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Part of Europe’s temperate species survived the Pleistocene glacial cycles in refugia north of the Mediterranean peninsulas. For one such extra-Mediterranean refugia, the Carpathians, an intricate ‘refugia-within-refugia’ scenario has been suggested, involving species surviving in multiple discrete glacial refugia. We test the Carpathian refugia-within-refugia hypothesis, employing genome-wide multilocus data sets for two newt species (Triturus cristatus and Lissotriton montandoni). We first use Bayesian clustering to delineate intraspecific evolutionary lineages. The number of intraspecific lineages identified, and the allocation of localities to these lineages, were used to construct testable hypotheses on the spatial arrangement of glacial refugia in both newt species. Next we employ approximate Bayesian computation to date whether these lineages are of Holocene (< 12 Ka) or Pleistocene (> 12 Ka) origin. We identify three intraspecific evolutionary lineages for T. cristatus and two for L. montandoni. For both newt species, intraspecific divergence is rooted in the Pleistocene, in line with species survival in distinct range fragments during the last glacial period. Hence, our findings firmly support the Carpathian refugia-within-refugia hypothesis. Furthermore, we show that mitochondrial DNA overestimates the age of intraspecific evolutionary lineages and we urge caution in basing refugia-within-refugia scenarios on mitochondrial DNA alone

The Carpathians hosted extra-Mediterranean refugia-within-refugia during the Pleistocene Ice Age: genomic evidence from two newt genera