Due to anthropogenic influences, habitat degradation and a continuous loss of biodiversity in freshwater ecosystems are occurring on a large scale, while these ecosystems constitute invaluable natural resources. Therefore, it is essential to study and monitor freshwater ecosystems to guide conservation efforts. Freshwater ecosystems are one of the less-studied fields in Georgia. Studies about the species distribution of many taxa and/or regions carried out during the last century have not been updated for decades. Here, we report the results of an environmental DNA (eDNA) metabarcoding exercise, based on samples collected from the Rioni River, a tributary to the Black Sea and a crucial aquatic ecosystem regionally and globally. The only comprehensive review of the fish of the Rioni River dates back to 1956. We compared the eDNA-based taxonomic composition to the known faunal composition within the Rioni River and found that the eDNA-based taxonomic coverage approached 75% of the expected total fish fauna. A number of new species occurrences were also found, including the first detection of three invasive alien species (Carassius gibelio, Pseudorasbora parva, Rhinogobius lindbergi) in the Rionis River Basin and a new country record of the ninespine stickleback (genus Pungitius) for Georgia. In spite of the usefulness of the eDNA metabarcoding approach, the sparsity of the fish DNA barcode reference library for the region emerged as a limitation to this study. However, our findings still represent a great leap forward in updating fish status on the Rioni River and testing the effectiveness of the eDNA sampling for aquatic species.
