Multilocus Analysis of Phylogenetic Relationships in the Crocidura suaveolens Sensu Lato Species Complex: A Comparison with Mitochondrial Data.

Multilocus analysis was for the first time used to study the phylogeny of the Crocidura suaveolens s. l. species complex. Sequencing data for 16 nuclear genes indicated that several distinct forms exist within the species complex. The structure of the complex did generally not contradict its mitochondrial phylogeny. Siberian shrew showed certain specificity of the nuclear genome, but the degree of its genetic differentiation did not correspond to the species level. Relationships of Crocidura aff. suaveolens from South Gansu and Sichuan with other forms of the species complex were clarified. Shrews from Buryatia and Khentei also belong to this form, but their mtDNA apparently introgressed from C. shantungensis in the past. Hybridization of C. suaveolens s. str. with C. aff. suaveolens and C. güeldenstaedtii occurred recently. Due to multiple introgression events in the history of C. suaveolens s. l., a far larger set of loci is necessary for the analysis of the phylogenetic relationships between its forms.

Phylogeny of the Dipus sagitta Species Complex by Nuclear Gene Sequences.

The northern three-toed jerboa Dipus sagitta had long been considered to be a single polytypic species. High genetic diversity of D. sagitta was earlier revealed on the basis of several mitochondrial and nuclear genes, and several separate species were hypothesized to occur within the taxon. However, the relationships between phylogenetic lineages have not been established because of the small sample size of nuclear genes. In the present work, a far larger set of nuclear DNA loci was used, and thus, a higher resolution of the phylogenetic tree was achieved for ten D. sagitta forms. The structure revealed for the species mainly confirmed the topology and relationships of the mtDNA lineages. Yet the mitochondrial and nuclear phylogenies were not completely consistent. Some of the D. sagitta genetic lineages were therefore assumed to be a product of reticular evolutionary processes. The taxon was concluded to be the diverse species complex D. sagitta sensu lato, in which long-diverged lineages are not always reproductively isolated.

Phylogeography of the Steppe Bat, Myotis davidii (Chiroptera; Vespertilionidae) in Eastern Part of the Species Range.

Phylogeography of the steppe bat, Myotis davidii in the eastern part of its broad range, was explored for the first time using mitochondrial genetic markers. The presence of two main intraspecific clades, Eastern and Western, was confirmed. Definite inner structure inside the Eastern group was shown. We discovered genetic diversity hotspot in northwestern Mongolia and neighboring regions, where highly divergent haplotypes are found. Presumably, this can be explained by Pleistocene refugial structure shaped by the ridges of the Mongolian Altai. The haplogroups from the southeast of Mongolia and Transbaikalia found to be related, while populations of the Kerulen valley located between these regions carry more distant haplotypes.

An Attempt to Reconstruct the Phylogeny of the Hipposideros Leaf-nosed Bats Based on Nuclear Gene Markers.

The genus Hipposideros is one of the largest bat genera. The latest research on its Asian representatives has been done only with mitochondrial or sole nuclear markers. In the present study we compare the results of phylogenetic reconstructions, based on mitochondrial, on single nuclear genes and on multiple nuclear markers analyzed using the MRP algorithm (Matrix representation with parsimony) for representative species from all the main species groups. MRP analysis provides the most supported and well-interpretable phylogeny. Our data indicate monophyly of the large Asian leaf-nosed bats which suggests they are an independent taxon of the subgenus rank, as well as monophyly of several species complexes which are paraphyletic according to mtDNA analysis.

Is the Center of Origin of Long-tailed Hamster Cricetulus longicaudatus Milne-Edwards 1867 (Rodentia, Cricetidae) Located in Tibet?

First data on morphological and mitochondrial variation in the long-tailed hamster Cricetulus longicaudatus is presented. In contrast to genetically monomorphic populations of Mongolia and Tuva, the northeastern part of the Qinghai-Tibetian Plateau was found to harbor several divergent mtDNA lineages. This pattern suggests a recent expansion of the long-tailed hamster to the northern part of its recent range, which started from Tibet, presumably, in the late Middle Pleistocene. Several populations from the northern edge of the species range were found to be morphologically but not genetically distinct. The apparent disagreement between genetic and morphological data can be explained by rapid morphological evolution in peripheral isolates.

[Microsatellite loci variation and investigation of gene flow between two karyoforms of Cricetulus barabensis sensu lato (Rodentia, Cricetidae)].

We examine the diversity of six microsatellite loci and partial RAG1 exon of "barabensis" and "pseudogriseus" karyoforms in Cricetulus barabensis sensu lato species complex. A total of 435 specimens from 68 localities ranging from Altai to the Far East are investigated. The results of the population structure analysis (factor analysis and NJ tree based on Nei genetic distances) support subdivision into two well-differentiated clusters corresponding to the two karyoforms. These karyoforms are also well differentiated by the level of microsatellite variability. In several "barabensis" specimens, we found microsatellite alleles that are common in "pseudogriseus" populations but are otherwise absent in "barabensis." Most of these specimens originate from a single population in one of the zones of potential contact between karyoforms, Kharkhorin in Central Mongolia. These molecular results are consistent with previously published karyological data in suggesting that rare hybridization events between the two chromosomal races occur in nature.

Unexpectedly high genetic diversity of the asiatic short-tailed shrews Blarinella (Mammalia, Lipotyphla, Soricidae).

Genetic diversity of the genus Blarinella was studied using one mitochondrial and four nuclear genes as examples. The first genotyping of shrews from the southeast of Gansu province (China), which is a typical habitat of B. griselda, has been performed. Molecular genetic analysis showed that Blarinella sp. from southern Gansu stand apart among the other representatives of the genus and can be assigned to neither of the known species.

Phylogenetic position of the Gansu mole Scapanulus oweni Thomas, 1912 and the relationships between strictly fossorial tribes of the family talpidae.

The results of the first molecular study focused on the phylogenetic position of the Gansu mole, Scapanulus oweni are presented. The analysis based on sequences of the mitochondrial cytb gene and five nuclear genes supports the monophyly of the Scalopini tribe including S. oweni and shows that two highly fossorial talpid tribes, Talpini and Scalopini, are not immediate sister taxa. These results highlight the role of morphological parallelism as a potential source of conflict between molecular and morphology-based phylogenies in Talpidae.

[Differentiation of tortoises of the genera Testudo and Agrionemys (Testudinidae) based on the polymorphism of nuclear and mitochondrial markers].

Based on polymorphism of the 12S rRNA gene and RAPD markers, differentiation of 122 tortoise individuals belonging to the three species of genus Testudo (T. kleinmanni, T. marginata, and T. graeca), six subspecies of T. graeca (T. g. nikolskii, T. g. pallasi, T. g. armeniaca, T. g. zarudnyi, T. g. terrestris, T. g. ibera), and two subspecies of the Central Asian tortoise Agrionenemys horsfieldii (A. h. horsfieldii, A. h. kazakhstanica) was performed. For comparison, 32 known sequences of 12S rRNA gene (392 bp) from tortoises of the two genera inhabiting the territories of Europe, Asia, and Africa were used. In the populations of A. horsfieldii, a total of six haplotypes; including three newly described variants, were identified. In the examined tortoises of the genus Testudo, eleven 12S rRNA haplotypes were identified. One new haplotype was detected in T. kleinmanni. Among the eight subspecies of T. graeca, eight haplotypes were identified, with four newly described ones. The reported RAPD markers generally supported the reconstructions obtained with the use of the mitochondrial marker. Similarly to the 12S rRNA-based reconstructions, two independent clusters included representatives of the two genera, Agrionemys and Testudio. Among the latter, representatives of T. marginata and T. kleinmanni, as well as T. graeca, with high statistical support values, formed two reciprocally monophyletic groups. Compared to the mitochondrial markers, RAPDs more statisticallysignificantly discriminated the sample of T. g. terrestris and the four subspecies, T. g. ibera, T. g. armeniaca, T. g. pallasi, and T. g. nikolskii. In almost all cases except the representatives of T. g. ibera, the representatives of each of four subspecies formed individual subclusters. The geographical haplotype distribution patterns and possible evolutionary scenario of the origin and dispersal of tortoises of the two genera are discussed.

[Genetic diversity of Chionomys genus (Mammalia, Arvicolinae) and comparative phylogeography of snow voles].

In the present study, the genetic polymorphism of the Chionomys genus was examined based on the sequencing of the mitochondrial cytb gene and two nuclear exons, including CHR exon 10 and BRCA1 exon 11. The distinct subdivision of the genus of snow voles into five lineages, including Ch. nivalis, Ch. gud, Ch. roberti, and Ch. aff. nivalis from Turkey, as well as Ch. aff. gud from Turkey, was demonstrated. The branching order in the trees constructed based on the data for different genes was ambiguous, which was probably the consequence of recent and rapid radiation of the major lineages from a common ancestor. However, the data of the mitochondrial and nuclear gene analyses definitely indicated that the genetic and taxonomic diversity of the Chionomys genus was higher than it was expected before. The genetic divergence of some populations was so deep that they probably deserved the statuses of independent species. Despite that the range of the European snow vole Ch. nivalis is larger and more fragmented than the Gudaur vole Ch, gud, the latter species with its relatively small range, which is limited to the Caucasian and Pontic Mountains, was characterized by a similarly expressed phylogenetic structure. At the same time, Robert's vole Ch. roberti was less structured genetically than the first two species. The data obtained supported the Near Eastern, rather than the European origin of the Chionomys genus.

[Karyotypes divergence in superspecies complex Cricetulus barabensis sensu lato and their interrelationships in natural contact zones].

Interrelationships between three chromosomal forms of striped hamsters belonging to the superspecies complex Cricetulus barabensis sensu lato (Cricetidae, Rodentia), namely 'barabensis', 'pseudogriseus', and 'griseus', are examined from the standpoint of different biological/genetic species concepts. Craniometric data suggest that differences between the karyotypes are rather of subspecies level. Cytogenetic differences between the karyotypes are also not great and correspond to the level of chromosomal races within a single species. The analysis of mitochondrial gene for cytochrome b (cytb) indicates the level of differences between forms of about 2.3-4.1%. Such a level may correspond to intraspecific differences as well as to differences between allied species. Experiments on hybridization resulted in hybrid litters obtained in different mating combinations of karyotypes. Both hybrid males and females fertility was confirmed, too. In nature, there exist several zones where contact between karyotypes 'barabensis' and 'pseudogriseus' is possible. Within one of such zones, in Central Mongolia, two males with atypical karyotype were detected by use of chromosome analysis. With chromosomes number and form, this karyotype may correspond to the hybrid between second and subsequent generations. However, typing of gene cytb and the intron localized in Y-chromosome (DBY1) did not reveal any traces of recent or ancient hybridization. In the other zone where contact between the karyotypes is possible, in South Buryatia, 'barabensis' and 'pseudogriseus' populations are separated by the River Chika and the distance between them does not exceed 5 km. Nevertheless, no hamsters with non-standard karyotypes and no traces of gene flow are detected by karyological and molecular analyses of these populations. The results of Tajima's test, analysis of pairwise differences distribution and indices of nucleotide diversity obtained in the course of examining gene cytb full nucleotide sequence all suggest that both contact zones studied have formed long ago and the absence of gene flow cannot be explained by their forming in the recent past. Thus, the karyotypes studied appear to be evolutionary lines developing independently from each other.

[Molecular differentiation and taxonomy of the sunwatcher toadheaded agama species complex Phrynocephalus superspecies helioscopus (Pallas, 1771) (reptilia: agamidae)].

Lizards of the sunwatcher toad-headed agama species complex Phrynocephalus superspecies helioscopus, mostly distributed in Middle Asia and Middle East, were examined using analysis of variation at the mitochondrial cytochrome oxidase c subunit I gene fragment and fingerprint analysis of nuclear DNA (inter-SINE PCR technique). A total of 86 individual tissue samples from 53 populations, to the full extent representing different parts of the species complex range, were subjected to molecular genetic examination, and surprisingly deep differentiation was revealed. The populations analyzed split into 12 isolated phylogroups, many of which were characterized by a narrow range and genetic isolation. Monophyly of sunwatcher (Ph. helioscopus) and Persian (Ph. persicus) toad-headed agamas was confirmed. However, both of these species probably represent the species complexes. Zoogeography of Middle Asiais discussed.

Molecular systematics and evolutionary biogeography of the genus Talpa (Soricomorpha: Talpidae).

The range of the genus Talpa covers almost all Europe up to Western Asia. This genus has never been the object of comprehensive systematic studies using molecular and genetic techniques, such that the evolutionary relationships among species remain unclear. Talpa shows high levels of endemism, and the influence of past glaciation cycles on the distribution pattern of several species has been hypothesized. In this work, we assessed the molecular systematics of the genus using the mitochondrial gene cytochrome b from eight of the nine extant species of Talpa moles. Furthermore, molecular clock estimations were used to hypothesize a biogeographic scenario in concordance with fossil data. Results suggest a monophyletic origin of the genus and a common ancestor for the western European moles T. europaea, T. caeca, T. romana and T. occidentalis. The eastern species T. altaica and T. caucasica are basally divergent. The estimated ages of divergence among lineages are in accordance with a Miocene origin of the extant moles. The genus likely originated in Asia, spreading into Europe during the Pliocene. The evolution of moles appears to have been driven by changes in moisture levels that influenced extinction and speciation events during the Miocene and the Pliocene. Pleistocene climatic oscillations likely caused the range shrinkages and expansions that led to the current distribution pattern of most Talpa species.

[Phylogeny and systematics of marmots inferred from inter-SINE PCR data].

Phylogenetic and taxonomic relationships in the genus Marmota were examined using inter-SINE PCR. The primers used were complementary to the consensus sequences of two short retroposons, MIR and B1-dID. The results suggest long-term genetic isolation of Nearctic and Palearctic marmots, but do not support subgeneric subdivision because of relatively low genetic differences between the marmot groups. Confirmation was received for the isolation of bobak and camtschatica, but not the caudata intrageneric species groups. Based on comparison of the mitochondrial and nuclear genome differences, the possibility of ancient hybridization between M. menzbieri and M. caudata was recognized. Species independence of M kastschenkoi within the suggested superspecies of M. baibacina was supported.

[Supraspecies relationships in the subfamily (Rodentia, Cricetidae, Arvicolinae): unexpexted result of nuclear genes analysis].

Phylogenetic analysis of supraspecies relationships in one of the most young and species rich group of myomorph rodents - subfamily Arvicolinae was carried out on the base of two nuclear genes. Results have shown that mole-voles - Ellobiusini, steppe voles - (Lagurini) and grey voles (Arvicolini) are sister groups. This divergence is the most late, third wave of radiation within the family. The sister clade to this group is the tribe of red-back voles - Myodini (=Clethrionomini) - "second radiation". The order of divergence for earliest radiation remains still unresolved (Ondatrini, Prometheomyini, Dicrostonychini, Lemmini). New data on the close relationships of mole voles, grey voles and steppe voles are unexpected one and contradict to the conventional views. The latter ideas on the significant ancientry and separation of Ellobiusini from all other voles is based on extreme simplicity of their rooted molars and very peculiar structure of the skull and postcranial skeleton. However, many of these characters most likely indicate on significant degree of adaptation to the subterranean life and have no phylogenetic signal.

[Taxonomic position of Afghan vole (subgenus Blanfordimys) by sequencing the mitochondrial cytb gene].

It is rather difficult to construct a system of gray voles of the tribe Microtini by a set of morphological and karyological characters because form generation is mosaic at these organization levels. The sequence of the mitochondrial cytochrome b gene was used to study the phylogenetic relationships and taxonomic position of the Central Asian subgenus Blanfordimys. Afghan vole Microtus (Blanfordimys) afghanus and Bucharian vole M. (Blanfordimys) bucharensis clustered with Pamir vole M. (Neodon) juldaschi, which is conventionally assigned to another subgenus. The last two species proved to be significantly closer to each other than either of them was to M. (Blanfordimys) afghanus, which disagrees with the monophyletic origin accepted for Blanfordimys. The genetic distances between the species of the subgenus Blanfordmys and M. juldaschi were comparable with the distances between the sister subgenera Microtus s. str. and Sumeriomys or Pallasiinus and Alexandromys and with the basal divergence of supraspecific clades in the subgenus Terricola. It was assumed that a special Central Asian group of species exists within the tribe Microtini and includes species of the subgenus Blanfordimys and Ml juldaschi and that the subgenera Neodon and Blanfordimys should be revised.

[The first genetic evidence of hybridization between west European and northern white-breasted hedgehogs (Erinaceus europaeus and E. roumanicus) in Moscow Region].

The complex genetic examination of hedgehogs from the vicinity of the village of Nikolina Gora (Moscow region, Odintsovskii district) showed both Erinaceus europaeus and E. roumanicus in the sample. One of the hedgehogs was designated as E. roumanicus by the nucleotide sequence of 1 TTR intron but possessed mitochondrial DNA of E. europeus. Only one of the chromosomal pairs that differ in E. europeus and E. roumanicus was heteromorphic in this specimen. Its hybridous origin as the offspring of one or several back-crosses between F1 hybrid and E. roumanicus was suggested.

[Analysis of DNA of higher primates using inter-SINE PCR].

Two types (MIR and Alu) of short interspersed repeated DNA sequences (SINEs) were used for analysis of genetic relationships among higher primates, and for detection of polymorphism in human genomic DNA. The DNA regions located between the neighboring copies of these SINEs were amplified in polymerase chain reaction with primers complementary to the MIR and Alu consensus sequences (inter-SINE PCR). Comparison of the sets of amplified DNA fragments for different species or individuals provides evaluation of the relationships among them. Using inter-MIR PCR technique, the relationships among the higher primates of the infraorder Catarrhini reported elsewhere were confirmed, pointing to the efficiency of the method for phylogenetic studies. No human DNA polymorphism was revealed with the help of inter-MIR PCR. This polymorphism was detected by means of inter-Alu PCR, which is probably associated with the continuing amplification of Alu elements in human genome.

Molecular phylogenetics and taxonomy of dwarf hamsters Cricetulus Milne-Edwards, 1867 (Cricetidae, Rodentia): description of a new genus and reinstatement of another.

The taxonomy of the genus Cricetulus has been controversial. The phylogenetic relationships both within the genus and among Cricetulus lineages and other genera were examined using a set of five nuclear and two mitochondrial genes. The results demonstrate that Cricetulus in its current treatment is a polyphyletic assemblage because the subgenus Urocricetus is phylogenetically unrelated to all other Cricetulus and is a distant sister group to Phodopus. The grey hamster (C. migratorius) proved to be closer to Cricetus and Allocricetulus than to Cricetulus proper, which includes C. barabensis C. sokolovi and C. longicaudatus. Based on these results Urocricetus is elevated to the rank of a full genus and a new genus Nothocricetulus gen.nov. is described for the grey hamster.