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.

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.

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.

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Ireland, being an island situated on Europe's western seaboard, has a fewer number of native species than mainland European Union Member States (MS). Increased numbers of vectors and pathways have reduced the island's biotic isolation, increasing the risk of new introductions and their associated impacts on native biodiversity. It is likely that these risks are greater here than they are in continental MSs, where the native biodiversity is richer. A horizon scanning approach was used to identify the most likely invasive alien species (IAS) (with the potential to impact biodiversity) to arrive on the island of Ireland within the next ten years. To achieve this, we used a consensus-based approach, whereby expert opinion and discussion groups were utilised to establish and rank a list of 40 species of the most likely terrestrial, freshwater and marine IAS to arrive on the island of Ireland within the decade 2017-2027. The list of 40 included 18 freshwater, 15 terrestrial and seven marine IAS. Crustacean species (freshwater and marine) were taxonomically dominant (11 out of 40); this reflects their multiple pathways of introduction, their ability to act as ecosystem engineers and their resulting high impacts on biodiversity. Freshwater species dominated the top ten IAS (seven species out of ten), with the signal crayfish (Pacifastacus leniusculus) highlighted as the most likely species to arrive and establish in freshwaters, while roe deer (Capreolus capreolus) (second) and the warm-water barnacle (Hesperibalanus fallax) (fifth), were the most likely terrestrial and marine invaders. This evidence-based list provides important information to the relevant statutory agencies in both the Republic of Ireland and Northern Ireland to prioritise the prevention of the most likely invaders and aid in compliance with legislation, in particular the EU Regulation on Invasive Alien Species (EU 1143/2014). Targeted biosecurity in both jurisdictions is urgently required in order to manage the pathways and vectors of arrival, and is vital to maintaining native biodiversity on the island of Ireland.

Biomonitoring of complex heterogeneous environments is highly challenging. Fish in deep water bodies occupy different habitats, therefore a combination of survey methods has traditionally been used. Environmental DNA (eDNA) metabarcoding is a novel monitoring tool that can overcome spatial heterogeneity in a highly sensitive and entirely non-invasive manner. However, taxon detection probability is dependent on real-time environmental variables. In this study, three reservoirs were sampled in two seasons using a spatiotemporally distributed sampling design covering major environmental gradients. In all sampling campaigns, 31 fish taxa were detected which exceeded expectations. Data reliability was confirmed by a tight positive correlation between individual taxon scores derived from gillnet sampling and eDNA site occupancy. Analyses confirmed anticipated trends, such as the highest number of taxa were observed in the largest water body, and more taxa were detected in inflows and littoral regions compared to open water. The most important factors for fish distribution were temperature, age and trophic status (expressed as total Chlorophyll a concentration) of water bodies. Taxon detection reflected ecological niches of individual species, e.g. warm water wels catfish (Silurus glanis) and cold water salmonids. This study provides further evidence that eDNA metabarcoding is suitable for ecological study in heterogeneous environments and may substitute conventional fish sampling techniques.

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 extraction 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.

Marine sponges have recently been recognized 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 optimized 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 preserva-