Unsupervised biological integrity assessment by eDNA biomonitoring of multi-trophic aquatic taxa

The biological integrity of global freshwater ecosystems is threatened by ever-increasing environmental stressors due to increased human activities, such as land-use change, eutrophication, toxic pollutants, overfishing, and exploitation. Traditional ecological assessments of lake or riverine ecosystems often require human supervision of a pre-selected reference area, using the current state of the reference area as the expected state. However, selecting an appropriate reference area has become increasingly difficult with the expansion of human activities. Here, an unsupervised biological integrity assessment framework based on environmental DNA metabarcoding without a prior reference area is proposed. Taxon richness, species dominance, co-occurrence network density, and phylogenetic distance were used to assess the aquatic communities in the Taihu Lake basin. Multi-gene metabarcoding revealed comprehensive biodiversity at multiple trophic levels including algae, protists, zooplankton, and fish. Fish sequences were mainly derived from 12S, zooplankton mainly from mitochondrial cytochrome C oxidase subunit I, and algae and protists mainly from 18S. There were significant differences in community composition among lakes, rivers, and reservoirs but no significant differences in the four fundamental biological indicators. The algal and zooplankton integrities were positively correlated with protist and fish integrities, respectively. Additionally, the algal integrity of lakes was found to be significantly lower than that of rivers. The unsupervised assessment framework proposed in this study allows different ecosystems, including the same ecosystem in different seasons, to adopt the same indicators and assessment methods, which is more convenient for environmental management and decision-making.