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.
This is a preliminary and exploratory study of cranial variation in European populations of grayling. We investigated the correspondence between size/shape variation of the dorsal (dc), ventral (vc) and occipital (oc) cranium and phylogenetic relationships (inferred from mitochondrial control region – mtDNA cr and microsatellite dna data) of six grayling populations: three from Balkan phylogenetic clade and two from Caspian phylogenetic clade of the European grayling Thymallus thymallus and one population of the Adriatic grayling Thymallus aeliani, which until recently was considered the Adriatic phylogenetic clade of T. thymallus. Significant size and shape differences were found between populations in all three cranial views. However, significant size-related shape variation (allometry) was found for dc and vc, but not for oc. The size variation of each cranial view does not contain phylogenetic signal, but size variation of oc is consistent with genetic variation inferred from microsatellite dna. Regarding shape variation, a significant phylogenetic signal was detected only for oc, and only the shape variation of oc is consistent with the genetic variation inferred from the mtDNA cr. Moreover, the Adriatic grayling T. aeliani (Soča population) was clearly separated from the three T. thymallus populations of the Balkan phylogenetic clade and the two T. thymallus populations of the Caspian phylogenetic clade only at the level of oc. Thus, our results suggest that different cranial regions differ in allometry, reflect phylo(genetic) relationships differently, and exhibit differences in ecophenotypic plasticity, with oc seeming best suited to represent the phylogenetic relationships of the grayling populations studied.