Aquatic invertebrate diversity profiling in heterogeneous wetland habitats by environmental DNA metabarcoding

Invertebrates play vital roles in maintaining biodiversity and food web structure. However, it is difficult to identify invertebrate taxa across complex habitats due to the limitation of traditional morphology. In subtropical wetlands, environmental DNA (eDNA) metabarcoding was used to characterize the composition and diversity of aquatic invertebrates and analyze the environmental impacts on invertebrate community structure. According to the relative abundance (%) of invertebrate OTU richness, 30 sampling sites in wetlands were clustered into six zones, which exhibited significant spatial (i.e., wetland type-specific) differences in taxonomic composition. The relative OTU abundance (%) at the phylum level showed that Cnidaria (48.5%) > Porifera (19.4%) > Rotifera (11.0%) > Mollusca (9.2%) > Annelida (5.3 +/- 2.8%) > others (less than 2.2%). Of the five alpha diversity in-dexes, 'Simpson' was the most effective index to distinguish the spatial differences in invertebrate diversity, and the class-level alpha diversity showed higher recognition than other taxonomic levels. The class-level biomarkers that could indicate the habitat-specific composition of local invertebrates were Insecta, Cnidaria, Hydrozoa, and Gastropoda in the inflow river; Ascidiacea and Demospongiae in the fluvial wetland; Clitellata, Gymnolaemata, and Monogononta in the lacustrine wetland; and Echinoidea in the estuarine wetland. The associations between environmental factors and invertebrate OTU richness based on redundancy analysis showed the taxon-level tendency of phylum (78.5%) > class (73.4%) > order (69.3%) > family (64.9%) > genus (61.2%). Our re-sults demonstrated that aquatic invertebrate diversity profiling by environmental DNA metabarcoding can effectively reflect the composition, diversity and biomarkers of invertebrate communities. In the future, because of its great application potential, the eDNA technique may play an important role in biomonitoring complex water environments.