Targeted eDNA Metabarcoding Reveals New Populations of a Range-Limited Stonefly.

Understanding the geographic distributions of rare species can be crucial for conservation management. New environmental DNA (eDNA) technologies offer the potential to efficiently document the distributions of endangered species, but to date, such screening has focused largely on vertebrate taxa. Here we use freshwater eDNA to assess the geographic distribution of the Maungatua stonefly, , a flightless insect previously known from only a handful of streams draining a 4-km section of the Maungatua mountain range in southern New Zealand. We analyzed freshwater eDNA from 12 stream localities across the Maungatua range. Screening with commercial eDNA COI primers failed to detect the focal species . However, newly designed species-specific primers detected this taxon from four adjacent east-flowing streams known to contain , and two streams from which it had not previously been detected. Subsequent manual surveys confirmed the presence of two newly discovered populations, with COI barcoding revealing that they together represent a previously unknown, genetically divergent subclade. Our results illustrate the potential of eDNA metabarcoding to help delineate the geographic ranges of rare taxa, and highlight the importance of primer specificity when screening for rare taxa. These findings also have considerable implications for commercial companies offering biodiversity and stream health eDNA services targeting invertebrates.