Revision of the species composition of the wood mice from the genus <Emphasis Type="Italic">Sylvaemus</Emphasis> from the territory of Rostov oblast using karyological,allozyme and molecular genetic analysis

Using karyological, allozyme, and molecular genetic analysis, habitation of the four Sylvaemus wood mice species, pygmy wood mouse (S. uralensis), wood mouse (S. sylvaticus), yellow-necked mouse (S. flavicollis), and yellow-bellied mouse (S. fulvipectus) in Rostov oblast was demonstrated. Sylvaemus uralensis was distributed nearly over the whole territory of the oblast; S. sylvaticus was found in the central and western parts of the oblast on the right bank area of Don River; S. flavicollis inhabited northern and central parts of Rostov oblast on the right bank area of Don River; S. fulvipectus was found in the southern parts of the oblast, in the left bank area of Don River. Using the chromosome C-banding technique, it was demonstrated that the pygmy wood mice living in Rostov oblast in the right bank areas of Manych River and Don River in its low course, belonged to the eastern European chromosomal form of S. uralensis. The pygmy wood mice from the outskirts of the town of Salsk, the left bank area of Manych River, were probably hybrids between eastern European and southern European chromosomal forms. Based on the mitochondrial DNA cytochrome b gene fragment sequencing and chromosome C-banding, it was suggested that the wood mice inhabiting Rostov oblast belonged to the southern lineage of S. sylvaticus, living on Apennine Peninsula, Balkan Peninsula, and nearly throughout Ukraine.     

Genetic variation and differentiation of wood mice from the genus Sylvaemus inferred from sequencing of the cytochrome oxidase subunit 1 gene fragment

To ascertain intra- and interspecific differentiation patterns of some Sylvaemus wood mice species (S. uralensis, S. sylvaticus, S. ponticus, S. flavicollis, and S. fulvipectus), sequence variation of the mitochondrial cytochrome oxidase subunit I gene (COI) fragment (654 bp) was analyzed and the data obtained using several molecular genetic markers were compared. Distinct isolation of all Sylvaemus species (including closely related allopatric S. flavicollis and S. ponticus), as well as of the European and Asian races of pygmy wood mouse S. uralensis at the COI gene was demonstrated. However, genetic differences of the Sylvaemus species were 1.5 times and more higher than the distance (D) between the races of S. uralenciis. This finding provides no ample grounds to treat the latter as the independent species. The only specimen of Pamir-Alay subspecies S. uralensis pallipes examined showed closest relatedness to to the Asian race, although was rather distant from it (D = 0.038). No reliable isolation of the eastern European and southern European chromosomal forms, representing the European race of S. uralensis, as well as of their presumptive hybrids from the outskirts of the city of Sal'sk, Rostov region, at the COI gene was revealed. A hybrid origin of the populations of pygmy wood mouse from the outskirts of the Talapker railway station, Novovarshavsky district, Omsk region, was confirmed. In preliminary studies, based on karyotypic characters, these populations were diagnosed as distant hybrids of the eastern European chromosomal form and the Asian race. In yellow-necked wood mouse S. flavicollis from the territory of Russia and Ukraine, weak differentiation into northern and southern lineages (with mean genetic distance between them of 0.020) was observed. Considerably different relative genetic distances between the races of S. uralensis and the S. flavicollis--S. ponticus species pair, inferred from the mitochondrial cytochrome oxidase and cytochrome b gene data, indicated that the rates of evolution of different mitochondrial genome regions could be very different. It is suggested that transformations of the cytochrome b gene, or at least its part, were irregular in time and/or in different phyletic lineages (i.e., accelerated upon the formation of pygmy wood mouse races, and delayed upon the establishment of S. flavicollis and S. ponticus).     

On the taxonomic rank of ciscaucasicus and its relationships with the pygmy wood mouse Sylvaemus uralensis inferred from the mtDNA cytochrome b gene sequence

To specify the taxonomic rank of form ciscaucasoides (independent species Sylvaemus ciscaucasoides, or intraspecific form of pygmy wood mouse, S. uralensis), a 402-bp the mtDNA cytochrome b gene fragment (402 bp) was examined in S. ciscaucasoides individuals from six geographic localities of the Caucasus and Ciscaucasus, (Krasnodar krai and Adygeya Republic) and 17 S. uralensis individuals from seven localities of the Russian Plai (Saratov oblast, Smolensk oblast, Voronezh oblast, Tula oblast, Moscow oblast, and Tver' oblast). For comparison, the cytochrome b gene was partly sequenced in the samples of yellow necked, S. flavicollis (n = 2, Samara oblast), and Caucasian, S. ponticus (n = 6, Krasnodar krai), wood mice. One Mus musculus specimen from Western Europe, whose nucleotide sequences were deposed in the GenBank, was used as intergeneric outgroup. Phylogenetic trees for the forms examined were constructed based on the mtDNA sequence variation and using the neighbor joining and maximum parsimony methods. The network of the cytochrome b haplotypes was also constructed. The level of genetic divergence was evaluated using Kimura's two-parameter algorithm. Based on the data on the sequence variation in a 402-bp mtDNA cytochrome b gene fragment, the hypothesis on the species status of the ciscaucasicus form was. The mean intergroup distances (d) between the geographic groups of S. uralensis varied from 0.0036 to 0.0152. At the same time, the distances between the pygmy wood mice and the group of S.flavicollis-S. ponticus varies in the range from 0.0860 to 0.0935, and the level of intergeneric genetic differentiation (Sylvaemus-Mus) is higher than the latter index (d = 0.142). Ciscaucasoides should be considered as geographic substitution form of S. uralensis. Furthermore, the Caucasian populations of S. uralensis (= ciscaucasoides) were characterized by a threefold lower value of intergroup genetic divergence (d = 0.0062) than the East European populations (d= 0.0179). This finding pointed to some isolation of Caucasian populations of pygmy wood mouse and depletion of their gene pool. However other molecular genetic data (similarity of nucleotide composition and consistence of the levels of intra- and intergroup distances) suggest the absence of geographic subdivision between Caucasian and East European populations ofS. uralensis relative to the molecular marker examined.     

Comparative FISH analysis of C-positive regions of chromosomes of wood mice (Rodentia, Muridae, Sylvaemus)

The homology of DNA of C-positive centromeric regions of chromosomes in wood mice of the genus Sylvaemus (S. uralensis, S. fulvipectus, S. sylvaticus, S. flavicollis, and S. ponticus) was estimated for the first time. DNA probes were generated by microdissection from the centromeric regions of individual autosomes of each species, and their fluorescence in situ hybridization (FISH) with metaphase chromosomes of representatives of all studied wood mouse species was carried out. Unlike in the chromosomal forms and races of S. uralensis, changes in the DNA composition of the chromosomal centromeric regions in the wood mouse species of the genus Sylvaemus (including closely related S. flavicollis and S. ponticus) are both quantitative and qualitative. The patterns of FISH signals after in situ hybridization of the microdissection DNA probes with chromosomes of the species involved in the study demonstrate significant differences between C-positive regions of wood mouse chromosomes in the copy number and the level of homology of repetitive sequences as well as in the localization of homologous repetitive sequences. It was shown that C-positive regions of wood mouse chromosomes can contain both homologous and distinct sets of repetitive sequences. Regions enriched with homologous repeats were detected either directly in C-positive regions of individual chromosomes or only on the short arms of acrocentrics, or at the boundary of C-positive and C-negative regions.     

Allozyme variation of the pygmy wood mouse Sylvaemus uralensis (Rodentia, Muridae) and estimation of the divergence of its chromosome forms

The genetic divergence between the eastern European, southern European, and Asian chromosome forms of the pygmy wood mouse Sylvaemus uralensis, whose karyotypes differ from one another in the amount of pericentromeric heterochromatin, has been reevaluated using allozyme analysis. In general, Asian S. uralensis living in eastern Kazakhstan, eastern Turkmenistan (the Kugitang Ridge), and Uzbekistan are more monomorphic than European populations of this species. However, the allozyme differences between all chromosome forms of the pygmy wood mouse is comparable with the interpopulation differences within each form and are an order of magnitude smaller than those between "good" species of the genus Sylvaemus. Thus, the chromosome forms of S. uralensis cannot be considered to be separate species. The concept of races as large population groups that have not diverged enough to regard them as species but differ from one another in some genetic characters is used to describe the differentiation of S. uralensis forms more adequately. The currently available evidence suggests the existence of two S. uralensis races, the Asian and the European ones, and two chromosome forms (eastern and western) of the European race. The possible historical factors that have determined the formation of the races of the pygmy wood mouse are considered. According to the most plausible hypothesis, the shift and fragmentation of the broad-leaved forest zone during the most recent glacial period (late Pleistocene) were the crucial factors of the formation of these races, because they resulted in a prolonged isolation of the European and Asian population groups of S. uralensis from each other.     

Variability in size of the nuclear genome in pygmy wood mouse Sylvaemus uralensis (Rodentia, Muridae)

Earlier, in an integral genetic study, the Asian and European races were distinguished within the species Sylvaemus uralensis (pygmy wood mouse) and the European race was divided into the East European and South European forms. Each of these groups differed from the others, in particular, in the quantity of the centromeric heterochromatin in karyotypes of the animals. To establish the pattern of its changes in S. uralensis, in the present study the DNA content in splenocyte nuclei in all races and forms of pygmy wood mice was assessed using DNA flow cytometry. The heterochromatin amount in karyotypes and genome size were shown to be correlated. The East European chromosomal race of S. uralensis (Central Chernozem and Non-Chernozem regions of Russia, Crimea Peninsula, Middle Volga region, and Southern Ural) and the Asian race of this species (East Kazakhstan, Uzbekistan, and East Turkmenistan), which have respectively the highest and the lowest amounts of centromeric heterochromatin in the karyotype, exhibit the greatest difference in the DNA content in the genome. On average, the difference is approximately 8% in males and 6.7% in females; in both cases, the ranges of variability were distinctly different. Against the general background of the trait variation, the Asian race, whose members have the smallest DNA amount in their cells, looks homogeneous. The genome of the South European chromosomal form of S. uralensis (Caucasus, Transcaucasia, Carpathians, and Balkan Peninsula), which exhibits an intermediate content of the centromeric heterochromatin in the karyotype, is smaller that the genome of the East European race (by 3.2% in the group of males and by 1.9%, in the group of females), but larger than that of the Asian race (by 5% in either sex). Thus, the variability of size of centromeric C-blocks in pygmy wood mouse is likely to be associated with elimination (or, conversely, an increase in the amount) of the genetically inert chromatin. It is suggested that a significant contribution to the variability of genome size in S. uralensis is made by heterochromosomes, or, more precisely, their variable regions, which seem to be largely heterochromatic.     

A phylogenetic study of the genus Apodemus by sequencing the mitochondrial DNA control region

The sequences of the mitochondrial DNA control region (D-loop) and flanking tRNA genes (about 1000 bp) of 20 samples of wood mice (genus Apodemus ) were analyzed in order to clarify the relationships between different species belonging to the genus. The phylogenetic trees obtained using different methods showed similar topologies with distinct Karstomys ( Apodemus epimelas and Apodemus mystacinus ) and Sylvaemus ( Apodemus alpicola , Apodemus flavicollis , Apodemus hermonensis , Apodemus sylvaticus and Apodemus uralensis ) subtrees. Within Sylvaemus all species appeared to be closely related to each other, probably as result of a bush-like radiation event. Nevertheless, A. hermonensis seemed to be the first diverging branch followed by A. sylvaticus ; A. alpicola and A. flavicollis appeared to be very closely related. Three individuals of uncertain taxonomical status were included in the analysis: hypotheses as to their status are discussed. Further phylogenetic analysis was carried out combining the D-loop sequences of part of the samples of certain taxonomical status with 12S rRNA and cytochrome b sequences obtained by other researchers. Furthermore, I present a structure analysis of the D-loop in Apodemus as compared other rodent species.     

Genetic variation and differentiation of wood mice from the genus <Emphasis Type="Italic">Sylvaemus</Emphasis> inferred from sequencing of the cytochrome oxidase subunit 1 gene fragment

To ascertain intra- and interspecific differentiation patterns of some Sylvaemus wood mice species (S. uralensis, S. sylvaticus, S. ponticus, S. flavicollis, and S. fulvipectus), sequence variation of the fragment (654 bp) of the mitochondrial cytochrome oxidase subunit I gene (COI) was analyzed and the data obtained using several molecular genetic markers were compared. Distinct isolation of all Sylvaemus species (including closely related allopatric S. flavicollis and S. ponticus), as well as of the European and Asian races of pygmy wood mouse S. uralensis at the COI gene was demonstrated. However, genetic differences of the Sylvaemus species were 1.5 times and more higher than the distance (D) between the races of S. uralensis. This finding provides no ample grounds to treat them as the independent species. The only examined specimen of Pamir-Alay subspecies S. uralensis pallipes showed closest relatedness to to the Asian race, although was rather distant from it (D = 0.038). No reliable isolation of the eastern European and southern European chromosomal forms, representing the European race of S. uralensis, as well as of their presumptive hybrids from the out-skirts of the city of Sal’sk, Rostov region, at the COI gene was revealed. A hybrid origin of the population of pygmy wood mouse from the outskirts of the Talapker railway station, Novovarshavsky district, Omsk region, was confirmed (in preliminary studies, based on karyotypic characters, these specimens were diagnosed as distant hybrids of the eastern European chromosomal form and the Asian race). In yellow-necked wood mouse S. flavicollis from the territory of Russia and Ukraine, weak differentiation into northern and southern lineages (with mean genetic distance between them of 0.020) was observed. Considerably different relative genetic distances between the races of S. uralensis and the S. flavicollis-S. ponticus species pair, inferred from the mitochondrial cytochrome oxidase and cytochrome b genes data, indicated that the rates of evolution of the mitochondrial genome regions could be very dissimilar. It is suggested that transformations of the cytochrome b gene, or at least its part, were irregular in time and/or in different phyletic lineages (i.e., accelerated upon the formation of pygmy wood mouse races, and delayed upon the establishment of S. flavicollis and S. ponticus).     

Comparative FISH analysis of C-positive blocks of centromeric chromosomal regions of pygmy wood mice <Emphasis Type="Italic">Sylvaemus uralensis</Emphasis> (Rodentia,Muridae)

The composition and homology of centromeric heterochromatin DNA has been compared in representatives of the Asian race and two chromosomal forms (Eastern European and Southern European) of the European race of the pygmy wood mouse Sylvaemus uralensis by means of in situ hybridization with metaphase chromosomes of microdissection DNA probes obtained from centromeric C-blocks of mice of the Southern European chromosomal form and the Asian race. Joint hybridization of both DNA probes yielded all possible variants of centromeric regions in terms of the presence of repetitive sequences homologous to those of some or another dissection region, which indicates a diversity of centromeric regions differing in DNA composition. However, most variations of the fluorescent in situ hybridization (FISH) patterns are apparently related to quantitative differences of repetitive elements of the genome. Experiments with the DNA probe obtained from the genome of the Southern European form of the pygmy wood mouse have shown that the number of intense FISH signals roughly corresponds to the number of large C-segments in representatives of the European race, which is characterized by a large amount of the centromeric C-heterochromatin in the karyotype. However, intense signals have been also detected in experiments on hybridization of this probe with chromosomes of representatives of the Asian race, which has no large C-blocks in the karyotype; thus, DNA sequences homologous to heterochromatic ones are also present in nonheterochromatic regions adjacent to C-segments. Despite the variations of the numbers of both intense and weak FISH signals, all chromosomal forms/races of S. uralensis significantly differ of the samples from one another in these characters. The number of intense FISH signals in DNA in pygmy wood mice of the samples from eastern Turkmenistan (the Kugitang ridge) and southern Omsk oblast (the vicinity of the Talapker railway station) was intermediate between those in the European and Asian races, which is apparently related to a hybrid origin of these populations (the hybridization having occurred long ago in the former case and recently in the latter case).     

Allozyme Variation of the Pygmy Wood Mouse Sylvaemus uralensis (Rodentia,Muridae) and Estimation of the Divergence of Its Chromosome Forms

The genetic divergence between the eastern European, southern European, and Asian chromosome forms of the pygmy wood mouse Sylvaemus uralensis, whose karyotypes differ from one another in the amount of centromeric heterochromatin, has been reevaluated using allozyme analysis. In general, Asian chromosome forms in S. uralensis living in eastern Kazakhstan, eastern Turkmenistan (the Kugitang Ridge), and Uzbekistan are more monomorphic than European populations of this species. However, the allozyme differences between all chromosome forms of the pygmy wood mouse are comparable with the interpopulation differences within each form and are an order of magnitude smaller than those between good species of the genus Sylvaemus. Thus, the chromosome forms of S. uralensis cannot be considered to be separate species. The concept of races as large population groups that have not diverged enough to regard them as species but differ from one another in some genetic characters is used to describe the differentiation of S. uralensis forms more adequately. The currently available evidence suggests the existence of two S. uralensis races, the Asian and the European ones, and two chromosome forms (eastern and southern) of the European race. The possible historical factors that have determined the formation of the races of the pygmy wood mouse are considered. According to the most plausible hypothesis, the shift and fragmentation of the broad-leaved forest zone during the most recent glacial period (late Pleistocene) were the crucial factors of the formation of these races, because they resulted in a prolonged isolation of the European and Asian population groups ofS. uralensis from each other.     

Small mammal populations in relation to hedgerow structure in an arable landscape

The population ecology of small mammals in hedgerows in arable farmland in eastern England is described. Features of hedgerows of importance to individual species are examined. Some 97% of the total 3042 mammals captured were wood mouse Apodemus sylvaticus , yellow-necked mouse Apodemus flavicollis , bank vole Clethrionomys glareolus and common shrew Sorex araneus . Small numbers of harvest mice Micromys minutus , field voles Microtus agrestis , pygmy shrews Sorex minutus and water shrews Neomys fodiens were also caught. Wood mouse, the most numerous species, showed a typical pattern of large numbers in autumn and winter, followed by a simultaneous decline over all hedges in early spring. Population changes were less clear in yellow-necked mouse and bank vole but the yellow-necked mouse was more scarce in the second year of study. Common shrews were most numerous in summer and declined rapidly in autumn. Hedgerow coppicing had a marked effect on yellow-necked mouse numbers but not on wood mouse. In an extensive survey of mammal numbers in relation to hedgerow features, ground cover was found to be the single largest factor influencing size of bank vole populations. Hedgerow condition (lack of gaps) was important to yellow-necked mice, which thrived only in well-established hedgerows. Wood mice appeared little influenced by the characteristics of the hedge. Common shrews were more abundant in hedgerows with adjacent permanent water.     

On the taxonomic rank of <Emphasis Type="Italic">ciscaucasicus</Emphasis> and its relationships with the pygmy wood mouse <Emphasis Type="Italic">Sylvaemus uralensis</Emphasis> inferred from the mtDNA cytochrome b gene sequence

To specify the taxonomic rank of form ciscaucasicus (independent species Sylvaemus ciscaucasicus, or intraspecific form of pygmy wood mouse, S. uralensis), a 402-bp the mtDNA cytochrome b gene fragment (402 bp) was examined in ciscaucasicus individuals from six geographic localities of the Caucasus and Ciscaucasus (Krasnodar krai and Adygeya Republic) and 17 S. uralensis individuals from seven localities of the Russian Plain (Saratov oblast, Smolensk oblast, Voronezh oblast, Tula oblast, Moscow oblast, Tver’ oblast, and northern Krasnodar krai). For comparison, the cytochrome b gene was partly sequenced in the samples of yellow necked, S. flavicollis (n = 2, Samara oblast), and Caucasian, S. ponticus (n = 6, Krasnodar krai), wood mice. One Mus musculus specimen from Western Europe, whose nucleotide sequences were deposed in the GenBank, was used as intergeneric outgroup. Phylogenetic trees for the forms examined were constructed based on the mtDNA sequence variation and using the neighbor joining and maximum parsimony methods. The network of the cytochrome b haplotypes was also constructed. The level of genetic divergence was evaluated using Kimura’s two-parameter algorithm. Based on the data on the sequence variation in a 402-bp mtDNA cytochrome b gene fragment, the hypothesis on the species status of the ciscaucasicus form was. The mean intergroup distances (d) between the geographic groups of S. uralensis varied from 0.0036 to 0.0152. At the same time, the distances between the pygmy wood mice and the group of S. flavicollis-S. ponticus varies in the range from 0.0860 to 0.0935, and the level of intergeneric genetic differentiation (Sylvaemus-Mus) is higher than the latter index (d = 0.142). Ciscaucasicus should be considered as geographic substitution form of S. uralensis. Furthermore, the Caucasian populations of S. uralensis (= ciscaucasicus) were characterized by a threefold lower value of intergroup genetic divergence (d = 0.0062) than the East European populations (d = 0.0179). This finding pointed to some isolation of Caucasian populations of pygmy wood mouse and depletion of their gene pool. However other molecular genetic data (similarity of nucleotide composition and consistence of the levels of intra-and intergroup distances) suggest the absence of geographic subdivision between Caucasian and East European populations of S. uralensis relative to the molecular marker examined.     

Comparative FISH analysis of C-positive regions of chromosomes of wood mice (Rodentia,Muridae, <Emphasis Type="Italic">Sylvaemus</Emphasis>)

The homology of DNA of C-positive centromeric regions of chromosomes in wood mice of the genus Sylvaemus (S. uralensis, S. fulvipectus, S. sylvaticus, S. flavicollis, and S. ponticus) was estimated for the first time. DNA probes were generated by microdissection from the centromeric regions of individual autosomes of each species, and their fluorescence in situ hybridization (FISH) with metaphase chromosomes of representatives of all studied wood mouse species was carried out. Unlike in the chromosomal forms and races of S. uralensis, changes in the DNA composition of the chromosomal centromeric regions in the wood mouse species of the genus Sylvaemus (including closely related S. flavicollis and S. ponticus) are both quantitative and qualitative. The patterns of FISH signals after in situ hybridization of the microdissection DNA probes with chromosomes of the species involved in the study demonstrate significant differences between C-positive regions of wood mouse chromosomes in the copy number and the level of homology of repetitive sequences as well as in the localization of homologous repetitive sequences. It was shown that C-positive regions of wood mouse chromosomes can contain both homologous and distinct sets of repetitive sequences. Regions enriched with homologous repeats were detected either directly in C-positive regions of individual chromosomes or only on the short arms of acrocentrics, or at the boundary of C-positive and C-negative regions.     

Phylogeny of the genus Apodemus with a special emphasis on the subgenus Sylvaemus using the nuclear IRBP gene and two mitochondrial markers: cytochrome b and 12S rRNA

Phylogenetic relationships among 17 extant species of Murinae, with special reference to the genus Apodemus, were investigated using sequence data from the nuclear protein-coding gene IRBP (15 species) and the two mitochondrial genes cytochrome b and 12S rRNA (17 species). The analysis of the three genes does not resolve the relationships between Mus, Apodemus, and Rattus but separates Micromys from these three genera. The analysis of the two mitochondrial regions supported an association between Apodemus and Tokudaia and indicated that these two genera are more closely related to Mus than to Rattus or Micromys. Within Apodemus, the mitochondrial data sets indicated that 8 of the 9 species analyzed can be sorted into two main groups: an Apodemus group, with A. agrarius, semotus, and peninsulae, and a Sylvaemus group, with uralensis, flavicollis, alpicola, sylvaticus, and hermonensis. The position of Apodemus mystacinus is ambiguous and might be either included in Sylvaemus or considered a distinct subgenus, Karstomys, more closely related to Sylvaemus than to Apodemus. Estimation of the divergence time for these taxa suggests a separation between 7 and 8 My ago for the three groups (mystacinus and the two subgenera Apodemus and Sylvaemus). Within each subgenus, divergence times are between 5.4 and 6 My for Apodemus and between 2.2 and 3.5 My for Sylvaemus and mystacinus.     

Genetic relationships between European and Transcaucasian mice of the genus Apodemus Kaup]

Genetic relations between four European mice species of the genus Apodemus (Apodemus sylvaticus, A. flavicollis, A. microps and A. falzfeini) and five Transcaucasian ones (A. mystacinus, A. microps, and the forms No 2, 3p, 3f) were studied for 37 biochemical loci. Close genetic relations were demonstrated between the mice of subgenus Sylvaemus and A. mystacinus. A. microps and A. falzfeini from the Caucasus were shown conspecific to these species from the Ukraine and the presence of the two new different species--the form No 2 and 3p in the Caucasus was established.     

Absence of spermatozoal CD46 protein expression and associated rapid acrosome reaction rate in striped field mice (Apodemus agrarius)

Background  

In rodents, the cell surface complement regulatory protein CD46 is expressed solely on the spermatozoal acrosome membrane. Ablation of the CD46 gene is associated with a faster acrosome reaction. Sperm from Apodemus flavicollis (yellow-necked field mice), A. microps (pygmy field mice) and A. sylvaticus (European wood mice) fail to express CD46 protein and exhibit a more rapid acrosome reaction rate than Mus (house mice) or BALB/c mice. A. agrarius (striped field mice) belong to a different Apodemus subgenus and have pronounced promiscuity and large relative testis size. The aim of this study was to determine whether A. agrarius sperm fail to express CD46 protein and, if so, whether A. agrarius have a faster acrosome reaction than Mus.     

Molecular phylogeny and taxonomy of wood mice (genus Apodemus Kaup, 1829) based on complete mtDNA cytochrome b sequences, with emphasis on Chinese species

Phylogenetic relationships among 15 species of wood mice (genus Apodemus) were reconstructed to explore some long-standing taxonomic problems. The results provided support for the monophyly of the genus Apodemus, but could not reject the hypothesis of paraphyly for this genus. Our data divided the 15 species into four major groups: (1) the Sylvaemus group (A. sylvaticus, A. flavicollis, A. alpicola, and A. uralensis), (2) the Apodemus group (A. peninsulae, A. chevreri, A. agrarius, A. speciosus, A. draco, A. ilex, A. semotus, A. latronum, and A. mystacinus), (3) A. argenteus, and (4) A. gurkha. Our results also suggested that orestes should be a valid subspecies of A. draco rather than an independent species; in contrast, A. ilex from Yunnan may be regarded as a separate species rather than a synonym of orestes or draco. The species level status of A. latronum, tscherga as synonyms of A. uralensis, and A. chevrieri as a valid species and the closest sibling species of A. agrarius were further corroborated by our data. Applying a molecular clock with the divergences of Mus and Rattus set at 12 million years ago (Mya) as a calibration point, it was estimated that five old lineages (A. mystacinus and four major groups above) diverged in the late Miocene (7.82-12.74 Mya). Then the Apodemus group (excluding A. mystacinus) split into two subgroups: agrarius and draco, at about 7.17-9.95 Mya. Four species of the Sylvaemus group were estimated to diverge at about 2.92-5.21 Mya. The Hengduan Mountains Region was hypothesized to have played important roles in Apodemus evolutionary histories since the Pleistocene.     

Tick-borne zoonotic bacteria in wild and domestic small mammals in northern Spain

The prevalence and diversity of tick-borne zoonotic bacteria (Borrelia spp., Anaplasma phagocytophilum, Coxiella burnetii, and spotted fever group rickettsiae) infecting 253 small mammals captured in the Basque Country (Spain) were assessed using PCR and reverse line blot hybridization. Trapping sites were selected around sheep farms (study 1, 2000 to 2002) and recreational parks (study 2, 2003 to 2005). The majority of the studied mammals (162) were wood mice (Apodemus sylvaticus), but six other different species were also analyzed: yellow-necked mice (Apodemus flavicollis), shrews (Crocidura russula and Sorex coronatus), bank voles (Clethrionomys glareolus), domestic mice (Mus domesticus), and moles (Talpa europaea). The results showed an infection rate ranging from 10.7% to 68.8%, depending on the small mammal species. One C. russula shrew and one A. sylvaticus mouse gave positive reactions for A. phagocytophilum, and C. burnetii was detected in two domestic mice and one A. sylvaticus mouse in a farm. The DNA of Borrelia spp. was detected in 67 animals (26.5%), most of them presenting positive hybridization with the probe for Borrelia sp. strain R57, the new Borrelia species previously detected in small mammals in our region. Furthermore, a second PCR and reverse line blot hybridization specific for B. burgdorferi sensu lato revealed the presence of Borrelia afzelii in 6.3% of C. glareolus voles and 14.3% of S. coronatus shrews. All small mammals were negative for spotted fever group rickettsiae. These results highlight the relevance of small mammals as reservoirs of some zoonotic bacteria.     

Wild small mammals are the reservoir hosts of the Bartonella genus bacteria in the south of Moscow region

A total of 103 blood samples collected from wild small mammals captured in the Prioksko-Terrasny Reserve on the south of Moscow region were studied to determine the bartonellae prevalence. The examined species were the yellow-necked mice Apodemus flavicollis (35 samples), the European wood mouse Apodemus uralensis (10 samples), the bank vole Clethrionomys glareolus (51 samples), the house mouse Mus musculus (3 samples), the common vole Microtus arvalis (2 samples), and the shrew Sorex araneus (2 samples). Initially, we obtained 76 bacterial Bartonella-like isolates after plating onto the surface of the solid nutrient media. 66 of them were PCR-positive at least for three of four targets, gltA, ftsZ, ribC and 16S RNA. Thus, the percentage of the infection in the studied community was 64%. Subsequent RFLP assay showed that obtained isolates belonged to the Bartonella grahamii and/or B. taylorii species. In 7 cases we found both bartonellae species in one animal. These data were confirmed by direct sequencing of four ftsZ, four ribC and two gltA amplicons. According to our data, there is no any marked host specificity for these bartonellae species. Now we have laid the bartonellae strain collection consisting of 31 isolates. To our knowledge, this is the first investigation of the bartonellae prevalence in wild small mammals performed in Russia. The comparison of our data with those obtained by European researchers and issues of coinfection by different bartonellae species and host specificity are discussed.