An integrative analysis of an isolated population of Harting’s vole (Microtus hartingi) from the Eastern Rhodope Mountains (Bulgaria) was carried out by morphological and morphometric methods, computed tomography, Cytb variation data, and experimental hybridization. Substantial changes in the development of the skull and teeth were found. Nevertheless, those voles can live to the senex stage. A phylogenetic reconstruction based on Cytb sequences showed that M. hartingi from the Eastern Rhodopes forms a separate clade, which is a sister clade to the voles from Northeastern Greece (also from the foothills of the Rhodopes). M. hartingi from the Rhodopes is mostly isolated reproductively from M. h. ankaraensis from Anatolia (Turkey), as evidenced by a decrease in the proportion of pairs that started breeding, relatively high mortality of pups, and increased sterility of hybrid males. Possible time of isolation of the Rhodopean population and consequences of the “bottleneck” effect on its current state are discussed. At the same time, we believe that, at present, it is impossible to determine its taxonomic status. It is necessary to recognize M. hartingi from the Bulgarian Rhodopes as an endangered population and to include it in regional Red Lists of Bulgaria.

The adaptive effects of inbreeding are understudied, but accumulating evidence suggests that it plays a major adaptive role in speciation, as populations with a history of inbreeding are more prone to it. Differences in inbreeding tolerance in two populations of Microtus hartingi voles were studied through a series of experiments on reproductive behaviour and partner choice. Modelling an artificial polygyny via the formation of trios composed of a brother, sister, and an unrelated female has shown an uneven attitude towards inbreeding, which was closely linked to the reproduction strategy: voles of the Rhodopean population (RP) would reproduce communally with only a moderate decrease in reproduction success, while in monogamous M. hartingi ankaraensis voles from Central Anatolia (CAP), the reproductive success dropped almost to zero due to severe social stress resulting in heavily antagonistic behaviour between females; aggression levels rose with maturation. Studies of reproduction in pairs of relatives revealed the absence of inbreeding avoidance in the polygynous population (RP) and a sharp decrease in reproduction probability in the monogamous subspecies (CAP). Olfactory three-choice behavioural tests have proven that voles do not choose the smell randomly, juveniles do not have partner preferences, and adults choose the unrelated individual’s odour. RP voles prefer to mate with kin first, whereas CAP voles mate with non-kin first. The differences in reproductive strategy might have evolved due to habitat fragmentation and restriction of natal dispersal in the RP which has been isolated from the ancient Anatolian Microtus hartingi population since the Pleistocene. The adaptation to communal reproduction via kin selection resulted in increased inbreeding tolerance.

We analyzed the cranium dorsal projection and the mandible lateral projection in bone specimens from five Microtus guentheri and Microtus hartingi forms by geometric morphometrics (GM) methods (generalized Procrustes analysis, principal component analysis, canonical variance analysis, and discriminant function analysis). Analyses of the linear size and shapes of the cranium and lower jaw showed clear-cut differentiation among the forms into an eastern cluster and western cluster, matching M. guentheri and M. hartingi, respectively. Differences were revealed both between two subspecies of M. guentheri and between the subspecies M. h. strandzensis and Rhodopean M. hartingi, whose subspecies status has not yet been determined. M. h. ankaraensis bone specimens differ in many parameters of GM from the studied European specimens and to a lesser extent from M. g. guentheri and M. g. philistinus. Calculated morpho-ecological indices of the lower jaw revealed significant differences among all these forms, thereby possibly indicating adaptation of each to a specific habitat and dietary habits. Because of the emergence of impenetrable barriers for voles (the Anatolian Diagonal in the east and the Dardanelles and Bosporus in the west), the resultant vole groups have evolved independently.

Successful reproduction of two subspecies of Harting’s vole in monogamous pairs (monogamous groups, 35 pairs for each) was shown. The results obtained in modeling the experimental polygyny revealed a different attitude of females of the two subspecies to the formation of communal groups. In polygynous groups of M. h. lydius, regardless of the relationship of the females (35 groups consisting of sisters and 20 groups of unrelated females), the intensity of reproduction and the proportion of successfully reared pups decreased significantly compared to the monogamous groups. With high energy costs for reproduction, there were significantly fewer reared pups per mother giving birth than in the monogamous groups, thus suggesting the predominance of monogamy in this subspecies. On the contrary, related females of M. h. hartingi bred successfully in communal groups (35 groups of sisters), demonstrating a high tolerance, a tendency to co-rearing and feeding the pups, and low infant mortality. In polygynous groups consisting of unrelated females (20 groups), reproductive success decreased due to increased infant mortality. Possible benefits of communal reproduction in this subspecies are discussed, as well as the likely relationship with habitat fragmentation under such conditions and with a lack of vacancies for the dispersal of young animals, which may be an important adaptation for this subspecies of voles.