Biodiversity and spatial distribution of ascidian using environmental DNA metabarcoding

Monitoring studies are necessary to understand the biodiversity of marine ecosystems and are useful for iden-tifying and managing rare or invasive species. Because monitoring has traditionally relied only on visual surveys (e.g., trapping, netting, electrofishing, and SCUBA diving) with limited time and physical resources, environ-mental DNA (eDNA) analysis is being applied as an efficient monitoring method. This study compared whether the eDNA metabarcoding technique can replace the traditional visual survey in an ascidian fauna study. We designed ascidian-specific primers and identified a clear gap (3.75%) by barcoding gap analysis. Then, we collected seawater samples for eDNA analysis during the summer (August-September) of 2021 at three sites (Mokpo, Yeosu, and Uljin) in South Korea. In the survey sites of this study, 25 species were observed through literature and visual survey, among which 9 species were detected by metabarcoding and 16 species were not detected. On the other hand, 10 species were detected only by metabarcoding, and one of them was identified as Pyura mirabilis, an unrecorded species in South Korea. This study succeeded in detecting cryptic or rare species with one seawater collection, which can be used to determine their unexplored habitat. Therefore, we conclude that monitoring using eDNA is more efficient than visual surveys for detecting rare or cryptic ascidian species. We also suggest that, when combined with traditional monitoring methods, it could be a tool to complement ascidian fauna studies.