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The rich genetic and phenotypic diversity of species complexes is best recognized through formal taxonomic naming, but one must first assess the evolutionary history of phylogeographic lineages to identify and delimit candidate taxa. Using genomic markers, mitochondrial DNA barcoding, and morphometric analyses, we examined lineage diversity and distribution in the Iberian endemic frog Rana parvipalmata. We confirmed two deep phylogeographic lineages, one relatively homogenous genetically, found in Asturias and adjacent areas (T2), and one more fragmented and locally genetically impoverished, restricted to Galicia (T1). Analyses of their hybrid zone suggested a shallow transition characterized by far-ranging admixture, which was modelled by a wide geographic cline (~60 km for the genome average) and no obvious barrier loci (i.e. loci showing disproportionally restricted introgression). The relatively young T1 and T2 have thus remained reproductively compatible, which argues against their distinction as biological species, and we accordingly describe T2 as a new subspecies, Rana parvipalmata asturiensis ssp. nov. Remarkably, we highlight striking discordances between mitochondrial and nuclear distributions across their hybrid zones, as well as between their genetic and morphological differentiation. Our study illustrates how genomic-based phylogeographic frameworks can help decipher the high genetic and phenotypic variation of species complexes and substantiate the taxonomic assessment of candidate lineages. [ABSTRACT FROM AUTHOR]

Amphibians often show complex histories of intraspecific and interspecific genetic introgression, which might differ in mitochondrial and nuclear genes. In our study of the genetic differentiation of the European common frog, Rana temporaria (159 specimens from 23 populations were analyzed for 24 presumptive allozyme loci; 82 specimens were sequenced for a 540-bp fragment of the mitochondrial 16S rRNA gene), multilocus correspondence analysis (CA) and Bayesian assignment tests of the nuclear data were concordant in identifying 2 population groups corresponding to 1) the Pyrenees in the east and 2) the Galicia and Asturias regions in the west, the latter corresponding to the subspecies R. temporaria parvipalmata. Geographically intermediate populations were genetically intermediate in the allozyme CA and, less clearly in the Bayesian assignment, with mitochondrial haplotypes exclusively belonging to the parvipalmata group. This indicates different degrees of introgression in the mitochondrial and nuclear genomes. Although Pyrenean high-altitude populations are morphologically distinct from low-altitude populations, these 2 groups were not separate clusters in any analysis. This suggests that the morphological differences may be due to fast adaptation to elevational gradients, likely under maintenance of gene flow, and that the underlying genetic changes are not detectable by the analyzed markers. We argue that a parsimonious explanation for the observed pattern along the east–west axis in northern Spain may be competition between invading and resident populations, with no need to invoke selection. However, in order to conclusively rule out selective processes, additional and finer scale data are required to test for asymmetric mating preference/behaviour, sex-biased gene flow, or sex-biased survival of potential hybrids. [ABSTRACT FROM PUBLISHER]

Based on DNA sequences of fragments of the mitochondrial COI and 16S rRNA genes we provide a first assessment of genetic variability of Iberian treefrog populations that have been historically allocated to Hyla arborea, partly as subspecies H. a. molleri. Our data from 147 specimens and 33 populations confirm strong divergence between these frogs and Central European H. arborea but relatively low differentiation across their range, supporting their status as a separate species, H. molleri. Preliminary phylogeographic data indicate a possible weak genetic differentiation of populations from the northern coast of the region of Galicia. We suggest inclusion of nuclear markers and an extension of the sampling into the coastal regions of Asturias and Cantabria, as well as the identification of the contact zone between H. molleri and H. arborea in either the Spanish Basque country or in France, as priorities for future research on this species. [ABSTRACT FROM AUTHOR]

We analysed the DNA sequences of the mitochondrial cytochrome b gene for 49 specimens of the Western spadefoot toad, Pelobates cultripes, from Spain, and one sample from France, with a focus on populations from Galicia in northwestern Spain. Our results point to a generally low mitochondrial differentiation of the species across its distributional range although some phylogeographic structure was detected. Galician populations from coastal localities, from the A Limia region, and from Monforte appeared to be closely related, whereas the only available sample from the relict Galician population from near O Barco de Valdeorras has the same haplotype as the samples from Burgos, in central Spain. [ABSTRACT FROM AUTHOR]

We determined DNA sequences of the mitochondrial cytochrome b gene for 44 specimens of the common frog, Rana temporaria parvipalmata, from 14 localities in Galicia, northwestern Spain. We found a low differentiation among all studied individuals, with a maximum of four mutational steps between haplotypes, including those from the southernmost isolated populations on the Manzaneda-Queixa and Trevinca-Segundeira massifs which thus probably originated by a rather recent colonization and likely maintained intermittent gene flow to more northern populations during climatically colder, glacial periods with a higher habitat connectivity. [ABSTRACT FROM AUTHOR]

Subdivided Pleistocene glacial refugia, best known as "refugia within refugia", provided opportunities for diverging populations to evolve into incipient species and/or to hybridize and merge following range shifts tracking the climatic fluctuations, potentially promoting extensive cytonuclear discordances and "ghost" mtDNA lineages. Here, we tested which of these opposing evolutionary outcomes prevails in northern Iberian areas hosting multiple historical refugia of common frogs (Rana cf. temporaria), based on a genomic phylogeography approach (mtDNA barcoding and RAD-sequencing). We found evidence for both incipient speciation events and massive cytonuclear discordances. On the one hand, populations from northwestern Spain (Galicia and Asturias, assigned to the regional endemic R. parvipalmata), are deeply-diverged at mitochondrial and nuclear genomes (~4 My of independent evolution), and barely admix with northeastern populations (assigned to R. temporaria sensu stricto) across a narrow hybrid zone (~25 km) located in the Cantabrian Mountains, suggesting that they represent distinct species. On the other hand, the most divergent mtDNA clade, widespread in Cantabria and the Basque country, shares its nuclear genome with other R. temporaria s. s. lineages. Patterns of population expansions and isolation-by-distance among these populations are consistent with past mitochondrial capture and/or drift in generating and maintaining this ghost mitochondrial lineage. This remarkable case study emphasizes the complex evolutionary history that shaped the present genetic diversity of refugial populations, and stresses the need to revisit their phylogeography by genomic approaches, in order to make informed taxonomic inferences., (© 2020 John Wiley & Sons Ltd.)