Ecomorphological divergence and lack of gene flow in two sympatric Balkan slow worms (Squamata: Anguidae)

Abstract The slow-worm lizards of the genus Anguis show significant morphological, behavioural and ecological uniformity owing to their limbless, elongated bodies and semifossorial lifestyle. This makes the delimitation of species challenging. Five monophyletic, deeply divergent and mostly parapatric mitochondrial lineages are currently recognized, although hybridization occurs along contact zones. In the southern edge of the range of the genus, two endemic Balkan Anguis (Anguis graeca and Anguis cephallonica) share a broad area of co-distribution and intermediate morphological characteristics. Expecting to retrieve hybrids within the overlap in their distributions, we used genome-wide single nucleotide polymorphisms but did not recover any evidence of interspecific gene flow. Within each species, genomic data revealed shallow population structuring influenced by spatial isolation, with the exception of the southernmost population of A. cephallonica from the Mani Peninsula. Species distribution modelling showed a lack of strong niche overlap and specific environmental preferences; both species showed a precipitation dependence, but A. cephallonica was better acclimatized to high temperatures. Finally, geometric morphometrics of the head shape suggested morphological divergence, mostly seen in the elongation of the head of A. cephallonica. All the evidence supports a long and complete reproductive isolation between the two southern slow worms and raises questions about how ecomorphological divergence might have acted to promote their speciation.