Metabarcoding assays for the detection of freshwater mussels (Unionida) with environmental DNA

Abstract

Freshwater mussels of the order Unionida are a widely distributed taxon that are important in maintaining freshwater ecosystems and are also highly imperiled throughout the world. Monitoring of mussel populations with environmental DNA (eDNA) is an attractive alternative to traditional methods because it is noninvasive and requires less labor and taxonomic knowledge from field personnel. We developed eDNA metabarcoding assays specific to freshwater mussels and tested them at six sites in the Clinch River, located in the southeastern United States. Our objective was to determine the utility of eDNA metabarcoding for future monitoring of mussel populations and restoration efforts in this watershed. Two metabarcoding assays that target the mitochondrial DNA regions of the cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit (ND1) genes were developed and tested. Our assays appear to be order specific, amplifying members from the two families found in North America, Unionidae and Margaritiferidae, while not amplifying nontarget fish or other bivalve species. From the field collected samples, our assays together detected 19 species, eight of which are listed as federally endangered. The assays also detected 42%, 58%, and 54% of the species identified by recent quantitative visual mussel surveys at three sampling sites. Increased sampling effort by processing a greater water volume or number of samples will likely increase species detections. These eDNA metabarcoding assays may enable enhanced monitoring of freshwater mussel assemblages and subsequently inform conservation efforts.

Community eDNA metabarcoding as a detection tool for documenting freshwater mussel (Unionidae) species assemblages

“…The fact that the majority of reads were lost based on amplicon length, coupled with the absence of any non-unionid species detections that met our match and count thresholds, likely indicates that many non-target amplifications resulted from primer annealing sites outside the target region for multiple taxa. Our results are comparable to Klymus et al, (2017), in which ~0.13%-99.56% (average 34.46%) of the total reads from eDNA samples, not including blanks, were assigned an OTU at 97% identity, and Klymus et al, (2020) in which 55% and 49% of total reads were assigned to species level for COI and ND1, respectively. Further testing is needed to investigate whether the sequences shorter or longer than 180-200 bp are primers, sequences that do not match to any database sequence, or sequences from target or non-target organisms that match to database sequences lower than 98% match.…”

“…With the exception of E. dariensis, species identified from sequence data that are not known to occur in Ontario had the lowest read counts within sites where they were detected (Figure 3), suggesting either undetected taxa or spurious detections. Using a higher minimum read count threshold could help reduce potential false but also risk increasing false negative results for low-abundance species (Klymus et al, 2020).…”

“…While useful for detecting individual species of interest, targeted single-species assays are inherently blind to the presence of other taxa. Community eDNA or eDNA metabarcoding offers a more broadly effective approach for community assessment, as it does not require a priori assessment of which species are most likely to occur and enables simultaneous testing for evidence of multiple species (Klymus et al, 2020;Prié et al, 2020;Taberlet et al, 2018).…”

The future of biodiversity monitoring and conservation utilizing environmental DNA

“…Concurrent larval fish sampling by netting produced equivalent estimates of species richness and diversity with eDNA metabarcoding with 75% agreement. Klymus et al (2020) developed two metabarcoding assays for freshwater mussels for future monitoring of the diverse and unique mussel populations in Clinch River watershed in the southeastern United States. The assays detected 19 species including eight federally endangered species.…”

Airborne Environmental DNA Metabarcoding Detects More Diversity, More Efficiently, Than a Traditional Plant Community Survey

“…If any ASVs had a 100% match to multiple species in the same genus, the ASV was assigned to the genus level (41). For ASVs that had multiple 100% genera or species matches, all were kept following the recommendation of Klymus et al (42). The species and genus results were then examined through the United States Department of Agriculture Plants Database (43) to classify the species or genus as being either within our study site (i.e., Texas Tech Native Rangeland), local county (Lubbock County), state (Texas), country (United States), or the World.…”