DNA barcoding reference dataset revealed overlooked diversity and clarified biogeography of fishes in the largest European river system – Volga River

This study represents the first thorough genetic assessment of freshwater fishes from the Volga River Basin – the longest (3,690 km) river in Europe. DNA barcode sequences ( COI ) were studied in 84 species (approx. 85% of all diversity) belonging to 57 genera and 25 families. In total, 1,327 DNA barcodes were used to: (i) build a DNA barcode reference library and check for the presence of a barcoding gap; ii) assess the genetic and taxonomic diversity of Volga fishes and iii) clarify the biogeography of Volga fishes. DNA barcodes are reliable means for the identification of over 95% of analysed species showing barcode gaps. An overlap between intra- and interspecific genetic distances was present in four species of genera Leuciscus and Rutilus . DNA barcoding identified seven new species for the Volga. Amongst these, two are aggressive alien species ( Hemiculter leucisculus and Pseudorasbora parva ), while five are indigenous to the region and belong to the genera Salmo , Leuciscus , Gobio and Cobitis from neighbouring basins, which naturally or human-aided colonised the Volga Basin. Moreover, three new phylogenetic lineages were discovered in the genera Leucaspius , Phoxinus and Proterorhinus . While the fishes from sub-basins of the Volga usually do not demonstrate sorting of the haplotypes, the more ancient Kama displays regional endemism for rheophilic species (e.g. Barbatula , Cottus ). Our results suggest the Kama might serve as refuge during Pleistocene climate oscillations. In summary, the generated COI dataset fills the existing gap in COI database of fishes inhabiting freshwaters of Eastern Europe.