Development of genetic tools to detect three New Zealand indigenous freshwater mussels in environmental DNA

Abstract

As “ecological engineers”, freshwater mussels are key components in the freshwater ecosystem. However, surveying mussels can be challenging since mussel populations may be difficult to locate. Conventional methods for identifying mussel species are also problematic and require expertise since mussels are often morphologically cryptic. The same problems apply to New Zealand indigenous freshwater mussels. New Zealand had long been considered to have two indigenous species, Echyridella menziesii and Echyridella aucklandica, until a recent revision based on molecular evidence recognised a new species, Echyridella onekaka, which was previously mis-identified as Echyridella menziesii. Results from molecular studies have placed all three freshwater mussel species under a single genus, Echyridella, specific to New Zealand. For conservation and ecosystem management, a fast and reliable method for surveying and identifying New Zealand mussels is required. This research aims to develop molecular tools that utilise environmental DNA (eDNA) collected from freshwater environments to detect mussel species. These tools target a widely used DNA marker, cytochrome c oxidase subunit I (COI), for species identification. The COI targets in eDNA samples are detected by digital polymerase chain reaction (dPCR) using genus-specific primers and species-specific TaqMan probes. Three species-specific assays were developed, followed by assessment of specificity and sensitivity. Assay for E. aucklandica showed high specificity and tolerance to inhibition from high levels of non-target DNA. A trial of duplexing simultaneously assays detecting E. onekaka and E. aucklandica also showed promising results. With further assessment of assays using in vivo and in situ environmental samples in the future, these techniques hold great promise for being a rapid and cost-effective method for surveying and identifying New Zealand freshwater mussels. Additionally, to increase detection rate when target concentration in eDNA is extremely low, known mitochondrial sequences for all three species were extended through PCR and de novo sequencing to allow future manufacturing of a customised commercial target DNA enrichment kit (MYbaits). This work is necessary since known mitochondrial genomic sequences for these three species are too short to generate effective MYbaits oligos. Consensus primers were designed based on available complete mitochondrial genomes of closely related mussel species. Most consensus primers successfully amplified in PCR, and about 1700bp have been sequenced so far. Once the sequencing of extended region is completed, a MYbaits kit will be manufactured for future research.