Phylogeny of slug species of the genus Arion: evidence of monophyly of Iberian endemics and of the existence of relict species in Pyrenean refuges

The Iberian Peninsula contains the majority of the Paleartic land slug species of the genus Arion, which exhibits diverse taxonomic problems. The present study investigated Arion taxonomy on the basis of analyses of the mitochondrial ND1 gene and nuclear internal transcribed spacer 1 (ITS1) sequences. The Iberian endemic species were monophyletically clustered in two divergent sister clades. The topotype specimens of Arion lusitanicus and the closely related species Arion nobrei and Arion fuligineus, as well as Arion hispanicus and Arion flagellus, were grouped into an ‘Atlantic’ clade, whereas Arion baeticus, Arion gilvus, Arion anguloi, Arion wiktori and Arion paularensis were included in a ‘Continental–Mediterranean’ clade. Calibration of mutation rate in the ND1 gene suggested that the divergence of these two clades occurred around the Pliocene–Pleistocene boundary, with subsequent speciation events during the Pleistocene. A group of ancestral and divergent endemic species with distribution centred in the Pyrenean mountain range (Arion molinae, Arion lizarrusti, Arion antrhacius and Arion iratii) arose in the Pliocene and survived through the Pleistocene in geographically confined small populations. Arion lusitanicus showed up to be polyphyletic: specimens, sampled outside the geographic range of the topotype in the north‐western Iberian Peninsula, were included in a non‐monophyletic clade together with the widely distributed species Arion ater and Arion rufus. The divergent species with a wide European distribution (Arion subfuscus, Arion hortensis, Arion fagophilus and Arion intermedius) were located in basal positions in all topologies. The evolutionary history of these slug species (highly sensitive to climatic factors, with capacity for both outcrossing and selfing, and with low dispersal ability) appears to have been moulded by Pliocene–Pleistocene climate events and by the rugged topography of southern Europe, giving rise to repeated cycles of population isolation during periods of glaciation alternating with interglacial expansions limited by geographic barriers.