A new PCR-RFLP-based method for an easier systematic affiliation of European water frogs

We describe a non-invasive, PCR-RFLP-based method that allows reliable determination of the European water frog species Pelophylax lessonae and Pelophylax ridibundus and the hybrid form Pelophylax esculentus. Maximum-likelihood analysis of ITS2 sequences revealed two robust monophyletic clades corresponding to water frogs of the P. lessonae and P. ridibundus groups. Three restriction enzymes (KpnI, HaeII, and SmaI) were used to digest three conserved ITS2 domains. Taxonomic identification was unambiguous; the three restriction enzymes gave the same results. A French reference sample was identified using allozyme electrophoresis. Our PCR-RFLP method confirmed circa 83% of identification of the allozyme method. We conclude that the difference between identifications was caused by introgression.