Paleohydrological dynamics are well‐documented for European river systems, promoting shifting phases of isolation and connectivity of their aquatic fauna. These conditions coupled with high rates of hybridisation found in freshwater fishes may introduce considerable complexity and potential mito‐nuclear discordance of phylogenetic patterns. We evaluate this hypothesis using the first large‐scale analysis of nuclear SNPs in European species of grayling (Thymallus) compared to mtDNA data with the aim of reassessing the evolutionary history of this group of rheophilic fishes.

Freshwater systems in Europe.

Based on mitochondrial (mitogenomes, control region) and nuclear (ddRADseq) data, we applied population‐genetic, phylogenetic, and biogeographic tools to evaluate lineage diversity in the context of paleohydrological alterations.

The results corroborated previously recognised high levels of lineage diversity, but revealed several cases of mito‐nuclear discordance and signals of both historical (natural) and human‐mediated introgression among major inter‐ and intraspecific lineages of Thymallus in Europe. A time‐calibrated phylogeny and ancestral area estimation, based on nuclear SNP data, supported a late Pliocene diversification of the genus in Europe and suggested an early colonisation of the Black Sea basin with subsequent dispersal into Central and Western Europe.

The genetic structure of Thymallus in Europe recovered by nuclear SNPs contrasts considerably with that supported by mtDNA. Several instances of mito‐nuclear discordance underscore frequent contact of allopatric lineages in a dynamic paleohydrological landscape and reveal the weakness of basing both taxonomic and conservation decisions on inferences based on mtDNA alone. The Danube and Rhine drainages were inferred as important zones of contact between divergent phylogeographic lineages. Additionally, our data cast doubt on the genetic integrity of the endangered T. aeliani. Its divergence from T. thymallus, using nuclear SNPs, appears minimal as samples of T. aeliani group within Danubian lineages, despite carrying highly divergent reciprocally monophyletic mtDNA.