The sibling species Microtus thomasi and M. atticus represent probably the highest karyotypic diversity within the genus Microtus and are an interesting model for chromosomal evolution studies. In addition to variation in autosomes, they show a high intraspecific variation in the size and morphology of both sex chromosomes. We analyzed individuals with different sex chromosome constitutions using 3 painting probes, 2 from Y chromosome variants and 1 from the small arm of the submetacentric X chromosome. Our comparative painting approach uncovered 12 variants of Y and 14 variants of X chromosomes, which demonstrates that the polymorphism of sex chromosomes is substantially larger than previously reported. We suggest that 2 main processes are responsible for this sex chromosome polymorphism: change of morphology from acrocentric to submetacentric or metacentric chromosomes and increase in size due to accumulation of repetitive DNA sequences, generating heterochromatic blocks. Strong genetic drift in small and fragmented populations of these 2 species could be related to the origin and maintenance of the large polymorphism of sex chromosomes. We proposed that a similar polymorphism variation combined with random drift fixing the biggest sex chromosomes could have occurred in the origin of some of the actual Microtus species with giant sex chromosomes.

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