Parsley frogs (Pelodytes) comprise the only genus in the family Pelodytidae, an ancient anuran lineage that split from their closest relatives over 140 million years ago. Pelodytes is a Palearctic group restricted to Western Eurasia including three extant species: the eastern species P. caucasicus, endemic to the Caucasus area, and two closely related species inhabiting Western Europe: the Iberian endemic P. ibericus and the more widespread P. punctatus. Previous studies based on mitochondrial and nuclear DNA markers have revealed the existence of two additional lineages of Parsley frogs in the Iberian Peninsula, which have been flagged as candidate species. Here, we integrate novel molecular, morphological and bioacoustical data to assess the differentiation of the four western Parsley frog lineages. Species trees and Bayesian population assignment analyses based on nuclear markers confirm previous studies and concordantly delineate four parapatric lineages with narrow hybrid zones. Mitochondrial divergence is low (< 2% pairwise distances in the 16S rRNA gene), in line with previously reported low mitochondrial substitution rates in non-neobatrachian frogs. Based on concordance between mitochondrial and nuclear markers, we conclude that four species of Parsley frogs occur in Western Europe: Pelodytes punctatus, distributed from northern Italy to northeastern Spain; Pelodytes ibericus, inhabiting southern Spain and southern Portugal; Pelodytes atlanticus sp. nov., from the Portuguese Atlantic coast; and Pelodytes hespericus sp. nov., occurring in central and eastern Spain. However, bioacoustical and morphological differentiation of these species is low, with no obvious and qualitative diagnostic characters allowing full species discrimination. Differences in the relative size of metacarpal tubercles exist but this character is variable. Pelodytes ibericus and Pelodytes atlanticus are smaller than the other two species, and P. ibericus has shorter limbs and various distinctive osteological characters. Bioacoustically, the pattern by which two different note types are combined in advertisement calls separates P. hespericus from the remaining species. Despite these differences, we emphasize that the taxonomic status of all four western Parsley frogs requires additional investigation, especially the patterns of genetic admixture across contact zones. While a status of separate species best conforms to the currently available data, alternative hypotheses are also discussed.

Species delineation is a central topic in evolutionary biology, with current efforts focused on developing efficient analytical tools to extract the most information from molecular data and provide objective and repeatable results. In this paper we use a multilocus dataset (mtDNA and two nuclear markers) in a geographically comprehensive population sample across Iberia and Western Europe to delineate candidate species in a morphologically cryptic species group, Parsley frogs (genus Pelodytes). Pelodytes is the sole extant representative of an ancient, historically widely distributed anuran clade that currently includes three species: P. caucasicus in the Caucasus; P. punctatus in Western Europe, from Portugal to North-Western Italy; and P. ibericus in Southern Iberia. Phylogenetic analyses recovered four major well-supported haplotype clades in Western Europe, corresponding to well demarcated geographical subdivisions and exhibiting contrasting demographic histories. Splitting times date back to the Plio-Pleistocene and are very close in time. Species-tree analyses recovered one of these species lineages, corresponding to P. ibericus (lineage B), as the sister taxon to the other three major species lineages, distributed respectively in: western Iberian Peninsula, along the Atlantic coast and part of central Portugal (lineage A); Central and Eastern Spain (lineage C); and North-eastern Spain, France and North-western Italy (lineage D). The latter is in turn subdivided into two sub-clades, one in SE France and NW Italy and the other one from NE Spain to NW France, suggesting the existence of a Mediterranean-Atlantic corridor along the Garonne river. An information theory-based validation approach implemented in SpedeSTEM supports an arrangement of four candidate species, suggesting the need for a taxonomic revision of Western European Pelodytes.

Abstract. Painted frogs (Discoglossus) contain five to six species of Western Palearctic anurans that are mainly distributed in allopatry. We here provide the first comprehensive assessment of the phylogeography of the Moroccan species D. scovazzi and geographically characterize its contact zone with D. pictus in Eastern Morocco. Discoglossus scovazzi shows, in general, a weak phylogeographic structure across Morocco on the basis of mitochondrial DNA sequences of the cytochrome b gene, with only populations centered in the Atlas Mountains characterized by the presence of slightly divergent haplotypes. In eastern Morocco, all populations east of the Moulouya River were clearly assignable to D. pictus. This species was also found along the Mediterranean coast west of the Moulouya, in the cities of Nador and Melilla, suggesting that not the river itself but the wide arid valley extending along much of the river (except close to the estuary) acts as a possible distributional barrier to these frogs. No sympatry of D. scovazzi with D. pictus was observed, and all specimens were concordantly assigned to either species by DNA sequences of cytochrome b and of the nuclear marker RAG1. Species distribution models of the two taxa show largely overlapping areas of suitable habitat, and the two species' niches are significantly more similar than would be expected given the underlying environmental differences between the regions in which they occur. Comparative data are also presented from the southern Iberian contact zone of D. galganoi galganoi and D. g. jeanneae. These taxa showed less clear-cut distributional borders, extensively shared RAG1 haplotypes, and had instances of sympatric occurrence on the basis of cytochrome b haplotypes, in agreement with the hypothesis of a yet incomplete speciation. In this wide contact zone area we found mitochondrial sequences containing double peaks in electropherograms, suggesting nuclear pseudogenes or (less likely) heteroplasmy, possibly related to the ongoing admixture among the lineages.