Speciation on the Roof of the World: Parallel Fast Evolution of Cryptic Mole Vole Species in the Pamir-Alay-Tien Shan Region.

Speciation is not always accompanied by morphological changes; numerous cryptic closely related species were revealed using genetic methods. In natural populations of Ellobius tancrei (2n = 54-30) and E. alaicus (2n = 52-48) of the Pamir-Alay and Tien Shan, the chromosomal variability due to Robertsonian translocations has been revealed. Here, by comprehensive genetic analysis (karyological analyses as well as sequencing of mitochondrial genes, cytb and COI, and nuclear genes, XIST and IRBP) of E. alaicus and E. tancrei samples from the Inner Tien Shan, the Alay Valley, and the Pamir-Alay, we demonstrated fast and independent diversification of these species. We described an incompletely consistent polymorphism of the mitochondrial and nuclear markers, which arose presumably because of habitat fragmentation in the highlands, rapid karyotype changes, and hybridization of different intraspecific varieties and species. The most intriguing results are a low level of genetic distances calculated from mitochondrial and nuclear genes between some phylogenetic lines of E. tancrei and E. alaicus, as well significant species-specific chromosome variability in both species. The chromosomal rearrangements are what most clearly define species specificity and provide further diversification. The "mosaicism" and inconsistency in polymorphism patterns are evidence of rapid speciation in these mammals.

Nonhomologous Chromosome Interactions in Prophase I: Dynamics of Bizarre Meiotic Contacts in the Alay Mole Vole Ellobius alaicus (Mammalia, Rodentia).

Nonhomologous chromosome interactions take place in both somatic and meiotic cells. Prior to this study, we had discovered special contacts through the SYCP3 (synaptonemal complex protein 3) filament between the short arms of nonhomologous acrocentrics at the pachytene stage in the Alay mole vole, and these contacts demonstrate several patterns from proximity to the complete fusion stage. Here, we investigated the nonhomologous chromosome contacts in meiotic prophase I. It turned out that such contacts do not introduce changes into the classic distribution of DNA double-strand breaks. It is noteworthy that not all meiotic contacts were localized in the H3k9me3-positive heterochromatic environment. Both in the mid zygotene and in the early-mid diplotene, three types of contacts (proximity, touching, and anchoring/tethering) were observed, whereas fusion seems to be characteristic only for pachytene. The number of contacts in the mid pachytene is significantly higher than that in the zygotene, and the distance between centromeres in nonhomologous contacts is also the smallest in mid pachytene for all types of contacts. Thus, this work provides a new insight into the behavior of meiotic contacts during prophase I and points to avenues of further research.

Geographic Mosaic of Extensive Genetic Variations in Subterranean Mole Voles Ellobius alaicus as a Consequence of Habitat Fragmentation and Hybridization.

Restricted mobility, sociality, and high inbreeding-characteristic for subterranean mammals-lead to rapid changes in their genome structure. Up to now, the Alay mole vole Ellobius alaicus was a data-deficient species; its spatial and phylogenetic relationships with a sibling species, E. tancrei, were not clarified. We carried out a genetic analysis including differential G-banding of chromosomes and mitochondrial (cytb) and nuclear gene (XIST and IRBP) sequencing. The phylogenetic reconstruction based on cytb represented the expected phylogenetic relationships of two species. Using the XIST, we revealed two new lineages among E. alaicus from the Alay Valley (Southern Kyrgyzstan). Analysis of IRBP demonstrated presence of the specific genotype in most of E. alaicus specimens, but also revealed the haplotype, typical for E. tancrei, in some Alay mole voles. The results may be explained as persistence of ancestral gene polymorphism in E. alaicus or limited interspecific hybridization with E. tancrei. Several chromosomal forms were revealed in E. alaicus in the Alay Valley. We propose that 'mosaic' genetic polymorphism might appear in E. alaicus due to fragmentation of their habitats in highlands of the Alay Valley, Tien Shan, and Pamir-Alay as well as due to hybridization with E. tancrei or persistence of ancestral polymorphisms.

Sex differences in the meiotic behavior of an XX sex chromosome pair in males and females of the mole vole Ellobius tancrei: turning an X into a Y chromosome?

Sex determination in mammals is usually provided by a pair of chromosomes, XX in females and XY in males. Mole voles of the genus Ellobius are exceptions to this rule. In Ellobius tancrei, both males and females have a pair of XX chromosomes that are indistinguishable from each other in somatic cells. Nevertheless, several studies on Ellobius have reported that the two X chromosomes may have a differential organization and behavior during male meiosis. It has not yet been demonstrated if these differences also appear in female meiosis. To test this hypothesis, we have performed a comparative study of chromosome synapsis, recombination, and histone modifications during male and female meiosis in E. tancrei. We observed that synapsis between the two X chromosomes is limited to the short distal (telomeric) regions of the chromosomes in males, leaving the central region completely unsynapsed. This uneven behavior of sex chromosomes during male meiosis is accompanied by structural modifications of one of the X chromosomes, whose axial element tends to appear fragmented, accumulates the heterochromatin mark H3K9me3, and is associated with a specific nuclear body that accumulates epigenetic marks and proteins such as SUMO-1 and centromeric proteins but excludes others such as H3K4me, ubiH2A, and γH2AX. Unexpectedly, sex chromosome synapsis is delayed in female meiosis, leaving the central region unsynapsed during early pachytene. This region accumulates γH2AX up to the stage in which synapsis is completed. However, there are no structural or epigenetic differences similar to those found in males in either of the two X chromosomes. Finally, we observed that recombination in the sex chromosomes is restricted in both sexes. In males, crossover-associated MLH1 foci are located exclusively in the distal regions, indicating incipient differentiation of one of the sex chromosomes into a neo-Y. Notably, in female meiosis, the central region of the X chromosome is also devoid of MLH1 foci, revealing a lack of recombination, possibly due to insufficient homology. Overall, these results reveal new clues about the origin and evolution of sex chromosomes.

Kinase CDK2 in Mammalian Meiotic Prophase I: Screening for Hetero- and Homomorphic Sex Chromosomes.

Cyclin-dependent kinases (CDKs) are crucial regulators of the eukaryotic cell cycle. The critical role of CDK2 in the progression of meiosis was demonstrated in a single mammalian species, the mouse. We used immunocytochemistry to study the localization of CDK2 during meiosis in seven rodent species that possess hetero- and homomorphic male sex chromosomes. To compare the distribution of CDK2 in XY and XX male sex chromosomes, we performed multi-round immunostaining of a number of marker proteins in meiotic chromosomes of the rat and subterranean mole voles. Antibodies to the following proteins were used: RAD51, a member of the double-stranded DNA break repair machinery; MLH1, a component of the DNA mismatch repair system; and SUN1, which is involved in the connection between the meiotic telomeres and nuclear envelope, alongside the synaptic protein SYCP3 and kinetochore marker CREST. Using an enhanced protocol, we were able to assess the distribution of as many as four separate proteins in the same meiotic cell. We showed that during prophase I, CDK2 localizes to telomeric and interstitial regions of autosomes in all species investigated (rat, vole, hamster, subterranean mole voles, and mole rats). In sex bivalents following synaptic specificity, the CDK2 signals were distributed in three different modes. In the XY bivalent in the rat and mole rat, we detected numerous CDK2 signals in asynaptic regions and a single CDK2 focus on synaptic segments, similar to the mouse sex chromosomes. In the mole voles, which have unique XX sex chromosomes in males, CDK2 signals were nevertheless distributed similarly to the rat XY sex chromosomes. In the vole, sex chromosomes did not synapse, but demonstrated CDK2 signals of varying intensity, similar to the rat X and Y chromosomes. In female mole voles, the XX bivalent had CDK2 pattern similar to autosomes of all species. In the hamster, CDK2 signals were revealed in telomeric regions in the short synaptic segment of the sex bivalent. We found that CDK2 signals colocalize with SUN1 and MLH1 signals in meiotic chromosomes in rats and mole voles, similar to the mouse. The difference in CDK2 manifestation at the prophase I sex chromosomes can be considered an example of the rapid chromosome evolution in mammals.

Meiotic Nuclear Architecture in Distinct Mole Vole Hybrids with Robertsonian Translocations: Chromosome Chains, Stretched Centromeres, and Distorted Recombination.

Genome functioning in hybrids faces inconsistency. This mismatch is manifested clearly in meiosis during chromosome synapsis and recombination. Species with chromosomal variability can be a model for exploring genomic battles with high visibility due to the use of advanced immunocytochemical methods. We studied synaptonemal complexes (SC) and prophase I processes in 44-chromosome intraspecific (Ellobius tancrei × E. tancrei) and interspecific (Ellobius talpinus × E. tancrei) hybrid mole voles heterozygous for 10 Robertsonian translocations. The same pachytene failures were found for both types of hybrids. In the intraspecific hybrid, the chains were visible in the pachytene stage, then 10 closed SC trivalents formed in the late pachytene and diplotene stage. In the interspecific hybrid, as a rule, SC trivalents composed the SC chains and rarely could form closed configurations. Metacentrics involved with SC trivalents had stretched centromeres in interspecific hybrids. Linkage between neighboring SC trivalents was maintained by stretched centromeric regions of acrocentrics. This centromeric plasticity in structure and dynamics of SC trivalents was found for the first time. We assume that stretched centromeres were a marker of altered nuclear architecture in heterozygotes due to differences in the ancestral chromosomal territories of the parental species. Restructuring of the intranuclear organization and meiotic disturbances can contribute to the sterility of interspecific hybrids, and lead to the reproductive isolation of studied species.

Meiotic Chromosome Contacts as a Plausible Prelude for Robertsonian Translocations.

Robertsonian translocations are common chromosomal alterations. Chromosome variability affects human health and natural evolution. Despite the significance of such mutations, no mechanisms explaining the emergence of such translocations have yet been demonstrated. Several models have explored possible changes in interphase nuclei. Evidence for non-homologous chromosomes end joining in meiosis is scarce, and is often limited to uncovering mechanisms in damaged cells only. This study presents a primarily qualitative analysis of contacts of non-homologous chromosomes by short arms, during meiotic prophase I in the mole vole, Ellobius alaicus, a species with a variable karyotype, due to Robertsonian translocations. Immunocytochemical staining of spermatocytes demonstrated the presence of four contact types for non-homologous chromosomes in meiotic prophase I: (1) proximity, (2) touching, (3) anchoring/tethering, and (4) fusion. Our results suggest distinct mechanisms for chromosomal interactions in meiosis. Thus, we propose to change the translocation mechanism model from 'contact first' to 'contact first in meiosis'.

The complete mitochondrial genomes of three Ellobius mole vole species (Rodentia: Arvicolinae).

The subterranean voles of the genus Ellobius are species of subfamily Arvicolinae well adapted to underground life. In this paper, we report the assemblies of complete mitochondrial genomes for three mole voles from genus Ellobius - northern mole vole Ellobius talpinus (16,376 bp), transcaucasian mole vole E. lutescens (16,540 bp), and southern mole vole E. fuscocapillus (16,388 bp). Each of three mitogenomes encode for 12S and 16S rRNAs, 22 tRNAs, 13 protein-coding genes, and D-loop in the characteristic arrangement of subfamily Arvicolinae (Rodentia: Cricetidae). This study verifies the evolutionary status of subgenera Bramus and Ellobius within the genus Ellobius at the molecular level. The mitochondrial genome would be a significant supplement for the Ellobius genetic background. The three Ellobius species formed a monophyletic group with the high bootstrap value (100%) in all examinations.

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

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.

Fathers and sons: Physiological stress in male Zaisan mole voles, Ellobius tancrei.

The social environment can be stressful for at least some group members, resulting in elevated levels of glucocorticoid stress hormones (GC). Patterns of the relationships between social rank and GC levels vary between species. In carnivores, primates and birds that live in permanent cooperative groups, helpers do not usually display physiological indicators of stress. Very little is known about status-related GC differences within cooperative groups of rodents. In this laboratory study, we compared GC concentrations in dominant (fathers) and subordinate (natal sons) males of a cooperative subterranean vole, Ellobius tancrei. The assessment of adrenocortical activity by measuring urine glucocorticoid metabolites (UGM) was previously validated for this species through an ACTH challenge test. We observed clear peaks of UGM in the second or third urine samples taken after the administration of ACTH (lag time equal to 2.5-3 h). Thus, UGM is suitable to estimate physiological stress in Ellobius. Postpubertal sons living in natal groups had significantly higher UGM concentrations than their fathers. The average UGM levels of sons were positively associated with their ages and paternal body masses, and negatively associated with paternal ages. Hence, son-father interactions rather than just younger ages of sons appear to contribute to GC differences. The revealed pattern was not consistent with that reported for most cooperative species from other taxa, highlighting the importance of comparative studies.

Chromosomal Evolution in Mole Voles Ellobius (Cricetidae, Rodentia): Bizarre Sex Chromosomes, Variable Autosomes and Meiosis.

This study reports on extensive experimental material covering more than 30 years of studying the genetics of mole voles. Sex chromosomes of Ellobius demonstrate an extraordinary case of mammalian sex chromosomes evolution. Five species of mole voles own three types of sex chromosomes; typical for placentals: XY♂/XX♀; and atypical X0♂/X0♀; or XX♂/XX♀. Mechanisms of sex determination in all Ellobius species remain enigmatic. It was supposed that the Y chromosome was lost twice and independently in subgenera Bramus and Ellobius. Previous to the Y being lost, the X chromosome in distinct species obtained some parts of the Y chromosome, with or without Sry, and accumulated one or several copies of the Eif2s3y gene. Along with enormous variations of sex chromosomes, genes of sex determination pathway and autosomes, and five mole vole species demonstrate ability to establish different meiotic mechanisms, which stabilize their genetic systems and make it possible to overcome the evolutionary deadlocks.

Nasal aerodynamics protects brain and lung from inhaled dust in subterranean diggers, Ellobius talpinus.

Inhalation of air-dispersed sub-micrometre and nano-sized particles presents a risk factor for animal and human health. Here, we show that nasal aerodynamics plays a pivotal role in the protection of the subterranean mole vole Ellobius talpinus from an increased exposure to nano-aerosols. Quantitative simulation of particle flow has shown that their deposition on the total surface of the nasal cavity is higher in the mole vole than in a terrestrial rodent Mus musculus (mouse), but lower on the olfactory epithelium. In agreement with simulation results, we found a reduced accumulation of manganese in olfactory bulbs of mole voles in comparison with mice after the inhalation of nano-sized MnCl2 aerosols. We ruled out the possibility that this reduction is owing to a lower transportation from epithelium to brain in the mole vole as intranasal instillations of MnCl2 solution and hydrated nanoparticles of manganese oxide MnO · (H2O)x revealed similar uptake rates for both species. Together, we conclude that nasal geometry contributes to the protection of brain and lung from accumulation of air-dispersed particles in mole voles.

A new form of the mole vole Ellobius tancrei Blasius, 1884 (Mammalia, Rodentia) with the lowest chromosome number.

The subterranean mole vole, Ellobius tancrei, with aspecific variability in autosomes (2n = 31-54) and unusual sex chromosomes (XX in males and females), represents an amazing model for studying the role of chromosome changes in speciation. New materials from the upper reaches of the Surkhob River in the Pamiro-Alay mountains resulted in the discovery of a new form with 2n = 30. The application of Zoo-FISH and G-banding methods allowed the detection of 13 pairs of autosomes as Robertsonian metacentrics originated after fusions of acrocentrics of an assumed ancestral karyotype of Ellobius tancrei with 2n = 54. The sex chromosomes (XX, in both sexes) and one pair of acrocentric autosomes are the only acrocentrics in this karyotype, and the set with 2n = 30 possesses the lowest possible chromosome number among populations of Ellobius tancrei.