Historical factors (colonization scenarios, demographic oscillations) and contemporary processes (population connectivity, current population size) largely contribute to shaping species’ present-day genetic diversity and structure. In this study, we use a combination of mitochondrial and nuclear DNA markers to understand the role of Quaternary climatic oscillations and present-day gene flow dynamics in determining the genetic diversity and structure of the newt Calotriton asper (Al. Dugès, 1852), endemic to the Pyrenees. Mitochondrial DNA did not show a clear phylogeographic pattern and presented low levels of variation. In contrast, microsatellites revealed five major genetic lineages with admixture patterns at their boundaries. Approximate Bayesian computation analyses and linear models indicated that the five lineages likely underwent separate evolutionary histories and can be tracked back to distinct glacial refugia. Lineage differentiation started around the Last Glacial Maximum at three focal areas (western, central and eastern Pyrenees) and extended through the end of the Last Glacial Period in the central Pyrenees, where it led to the formation of two more lineages. Our data revealed no evidence of recent dispersal between lineages, whereas borders likely represent zones of secondary contact following expansion from multiple refugia. Finally, we did not find genetic evidence of sex-biased dispersal. This work highlights the importance of integrating past evolutionary processes and present-day gene flow and dispersal dynamics, together with multilocus approaches, to gain insights into what shaped the current genetic attributes of amphibians living in montane habitats.

Sampling was conducted in the period 2004-2017 across the whole Pyrenees, encompassing most of the species range. DNA was sampled via buccal swab or toe clipping of metamorphosed individuals. Samples were preserved in EDTA or absolute ethanol and stored at -20°C until DNA extraction. Genomic DNA was extracted using QIAGEN DNeasy Blood and Tissue Kit (Qiagen^TM, Hilden, Germany) according to the manufacturer’s protocol, or following the HotSHOT method (Montero‐Pau, Gómez, & Muñoz, 2008), in a total volume of 100 µl.

A fragment of the cytochrome b (cyt-b) gene was sequenced from 258 individuals from 59 sampling sites. We amplified a fragment of 374 bp using primers Cytb1EuprF and Cytb2EuprR (Carranza & Amat, 2005). Amplification conditions were those described in Carranza, Arnold, Mateo, and López-Jurado (2000). Sequences were aligned using the ClustalW algorithm in MEGA 7 (Kumar, Stecher, & Tamura, 2016).

A total of 1,299 individuals from 96 sampling sites were genotyped for a set of 17 microsatellite loci combined in three multiplexes (Drechsler et al., 2013). Fragments were sized with LIZ-500 size standard and binned using either GeneMapper v4.0 (Applied Biosystems) or Geneious 11.0.5 (Kearse et al., 2012). Only individuals that could be scored in a reliable manner for at least 15 loci were included in the analyses.