Unlocking karst biodiversity with eDNA: a validated qPCR toolkit for discovery and monitoring the worlds largest cavefish radiation (Cyprinidae: Sinocyclocheilus)

Environmental DNA (eDNA) enables non-invasive detection of aquatic species through trace genetic material, but uncertainties regarding DNA persistence, detection accuracy, and the limited taxonomic resolution of stygian fauna have constrained its use in subterranean aquatic environments. The species-rich cavefish genus Sinocyclocheilus, endemic to the karst landscapes of southwestern China, offers an excellent model for understanding these issues. With over eighty species, many of which are endemics, and many still being described, this lineage is taxonomically rich, poorly understood, and highly threatened. However, access to these deep habitats remains challenging, making eDNA detection one of the most practical methods for documenting and monitoring them. We developed and validated a qPCR assay targeting a hypervariable region of the mitochondrial 16S rRNA gene, allowing for genus-specific detection and species-level identification. Laboratory and field evaluations involving 47 representative species confirmed high specificity and a detection limit of around 20 DNA copies per reaction (R{superscript 2} = 0.995). Controlled degradation studies indicated that temperature and pH strongly affect DNA persistence. Field surveys across 47 karst sites (37 caves and 10 surface sites) detected Sinocyclocheilus eDNA in only 33 caves. Dideoxy sequencing of qPCR amplicons provided species-level identification and phylogenetic validation, including detection of a new species. At three cave sites yielding multiple qPCR detections, 16S metabarcoding enabled species discrimination. By integrating qPCR assay validation, eDNA degradation modelling, hierarchical genetic identification via dideoxy sequencing, and selective NGS metabarcoding, this study presents a strategic framework for applying eDNA methods in subterranean environments.