Next-generation monitoring of aquatic biodiversity using environmental DNA metabarcoding.

Global biodiversity in freshwater and the oceans is declining at high rates. Reliable tools for assessing and monitoring aquatic biodiversity, especially for rare and secretive species, are important for efficient and timely management. Recent advances in DNA sequencing have provided a new tool for species detection from DNA present in the environment. In this study, we tested whether an environmental DNA (eDNA) metabarcoding approach, using water samples, can be used for addressing significant questions in ecology and conservation. Two key aquatic vertebrate groups were targeted: amphibians and bony fish. The reliability of this method was cautiously validated in silico, in vitro and in situ. When compared with traditional surveys or historical data, eDNA metabarcoding showed a much better detection probability overall. For amphibians, the detection probability with eDNA metabarcoding was 0.97 (CI = 0.90-0.99) vs. 0.58 (CI = 0.50-0.63) for traditional surveys. For fish, in 89% of the studied sites, the number of taxa detected using the eDNA metabarcoding approach was higher or identical to the number detected using traditional methods. We argue that the proposed DNA-based approach has the potential to become the next-generation tool for ecological studies and standardized biodiversity monitoring in a wide range of aquatic ecosystems.

Identification of ecological thresholds from variations in phytoplankton communities among lakes: contribution to the definition of environmental standards.

In aquatic ecosystems, the identification of ecological thresholds may be useful for managers as it can help to diagnose ecosystem health and to identify key levers to enable the success of preservation and restoration measures. A recent statistical method, gradient forest, based on random forests, was used to detect thresholds of phytoplankton community change in lakes along different environmental gradients. It performs exploratory analyses of multivariate biological and environmental data to estimate the location and importance of community thresholds along gradients. The method was applied to a data set of 224 French lakes which were characterized by 29 environmental variables and the mean abundances of 196 phytoplankton species. Results showed the high importance of geographic variables for the prediction of species abundances at the scale of the study. A second analysis was performed on a subset of lakes defined by geographic thresholds and presenting a higher biological homogeneity. Community thresholds were identified for the most important physico-chemical variables including water transparency, total phosphorus, ammonia, nitrates, and dissolved organic carbon. Gradient forest appeared as a powerful method at a first exploratory step, to detect ecological thresholds at large spatial scale. The thresholds that were identified here must be reinforced by the separate analysis of other aquatic communities and may be used then to set protective environmental standards after consideration of natural variability among lakes.

Distribution of pesticides and some of their transformation products in a small lentic waterbody: Fish, water, and sediment contamination in an agricultural watershed.

More than 20 years after the Water Framework Directive was adopted, there are still major gaps in the sanitary status of small rivers and waterbodies at the head of basins. These small streams supply water to a large number of wetlands that support a rich biodiversity. Many of these waterbodies are fishponds whose production is destined for human consumption or for the restocking of other aquatic environments. However, these ecosystems are exposed to contaminants, including pesticides and their transformation products. This work aims to provide information on the distribution, diversity, and concentrations of agricultural contaminants in abiotic and biotic compartments from a fishpond located at the head of watersheds. A total of 20 pesticides and 20 transformation products were analyzed by HPLC-ESI-MS/MS in water and sediment sampled monthly throughout a fish production cycle, and in three fish species at the beginning and end of the cycle. The highest mean concentrations were found for metazachlor-OXA (519.48 ± 56.52 ng.L-1) in water and benzamide (4.23 ± 0.17 ng g-1 dry wt.) in sediment. Up to 20 contaminants were detected per water sample and 26 per sediment sample. The transformation products of atrazine (banned in Europe since 2003 but still widely used in other parts of the world), flufenacet, imidacloprid (banned in France since 2018), metazachlor, and metolachlor were more concentrated than their parent compounds. Fewer contaminants were detected in fish and principally prosulfocarb accumulated in organisms during the cycle. Our work brings innovative data on the contamination of small waterbodies located at the head of a basin. The transformation products with the highest frequency of occurrence and concentrations should be prioritized for further environmental monitoring studies, and specific toxicity thresholds should be defined. Few contaminants were found in fish, but the results challenge the widely use of prosulfocarb.

Spatio-temporal variability of eDNA signal and its implication for fish monitoring in lakes.

Environmental DNA (eDNA) metabarcoding is revolutionizing the monitoring of aquatic biodiversity. The use of eDNA has the potential to enable non-invasive, cost-effective, time-efficient and high-sensitivity monitoring of fish assemblages. Although the capacity of eDNA metabarcoding to describe fish assemblages is recognised, research efforts are still needed to better assess the spatial and temporal variability of the eDNA signal and to ultimately design an optimal sampling strategy for eDNA monitoring. In this context, we sampled three different lakes (a dam reservoir, a shallow eutrophic lake and a deep oligotrophic lake) every 6 weeks for 1 year. We performed four types of sampling for each lake (integrative sampling of sub-surface water along transects on the left shore, the right shore and above the deepest zone, and point sampling in deeper layers near the lake bottom) to explore the spatial variability of the eDNA signal at the lake scale over a period of 1 year. A metabarcoding approach was applied to analyse the 92 eDNA samples in order to obtain fish species inventories which were compared with traditional fish monitoring methods (standardized gillnet samplings). Several species known to be present in these lakes were only detected by eDNA, confirming the higher sensitivity of this technique in comparison with gillnetting. The eDNA signal varied spatially, with shoreline samples being richer in species than the other samples. Furthermore, deep-water samplings appeared to be non-relevant for regularly mixed lakes, where the eDNA signal was homogeneously distributed. These results also demonstrate a clear temporal variability of the eDNA signal that seems to be related to species phenology, with most of the species detected in spring during the spawning period on shores, but also a peak of detection in winter for salmonid and coregonid species during their reproduction period. These results contribute to our understanding of the spatio-temporal distribution of eDNA in lakes and allow us to provide methodological recommendations regarding where and when to sample eDNA for fish monitoring in lakes.

Ecosystem maturity modulates greenhouse gases fluxes from artificial lakes.

Lentic ecosystems play a major role in the global carbon cycling but the understanding of the environmental determinants of lake metabolism is still limited, notably in small artificial lakes. Here the effects of environmental conditions on lake metabolism and CO2 and CH4 emissions were quantified in 11 small artificial gravel pit lakes covering a gradient of ecosystem maturity, ranging from young oligotrophic to older, hypereutrophic lakes. The diffusive fluxes of CO2 and CH4 ranged from -30.10 to 37.78 mmol m-2 d-1 and from 3.05 to 25.45 mmol m-2 d-1 across gravel pit lakes, respectively. Nutrients and chlorophyll a concentrations were negatively correlated with CO2 concentrations and emissions but positively correlated with CH4 concentrations and emissions from lakes. These findings indicate that, as they mature, gravel pit lakes switch from heterotrophic to autotrophic-based metabolism and hence turn into CO2-sinks. In contrast, the emission of CH4 increased along the maturity gradient. As a result, eutrophication occurring during ecosystem maturity increased net emissions in terms of climate impact (CO2 equivalent) due to the higher contribution of CH4 emissions. Overall, mean CO2equivalent emission was 7.9 g m-2 d-1, a value 3.7 and 4.7 times higher than values previously reported in temperate lakes and reservoirs, respectively. While previous studies reported that lakes represent emitters of C to the atmosphere, this study highlights that eutrophication may reverse lake contribution to global C budgets. However, this finding is to be balanced with the fact that eutrophication also increased CH4 emissions and hence, enhanced the potential impact of these ecosystems on climate. Implementing mitigation strategies for maintaining intermediate levels of maturity is therefore needed to limit the impacts of small artificial waterbodies on climate. This could be facilitated by their small size and should be planned at the earliest stages of artificial lake construction.

Scale dependency in the hydromorphological control of a stream ecosystem functioning.

Physical habitat degradation is prevalent in river ecosystems. Although still little is known about the ecological consequences of altered hydromorphology, understanding the factors at play can contribute to sustainable environmental management. In this study we aimed to identify the hydromorphological features controlling a key ecosystem function and the spatial scales where such linkages operate. As hydromorphological and chemical pressures often occur in parallel, we examined the relative importance of hydromorphological and chemical factors as determinants of leaf breakdown. Leaf breakdown assays were investigated at 82 sites of rivers throughout the French territory. Leaf breakdown data were then crossed with data on water quality and with a multi-scale hydromorphological assessment (i.e. upstream catchment, river segment, reach and habitat) when quantitative data were available. Microbial and total leaf breakdown rates exhibited differential responses to both hydromorphological and chemical alterations. Relationships between the chemical quality of the water and leaf breakdown were weak, while hydromorphological integrity explained independently up to 84.2% of leaf breakdown. Hydrological and morphological parameters were the main predictors of microbial leaf breakdown, whereas hydrological parameters had a major effect on total leaf breakdown, particularly at large scales, while morphological parameters were important at smaller scales. Microbial leaf breakdown were best predicted by hydromorphological features defined at the upstream catchment level whereas total leaf breakdown were best predicted by reach and habitat level geomorphic variables. This study demonstrates the use of leaf breakdown in a biomonitoring context and the importance of hydromorphological integrity for the functioning of running water. It provides new insights for environmental decision-makers to identify the management and restoration actions that have to be undertaken including the hydromorphogical features that should be kept in minimal maintenance to support leaf breakdown.