybridization. Mosquitoes of two genera, the species of one of them being sibling species, were investigated. The content of all repeated families is the same both in sibling species and in species of different genera DNA. The percent of homologous sequences is low as compared to the high thermal stability of heterologous duplexes both in sibling species DNA and in different genera DNA. Restriction analysis of DNA and blot-hybridization with 35S repeated fraction revealed certain specific families of repeated sequences in the DNA of sibling species and of different genera of mosquitoes.

[Formation of the genome of the alligator gar Lepisosteus osseus (Ganoidomorpha) genome]. / Stroenie genoma pantsirnoi shchuki Lepisosteus osseus (kostnye ganoidy).

Genome structure of the alligator gar was studied by means of a comparison of reassociation kinetics of short and long DNA fragments, an estimation of hyperchromicity of reassociated repetitive DNA as a function of fragments length, and length estimation of S1-resistant duplexes by gel filtration. It was shown that most of the repeated sequences in the alligator gar DNA are no less than 2000 b.p. long and weakly divergent. Little or no interspersion of unique and short repeated sequences were observed in this genome. No highly divergent repeats were found in the alligator gar genome.

[Comparison of the genome of the alligator gar with the genomes of several other fish]. / Sravnenie genoma pantsirnoi shchuki s genomami nekotorykh drugikh ryb.

Hybridization of alligator gar (Lepisosteus osseus, Lepisosteiformes, Ganoidomorpha) [125I]- or [3H]DNA fractions with DNAs of more or less phylogenetically related fishes was studied. Almost all of the repeated and unique sequences of alligator gar DNA and DNA of the spotted gar (from the same genus) are highly homologous (1-2% of nucleotide substitutions). The degrees of homology between repeated and unique sequences of alligator gar DNA and DNAs of the representatives of Acipenseriformes (the same super-order Ganoidomorpha), Latimeria chalumnae (another subclass, Sarcopterygii) and a shark (another class, Chondrichthyes) are of the same order, and the levels of divergency of their DNAs sequences are similar. These results demonstrate, that the joining of Lepisosteiformes and Acipenseriformes in one and the same group of Ganoidomorpha is artificial, and that the superclass of fishes, Pisces, includes more taxons of the class rank then it has been taken in theory.

[Divergence of unique and repetitive sequences in the genomes of fish]. / Divergentsiia unikal'ykh i povtoriaiushchikhsia posledovatel' "nostei v genomakh ryb.

The sterlet (Acipenser acipenser) genome was compared with the genome of other fishes by molecular hybridization of its fractions. The genomes of all fishes of the family Acipenseridae are represented by the same sets of repetitive and single copy sequences. Single copy sequences contain 1.5-2.7% nucleotide mismatches, and repetitive sequences 0-2.6%, so the divergency rate of single copy sequences in this family is higher that of the repetitive sequences. The rates of evolution of the repetitive sequences with different intragenomic divergency are the same. The great similarity in the structure of genomes of Acipenseridae is the molecular basis for the formation of vital fertile hybrids between these fishes. The genomes of Acipenseridae contain some sequences homologous to teleostean and chondrostean fishes DNA. These sequences are 5% of single copy and 10-25% of repetitive sequences and are the most conservative sequences in the fishes genomes.

[Organization of nucleotide sequences in the genome of the sterlet Acipenser acipenser]. / Organizatsiia nukleotidnykh posledovatel' "nostei v genome sterliadi Acipenser acipenser.

Three fractions of repeated sequences with different thermal stability after reassociation and zero time and single copy fractions as well were isolated from a sterlet (acipenser acipenser) genome. Repeated sequences with different thermal stabilities are organized as homogeneous families not reassociating with one another when being repeatedly annealed. These fractions consist of the similar kinetic components; the exception is the slowly reassociating component which is found only in the low temperature fraction. The sterlet repeated sequences were also fractionated according to their length on agarose A-50 columns. The long (no less than 1350 nucleotides) sequences are heterogeneous by extent of their divergency, some part of them being highly divergent and contain sequences that are homologous to repeated sequences with low thermal stability. A suggestion is made that long and short repeated sequences in the sterlet genome are partly shared. The short (about 400-600 nucleotides) repeated sequences in the sterlet genome are interspersed within single copy ones.