Difficulties in species identification in water frogs (genus Pelophylax) using morphological and molecular markers in The Netherlands

Several molecular protocols have been published to rapidly identify water frog species (genus Pelophylax) at low costs by genotyping without sequencing. Here, we compare identifications based on one morphological character (the shape of the metatarsal tubercle) with identifications based on one mitochondrial (COI) and one nuclear marker (SAI-1) – for which the PCR products shows species-diagnostic length polymorphism – for three populations (19 individuals) representative of P. lessonae, P. ridibundus, and their hybrid P. esculentus; the three species present in The Netherlands. Species identification based on the shape of the metatarsal tubercle is consistent with the known distribution of water frogs in the country. However, species identification based on genetic markers yielded discordant patterns, namely that P. lessonae occurs in the western Netherlands, where it is presumably absent. We discuss potential natural (introgression of mtDNA) and technical (unsuitability of SAI-1 for species identification in The Netherlands) reasons that could explain the discrepancies. Before reliable molecular identification based on genome-wide loci data becomes easily accessible, morphological and acoustic characteristics remain the most reliable way to identify Pelophylax populations in The Netherlands.