We present a phylogenetic investigation of the Northern Clade, the major monophyletic clade within the freshwater fish family Cobitidae, one of the most prominent families of freshwater fishes found in Asian and European waters. Phylogenetic reconstructions based on the cytochrome b and RAG-1 genes show the genera Microcobitis, Sabanejewia, Koreocobitis and Kichulchoia as monophyletic groups. These reconstructions also show a Cobitis sensu lato and a Misgurnus sensu lato group. The Cobitis sensu lato group includes all species of Cobitis, Iksookimia, Niwaella and Kichulchoia, while the Misgurnus sensu lato group includes Misgurnus, Paramisgurnus and Koreocobitis. Although the monophyly of both the Cobitis sensu lato and Misgurnus sensu lato groups is supported, relationships within the groups are incongruent with current generic definitions. The absence of monophyly of most genera included in the Cobitis sensu lato group (Cobitis, Iksookimia and Niwaella) or their low genetic differentiation (Kichuchoia) supports their consideration as synonyms of Cobitis. Molecular phylogenies indicate that the Asian species of Misgurnus experienced a mitochondrial introgression from a lineage of Cobitis. We also find two nuclear haplotypes in the same Cobitis species from the Adriatic area that, in the absence of morphological differentiation, may indicate molecular introgression. Most lineages within the Northern Clade consist of species found in East Asia. However, some lineages also contain species from Europe and Asia Minor. The phylogenetic relationships presented here are consistent with previous studies suggesting an East Asian origin of the Northern Clade. According to the current distributions and phylogenetic relationships of the Misgurnus sensu lato and Cobitis clade lineages, particularly of M. fossilis and C. melanoleuca, the range expansion of East Asian species into Europe was most likely via Siberia into Northern and Central Europe. Phylogenetic analyses also show that the Cobitis sensu lato group consists of two clear subgroups (I and II), each presenting geographical differences. Subgroup I is distributed exclusively in East Asian drainages with an Eastern European offshoot (C. melanoleuca), whereas Subgroup II includes species widespread throughout Europe (including the Mediterranean), Asia Minor, the Black Sea and the Caucasus, with some lineages related to species restricted to East Asia.

Accurate determination of species diversity in areas of high endemicity, particularly those lacking comprehensive systematic knowledge, represents a challenge for both taxonomists and conservationists. This need is particularly evident in areas greatly affected by anthropogenic disturbances such as the Eastern Mediterranean and its freshwater environments. To improve our knowledge of Eastern Mediterranean freshwater fishes, we phylogenetically studied Western Palearctic Cobitis species, focusing on those found in Turkey. Overall, our results provide a robust framework to assess the number of species of Cobitis. Phylogenetic reconstructions based on mitochondrial (cyt b) and nuclear (RAG1) sequences show seven major clades (Clades 1–7) grouping all Western Palearctic Cobitis species, except C. melanoleuca. In general, each major clade comprises Cobitis species that inhabit geographically close areas and have similar secondary sexual characters. Multiple divergent lineages were identified in our analyses, some of which were highly divergent such as the ones inhabiting Turkish freshwaters. Moreover, in some analyses, several of the identified lineages were incongruent with a priori defined species. Furthermore, our analyses identified eight potentially new candidate species, six that had been suggested in previous studies and two that are reported here for the first time. Our results reveal Turkey as the area with the greatest diversity of spined loaches in the Mediterranean.