Diversity of brown trout, Salmo trutta (Actinopterygii: Salmoniformes: Salmonidae), in the Danube River basin of Croatia revealed by mitochondrial DNA

Abstract

Background. The molecular diversity of brown trout, Salmo trutta Linnaeus, 1758, has been poorly studied in Croatia. The control region of mitochondrial DNA (CR mtDNA) is in addition to other molecular markers a reliable for identifying phylogenetic lineages (haplogroups) and haplotypes of brown trout. Based on analyses of the control region of mitochondrial DNA several major brown trout phylogenetic lineages were identified of which the Danubian (DA) haplotypes, though not all, are considered native to Croatian rivers belonging to the Danube basin. The introduction of allochthonous haplotypes into natural streams seriously threatens the genetic diversity of this species. Therefore, the aim of this study was to map brown trout populations inhabiting Croatian rivers of the Danube River basin and to investigate their molecular diversity and phylogeographic patterns of the established haplotypes. Materials and methods. Anal fin tissue was taken from 141 specimens of brown trout in 14 localities in the protected areas of Croatia, situated in the mountainous regions of Gorski Kotar, Žumberak, as well as Mountain Papuk in the western Slavonia. The total DNA was extracted and then the amplification of the mtDNA control region was carried out using primers Trutta-mt-F and HN20. Amplification of the 440 bp long region of the LDH-C1 gene locus was done using primers Ldhxon3F and Ldhxon4R. Amplified LDH-C* fragments were used for Restriction Fragment Length Polymorphism (RFLP) analysis using BselI restriction enzyme. Results. Analysis of the CR mtDNA revealed the presence of two phylogenetic lineages, the DA and the Atlantic (AT). Haplotypes Da1, Da2, and Da22 were recorded within the DA lineage and At1 was recorded within the AT haplogroup. Two new haplotypes were described for the first time in this study and are named Da1f and Da1g. Restriction analysis of the lactate dehydrogenase gene locus revealed a high degree of hybridization between brown trout of DA and AT haplogroups. Conclusion. The results of this study confirmed the complex molecular diversity of brown trout and the high degree of the introduction of non-native haplogroups into rivers of the Danube basin in Croatia. Conservation of native brown trout populations has become evident, as introduced allochthonous DA and AT haplogroups severely disrupt the indigenous brown trout stock.

Keywords

• trout
• Croatia
• mtDNA
• haplotype diversity
• conser