Distribution and evolution of the western European water frogs (genus Pelophylax) from Catalonia, northeastern Spain.

European water frogs from the genus Pelophylax are particular among amphibians as they can produce hybrids (named kleptons) reproducing by hybridogenesis. Four klepton species have been described in Europe: Pelophylax kl. esculentus, P. kl. hispanicus, P. kl. grafi, and the putative klepton PK. While most of these kleptons originated naturally in areas where the parental species occur in sympatry, human-mediated translocations of water frogs across Europe have altered this dynamic. As a result, several Pelophylax species and kleptons are now found outside their natural ranges, posing a threat to autochthonous water frogs. Additionally, the subtle morphological differences between Pelophylax species make hybrid identification and, thus, conservation difficult. In the present study, we analyzed 423 specimens from 54 populations sampled across Catalonia and implemented a two-step molecular method to identify all species of water frogs present in Catalonia. We also examined the mitochondrial genome of the hybrid Pelophylax kl. grafi to obtain new insights into their reproductive system and spatial structure. Despite the large number of samples analyzed, only the native P. perezi and its klepton P. kl. grafi were found, with the proportion of the latter being unexpectedly high. Results showed a high misidentification rate based on morphology compared to molecular methods, indicating that identification of P. kl. grafi through morphological characters is unreliable. Furthermore, the mitochondrial DNA of hybrid specimens entirely belonged to P. perezi and showed high intra-specific variability. This suggests either a single hybridization event involving a male P. ridibundus or P. kl. esculentus and a female P. perezi, or that P. ridibundus mitochondrial DNA has been eliminated from the P. kl. grafi germline by adaptive or non-adaptive processes. This study offers new insights into the distribution and composition of the North Iberian Pelophylax hybridogenetic complex, providing comprehensive sampling across one of the main entry points of the complex into the Iberian Peninsula.