Phylogeny of European Bullhead (Cottus sp.) in Western Balkans

European bullhead sensus lato is a species complex, in which lineage determination based on morphology has proven to be extremely difficult, probably due to low interspecific and high intraspeciefic morphological variation. Therefore, major evolutionary lineages across Europe have been determined based on molecular data, which also to some extent corresponds to known morphological variation. Cottus populations on the Balkan Peninsula have not yet been thoroughly studied. Thus we used molecular genetic approach (mtDNA-CR and nu-LSU) to evaluate evolutionary distinctive units of Cottus in the Western Balkans and compare them to other European lineages. We found presence of five distinct lineages. Populations corresponding to Cottus gobio clade inhabit tributaries of Danube River in southern Serbia, rivers of Adriatic drainage and Drava River. Interestingly, we found three additional Cottus linages in Sava River and its tributaries, differing considerably from “main” C. gobio. First, we confirmed morphologically determined Cottus meate from Upper Sava, with species range from the source of Sava River, including tributaries, to the inflow of Kolpa River. Second, we found a new lineage inhabiting the Kolpa and Una river systems. Third, lineage was found inhabiting Bosna and Vrbas rivers. High density of evolutionary distinctive lineages in such a small area is according to our knowledge exceptional for European bullhead and lines up with the general notion of the Balkans as major refuge and present day biodiversity hotspot. Our results correspond with two wave colonization theory of Europe by Cottus genus probosed by Volckaert, first possibly being in Pliocene and second during the Pleistocene, with Balkan Peninsula as starting point for both of them.