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Alternative methods for assessing habitat quality in freshwater systems“Water, water, everywhere…”. 71% of the earth’s surface is covered by water, freshwater representing 2.5% of it, and only 1% being accessible. Due, largely to a number of anthropogenic activities (pollution, habitats modification) coupled with the impacts of climate change, a dramatic decline in biodiversity is occurring across all earth’s ecosystems. Surprisingly, freshwater ecosystems receive considerably less attention than many other habitats and therefore, effective biodiversity monitoring programs are urgently needed to assess the health and state of the endangered and threatened species in these aquatic systems. Further, current techniques utilised to survey freshwater ecosystems are often considered ineffective, invasive, time consuming and biased. As a result, the implementation of molecular-based detection tools are attractive options as they are often shown to be more sensitive and cost effective. The use of environmental DNA (eDNA) detection is one such molecular tool which is showing promising results, due to its high reliability, sensitivity and non-invasiveness characters. However, recent studies have highlighted potential limitations associated with eDNA-based detection. Such limitations may lead to a decrease in the confidence of this method. The aim of this thesis was to investigate the use of eDNA-based detection across a number of species and a number of systems, all as a proxy of habitat quality. Stringent laboratory practices and validation guidelines were adhered to, allowing for reliable quality assessments of newly designed eDNA assays outlined in this thesis. Moreover, distinct controlled mesocosm experiments allowed the investigation of critical factors, part of the sampling method or analysis processes leading to an optimisation of eDNA collection and decreasing the rates of false negative results. Several comparison between traditional monitoring techniques and the novel assays were also performed aiding in the confidence of these new methods. Interestingly, the results obtained in this thesis shows a similar efficiency between traditional and eDNA-based methods for monitoring invasive species, but a higher efficiency of eDNA detection when detecting rare or low abundant organisms (i.e. those that are endangered or threatened). Furthermore, this thesis reports an extreme example where a species was found at a number of locations within a stretch of a river, yet undetected with the eDNA assay. In this chapter eDNA detection was only possible when I utilised ddPCR rather than qPCR (the more standard technique for assessing eDNA in any given system). Overall, eDNA detection was found to be an effective tool for assessing the presence of invasive and/or endangered species, increasing theknowledge on their distribution and the impact of future management plans. In this thesis, chapters 2, 3, 4, 5 and 6 are organised as case studies, aiming to highlight benefits and limitations of species-specific detection using eDNA.