Generating and testing ecological hypotheses at the pondscape with environmental DNA metabarcoding: A case study on a threatened amphibian

Background: Environmental DNA (eDNA) metabarcoding is revolutionizing biodiversity monitoring, but has unrealized potential for ecological hypothesis generation and testing. Aims: Here, we validate this potential in a large-scale analysis of vertebrate community data generated by eDNA metabarcoding of 532 UK ponds. Materials & Methods: We test biotic associations between the threatened great crested newt (Triturus cristatus) and other vertebrates as well as abiotic factors influencing T.cristatus detection at the pondscape. Furthermore, we test the status of T.cristatus as an umbrella species for pond conservation by assessing whether vertebrate species richness is greater in ponds with T.cristatus and higher T.cristatus Habitat Suitability Index (HSI) scores. Results: Triturus cristatus detection was positively correlated with amphibian and waterfowl species richness. Specifically, T.cristatus was positively associated with smooth newt (Lissotriton vulgaris), common coot (Fulica atra), and common moorhen (Gallinula chloropus), but negatively associated with common toad (Bufo bufo). Triturus cristatus detection did not significantly decrease as fish species richness increased, but negative associations with common carp (Cyprinus carpio), three-spined stickleback (Gasterosteus aculeatus), and ninespine stickleback (Pungitius pungitius) were identified. Triturus cristatus detection was negatively correlated with mammal species richness, and T.cristatus was negatively associated with gray squirrel (Sciurus carolinensis). Triturus cristatus detection was negatively correlated with larger pond area, presence of inflow, and higher percentage of shading, but positively correlated with HSI score, supporting its application to T.cristatus survey. Vertebrate species richness was significantly higher in T.cristatus ponds and broadly increased as T.cristatus HSI scores increased. Discussion: We reaffirm reported associations (e.g., T.cristatus preference for smaller ponds) but also provide novel insights, including a negative effect of pond inflow on T.cristatus. Conclusion: Our findings demonstrate the prospects of eDNA metabarcoding for ecological hypothesis generation and testing at landscape scale, and dramatic enhancement of freshwater conservation, management, monitoring, and research

Navigating the trade-offs between environmental DNA and conventional field surveys for improved amphibian monitoring

The need for efficient, accurate biodiversity monitoring is growing, especially for globally imperiled taxa, such as amphibians. Environmental DNA (eDNA) analysis holds enormous potential for enhancing monitoring programs, but as this tool is increasingly adopted, it is imperative for users to understand its potential benefits and shortcomings. We conducted a comparative study to evaluate the efficacy of two eDNA methodologies (quantitative (q)PCR and metabarcoding) and conventional field sampling approaches (seining, dipnetting, and visual encounter surveys) in a system of 20 ponds containing six different amphibian species. Using an occupancy modeling framework, we estimated differences in detection sensitivity across methods, with a focus on how eDNA survey design could be further optimized. Overall, both metabarcoding and qPCR were competitive with or improved upon conventional methods. Specifically, qPCR (species-specific approach) was the most effective technique for detecting two rare species, the California tiger salamander (Ambystoma californiense) and California red-legged frog (Rana draytonii), with a detection probability of &gt;0.80 per survey. Metabarcoding (community approach) estimated amphibian diversity with comparable rates to field techniques on average, and detected an additional 41 vertebrate taxa. However, for two abundant species (western toads, Anaxyrus boreas, and Pacific chorus frogs, Pseudacris regilla), field techniques outperformed metabarcoding, especially as individuals metamorphosed. Our results indicate that eDNA approaches would be most effective when paired with visual encounter surveys to detect terrestrial life stages, and that more optimization, specifically primer choice and validation, is needed. By comparing methods across a diverse set of ponds and species, we provide guidance for future studies integrating eDNA approaches into amphibian monitoring. &nbsp;</p

Terrestrial land cover shapes fish diversity in major subtropical rivers

Freshwater biodiversity is critically affected by human modifications of terrestrial land use and land cover (LULC). Yet, knowledge of the spatial extent and magnitude of LULC-aquatic biodiversity linkages is still surprisingly limited, impeding the implementation of optimal management strategies. Here, we compiled fish diversity data across a 160,000-km² subtropical river catchment in Thailand characterized by exceptional biodiversity yet intense anthropogenic alterations, and attributed fish species richness and community composition to contemporary terrestrial LULC across the catchment. We estimated a spatial range of LULC effects extending up to about 20 km upstream from sampling sites, and explained nearly 60% of the variance in the observed species richness, associated with major LULC categories including croplands, forest, and urban areas. We find that integrating both spatial range and magnitudes of LULC effects is needed to accurately predict fish species richness. Further, projected LULC changes showcase future gains and losses of fish species richness across the river network and offer a scalable basis for riverine biodiversity conservation and land management, allowing for potential mitigation of biodiversity loss in highly diverse yet data-deficient tropical to sub-tropical riverine habitats

Spatio-temporal monitoring of lake fish spawning activity using environmental DNA metabarcoding

Determining the timing and location of fish reproductive events is crucial for the implementation of correct management and conservation schemes. Conventional methods used to monitor these events are often unable to assess the spawning activity directly or can be invasive and therefore problematic. This is especially the case when threatened fish populations are the study subject, such as the Arctic charr (Salvelinus alpinus L.) populations in Windermere (Cumbria, UK). Arctic charr populations have been studied in this lake since the 1940s, and the locations and characteristics of spawning grounds have been described in detail using techniques such as hydroacoustics, as well as physical and visual surveys of the lake bottom. Here, in conjunction with established netting surveys, we added an environmental DNA (eDNA) metabarcoding approach to assess the spatial distribution of Arctic charr in the lake throughout the year to test whether this tool could allow us to identify spawning locations and activity. Sampling was carried out between October 2017 and July 2018 at three locations in the lake, covering putative and known spawning sites. eDNA metabarcoding provided accurate spatial and temporal characterization of Arctic charr spawning events. Peaks of Arctic charr relative read counts from eDNA metabarcoding were observed during the spawning season and at specific locations of both putative and known spawning sites. Net catches of mature Arctic charr individuals confirmed the association between the Arctic charr spawning activity and the peaks of eDNA metabarcoding relative read counts. This study demonstrates the ability of eDNA metabarcoding to effectively and efficiently characterize the spatial and temporal nature of fish spawning in lentic systems

Pond ecology and conservation: research priorities and knowledge gaps

Ponds are among the most biodiverse and ecologically important freshwater habitats globally and may provide a significant opportunity to mitigate anthropogenic pressures and reverse the decline of aquatic biodiversity. Ponds also provide important contributions to society through the provision of ecosystem services. Despite the ecological and societal importance of ponds, freshwater research, policy, and conservation have historically focused on larger water bodies, with significant gaps remaining in our understanding and conservation of pond ecosystems. In May 2019, pond researchers and practitioners participated in a workshop to tackle several pond ecology, conservation, and management issues. Nine research themes and 30 research questions were identified during and following the workshop to address knowledge gaps around: (1) pond habitat definition; (2) global and long-term data availability; (3) anthropogenic stressors; (4) aquatic–terrestrial interactions; (5) succession and disturbance; (6) freshwater connectivity; (7) pond monitoring and technological advances; (8) socio-economic factors; and (9) conservation, management, and policy. Key areas for the future inclusion of ponds in environmental and conservation policy were also discussed. Addressing gaps in our fundamental understanding of pond ecosystems will facilitate more effective research-led conservation and management of pondscapes, their inclusion in environmental policy, support the sustainability of ecosystem services, and help address many of the global threats driving the decline in freshwater biodiversity

Pond ecology and conservation: research priorities and knowledge gaps

Ponds are among the most biodiverse and ecologically important freshwater habitats globally and may provide a significant opportunity to mitigate anthropogenic pressures and reverse the decline of aquatic biodiversity. Ponds also provide important contributions to society through the provision of ecosystem services. Despite the ecological and societal importance of ponds, freshwater research, policy, and conservation have historically focused on larger water bodies, with significant gaps remaining in our understanding and conservation of pond ecosystems. In May 2019, pond researchers and practitioners participated in a workshop to tackle several pond ecology, conservation, and management issues. Nine research themes and 30 research questions were identified during and following the workshop to address knowledge gaps around: (1) pond habitat definition; (2) global and long-term data availability; (3) anthropogenic stressors; (4) aquatic–terrestrial interactions; (5) succession and disturbance; (6) freshwater connectivity; (7) pond monitoring and technological advances; (8) socio-economic factors; and (9) conservation, management, and policy. Key areas for the future inclusion of ponds in environmental and conservation policy were also discussed. Addressing gaps in our fundamental understanding of pond ecosystems will facilitate more effective research-led conservation and management of pondscapes, their inclusion in environmental policy, support the sustainability of ecosystem services, and help address many of the global threats driving the decline in freshwater biodiversity.Additional co-authors: James C. White, Robert A. Briers, Kate L. Mathers, Michael J. Jeffries, and Paul J. Woo

Environmental DNA (eDNA) metabarcoding of pond water as a tool to survey conservation and management priority mammals

Environmental DNA (eDNA) metabarcoding can identify terrestrial taxa utilising aquatic habitats alongside aquatic communities, but terrestrial species' eDNA dynamics are understudied. We evaluated eDNA metabarcoding for monitoring semi-aquatic and terrestrial mammals, specifically nine species of conservation or management concern, and examined spatiotemporal variation in mammal eDNA signals. We hypothesised eDNA signals would be stronger for semi-aquatic than terrestrial mammals, and at sites where individuals exhibited behaviours. In captivity, we sampled waterbodies at points where behaviours were observed (‘directed’ sampling) and at equidistant intervals along the shoreline (‘stratified’ sampling). We surveyed natural ponds (N = 6) where focal species were present using stratified water sampling, camera traps, and field signs. eDNA samples were metabarcoded using vertebrate-specific primers. All focal species were detected in captivity. eDNA signal strength did not differ between directed and stratified samples across or within species, between semi-aquatic or terrestrial species, or according to behaviours. eDNA was evenly distributed in artificial waterbodies, but unevenly distributed in natural ponds. Survey methods deployed at natural ponds shared three species detections. Metabarcoding missed badger and red fox recorded by cameras and field signs, but detected small mammals these tools overlooked, e.g. water vole. Terrestrial mammal eDNA signals were weaker and detected less frequently than semi-aquatic mammal eDNA signals. eDNA metabarcoding could enhance mammal monitoring through large-scale, multi-species distribution assessment for priority and difficult to survey species, and provide early indication of range expansions or contractions. However, eDNA surveys need high spatiotemporal resolution and metabarcoding biases require further investigation before routine implementation

Assessment of habitat and survey criteria for the great crested newt (Triturus cristatus) in Scotland: a case study on a translocated population

The great crested newt Triturus cristatus has declined across its range due to habitat loss, motivating research into biotic and abiotic species determinants. However, research has focused on populations in England and mainland Europe. We examined habitat and survey criteria for great crested newts in Scotland, with focus on a large, translocated population. Adult counts throughout the breeding season were obtained annually using torchlight surveys, and Habitat Suitability Index (HSI) assessed at created ponds (N = 24) in 2006 (immediately post-translocation) and 2015 (9 years post-translocation). In 2006, ‘best case’ HSI scores were calculated to predict habitat suitability should great crested newts have unrestricted access to terrestrial habitat. Abiotic criteria included in and omitted from current great crested newt survey guidelines were assessed using data recorded in 2015. Some ponds had improved HSI scores in 2015, but overall failure to meet predicted scores suggests management is needed to improve habitat suitability. Great crested newt activity was positively associated with moon visibility and phase, air temperature, and pH, but negatively correlated with water clarity. Importantly, our results indicate there are abiotic determinants specific to Scottish great crested newts. Principally, survey temperature thresholds should be lowered to enable accurate census of Scottish populations

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570