Mammalian eDNA on herbaceous vegetation? Validating a qPCR assay for detection of an endangered rodent

Abstract

Vegetation is an underutilized medium for environmental DNA (eDNA) sampling.eDNA methods leveraging water as a substrate exclude application to many terrestrial species. The use of eDNA to detect small mammals can complement current survey approaches (live capturing, track plating, and camera trapping) while reducing risks to the animals. The endangered New Mexico meadow jumping mouse (Zapus hudsonius luteus) is specialized to herbaceous riparian zones, making it an ideal candidate for developing a terrestrial eDNA detection method. We developed a species-specific assay for quantitative real-time PCR, then tested the long-term persistence of jumping mouse eDNA on plant material using four herbaceous day nests collected three to six months after occupancy. We conducted a field trial using sterile cotton swabs at six locations along two occupied streams to evaluate our assay's capability to detect present-day eDNA. Each of 60 swabs was used to swab a 0.50 m 2 area along streamside transects that included vegetation such as forbs, grasses, and sedges. We also opportunistically swabbed plants (n = 9) following visual observation of jumping mice.We determined the limit of detection for both assays are fewer than eight copies per reaction. We detected eDNA in three of four nests. From field trial samples, we successfully detected the species from randomly swabbed vegetation (N = 3), and four of nine swabs from vegetation recently used by individuals. Further work is required to develop a robust survey method using this eDNA detection approach. Our study demonstrated that mammalian eDNA can persist on nest vegetation long after the animal was present, highlighting the promise of using eDNA from plants to detect rare or endangered terrestrial species.

Environmental DNA Metabarcoding: A Novel Contrivance for Documenting Terrestrial Biodiversity

“…Only a few studies have been able to trace animals using the eDNA technique from soil [ 97 ] in areas where the animals were previously present under controlled conditions like Safari Parks or Zoos or from natural zones where the species are reported [ 98 ]. Similarly, an investigation revealed that the endangered New Mexico meadow jumping mouse ( Zapus hudsonius luteus ) is a prime candidate for creating a terrestrial eDNA detection tool because it is restricted to herbaceous riparian zones [ 99 ]. More study is necessary to create a reliable survey technique employing this eDNA detection methodology.…”

“…Using eDNA from the soil to trace mammals [ 105 ] is not only environment dependent but also dependent on mammal abundance and size [ 106 ]. Hence, knowledge of the ecological behavior of the mammal is essential for the sampling design [ 99 ]. We anticipate that eDNA technology will be crucial in delivering quick and widespread insights into the population genetics of endangered and challenging-to-sample species worldwide [ 49 ].…”

Endangered Nectar-Feeding Bat Detected by Environmental DNA on Flowers

“…We used PrimerProspector v1.0.1 [ 57 ] to find all possible primer or probe sequences (ranging from 18 to 28 bp in length), favoring L. nivalis (100% similarity) over L. yerbabuenae . We filtered candidate primer and probe sequences based on melt temperature, sequence length, and GC content using open-source python scripts [ 11 ], custom R scripts, and the IDT OligoAnalyzer™ Tool ( ; accessed 26 August 2022). We calculated the maximum number of mismatches to non-target sequences ( L. yerbabuenae ) for primers and probes using SequenceMatcher software ( ; accessed 26 August 2022).…”

“…Terrestrial applications have received less attention [ 8 ], and most studies have involved water as a sampling source and have largely included species that are common or invasive (e.g., [ 9 , 10 ]). The utility of eDNA is due to its assay sensitivity, and there is great potential to detect rare or endangered terrestrial species with creative sampling methods [ 11 ]. Wildflowers have been used to sample the eDNA of terrestrial arthropod pollinators and predators [ 12 ], and hence may be useful for capturing DNA from vertebrate pollinators.…”

Environmental DNA as an emerging tool in botanical research

“…As applications for botanical eDNA continue to expand, new sources of eDNA are constantly being discovered. For example, plant species themselves have come into focus as a viable source of eDNA, either through washing plant material and collecting the runoff (Valentin et al, 2020) or directly swabbing plants to detect mammals or insects (Kudoh et al, 2020; Lyman et al, 2022).…”