Abstract
This study investigates the ecological connectivity and biodiversity of groundwater fauna within Gauteng’s dolomitic karst aquifers using environmental DNA (eDNA) metabarcoding. By targeting the mitochondrial COI gene across water and sediment samples from Bakwena and Sterkfontein caves, we identified 59 unique metazoan families spanning 12 phyla. Water provided a greater taxonomic coverage than sediment. β-diversity revealed strong site-specific assemblages with limited overlap, while shared taxa such as copepods, thrips and bat families suggest potential ecological linkages. Methodological constraints including incomplete reference databases, primer bias, and limited temporal replication impacted taxonomic resolution but did not obscure the utility of eDNA for subterranean biosurveillance. Findings support the hypothesis that karst aquifers host distinct, structured communities shaped by environmental filtering and dispersal limitation. This work establishes a molecular ecological biodiversity data baseline for future monitoring and underscores the importance of integrating eDNA into groundwater biodiversity conservation strategies.