Sex chromosome variability of Spermophilus citellus (Linnaeus, 1766) in the Southeastern part of the Balkan Peninsula

This work presents novel data that has allowed us to propose an evolutionary model for the karyotype of the European ground squirrel (Spermophilus citellus, L. 1766) based on sex chromosome variants. Routine C- and G-banding, specific fluorochrome staining (CMA3, DAPI) and FISH experiments were used to characterize the pattern of chromosomal variation in populations of S. citellus in the Southeastern part of the Balkan Peninsula, the ancestral area of the species with the highest genetic diversity. The established diploid chromosome number of S. citellus is 2n = 40. All autosomes are biarmed: 12 pairs of large to small submetacentrics, 5 pairs of large to small subtelocentrics, one medium-sized and one small pair of metacentrics. Three different morphologies are observed for both X and Y chromosomes. The X chromosomes are classified as acrocentric, subtelocentric, or submetacentric. In most of the individuals studied, the Y chromosome was dot-like, with the exception of a few presenting small or medium-sized acrocentrics and metacentrics. Blocks of heterochromatin of different sizes were found in the centromeric regions of all autosomes and X chromosomes, while all Y chromosome variants appeared to be entirely heterochromatic. CMA3 fluorescence was bright in the majority of the C-positive regions and in the acrocentric X chromosome. These results were supported by DAPI staining, which showed that the CMA3-positive sites were DAPI-negative and approximately of the same distinct sizes. In situ hybridization with the (TTAGGG)n probe produced a staining typical of telomeric repeats at the edges of all chromosomes. Notably, the Y chromosome showed stronger accumulation of telomeric repeats than that observed on other chromosomes. The nature of the C-, G- and fluorochrome staining of the sex chromosomes of S. citellus suggested that the amplification of constitutive heterochromatin and the pericentric inversions were likely sufficient to explain the differences between the morphology of sex chromosome. The observed sex chromosome variability reinforces the differences among the populations of the S. citellus in the Southeastern part of the Balkan Peninsula and correlate with specific geographical areas. These will be further investigated in order to fully evaluate the adaptive value of chromosome rearrangements.