Faith M. Walker

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.