[Molecular criteria in insects systematics: bar-coding gene COI range of variability as a taxonomic criterion for genus, tribe, and subfamily, with Chironominae and Orthocladiinae midges (Chironomidae, Diptera) as a case study].

Contemporary systematics of insects is based mainly on morphological traits. However, their usage is limited both by high variability and complications in comparisons of remote taxa due to low number of common traits. In whole, this leads to a somewhat subjective view when elaborating the system. Unlike morphological ones, molecular traits of taxa, revealed by use of marker genes such as gene cytochrome-c-oxidase I (COI), are less variable and more uniform, which allows them to be used as a criterion of genus, tribe, and subfamily for a wide range of organisms. Application of molecular criteria appears to be all the more important when constructing the system for groups of organisms with high morphological and specific diversity, such as midges (Chironomidae, Diptera). Last years, the DNA-sequence of gene COI is becoming widely used for species identification as a bar-coding one. Its use as a criterion for taxa of super-species level is hampered by its high nucleotide variability. We established the bounds of COI nucleotide and aminoacid divergence between midge species of Chironominae subfamily belonging to the same genus, same tribe, different tribes, as well as between species of Chironominae and Orthocladiinae subfamilies. It is shown that the level of aminoacid divergence reflects molecular boundaries of genus and tribe better than nucleotide one. It can be stated that if the level of aminoacid divergence falls within the limits from 0 to 1.7% then a pair of species compared belongs to the same genus; if it falls within the limits from 1.7 to 4.0% then they belong to the same tribe; within the limits from 4.6 to 6.3%--to different tribes; if it exceeds 7.9%--to different subfamilies. The accuracy of identification when using these ranges turns out to be not less than 75%. In this regard, bounds of COI sequence aminoacid divergence may be used as taxonomic criteria for midge genus, tribe or subfamily.

[Evolutional principles of homology in regulatory genes of myogenesis].

Analysis of early steps in muscular system development of invertebrates and vertebrates shows that early steps of myogenesis are regulated by genes-orthologs mainly belonging to two families, Pax and bHLH. In the majority of the following organisms, muscles formation (steps of determination and the earliest steps of myogenesis) is regulated by genes orthologs Pax3 which belong to the family Pax: nematodes (Caenorhabditis elegans, Pristionchus pacificus), insects (Drosophila melanogaster), echinoderms (Strongylocentrotus purpuratus), sea squirts (Ciona intestinalis, Holocynthia roretzi), fishes (Danio rerio), amphibians (Xenopus laevis), birds, and mammals (mouse, rat). The nematode C. elegans is an exception since formation of its muscles in this period is regulated by homeobox gene Pal-1 belonging to the family Caudal. The sea squirt C. intestinalis is also an exception because the earliest steps of development involved in further muscle formation are accompanied by activation of the gene CiSna (snail) (gene family basic Zinc finger). The next steps of myogenesis in all analyzed species are regulated by genes orthologs belonging to the family of transcriptional factors bHLH. They along with genes Pax3 are characterized by a high extent of homology in all studied groups of animals.

[Population structure of beaked redfish (Sebastes mentella Travin, 1951) in the Irminger Sea and adjacent waters inferred from microsatellite data].

Samples of beaked redfish from the Irminger Sea and adjacent waters were examined for polymorphism at ten microsatellite loci. The strategy of the material collection enabled investigation of geographic, bathymetric, and temporal variation of this species. The results did not support the evidence on spatial differentiation and temporal stability of the species distribution, favoring the idea that the water area examined was inhabited by a single pelagic population of beaked redfish.

[Molecular phylogeny and the time of divergence of minges (Chironomidae, Nematocera, Diptera) inferred from a partial nucleotide sequence of the cytochrome oxidase I gene (COI)].

This is the first study to infer the phylogenetic structure of minges of the subfamily Chironominae from the amino acid sequence of cytochrome oxidase I (COI). The subdivision of Chironominae into two tribes, Chironomini and Tanytarsini, has been confirmed. The segregation of the genera Pseudochironomus and Riethia into a separate tribe has not been confirmed. Stenochironomus gibbus forms a branch considerably deviating from the subfamily Chironominae. The genus Micropsectra is formed by a large polyphyletic cluster that also includes the genera Virgotanytharsus, Reotanytharsus, Kenopsectra, and Parapsectra. Tanytarsus is the basal genus of the tribe Tanytarsini. The times of divergence of the main taxa of Chironominae have been estimated. The calculated time of divergence of the genus Chironomus disproves the assumption that it is phylogenetically old.

[Comparative analysis of STR and SNP polymorphism in the populations of sockeye salmon (Oncorhynchus nerka) from Eastern and Western Kamchatka].

Sockeye salmon samples from five largest lacustrine-riverine systems of Kamchatka Peninsula were tested for polymorphism at six microsatellite (STR) and five single nucleotide polymorphism (SNP) loci. Statistically significant genetic differentiation among local populations from this part of the species range examined was demonstrated. The data presented point to pronounced genetic divergence of the populations from two geographical regions, Eastern and Western Kamchatka. For sockeye salmon, the individual identification test accuracy was higher for microsatellites compared to similar number of SNP markers. Pooling of the STR and SNP allele frequency data sets provided the highest accuracy of the individual fish population assignment.

[Intraspecific genetic polymorphism of Russian sturgeon Acipencer gueldenstaedtii].

Three populations (Azov, Caspian, and Black Sea) of Russian sturgeon Acipenser queldenstaedtii were tested for polymorphism at nuclear (RAPD and microsatellites) and mitochondrial (PCR identification of two mito-types) markers. In addition, morphometric analysis of he representatives of Azov population was carried out. According to the morphological characters, the Black Sea population occupied an intermediate position between the Caspian and Azov populations, reflecting the phylogeography of this species. In agreement with the morphometric data, genetic distances (the data of STR analysis) also placed the Black Sea population between the Caspian and Azov populations (FST = 0.058 and 0.043). The genetic distance between the Azov and Caspian population was somewhat higher (FST = 0.070). The highest allelic polymorphism at four microsatellite loci was found observed in Caspian population, while the lowest polymorphism was in the Sea of Azov. RAPD analysis distinguished high polymorphism within the populations, although it was not feasible for interpopulation analysis. Using the method differentiating the "baerii-like" and typical "gueldenstaedtii" mitotypes, the absence of the "baerii-like" marker in the Black Sea population was demonstrated. The frequency of this marker in Caspian and Azov populations constituted 31.1 and 1.8%, respectively. Possible evolutionary reasons for the interpopulation differences observed are discussed.

[Comparison of Chironomus usenicus and Chironomus curabilis with species of the group plumosus (Diptera) inferred from the mitochondrial DNA gene COI and by the polytene chromosomes banding pattern].

The sequence of a 595-bp fragment of the mitochondrial COI gene was determined for the species Chironomus usenicus and Chironomus curabilis of the genus Chironomus. Phylogenetic reconstructions based on the analysis of the COI gene sequence coincide on the whole with cytogenetic data, permitting Ch. usenicus and Ch. curabilis to be regarded as members of the group plumosus. Chironomus usenicus and Ch. plumosus have identical COI gene sequences. Two hypotheses explaining this identity are considered: inheritance of mtDNA from one of the parental species during hybridogenesis and horizontal transfer of mitochondrial genes.

[Analysis of expression of heavy myosin chains during in vitro differentiation of satellite cells and myoblasts derived from rat skeletal muscles].

Differentiation of cultured myogenic progenitor cells (satellite cells and mononucleated myoblasts) derived from hindlimb muscles of rat embryos and newborn animals was studied. Immunocytochemical methods and PCR analysis revealed expression of heavy myosin chains at the earliest stages of myogenesis (in mononucleated myoblasts). Expression of the gene encoding the embryonic form of myosin and a low level of expression of the gene encoding perinatal myosin in cultured progenitor cells derived from embryonic muscles was detected by PCR. Cells derived from muscles of newborn animals also expressed these two myosin forms, though at a lower level. The progenitor cells derived from muscles of rat embryos and newborn animals were found to express myosin 2a, which is characteristic of fast-twitch definitive muscle fibers.

[Polymorphism of the mitochondrial DNA control region in eight sturgeon species and development of a system for DNA-based species identification].

Intraspecific and interspecific nucleotide sequence variations of the mtDNA control region (D-loop) were studied with mtDNAs isolated from tissue specimens of more than 1400 sturgeons of nine species: Russian sturgeon Acipenser gueldenstaedtii, Persian sturgeon A. persicus, Siberian sturgeon A. baerii, Amur sturgeon A. schrenkii, Fringebarbel sturgeon A. nudiventris, sterlet A. ruthenus, stellate sturgeon A. stellatus, beluga Huso huso, and kaluga H. dauricus. The results were used to analyze the interspecific variation of the mtDNA control region in the given set of species and to develop a test system of ten species-specific primers, which allowed genetic identification intravital tissue specimens, spawn, and food products of eight species. The system proved suitable for multiplex PCR. A method was developed for the first time to reliably differentiate the A. baerii mitotype and the baerii-like mitotype of A. gueldenstaedtii. It was found that, although genetically separate, A. gueldenstaedtii and A. persicus are relatively young species and have common mitochondrial haplotypes, precluding their identification via mtDNA analysis alone. To develop a system for species identification of A. gueldenstaedtii and A. persicus, it is necessary to study the polymorphism of nuclear markers.

[Red king crab (Paralithodes camtschaticus) in the Barents Sea: a comparative study of introduced and native populations].

Red king crab (Paralithodes camtschaticus) was introduced into the Barents Sea in the 1960-1970s. Its present habitation area spans on the coastal zone from Hammerfest (Northern Norway) to the Barents Sea Funnel in the north-east of the Kola Peninsula. We studied the polymorphism of a mitochondrial gene encoding cytochrome oxidase (COI) and five nuclear microsatellite loci in four samples from the Barents Sea and two donor populations from the Western Kamchatka and Primorye. No decrease in the genetic diversity of the introduced populations was detected. Microsatellite assay demonstrated that the sample from Varrangerfjord was distinct from the rest five populations. However, no significant differences between the rest samples were found. Possible reasons underlying this phenomenon are discussed.

[Dynamics of mitochondrial polymorphism in a natural population of Drosophila littoralis].

During seven years, we observed stable mtDNA polymorphism in a local population of Drosophila littoralis. Using RFLP, a number of mitochondrial haplotypes were revealed, two of which were the core and in condition of stable equilibrium. To explain the absence of fixation of one haplotype, we checked a hypothesis that the D. littoralis population had a complex structure, being subdivided into several partially isolated races existing on the same territory. Analysis of highly hypervariable nuclear sequence of retrotransposons Tv1 showed positive correlation of the mitochondrial haplotype with a particular allelic form of Tv1. This supports the proposal that the D. littoralis natural population forms the population system consisting of genetically differentiated races.

[Variation of the mitochondrial DNA control region in the populations of southern form of Dolly Varden (Salvelinus malma krascheninnikovi) from Sakhalin].

Analysis of a 551-bp segment of the mitochondrial DNA control region in 23 individuals from nine populations of Dolly Varden from Sakhalin and three individuals from the Shikaribetsu Lake (Hokkaido) revealed the presence of seven haplotypes of southern form, along with one haplotype of northern form of Dolly Varden. All seven haplotypes of southern Dolly Varden were earlier described in the populations from Hokkaido. Nested analysis of molecular variance (AMOVA) based on the haplotype frequencies, performed using literature data, suggested that, during the glacial epoch, there were three regional population groups of Dolly Varden (from eastern and western coasts of Sakhalin, and from Southern Primorye). Population groups from Sakhalin and Primorye were clearly separated. The differences between two Sakhalin population groups in the mtDNA haplotype frequencies were not statistically significant. However, relative to the earlier obtained data on microsatellite loci, these differences were statistically significant. For the populations of Sakhalin Dolly Varden, the data on mitochondrial and microsatellite DNA variation supplement each other.

[Molecular phylogeny of gastrotricha based on 18S rRNA genes comparison: rejection of hypothesis of relatedness with nematodes].

Gastrotrichs are meiobenthic free-living aquatic worms whose phylogenetic and intra-group relationships remain unclear despite some attempts to resolve them on the base of morphology or molecules. In this study we analysed complete sequences of the 18S rRNA gene of 15 taxa (8 new and 7 published) to test numerous hypotheses on gastrotrich phylogeny and to verify whether controversial interrelationships from previous molecular data could be due to the short region available for analysis and the poor taxa sampling. Data were analysed using both maximum likelihood and Bayesian inference. Results obtained suggest that gastrotrichs, together with Gnathostomulida, Plathelminthes, Syndermata (Rotifera + Acanthocephala), Nemertea and Lophotrochozoa, comprise a clade Spiralia. Statistical tests reject phylogenetic hypotheses regarding Gastrotricha as close relatives of Nematoda and other Ecdysozoa or placing them at the base of bilaterian tree close to acoels and nemertodermatides. Within Gastrotricha, Chaetonotida and Macrodasyida comprise two well supported clades. Our analysis confirmed the monophyly of the Chaetonotidae and Xenotrichulidae within Chaetonida as well as Turbanellidae and Thaumastodermatidae within Macrodasyida. Mesodasys is a sister group of the Turbanellidae, and Lepidodasyidae appears to be a polyphyletic group as Cephalodasys forms a separate lineage at the base of macrodasyids, whereas Lepidodasys groups with Neodasys between Thaumastodermatidae and Turbanellidae. To infer a more reliable Gastrotricha phylogeny many species and additional genes should be involved in future analyses.

[Comparative structural analysis of myosin light chains and gene duplication in fish].

Origin and structure of myosin light chain (MLC) proteins have been studied by comparative analysis of fish mlc1, mlc2, and mlc3 genes encoding MLC1, MLC2, and MLC3, respectively. The exon-intron structure of these genes has been analyzed in zebrafish Danio rerio, loach Misgurnus fossilis, fugu Takifugu rubripes, and Nile puffer Tetraodon fahaka. We propose that mlc1 and mlc3 are homologues genes originated by fish-specific whole genome duplication (paralogs). This is supported by high sequence similarity between mlc1 and mlc3 as well as by the exon-intron structure of these genes and their localization on different chromosomes. Exons 2 to 5 of mlc1 and mlc3 are highly conserved and have similar splicing sites. A paralog gene of mlc2 resulting from a similar duplication event has been identified in zebrafish genome. Expression of mlc2 paralog is limited to the larval stages of Danio rerio and to regenerating tissues of the adult fish. There is a possibility that the paralog of mlc2 encodes larval myosin light chain protein (larval MLC) previously reported in a number of fish species.

[Ontogenetic and phylogenetic analysis of myosin light chain proteins from skeletal muscles of loach Misgurnus fossilis].

mRNAs of all three types of myosin light chain proteins are expressed in skeletal muscles of both larval and adult stages of loach Misgurnus fossilis (Cobitidae) and these proteins are encoded by different genes (mlc1, mlc2, and mlc3). No difference was revealed between transcripts from larval stage and adult fish for all three mlc proteins. Our approach (RT-PCR with fish-specific mlc1, mlc2, and mlc3 primers) failed to reveal the larval form of myosin light chain protein found previously by protein electrophoresis of loach fry muscle extract. Comparative analysis of the protein structure shows high homology of MLC1 and MLC3 proteins sharing a large EF-hand calcium-binding domain. Phylogenetic analysis of MLC1 from skeletal muscles of fish and other vertebrate species is concordant with the traditional phylogeny of the group. Within the Teleostei, loach MLC1 had the highest homology with other Cyprinidae, and least with Salmonidae fishes.

[Variation of 3'-terminal fragment of 16S rRNA gene in closely related species of Drosophila virilis group].

Primary sequence of 3-terminal fragment of the mitochondrial 16S rRNA gene has been determined in 12 Drosophila species of the virilis group. The functionally important elements in secondary structure of the RNA product were defined. The region corresponding to the peptidyltransferase center has been localized. Variation of the 3'-terminal region of 16S rRNA gene has been described in 12 species of the virilis group. Phylogeny of the Drosophila virilis species group is discussed.