Development of species-specific primers with potential for amplifying eDNA from imperilled freshwater unionid mussels

Abstract

Environmental DNA (eDNA) is emerging as a potentially powerful tool for inferring species' presence, and hence occupancy, from DNA that is shed into environmental samples such as water. Although eDNA screening has been used to detect DNA from a variety of taxonomic groups, it has not yet been used to identify DNA from species with numerous potentially sympatric confamilial species, a situation that may preclude the development of species-specific markers. There are 41 native freshwater mussel species (Unionidae) in Ontario, Canada. Many of these are potentially sympatric, and 14 species have been formally assessed as endangered, threatened, or special concern. We investigated whether there was sufficient variation within the cytochrome oxidase region (COI) to develop species-specific eDNA markers for at-risk unionids. We developed 32 COI markers for eight unionid species, and tested each of these on the target species plus 29 potentially sympatric unionid taxa. Six of these markers amplified DNA only from the intended target species. We then extracted and amplified mussel eDNA from rearing-tank water samples. We conclude that despite high species diversity, it should be possible to develop eDNA COI markers and screen water samples for habitat occupancy by unionid mussels.

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

“…In conclusion, our work adds to the growing literature which points to the suitability of eDNA analyses for monitoring mussel populations (Belle et al., 2019; Cho et al., 2016; Currier et al., 2018; Gasparini et al., 2020; Prié et al., 2020). The study verified two Unionida‐specific metabarcoding assays suitable for freshwater mussel monitoring through eDNA sampling.…”

“…We developed the first primer set based on the work of Cho et al. (2016) who created a number of species‐specific primer sets for Canadian freshwater mussels using the COI gene region. Among the primers they developed was a pair ( Pfa COI2) which amplified 28 of the 30 species they tested, including 17 species that are found in the Clinch River system.…”

“…The two main strategies for species detection from eDNA are a targeted approach using PCR amplification from species‐specific primers and a multispecies approach known as metabarcoding, which involves high‐throughput sequencing of all the products of amplification from “universal” primers developed for a taxon of interest. Analysis of eDNA has been suggested as an additional tool for monitoring mussels, especially rare and endangered species (Cho et al., 2016; Gasparini et al., 2020; Prié et al., 2020). Monitoring of mussels with eDNA offers advantages of being less labor‐intensive, less invasive for the organisms and their habitat, and potentially detecting cryptic individuals or life stages, or animals living in inaccessible habitats (Stoeckle et al., 2016).…”

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

“…Mussels were equally detectable from all eDNA samples regardless of sampling depth. This work contributes to a growing body of literature investigating the efficacy of eDNA sampling for the detection of invertebrates and endangered species (Carlsson et al, ; Cho, Morris, Wilson, & Freeland, ; Deiner & Altermatt, ; Goldberg, Sepulveda, Ray, Baumgardt, & Waits, ; Lacoursière‐Roussel, Dubois, Normandeau, & Bernatchez, ; Stoeckle et al, ; Thomsen et al, ) and demonstrates the utility of eDNA sampling for the detection of multiple unionid mussel species at risk in natural habitats.…”

Monitoring for freshwater mussel presence in rivers using environmental DNA

“…In lieu of having physical survey data on freshwater mussel presence, the eDNA method can provide information on the distribution of freshwater mussels in waterbodies worldwide. Literature using eDNA as a method to detect freshwater mussels is increasing (Carlsson et al 2017;Cho et al, 2016;Currier et al, 2018;Deiner & Altermatt, 2014;Dysthe et al, 2018;Sansom & Sassoubre, 2017;Stoeckle et al 2015). Yet, there are currently no best practices for effectively sampling freshwater mussels in riverine environments.…”