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. 90% of all diversity) belonging to 57 genera and 25 families. Totally 1,327 DNA barcodes (1,151 sequences from 202 localities generated by this study) 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 analyzed 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. Among these are two aggressive alien species ( Hemiculter leucisculus and Pseudorasbora parva ), while five are aboriginal species in genera Salmo , Leuciscus , Gobio , and Cobitis from neighboring basins, which naturally or human-aided colonized the Volga basin. Moreover, three new phylogenetic lineages (candidate species) were discovered – specifically, in the genera Leucaspius , Phoxinus , and Proterorhinus . While the fish fauna of the Volga is highly mixed, the more ancient Kama displays regional endemism for rheophilic species (e.g., Barbatula, Cottus ). Likely, the Kama served as refuge during Pleistocene climate oscillations. In sum, generated COI dataset fills the existing gap in COI database of fishes inhabiting freshwaters of Eastern Europe and will be highly useful in future studies.