| Summary: | Alien fish introductions, conducted towards the ichthyofauna enhancement in local drainages, have been occasionally proved harmful for the indigenous freshwater fish populations. The present study was designed to assess the impact of stocking activities, carried out in the past decades with fingerlings originating from Acheloos river hatcheries, on the native trout (<i>Salmo</i> sp.) populations of Nestos River, Greece. Trout specimens collected from several tributaries of Nestos River and were analyzed by means of PCR-RFLP and sequencing targeting the mitochondrial ND5-ND6 genes and the entire control region, respectively. It should be mentioned that trouts from Acheloos mainly belong to the <i>marmoratus</i> mitochondrial DNA (mtDNA) lineage, while the autochthonous trouts from Nestos belong to the Adriatic lineage. Both methodologies demonstrated that most samples from the three tributaries located at the lower part of Nestos constitute offspring of the fingerlings transferred from Acheloos hatcheries. Therefore, these tributaries have been strongly affected by stocking activities with a potential complete loss of their autochthonous trout. On the other hand, it seems that trout populations from higher altitude tributaries have not been affected by stockings. Hence, efforts should be undertaken in order to prevent the prevalence of the non-indigenous translocated <i>Salmo</i> in higher altitude tributaries, in conjunction with a management plan designed for the total trout populations from the area, speaking of which it has been recently included to the National Park of Rodopi Mountains.
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