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

Abstract

Documenting species occurrences and habitat occupancy of unionid mussels can often be challenging. Environmental DNA (eDNA) has been shown to be a reliable tool for detecting unionids with comparable or greater sensitivity than conventional sampling and has the added advantages of not disturbing individuals or occupied habitats.However, single-species eDNA assays are limited to targeting individual species of interest and are functionally blind to the presence of other species. Community eDNA assays have the potential to characterize local species assemblages simultaneously but are currently less extensively developed and implemented than single-species eDNA testing. We tested the effectiveness of community eDNA markers to identify unionid species assemblages, using two overlapping conserved primers that target the maternal mitochondrial 16S rDNA region. Both primer sets were optimized using three mock communities and successfully amplified 62.5%-81.6% of species with largely consistent results between the primer sets. Following optimization, eDNA from water samples from 24 reference sites with known mussel communities was amplified and sequenced to quantify species richness and diversity within and among sites. Metabarcoding results from the monitoring sites largely mirrored those from the mock communities, with >80% of species detections identified by both assays.The results were broadly consistent with species data from quadrat-based manual field surveys, although both community eDNA and conventional sampling detected some species that the other method did not. These results demonstrate that community eDNA assays using conserved primers and next-generation sequencing have the potential to simultaneously target eDNA from multiple unionid species and provide a powerful tool for complementing or augmenting conventional field surveys to characterize and monitor unionid species assemblages.

Toward quantitative metabarcoding

“…Read counts following amplification and sequencing invariably fail to match – or often, even approximate – the mock-community DNA starting proportions. For example, relative read counts deviate strongly from the mock communities created for freshwater mussels (Coghlan et al 2021), freshwater invertebrates (Fernández et al 2018), arthropods (Piñol et al 2015, Krehenwinkel et al 2017), freshwater fish (Hänfling et al 2016, Rivera et al 2021), marine vertebrates (Port et al 2016, Andruszkiewicz et al 2017), fungi (Adams et al 2013, De Filippis et al 2017, Palmer et al 2018), diet studies from a range of organisms (Ford et al 2016, Thomas et al 2016, Ando et al 2020, Tournayre et al 2020), and microbiome studies (McLaren et al 2019, Silverman et al 2021). Beyond their obvious taxonomic diversity, these analyses span a wide range of methodological implementations (i.e.…”

Evaluating environmental DNA metabarcoding as a survey tool for unionid mussel assessments

“…The high‐level of species agreement is similar to Prié et al (2020) who designed the current assay for use on western Palearctic communities, and found eDNA detected all, and often more species than field surveys throughout France. Additionally, a second assay, likewise targeting the 16S gene region, recovered >80% of species that were found in conventional surveys from waterbodies across southern Ontario (Coghlan et al, 2021). In the present study, species accumulation curves suggest eDNA sampling provided better representation of species richness with fewer total sites required.…”

“…Additionally, if multiple MOTUs assigned to the same taxonomy, they were inspected and removed or collapsed into a single MOTU to obtain a final matrix of read counts per taxa. Some eDNA metabarcoding studies have incorporated a read cut-off threshold in an attempt to remove potential sequencing error resulting from tag-jumping or index-hopping (Snyder et al, 2020;Klymus et al, 2021), while others have analysed the complete dataset by including rare MOTUs (Marshall and Stepien, 2020;Coghlan et al, 2021). We evaluated the increase in eDNA false negative detections when the dataset was analysed with three bioinformatic filters compared to the full unfiltered dataset.…”

“…However, considering the high diversity of unionid communities within North American waterways, the use of single-species qPCR analysis is highly inefficient for community-level description of potentially diverse mussel beds. Therefore, an eDNA metabarcoding approach, which employs amplicon-based high-throughput sequencing (HTS) to simultaneously identify multiple taxa, has been used to detect bivalve communities in river systems (Klymus et al, 2017(Klymus et al, , 2021Prié et al, 2020;Coghlan et al, 2021). Metabarcoding can describe diverse mussel beds, including the detection of threatened and endangered species, aiding in environmental impact assessments and evaluating conservation efforts of freshwater mussels.…”

“…This delayed exposure of substrate allowed search teams to visually identify and enumerate mussels exposed as water receded, thereby providing a detailed description of mussel community composition. While previous studies have compared eDNA metabarcoding datasets with conventional unionid survey methods (Prié et al, 2020;Coghlan et al, 2021;Klymus et al, 2021), the staged dewatering in the present study provides the most detailed and informative analysis of unionid detection from eDNA to date.…”