Mitochondrial ghost lineages blur phylogeography and taxonomy of Natrix helvetica and N. natrix in Italy and Corsica

Grass snakes are widely distributed across the Western Palearctic. Recent phylogeographic studies provided evidence that three distinct parapatric species exist. Two of these occur in Italy, Natrix helvetica and N. natrix, and a contact zone between both taxa has been suggested for north-eastern Italy. Moreover, previous investigations revealed for the Italian Peninsula a complex phylogeographic structure. Using mtDNA sequences and microsatellite loci, we examined the situation for mainland Italy, Sicily, Sardinia, and Corsica. Our study confirmed the occurrence of N. natrix in north-eastern Italy. Cline analyses revealed limited gene flow between N. helvetica and N. natrix across a narrow hybrid zone. Within N. helvetica, conflicting patterns of mitochondrial and nuclear genomic differentiation were revealed. Three nuclear genomic clusters were found; one of them corresponded to no fewer than five distinct and, in part, deeply divergent and ancient mitochondrial lineages from mainland Italy and Sicily. This cluster was paraphyletic with respect to the two remaining mitochondrial lineages, each of which matched with another nuclear genomic cluster (one from Corsica plus Sardinia and another one from western Europe north of the Alps). This unexpected pattern most likely results from mainly male-mediated gene flow and female philopatry combined with population-density-dependent processes such as ‘high-density blocking’. With respect to taxonomy, we propose to synonymize N. h. lanzai Kramer, 1970 with N. h. sicula (Cuvier, 1829), acknowledging their lacking nuclear genomic differentiation. The studied hybrid zone of N. h. helvetica and N. h. sicula in Italy is wide, with a smooth cline for nuclear markers, supporting their subspecies status. We found no evidence for the distinctiveness of the two subspecies from Corsica (N. h. corsa) and Sardinia (N. h. cetti), suggesting their synonymy, but refrain from taxonomic conclusions because of small sample sizes and the endangered status of the Sardinian taxon.