Alternative methods for assessing habitat quality in freshwater systems

“Water, water, everywhere…”. 71% of the earth’s surface is covered by water, freshwater representing 2.5% of it, and only 1% being accessible. Due, largely to a number of anthropogenic activities (pollution, habitats modification) coupled with the impacts of climate change, a dramatic decline in biodiversity is occurring across all earth’s ecosystems. Surprisingly, freshwater ecosystems receive considerably less attention than many other habitats and therefore, effective biodiversity monitoring programs are urgently needed to assess the health and state of the endangered and threatened species in these aquatic systems. Further, current techniques utilised to survey freshwater ecosystems are often considered ineffective, invasive, time consuming and biased. As a result, the implementation of molecular-based detection tools are attractive options as they are often shown to be more sensitive and cost effective. The use of environmental DNA (eDNA) detection is one such molecular tool which is showing promising results, due to its high reliability, sensitivity and non-invasiveness characters. However, recent studies have highlighted potential limitations associated with eDNA-based detection. Such limitations may lead to a decrease in the confidence of this method. The aim of this thesis was to investigate the use of eDNA-based detection across a number of species and a number of systems, all as a proxy of habitat quality. Stringent laboratory practices and validation guidelines were adhered to, allowing for reliable quality assessments of newly designed eDNA assays outlined in this thesis. Moreover, distinct controlled mesocosm experiments allowed the investigation of critical factors, part of the sampling method or analysis processes leading to an optimisation of eDNA collection and decreasing the rates of false negative results. Several comparison between traditional monitoring techniques and the novel assays were also performed aiding in the confidence of these new methods. Interestingly, the results obtained in this thesis shows a similar efficiency between traditional and eDNA-based methods for monitoring invasive species, but a higher efficiency of eDNA detection when detecting rare or low abundant organisms (i.e. those that are endangered or threatened). Furthermore, this thesis reports an extreme example where a species was found at a number of locations within a stretch of a river, yet undetected with the eDNA assay. In this chapter eDNA detection was only possible when I utilised ddPCR rather than qPCR (the more standard technique for assessing eDNA in any given system). Overall, eDNA detection was found to be an effective tool for assessing the presence of invasive and/or endangered species, increasing theknowledge on their distribution and the impact of future management plans. In this thesis, chapters 2, 3, 4, 5 and 6 are organised as case studies, aiming to highlight benefits and limitations of species-specific detection using eDNA.Surescreen Scientifics Lt

Rediscovery of the critically endangered ‘scarce yellow sally stonefly’ Isogenus nubecula in United Kingdom after a 22 year period of absence.

The critically endangered ‘scarce yellow sally stonefly’ Isogenus nubecula (Newman, 1833) (Plecoptera: Perlodidae) was rediscovered in the United Kingdom (UK) in 2017. This rediscovery comes after a 22-year period of absence despite numerous surveys since its last record in 1995. This species is one of the rarest stoneflies in the UK and Europe and its rediscovery is of international significance, being the westernmost point in Europe where the species is found, with the next nearest populations occurring in Austria and western Hungary, Slovakia, and central Sweden. The species is classed as pRDB2 (vulnerable), however is not listed in the British Red Data Book despite only being present (as far as records detail) in one river, the River Dee in North Wales, UK. Only fourteen individuals were caught and the need for conservation of this rare stonefly is therefore of paramount importance. We have made recommendations for the need to increase survey effort using environmental DNA (eDNA) techniques in order to fully understand the species range in this river and those in the surrounding area. The DNA sequence of I. nubecula has been uploaded on GenBank for further genetic studies. Captive rearing could also be explored with possible reintroductions to sites within its former UK range

Plant biodiversity assessment through soil eDNA reflects temporal and local diversity

1. Several studies have shown the potential of eDNA-based proxies for plant identification, but little is known about their spatial and temporal resolution. This limits its use for plant biodiversity assessments and monitoring of vegetation responses to environmental changes. Here we calibrate the temporal and spatial plant signals detected with soil eDNA surveys by comparing with a standard visual above-ground vegetation survey. 2. Our approach compares vegetation in an old-growth boreal forest in southern Norway, surveyed in 100 permanent 1-m2 plots seven times over a 30-year period, with a single soil eDNA metabarcoding-based survey from soil samples collected at the same 100 plots in the year of the last vegetation survey. 3. On average, 60% and 10% of the vascular plants and bryophytes recorded across all vegetation surveys were detected by soil eDNA. Taxa detected by soil eDNA were more representative for the local taxa pool than for the specific plot, and corresponded to those surveyed over the 30-year period although most closely matched the current taxa composition. Soil eDNA detected abundant taxa better than rare ones although both rare taxa and taxa unrecorded by the visual survey were detected. 4. Our study highlights the potential of soil eDNA assessments for monitoring of vegetation responses over broad spatial and temporal scales. The method's ability to detect abundant taxa makes it suitable for assessment of vegetation composition in a specific area and for broad-scale plant diversity assessments

First detection of a highly invasive freshwater amphipod (Crangonyx floridanus) in the United Kingdom

The freshwater gammarid, Crangonyx floridanus, originates from North America but has invaded and subsequently spread rapidly throughout Japan. We provide here the first genetic and microscopic evidence that C. floridanus has now also reached the United Kingdom. We found this species in two locations separated by more than 200 km (Lake Windermere in the North of the UK and Smestow Brook, West Midlands). The current distribution of C. floridanus is currently unknown, however, both sites are well connected to other river and canal systems. Therefore, the chance of further spread is high. Genetic analyses of C. floridanus indicate that British inland waters are colonised by the same lineage, which invaded Japan. We recommend further work to assess the distribution of this species and its impact on the local fauna and flora

Improving the reliability of eDNA data interpretation

Global declines in biodiversity highlight the need to effectively monitor the density and distribution of threatened species. In recent years, molecular survey methods detecting DNA released by target‐species into their environment (eDNA) have been rapidly on the rise. Despite providing new, cost‐effective tools for conservation, eDNA‐based methods are prone to errors. Best field and laboratory practices can mitigate some, but the risks of errors cannot be eliminated and need to be accounted for. Here, we synthesize recent advances in data processing tools that increase the reliability of interpretations drawn from eDNA data. We review advances in occupancy models to consider spatial data‐structures and simultaneously assess rates of false positive and negative results. Further, we introduce process‐based models and the integration of metabarcoding data as complementing approaches to increase the reliability of target‐species assessments. These tools will be most effective when capitalizing on multi‐source data sets collating eDNA with classical survey and citizen‐science approaches, paving the way for more robust decision‐making processes in conservation planning.GCR

Complementary authentication of Chinese herbal products to treat endometriosis using DNA metabarcoding and HPTLC shows a high level of variability

Traditional Chinese Medicine (TCM) is popular for the treatment of endometriosis, a complex gynecological disease that affects 10% of women globally. The growing market for TCMs has yielded a significant incentive for product adulteration, and although emerging technologies show promise to improve their quality control, many challenges remain. We tested the authenticity of two traditional Chinese herbal formulae used in women’s healthcare for the treatment of endometriosis, known as Gui Zhi Fu Ling Wan (FL) and Ge Xia Zhu Yu Tang (GX). Dual-locus DNA metabarcoding analysis coupled with high-performance thin-layer chromatography (HPTLC) were used to authenticate 19 FL and six GX commercial herbal products, as well as three ad hoc prepared artificial mixtures. HPTLC was able to detect most of the expected ingredients via comparative component analysis. DNA metabarcoding was able to detect an unexpected species diversity in the products, including 38 unexpected taxa. Chromatography has a resolution for all species indirectly through the identification of marker compounds for the different species ingredients. Metabarcoding on the other hand yields an overview of species diversity in each sample, but interpretation of the results can be challenging. Detected species might not be present in quantities that matter, and without validated quantification, some detected species can be hard to interpret. Comparative analysis of the two analytical approaches also reveals that DNA for species might be absent or too fragmented to amplify as the relevant chemical marker compounds can be detected but no amplicons are assigned to the same species. Our study emphasizes that integrating DNA metabarcoding with phytochemical analysis brings valuable data for the comprehensive authentication of Traditional Chinese Medicines ensuring their quality and safe use

Trade-Offs Between Reducing Complex Terminology and Producing Accurate Interpretations from Environmental DNA: Comment on “Environmental DNA: What\u27s behind the term?” by Pawlowski et al., (2020)

In a recent paper, “Environmental DNA: What\u27s behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring,” Pawlowski et al. argue that the term eDNA should be used to refer to the pool of DNA isolated from environmental samples, as opposed to only extra-organismal DNA from macro-organisms. We agree with this view. However, we are concerned that their proposed two-level terminology specifying sampling environment and targeted taxa is overly simplistic and might hinder rather than improve clear communication about environmental DNA and its use in biomonitoring. This terminology is based on categories that are often difficult to assign and uninformative, and it overlooks a fundamental distinction within eDNA: the type of DNA (organismal or extra-organismal) from which ecological interpretations are derived

Kunnskapsstatus for bruk av molekylære verktøy i kartlegging og overvåkning av biologisk mangfold i marine miljø

Source at https://www.miljodirektoratet.no/publikasjoner/2021/juni-2021/kunnskapsstatus-for-bruk-av-molekylare-verktoy-i-kartlegging-og-overvakning-av-biologisk-mangfold-i-marine-miljo/Denne rapporten er bestilt av Miljødirektoratet fra NorBOL (Norwegian Barcode of Life), et nasjonalt nettverk av forskningsinstitusjoner som koordineres av NTNU Vitenskapsmuseet. Universitetsmuseet i Bergen har hatt prosjektledelsen. Rapporten gir en oppsummering av dagens status på DNA-basert metodikk som verktøy i kartlegging og overvåking av biologisk mangfold i det marine miljø. Fokus har vært på innsamlingsmetodikk og protokoller for DNA-analyser av arter og artsgrupper. Metodikken er evaluert opp mot kartleggings- og overvåkingsaktivitet i regi av Miljødirektoratet, og kommer med anbefalinger for videre framdrift for å ta metodikken i bruk i nasjonal kartlegging og overvåking. Rapporten har også en gjennomgang og evaluering av eksisterende referansemateriale og referansesekvenser for relevante arter og artsgrupper som inngår i Miljødirektoratets kartleggings- og overvåkingsaktivitet

The use of multiple markers and internal positive controls significantly improves species eDNA detection rates and data reliability

In recent years, environmental DNA analyses became increasingly integrated to detect and monitor the presence and abundance of rare organisms, especially in inaccessible aquatic habitats. Although it is generally proven that detection probabilities of eDNA surveys exceed those obtained via conventional techniques, these molecular approaches are, however, also subjected to detection limitations and levels of uncertainty. Besides improvements that can be made in terms of sampling design, volumes of filtered water, and the effective quantity of DNA that is finally analysed, the sensitivity of eDNA surveys is inherently determined by the number of target eDNA copies suspended in the water column. Here we show that multiplexing different primer/probe assays for the same species, but targeting amplicons situated at different loci, is a surprisingly overlooked aspect that can substantially contribute to reduce these limitations and increase the sensitivity of single-species detections. By empirically testing a large number of natural eDNA samples via ddPCR, we reveal that the use of multiple markers can significantly lower the LOD and LOQ of rare and elusive species, such as the invasive American bullfrog and the endangered European weather loach in a variety of different water bodies, such as ponds, lakes, streams, canals, etc. Especially at very low eDNA concentrations of both target species, our results showed that analysing mulitple loci significantly increased detection probabilities and lowered stochasticity effects, and thus ultimately reduces PCR costs when analysed in multiplex. The validation and use of more than one assay taregtting a single species, may further increase the confidence of positive detections. Finally, we illustrate that the implementation of internal positive controls (IPC's), is an absolute must for accurate validation of eDNA workflows and reliable interpretation of the generated data. IPC’s not only help to track down degraded and inhibited samples, to avoid false-negative detections, it also offers insights into extraction efficiency, indispensable for accurate quantification of population densities. Overall, our findings provide strong support that the multiplexing of multiple markers on different loci in combination with the use of internal positive controls ensures increased detection rates at very low eDNA concentrations and generates more robust and reliable data