A treatise about reliability in dating events of evolutionary history of brown trout Salmo cf. trutta (Actinopterygii) at Western Balkans: Impassable barriers, isolation of populations and assistance of geological timeframe
Authors: Marić Ana , Srećković Batoćanin Danica , Škraba Jurlina Dubravka , Brkušanin Miloš , Karanović Jelena , Kanjuh Tamara , Nikolić Vera , Mrdak Danilo , Simonović Predrag 
Published: February 2023

A pool of data already existing about D-loop, i.e., the Control Region (CR) haplotypes of the mitochondrial DNA (mtDNA) of brown trout, Salmo trutta Linnaeus, 1758, tentative Adriatic trout Salmo farioides Karaman, 1938, and tentative Macedonian trout, Salmo macedonicus (Karaman, 1924), and their reconstructed phylogeography makes a good starting point for resolving their evolutionary history. That includes the dating of particular events in it. The events have hitherto been dated using the method of a molecular clock. Various calibrations were applied for the mutation rate, owing to the incongruence between the time of divergence that various authors notified and general knowledge about events in geological history and the periods in which they occurred in the Mediterranean region. Since geological history events were mandatory for setting the scene for the evolutionary history of brown trout, the incongruence between them has questioned the molecular clock calibration’s validity. From results about both the phylogeography and phylogenetic relations between native haplotypes (both partial and whole CR sequences) and the population genetics that characterized particular populations, we calculated the time of divergence between haplotypes in the regions of the western part of the Balkans: Iron Gate broader area in eastern Serbia, continental Montenegro and south-eastern Serbia. The distinct status of adjacent populations was verified by frequencies of microsatellites’ alleles and the STRUCTURE analysis that examined the significance of differences between them. In particular, we examined the populations that were clearly separated either by physical barriers, such as a waterfall in eastern Serbia (e.g., the upper and lower River Rečka supplemented by nearby rivers Vratna and Zamna), or by underground drops in Montenegro (e.g., upper and lower River Zeta, and rivers Nožica and lower River Mrtvica as isolated counterparts). We used the so far most common substitution rate of 1% in a million years’ (MY) period. The divergence times we obtained were compared to the events known for the region from available geological history data. There was a fairly good congruence between the dating obtained by the molecular clock method and that by geological history where the advanced, i.e., modern haplotypes, were concerned. In contrast, the congruence was worse for dating of divergence when more ancient haplotypes were in question, being much better if the mutational rate would be decreased to lower rates. That supported results both from the Rate Correlation Test about the independence of evolutionary rates in different lineages of brown trout, and from the Molecular Clock Test, which revealed that the evolutionary rate throughout the phylogenetic tree is not equal. That implies a difference in the speed of evolution in them, which was likely slower and faster, in the ancient, pre-Pleistocene haplotypes and the advanced, Pleistocene ones, respectively. The setting of the variable, or non-linear (i.e., logarithmic) speed of evolving seems helpful, since the early cladogenesis with the dominance of mutations was most likely combined afterwards with the acting of other evolutionary mechanisms, especially of genetic drift in populations that passed through the bottleneck episodes of the abrupt decrease in population size during the unfavourable periods of their evolutionary history.