Abstract
BackgroundThe structure and diversity of grayling (Thymallus thymallus) populations have been well studied in most of its native habitat; however the southernmost populations of the Balkan Peninsula remain largely unexplored. The purpose of this study was to assess the genetic diversity of Serbian grayling populations, detect the impact of stocking and provide guidelines for conservation and management.MethodsEighty grayling individuals were collected from four rivers (Ibar, Lim, Drina and Rzav). The mitochondrial DNA control region (CR; 595 bp of the 3'end and 74 bp of flanking tRNA) and the ATP6 gene (630 bp fragment) were sequenced for 20 individuals (five from each locality). In addition, all individuals were genotyped with 12 microsatellite loci. The diversity and structure of the populations as well as the recent and ancient population declines were studied using specialized software.ResultsWe detected three new haplotypes in the mtDNA CR and four haplotypes in the ATP6 gene of which three had not been described before. Previously, one CR haplotype and two ATP6 gene haplotypes had been identified as allochthonous, originating from Slovenia. Reconstruction of phylogenetic relations placed the remaining two CR haplotypes from the River Danube drainage of Serbia into a new clade, which is related to the previously described sister Slovenian clade. These two clades form a new Balkan clade. Microsatellite marker analysis showed that all four populations are genetically distinct from each other without any sign of intra-population structure, although stocking of the most diverse population (Drina River) was confirmed by mtDNA analysis. Recent and historical population declines of Serbian grayling do not differ from those of other European populations.ConclusionsOur study shows that (1) the Ibar, Lim and Drina Rivers grayling populations are genetically distinct from populations outside of Serbia and thus should be managed as native populations in spite of some introgression in the Drina River population and (2) the Rzav River population is not appropriate for further stocking activities since it originates from stocked Slovenian grayling. However, the Rzav River population does not represent an immediate danger to other populations because it is physically isolated from these.
Highlights
The structure and diversity of grayling (Thymallus thymallus) populations have been well studied in most of its native habitat; the southernmost populations of the Balkan Peninsula remain largely unexplored
Based on control region (CR) mtDNA sequence analyses and calibration of molecular clock applied to the nucleotide divergence of these sequences between the major grayling clades with a CR mutation rate of 1% per million years (MY), Froufe et al [4] have dated the colonization of Europe to the Pliocene-Pleistocene boundary around 4.6 to 1.6 million years ago (MYA), far before the onset of Pleistocene ice age
The main goal of the present study was to investigate the genetic diversity of grayling populations in Serbia, using two mtDNA loci (CR and ATP6), in order to clarify the phylogeography of grayling populations in this previously unstudied part of its native range
Summary
The structure and diversity of grayling (Thymallus thymallus) populations have been well studied in most of its native habitat; the southernmost populations of the Balkan Peninsula remain largely unexplored. Numerous DNA marker-based studies on population genetic structure, phylogeography and phylogeny of European grayling are available for various geographic regions The assumed refugial region for (i) the Northern/Northeastern-European clade was the area north of the Caspian and Black Seas, (ii) the CentralEastern European clade, the ice-free tributaries of Vistula and Elbe Rivers, (iii) the Central-Western Europe, the ice-free tributaries of Rhine, Main and upper Danube, and (iv) the Danubian clades, the lower Danube drainage area, i.e., in the Balkan Peninsula [19]. Koskinen et al [6] have revealed that a substantial proportion of molecular variation (44%) in European grayling exits between populations, whereas Gum et al [7,16] have revealed that about 25% of the total genetic variation is explained by differences between major drainage systems, about 11 to 20% by differences between populations within drainages and about 57 to 64% by differences within populations
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