Rapid chromosomal evolution in enigmatic mammal with XX in both sexes, the Alay mole vole Ellobiusalaicus Vorontsov et al., 1969 (Mammalia, Rodentia)

Abstract Evolutionary history and taxonomic position for cryptic species may be clarified by using molecular and cytogenetic methods. The subterranean rodent, the Alay mole vole Ellobiusalaicus Vorontsov et al., 1969 is one of three sibling species constituting the subgenus Ellobius Fischer, 1814, all of which lost the Y chromosome and obtained isomorphic XX sex chromosomes in both males and females. E.alaicus is evaluated by IUCN as a data deficient species because their distribution, biology, and genetics are almost unknown. We revealed specific karyotypic variability (2n = 52–48) in E.alaicus due to different Robertsonian translocations (Rbs). Two variants of hybrids (2n = 53, different Rbs) with E.tancrei Blasius, 1884 were found at the Northern slopes of the Alay Ridge and in the Naryn district, Kyrgyzstan. We described the sudden change in chromosome numbers from 2n = 50 to 48 and specific karyotype structure for mole voles, which inhabit the entrance to the Alay Valley (Tajikistan), and revealed their affiliation as E.alaicus by cytochrome b and fragments of nuclear XIST and Rspo1 genes sequencing. To date, it is possible to expand the range of E.alaicus from the Alay Valley (South Kyrgyzstan) up to the Ferghana Ridge and the Naryn Basin, Tien Shan at the north-east and to the Pamir-Alay Mountains (Tajikistan) at the west. The closeness of E.tancrei and E.alaicus is supported, whereas specific chromosome and molecular changes, as well as geographic distribution, verified the species status for E.alaicus. The case of Ellobius species accented an unevenness in rates of chromosome and nucleotide changes along with morphological similarity, which is emblematic for cryptic species.


Introduction
An origin of species due to chromosome changes is still debatable (King 1993, Castiglia 2014, Dobigny et al. 2017. The problem of chromosomal speciation is closely connected with the phenomenon of sibling species. Mole voles of the genus Ellobius Fischer, 1814, and some other rodents, such as Mus Linnaeus, 1758, Nannomys Peters, 1876 (Gropp et al. 1972, Capanna et al. 1976, Capanna and Castiglia 2004, Veyrunes et al. 2010, Garagna et al. 2014, and subterranean Spalax Guldenstaedt, 1770, Fukomys Kock et al., 2006, Ctenomys Blainville, 1826etc. (Wahrman et al. 1969, Nevo et al. 2000, Van Daele et al. 2007, Deuve et al. 2008, Kryštufek et al. 2012, Buschiazzo et al. 2018, demonstrate a broad chromosome variability at the species and intraspecies levels without morphological differences . The lack of clear morphological characters, by which specimens can be easily distinguished in museum collections, as well as in nature, makes such species problematic for study and protection. New molecular methods, especially DNA sequencing and cross-species chromosome painting, can be a precise approach for studying the most intriguing groups (Graphodatsky et al. 2011).
The genus Ellobius divides into two subgenera: Bramus Pomel, 1892 and Ellobius Fischer, 1814 (Musser, Carleton 2005). The subgenus Bramus includes two species: E. fuscocapillus Blyth, 1843 (2n = 36, XX♀-XY♂), and E. lutescens Thomas, 1897 (2n = 17, X0♀-X0♂) (Matthey 1953, Vorontsov et al. 1969, Lyapunova, Vorontsov 1978. Species of the subgenus Ellobius (E. talpinus Pallas, 1770, E. tancrei Blasius, 1884, and E. alaicus Vorontsov et al. 1969) are cryptic ones, indistinguishable by morphological features (Yakimenko and Vorontsov 1982), the main diagnostic features are distant karyotypes. E. talpinus, E. tancrei, and E. alaicus are unique in mammals. Along with autosomal changes, the species lost the Y chromosome, the Sry gene, and obtained isomorphic XX chromosomes in both males and females (Lyapunova and Vorontsov 1978, Kolomiets et al. 1991, Just et al. 1995, Bakloushinskaya et al. 2012, Bakloushinskaya and Matveevsky 2018. The study of E. lutescens and E. talpinus whole genomes was not able to reveal any sex determining factors (Mulugeta et al. 2016). The first signs of sex chromosomes heteromorphism in E. talpinus and E. tancrei were observed in the meiotic behaviour of XX chromosomes in males (Kolomiets et al. 1991, Matveevsky et al. 2016. The northern mole vole, E. talpinus, with 2n = NF = 54 (Ivanov 1967, has no described chromosomal variability, but significant mtDNA vari-ability was revealed recently along its wide range (Bogdanov et al. 2015). The eastern mole vole, E. tancrei has stable 2n = 54, NF = 56 in most of its range, and demonstrates enormous karyotype variability (2n = 54-30) in the Pamir-Alay region (Vorontsov and Radzhabli 1967, Bakloushinskaya et al. 2013. The third species was described first as a chromosomal form of E. talpinus sensu lato (a chromosomal form of E. tancrei from the modern point of view) with one pair of large metacentric chromosomes and small submetacentrics, specific 2n = 52, NF = 56 (Vorontsov and Radzhabli 1967), and later it was designated as the Alay mole vole E. alaicus (Vorontsov et al. 1969, Lyapunova andVorontsov 1978). The Alay Valley, the terra typica of the Alay mole vole, extending appr. 180 km from Tajikistan in the west to China in the east between two mountain systems: the Tien Shan and the Pamir. Range of the species was limited to the Alay Valley and the Northern slopes of the Alay Ridge, Tien-Shan (Kyrgyzstan). E. alaicus was listed by IUCN as data deficient species; cytogenetic data are scarce, no molecular study has been made ever (Gerrie and Kennerley 2016).
We studied the G-band structure of the E. alaicus karyotype previously and described a morphological homology for one pair of large metacentrics of the species to the Robertsonian metacentrics of E. tancrei from the Pamir-Alay (Bakloushinskaya 2003). We also discovered different forms of E. alaicus and their hybrids with E. tancrei with 2n = 50-53 from other parts of the Inner Tien-Shan (Lyapunova et al. 1985, Bakloushinskaya, Lyapunova 2003. But the study was incomplete, and application of modern cytogenetical and molecular techniques is required to confirm the karyotype structure, validity of E. alaicus as a species and its distribution. The main objectives of this study were to reveal the chromosomal variability in E. alaicus and prove species affiliations for mole voles from adjacent to the Alay Valley territories of the Inner Tien-Shan and the Pamir-Alay Mountains. To bring a phylogenetic framework to the delimiting species, we examined the phylogeny of the subgenus Ellobius using the mitochondrial DNA marker, complete cytochrome b gene, cytb, and two nuclear DNA markers, fragments of the XIST (X-inactive specific transcript) and Rspo1 (R-spondin 1) genes.

Material and methods
We analyzed karyotypes or cytb structure, or both, of 116 specimens of E. alaicus and E. tancrei mole voles from 27 localities across the Alay Valley and adjacent territories, as well as 7 E. talpinus specimens from 6 localities of Russia (Fig. 1, Table 1). Fragments of the XIST and Rspo1 genes were studied for nine specimens of three species.

Samples
We used samples from the Joint collection of wildlife tissues for fundamental, applied and environmental researches of the Koltzov Institute of Developmental Biology RAS, the state registration number AAAA-A16-116120810085-1, which is a part of the Core Centrum of the Koltzov Institute of Developmental Biology RAS, the state registration number 6868145. Tissues and chromosome suspensions were collected during our field trips in 1981-1983, 2008, 2010, 2013, and 2015-2018. For cytb sequencing we also used dried skins of specimens S132130*, S132131*, S132133*, S132135* deposited to the Zoological Museum of Lomonosov Moscow State University (Table  1) and originated from the terra typica of the Alay mole vole.
Animals were treated according to established international protocols, as in the Guidelines for Humane Endpoints for Animals Used in Biomedical Research. All the experimental protocols were approved by the Ethics Committees for Animal Research of the Koltzov Institute of Developmental Biology RAS in accordance with the Regulations for Laboratory Practice in the Russian Federation. All efforts were made to minimize animal suffering.

Mitotic and meiotic chromosomes
Chromosomes from bone marrow were prepared according to Ford and Hamerton (1956) for all animals listed with chromosome numbers in Table 1. G-banding was achieved using trypsin digestion (Seabright 1971). Samples from 3 animals (25610, 25611, 25612, Table 1) were used for tissue culture Galleni 1991, Romanenko et al. 2015). All cell lines were retrieved from the IMCB SB RAS cell bank ("The general collection of cell cultures", № 0310-2016-0002). Full sets of paints derived from flow-sorted chromosomes of the field vole Microtus agrestis Linnaeus, 1761 were used . FISH was performed according to previously published protocols (Yang et al. 1999, Graphodatsky et al. 2000. G-banding was carried out for metaphase chromosomes prior to FISH. The same procedures were used previously for specimens from localities 11, 12, 16, 17, and 18 (Bakloushinskaya et al. 2010, Matveevsky et al. 2015. It was not possible to use Zoo-FISH on material gathered in the 1980s, but the pictures of G-banded karyotypes were suitable for comparative analyses. Karyological data, obtained from 1981 to 2008, were re-examined in accordance with a new nomenclature for the Rb translocations in E. tancrei (Bakloushinskaya et al. 2012(Bakloushinskaya et al. , 2013. In total, we studied chromosomes for 114 specimens of E. alaicus, E. tancrei and E. talpinus. Images were captured using VideoTesT-FISH 2.0. and VideoTesT-Karyo 3.1. (Imicrotec) or Case Data Manager 6.0 (Applied Spectral Imaging Inc., ASI) software with either ProgRes CCD (Jenoptik) or ASI CCD camera, respectively, mounted on an Axioskop 2 plus (Zeiss) microscope with filter sets for DAPI, FITC, and rhodamine. Hybridization signals were assigned to specific chromosome regions defined by GTGbanding patterns previously captured with the CCD camera. Routine and G-banded plates were captured with a CMOS camera, mounted on an Axioskop 40 (Zeiss) microscope. Images were processed using Paint Shop Pro X2 (Corel).
The suspensions and spreads of spermatocytes of two E. alaicus males (27024, 27025) were made as described by Kolomiets et al. (2010). Immunostaining was de-signed as in our previous studies , Matveevsky et al. 2016. Synaptonemal complexes (SC) and centromeres in pachytene spermatocytes were detected using antibodies to axial SC elements -SYCP3 (Abcam, UK) and the kinetochores (CREST, Fitzgerald Industries International, USA). The slides were analyzed with an Axioimager D1 microscope (Carl Zeiss, Jena, Germany). Images were processed using Adobe Photoshop CS3 Extended.

cytb sequencing
Total DNA was isolated by phenol-chloroform deproteinisation after treatment of shredded tissues with proteinase K (Sambrook et al. 1989). The primers used for amplification and sequencing of the complete cytb gene (1143 bp) in species of the Ellobius subgenus are listed in Table 2. Polymerase chain reaction (PCR) was carried out in a mixture containing 25-50 ng DNA, 2 µl 10×Taq-buffer, 1.6 µl 2.5 mM dNTP solution, 4 pM of each primer, 1 unit of Taq-polymerase, and deionized water to a final volume of 20 µL. Amplification was as follows: preheating at 94 °C for 3 min, then 35 cycles in a sequential mode of 30 s at 94 °C, 1 min at 55 or 57 °C depending on the applied pair of primers, and 1 min at 72 °C; the reaction was completed by a single final elongation of PCR products at 72 °C for 6 min. Automatic sequencing was carried out using a PRISM BigDye TM Terminator v. 3.1 kit (ABI, United States) with ABI 3500 genetic analyzer at the Core Centrum of the Koltzov Institute of Developmental Biology RAS.

XIST (X-inactive specific transcript) and Rspo1 (R-spondin 1) sequencing
Fragments of XIST gene (449 bp including deletions/insertions) and one exon and one intron of Rspo1 gene (816 bp) were sequenced for nine animals (Table 1). PCR was carried out in a mixture containing 35-50 ng DNA, 2 µl 10×Taq-buffer, 1.6 µl 2.5 mM dNTP solution, 4 pM of each primer, 1 unit of Taq-polymerase, and deionized water to a final volume of 20 µL. Amplification was as follows: preheating at 94 °C for 3 min, then 35 cycles in a sequential mode of 30 s at 94 °C, 1 min at 63 °C (in case of XIST) or 67 °C (Rspo1), and 1 min at 72 °C; the reaction was completed by a single final elongation of PCR products at 72 °C for 6 min. For Rspo1 gene analysis, we conducted second PCR with a PRISM®BigDye TM Terminator v. 3.1 kit using two internal primers to the PCR product obtained by first amplification. All primers are listed in Table 3

Molecular evolutionary analyses
DNA sequences were aligned using the MUSCLE algorithm (Edgar 2004) in MEGA X software (Kumar et al. 2018). Maximum likelihood analyses and calculation of genetic distances (D) were carried out in MEGA X software using the TN93+G model of DNA substitution (Tamura-Nei model, Gamma distributed) for cytb and Jukes-Cantor model (Jukes, Cantor 1969) for concatenated sequences of XIST and Rspo1 genes according to modeltest, with statistical support for internodes tested by bootstrapping in 1,000 replications. Bayesian inference for cytb sequences was additionally evaluated in MrBayes ver. 3.2 (Ronquist et al. 2012); analyses were run for 1 million generations with Markov chains sampled every 1000 generations, 25% trees were discarded ('burn-in') and node support was assessed with posterior probabilities. Final images of phylogenetic trees were rendered in FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/) and Inkspace (https://inkscape.org/).

Karyotyping
The main result was a discovery of specific chromosome variability in E. alaicus, with 2n varying from 52 to 48 chromosomes. For mole voles from the Alay Ridge, the Naryn Valley, and the Aksai River Valley (localities # 3, 5, 6, Fig. 1 (Fig. 3a), were found at the northern slopes of the Alay Ridge (# 3), which marks the species contact zone. The Ferghana Ridge separates the Alay Valley from the Chatyr-Kel' Lake Basin, the Aksai River Valley, and the Naryn Valley, one of the largest within the Inner Tien Shan. Fascinating data were obtained for animals inhabiting the Chatyr-Kel' Lake surrounds and the Naryn district (localities # 4 and 6, Table 1, Fig. 1), where we found Alay mole voles with 2n = 50, and heterozygotes with 2n = 51, which are presumed hybrids with typical E. alaicus, 2n = 52 (Figs 2b, 3c). Chromosomal number in animals with 2n = 50 was decreased because of another translocation, the Rb(1.3). Nevertheless, in the Aksai River Valley, the typical E. alaicus with 2n = 52 [2 Rb(2.11)] were found (locality # 5, Table 1, Fig. 1).
Two heterozygous karyotypes with 2n = 53 due to the presence of different Rb metacentrics were found. In point # 3, we found animals with 2n = 53 and 1 Rb(2.11), which are hybrids of E. alaicus and E. tancrei (Fig. 3a). Mole voles with 2n = 53 from the Naryn district (#7, Fig. 1, Table 1) had another translocation, 1 Rb(1.3) (Fig. 3b). Table 2. Primers, which were used for amplification and sequencing of cytb gene in mole voles of the Ellobius subgenus. Primers Eta_CytbF1, and VOLE14 were used to amplify the full cytb gene with flanked fragments of mtDNA; all other primers correspond to various internal areas of cytb gene, the position of their 5'-end nucleotide from the start of cytb gene is in parentheses.

Species
Primer designation Nucleotide sequence of primer (5'-3')   We were not able to find animals with 2n = 52 and 2 Rb(1.3), but probably they inhabit an extensive unstudied area in the Naryn Valley, between points #6 and 7. The most surprising data we revealed for animals from the Pamir-Alay mountains, Tajikistan, (# 8, Fig. 1, Table 1). In 1981, we got Alay mole voles from there for breeding and karyotyping; two animals had 2n = 50, and one was a somatic mosaic, 2n = 50-51. Their karyotypes included 2 Rb(2.11) and 1-2 Rb(4.9); the last one was heterozygous in the mosaic specimen (Fig. 2c). After almost 30 years (in 2010) we caught animals with 2n = 48 at the same locality, and one mole vole with the same karyotype at the opposite bank of the Kyzyl-Suu River (locality # 9, Fig. 1, Table 1). Their karyotypes contained one more pair of Rb metacentrics, Rb(3.10). The entire set of Rbs was 2 Rb(2.11), 2 Rb(4.9),  Table 1. Localities 23-27 are outside the map.
2 Rb(3.10), all of which were confirmed by chromosome painting for specimens 25610, 25611, 25612 (Figs 4, 5). The 21 MAG (Microtus agrestis) autosomal probes revealed 35 conserved segments in the mole voles' genome, which corresponds to the genome composition of the typical E. tancrei, 2n = 54 (Bakloushinskaya et al. 2012), and its form with the lowest chromosome number, 2n = 30 (Bakloushinskaya et al. 2013). The MAG X chromosome probe produced signals on both male and female X chromosomes; the MAG Y probes did not demonstrate any specific signal. Therefore, we suppose that E. alaicus has the same isomorphic sex chromosomes, XX in both sexes, as E. talpinus and E. tancrei.

Synaptonemal complexes
In the 48-chromosomal form of E. alaicus the 23 bivalents (including 19 acrocentric SC, four bi-armed SC), and sex (XX) bivalent were formed in spermatocytes at the pachytene stage (Fig. 6). Male XX bivalent was shifted to the periphery of the meiotic nucleus and had two short distal synaptic segments and an extended asynaptic region, which is typical for mammals.
Our data on cytb revealed a significant range of interspecies genetic distances, which are moderate for E. alaicus -E. tancrei (D = 0.0256), and quite high for E. alaicus -E. talpinus   (D = 0.0799), E. tancrei -E. talpinus (D = 0.0839). Thus, E. alaicus and E. tancrei are the most closely related species that coincides with results of chromosomal analysis too. E. talpinus demonstrated high intraspecific differentiation (D value averages 0.033), as we described earlier (Bogdanov et al. 2015). We also evaluated the physical differences between sequences using uncorrected so-called p distances: E. alaicus -E. tancrei p = 0.0243, E. alaicus -E. talpinus p = 0.0688, E. tancrei -E. talpinus p = 0.0715. P distances were lower if compare with genetic distances (D), calculated using the TN93+G model, but even in the case of E. alaicus -E. tancrei, p distance was more than 2%. It had a high probability of being indicative of valid species (Bradley and Baker 2001).
The evolutionary history of the subgenus Ellobius was also inferred by using the concatenated sequences of nuclear XIST (449 bp) and Rspo1 (1203 bp) genes, 1652 bp in total. The analysis showed the existence of "fixed" nucleotide substitutions and the species-specific clustering for three Ellobius species despite the genetic distances were rather low: D = 0.003 for E. alaicus -E. tancrei, D = 0.006 for E. alaicus -E. talpinus and D = 0.004 for E. tancrei -E. talpinus. As a result, the species relationships were proven by analyses of mitochondrial and nuclear DNA markers. It is noticeable that nuclear genes variability indicates more significant intraspecific differentiation for E. tancrei compared with results of cytb analysis. Thus, differences between the specimen from  Tashkent vicinities, which could not be assigned to any of the two clades in this analysis, and Tajikistan eastern mole voles (D = 0.002) reach up to a half and even more of interspecific distance in E. tancrei -E. talpinus and E. alaicus -E. tancrei (Fig. 8).

Discussion
A few studies dealt with Ellobius molecular phylogeny before. Conroy and Cook (1999) studied cytb of two species, E. fuscocapillus and E. tancrei, and their position in the Arvicolinae tree appeared to be unstable in different models. Data on variations of short fragments of nuclear genes (partial LCAT and exon 10 GHR) in E. talpinus and E. tancrei contradicted the conventional view that Ellobius is an ancient group because of simplicity of rooted molars and the peculiar structure of the skull (Abramson et al. 2009). Fabre et al. (2012) re-analyzed these data among others for comparative meta-analyses of the rodent diversity and phylogeny without special attention to Ellobiini. Nevertheless, the genus Ellobius appears to be a young group; its morphological characters indicate adaptation to subterranean life and provide no phylogenetic signal. E. talpinus and E. tancrei separated not earlier than the latest Pliocene and Early Pleistocene between ca. 2.1-1.0 Ma (Abramson et al. 2009). The phyletic lineage leading to the recent E. talpinus includes at least two chronospecies: Late Pliocene-Early Pleistocene E. kujalnikensis and early Middle Pleistocene E. melitopoliensis; E. talpinus was recognized from the late Middle Pleistocene (Tesakov 2009). There are no such data for E. tancrei and E. alaicus.
Here, for the first time, we demonstrated data on molecular, mitochondrial (cytb) and nuclear (XIST and Rspo1 fragments) specificity of E. alaicus. The cytb variability in the subgenus Ellobius, which we demonstrated here, is comparable and even higher than in Ctenomys, subterranean rodents with numerous species-specific chromosome changes (Buschiazzo et al. 2018). In Ctenomys genetic distances, calculated on cytb gene, range from 0 to 2.28%, whereas 2n varies from 41 to 70, and autosomal fundamental numbers (NFa) from 72 to 84. Nevertheless, cytb appears to be more informative for phylogenetic reconstructions compared to nuclear markers. Published data on partial sequences of XIST and Sox9 revealed no differences for E. talpinus and E. tancrei , Bagheri-Fam et al. 2012. Our data on fragments of Eif2s3x and Eif2s3y for E. talpinus, E. tancrei, and E. alaicus also reveal no changes in the exonic part of the genes (Matveevsky et al. 2017). The cryptic Ellobius species are rather young ones, so this may be why nuclear DNA markers were insufficient. However, our new data on XIST and Rspo1 variability demonstrated apparent clustering for all species of the Ellobius subgenus despite interspecific genetic distances were rather low and relatively high difference of E. tancrei specimens from Tajikistan and Uzbekistan, as nuclear markers of the latest (specimen 25159) could not be assigned to any of the two clades.
Originally, E. alaicus was described as a species with specific karyotype structure, including a pair of very large bi-armed chromosomes (Vorontsov et al. 1969, Lyapunova, Vorontsov 1978. Now we proved, that this Rb(2.11) metacentric is the same as in the E. tancrei forms with 2n = 30 and 2n = 48 from the northern bank of the Surkhob River (Bakloushinskaya et al. 2013, but not the Rb(2.18) as in the form with 2n = 50 from the opposite bank of the river. Moreover, translocations Rb(1.3), Rb(4.9), and Rb(3.10) were revealed in the Alay mole voles only. Thus, the Alay mole vole generated a distinctive Robertsonian variability with special structure that highlights genetic distinctness of this species compared to E. tancrei. No specimens with 2n = 52 and a single pair of Rb(2.11) were found among over 400 studied E. tancrei with Rb translocations (Bakloushinskaya, Lyapunova 2003). Probably, the translocation Rb(2.11) originated independently in E. alaicus and E. tancrei. The results of the phylogenetic analyses support this assumption because both ML and BI trees demonstrated distant positions for E. alaicus and E. tancrei specimens carrying Rb(2.11). Their relationships were established indirectly through Uzbekistan and South-West Tajikistan populations of E. tancrei, which have no any Robertsonian translocations (Fig. 7).
Here, we demonstrated, that the synapsis and behaviour of E. alaicus (2n = 48) meiotic chromosomes were very similar to E. tancrei and E. talpinus ones (Kolomiets et al.   Bakloushinskaya. 1991, Bakloushinskaya et al. 2012, Matveevsky et al. 2016. Isomorphic sex chromosomes exhibit a functional heteromorphism in the meiotic prophase I in all three species, that is a unique case for mammals. Therefore, characteristic nucleotide substitutions in mitochondrial and nuclear genes, distinct Rbs variability and independent origin of typical for E. alaicus translocation Rb(2.11) support the species status of the Alay mole vole notwithstanding the closeness to E. tancrei.
As we mentioned previously (Bogdanov et al. 2015), the differentiation of wideranging steppe species E. talpinus has occurred because of isolation due to geographic barriers, for example, large rivers such as the Volga River and the Irtysh River. E. tancrei and E. alaicus inhabit mountainous steppes and alpine meadows. Mountain ranges might be the most important geographic barriers for the spreading of mole voles because the animals do not inhabit mountains higher than 3500-4000 m above sea level. In the Tien Shan, the Pamir and the Pamir-Alay a distribution of mole voles should be sporadic because suitable habitats are mosaic. The complex orography of the regions may be a main source for geographical separation and ensuing fixation of the chromosomal forms (Bush et al. 1977). The situation is further complicated by the rapid change in the landscape due to neotectonic activity. The Alay Valley is an asymmetric intra-montane sedimentary basin with an average elevation of 2700 m, which formed in response to the convergence between India and Eurasia during the late Cenozoic (Coutand et al. 2002). The Pamir continues to move northward with a large fraction absorbed near the Alay Valley. The highest observed rate of the North-South convergence is between 10 and 15 mm/year as derived from Global Positioning System (GPS) measurements (Zubovich et al. 2016). The Pamir-Tien Shan region accommodates a high deformation over a short distance and is capable of producing magnitude 7 earthquakes in nearly decadal repeat times (Storchak et al. 2013). The last large seismic event was the 2008 magnitude 6.6 Nura earthquake with an epicenter just east of the Alay Valley (Sippl et al. 2014). Large earthquakes, which appeared to be in the Tien Shan and the Pamir, can trigger landslides (Havenith et al. 2003). Mudflows and landslides may quickly separate habitats of subterranean mole voles (Vorontsov and Lyapunova 1984). All three E. alaicus forms (2n = 52, 50 and 48) live in valleys, which are bordered by the mountain ranges. The evident pathways for mole voles spreading are the river banks in canyons crossing the ridges. Mole voles have a complex system of burrows, with at least three horizontal levels and numerous vertical connecting tunnels. But sometimes, most often at night, the animals run out onto the surface and move quickly over the ground. They probably can use human-made bridges, which are often destroyed by flows; new bridges may open a new route for mole voles. The suggestion was inspired when bursts of variations in chromosome numbers in mole voles from the opposite banks of the Vakhsh River were discovered at places close to bridges . In some cases, as when mole voles inhabit opposite banks of the Kyzyl-Suu River in a deep canyon (localities # 8,9,Figs 1,9,10), we can only explain how animals cross a mountain river if we assume that they use human-made bridges.
Despite a complex relief of the region, the geographical barriers are not as strong as genomic ones. We revealed no signs of hybridization in neighbor populations of E. alaicus and E. tancrei yet, i.e. between E. alaicus (2n = 48, locality #8, Fig. 1) and E. tancrei (2n = 30, locality # 16, Fig. 1) or E. alaicus (2n = 48, locality #12, Fig. 1) and E. tancrei (2n = 54, locality # 13, Fig. 1). There are no geographical barriers preventing active contact between these populations in about ten or even few kilometers. In such cases, the assumption that genomic (chromosomal) reorganization in mammals is often rapid (Vorontsov, Lyapunova 1989, Bakloushinskaya 2016, Dobigny et al. 2017) seems plausible, if one considers that polymorphism for isolation traits segregates within populations with different genetic compositions and ecological settings. If we assume that loci, which may contribute to a reproductive barrier, are dispersed throughout the genome, and intragenomic interactions that arise from genetic pathways can maintain species-specific differences (Lindtke and Buerkle 2015, Payseur and Rieseberg 2016), we can consider speciation starting with chromosome changes as a reliable and fast way of speciation.

Conclusion
The study of E. alaicus demonstrates that the difficulty of species delimitation due to lack of morphological differences might be resolved by using chromosomal and molecular markers.
We assumed, that the independent emergence of Robertsonian translocation Rb(2.11) was crucial for the divergence of ancestors of E. alaicus and E. tancrei, which both developed specific karyotypic variability, more extensive in E. tancrei (2n = 54-30) but distinct due to non-homological (except Rb(2.11)) translocations in E. alaicus (2n = 52-48). Notwithstanding, the closeness of species, which was demonstrated here by studying mitochondrial DNA (cytb) and fragments of two nuclear genes, determines the possibility of sporadic hybridization at the zones of species contacts. Using different cytogenetic methods, G-banding and chromosome painting, along with by cytb, XIST, and Rspo1 genes sequencing allowed us to expand the range of E. alaicus from the terra typica, the Alay Valley (South Kyrgyzstan) up to the Ferghana Ridge and the Naryn Basin, Tien Shan at the north-east and to the Pamir-Alay Mountains (Tajikistan) at the west.