Validation of environmental DNA (eDNA) as a detection tool for at‐risk freshwater pearly mussel species (Bivalvia: Unionidae)

Abstract

1. Documenting the occurrence and habitat occupancy of rare aquatic species is an ongoing challenge for conservation. Characterization of environmental DNA (eDNA) from bulk water samples has emerged as a powerful tool to infer species presence or absence without the need to observe or handle organisms.2. Previous eDNA studies have yet to develop species-specific markers that target taxa with many potentially sympatric confamilials. Forty-one freshwater pearly mussel species (Unionidae) are found in southern Ontario, Canada, with many of these listed as threatened, endangered, or of conservation concern; however, locating populations for protection can be challenging owing to morphological crypsis and species scarcity.3. Species-specific eDNA markers were developed to target four unionid species. Following in silico and in vitro validation, markers were validated in the field by comparing eDNA results from water samples to detections based on quadrat sampling. 4. Target species were detected by eDNA sampling at all sites where they had previously been located by quadrat sampling. 5. The paired sampling design showed that species-specific markers can be designed even within speciose families, and that eDNA detection of mussels is at least as sensitive as quadrat sampling. Furthermore, detection probabilities were not affected by sampling depth, and eDNA concentrations were positively correlated with mussel densities. 6. These findings confirm that eDNA assays are a valuable complement to traditional methods for locating and managing imperilled unionid populations.

Downstream transport and seasonal variation in freshwater pearl mussel (Margaritifera margaritifera) eDNA concentration

“…Our results have important implications for monitoring FPM with eDNA. Together with previous studies (Carlsson et al, 2017;Currier et al, 2018;Sansom & Sassoubre, 2017), our results show that larger freshwater mussel aggregations can be reliably detected with eDNA. At the same time, efficient downstream transport, strong seasonal variation in eDNA concentrations and limits in the detection of small mussel aggregations emphasize that surveys need to be carefully adjusted to the study aims.…”

“…Previous studies on the detection of freshwater mussel eDNA in natural systems have either targeted much larger mussel aggregations (Stoeckle et al, 2016) or do not report individual counts (Currier et al, 2018;Deiner & Altermatt, 2014;Dysthe et al, 2018;Sansom & Sassoubre, 2017 for example different eDNA shedding rates, but high-detection rates have been found for very low densities, such as less than one fish per 100 m river (Wilcox et al, 2016). While this is a simplification, the expected concentrations fit well with our observed concentrations from eDNA PCR-amplifications.…”

“…Notably, models on decay and accumulation of eDNA would be needed to predict eDNA concentrations along larger river distances and with more complex distributions of mussels. Previous studies on the detection of freshwater mussel eDNA in natural systems have either targeted much larger mussel aggregations (Stoeckle et al, 2016) or do not report individual counts (Currier et al, 2018;Deiner & Altermatt, 2014;Dysthe et al, 2018;Sansom & Sassoubre, 2017). Carlsson et al (2017) (Wilcox et al, 2016).…”

“…Visual searches for mussels can be hampered when adult individuals are partly or fully buried in the substratum or when the visibility is poor. Environmental DNA is a highly promising tool for monitoring freshwater mussels and previous studies revealed that freshwater mussels shed DNA that can be detected in water samples (Carlsson et al, 2017;Currier, Morris, Wilson, & Freeland, 2018;Deiner & Altermatt, 2014;Dysthe et al, 2018;Sansom & Sassoubre, 2017;Stoeckle, Kuehn, & Geist, 2016). However, empirical work needs to reveal how eDNA concentrations are affected by stream characteristics such as downstream transport and by seasonal variation.…”

Detecting the undetectable: Characterization, optimization, and validation of an eDNA detection assay for the federally endangered dwarf wedgemussel, Alasmidonta heterodon (Bivalvia: Unionoida)

“…Most often this has been achieved by incorporating into the primers degenerate (multiple) bases at variable positions during synthesis to force a match in a proportion of the primers with the polymorphic site. Recently, Currier et al () used this approach to study pearly mussel species. Some authors, however, have used ‘cocktails’ of individually synthesized oligonucleotides (each matching a variant of the target) to achieve the same goal (Gijavanekar et al, ; Heckenhauer, Barfuss, & Samuel, ; Lobo et al, ).…”