Cave gudgeon (Milyeringa spp.) eDNA assay for rapid detection in the dark

Australia has two eyeless cave gudgeons ( Milyeringa justitia and M. veritas ) which are restricted to subterranean waters and listed as vulnerable and endangered. Despite being a priority conservation target species, they are inadequately monitored as their restricted distribution in groundwater ecosystems impedes conventional surveying methods, constraining effective monitoring and conservation. As part of ongoing efforts to improve monitoring and conservation of these subterranean fish species, we developed and validated a genus-specific eDNA assay for detecting species of Milyeringa . Mitochondrial genomes were sequenced and assembled to inform PCR assay design, which was then validated in-silico , in-vitro , and in-situ . The assay demonstrated high specificity and sensitivity, particularly for M. justitia (Limit of Detection, LOD: 1.36 copies per reaction), and successfully detected M. veritas (LOD: 12.8 copies per reaction) with moderate sensitivity in groundwater samples from Cape Range. No PCR amplification occurred in non-target surface water samples. This assay provides a non-invasive, robust tool for conservation monitoring cryptic subterranean fishes, supporting conservation management in habitats where traditional survey methods are impractical.