Molecular detection of hybridogenetic pattern in the Pelophylax esculentus complex in Italy

Hybridogenesis is an extremely rare form of sexual parasitism. A case in point is the Pelophylax esculentus complex that is composed by three morphologically cryptic species: the parental P. lessonae and P. ridibundus, and their fertile hybrid P. kl. esculentus. As the hybridogenetic condition depends on the backcross of the hybrid, which in its gametes is able to only transmit the “ridibundus” genome excluding the P. lessonae-DNA, this system can only exist in the absence of P. ridibundus. Nowadays, translocation of alien waterfrogs of the P. ridibundus clade from eastern and western Europe is threatening the persistence of this hybridogenetic system in the Po Plain, leading to the extinction of P. lessonae, the reduction of P. esculentus hybrids, and the establishment of new hybrid lineages with unknown reproductive outcome. From an evolutionary point of view, this scenario offers the opportunity to investigate the effects of alien genome introgression among species and the mechanisms beneath the hybridogenetic complex. Our aim is to study hybridization and genome exclusion adopting a two-way integrative approach that combines whole genome sequencing, through ddRAD-seq (double digested Restriction-site-Associated DNA sequencing), and GISH (Genomic In Situ Hybridization) techniques. To do so we identified pure parental lineages with disagnostic mtDNA and SSR markers, and crossed them by in vitro fertilizations. Obtained tadpoles have been reared indoor until they reached the gametogenesis stage and then analyzed with the approach upmentioned. In vitro crossing will allow us to compare gDNA information of hybrid offspring and its direct parents. Also, GISH with parental genomic DNA probes will allow us to confirm and strengthen the results. The results will represent a unique resource to study how hybridogenesis works, and to investigate the effects of alien species introduction, thus providing important information to draw up conservation guidelines.