Evaluation the influences of different sampling strategies and DNA extracting methods on eDNA metabarcoding: A case study of fish diversity monitoring in an estuarine ecosystem

Environment DNA (eDNA) integrate with metabarcoding theory has been increasingly used to evaluate fish diversity and recognized as a promising and powerful tool for species composition characterization and biodiversity assessment in aquatic ecosystems. But there are no standardized eDNA monitoring programmes for the study of fish diversity. Detail evaluations of methods concerning sampling strategies and laboratory workflow in eDNA metabarcoding need to be done before application. In the study, two sampling treatments, three filtering strategies and three DNA extracting methods were assessed and compared to establish a suitable approach of eDNA-based fish diversity monitoring in an estuarine ecosystem, Pearl River Estuary (PRE). The sampling treatment of direct filtration was considered as more suitable for eDNA metabarcoding in PRE. 1000 mL filtered water showed high fish diversity and provided higher OTU numbers than the other two filtering strategies, suggesting that 1000 mL water samples were sufficient to investigate fish diversity in PRE. The extraction of DNeasy Blood and Tissue kit (BT) not only gained higher DNA yields, OTU numbers and Shannon index, but also generated more homogeneous and consistent community composition among replicates. Collectively, our results support that the sampling strategy of directly filtering 1000 mL water and BT extraction in eDNA metabarcoding procedure is the appropriate methodological choice for the fish community survey in PRE. Furthermore, each protocol of eDNA metabarcoding behaved more sensitive than bottom trawling for fish surveys in PRE, and eDNA metabarcoding combined with traditional survey reveal more taxonomy diversity. However, we found that different sampling strategies and DNA extraction combinations can provide inconsistent biodiversity detection. Thus, combined traditional method with eDNA analysis could effectively enhance the accuracy, and methodological decisions related to eDNA metabarcoding should be cautiously made during the fish community monitoring in the estuarine ecosystem.