A search for Y-chromosomal species-specific markers and their use for hybridization analysis in ground squirrels (Spermophilus: Rodentia, sciuridae)

In four ground squirrel species from the Volga region—yellow (Spermophilus fulvus), russet (S. major), little (S. pygmaeus), and speckled (S. suslicus)—four hybridization variants (major/fulvus, major/pygmaeus, major/suslicus, and pygmaeus/suslicus) have been reliably described. Earlier we have shown that populations of S. major from the Volga region were characterized by wide introgression of mtDNA from S. fulvus and S. pygmaeus, which probably, resulted from ancient hybridization [5]. In this study, the same populations were used to analyze the introgression of the Y chromosome, which (unlike mtDNA) is paternally inherited. Three genes, ZfY, SRY, and SmcY were tested as Y-chromosomal candidate markers. It was demonstrated that Y chromosome of ground squirrels lacked the ZfY gene, while its homologous structure, ZfY(X), was presumably linked to the X chromosome. The SRY region examined was rather conservative. In particular, the sequences determined in S. major and S. fulvus were identical, while three out of four substitutions found in S. pygmaeus were located in the coding region. The SmcY gene was found to be the most suitable marker, providing distinguishing of all of the four ground squirrel species by nine nucleotide substitutions. Introgression at the Y chromosome was observed only in two cases: in one S. major individual (out of 51 phenotypically pure animals) caught in the major/fulvus sympatry zone, and in four (one litter) out of fourteen S. fulvus individuals caught in close vicinity of the sympatry zone of these two species. Among 28 S. pymaeus and 9 S. suslicus individuals, no foreign SmcY genes were detected. Two colonies of the “hybrid swarm” type were examined with eight major/suslicus hybrids analyzed in the first and seventeen major/fulvus hybrids in the second colony. The prevalence of the S. major paternal lineages was observed in both colonies (87.5 and 82.4%, respectively). The data obtained suggest that compared to wide mtDNA introgression, introgression of Y chromosome in the Volga region ground squirrels is statistically significantly less frequent event.     

Study of hybridization in four species of ground squirrels (spermophilus: Rodentia,Sciuridae) by molecular genetic methods

Four species of ground squirrel--yellow (Spermophilus fulvus), russet (S. major), small (S. pygmaeus), and spotted (S. suslicus)--occur in the Volga region. Between S. major and S. pigmaeus, S. major and S. fulvus, and S. major and S. suslicus, sporadic hybridization was reported. Using sequencing and restriction analysis, we have examined the mtDNA C region in 13 yellow, 60 russet, 61 small, 45 spotted ground squirrels, and 9 phenotypic hybrids between these species. It was shown that 43% of S. major individuals had "alien" mitotypes typical of S. fulvus and S. pygmaeus. Alien mitotypes occurred both within and outside sympatric zones. No alien mitotypes were found in 119 animals of the other three species, which suggests that only one parental species (S. major) predominantly participates in backcrosses. Phenotypic hybrids S. fulvus x S. major and S. major x S. pygmaeus) were reliably identified using RAPD-PCR of nuclear DNA. However, we could find no significant traces of hybridization in S. major with alien mitotypes. Analysis of p53 pseudogenes of S. major and S. fulvus that were for the first time described in the present study produced similar results: 59 out of 60 individuals of S. major (including S. major with S. fulvus mitotypes) had only the pseudogene variant specific for S. major. This situation is possible even at low hybridization frequencies (less than 1% according to field observations and 1.4 to 2.7% according to nuclear DNA analysis) if dispersal of S. major from the sympatric zones mainly involved animals that obtained alien mtDNA via backcrossing. The prevalence of animals with alien mitotypes in some S. major populations can be explained by the founder effect. Further studies based on large samples are required for clarifying the discrepancies between mitochondrial and nuclear DNA data.     

Genetic evidence of extensive introgression of short-tailed ground squirrel genes in a hybridization zone of Spermophilus major and S. erythrogenys, inferred from sequencing of the mtDNA cytochrome b gene

We have completely sequenced the mtDNA cytochrome b gene of ground squirrels from the zone of overlapping ranges of Spermophilus major and S. erythrogenys in the Tobol-Ishim interfluve, which is a putative hybridization zone of these species. The results of the sequencing showed extensive introgression of mtDNA genes of the short-tailed ground squirrel S. e. brevicauda, whose haplotype had fully replaced the S. major haplotype. All of the ground squirrels from the Tobol-Ishim interfluve had a variant of the S. e. brevicauda mtDNA haplotype that was specific for this zone. On average, 119 substitutions (10.44%) were found between S. major from Ul'yanovsk oblast and S. e. brevicauda from the northern Kazakhstan, the mean genetic distance (D) between them being 0.115, which conforms to the corresponding parameters for the S. e. brevicauda-S. pygmaeus pair (122 substitutions, D = 118). Insignificant differences (seven substitutions, D = 0.043) were found between the S. major and S. pygmaeus haplotypes, which suggest that these species have similar mitochondrial haplotypes. Five to ten nucleotide substitutions (0.44--0.88%) were detected between the animals from the Tobol--Ishim interfluve and S. e. brevicauda. The mtDNA haplotype divergence D within the genus Spermophilus (ten species) for all codon positions ranged from 0.035 to 0.158. Phylogenetic reconstructions (MP, ML, and NJ trees) showed two well-differentiated clusters with high bootstrap support. However, there was different branching topology within the cluster and their species composition varied. The maximum likelihood tree, ML, differentiating the species into two subgenera, Citellus and Colobotis, most reliably reflected taxonomic relationships of the species from the genus Spermophilus, inferred from morphological and genetic biochemical data. The morphologically pure S. major (subgenus Colobotis) animals, used in the analysis, proved to carry the haplotype of another species, S. pygmaeus (subgenus Citellus). This poses a question on the existence of the specific haplotype of S. major, the reason of its replacement by haplotype of other species, and possible consequences of this phenomenon for survival of the species.     

EVOLUTIONARY RELATIONSHIPS AMONG COLONIES OF COLUMBIAN GROUND SQUIRRELS AS SHOWN BY MITOCHONDRIAL DNA

Mitochondrial DNA (mtDNA) from 71 Columbian ground squirrels (Spermophilus columbianus) collected in 12 locations in western Canada were assayed for restriction-site variation with 10 endonucleases. Five of these endonucleases revealed variant patterns, and the composite genotypes were used to develop a linear transformation series among the mtDNA genotypes. Two of the four clones had a wide distribution, while the remaining two clones were geographically restricted. The mtDNA of Columbian ground squirrels was also compared to two other species of Sciuridae: Richardson's ground squirrels (S. richardsonii) and Arctic ground squirrels (S. parryii). Calculation of divergences from fragment length and restriction-site data indicated that Arctic ground squirrels and Richardson's ground squirrels were more closely related to each other than either was to Columbian ground squirrels. The transformation series among clones within the Columbian ground squirrels was rooted using Richardson's and Arctic ground squirrels as out-groups. From these data, we conclude that the colonization by female founders of Columbian ground squirrel populations occurred after deglaciation along the eastern ranges of the Rocky Mountains, while colonies on the western ranges may have been present before extensive deglaciation occurred, having existed in refugia in northwestern Alberta.     

Implications of Hybridization,NUMTs, and Overlooked Diversity for DNA Barcoding of Eurasian Ground Squirrels

The utility of DNA Barcoding for species identification and discovery has catalyzed a concerted effort to build the global reference library; however, many animal groups of economical or conservational importance remain poorly represented. This study aims to contribute DNA barcode records for all ground squirrel species (Xerinae, Sciuridae, Rodentia) inhabiting Eurasia and to test efficiency of this approach for species discrimination. Cytochrome c oxidase subunit 1 (COI) gene sequences were obtained for 97 individuals representing 16 ground squirrel species of which 12 were correctly identified. Taxonomic allocation of some specimens within four species was complicated by geographically restricted mtDNA introgression. Exclusion of individuals with introgressed mtDNA allowed reaching a 91.6% identification success rate. Significant COI divergence (3.5–4.4%) was observed within the most widespread ground squirrel species (Spermophilus erythrogenys, S. pygmaeus, S. suslicus, Urocitellus undulatus), suggesting the presence of cryptic species. A single putative NUMT (nuclear mitochondrial pseudogene) sequence was recovered during molecular analysis; mitochondrial COI from this sample was amplified following re-extraction of DNA. Our data show high discrimination ability of 100 bp COI fragments for Eurasian ground squirrels (84.3%) with no incorrect assessments, underscoring the potential utility of the existing reference librariy for the development of diagnostic ‘mini-barcodes’.     

Comparison of Y chromosome and mtDNA phylogenies leads to unique inferences of macaque evolutionary history

We report here the results of one of the first analyses to use male-specific nuclear markers in elucidating primate phylogenetic relationships at the intrageneric level. Two closely linked Y chromosome markers, TSPY and SRY, were sequenced for a total of 3100 bases. Forty-four macaques, representing 18 of the 19 recognized species, were sequenced for the full 3.1 kb, as was 1 individual from each of the following outgroup genera: Papio, Theropithecus, Mandrillus, Allenopithecus,Cercopithecus, Trachypithecus, Presbytis, and Homo. In contrast to recent mtDNA phylogenies, Y chromosome loci support four monophyletic species groups, including a sinica group containing M. arctoides-a classification largely congruent with those of Fooden and Delson. Comparison of mtDNA and Y chromosome phylogenies highlight (1) a potential hybrid origin of Macaca arctoides from M. fascicularis and proto-M. assamensis/thibetana and (2) cases of mitochondrial paraphyly in macaque species whose Y chromosome lineages are monophyletic-a probable evolutionary consequence of philopatric females vs dispersing males. These results raise the question of whether a phylogenetic tree should be a topology of species origins or a depiction of more current species relationships, including subsequent episodes of introgression.     

Strongyloides robustus and the northern sympatric populations of northern (Glaucomys sabrinus) and southern (G. volans) flying squirrels

Within North America, northern (Glaucomys sabrinus) and southern (Glaucomys volans) flying squirrels occupy distinct ranges with limited overlap. Sympatry in northern latitudes coincides with northern hardwood vegetation from Minnesota to New England. Strongyloides robustus is an intestinal parasite that infects both species but appears to be deleterious only to northern flying squirrels. As a result, S. robustus could be a critical determinant of flying squirrel population characteristics in at least some areas of sympatry. However, cold weather could potentially limit the distribution of S. robustus in northern climates. Therefore, we assessed fecal samples from both flying squirrel species to determine the presence of the nematode in Wisconsin. Strongyloides robustus was found in 12 flying squirrel scat samples and infected 52% of southern flying squirrels and 11% of northern flying squirrels. Prevalence of S. robustus infection for northern flying squirrels was substantially lower than previously reported from more southern regions. This is the northernmost documentation of S. robustus in flying squirrels and the first documentation of S. robustus parasitizing flying squirrels in Wisconsin.     

Erythrocyte 2,3-diphosphoglycerate and hemoglobin electrophoretic characteristics in rodent adaptation to high mountains

Adaptation of the erythropoetic system to hypoxia was shown to be different in different populations of montaine rodents of the Central Caucasus. One type of adaptation includes the increase in 2,3-diphosphoglycerate content of erythrocytes, the hemoglobin structure remaining apparently unchanged. This pattern was found in two groups of animals -- in one population of the ground squirrel Citellus suslicus whose ancestors were introduced to the mountains some 25 years ago, and in the wood mouse Apodemus sylvaticus which is a usual inhabitant of highland, although it does not form there any isolated populations. The other type of adaptation was found in the ground squirrel Citellus pygmaeus musicus, which is native to high altitude. The content of 2,3-diphosphoglycerate in the erythrocytes in this animal is the same as in the sea-level subspecies C. p. planicola, whereas the electrophoretic picture of hemoglobin in these two subspecies was found to be different.     

Reduced introgression of the Y chromosome between subspecies of the European rabbit (Oryctolagus cuniculus) in the Iberian Peninsula

The role of the Y chromosome in speciation is unclear. Hybrid zones provide natural arenas for studying speciation, as differential introgression of markers may reveal selection acting against incompatibilities. Two subspecies of the European rabbit (Oryctolagus cuniculus) form a hybrid zone in the Iberian Peninsula. Previous work on mitochondrial DNA (mtDNA), Y- and X-linked loci revealed the existence of two divergent lineages in the rabbit genome and that these lineages are largely subspecies-specific for mtDNA and two X-linked loci. Here we investigated the geographic distribution of the two Y chromosome lineages by genotyping two diagnostic single nucleotide polymorphisms in a sample of 353 male rabbits representing both subspecies, and found that Y chromosome lineages are also largely subspecies-specific. We then sequenced three autosomal loci and discovered considerable variation in levels of differentiation at these loci. Finally, we compared estimates of population differentiation between rabbit subspecies at 26 markers and found a surprising bimodal distribution of F(ST)values. The vast majority of loci showed little or no differentiation between rabbit subspecies while a few loci, including the SRY gene, showed little or no introgression across the hybrid zone. Estimates of population differentiation for the Y chromosome were surprisingly high given that there is male-biased dispersal in rabbits. Taken together, these data indicate that there is a clear dichotomy in the rabbit genome and that some loci remain highly differentiated despite extensive gene flow following secondary contact.     

Karyotypic evolution of the family Sciuridae: inferences from the genome organizations of ground squirrels

Cross-species chromosome painting has made a great contribution to our understanding of the evolution of karyotypes and genome organizations of mammals. Several recent papers of comparative painting between tree and flying squirrels have shed some light on the evolution of the family Sciuridae and the order Rodentia. In the present study we have extended the comparative painting to the Himalayan marmot (Marmotahimalayana) and the African ground squirrel (Xerus cf. erythropus), i.e. representative species from another important squirrel group--the ground squirrels--, and have established genome-wide comparative chromosome maps between human, eastern gray squirrel, and these two ground squirrels. The results show that 1) the squirrels so far studied all have conserved karyotypes that resemble the ancestral karyotype of the order Rodentia; 2) the African ground squirrels could have retained the ancestral karyotype of the family Sciuridae. Furthermore, we have mapped the evolutionary rearrangements onto a molecular-based consensus phylogenetic tree of the family Sciuridae.     

Phylogenies using mtDNA and SRY provide evidence for male-mediated introgression in Asian domestic cattle

Using nucleotide sequences of the mitochondrial DNA (mtDNA) cytochrome b and SRY genes, we examined the genetic status of two major groups of domestic cattle, the humpless taurine (Bos taurus) and humped zebu (B. indicus), using 10 cattle populations in Asia. Several sequence polymorphisms specific for each major group were found, although the frequency of these polymorphisms varied in each population. Six major mtDNA-SRY composite types were observed. The Mishima, Mongolian, Korean, Chinese Yellow and Sri Lanka cattle populations had a full match between the mtDNA and SRY sequences, specifically the taurine/taurine type or zebu/zebu type. A non-match type (zebu/taurine type) was found at a high frequency in the Bangladesh (83.4%) and Nepal populations (83.3%). Our results suggest that these non-match type populations developed from genetic hybridization of different strains. Also, the domestication history of modern Asian domestic cattle could be explained by male-mediated introgression. Additionally, our results suggest the occurrence of introgression of mtDNA from other Bibos or Poephagus species into native cattle populations. The existence of other mtDNA-SRY composite types, such as the Bali-zebu and yak-zebu types in Indonesia (85.7%) and Nepal (16.7%), respectively, suggests that genetic introgression also occurred from other genera into domestic cattle during the process of domestication.     

Multigenic and morphometric differentiation of ground squirrels (Spermophilus, Scuiridae, Rodentia) in Turkey, with a description of a new species

This study reports on the molecular phylogenetics of ground squirrels, genus Spermophilus, in Turkey using cytochrome b (1140bp), part of the D-loop and flanking tRNAs (572bp), X chromosome (867-1051bp) and Y chromosome (983-989bp) DNA sequences. Individuals also were characterized by karyotype and with geometric morphometric analyses of mandibles and skulls. Two hundred fourteen individuals from 91 localities were studied. All the data support the recognition of a new species in SW Anatolia: the Taurus ground squirrel Spermophilus taurensis sp. nov. The new species has a small distribution in the Taurus Mountains in an area that is a hotspot for biodiversity. Molecular clock analysis suggests that the new species diverged from the European ground squirrel, Spermophilus citellus, about 2.5 million years ago and that the ancestor of these two species diverged from the widespread Anatolian ground squirrel, Spermophilus xanthoprymnus, about 5 million years ago. Morphometric differentiation in skull and mandible shape among the three species is incomplete, but statistically significant. S. xanthoprymnus is subdivided into five cytochrome b phylogroups and we use these data to infer the location of glacial refugia where the species lived during the last glacial maximum. This study illustrates the potential of combined molecular and morphometric studies to uncover new Anatolian species and to reconstruct their phylogeographic history. The new species is important for squirrel taxonomy and for understanding Eurasian mammal evolution.     

The intraspecies variability of <Emphasis Type="Italic">Spermophilus relictus</Emphasis> sensu lato Kaschkarov 1923 (Sciuridae,Rodentia)

The intraspecies variability of Spermophilus relictus sensu lato was studied based on 27 measurements of skulls from 67 specimens of relict ground squirrels (S. relictus) and 66 specimens of Tien Shan ground squirrels (S. ralli), as well as six specimens of the relict ground squirrel from the Gissar Ridge (Tien Shan). A colorimetric analysis of skins of relict ground squirrels (19 specimens, including three individuals from the Gissar Ridge) and Tien Shan ground squirrels (19 specimens) was made. Significant intraspecies variability was found in the relict and Issyk-Kul ground squirrels, whereas the interspecies differences were small, raising questions about the species independence of the Tien Shan ground squirrel (S. ralli).     

Differential introgression of uniparentally inherited markers in bison populations with hybrid ancestries

Historical hybridization between Bison bison (bison) and Bos taurus (cattle) has been well documented and resulted in cattle mitochondrial DNA (mtDNA) introgression, previously identified in six different bison populations. In order to examine Y chromosome introgression, a microsatellite marker (BYM-1) with non-overlapping allele size distributions in bison and cattle was isolated from a bacterial artificial chromosome (BAC) clone, and was physically assigned to the Y chromosome by fluorescence in situ hybridization. BYM-1 genotypes for a sample of 143 male bison from 10 populations, including all six populations where cattle mtDNA haplotypes were previously identified, indicated that cattle Y chromosome introgression had not occurred in these bison populations. The differential permeability of uniparentally inherited markers to introgression is consistent with observations of sterility among first generation hybrid males and a sexual asymmetry in the direction of hybridization favouring matings between male bison and female cattle.     

Phylogeny of mitochondrial DNA clones in tassel-eared squirrels Sciurus aberti

The tassel-eared squirrel, Sciurus aberti , includes six subspecies which occupy restrictive and apparently identical habitats in Ponderosa pine forests in the south-western United States and Mexico; the strict habitat requirement of this species is based on dietary requirements which are only fulfilled in these forests. To examine evolutionary relationships among certain subspecies of S. aberti , we obtained estimates of nucleotide diversity within subspecies as well as nucleotide divergence between subspecies using mitochondrial DNA (mtDNA) analysis. Restriction site polymorphisms were identified in samples of the four US subspecies: S. a. aberti (Abert), S. a. kaibabensis (Kaibab), S. a. ferreus (Ferreus), and S. a. chuscensis (Chuska) Fourteen mtDNA clones were resolved that were, with one exception, uniquely subspecific. Dendrograms constructed by neighbour-joining and maximum parsimony methods revealed two major assemblages: (1) an Abert/Kaibab group; and (2) a Ferreus/Chuska group. The Abert vs. Ferreus clones exhibited the greatest net nucleotide divergence, with a lineage separation estimate approximating 572 000 years ago assuming a nucleotide substitution rate of 7.15 × 10^-9/year/site. Five out of ten Chuska squirrels shared a clone with one Abert sample; the relative sizes of these two populations and their respective ranges as well as their close proximity support the proposal for relatively recent intermixing of Abert and Chuska populations resulting in what appears to be Abert → Chuska migration. Nucleotide diversity within subspecies ranked as Kaibab < Ferreus < Abert < Chuska; the relatively high diversity for the Chuska sample is based on the apparent introgression of Abert mtDNA. The relative diversity exhibited by Kaibab, Ferreus and Aberti samples corresponds to the range size of the respective subspecies.     

Bidirectional introgressive hybridization between Lepus capensis and Lepus yarkandensis

Lepus capensis and Lepus yarkandensis are two well-established hare species with different habitat preferences and obvious morphological distinctions. L. capensis is common and widespread while L. yarkandensis is an endemic species with a restricted distribution in the Tarim Basin of western China. During field sampling, individuals with a distinct phenotype were found in the contact zone between the two species. To understand the origin of these unclassified individuals, we analyzed morphological and genetic data from 700 individuals including L. yarkandensis, L. capensis and the unclassified individuals. Morphological analyses of the unclassified individuals revealed that they lie between L. yarkandensis and L. capensis. Genetic analyses based on species-specific molecular markers (mitochondrial control region and SRY) showed that the unclassified populations have mtDNA and SRY genes from both L. capensis and L. yarkandensis, suggesting that the unclassified populations result from hybridization between the two species. Interestingly, many identical and/or very similar mtDNA haplotypes were shared between L. capensis populations and L. yarkandensis populations close to their contact zones, which further suggests the existence of extensive bidirectional mtDNA introgression. Similarly, we found evidence for SRY introgression, though it existed at a lower level compared to mtDNA introgression.     

Content of DSIP, enkephalins and ACTH in some tissues of active and hibernating ground squirrels (Citellus suslicus)

Seasonal changes in the amount of DSIP-like material have been measured in extracts from the brain of Citellus suslicus. 2. Analysis of extracts from the brain of deeply sleeping ground squirrels shows that the level of enkephalin-like material reliably increases while that of ACTH-like material decreases. 3. It is concluded that the opiate brain activation system is among the factors participating in the induction of hibernation in ground squirrels.     

The evolving male: spinner dolphin (Stenella longirostris) ecotypes are divergent at Y chromosome but not mtDNA or autosomal markers

The susceptibility of the Y chromosome to sexual selection may make this chromosome an important player in the formation of reproductive isolating barriers, and ultimately speciation. Here, we investigate the role of the Y chromosome in phenotypic divergence and reproductive isolation of spinner dolphin (Stenella longirostris) ecotypes. This species contains six known ecotypes (grouped into four subspecies) that exhibit striking differences in morphology, habitat and mating system, despite having adjacent or overlapping ranges and little genetic divergence at previously studied mtDNA and autosomal markers. We examined the phylogeographic structure for all six ecotypes across the species range (n = 261, 17 geographic locations) using DNA sequences from three Y chromosome markers, two maternally inherited mitochondrial (mtDNA) markers, and a biparentally inherited autosomal intron. mtDNA and autosomal analyses revealed low divergence (most Φ_ST values <0.1) between ecotypes and geographic regions, concordant with previous studies. In contrast, Y intron analyses revealed fixed differences amongst the three most phenotypically divergent groups: S. l. longirostris vs. S. l. roseiventris vs. combined S. l. orientalis/S. l. centroamericana/Tres Marias ecotypes). Another ecotype (whitebelly), previously postulated to be a hybrid between the two phenotypically most divergent ecotypes, had Y haplotypes from both putative parent ecotypes, supporting a hybrid designation. Reduced introgression of the Y chromosome has previously been observed in other organisms ranging from insects to terrestrial mammals, and here we demonstrate this phenomenon in a marine mammal with high dispersal capabilities. These results indicate that reduced introgression of the Y chromosome occurs in a wide taxonomic range of organisms and support the growing body of evidence that rapid evolution of the Y chromosome is important in evolutionary diversification.     

Additional data for nuclear DNA give new insights into the phylogenetic position of Sorex granarius within the Sorex araneus group

Many species contain genetic lineages that are phylogenetically intermixed with those of other species. In the Sorex araneus group, previous results based on mtDNA and Y chromosome sequence data showed an incongruent position of Sorex granarius within this group. In this study, we explored the relationship between species within the S. araneus group, aiming to resolve the particular position of S. granarius. In this context, we sequenced a total of 2447 base pairs (bp) of X-linked and nuclear genes from 47 individuals of the S. araneus group. The same taxa were also analyzed within a Bayesian framework with nine autosomal microsatellites. These analyses revealed that all markers apart from mtDNA showed similar patterns, suggesting that the problematic position of S. granarius is best explained by an incongruent behavior by mtDNA. Given their close phylogenetic relationship and their close geographic distribution, the most likely explanation for this pattern is past mtDNA introgression from S. araneus race Carlit to S. granarius.     

Genetic evidence of extensive introgression of short-tailed ground squirrel genes in a hybridization zone of Spermophilus major and S. erythrogenys, inferred from sequencing of the mtDNA cytochrome b gene

We have completely sequenced the mtDNA cytochrome b gene of ground squirrels from the zone of overlapping ranges of Spermophilus major and S. erythrogenys in the Tobol-Ishim interfluve, which is a putative hybridization zone of these species. The results of the sequencing showed extensive introgression of mtDNA genes of the short-tailed ground squirrel S. e. brevicauda, whose haplotype had fully replaced the S. major haplotype. All of the ground squirrels from the Tobol-Ishim interfluve had a variant of the S. e. brevicauda mtDNA haplotype that was specific for this zone. On average, 119 substitutions (10.44%) were found between S. major from Ul'yanovsk oblast and S. e. brevicauda from the northern Kazakhstan, the mean genetic distance (D) between them being 0.115, which conforms to the corresponding parameters for the S. e. brevicauda-S. pygmaeus pair (122 substitutions, D = 0.118). Insignificant differences (seven substitutions, D = 0.043) were found between the S. major and S. pygmaeus haplotypes, which suggest that these species have similar mitochondrial haplotypes. Five to ten nucleotide substitutions (0.44–0.88%) were detected between the animals from the Tobol-Ishim interfluve and S. e. brevicauda. The mtDNA haplotype divergence D within the genus Spermophilus (ten species) for all codon positions ranged from 0.035 to 0.158. Phylogenetic reconstructions (MP, ML, and NJ trees) showed two well-differentiated clusters with high bootstrap support. However, there was different branching topology within the cluster and their species composition varied. The maximum likelihood tree, ML, differentiating the species into two subgenera, Citellus and Colobotis, most reliably reflected taxonomic relationships of the species from the genus Spermophilus, inferred from morphological and genetic biochemical data. The morphologically pure S. major (subgenus Colobotis) animals, used in the analysis, proved to carry the haplotype of another species, S. pygmaeus (subgenus Citellus). This poses a question on the existence of the specific haplotype of S. major, the reason of its replacement by haplotype of other species, and possible consequences of this phenomenon for survival of the species.