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Measuring global fish species richness with eDNA metabarcoding



Despite mounting threats to global freshwater and marine biodiversity, including climate change, habitat alteration, overharvesting and pollution, we struggle to know which species are present below the water's surface that are suffering from these stressors. However, the idea that a water sample containing environmental DNA (eDNA) can be screened using high‐throughput sequencing and bioinformatics to reveal the identity of aquatic species is a revolutionary advance for studying the patterns of species extirpation, invasive species establishment and the dynamics of species richness. To date, many of the critical tests of fisheries diversity using this metabarcoding approach have been conducted in lower diversity systems (<40 fish species), but in this issue of Molecular Ecology Resources, Cilleros et al. (2018) described their eDNA application in the species‐rich French Guiana fishery (>200 fish species) and showed the greater potential and some limitations of using eDNA in species‐rich environments.
Measuring global fish species richness with eDNA
Christopher L. Jerde
Emily A. Wilson
Terra L. Dressler
Marine Science Institute, University of
California, Santa Barbara, California
Department of Ecology, Evolution, and
Marine Biology, University of California,
Santa Barbara, California
Christopher L. Jerde, Marine Science
Institute, University of California, Santa
Barbara, CA.
Funding information
United States Agency for International
Development, Grant/Award Number: AID-
Despite mounting threats to global freshwater and marine biodiversity, including cli-
mate change, habitat alteration, overharvesting and pollution, we struggle to know
which species are present below the water's surface that are suffering from these
stressors. However, the idea that a water sample containing environmental DNA
(eDNA) can be screened using highthroughput sequencing and bioinformatics to
reveal the identity of aquatic species is a revolutionary advance for studying the
patterns of species extirpation, invasive species establishment and the dynamics of
species richness. To date, many of the critical tests of fisheries diversity using this
metabarcoding approach have been conducted in lower diversity systems (<40 fish
species), but in this issue of Molecular Ecology Resources, Cilleros et al. (2018)
described their eDNA application in the speciesrich French Guiana fishery (>200
fish species) and showed the greater potential and some limitations of using eDNA
in speciesrich environments.
biodiversity, environmental DNA, freshwater, marine
We performed a literature search in Google Scholar and Web of
Science to collect published papers using the metabarcoding
approach to estimate fish biodiversity. Key words used in the search
included variations of three terms: environmental DNA,”“metabar-
codingand fish.Only papers that attempted to estimate species
richness of fishes in natural systems were included in the analysis.
Since the first efforts (Thomsen, Kielgast, Iversen, Møller et al.,
2012; Thomsen, Kielgast, Iversen, Wiuf et al., 2012), over 24 studies
(53 unique observations) have been conducted in freshwater (Fig-
ure 1a; n= 46) and marine (Figure 1b; n= 7) systems. While the few
marine studies have ranged in measured species richness from 15 to
128 fish species, freshwater studies have all been less than 93 fish
species (Miya et al., 2015)that is until Cilleros et al. (2018) con-
ducted a study in a system with an estimated fish species richness
of 132.
So why have not metabarcoding approaches been applied to crit-
ical biodiversity regions? Many existing studies (38 of 53 unique
observations) have either concurrently measured species richness
using traditional fisheries capture methods (nets, electrofishing, etc.)
or formed a baseline from historical records from which to compare
results. In part, the metabarcoding approach is still working through
proofofconcept development and demonstrating that eDNArecov-
ered species richness estimates are similar to traditional methods
(Olds et al., 2016). While many of the temperate freshwater diversity
studies have agreement in estimates of species richness and the spe-
cies identity recovered, Cilleros et al. (2018) noted that agreement in
species identity between the piscicide (chemical used to kill fish)
effort and metabarcoding was lacking in the speciesrich system. The
disparity has multiple explanations ranging from the timing between
eDNA and piscicide efforts, the geographic extent from which
eDNAbased inferences are made compared to the piscicide treat-
ment, the incomplete inventories of fish species using traditional
methods or historical records, the inability of a single eDNA marker
to adequately discriminate between closely related species and the
Received: 9 June 2018
Accepted: 26 June 2018
DOI: 10.1111/1755-0998.12929
Mol Ecol Resour. 2019;19:1922. ©2019 John Wiley & Sons Ltd
need for more fish sequences placed into genetic repositories to
provide reliable reference data. Yet, like Cilleros et al. (2018), we are
optimistic that many of these sources of uncertainty are actively
being addressed with methodological improvements, better experi-
mental design and the populating of databases with the genetic sig-
natures of additional fish species.
The georeferenced locations of published studies reveal that
metabarcoding research for freshwater fish has been concentrated in
wellstudied, temperate biomes and along the coastline of marine
systems (Figure 1c). The Pearson correlation between observed
freshwater species richness from metabarcoding and the minimum
projected species richness from the map is r=0.39 (p<0.01). While
FIGURE 1 Distribution of observed fish species richness for (a) freshwater and (b) marine systems and the (c) georeferenced location of the
studies. The Cilleros et al. (2018) study is the first to use the metabarcoding approach in a highdiversity, freshwater system. For panel (c), the
basic world biomes were delineated using a map layer generated from Olson et al. (2001) and was used to visualize tundra (light blue), boreal
forest (dark blue), temperate forest (green), tropical forest (purple), savannah (yellow) and desert (orange). An additional layer was used to
visualize current estimations of worldwide freshwater biodiversity (Abell et al., 2008). This layer was colourscaled so that areas of high
biodiversity are represented by dark shading and areas with low biodiversity are represented by light shading. Circles represent the
georeferenced location of metabarcoding studies for freshwater (red) and marine (yellow) systems and sizescaled according to the number of
species detected by eDNA analysis (largest points = highest number of species detected)
FIGURE 2 Freshwater systems, like the commercial fisheries on the (left) Tonle Sap River, Cambodia, have historical records of over 900
fish species but have been understudied using the metabarcoding approach. Restorations efforts, such as the (right) Los Angeles River, USA,
have far fewer fish species, but may also benefit from metabarcoding approaches by providing greater geographic coverage and detecting rare
and/or unexpected species
there is considerable uncertainty, this positive relationship, along
with the growing interest and application of eDNArelated surveil-
lance efforts (Valentini et al., 2016), implies we are on the cusp of
producing a more cultivated mapping of global freshwater biodiver-
sity. However, there have been disproportionally few or no studies
in freshwater fish biodiversity hot spots or open oceans, where esti-
mating localized biodiversity using any detection method is difficult
and where information is needed to measure the impact of growing
environmental threats.
So where specifically do we need better fish species richness
measurements to form baselines? Like the French Guiana fishery
(Cilleros et al., 2018), eDNA methods should be deployed in systems
likely to experience changes from immediate threats. The Mekong
River Basin is a biodiversity hot spot with over 900 species of fresh-
water, brackish and marine fish supporting the diets over 100 differ-
ent ethnic groups in seven countries (ValboJørgensen, Coates, &
Hortle, 2009) with a cumulative population of over 60 million peo-
ple. The scheduled completion of dams on the tributaries of the
Mekong River will likely have disastrous impacts on fish biodiversity
and food security (Ziv, Baran, Nam, RodríguezIturbe, & Levin, 2012;
Figure 2). While traditional fish capture methods, such as nets or
hook and line fishing, will provide useful estimates of species rich-
ness, the metabarcoding approach may complement these
approaches by detecting overlooked species that are missed due to
low capture probably resulting from any number of factors, including
bias size selection of gear (Millar & Fryer, 1999), feeding and move-
ment behaviour of the fish, or rarity within the system. In addition,
as has been demonstrated for eDNA application for singlespecies
detection (Tucker et al., 2016), metabarcoding could provide broader
geographic coverage of species presence, and the eDNA approach
will not cause damage or death to any captured rare species through
direct handling.
In contrast, there are systems where changing policies regarding
water flow and restoration are leading to potential increases in spe-
cies richness. The concretelined Los Angeles River (Figure 2), a once
relatively dead river with respect to fish biodiversity (Gumprecht,
2001), is being restored with particular attention to removing barri-
ers and providing seminatural riffles and pools for potential Southern
Pacific steelhead trout (Oncorhynchus mykiss) migration to upper
headwaters, yet very little monitoring beyond common species
caught by anglers is being conducted in the river or the upper head-
waters. The metabarcoding approach could provide routine monitor-
ing to measure the effectiveness of restoration efforts, particularly
for rare or elusive species (Gangloff, Edgar, & Wilson, 2016).
We have learned from Cilleros et al. (2018) that the metabarcod-
ing approach is applicable to more diverse systems and can comple-
ment traditional fisheries approaches. The groundtruthing in 36
biodiversitylimited study areas has been successful in building confi-
dence that metabarcoding reveals estimates of species richness on
par with, and potentially better than (Olds et al., 2016), traditional
fisheries methods, and now, it is time to expand into more biologi-
cally diverse areas of conservation concern. Clearly, we should fur-
ther develop our eDNA collection, genetic sequencing and
bioinformatic screening components of the metabarcoding approach,
but if we wait for the methods to be further improved before
deploying in areas of conservation concern with greater species rich-
ness, we will miss global opportunities to motivate the protection of
rare species and prevent fishery collapses.
C. L. J. is interested in the application of quantitative tools for
resource management of rare species and estimation of species rich-
ness. This work supported by USAID (AIDOAAA1600057). E. A.
W. is interested in the effects of invasive species on restoration and
STEM education to promote scientific literacy. T. L. D. is interested
in the persistence of fish populations in extreme conditions near
their native range limits, in particular those affected by wildfire.
Christopher L. Jerde
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How to cite this article: Jerde CL, Wilson EA, Dressler TL.
Measuring global fish species richness with eDNA
metabarcoding. Mol Ecol Resour. 2019;19:1922.
... Environmental DNA (eDNA) has become a valuable tool for detecting organisms from all domains of life in the environment without visual or auditory observations. Molecular metabarcoding of eDNA is now used routinely to survey community composition and estimate diversity in freshwater and marine habitats (e.g., Berry et al., 2019;Jerde et al., 2019;Stoeckle et al., 2020). However, the development of optimal sampling protocols is crucial for accurate and reproducible eDNA results (Deiner et al., 2015) that can be integrated globally (Chavez et al., 2021). ...
... Despite their potential, the scale and pace of eDNA metabarcoding studies in the ocean have lagged behind those of freshwater environments (Beng & Corlett, 2020;Jerde et al., 2019). This discrepancy is largely due to the scale differences between the two environments; access to marine environments requires expensive oceanographic research vessels that sample over considerable temporal (months) and spatial (hundreds of square kilometers and multiple depths) scales (McClenaghan et al., 2020). ...
Full-text available
Environmental DNA (eDNA) is an emerging and powerful method for use in marine research, conservation, and management, yet time‐ and resource‐intensive protocols limit the scale of implementation. Long‐range autonomous underwater vehicles equipped with autonomous environmental sample processors (LRAUV‐ESPs) provide a new means for scaling up marine eDNA sample collection and processing. Here, we used eDNA metabarcoding of four marker genes (mitochondrial 12S rRNA, bacterial and archaeal 16S rRNA, nuclear 18S rRNA, and mitochondrial COI), which encompass the diversity of marine species from microbes to vertebrates, to demonstrate the efficacy of an LRAUV‐ESP in sampling eDNA and assessing community structure in the Monterey Bay National Marine Sanctuary. The sequencing results from samples that were autonomously collected were comparable with those collected from a ship at similar locations, times, and depths, supporting previous results that found no significant differences using targeted qPCR. This study demonstrates the potential of equipping autonomous underwater vehicles with ESPs to greatly expand the scale of eDNA sample collection and processing and provide much needed information regarding the changing spatial and temporal patterns of marine biodiversity, especially in many data‐poor regions of the world's oceans. Long‐range autonomous underwater vehicles equipped with autonomous environmental sample processors provide a new means for scaling up marine eDNA research. This comparative study provides evidence that autonomous eDNA sampling compares favorably to commonly used shipboard methods. Autonomous eDNA sampling has the potential to expand eDNA biomonitoring in space and time to help inform aquatic conservation and management.
... Hence, it could be incorporated into stock assessment programs to provide a less costly method that can cover a broad geographic space, is non-lethal for the fish and not damaging for its environment in comparison with standardized trawling efforts. Erroneous detections, or false positives, may complexify management decisions, because "there is no fish in hand" and contribute to current skepticism on the use of eDNA for such purposes (Jerde;Wilson & Dressler, 2019). However, further research will continue to further minimize this uncertainty linked to the nature of eDNA technologies. ...
... Hence, it could be incorporated into stock assessment programs to provide a less costly method that can cover a broad geographic space, is non-lethal for the fish and not damaging for its environment in comparison with standardized trawling efforts. Erroneous detections, or false positives, may complexify management decisions, because "there is no fish in hand" and contribute to current skepticism on the use of eDNA for such purposes (Jerde;Wilson & Dressler, 2019). However, further research will continue to further minimize this uncertainty linked to the nature of eDNA technologies. ...
Full-text available
Environmental DNA (eDNA) studies have burgeoned over the last two decades and the application of eDNA has increased exponentially since 2010, albeit at a slower pace in the marine system. We provide a literature overview on marine metazoan eDNA studies and assess recent achievements in answering questions related to species distributions, biodiversity and biomass. We investigate which are the better studied taxonomic groups, geographic regions and the genetic markers used. We evaluate the use of eDNA for addressing ecological and environmental issues through food web, ecotoxicological, surveillance and management studies. Based on this state of the art, we highlight exciting prospects of eDNA for marine time series, population genetic studies, the use of natural sampler DNA, and eDNA data for building trophic networks and ecosystem models. We discuss the current limitations, in terms of marker choice and incompleteness of reference databases. We also present recent advances using experiments and modeling to better understand persistence, decay and dispersal of eDNA in coastal and oceanic systems. Finally, we explore promising avenues for marine eDNA research, including autonomous or passive eDNA sampling, as well as the combined applications of eDNA with different surveillance methods and further molecular advances. Keywords: environmental DNA, DNA metabarcoding, marine metazoa, biodiversity, population genetics, natural sampler DNA, diet analysis.
... for example, detection of killer whales was possible for up to 2 hours after animals left the area (Baker et al., 2018), and detection of species richness of shark species was increased by metabarcoding of eDNA over traditional methods (Boussarie et al., 2018). When compared to other methods of marine diversity measurements, eDNA appears to be comparable or more sensitive at times but may be limited in other cases (Boussarie et al., 2018;Jerde et al., 2019). Even so, a complete understanding of the strengths and limitations of eDNA in varying marine ecosystems and across species is still being worked out (Cristescu & Hebert, 2018;Hansen et al., 2018;Jerde et al., 2019). ...
... When compared to other methods of marine diversity measurements, eDNA appears to be comparable or more sensitive at times but may be limited in other cases (Boussarie et al., 2018;Jerde et al., 2019). Even so, a complete understanding of the strengths and limitations of eDNA in varying marine ecosystems and across species is still being worked out (Cristescu & Hebert, 2018;Hansen et al., 2018;Jerde et al., 2019). Environmental DNA sampling in marine ecosystems has been limited by the necessity of humans to collect water samples, particularly in deep waters. ...
Full-text available
Biodiversity must be documented before it can be conserved. However, it may be difficult to document species with few individuals (Thompson, 2013; Goldberg et al., 2016), thus it requires a multitude of tools to detect species that occur in low numbers or are elusive (see the various chapters in this volume). One tool that has become useful for conservation efforts utilizes environmental DNA, which is DNA shed into the environment by organisms (eDNA; Taberlet et al., 2018). Typically this involves taking environmental samples such as soil, water, air, or using biological surrogates for sampling biodiversity (e.g. leeches, sponges, carrion flies, etc.; Schnell et al., 2012; Calvignac-Spencer et al., 2013; Lynggaard et al., 2019; Mariani et al., 2019) and using laboratory approaches to concentrate, isolate, and test for target DNA through polymerase chain reaction (PCR) amplification (Taberlet et al., 2018). The utilization of eDNA for species detection is part of a larger field of non-invasive DNA sampling, which more broadly includes collecting DNA passively from wildlife, through collection of faeces, saliva, feathers, hair, or other methods of sampling shed DNA. Environmental DNA has been used to document presence/absence of a target species (Ficetola et al., 2008a, 2008b; Himter et al., 2017) or to quantify relative abundance for biodiversity from varied environments such as the arctic (e.g. Leduc et al., 2019; Von Duyke et al., 2019), marine (e.g. Port et al., 2016; Jo et al, 2017; Stoeckle et al., 2018), freshwater (e.g. Lacoursi^re-Roussel et al., 2016; Doi et al., 2017), and tropical (e.g. Schnell et al., 2012; Gogarten et al, 2020) ecosystems. The application of this technology includes the detection of invasive species, pathogens (including DNA and RNA), species of conservation concern, and biodiversity (Acevedo-Whitehouse et al., 2010; Rees et al., 2014; Sakai et al, 2019). In this world -of 'fast-paced technological advances, not all new methods prove useful in an applied context. Although eDNA has not been used regularly in biodiversity conservation for more than a decade, it has proven to be an extremely practical and informative tool. The utility of eDNA is supported by ongoing advancements and development of novel applications. There is no easy way to standardize the application or methods of eDNA as the conservation question, and the target system must drive the selection of a range of options at every step. However, guidelines now exist for the best practices of optimizing a sampling scheme and sample processing for eDNA applications (Goldberg et al., 2016; Jeunen et al., 2019; Klymus et al., 2020; The eDNA Society, 2019; Shu et al, 2020). Further, the ranks of experienced eDNA practitioners have expanded globally; thus, it is fairly easy to find expert consultation. Therefore, it is now practical and prudent to adopt eDNA in the service of biodiversity conservation efforts.
... for example, detection of killer whales was possible for up to 2 hours after animals left the area (Baker et al., 2018), and detection of species richness of shark species was increased by metabarcoding of eDNA over traditional methods (Boussarie et al., 2018). When compared to other methods of marine diversity measurements, eDNA appears to be comparable or more sensitive at times but may be limited in other cases (Boussarie et al., 2018;Jerde et al., 2019). Even so, a complete understanding of the strengths and limitations of eDNA in varying marine ecosystems and across species is still being worked out (Cristescu & Hebert, 2018;Hansen et al., 2018;Jerde et al., 2019). ...
... When compared to other methods of marine diversity measurements, eDNA appears to be comparable or more sensitive at times but may be limited in other cases (Boussarie et al., 2018;Jerde et al., 2019). Even so, a complete understanding of the strengths and limitations of eDNA in varying marine ecosystems and across species is still being worked out (Cristescu & Hebert, 2018;Hansen et al., 2018;Jerde et al., 2019). Environmental DNA sampling in marine ecosystems has been limited by the necessity of humans to collect water samples, particularly in deep waters. ...
Detection and monitoring of wildlife species of concern is a costly and time-consuming challenge that is critical to the management of such species. Tools such as lures and traps can cause unnecessary stress or other health impacts to sensitive species. Development and refinement of tools that provide means to detect rare and elusive species without requiring contact with them reduce such impacts. Further, the potential of detection after the target species has moved on from a sampling site could allow for higher potential for detection of rare species. The ability to amplify DNA from environmental samples (e.g. water, soil, air, and other substrates) has provided a non-invasive method for detection of rare or elusive species while reducing negative impacts to wildlife. Like other non-invasive methods, such as cameras, there are methodological pitfalls associated with environmental DNA (eDNA) sampling to consider. Each study system will provide unique challenges to adequate eDNA sampling. Thus, pilot studies are critical for successful implementation of a larger-scale detection and monitoring study. This chapter will describe the benefits and challenges of using eDNA, detail types of eDNA sampling, and provide guidance on designing appropriate study design and sampling schemes. Empirical studies using eDNA applied to wildlife conservation efforts will be highlighted and discussed.
... material 2: Table S2). To the best of our knowledge, the observed species richness (140 spp.) is the highest in coastal marine environments as revealed by fish eDNA metabarcoding, except for that of coral reefs (Jerde et al. 2019;Miya 2022). We acknowledge that greater survey effort (e.g., collecting more field samples of larger volume at each site) has been shown to increase the probability of detecting fish DNA, reducing the impact of false negatives and improving confidence in the eDNA metabarcoding approach (Ficetola et al. 2015;Pawlowski et al. 2018;Doi et al. 2019). ...
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To test the feasibility of a citizen science program for fish eDNA metabarcoding in coastal marine environments, we recruited six groups of voluntary citizens for a science education course at a natural history museum. We held a seminar on eDNA and a workshop for seawater sampling and on-site filtration using syringes and filter cartridges for the participants. After that, they selected single survey sites following the guidelines for conducting a safe field trip. They performed seawater sampling and on-site filtration at these sites during their summer holidays. The six selected sites unexpectedly included diverse coastal habitats within a 40 km radius, located at temperate latitudes in central Japan (~35°N). After the field trips, they returned filtered cartridges to the museum, and we extracted eDNA from the filters. We performed fish eDNA metabarcoding, along with data analysis. Consequently, we identified 140 fish species across 66 families and 118 genera from the six samples, with species richness ranging from 14 to 66. Despite its limited sample size, such a diverse taxonomic range of fish species exhibited spatial biodiversity patterns within the region, which are consistent with species distribution. These include north-south and urbanization gradients of species richness, geographic structure of the fish communities, and varying salinity preferences of the component species. This case study demonstrates the potential of fish eDNA metabarcoding as an educational and scientific tool to raise public awareness and perform large-scale citizen science initiatives encompassing regional, national, or global fauna.
... Among the main benefits of using eDNA as a monitoring tool are the fact that it is an indirect non-invasive technique (i.e., no need to capture the target organism) and it does not require specialist taxonomic expertise to detect taxa across the tree of life (Goricki et al., 2017;Stefanni et al., 2018), though the latter strongly depends on availability of comprehensive reference DNA databases (as further discussed below). Once an environmental sample such as water, biofilm or sediment is acquired , the collected eDNA can be queried either by using "universal" markers targeting whole communities by means of HTS (Jerde et al., 2019), or by targeted species-specific assays usually performed by real-time quantitative PCR (qPCR) or digital PCR (dPCR) (Goldberg et al., 2016). The effectiveness of both approaches depends on the availability of reference data, for taxonomy identification of sequenced reads with eDNA metabarcoding and for the development of species-specific assays with the targeted approach. ...
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Deep-sea ecosystems are reservoirs of biodiversity that are largely unexplored, but their exploration and biodiscovery are becoming a reality thanks to biotechnological advances (e.g., omics technologies) and their integration in an expanding network of marine infrastructures for the exploration of the seas, such as cabled observatories. While still in its infancy, the application of environmental DNA (eDNA) metabarcoding approaches is revolutionizing marine biodiversity monitoring capability. Indeed, the analysis of eDNA in conjunction with the collection of multidisciplinary optoacoustic and environmental data, can provide a more comprehensive monitoring of deep-sea biodiversity. Here, we describe the potential for acquiring eDNA as a core component for the expanding ecological monitoring capabilities through cabled observatories and their docked Internet Operated Vehicles (IOVs), such as crawlers. Furthermore, we provide a critical overview of four areas of development: (i) Integrating eDNA with optoacoustic imaging; (ii) Development of eDNA repositories and cross-linking with other biodiversity databases; (iii) Artificial Intelligence for eDNA analyses and integration with imaging data; and (iv) Benefits of eDNA augmented observatories for the conservation and sustainable management of deep-sea biodiversity. Finally, we discuss the technical limitations and recommendations for future eDNA monitoring of the deep-sea. It is hoped that this review will frame the future direction of an exciting journey of biodiscovery in remote and yet vulnerable areas of our planet, with the overall aim to understand deep-sea biodiversity and hence manage and protect vital marine resources.
... This is particularly valuable for rare or vulnerable species (Storer et al., 2019). Successful gNIS or eDNA sampling has been described across a wide range of species, including mammals (De Padgett-Stewart et al., 2016), birds (Miño & Del Lama, 2009;Neice & McRae, 2021), reptiles (Hu & Wu, 2008), amphibians (Eiler et al., 2018;Olson et al., 2012), fish (Jerde et al., 2019;Lieber et al., 2013), and insects (Storer et al., 2019;Uchida et al., 2020). ...
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Effective conservation requires accurate data on population genetic diversity, inbreeding, and genetic structure. Increasingly, scientists are adopting genetic non-invasive sampling (gNIS) as a cost-effective population-wide genetic monitoring approach. gNIS has, however, known limitations which may impact the accuracy of downstream genetic analyses. Here, using high-quality single nucleotide polymorphism (SNP) data from blood/tissue sampling of a free-ranging koala population (n = 430), we investigated how the reduced SNP panel size and call rate typical of genetic non-invasive samples (derived from experimental and field trials) impacts the accuracy of genetic measures, and also the effect of sampling intensity on these measures. We found that gNIS at small sample sizes (14% of population) can provide accurate population diversity measures, but slightly underestimated population inbreeding coefficients. Accurate measures of internal relatedness required at least 33% of the population to be sampled. Accurate geographic and genetic spatial autocorrelation analysis requires between 28% and 51% of the population to be sampled. We show that gNIS at low sample sizes can provide a powerful tool to aid conservation decision-making and provide recommendations for researchers looking to apply these techniques to free-ranging systems.
... This study is an excellent example of a well designed and implemented eDNA research effort and has been followed by several other studies that have highlighted the importance of fisheries eDNA (Jerde et al., 2019;Miya et al., 2020). Additional work will look to improve on the ability to calculate abundances of different fish species from eDNA by incorporating biomass information directly from individual samples, such as proposed recently by Yates et al. (2020). ...
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Aim: The aim of this chapter is to introduce the concepts and applications of environmental DNA (eDNA) for species detection and biomonitoring of freshwater ecosystems. Environmental assessment of inland waters is currently undergoing a revolution due to the increased utilization of eDNA and major advancements in molecular techniques. Several aspects of ecology and conservation biology across the academic, private and government sectors are already utilizing eDNA-based approaches with more applications being rapidly developed. Therefore, this chapter disseminates current fundamental understanding of the dynamics and applications of eDNA in freshwater environments. Main concepts covered: Environmental DNA (eDNA) is DNA extracted from environmental samples without targeting a particular organism or group of organisms. Within the realm of inland waters, eDNA samples typically include water, sediment or biofilm samples, though there is potential for other environmental sources. The ecology of eDNA (e.g., transport, degradation rate, molecular state) greatly affects the detectability of eDNA, as well as the interpretation of the results drawn from eDNA-based assessment. In particular, understanding the difference between eDNA detection dynamics in lotic (e.g., rivers and streams) versus lentic (e.g., ponds and lakes) environments is key to understanding and applying eDNA-derived information. Main methods covered: A major characteristic of eDNA-based research is the non-targeted aspect of species and community detection. It is therefore paramount that an understanding of the sampling and DNA extraction methods are outlined, and that concerns regarding potential inhibition (false negatives) and contamination sources (false positives) are addressed. The use of eDNA also requires additional experimental design considerations, particularly regarding replication and spatial resolution, due to the need to cross validate findings and the increased complexity of the data created compared to traditional taxonomic-based approaches. Currently, eDNA-based research can be divided into two main groups, population- and community-based analyzes. Population-based analyzes rely primarily on single-targeted (i.e., species) methods such as qPCR, which are lower cost and easier for smaller institutes or individuals to independently implement. Community-based approaches rely largely on high throughput sequencing (HTS) and require additional molecular and bioinformatics specialization and support, but result in greater potential for data generation and analytical power, given the proper study design. Future eDNA work will include applying PCR free-based methods to population analyzes and combining multi-dimensional environmental data for environmental community analyzes. Other advances in eDNA research may look to assess the transcriptional profiles of eDNA samples to assess functional community diversity. Conclusion/Outlook: This chapter provides an overview of current molecular and eDNA-based approaches for inland water assessment. There are many aspects of eDNA that are still largely unknown, but the ability to apply standardized non-invasive sampling with high throughput data is hard to ignore in the modern age.
... Despite the problems inherent in the development of new methodologies, eDNA technology and associated bioinformatics are evolving at accelerated rates and will soon play a central role in the inventory of fish diversity 6,28,33,34 . Freshwater ecosystems, many of which are poorly explored, are under severe and fast-paced threats due to anthropogenic activities 35 . ...
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Ichthyological surveys have traditionally been conducted using whole-specimen, capture-based sampling with varied but conventional fishing gear. Recently, environmental DNA (eDNA) metabarcoding has emerged as a complementary, and possible alternative, approach to whole-specimen methodologies. In the tropics, where much of the diversity remains undescribed, vast reaches continue unexplored, and anthropogenic activities are constant threats; there have been few eDNA attempts for ichthyological inventories. We tested the discriminatory power of eDNA using MiFish primers with existing public reference libraries and compared this with capture-based methods in two distinct ecosystems in the megadiverse Amazon basin. In our study, eDNA provided an accurate snapshot of the fishes at higher taxonomic levels and corroborated its effectiveness to detect specialized fish assemblages. Some flaws in fish metabarcoding studies are routine issues addressed in natural history museums. Thus, by expanding their archives and adopting a series of initiatives linking collection-based research, training and outreach, natural history museums can enable the effective use of eDNA to survey Earth’s hotspots of biodiversity before taxa go extinct. Our project surveying poorly explored rivers and using DNA vouchered archives to build metabarcoding libraries for Neotropical fishes can serve as a model of this protocol.
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To date, more than 2400 valid fish species have been recorded in the Amazon basin. However , some regions remain poorly documented. This is the case in the Beni basin and in particular in one of its main sub-basins, the Tuichi, an Andean foothills rivers flowing through the Madidi National Park in the Bolivian Amazonia. The knowledge of its ichthyological diversity is, however, essential for the management and protection of aquatic ecosystems, which are threatened by the development of infrastructures (dams, factories and cities), mining and deforestation. Environmental DNA (eDNA) has been relatively little used so far in the Amazon basin. We sampled eDNA from water in 34 sites in lakes and rivers in the Beni basin including 22 sites in the Tuichi sub-basin, during the dry season. To assess the biogeographical patterns of the amazonian ichthyofauna, we implemented a metabarcoding approach using two pairs of specific primers designed and developed in our laboratory to amplify two partially overlapping CO1 fragments, one of 185bp and another of 285bp. We detected 252 fish taxa (207 at species level) among which 57 are newly identified for the Beni watershed. Species compositions are significantly different between lakes and rivers but also between rivers according to their hydrographic rank and altitude. Furthermore, the diversity patterns are related to the different hydro-ecoregions through which the Tuichi flows. The eDNA approach makes it possible to identify and complete the inventory of the ichthyofauna in this still poorly documented Amazon basin. However, taxonomic identification remains constrained by the lack of reference barcodes in public databases and does not allow the assignment of all OTUs. Our results can be taken into account in conservation and management strategies and could serve as a base-line for future studies, including on other Andean tributaries. PLOS ONE PLOS ONE |
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Determining the species compositions of local assemblages is a prerequisite to understanding how anthropogenic disturbances affect biodiversity. However, biodiversity measurements often remain incomplete due to the limited efficiency of sampling methods. This is particularly true in freshwater tropical environments that host rich fish assemblages, for which assessments are uncertain and often rely on destructive methods. Developing an efficient and non‐destructive method to assess biodiversity in tropical freshwaters is highly important. In this study, we tested the efficiency of environmental DNA (eDNA) metabarcoding to assess the fish diversity of 39 Guianese sites. We compared the diversity and composition of assemblages obtained using traditional and metabarcoding methods. More than 7,000 individual fish belonging to 203 Guianese fish species were collected by traditional sampling methods, and ~17 million reads were produced by metabarcoding, among which ~8 million reads were assigned to 148 fish taxonomic units, including 132 fish species. The two methods detected a similar number of species at each site, but the species identities partially matched. The assemblage compositions from the different drainage basins were better discriminated using metabarcoding, revealing that while traditional methods provide a more complete but spatially limited inventory of fish assemblages, metabarcoding provides a more partial but spatially extensive inventory. eDNA metabarcoding can therefore be used for rapid and large‐scale biodiversity assessments, while at a local scale, the two approaches are complementary and enable an understanding of realistic fish biodiversity. This article is protected by copyright. All rights reserved.
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Environmental DNA (eDNA) metabarcoding has been increasingly applied to biodiversity surveys in stream ecosystems. In stream networks, the accuracy of eDNA-based biodiversity assessment depends on whether the upstream eDNA influx affects downstream detection. Biodiversity assessment in low-discharge streams should be less influenced by eDNA transport than in high-discharge streams. We estimated α- and β-diversity of the fish community from eDNA samples collected in a small Michigan (USA) stream from its headwaters to its confluence with a larger river. We found that α-diversity increased from upstream to downstream and, as predicted, we found a significant positive correlation between β-diversity and physical distance (stream length) between locations indicating species turnover along the longitudinal stream gradient. Sample replicates and different genetic markers showed similar species composition, supporting the consistency of the eDNA metabarcoding approach to estimate α- and β-diversity of fishes in low-discharge streams.
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We present a performance evaluation of environmental DNA (eDNA) metabarcoding with MiFish-U/E primers to investigate local and regional diversities of stream fish species to examine potential effectiveness, limits and future remedies of this technique in large-scale monitoring. We hypothesised that eDNA inferences are more consistent with fish assemblages observed upstream than downstream due to a directional flow of river water. River water was sampled at 102 sites in 51 rivers around Lake Biwa in the central part of Honshu Island, Japan, within 10 person-days, and fish species compositions inferred from eDNA and existing observational data were compared. Observation sites were chosen from the observational data that were within a certain distance (buffer range) of a water-sampling site along a river trajectory. The hypothesis of the detection bias of eDNA towards upstream assemblage was tested by comparing results with all of the observational data, data from a higher elevation and data from a lower elevation. The Jaccard dissimilarity index was plotted between the observational data and the eDNA estimates against the buffer range; the buffer range with minimum dissimilarity was chosen. When using existing observational data from within 6 km upstream of the eDNA sampling sites, the eDNA results were the most consistent with the observational data and inferred 88.6% of the species reported (38/44), as well as two additional species. eDNA results also showed patterns consistent with known upstream-downstream turnover of related species and biogeographical assemblage patterns of certain species. Our 10-person-days survey using the metabarcoding technique enabled us to obtain as much regional fish diversity data including the hypothesised pattern of eDNA detection with an upstream bias as the accumulated observational data obtained through greater amounts of time, money and labour. The problems regarding false-positive/negative detection were suggested in our survey; however, these should be decreased or removed by modifying the sampling methods and experimental procedures in future works. Therefore, we concluded this new tool to enable monitoring that has never been implemented, such as cross-nation, and even whole-Earth monitoring with the data at yearly, seasonal or finer temporal scales.
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Environmental DNA (eDNA) metabarcoding has been used increasingly to assess biodiversity of aquatic vertebrates. However, there still remains to be developed a sampling design of eDNA metabarcoding that can ensure high detection rates of species with minimum total survey effort, especially for large-scale surveys of aquatic organisms. We here tested whether pooling of eDNA samples can be used to evaluate biodiversity of freshwater fishes in four satellite lakes of Lake Biwa, Japan. Fish communities detected by eDNA metabarcoding of the mitochondrial 12S region were compared between the individual and pooled samples. In the individual samples, 31, 22, 33, and 31 fish lineages (proxies for species) were observed at the respective sites, within which moderate spatial autocorrelation existed. In the pooled samples, 30, 20, 29, and 27, lineages were detected, respectively, even after 15 PCR replicates. Lineages accounting for < 0.05% of the total read count of each site’s individual samples were mostly undetectable in the pooled samples. Moreover, fish communities detected were similar among PCR replicates in the pooled samples. Because of the decreased detection rates, the pooling strategy is unsuitable for estimating fish species richness. However, this procedure is useful potentially for among-site comparison of representative fish communities.
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Relatively small volumes of water may contain sufficient environmental DNA (eDNA) to detect target aquatic organisms via genetic sequencing. We therefore assessed the utility of eDNA to document the diversity of coral reef fishes in the central Red Sea. DNA from seawater samples was extracted, amplified using fish-specific 16S mitochondrial DNA primers, and sequenced using a metabarcoding workflow. DNA sequences were assigned to taxa using available genetic repositories or custom genetic databases generated from reference fishes. Our approach revealed a diversity of conspicuous, cryptobenthic, and commercially relevant reef fish at the genus level, with select genera in the family Labridae over-represented. Our approach, however, failed to capture a significant fraction of the fish fauna known to inhabit the Red Sea, which we attribute to limited spatial sampling, amplification stochasticity, and an apparent lack of sequencing depth. Given an increase in fish species descriptions, completeness of taxonomic checklists, and improvement in species-level assignment with custom genetic databases as shown here, we suggest that the Red Sea region may be ideal for further testing of the eDNA approach.
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Coastal marine fish populations are in decline due to overfishing, habitat destruction, climate change and invasive species. Seasonal monitoring is important for detecting temporal changes in the composition of fish communities, but current monitoring is often non-existent or limited to annual or semi-annual surveys. In the present study, we investigate the potential of using environmental DNA (eDNA) metabarcoding of seawater samples to detect the seasonal changes in a coastal marine fish community. Water sampling and snorkelling visual census were performed over 1 year (from 23rd of August 2013 to 11th of August 2014) at a temperate coastal habitat in Denmark (55°45′39″N, 12°35′59″E) and compared to long-term data collected over a 7-year period. We used Illumina sequencing of PCR products to demonstrate that seawater eDNA showed compositional changes in accordance with seasonal changes in the fish community. The vast majority of fish diversity observed in the study area by snorkelling was recovered from sequencing, although the overlap between methods varied widely among sampling events. In total, 24 taxa were detected by both methods, while five taxa were only detected using eDNA and three taxa were only detected by snorkelling. A limitation of the applied primers was the lack of resolution to species level in a few diverse families, and varying sequencing depth between samples represents a potential bias. However, our study demonstrates the utility of eDNA for recovering seasonal variation in marine fish communities, knowledge of which is essential for standardised long-term monitoring of marine biodiversity.
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The difficulty of censusing marine animal populations hampers effective ocean management. Analyzing water for DNA traces shed by organisms may aid assessment. Here we tested aquatic environmental DNA (eDNA) as an indicator of fish presence in the lower Hudson River estuary. A checklist of local marine fish and their relative abundance was prepared by compiling 12 traditional surveys conducted between 1988–2015. To improve eDNA identification success, 31 specimens representing 18 marine fish species were sequenced for two mitochondrial gene regions, boosting coverage of the 12S eDNA target sequence to 80% of local taxa. We collected 76 one-liter shoreline surface water samples at two contrasting estuary locations over six months beginning in January 2016. eDNA was amplified with vertebrate-specific 12S primers. Bioinformatic analysis of amplified DNA, using a reference library of GenBank and our newly generated 12S sequences, detected most (81%) locally abundant or common species and relatively few (23%) uncommon taxa, and corresponded to seasonal presence and habitat preference as determined by traditional surveys. Approximately 2% of fish reads were commonly consumed species that are rare or absent in local waters, consistent with wastewater input. Freshwater species were rarely detected despite Hudson River inflow. These results support further exploration and suggest eDNA will facilitate fine-scale geographic and temporal mapping of marine fish populations at relatively low cost.
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Species richness is a metric of biodiversity that represents the number of species present in a community. Traditional fisheries assessments that rely on capture of organisms often underestimate true species richness. Environmental DNA (eDNA) metabarcoding is an alternative tool that infers species richness by collecting and sequencing DNA present in the ecosystem. Our objective was to determine how spatial distribution of samples and “bioinformatic stringency” affected eDNA-metabarcoding estimates of species richness compared with capture-based estimates in a 2.2 ha reservoir. When bioinformatic criteria required species to be detected only in a single sample, eDNA metabarcoding detected all species captured with traditional methods plus an additional 11 noncaptured species. However, when we required species to be detected with multiple markers and in multiple samples, eDNA metabarcoding detected only seven of the captured species. Our analysis of the spatial patterns of species detection indicated that eDNA was distributed relatively homogeneously throughout the reservoir, except near the inflowing stream. We suggest that interpretation of eDNA metabarcoding data must consider the potential effects of water body type, spatial resolution, and bioinformatic stringency.
Effective ecosystem conservation and resource management require quantitative monitoring of biodiversity, including accurate descriptions of species composition and temporal variations of species abundance. Accordingly, quantitative monitoring of biodiversity has been performed for many ecosystems, but it is often time-and effort-consuming and costly. Recent studies have shown that environmental DNA (eDNA), which is released to the environment from macro-organisms living in a habitat, contains information about species identity and abundance. Thus, analysing eDNA would be a promising approach for more efficient biodiversity monitoring. In the present study, internal standard DNAs (i.e. known amounts of short DNA fragments from fish species that have never been observed in a sampling area) were added to eDNA samples, which were collected weekly from a coastal marine ecosystem in Maizuru Bay, Japan (from April 2015 to March 2016) and metabarcoding analysis was performed using Illumina MiSeq to simultaneously identify fish species and quantify fish eDNA copy numbers. A correction equation was obtained for each sample using the relationship between the number of sequence reads and the added amount of the standard DNAs and this equation was used to estimate the copy numbers from the sequence reads of non-standard fish eDNA. The calculated copy numbers showed significant positive correlation with those determined by quantitative PCR, suggesting that eDNA metabarcoding with standard DNA enabled useful quantifications of eDNA. Furthermore, for samples that show a high level of PCR inhibition, this method might allow more accurate quantification than qPCR because the correction equations generated using internal standard DNAs would include the effect of PCR inhibition. A single run of Illumina MiSeq produced >70 quantitative fish eDNA time series in this study, showing that this method could contribute to more efficient and quantitative monitoring of biodiversity.
The extraction and characterization of DNA from aquatic environmental samples offers an alternative, non-invasive approach for the detection of rare species. Environmental DNA, coupled with PCR and next-generation sequencing (“metabarcoding”), has proven to be very sensitive for the detection of rare aquatic species. Our study used a custom designed group-specific primer set and next-generation sequencing for the detection of three species at risk; (Eastern Sand Darter, Ammocrypta pellucida; Northern Madtom, Noturus stigmosus; and Silver Shiner, Notropis photogenis), one invasive species (Round Goby, Neogobius melanostomus) and an additional 78 native species from two large Great Lakes tributary rivers in southern Ontario, Canada; the Grand River and the Sydenham River. Out of 82 fish species detected in both rivers using capture-based and eDNA methods, our eDNA method detected 86.2% and 72.0% of the fish species in the Grand River and the Sydenham River, respectively, which included our four target species. Our analyses also identified significant positive and negative species co-occurrence patterns between our target species and other identified species. Our results demonstrate that eDNA metabarcoding that targets the fish community as well as individual species of interest provides a better understanding of factors affecting the target species spatial distribution in an ecosystem than possible with only target species data. Additionally, eDNA is easily implemented as an initial survey tool, or alongside capture-based methods, for improved mapping of species distribution patterns.