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.

Application of the geometric morphometrics approach in the discrimination of morphological traits between brown trout lineages in the Danube Basin of Croatia

“…In future study, the inclusion of landmarks to examine the lower jaw and head depth could give additional insight into the ecologies of the lineages, as head morphology can reflect trophic variation (Wainwright et al, 1991;Piggott et al, 2018), and these additional landmarks could better explain the largest Procrustes variation within the Atlantic lineage. Kanjuh et al (2020) found that none of the sampled streams included in this study contained completely pure Danube trout lineage populations, with at least one hybrid or Atlantic lineage individual with Atlantic trout lineage genes detected at each site. The present study indicated a difference in shape between trout belonging to the Danubian and Atlantic lineages.…”

“…This lower variation in body height could be additional proof of the influence of fish farm origin on the shape of all Atlantic specimens in this study. The hybrids analysed here share genes with Danubian trout (Kanjuh et al, 2020), and moreover they hatched and evolved in the same habitats as pure Danubian trout. As such, adaptations to the same habitats could be similar due to their high phenotypic plasticity, explaining why the shape of hybrids did not differ significantly from the shape of pure Danubian populations.…”

“…Photographs were taken by a digital camera mounted on a camera stand, at a sufficient distance from the fish (650 mm) to reduce the parallax effect (Fruciano, 2016). Prior to GM analyses, molecular phylogenetic analysis using control region mitochondrial DNA was conducted on the same samples (see Kanjuh et al, 2020 for details), which revealed the presence of two phylogenetic lineages, Danubian and Atlantic, while restriction analysis of the lactate dehydrogenase gene locus revealed a high degree of hybridization between them. These results enabled the a priori classification of specimens into three groups: Danubian lineage, Atlantic lineage, and their hybrids (Tab.…”

“…The selection of landmarks and semi-Table 1. Sampling locations with coordinates and number of sampled trout specimen with affiliation to lineages as determined by molecular methods (sensu Kanjuh et al, 2020) and sex ratio (n = number of individuals; TL min = minimal total length in cm; TL max = maximal total length in cm; F = female; M = male).…”

“…Historically, the Atlantic lineage of brown trout was introduced into suitable habitats throughout the region as stocking material from fish farms, and this activity appears to be ongoing (Piria et al, 2020;Ivić et al, 2021). Cross-breeding between introduced Atlantic and native lineages has recently been detected in the region through molecular analyses, forming hybrid populations that pose a threat to native genetic diversity (Kanjuh et al, 2020;Škraba Jurlina et al, 2020;Ivić et al, 2021). This interspecific hybridization caused by natural or anthropogenic events is a common phenomenon in freshwater fishes (Scribner et al, 2001), and has been described in spined loaches (Vasil'eva and Vasil'ev, 2019), barbels (Khaefi et al, 2018) and bitterling (Uemura et al, 2018).…”