A new perspective on the molecular dating of the brown trout complex with an extended phylogeographic information on the species in Serbia

To elucidate the historical biogeography of a species, the patterns of population divergence must be understood, and the evolutionary history of the species must be accurately known. For brown trout (Salmo trutta complex), estimating divergence times remains a challenge due to the lack of well-defined time calibration points and insufficient phylogeographic coverage in previous studies. The present work aims to improve molecular dating of mitochondrial control region sequences by using a multicalibration framework based on the latest paleogeological evidence for dating the origin of Lake Ohrid and two available Salmo fossils, including the overlooked Salmo immigratus. Our results clearly show that, contrary to common belief, the major divisions within the brown trout occurred in the Late Pliocene, not the Pleistocene. The Pliocene origin suggests that the brown trout lineages did not form because of geo(hydro)morphological changes during glaciation cycles but may be the result of orogeny and drainage evolution. In addition, increased sampling, particularly in Serbia, led to the identification of a new haplogroup (da-int) occupying an intermediate position with respect to da-es and da-bs haplogroups. While the control region can delineate brown trout lineages, its phylogenetic resolution is limited, so even extensive sampling could not further resolve the lineage level polytomies.