Karyotype stasis but species-specific repetitive DNA patterns in Anguis lizards (Anguidae), in the evolutionary framework of Anguiformes

Karyotype divergence may strongly affect the rate of hybridization between species in their secondary contact zones. Slow worms ( Anguis , Anguidae) are morphologically relatively cryptic legless lizards representing two evolutionary lineages, A. cephallonica from the southernmost Balkans, and the A. fragilis species complex (comprising two sister-species pairs A. fragilis  +  A. veronensis and A. colchica  +  A. graeca ) distributed in the Western Palearctic. To identify their level of chromosomal variation, we surveyed karyotype of all species except formerly studied A. veronensis and included Pseudopus apodus as an outgroup. We applied conventional and molecular cytogenetic methods and whole-chromosome painting using macrochromosome probes from Varanus komodoensis and interpreted the results within the evolutionary framework of the common clade Anguiformes. Unlike New World anguids with remarkable karyotype variation, all Anguis species and P. apodus have conserved diploid chromosome number 2 n  = 44 (20 macrochromosomes, 24 microchromosomes) and morphology. The sister species A. colchica and A. graeca (divergence 4.4 Mya) displayed highly similar karyotype features within our sample. In contrast, despite the generally conserved chromosome morphology, the phylogenetically older A. cephallonica (divergence 12.0 Mya), and A. colchica versus A. fragilis (divergence 6.7 Mya) exhibited distinct patterns of constitutive heterochromatin distribution and telomeric repeat accumulation. The chromosome painting revealed that slow worm karyotype mostly evolved by fissions of ancestral macrochromosomes, which likely occurred already in an Anguis  +  Pseudopus common ancestor. Our findings thus demonstrate karyotype stasis in Anguis and Pseudopus for > 25 My, with fixed species-specific differences which may serve as cytotaxonomic markers useful in hybrid zone studies of slow worms.