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ABSTRACT
Environmental DNA (eDNA) integrated with metabarcoding is a promising and powerful tool for species composition and biodiversity assessment in aquatic ecosystems and is increasingly applied to evaluate fish diversity. To date, however, no standardized eDNA-based protocol has been established to monitor fish diversity. In this study, we investigated and compared two filtration methods and three DNA extraction methods using three filtration water volumes to determine a suitable approach for eDNA-based fish diversity monitoring in the Pearl River Estuary (PRE), a highly anthropogenically disturbed estuarine ecosystem. Compared to filtration-based precipitation, direct filtration was a more suitable method for eDNA metabarcoding in the PRE. The combined use of DNeasy Blood and Tissue Kit (BT) and traditional phenol/chloroform (PC) extraction produced higher DNA yields, amplicon sequence variants (ASVs), and Shannon diversity indices, and generated more homogeneous and consistent community composition among replicates. Compared to the other combined protocols, the PC and BT methods obtained better species detection, higher fish diversity, and greater consistency for the filtration water volumes of 1 000 and 2 000 mL, respectively. All eDNA metabarcoding protocols were more sensitive than bottom trawling in the PRE fish surveys and combining two techniques yielded greater taxonomic diversity. Furthermore, combining traditional methods with eDNA analysis enhanced accuracy. These results indicate that methodological decisions related to eDNA metabarcoding should be made with caution for fish community monitoring in estuarine ecosystems.
Keywords: eDNA metabarcoding; Fish diversity; Sampling strategies; DNA extraction; Estuarine ecosystem
INTRODUCTION
Accurately describing species composition is not only necessary for evaluating the characteristics of ecosystems but is also the cornerstone for establishing biodiversity protection systems (Blowes et al., 2019; Dixon et al., 2019). As one of the most productive ecosystems worldwide, nutrient-rich estuaries play important ecological functions and provide critical animal breeding and nursery habitats (Bhakta et al., 2019). Overfishing, pollutant discharge, and offshore construction have led to a significant deterioration in estuarine ecosystem functions, including rapid decreases in fish stock and biodiversity (Irfan & Alatawi, 2019; Yan et al., 2021). Fish assemblages are particularly sensitive to environmental stresses and are therefore considered good indicators of aquatic ecosystem health, even in highly modified and threatened estuaries (Santos et al., 2017; Zhou et al., 2019). Fish diversity is traditionally monitored through netting, trapping, and electrofishing. However,...