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Efficient DNA extraction is fundamental to molecular studies. However, commercial kits are expensive when a large number of samples need to be processed. Here we present a simple, modular and adaptable DNA extraction 'toolkit' for the isolation of high purity DNA from multiple sample types (modular universal DNA extraction method or Mu-DNA). We compare the performance of our method to that of widely used commercial kits across a range of soil, stool, tissue and water samples. Mu-DNA produced DNA extractions of similar or higher yield and purity to that of the commercial kits. As a proof of principle, we carried out replicate fish metabarcoding of aquatic eDNA extractions, which confirmed that the species detection efficiency of our method is similar to that of the most frequently used commercial kit. Our results demonstrate the reliability of Mu-DNA along with its modular adaptability to challenging sample types and sample collection methods. Mu-DNA can substantially reduce the costs and increase the scope of experiments in molecular studies.

23Environmental DNA (eDNA) metabarcoding can identify terrestrial taxa utilising aquatic habitats 24 alongside aquatic communities, but terrestrial species' eDNA dynamics are understudied. We 25 evaluated eDNA metabarcoding for monitoring semi-aquatic and terrestrial mammals, 26 specifically nine species of conservation or management concern, and examined 27 spatiotemporal variation in mammal eDNA signals. We hypothesised eDNA signals would be 28 stronger for semi-aquatic than terrestrial mammals, and at sites where individuals exhibited 29 behaviours. In captivity, we sampled waterbodies at points where behaviours were observed 30 ('directed' sampling) and at equidistant intervals along the shoreline ('stratified' sampling). We 31 surveyed natural ponds (N = 6) where focal species were present using stratified water 32 sampling, camera traps, and field signs. eDNA samples were metabarcoded using vertebrate-33 specific primers. All focal species were detected in captivity. eDNA signal strength did not differ 34 between directed and stratified samples across or within species, between semi-aquatic or 35 terrestrial species, or according to behaviours. eDNA was evenly distributed in artificial 36 waterbodies, but unevenly distributed in natural ponds. Survey methods deployed at natural 37 ponds shared three species detections. Metabarcoding missed badger and red fox recorded by 38 cameras and field signs, but detected small mammals these tools overlooked, e.g. water vole. 39Terrestrial mammal eDNA signals were weaker and detected less frequently than semi-aquatic 40 mammal eDNA signals. eDNA metabarcoding could enhance mammal monitoring through 41 large-scale, multi-species distribution assessment for priority and difficult to survey species, and 42 provide early indication of range expansions or contractions. However, eDNA surveys need high 43 3 spatiotemporal resolution and metabarcoding biases require further investigation before 44 routine implementation. 45 46

The sampling of environmental DNA (eDNA) coupled with cost-efficient and ever-advancing sequencing technology is propelling changes in biodiversity monitoring within aquatic ecosystems. Despite the increasing number of eDNA metabarcoding approaches, the ability to quantify species biomass and abundance in natural systems is still not fully understood. Previous studies have shown positive but sometimes weak correlations between abundance estimates from eDNA metabarcoding data and from conventional capture methods. As both methods have independent biases a lack of concordance is difficult to interpret. Here we tested whether read counts from eDNA metabarcoding provide accurate quantitative estimates of the absolute abundance of fish in holding ponds with known fish biomass and number of individuals. Environmental DNA samples were collected from two fishery ponds with high fish density and broad species diversity. In one pond, two different DNA capture strategies (on-site filtration with enclosed filters and three different preservation buffers versus lab filtration using open filters) were used to evaluate their performance in relation to fish community composition and biomass/abundance estimates. Fish species read counts were significantly correlated with both biomass and abundance, and this result, together with information on fish diversity, was repeatable when open or enclosed filters with different preservation buffers were used. This research demonstrates that eDNA metabarcoding provides accurate qualitative and quantitative information on fish communities in small ponds, and results are consistent between different methods of DNA capture. This method flexibility will be beneficial for future eDNA-based fish monitoring and their integration into fisheries management.

Background Environmental DNA (eDNA) metabarcoding is a promising tool for rapid, non‐invasive biodiversity monitoring. Aims In this study, eDNA metabarcoding is applied to explore the spatial and temporal distribution of fish communities in two aquaculture ponds and to evaluate the detection sensitivity of this tool for low‐density species alongside highly abundant species. Materials & Methods This study was carried out at two artificially stocked ponds with a high fish density following the introduction and removal of two rare fish species. Results & Discussion When two rare species were introduced and kept at a fixed location in the ponds, eDNA concentration (i.e., proportional read counts abundance) of the introduced species typically peaked after two days. The increase in eDNA concentration of the introduced fish after 43 hrs may have been caused by increased eDNA shedding rates as a result of fish being stressed by handling, as observed in other studies. Thereafter, it gradually declined and stabilised after six days. These findings are supported by the highest community dissimilarity of different sampling positions being observed on the second day after introduction, which then gradually decreased over time. On the sixth day, there was no longer a significant difference in community dissimilarity between sampling days. The introduced species were no longer detected at any sampling positions on 48 hrs after removal from the ponds. eDNA is found to decay faster in the field than in controlled conditions, which can be attributed to the complex effects of environmental conditions on eDNA persistence or resulting in the vertical transport of intracellular DNA and the extracellular DNA absorbed by particles in the sediment. The eDNA signal and detection probability of the introduced species were strongest near the keepnets, resulting in the highest community variance of different sampling events at this position. Thereafter, the eDNA signal significantly decreased with increasing distance, although the signal increased slightly again at 85 m position away from the keepnets. Conclusions Collectively, these findings reveal that eDNA distribution in lentic ecosystems is highly localised in space and time, which adds to the growing weight of evidence that eDNA signal provides a good approximation of the presence and distribution of species in ponds. Moreover, eDNA metabarcoding is a powerful tool for detection of rare species alongside more abundant species due to the use of generic PCR primers, and can enable monitoring of spatial and temporal community variance.

Although the spread of sushi restaurants in the European Union and United States is a relatively new phenomenon, they have rapidly become among the most popular food services globally. Recent studies indicate that they can be associated with very high levels (>70%) of fish species substitution. Based on indications that the European seafood retail sector may currently be under better control than its North American counterpart, here we investigated levels of seafood labelling accuracy in sushi bars and restaurants across England. We used the COI barcoding gene to screen samples of tuna, eel, and a variety of other products characterised by less visually distinctive ‘white flesh’. Moderate levels of substitution were found (10%), significantly lower than observed in North America, which lends support to the argument that public awareness, policy and governance of seafood labels is more effective in the European Union. Nevertheless, the results highlight that current labelling practice in UK restaurants lags behind the level of detail implemented in the retail sector, which hinders consumer choice, with potentially damaging economic, health and environmental consequences. Specifically, critically endangered species of tuna and eel continue to be sold without adequate information to consumers.

Large tropical and subtropical rivers are among the most biodiverse ecosystems worldwide, but also suffer from high anthropogenic pressures. These rivers are hitherto subject to little or no routine biomonitoring, which would be essential for identification of conservation areas of high importance. Here, we use a single environmental DNA multi-site sampling campaign across the 200,000 km2 Chao Phraya river basin, Thailand, to provide key information on fish diversity. We found a total of 108 fish taxa and identified key biodiversity patterns within the river network. By using hierarchical clustering, we grouped the fish communities of all sites across the catchment into distinct clusters. The clusters not only accurately matched the topology of the river network, but also revealed distinct groups of sites enabling informed conservation measures. Our study reveals novel opportunities of large-scale monitoring via eDNA to identify relevant areas within whole river catchments for conservation and habitat protection.

The sampling of environmental DNA (eDNA) coupled with cost-efficient and ever-advancing sequencing technology is propelling changes in biodiversity monitoring within aquatic ecosystems. Despite the growth of DNA metabarcoding approaches, the ability to quantify species biomass and abundance in natural systems remains a major challenge. Few studies have examined the association between eDNA metabarcoding data and biomass inferred by whole-organism sampling, mesocosms or mock communities, and the interpretation of sequencing reads as a measure of biomass or number of organisms is largely disputed.Here we tested whether read counts from eDNA metabarcoding provide accurate quantitative estimates of fish abundance in holding ponds with known fish biomass and number of individuals.eDNA samples were collected from two fishery ponds with high fish density and broad species diversity. In one pond, two different DNA capture strategies (on-site filtration with enclosed filters and three different preservation buffers versus lab filtration using open filters) were used to evaluate their performance in relation to fish community composition and biomass/abundance estimates. Fish species read counts were significantly correlated with both biomass and abundance, and this result, together with information on fish diversity, was repeatable when open or enclosed filters with different preservation buffers were used.This research demonstrates that eDNA metabarcoding provides accurate qualitative and quantitative information on fish communities in small ponds, and results are consistent between different methods of DNA capture. This method flexibility will be beneficial for future eDNA-based fish monitoring and their integration into fisheries management.