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Pressures on freshwater biodiversity in Southeast Asia are accelerating, yet the status and conservation needs of many of the region’s iconic fish species are poorly known. The Mekong is highly species diverse and supports four of the six largest freshwater fish globally, three of which, including Mekong giant catfish (Pangasianodon gigas), are Critically Endangered. Emerging environmental DNA (eDNA) techniques have potential for monitoring threatened freshwater biodiversity, yet have not been applied in complex and biodiverse tropical ecosystems such as the Mekong. We developed species-specific primers for amplifying Mekong giant catfish DNA. In situ validation demonstrated that the DNA amplification was successful for all samples taken in reservoirs with known presence of Mekong giant catfish independent of fish density. We collected water samples from six deep pools on the Mekong, identified through Local Ecological Knowledge, in Cambodia, Lao PDR, and Thailand. DNA was extracted and amplified from these samples using the designed primers and probes. Mekong giant catfish DNA was detected from one sample from the species’ presumed spawning grounds on the Mekong mainstream, near the border between northern Thailand and Lao PDR. eDNA sampling using species-specific primers has potential for surveying and monitoring poorly known species from complex tropical aquatic environments. However accounting for false absences is likely to be required for the method to function with precision when applied to extremely rare species that are highly dispersed within a large river system. We recommend that such approach be utilised more widely by freshwater conservation practitioners for specific applications. The method is best suited for baseline biodiversity assessments or to identify and prioritise locations for more rigorous sampling. Our methods are particularly relevant for systems or species with limited baseline data or with physical characteristics that logistically limit the application of conventional methods. Such attributes are typical of large tropical rivers such as the Mekong, Congo, or Amazon.

1. Global pressures on freshwater ecosystems are high and rising. Viewed primarily as a resource for humans, current practices of water use have led to catastrophic declines in freshwater species and the degradation of freshwater ecosystems, including their genetic and functional diversity. Approximately three-quarters of the world's inland wetlands have been lost, one-third of the 28 000 freshwater species assessed for the International Union for Conservation of Nature (IUCN) Red List are threatened with extinction, and freshwater vertebrate populations are undergoing declines that are more rapid than those of terrestrial and marine species. This global loss continues unchecked, despite the importance of freshwater ecosystems as a source of clean water, food, livelihoods, recreation, and inspiration. 2. The causes of these declines include hydrological alterations, habitat degradation and loss, overexploitation, invasive species, pollution, and the multiple impacts of climate change. Although there are policy initiatives that aim to protect freshwater life, these are rarely implemented with sufficient conviction and enforcement. Policies that focus on the development and management of fresh waters as a resource for people almost universally neglect the biodiversity that they contain. 3. Here we introduce the Alliance for Freshwater Life, a global initiative, uniting specialists in research, data synthesis, conservation, education and outreach, and policymaking. This expert network aims to provide the critical mass required for the effective representation of freshwater biodiversity at policy meetings, to develop solutions balancing the needs of development and conservation, and to better convey the important role freshwater ecosystems play in human well-being. Through this united effort we hope to reverse this tide of loss and decline in freshwater biodiversity. We introduce several short- and medium-term actions as examples for making positive change, and invite individuals, organizations, authorities, and governments to join the Alliance for Freshwater Life.

“Each year at the season of the falling of the waters, the people living in the vicinity of the Golden Basin, the home of the Pla Buk [Mekong giant catfish, Pangasianodon gigas], join together for the purpose of catching these fish…..The ceremonies connected with the taking of these fish are ancient and have been performed from time immemorial, and carried out once a year.” - F.H. Giles, 1935 This thesis was undertaken to test the potential of the relatively new survey technique of analyzing water samples which contain DNA that has been shed by organisms (environmental DNA) for the study of diversity and distributions of fishes inhabiting tropical and subtropical rivers. The purpose was to gain information on species richness, assemblages, and distributions; with the ultimate aim of improving conservation management of rare and endangered freshwater fishes. The limitations of conventional (observation-based) survey methods to study rare and threatened fishes in large tropical rivers motivated the choice of testing environmental DNA (eDNA) analysis approaches. Specifically, challenges encountered during prior work to conserve the Mekong giant catfish and other rare species fostered the goal of testing eDNA analysis methods. The Mekong giant catfish was once part of a fishery with substantial cultural and socioeconomic import, as described by Giles (1935). However; today the fishery and ceremony surrounding it no longer exists, as the species has become so rare that the capture of wild individuals is banned and it has been assessed as Critically Endangered by the IUCN Red List. The case of the Mekong River and giant catfish represents an archetypical example of the challenges and limitations of using conventional methods to study and conserve rare fishes in large tropical rivers. These rivers host the highest species diversity of any freshwater environment, yet are facing the highest rates of extinction. This dire situation is partially due to the difficulty of gaining access to data on spatiotemporal distributions that are needed to inform effective conservation actions. The relatively new technique of using eDNA to access data on species richness, assemblages and distributions may enable access to data from tropical rivers that are not accessible with conventional methods. This thesis compares the benefits and limitations of conventional survey methods with eDNA techniques, within the context of rare species in large rivers. It reviews the development and application of eDNA to study aquatic macroorganisms, specifically fishes. It describes the rapid growth in application of the technique through an analysis of published literature, and finds the majority of studies are focused on developing the methodology. This technique was tested on captive populations of Mekong giant catfish (Pangasianodon gigas) in Thailand, and then used to detect wild giant catfish in the Mekong River at locations where it has been captured in the past. The giant catfish was detected in one of six survey locations, and only one sample from the river. Given the developmental stage of the eDNA technique, the method was further tested in two smaller subtropical rivers with well-known fish species composition. Three different clade-specific (also termed ‘universal’ or ‘generic’) fish primers were used to see if they returned congruent results. Clade-specific primers are designed to amplify all species within a given group, such as bony fishes, without amplifying species outside that group, such as other vertebrates. It was found that each primer set had different biases and that eDNA samples did not cluster either by primer set or by location. A subsequent study analyzed whether such variance was related to the choice of sampling location within the river. Given the highly dynamic and heterogeneous nature of rivers, it is reasonable to assume that eDNA may not be equally distributed along a cross-section within a single hydraulic unit. High variation was found among samples collected at three different points along a cross-section in a pool and riffle in the Brisbane River, Queensland, Australia when using universal primers and eDNA metabarcoding to determine taxa assemblage. Finally, the method was tested on sediment cores collected in a subtropical embayment at the outflow of the Brisbane River to determine whether a chronology of catchment-scale assemblage of freshwater fish communities could be identified. The results were inconclusive, as primarily bacterial sequences were identified from high-throughput sequencing. These results could indicate that eDNA of macroorganisms is not preserved well enough for detection in subtropical sediment cores. This is consistent with the endosymbiont theory that states that mitochondria are descendent from bacteria that have been assimilated into another cell ( Sagan, 1967). This has implications for the design of eDNA experiments that target extremely low copy number template, because eDNA studies often target mitochondria rather than nuclear DNA. The conservation implications of the overall findings of this thesis highlight the fact that all survey methods face limitations when the target organisms are rare aquatic species. Conservation practitioners and resource managers must be creative and forward-thinking in order to gain data on the distribution of threatened species that are needed to inform effective conservation actions. The lack of such data is a contributing factor to the high rates of decline faced by freshwater biodiversity around the globe. The potential of eDNA tools as described in the literature’s early stages may have been somewhat overstated, given that the limitations of the method hadn’t yet been exhaustively tested. There is still a great deal to learn about how useful this method can be. The tool seems to work best when using species-specific primers, which amplify a single species, rather than clade-specific primers which can assess total species richness or assemblage through metabarcoding. The method needs further refinement, yet is currently most useful when applied in tandem with conventional sampling methods to survey areas that are difficult to access or for the case of large tropical rivers that have high spatial extent and little knowledge currently available regarding distributions of threatened species.