In semi-arid regions, such as Australia's Northern Territory wet-dry tropics, groundwater represents a critical freshwater resource supporting both anthropogenic needs and unique subterranean ecosystems. The Beetaloo Sub-basin, a 30,000 km² area highly prospective for shale gas extraction, overlies aquifer systems that harbor stygofauna—obligate groundwater-dwelling invertebrates—and diverse microbial communities. An improved understanding of these subterranean ecosystems is necessary for assessing the risks associated with onshore petroleum activities.
Here, we investigated the distribution and ecology of stygofauna from three seasonal surveys conducted between October 2022 and May 2024. A multi-methodological approach was employed, integrating physical nets, video borescope inspections, and environmental DNA (eDNA) metabarcoding to characterise water quality, microbial community structure, and faunal diversity. Stygofauna were detected in 15 of 33 surveyed bores. Taxonomic analysis identified seven distinct groups, dominated by crustaceans, including genetically distinct populations of the decapod shrimp Paratya australis. While water chemistry and depth (9–100 m) were poor predictors of stygofaunal presence, video evidence confirmed that shrimp were strongly associated with specific geological cavities and bore screenings.
Metagenomic analyses revealed a complex microbial landscape. In the Beetaloo sub-basin we observed stygofauna up to 100 m below the surface in waters depleted in dissolved organic carbon (<1 mg L⁻¹) coupled with the presence of diverse chemolithotrophic microorganisms. These results suggest that the oxidation of inorganic molecules by chemolithotrophs may serve as the primary energy pathway for invertebrates, forming the foundation of the subterranean food web. No single sampling method ensured detection of stygofauna, highlighting the necessity of integrated monitoring approaches. The discovery of genetically distinct populations suggests limited connectivity in some areas, increasing vulnerability to environmental disturbances. Our research provides a baseline for environmental impact assessments, underscoring that alterations to nutrient profiles or microbial structures could destabilise these cryptic ecosystems.