Integration of molecular, bioacoustical and morphological data reveals two new cryptic species of Pelodytes (Anura, Pelodytidae) from the Iberian Peninsula

Abstract

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.

Are glacial refugia hotspots of speciation and cytonuclear discordances? Answers from the genomic phylogeography of Spanish common frogs

“…This first result thus adds to a growing body of literature supporting that diverging lineages persisting in separate microrefugia can be on the path to speciation, despite occasional gene flow during secondary contacts (e.g., Díaz-Rodríguez et al, 2017;Dufresnes et al, 2018). For the cold-tolerant Rana frogs, this outcome is remarkable because Spanish populations expanded and thrived during the prolonged glacials rather than during the short interglacial cycles only (Figures 4 and 5; see also Galán et al, 2010 for the Galician populations of R. parvipalmata), thus experiencing longer opportunities for lineage fusion compared to species with Mediterranean affinities.…”

“…Accordingly, many closely-related endemics have been tentatively described from Mediterranean refugia (e.g., Díaz-Rodríguez et al, 2017;Dufresnes et al, 2018;Dufresnes, Strachinis, et al, 2019).…”

Phylogeography of a cryptic speciation continuum in Eurasian spadefoot toads (Pelobates)

“…As such, the age of the split between P. fuscus and P. vespertinus (~3 My from the RAD data), which show advanced (but not complete) reproductive isolation, can serve as a benchmark for the tempo of allopatric speciation in these toads. In other cryptic amphibian radiations, unimodal yet narrow transitions (<50 km) span from a similar age (2–3 My) in Pelodytes (Díaz‐Rodríguez et al, ) and Bufotes (Dufresnes et al, ), to divergence times twice as old, for example 5 My in Hyla (Dufresnes et al, ), and 4–7 My in Triturus (Arntzen et al, ; Wielstra, McCartney‐Meslstad, Arntzen, Butlin, & Shaffer, ). Beyond methodological differences such as sampling design, type of molecular markers, and calibration settings of molecular clocks, this overall variation also reflects stochastic effects on local hybrid zone dynamics (e.g., dispersal opportunities, local demographic events), which confound reproductive isolation.…”

Genomic Evidence for Cryptic Speciation in Tree Frogs From the Apennine Peninsula, With Description of Hyla perrini sp. nov

“…nov., in the next section. We recommend such multidisciplinary approaches for future taxonomic evaluation of cryptic lineages, as also conducted elsewhere (e.g., Diaz-Rodriguez et al, 2017). Particularly, we stress the need for genomic resources to resolve nuclear divergence and assess patterns of introgression, especially in young sibling species where traditional genetic markers (e.g., allozyme, microsatellite, or few nuclear sequences) might lead to erroneous conclusions.…”

“…In the Western Palearctic, this diversity was shaped by the combined actions of geological and climatic events (e.g., Mountain chain uplifts, the Messinian Salinity crisis, Quaternary glaciations), which have promoted allopatric diversifications between and within Mediterranean regions (e.g., Iberian, Italian, and Balkan Peninsulas, Northwestern Africa, Anatolia), and isolated offshore islands (e.g., Crete, Cyprus) (Schmitt, 2007;Dufresnes, 2019). Yet, the resulting lineages often remained remarkably cryptic, sharing similar ecologies and featuring no obvious phenotypic differentiation, be it morphology, coloration or behavior, despite millions of years of independent evolution (Bickford et al, 2007, e.g., Wielstra et al, 2013Diaz-Rodriguez et al, 2017).…”