The global biodiversity crisis represents one of humanity´s grand challenges. Yet, most species remain undescribed, while for the many known species, we lack fundamental understanding of their ecology. Especially the functions that different organisms have in natural ecosystems remains poorly studied. This raises a key question: do we truly understand ecosystems and their response to environmental change? This project will use advanced environmental DNA (eDNA) sequencing to analyze biodiversity in freshwater streams, coasts and grasslands in Denmark. The project will be one of the hereto most comprehensive eDNA approaches for studying species diversity and interactions. The project will explore how ecosystems might shift with environmental change and how DNA sequences can be used in monitoring ecosystem quality. Finally, it will establish best practices for future eDNA ecosystem monitoring, providing valuable insights for nature management and ecosystem health.