Hybrid swarm as a result of hybridization between two alien and two native water frog species (genus Pelophylax) in Central Croatia: Crna Mlaka fishpond acting as a species melting pot?

Spreading of alien water frog species (genus Pelophylax) has been reported across Europe, posing a severe risk for the replacement or genetic swamping of indigenous species. Crna Mlaka fishponds in Central Croatia were an experimental station for freshwater fish aquaculture during the first half of twentieth century and hosted fish from different parts of Balkans. Crna Mlaka fishponds are thus estimated to be at higher risk of non-native frogs, likely introduced with the aquaculture industry, causing introgressive hybridization and genetic admixture of native Pelophylax species. We used nuclear and mitochondrial DNA sequences, in combination with microsatellite markers, to characterize water frogs of Crna Mlaka. We detected alleles of two native species, the Eurasian marsh frog Pelophylax ridibundus (Pallas, 1771) and the pool frog Pelophylax lessonae (Camerano, 1882), as well as non-native Albanian pool frog Pelophylax shqipericus (Hotz, Uzzell, Gunther, Tunner et Heppich, 1987) and Balkan pool frog Pelophylax kurtmuelleri (Gayda, 1940). Moreover, we found five hybrid forms: P. kurtmuelleri x P. ridibundus, P. kurtmuelleri x P. shqipericus, P. lessonae x P. kurtmuelleri, P. ridibundus x P. shqipericus, and P. lessonae x P. shqipericus indicating the existence of a hybrid swarm. The last three hybrids have never been reported in natural populations. Interestingly, although expected, no P. kl. esculentus (P. ridibundus x P. lessonae) was detected. Additionally, water frogs with introgressed mtDNA were detected, which results in cyto-nuclear discordance. The results of this study suggest that this is a unique case of species melting pot, a hybrid swarm emerging from two alien and two native frog species resulting in more than 50% of water frogs at this location being hybrids and/or having introgressed mtDNA and the loss of the hybridogenetic reproductive system of Pelophylax kl. esculentus.