Detection of alien Pelophylax species in Sardinia (western Mediterranean, Italy).
Sardinian herpetofauna represent a valuable biodiversity resource in the Mediterranean Basin. Among amphibians, most of the taxa are indeed endemic to the island. In contrast, water frogs of the Pelophylax genus are considered allochthonous to Sardinia, and are traditionally assigned to the Italian pool frog (P. bergeri) and its hybridogenetic hybrid (P. kl. hispanicus). Members of this genus were recently found in certain sites in northern and southern Sardinia, and ascribed to the "ridibundus" group (the "Marsh frog" species complex). As the absence of clear phenotypic diagnostic features complicates the identification of distinct Pelophylax species, particularly European P. ridibundus, P. kurtmuelleri and several still undescribed "bedriagae" taxa, an informative diagnostic mitochondrial marker (ND3) was isolated in this work in order to detect the occurrence of distinct lineages in alien Sardinian populations. As water frogs are known to readily hybridize in the wild, two further diagnostic nuclear markers (SAI-1 and 4 microsatellite loci) were subsequently screened in order to exclude the occurrence of hybrid genotypes. According to our results, three distinct taxa were detected on the island, and assigned to P. kurtmuelleri (the Balkan frog), and two cryptic taxa of the "bedriagae" group. Nuclear markers only successfully amplified alleles diagnostic for the "ridibundus"-type (R) genome. Accordingly, we can exclude hybridization with the Italian pool frog already introduced on the island, although further studies are needed to better address the occurrence of new hybrid lineages stemming from the cross between the taxa detected in this work. As eggs and tadpoles have been detected in the field, alien populations can thus be considered successfully established in Sardinia. Our study revealed for the first time the occurrence of the alien Balkan frog in the western Mediterranean. It is worthy of note that this taxon shows a high invasion potential in mainland Europe. The establishment of alien Pelophylax populations in Sardinia may be favored by vacant niches. Nonetheless, the dry hot season could limit their expansion along rivers.