# Targeted eDNA Metabarcoding Reveals New Populations of a Range‐Limited Stonefly

## TL;DR

New eDNA methods helped find hidden populations of a rare stonefly in New Zealand, showing the power of DNA sampling for conservation.

## Contribution

Species-specific eDNA primers were developed and used to discover new populations of a rare stonefly.

## Key findings

- eDNA metabarcoding revealed two previously unknown populations of Z. maungatuaensis.
- New populations formed a genetically distinct subclade, indicating hidden diversity.
- Commercial eDNA primers failed to detect the species, highlighting the need for specificity.

## Abstract

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, Zelandoperla maungatuaensis, 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 Z. maungatuaensis. However, newly designed species‐specific primers detected this taxon from four adjacent east‐flowing streams known to contain Z. maungatuaensis, and two streams from which it had not previously been detected. Subsequent manual surveys confirmed the presence of two newly discovered Z. maungatuaensis 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.

We developed new species‐specific eDNA primers to detect Zelandoperla maungatuaensis, a rare, flightless insect restricted to a 4‐km stretch of the Maungatua range (South Island, New Zealand). eDNA metabarcoding confirmed that Z. maungatuaensis has a very narrow geographic range and revealed two previously unknown populations. Subsequent manual surveys validated the eDNA screening results, with additional COI sequencing revealing that these new populations form a distinct subclade.