## 3 Results and Discussion
Although haplotypes A, B, and C have not been described so far, they only differ by two to three nucleotide substitutions from the most consensus _Salmo trutta_ sequences obtained from the GenBank database (AY836330, AY836348, AY836349, [16]), reflecting pairwise sequence divergence lower than 0.01. Therefore, mtDNA markers do not support the classification of trout from the Greek part of Nestos River as a valid distinct species. However, since mtDNA represents only a small part of the eukaryotic genome, and particularly concerning _Salmo_ species where there are several cases of conflict between mitochondrial and nuclear DNA phylogenies, e.g., [4; 12; 18], further genetic analyses including _Salmo_ spp. populations from a wider geographical range, i.e., such as Balkan peninsula, using neutral nuclear markers such microsatellites or SNPs are required in order to clarify the taxonomic distinctiveness of Nestos trout. On the other hand, from an evolutionary point of view, it is genetic integrity rather than purely taxonomic status that is of interest. Further, it should be also pointed that for conservation purposes, it is worth considering a morphotype, such is the Nestos River trout, as a distinct species, with purpose not to be neglected [18]. In general, translocations of fish species towards the erroneous enhancement of local habitats, although often are characterized as of good intention, are considered among the deterministic threats causing extinction [19]. Apart from the obvious risk of pathogen introduction and infection of closely related species [20], the impact on ecosystem stability could be catastrophic. Introduced species may not only be antagonistic to the inhabitants of the new environment, but also may hybridize with local genetically closely related populations. For instance, at least four alien freshwater fish species, i.e., _Gambusia holbrooki_, _Carassus gibelio_, _Pseudorasbora parva_, and _Leponis gibbosus_ have currently widespread their distribution in Greek rivers and streams [21]. On account of anthropogenic intra or inter-specific hybridization within _Salmo_ trout populations, the real conservation status is often obscured [22]. Our results provide evidence that the trout introductions threatened the local trout populations with irreversible impacts at some streams. RFLP and sequencing analysis agreed that all the trout samples from Mousda stream and most of those from Arkoudrema-Dramal and from Vathyrema near Kalikarpo (Figure 1), which had the prevalent Acheloos genotype, constitute, namely, offspring of the fingerlings transferred in the 1970s and thereafter, the latest being in 2011 in Arkoudorema River, whereas no genotype that would indicate translocation of trouts from a different area was found. Hence, these tributaries have been severely affected by the stocking activities with a potential complete loss of the autochthonous trout particularly in the Mousda stream. On the contrary, local brown trout populations from higher altitude tributaries, i.e., Diavolorema, Farasino, Vathyrema near Elatia forest and Arkoudrema-Drama 2, probably have not been affected by the stocking activities, since there was no  
Figure 2: Maximum-likelihood consensus dendrogram depicting the genetic relationships among the four haplotypes observed in the current study, along with one marmoratus and one Adriatic sequence, obtained from GenBank. Bootstrap values greater than 60% are shown on the branches. specimen in which the Acheloos genotype was observed. Obviously, physical barriers, such as waterfalls, located between the lower and the higher parts of Vathyrema and Arkoudorema, did not allow the upstream movement of non-indigenous translocated trout, or their offspring, protecting in this way the unique genetic profile of the brown trout in these tributaries. Therefore, efforts should be undertaken in order to prevent the prevalence of the non-indigenous translocated _Salmo_ species in the aforementioned tributaries.