Materials and methods:The present study is the first to apply DNA barcoding on identifying 37 freshwater fish species from the rich Balkan ichthyofauna. Results: The results are highly successful since in most cases barcodes cluster according to species, in agreement with morphological taxonomic studies. This is also evident based on mean conspecific and congeneric Kimura two-parameter distance values. The 5.6-fold difference between these values is lower than previous barcoding studies, possibly due to the restricted samplings and the recent taxonomy reevaluation for several species. A number of species were identified, where future work is needed: For the species Scardinius erythrophthalmus, Perca fluviatilis, and Rutilus rutilus, the divergence values found among conspecific populations could warrant their placement into different species; for Barbus and Rhodeus populations, the reported interspecific distances found were lower than expected; and for Cobitis species, the application of barcoding seems problematic, due to their complicated reproduction. Conclusion: The extension of this work to other Greek or even Balkan freshwater systems should clarify the situation.
According to reports, the following 16 species have extended their distribution to other Mediterranean areas or have made a new appearance in other regions. The first category includes the following organisms: The rare and common Indo-Pacific seaweed Codium arabicum (Lebanese coasts), the acari Thalassarachna affinis (Marmara Sea), and the non-indigenous nudibranch Flabellina rubrolineata, which has been found in several areas of the Aegean Sea. In addition, the rare sea slug Thecacera pennigera (Mar Piccolo of Taranto), the fangtooth moray Enchelycore anatina (National Marine Park of Zakynthos, Ionian Sea), the carangid Seriola fasciata (Gulf of Antalya), Lagocephalus sceleratus (SE Ionian Sea), the reticulated leatherjacket Stephanolepis diaspros (Slovenia, N. Adriatic Sea), the marbled stingray, Dasyatis marmorata (NE Levantine), the starry smooth-hound Mustelus asterias (Iskenderun Bay, NE Mediterranean), the cephalopod Ommastrephes bartramii (Ionian Sea) have also been reported. The Atlantic crab Dyspanopeus sayi has expanded to many Italian areas and the blue crab Callinectes sapidus to a lake in N. Greece and in the S. Adriatic Sea. Finally, Farfantepenaeus aztecus has been found in the Ionian Sea, thus showing its wide expansion in the Mediterranean. The larval stages of Faccionella oxyrhyncha have been found, after many years, in the Aegean Sea and the first report of an existence on intersexual acari Litarachna duboscqi in Split (Adriatic Sea) was reported.
Biodiversity is a key factor for the functioning and efficiency of an ecosystem. Greece, though covering a relatively small surface area, hosts a great deal of species diversity. This is especially true for freshwater fishes. In recent years, the traditional methods of species identification have been supplemented by the use of molecular markers. The present study therefore aims to extensively produce DNA barcodes for Greek freshwater fish species and investigate thoroughly if the presently accepted species classification is in agreement with molecular data. A 624-bases long fragment of the COI gene was sequenced, from 406 freshwater fish specimens belonging to 24 genera and originating from 18 lake and river sites. These sequences were used along with 596 sequences from the same genera, recovered from BOLD, for the construction of phylogenetic trees and the estimation of genetic distances between individuals. In total, 1002 sequences belonging to 72 species were analyzed. The method was found to be effective for 55 of 72 studied species. 17 closely related species with low interspecific genetic distances were observed, for which further study is proposed. It should also be noted that, in four cases, cryptic diversity was observed, where groups originally identified as one species exhibited genetic distance great enough to be separated into discrete species. Region specific haplotypes were also detected within populations of 14 species, giving the possibility to identify even the geographic origin of a species. Our findings are discussed in the light of the rich history of the Balkan peninsula and provide a significant steppingstone for the further study of Greek and European freshwater fish biodiversity.
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