Rapid detection of subterranean fauna from passive sampling of groundwater eDNA

Groundwater is an essential source of freshwater that supports surface ecosystems as well as organisms adapted to living underground. The impacts of anthropogenic climate change, extraction, and pollution pose major threats to groundwater ecosystem health, prompting a need for efficient and reliable means to detect and monitor subterranean faunal communities. Conventional survey of subterranean fauna relies on the collection of organisms for morphological identification, which can be biased, labor intensive, and often indeterminate at lower taxonomic levels. Environmental DNA (eDNA)‐based methods can dramatically improve on stygofaunal surveys, but currently rely on time consuming active water filtration that limits the number of samples that can be processed. Passive eDNA sampling, which involves submersion of absorbent material (e.g. filter membrane, sponge etc.) into the sampled environment for a fixed period, has shown promise as a viable alternative to active filtration in aquatic ecosystems and may be applicable to groundwater systems. Here, we compared groundwater eDNA collected from active pump filtered water samples, membranes submerged in water for 10 min and 24 h, and haul‐net samples morphologically identified from bores at two geographically distinct locations in Western Australia. Our results show that while the relative abundance of eDNA in groundwater (measured through qPCR) is 100–800 times lower in passive samples, the species richness detected is comparable between passive and active eDNA collection. Additionally, standard metabarcoding assays (18S and COI) of passive eDNA samples detected most subterranean orders identified morphologically (12/17), and this proportion may be improved with increased sampling and application of DNA extraction methods that increase DNA yield. Our findings demonstrate that passive eDNA sample collection is a non‐invasive survey method with the potential to improve the efficiency and replication level of stygofaunal surveys but will benefit from further development. Groundwater is an essential source of freshwater that supports surface ecosystems as well as organisms adapted to living underground. In this study we compared groundwater environmental DNA collected from active pump filtered water samples (AFGW), membranes submerged in water, and samples of morphologically identified animals collected at the same sampling sites. Our findings demonstrate that passive eDNA sample collection (PS10 and PS24) is a no