Environmental DNA (eDNA) monitoring of priority conservation fish species in UK lentic ecosystems

Freshwater environments cover only a tiny fraction of the planet’s surface, yet they are biodiversity hotspots and support almost half of the global fish diversity, including globally and locally endangered species. In the UK, three species of national conservation value namely, Arctic charr (Salvelinus alpinus L.), European whitefish (Coregonus lavaretus L.) and vendace (Coregonus albula L.), are threatened throughout their limited distribution range by a number of human-driven environmental changes.To combat a further loss of these priority conservation fish, non-invasive, sensitive and reliable monitoring tools are required to assess their status and guide appropriate conservation measures. Advances in the field of environmental DNA (eDNA) have shown the potential of this tool to detect low-abundant species, however the actual application of eDNA methods (e.g. targeted and metabarcoding) for rare species monitoring has been mostly limited to the collection of presence/absence information.The scope of this thesis is to explore novel aspects of eDNA metabarcoding including the ability to estimate fish biomass/abundance, determine fish spawning and assess distribution and abundance of priority conservation fish across a variety of UK lentic systems. This thesis provides evidence that eDNA metabarcoding accurately reflects absolute fish biomass and abundance in high fish density ponds, and quantitative estimates are repeatable between different methods of DNA capture. This thesis demonstrates that eDNA metabarcoding determines location, timing and effort of Arctic charr spawning and can be as sensitive as quantitative PCR. Lastly, eDNA metabarcoding provides accurate information of Arctic charr, vendace and European whitefish distribution and abundance in UK lentic systems including sites of special conservation interest.The fish community information obtained from eDNA metabarcoding is, therefore, beneficial to predict future changes in distribution and abundance of priority conservation fish as well as to design appropriate management actions, and can enormously contribute to more efficient monitoring programmes in UK lentic systems

Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types

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

Isotopic Niche and Trophic Position of the Invasive Portunid Portunus segnis Forskål, (1775) in Elounda Bay (Crete Island, Eastern Mediterranean)

There is a growing recognition that an advanced understanding of the trophic characteristics of an invasive consumer can provide important information on its ecological impact. In recent years, the blue swimming crab Portunus segnis, one of the earliest Lessepsian invaders, has considerably expanded its distribution range in the Mediterranean Sea, yet, its trophic habits in invaded areas remain scarcely investigated. In this study, we used carbon and nitrogen stable isotopes analysis (SIA) to determine the trophic position and isotopic niche of the crab compared with other representatives of the flora and fauna occurring in Elounda Bay (Crete). P. segnis showed a trophic position of 3.9, higher than the values determined by SIA or conventional gut content analysis in other areas of the Mediterranean Sea or in the native range. Crab specimens showed a high inter-individual variability in both delta C-13 and delta N-15 values; further analysis indicated negligible differences in the isotopic niche of adult males and females. Conversely, delta N-15 values were significantly related to the size of the specimens, ultimately suggesting an ontogenetic dietary shift. This study represents the first attempt to characterize the trophic habits of the blue swimming crab in the context of an invaded food web and may contribute to the implementation of long-term management strategies of control and mitigation of its ecological impact

An individual-based dataset of carbon and nitrogen isotopic data of Callinectes sapidus in invaded Mediterranean waters

[EN] Background The characterisation of functional traits of non-indigenous and invasive species is crucial to assess their impact within invaded habitats. Successful biological invasions are often facilitated by the generalist diet of the invaders which can modify their trophic position and adapt to new ecosystems determining changes in their structure and functioning. Invasive crustaceans are an illustrative example of such mechanisms since their trophic habits can determine important ecological impacts on aquatic food webs. The Atlantic blue crab Callinectes sapidus is currently established and considered invasive in the Mediterranean Sea where it has been recorded for the first time between 1947 and 1949. In the last decade, the blue crab colonised most of the eastern and central Mediterranean Sea and the Black Sea and it is currently widening its distribution towards the western region of the basin. New information Stable isotope analysis is increasingly used to investigate the trophic habits of invasive marine species. Here, we collated individual measures of the blue crab d C and d N values and of its potential invertebrate prey into a geo-referenced dataset. The dataset includes 360 records with 236 isotopic values of the blue crab and 224 isotopic data of potential prey collected from five countries and 12 locations between 2014 and 2019. This dataset allows the estimation of the trophic position of the blue crab within a variety of invaded ecosystems, as well as advanced quantitative comparisons of the main features of its isotopic niche.Di Muri, C.; Rosati, I.; Bardelli, R.; Cilenti, L.; Veli, DL.; Falco, S.; Vizzini, S.... (2022). An individual-based dataset of carbon and nitrogen isotopic data of Callinectes sapidus in invaded Mediterranean waters. Biodiversity Data Journal (Online). 10:1-12. https://doi.org/10.3897/BDJ.10.e775161121

Read counts from environmental DNA (eDNA) metabarcoding reflect fish abundance and biomass in drained ponds.

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

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

Optimising species detection probability and sampling effort in lake fish eDNA surveys

Environmental DNA (eDNA) metabarcoding is transforming biodiversity monitoring in aquatic environments. Such an approach has been developed and deployed for monitoring lake fish communities in Great Britain, where the method has repeatedly shown a comparable or better performance than conventional approaches. Previous analyses indicated that 20 water samples per lake are sufficient to reliably estimate fish species richness, but it is unclear how reduced eDNA sampling effort affects richness, or other biodiversity estimates and metrics. As the number of samples strongly influences the cost of monitoring programmes, it is essential that sampling effort is optimised for a specific monitoring objective. The aim of this project was to explore the effect of reduced eDNA sampling effort on biodiversity metrics (namely species richness and community composition) using algorithmic and statistical resampling techniques of a data set from 101 lakes, covering a wide spectrum of lake types and ecological quality. The results showed that reliable estimation of lake fish species richness could, in fact, usually be achieved with a much lower number of samples. For example, in almost 90% of lakes, 95% of complete fish richness could be detected with only 10 water samples, regardless of lake area. Similarly, other measures of alpha and beta-diversity were not greatly affected by a reduction in sample size from 20 to 10 samples. We also found that there is no significant difference in detected species richness between shoreline and offshore sampling transects, allowing for simplified field logistics. This could potentially allow the effective sampling of a larger number of lakes within a given monitoring budget. However, rare species were more often missed with fewer samples, with potential implications for monitoring of invasive or endangered species. These results should inform the design of eDNA sampling strategies, so that these can be optimised to achieve specific monitoring goals

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