Hybrid Horizons: Screening Hybridisation Through Nuclear Environmental DNA.

Effective monitoring of hybrid zones is essential for understanding evolutionary dynamics and mitigating species loss caused by human-mediated hybridisation. Conventional methods rely on sampling numerous individuals, which is costly, time-consuming and often impractical for rare or elusive species. Environmental DNA (eDNA) offers a promising alternative for locating hybrid zones but requires the detection of nuclear eDNA, which is typically scarce in natural ecosystems. While a few recent studies have successfully recovered sufficient nuclear eDNA to assess intraspecific variation, its application in hybridisation studies remains untested. This study provides the first empirical validation that nuclear eDNA can screen for hybrid populations. We present an eDNA-based toolkit that employs Kompetitive Allele-Specific PCR (KASP) to genotype a panel of species-diagnostic unlinked nuclear SNPs without sequencing, mapping end-point fluorescence to a hybrid index that reflects ancestry levels at the population scale. In mesocosms housing different combinations of individuals from two crested newt species (Triturus ivanbureschi and T. macedonicus) and their captive-bred F1 hybrids, we compared eDNA-derived ancestry estimates with genotypes obtained from skin swabs of the same individuals placed in the mesocosms. eDNA-based ancestry estimates showed strong concordance with individual genotypes across two eDNA sampling concentrations. This approach represents a promising non-invasive, fast and cost-efficient screening tool, qualities that make it well suited to locate and track putative hybrid zones and a scalable complement to conventional sampling for biodiversity monitoring and conservation.