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Marine sponges have recently been recognised as natural samplers of environmental DNA (eDNA) due to their effective water filtration and their ubiquitous, sessile and regenerative nature. However, laboratory workflows for metabarcoding of sponge tissue have not been optimised to ensure that these natural samplers achieve their full potential for community survey. We used a phased approach to investigate the influence of DNA isolation procedures on the biodiversity information recovered from sponges. In Phase 1, we compared three treatments of residual ethanol preservative in sponge tissue alongside five DNA extraction protocols. The results of Phase 1 informed which ethanol treatment and DNA extraction protocol should be used in Phase 2, where we assessed the effect of starting tissue mass on extraction success and whether homogenisation of sponge tissue is required. Phase 1 results indicated that ethanol preservative may contain unique and/or additional biodiversity information to that present in sponge tissue, but blotting tissue dry generally recovered more taxa and generated more sequence reads from the wild sponge species. Tissue extraction protocols performed best in terms of DNA concentration, taxon richness and proportional read counts, but the non-commercial tissue protocol was selected for Phase 2 due to cost-efficiency and greater recovery of target taxa. In Phase 2 overall, we found that homogenisation may not be required for sponge tissue and more starting material does not necessarily improve taxon detection. These results combined provide an optimised DNA isolation procedure for sponges to enhance marine biodiversity assessment using natural sampler DNA metabarcoding.

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.

The early detection of invasive non-native species (INNS) is important to allow for informed management actions. Established monitoring methods require the collection or observation of specimens, which is unlikely at the beginning of an invasion when densities are likely to be low. Environmental DNA (eDNA) analysis is a highly promising technique for the detection of INNS – particularly during the early stages of an invasion. Here, we compared the use of traditional kick-net sampling with two eDNA approaches (targeted detection using both conventional and quantitative PCR, and passive detection via metabarcoding) for detection of quagga mussel, Dreissena rostriformis bugensis; a high priority INNS, along a density gradient on the River Wraysbury, UK. All three molecular tools outperformed traditional sampling in terms of detection. Conventional PCR and qPCR both had 100% detection rate in all samples, and outperformed metabarcoding when the target species was at low densities. Additionally, quagga mussel DNA copy number (qPCR) and read count (metabarcoding) had a significant negative relationship with distance from source population. Synthesis and application . All three molecular approaches were more sensitive than traditional kick-net sampling for the detection of the quagga mussel in flowing water, and both qPCR and metabarcoding enabled estimates of relative abundance. Targeted approaches were more sensitive than metabarcoding, but metabarcoding has the advantage of providing information on the wider community, and consequently impacts of INNS.

Environmental DNA (eDNA) analysis is a rapid, cost-effective, non-invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species-specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and ‘metabarcoding’ have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real-time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using High-Throughput Sequencing technology. With qPCR and a detection threshold of 1/12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4/12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species-specific surveys.