Cuevas, A., C. Patrelle, F. Ciavatti, T. Gendre, P. Sourrouille, P. Geniez, P. Doniol-Valcroze & P.-A. Crochet

A new PCR-RFLP method for the identification of parental and hybridogenetic western European Water Frogs, including the Pelophylax perezi-grafi system. pp. 218-230 plus Supplementary File 1 (xls), Supplementary File 2 (pdf), Supplementary File 3 (txt), Supplementary File 4 (txt), Supplementary File 5 (pdf), Supplementary File 6 (txt), Supplementary File 7 (txt), Supplementary File 8 (txt), Supplementary File 9 (pdf).

Abstract. Western European Water Frog species can be morphologically very similar, especially Pelophylax ridibun­dus and P. perezi. As these species, together with P. lessonae, produce hybridogenetic lineages with highly variable phenotypes, the morphological identification of taxa in this genus remains challenging. This is especially problematic in the current context of the spread of the invasive P. ridibundus complex in native populations across Western Europe. In this study, we present a novel molecular method for the identification of western European Water Frogs (genus Pelophylax) based on the PCR-RFLP genotyping of nuclear species-specific loci. Using a combination of restriction digestions of species-specific genetic markers, this method facilitates reliable specific identification in the Pelophylax perezi-grafi system (PG system) and the Pelophylax lessonae-esculentus system (LE system). Our method provides several advantages compared to other genetic techniques previously used. It relies on PCR amplification allowing non-invasive sampling followed by RFLP to assess diagnostic SNPs in the nuclear genome, rendering obsolete the need for sequencing. Lastly, it is easier to replicate than methods based on microsatellites. Importantly, this novel protocol allows the identification of all five taxa of Water Frogs that occur in Western Europe.

Key words. Amphibia, Anura, Ranidae, species identification, hybridogenetic species complex, conservation, invasive species.