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Map of all sampling sites within the main channel of the Murrumbidgee River (MR). Sampling sites are numbered from upstream (MR01) to downstream (MR05).
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Monitoring aquatic biodiversity through DNA extracted from environmental samples (eDNA) combined with high-throughput sequencing, commonly referred to as eDNA metabarcoding, is increasing in popularity within the scientific community. However, sampling strategies, laboratory protocols and analytical pipelines can influence the results of eDNA metab...
Citations
... Here, as the number of sampling sites increased, so did species richness, consistent with previous studies conducted in pond and riverine environments (Bylemans et al., 2018;Evans et al., 2017;Macher et al., 2021). This suggests that sampling at one site within a wetland is unlikely to yield a representative result, underscoring the importance of accounting for even small-scale (~1 km) spatial sensitivity. ...
Wetlands are ecologically and culturally significant ecosystems that are experiencing biodiversity declines globally. Biomonitoring techniques that use environmental DNA (eDNA) to detect and monitor biodiversity are well established in lake, riverine, and marine ecosystems. However, their use in wetlands requires further development due to the presence of sediments that block eDNA filters to limit water filtration, alongside a lack of standardized methodology. In this study, we examined eDNA dynamics to understand spatiotemporal biodiversity patterns in an Aotearoa New Zealand wetland and to optimize their application to wetland‐specific challenges. We sampled four sites across Opuatia Wetland at three time points during an austral spring. We conducted conventional taxonomic surveys, tested three different filter sizes (1.2 μm, 5 μm, and semi‐quantitative dacron filters), and assessed our ability to detect foreign DNA (from kea; Nestor notabilis) at different time points and distances post‐release. We found significant differences in DNA sequence composition across time and space, and when using different sized filters. eDNA data generally complemented (versus replaced) conventional survey and identification methods, with certain species only detected by one method or the other. Taxonomic resolution of conventional sampling and identification methods often exceeded that of eDNA. Foreign DNA was detectable 10 m from its release point for up to 1 week post‐release. Our results provide new considerations for future eDNA research in wetland environments, where rapid biomonitoring techniques are needed to support conservation and preservation.
... However, with increased PCR replication (≥7) the same confidence could have been attained with only three sample replicates (Figure 4). Other studies have highlighted the importance of both sample and PCR replication in reducing the risk of false negatives (Ficetola et al., 2015;Fukaya et al., 2022), with optimal strategies noted to vary between river morphologies (Bylemans et al., 2018). In this case study, the CIADM framework considered the levels of replication required to apply evidence-based assessment of eel presence and absence (with confidence levels) to inform prioritisation at water pumping stations. ...
Policy-driven decision-making is an important aspect of environmental management globally, often focused on protecting priority species. However, declining trends in freshwater biodiversity have resulted in a lack of up-to-date knowledge regarding the distribution of rare and elusive species. Furthermore, when dealing with priority species, it is sometimes more important to provide a confident assessment of absence, than merely confirm their presence. Without such confident assessments, resource intensive management plans may be misplaced, and not adequately targeted to conserve important remaining populations.
Here, we present a framework to estimate confidence in absence, referred to as “Confidence in Absence for Decision-Making” (CIADM), based on single-visit environmental DNA metabarcoding data obtained from water samples. It uses a case study of European eel presence / absence upstream of 44 water pumping stations, given their critically endangered status and the legislative drivers for remediation (EC Eel Regulation 1100/2007, Eels (England and Wales) Regulation 2009). Through a high degree of biological (sample) and technical (PCR) replication, we retrospectively assigned ‘confidence in absence’ values and proposed various strategies to achieve the required confidence levels in future surveys.
Our findings indicate that 17 out of 44 pumping stations tested positive for eel, and we were able to assign a >99% confidence level that the remaining 27 sites were negative for eel DNA at the time of sampling. Increasing both biological and technical replication increased ‘confidence in absence’ values. For example, using three PCR replicates per sample, required four replicate biological samples to achieve >95% and six to achieve >99% confidence in eel absence given non-detection. However, we estimate that by using seven PCR replicates per sample a >99% confidence in eel absence following non-detection could be achieved with only three replicate biological samples. Furthermore, we found that eel positive sites had significantly higher species richness, and fish communities differed between eel positive and eel negative sites.
This study highlights the importance of optimising workflow specific replication, and provides an adaptable framework to produce confidence estimations of priority species absence given non-detection.
... Among these molecular approaches, the analysis of environmental DNA (eDNA), which involves the examination of genetic material discharged into the environment (e.g., water, air, soil, and sediment) by organisms through their skin, saliva, and secretions, has proven to be particularly valuable. In recent years, eDNA metabarcoding has emerged as a highly sensitive method for species detection and biodiversity assessment, offering significant advantages over traditional fish-catching methods in terms of minimizing ecosystem disturbance (Bylemans et al., 2018;Doi et al., 2021;Fujii et al., 2019;Laramie et al., 2015;Thomsen & Willerslev, 2015). Consequently, it has been extensively and successfully employed in studies investigating fish diversity in various aquatic ecosystems, including the coast of Japan (Yamamoto et al., 2017), the Yangtze River (Xu & Chang, 2016;Qu et al., 2020;Wang et al., 2022;Cheng, Luo, Li, Zhang, Liu, et al., 2023), and the Wujiang River Cheng, Luo, Zhang, Li, Wang, et al., 2023). ...
Fish diversity plays a critical role in maintaining the balance of water ecosystems, especially in the Chongqing section of the National Nature Reserve for Rare and Endemic Fishes in the upper Yangtze River, which serves as an important habitat for rare and endemic fish, as well as an important channel for the replenishment of fishery resources in the Three Gorges Reservoir. Under a 10‐year ban on fishing in the Yangtze River basin, we investigate fish diversity and seasonal variation in the Reserve by using environmental DNA (eDNA) metabarcoding. We found fishes belonging to 85 genera, 24 families, and 8 orders in the Reserve. A comparison of eDNA metabarcoding results with the diversity of a recent fish catch revealed that eDNA metabarcoding not only enables rapid and efficient fish monitoring but also has a high sensitivity. Furthermore, the study demonstrates that eDNA metabarcoding can be used as a tool for monitoring seasonal variations of fish composition in freshwater ecosystems. The alpha and beta diversity analysis both showed compositional differences in the fish community in accordance with seasonal variations. In addition, changes in eDNA relative sequence abundance and the detection of fish species at different sampling sites may reflect shifts in habitat use and distribution. Thus, we provide detailed seasonal data on fish diversity in the Chongqing section of the Reserve. This will contribute to conservation and to the understanding of fish diversity and community dynamics in the Chongqing section of the Reserve.
... Ugyanakkor a jövőben várható, hogy az elektromos halászat mellett a halak megfogását nem igénylő, a vízben levő örökítőanyag-maradványok azonosításán alapuló ún. környezeti DNS módszerek (eDNA) kiegészítik (BYLEMANS et al. 2018;DEINER et al. 2021;RADINGER et al. 2019), avagy megfelelő technológiai fejlődés esetén akár részben ki is váltják az elektromos halászatot. ...
A cikk röviden összefoglalja a biodiverzitás monitorozásának mivoltját, jelentőségét, céljait. Emellett a halak biodiverzitási monitorozására vonatkozóan bemutat egy az eddigi hazai gyakorlaton és tapasztalatokon nyugvó alapszemléletbeli javaslatot. A cikk célja a monitorozás mint rendszerezett kutatásmódszertani eljárás jelentőségének megvilágítása a biodiverzitás pillanatnyi állapotának és időbeli változásának megismerésében, ami különösen a tudományos kutatásban kevéssé jártas, ugyanakkor a monitorozásokban közreműködő gyakorlati és döntéshozatali szakemberek számára lehet hasznos. Emellett a cikkben levő fogalomértelmezések a tudományos kutatók monitorozásról alkotott személetének egységesítését is támogathatják. A bemutatott elvek és szempontok így támpontot adhatnak a monitorozási programok tervezéséhez és fejlesztéséhez. Végül, a cikk érvel amellett, hogy a monitorozás egy erre a célra létesített intézményi keretben működhet igazán eredményesen.
This paper summarises the essence, importance, and objectives of biodiversity monitoring. Then, inspired by the previous practice and experiences gained so far, a basic concept for fish biodiversity monitoring is presented. The aim of this article is to highlight the importance of monitoring as a systematic scientific method in gathering knowledge on current status and temporal changes of biodiversity. The paper is dedicated primarily to the professionals of management and decision policy who are less interested in science. On the other hand, the conceptual definitions are aimed to promote the unified thinking of scientists on monitoring. The views framed by this basic concept can help design and improve monitoring programs. Finally, the paper argues that monitoring can really be effective if it is operated by a dedicated institute.
... However, this might not be achieved for environmental samples in which a lower reproducibility is expected. Therefore, sufficient field and laboratory replicates to maximize species detection and minimize stochastic sampling effects are recommended (Sato et al. 2017;Bylemans et al. 2018b;Macher et al. 2021b;Rojahn et al. 2021). Particularly the SeaDNA-mid primer pair might suffer from lower reproducibility for environmental samples due to the strong co-amplification. ...
Environmental DNA (eDNA) metabarcoding has become a powerful tool for examining fish communities. Prior to the introduction of eDNA-based assessments into regulatory monitoring contexts (e.g., EU Water Framework Directive), there is a demand for meth-odological standardization. To ensure methodical accuracy and to meet regulatory standards , various sampling, laboratory and bioinformatic workflows have been established. However, a crucial prerequisite for comprehensive fish monitoring is the choice of suitable primer pairs to accurately identify the fishes present in a given water body. Various fish-specific primer pairs targeting different genetic marker regions were published over the past decade. However, a dedicated study to evaluate the performance of frequently applied fish primer pairs to assess Central European fish species has not yet been conducted. Therefore, we created an artificial 'mock' community composed of DNA from 45 Central European fish species and examined the detection ability and reproducibility of five primer pairs. Our study highlights the effect of primer choice and bioinformat-ic filtering on the outcome of eDNA metabarcoding results. From the five primer pairs evaluated in our study the tele02 (12S gene) primer pair was the best choice for eDNA metabarcoding of Central European freshwater fish. Also, the MiFish-U (12S) and SeaD-NA-mid (COI) primer pairs displayed good detection ability and reproducibility. However, less specific primer pairs (i.e., targeting vertebrates) were found to be less reliable and generated high numbers of false-positive and false-negative detections. Our study illustrates how the careful selection of primer pairs and bioinformatic pipelines can make eDNA metabarcoding a more reliable tool for fish monitoring.
... Previous studies evaluating sequencing depth revealed that a minimum sequencing depth is needed to accurately describe the diversity using eDNA analysis, particularly in highly diverse communities (Doble et al., 2020;Smith & Peay, 2014; but see Bylemans, Gleeson, Lintermans, et al., 2018). Recently, Shirazi et al. (2021) found that sequence sample read depths and filtering thresholds impact alpha and beta diversity, suggesting that both of these experimental factors should be considered to improve reliability of results. ...
... This suggests that, at this species minimum read proportion threshold, the increase in sequencing depth did not improve species detection in our study lakes. This is in line with Bylemans, Gleeson, Lintermans, et al. (2018), who found that for low species-richness communities (14 species), increasing sequencing depth only increased species detection moderately. On the other hand, this is in contrast with habitats with exceptional diversities where a higher sequencing depth seems necessary to approach saturation of species (Doble et al., 2020). ...
... By equalizing sequencing depths, our results show that the expected increase of species richness when using multiple primer sets was modest. Bylemans, Gleeson, Lintermans, et al. (2018) found no considerable differences in the number of freshwater fishes recovered at different sequencing depths in the Murrumbidgee River in Australia. ...
For nearly 15 years now, environmental DNA has demonstrated its effectiveness in monitoring biodiversity. Methodological and technical improvements have significantly enhanced the field. However, the effect of factors such as sequence coverage, bioinformatic filtration, and primer choice have been less explored or need to be optimized according to specific survey objectives and study site characteristics. We evaluated these factors to help optimize monitoring fish biodiversity in North American temperate lakes. We sampled water for fish community eDNA analysis in 12 lakes from southwestern Québec, Canada. The lakes were selected to encompass a wide range of surface areas and species richness. We sampled water from a total of 520 sites (25–50 per lake) and analyzed three mitochondrial DNA regions (12S rRNA; 16S rRNA; and cytb) using NovaSeq sequencing. Our results, based on rarefied count matrices (from a sequencing depth of 100,000 to a minimum depth of 1000 reads per sample), showed that keeping only species in each sample if they represented at least one thousandth (species minimum read proportion threshold = 0.001) of the sample's reads was adequate to remove false positives and had a limited negative impact on true positives with low read counts. The sequencing depth was found to have a negligible impact on the accuracy of fish community assessment in a given lake. With the same sequencing depth and a complete local reference database for each primer set, a single primer set produced similar species richness medians than the combination of two or three primer sets. Overall, 12S and 16S detected more species and provided more consistent community profiles than cytb. Based on our observations, we suggest using the 12S MiFish‐U primer set and applying a minimum proportion of 0.001 reads per species and site to monitor north‐temperate lentic freshwater fish communities.
... Recently, eDNA metabarcoding has been acknowledged as an exceptionally useful and powerful tool for community surveys (Bylemans et al., 2018;Cilleros et al., 2019;Deiner et al., 2016Deiner et al., , 2017Fujii et al., 2019;Li et al., 2019;Sato et al., 2017). However, despite the growing number of eDNA metabarcoding studies, the performance of eDNA metabarcoding in broad scale (e.g., nationwide) surveys, with a few exceptions (Cilleros et al., 2019;Deiner et al., 2016;Stat et al., 2017), is yet to be sufficiently evaluated, quantitatively and statistically (Jerde et al., 2019). ...
... Consequently, the size of a lake ecosystem tends to influence the rate of detection by eDNA metabarcoding. With respect to rivers, Bylemans et al. (2018) found that river morphology influences the optimal sampling strategy for eDNA metabarcoding, and in the present study, we identified a similar effect of ecosystem morphology on eDNA sampling strategy in lakes. Considering the factors of sampling effort and expenditure, broad-scale surveys, such as nationwide surveys, tend to be constrained with respect to limited sample sizes; therefore, it may be important to assess the performance of eDNA metabarcoding-based limited sampling effort. ...
Although environmental DNA (eDNA) metabarcoding is an exceptionally useful and powerful tool for monitoring biodiversity, little is known about whether the traits of organisms and their ecosystem characteristics affect eDNA metabarcoding performance. Nationwide surveys can provide more detailed insights, yet such studies have rarely been conducted.
In order to evaluate eDNA metabarcoding performance in broad‐scale monitoring, we examined the effects of species ecological/biological traits and ecosystem characteristics on species detection rates and the implications for community analysis. In addition, we tested the effects of sample mixing and transportation methods, including cooling and freezing, on eDNA metabarcoding. On a nationwide scale, we conducted eDNA metabarcoding for fish communities in 18 Japanese lakes.
By comparing species records, we observed that certain traits, including body size, body shape, saltwater tolerance and habitat preference, influenced eDNA detection. In addition, the proportion of species detected decreased significantly with an increase in lake surface area owing to ecosystem size effect on species detection.
We conclude that species traits, including habitat preference, body size and ecosystem size, should be considered when assessing the eDNA metabarcoding performance in broad‐scale monitoring.
... To visualize the differences in fish assemblages detected by the two methods, a Venn plot was constructed to indicate the overlap of the species number between TFL and eDNA metabarcoding [54], and heat maps were plotted to present the variations in the dominant species of the fish assemblage based on the relative abundance of each species [55]. Nonmetric multidimensional scaling (nMDS) was applied to visually assess the similarity of fish assemblages among the three reservoirs based on Bray-Curtis similarity matrices [56]. ...
The terminal reservoirs of water transfer projects directly supply water for domestic, agricultural, and industrial applications, and the water quality of these reservoirs produce crucial effects on the achievement of project targets. Typically, fish assemblages are monitored as indicators of reservoir water quality, and can also be regulated for its improvement. In the present study, we compared traditional fish landing (TFL) and environmental DNA (eDNA) metabarcoding methods for monitoring fish assemblages in three terminal reservoirs of the East Route of the South-to-North Water Transfer Project, China. Results of TFL and eDNA showed similar assemblage structures and patterns of diversity and spatial distribution with obvious differences in fish composition across three examined reservoirs. Demersal and small fish were dominant in all reservoirs. In addition, a strong association between water transfer distance and assemblages and distribution of non-native fish was found. Our findings highlight the necessity of the fish assemblage monitoring and managing for water quality and revealed the impact of water diversion distance on the structure of fish assemblages and dispersal of alien species along the water transfer project.
... The observation that even the species included at the lowest amount of biomass, such as C. Carassius (1 individual of 14 g), are still detectable 4 km away from the source indicates that a stray guest hanging around somewhere in a river system can be detected with high sensitivity via eDNA metabarcoding. A true positive downstream detection of low biomass species as such is important, because previous metabarcoding studies have shown that a relatively high abundance of one species can negatively influence the detection of rare species (Bylemans et al., 2018). In the context of species diversity monitoring without a priori knowledge of the species composition, our results thus confirm that elusive species can still be detected. ...
Monitoring fish communities is central to the evaluation of ecological health of rivers. Both presence/absence of
fish species and their relative quantity in local fish assemblages are crucial parameters to measure. Fish communities
in lotic systems are traditionally monitored via electrofishing, characterized by a known limited efficiency
and high survey costs. Analysis of environmental DNA could serve as a non-destructive alternative for detection
and quantification of lotic fish communities, but this approach still requires further insights in practical sampling
schemes incorporating transport and dilution of the eDNA particles; optimization of predictive power and quality
assurance of the molecular detection method. Via a controlled cage experiment, we aim to extend the knowledge
on streamreach of eDNA in small rivers and large brooks, as laid out in the European Water Framework Directive's
water typology. Using a high and low source biomass in two river transects of a species-poor river characterized
by contrasting river discharge rates, we found strong and significant correlations between the eDNA relative
species abundances and the relative biomass per species in the cage community. Despite a decreasing correlation
over distance, the underlying community composition remained stable from 25 to 300 m, or up to 1 km
downstream of the eDNA source, depending on the river discharge rate. Such decrease in similarity between relative
source biomass and the corresponding eDNA-based community profile with increasing distance downstream
from the source, might be attributed to variation in species-specific eDNA persistence. Our findings offer crucial
insights on eDNA behaviour and characterization of riverine fish communities. We conclude that water sampled
from a relatively small river offers an adequate eDNA snapshot of the total fish community in the 300–1000 m
upstream transect. The potential application for other river systems is further discussed.
... These studies obtained sufficient species richness to depict seasonality and differences among habitats in the fish community. However, recent studies have suggested that filtering tens or hundreds of liters of water is required to completely detect the expected fish fauna at a site (Bessey et al., 2020;Bylemans et al., 2018;Cantera et al., 2019;Stauffer et al., 2021). However, although increasing the filtration volume per filter would certainly increase the probability of collecting rare eDNA, filtration of a massive amount of water through a filter with a small pore size (0.2-0.7 μm for fish eDNA surveys) is unrealistic due to filter clogging, labor intensity, and the high cost of filters. ...
Clarifying the effect of the sampling protocol on the detection of environmental DNA (eDNA) is essential for appropriately designing biodiversity research. However, technical issues influencing eDNA detection in the open ocean, which consists of water masses with varying environmental conditions, have not been thoroughly investigated. This study evaluated the sampling effort for the metabarcoding-based detection of fish eDNA using replicate sampling with filters of different pore sizes (0.22 and 0.45 μm) in the subtropical and subarctic northwestern Pacific Ocean and Arctic Chukchi Sea. The asymptotic analysis predicted that the accumulation curves for detected taxa did not saturate in most cases, indicating that our sampling effort (7 or 8 replicates, corresponding to 10.5-40 L of filtration in total) was insufficient to fully assess the species diversity in the open ocean and that tens of replicates or a substantial filtration volume were required. The Jaccard dissimilarities between filtration replicates were comparable with those between the filter types at any site. In subtropical and subarctic sites, turnover dominated the dissimilarity, suggesting that the filter pore size had a negligible effect. In contrast, nestedness dominated the dissimilarity in the Chukchi Sea, implying that the 0.22 μm filter could collect a broader range of eDNA than the 0.45 μm filter. Therefore, the effect of filter selection on the collection of fish eDNA likely varies depending on the region. These findings highlight the highly stochastic nature of fish eDNA collection in the open ocean and the difficulty of standardizing the sampling protocol across various water masses.
