Environmental DNA (eDNA) analysis is transforming biodiversity monitoring in aquatic ecosystems with immense potential to inform their conservation and management. eDNA analysis is rapid, non-invasive, cost-efficient, and often more accurate and sensitive than conventional monitoring tools for single species detection and community survey. Ponds are extremely diverse yet understudied freshwater habitats that require novel tools to enable comprehensive, systematic, long-term monitoring. eDNA monitoring could radically improve assessments of pond biodiversity, but the applications and methodical constraints of this tool in ponds are largely unexplored. In this thesis, eDNA analysis was examined as a tool for monitoring biodiversity associated with ponds, including aquatic, semi-aquatic, and terrestrial taxa. eDNA analysis using metabarcoding was shown to have comparable detection sensitivity for Triturus cristatus to targeted eDNA analysis using quantitative PCR, depending on species detection thresholds applied. Using the community data generated by this method comparison, eDNA metabarcoding was validated as a tool for ecological hypothesis testing, specifically biotic and abiotic determinants of T. cristatus and vertebrate species richness. A novel eDNA assay was designed and validated for targeted survey of the threatened Carassius carassius, a fish species characteristic of ponds. Furthermore, eDNA metabarcoding was compared to established methods of freshwater invertebrate assessment, and all methods used to evaluate the impact of stocking C. carassius for conservation purposes. Finally, eDNA metabarcoding was vindicated as a tool to monitor semi-aquatic and terrestrial mammals associated with ponds, and investigate the spatiotemporal distribution of their eDNA signals in these water bodies as a function of behaviour. These results combined emphasise the biological and scientific importance of ponds, and the prospects of eDNA analysis - targeted and community approaches - for enhanced conservation, management, monitoring, and research of these valuable ecosystems
