AbstractIn freshwater fish, processes of population divergence and speciation are often linked to the geomorphology of rivers and lakes that create barriers isolating populations. However, current geographical isolation does not necessarily imply total absence of gene flow during the divergence process. Here, we focused on four species of the genus Squalius in Portuguese rivers: S. carolitertii, S. pyrenaicus, S. aradensis and S. torgalensis. Previous studies based on eight nuclear and

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... rial markers revealed incongruent patterns, with nuclear loci suggesting that S. pyrenaicus was a paraphyletic group, since its northern populations were genetically closer to S. carolitertii than to other southern populations. Here, for the first time, we successfully applied a genomic approach to the study of the relationship between these species, using a Genotyping by Sequencing approach to obtain single nucleotide polymorphisms (SNPs). Our results revealed a species tree with two main lineages: (i) S. carolitertii and S. pyrenaicus; (ii) S. torgalensis and S. aradensis. Moreover, regarding S. carolitertii and S. pyrenaicus, we found evidence for past introgression between these two species in the northern part of S. pyrenaicus distribution. This introgression reconciles previous mitochondrial and nuclear incongruent results and explains the apparent paraphyly of S. pyrenaicus. Although we cannot distinguish a scenario of hybrid speciation from secondary contact, our estimates are consistent across models, suggesting that the northern populations of S. pyrenaicus received approximately 80% from S. carolitertii and 20% from southern S. pyrenaicus. This illustrates that even in freshwater species currently found in isolated river drainages, we are able to detect past gene flow events in present-day genomes, suggesting that speciation is more complex than simply allopatric.