Use of Droplet Digital PCR for Estimation of Fish Abundance and Biomass in Environmental DNA Surveys

An environmental DNA (eDNA) analysis method has been recently developed to estimate the distribution of aquatic animals by quantifying the number of target DNA copies with quantitative real-time PCR (qPCR). A new quantitative PCR technology, droplet digital PCR (ddPCR), partitions PCR reactions into thousands of droplets and detects the amplification in each droplet, thereby allowing direct quantification of target DNA. We evaluated the quantification accuracy of qPCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp. We found that ddPCR quantified the concentration of carp eDNA along with carp abundance and biomass more accurately than qPCR, especially at low eDNA concentrations. In addition, errors in the analysis were smaller in ddPCR than in qPCR. Thus, ddPCR is better suited to measure eDNA concentration in water, and it provides more accurate results for the abundance and biomass of the target species than qPCR. We also found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by using both ddPCR and qPCR; this suggests that abundance can be better estimated by the analysis of eDNA for species with fewer variations in body mass.     

Marine water environmental DNA metabarcoding provides a comprehensive fish diversity assessment and reveals spatial patterns in a large oceanic area

Current methods for monitoring marine fish (including bony fishes and elasmobranchs) diversity mostly rely on trawling surveys, which are invasive, costly, and time‐consuming. Moreover, these methods are selective, targeting a subset of species at the time, and can be inaccessible to certain areas. Here, we used environmental DNA (eDNA), the DNA present in the water column as part of shed cells, tissues, or mucus, to provide comprehensive information about fish diversity in a large marine area. Further, eDNA results were compared to the fish diversity obtained in pelagic trawls. A total of 44 5 L‐water samples were collected onboard a wide‐scale oceanographic survey covering about 120,000 square kilometers in Northeast Atlantic Ocean. A short region of the 12S rRNA gene was amplified and sequenced through metabarcoding generating almost 3.5 million quality‐filtered reads. Trawl and eDNA samples resulted in the same most abundant species (European anchovy, European pilchard, Atlantic mackerel, and blue whiting), but eDNA metabarcoding resulted in more detected bony fish and elasmobranch species (116) than trawling (16). Although an overall correlation between fishes biomass and number of reads was observed, some species deviated from the common trend, which could be explained by inherent biases of each of the methods. Species distribution patterns inferred from eDNA metabarcoding data coincided with current ecological knowledge of the species, suggesting that eDNA has the potential to draw sound ecological conclusions that can contribute to fish surveillance programs. Our results support eDNA metabarcoding for broad‐scale marine fish diversity monitoring in the context of Directives such as the Common Fisheries Policy or the Marine Strategy Framework Directive.     

Can size distributions of European lake fish communities be predicted by trophic positions of their fish species?

An organism''s body size plays an important role in ecological interactions such as predator–prey relationships. As predators are typically larger than their prey, this often leads to a strong positive relationship between body size and trophic position in aquatic ecosystems. The distribution of body sizes in a community can thus be an indicator of the strengths of predator–prey interactions. The aim of this study was to gain more insight into the relationship between fish body size distribution and trophic position in a wide range of European lakes. We used quantile regression to examine the relationship between fish species'' trophic position and their log‐transformed maximum body mass for 48 fish species found in 235 European lakes. Subsequently, we examined whether the slopes of the continuous community size distributions, estimated by maximum likelihood, were predicted by trophic position, predator–prey mass ratio (PPMR), or abundance (number per unit effort) of fish communities in these lakes. We found a positive linear relationship between species'' maximum body mass and average trophic position in fishes only for the 75% quantile, contrasting our expectation that species'' trophic position systematically increases with maximum body mass for fish species in European lakes. Consequently, the size spectrum slope was not related to the average community trophic position, but there were negative effects of community PPMR and total fish abundance on the size spectrum slope. We conclude that predator–prey interactions likely do not contribute strongly to shaping community size distributions in these lakes.     

Environmental variability uncovers disruptive effects of species' interactions on population dynamics

How species respond to changes in environmental variability has been shown for single species, but the question remains whether these results are transferable to species when incorporated in ecological communities. Here, we address this issue by analysing the same species exposed to a range of environmental variabilities when (i) isolated or (ii) embedded in a food web. We find that all species in food webs exposed to temporally uncorrelated environments (white noise) show the same type of dynamics as isolated species, whereas species in food webs exposed to positively autocorrelated environments (red noise) can respond completely differently compared with isolated species. This is owing to species following their equilibrium densities in a positively autocorrelated environment that in turn enables species–species interactions to come into play. Our results give new insights into species'' response to environmental variation. They especially highlight the importance of considering both species'' interactions and environmental autocorrelation when studying population dynamics in a fluctuating environment.     

Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA

Preserving biodiversity is a global challenge requiring data on species’ distribution and abundance over large geographic and temporal scales. However, traditional methods to survey mobile species’ distribution and abundance in marine environments are often inefficient, environmentally destructive, or resource‐intensive. Metabarcoding of environmental DNA (eDNA) offers a new means to assess biodiversity and on much larger scales, but adoption of this approach for surveying whole animal communities in large, dynamic aquatic systems has been slowed by significant unknowns surrounding error rates of detection and relevant spatial resolution of eDNA surveys. Here, we report the results of a 2.5 km eDNA transect surveying the vertebrate fauna present along a gradation of diverse marine habitats associated with a kelp forest ecosystem. Using PCR primers that target the mitochondrial 12S rRNA gene of marine fishes and mammals, we generated eDNA sequence data and compared it to simultaneous visual dive surveys. We find spatial concordance between individual species’ eDNA and visual survey trends, and that eDNA is able to distinguish vertebrate community assemblages from habitats separated by as little as ~60 m. eDNA reliably detected vertebrates with low false‐negative error rates (1/12 taxa) when compared to the surveys, and revealed cryptic species known to occupy the habitats but overlooked by visual methods. This study also presents an explicit accounting of false negatives and positives in metabarcoding data, which illustrate the influence of gene marker selection, replication, contamination, biases impacting eDNA count data and ecology of target species on eDNA detection rates in an open ecosystem.     

Estimating fish abundance and biomass from eDNA concentrations: variability among capture methods and environmental conditions

Environmental DNA (eDNA) promises to ease noninvasive quantification of fish biomass or abundance, but its integration within conservation and fisheries management is currently limited by a lack of understanding of the influence of eDNA collection method and environmental conditions on eDNA concentrations in water samples. Water temperature is known to influence the metabolism of fish and consequently could strongly affect eDNA release rate. As water temperature varies in temperate regions (both seasonally and geographically), the unknown effect of water temperature on eDNA concentrations poses practical limitations on quantifying fish populations using eDNA from water samples. This study aimed to clarify how water temperature and the eDNA capture method alter the relationships between eDNA concentration and fish abundance/biomass. Water samples (1 L) were collected from 30 aquaria including triplicate of 0, 5, 10, 15 and 20 Brook Charr specimens at two different temperatures (7 °C and 14 °C). Water samples were filtered with five different types of filters. The eDNA concentration obtained by quantitative PCR (qPCR) varied significantly with fish abundance and biomass and types of filters (mixed‐design ANOVA, P < 0.001). Results also show that fish released more eDNA in warm water than in cold water and that eDNA concentration better reflects fish abundance/biomass at high temperature. From a technical standpoint, higher levels of eDNA were captured with glass fibre (GF) filters than with mixed cellulose ester (MCE) filters and support the importance of adequate filters to quantify fish abundance based on the eDNA method. This study supports the importance of including water temperature in fish abundance/biomass prediction models based on eDNA.     

External morphology explains the success of biological invasions

Biological invasions have become major players in the current biodiversity crisis, but realistic tools to predict which species will establish successful populations are still unavailable. Here we present a novel approach that requires only a morphometric characterisation of the species. Using fish invasions of the Mediterranean, we show that the abundance of non‐indigenous fishes correlates with the location and relative size of occupied morphological space within the receiving pool of species. Those invaders that established abundant populations tended to be added outside or at the margins of the receiving morphospace, whereas non‐indigenous species morphologically similar to resident ones failed to develop large populations or even to establish themselves, probably because the available ecological niches were already occupied. Accepting that morphology is a proxy for a species' ecological position in a community, our findings are consistent with ideas advanced since Darwin's naturalisation hypothesis and provide a new warning signal to identify invaders and to recognise vulnerable communities.     

A Mechanistic Study of Plant and Microbial Controls over R* for Nitrogen in an Annual Grassland

Differences in species'' abilities to capture resources can drive competitive hierarchies, successional dynamics, community diversity, and invasions. To investigate mechanisms of resource competition within a nitrogen (N) limited California grassland community, we established a manipulative experiment using an R* framework. R* theory holds that better competitors within a N limited community should better depress available N in monoculture plots and obtain higher abundance in mixture plots. We asked whether (1) plant uptake or (2) plant species influences on microbial dynamics were the primary drivers of available soil N levels in this system where N structures plant communities. To disentangle the relative roles of plant uptake and microbially-mediated processes in resource competition, we quantified soil N dynamics as well as N pools in plant and microbial biomass in monoculture plots of 11 native or exotic annual grassland plants over one growing season. We found a negative correlation between plant N content and soil dissolved inorganic nitrogen (DIN, our measure of R*), suggesting that plant uptake drives R*. In contrast, we found no relationship between microbial biomass N or potential net N mineralization and DIN. We conclude that while plant-microbial interactions may have altered the overall quantity of N that plants take up, the relationship between species'' abundance and available N in monoculture was largely driven by plant N uptake in this first year of growth.     

Macroevolutionary consequences of profound climate change on niche evolution in marine molluscs over the past three million years

In order to predict the fate of biodiversity in a rapidly changing world, we must first understand how species adapt to new environmental conditions. The long-term evolutionary dynamics of species'' physiological tolerances to differing climatic regimes remain obscure. Here, we unite palaeontological and neontological data to analyse whether species'' environmental tolerances remain stable across 3 Myr of profound climatic changes using 10 phylogenetically, ecologically and developmentally diverse mollusc species from the Atlantic and Gulf Coastal Plains, USA. We additionally investigate whether these species'' upper and lower thermal tolerances are constrained across this interval. We find that these species'' environmental preferences are stable across the duration of their lifetimes, even when faced with significant environmental perturbations. The results suggest that species will respond to current and future warming either by altering distributions to track suitable habitat or, if the pace of change is too rapid, by going extinct. Our findings also support methods that project species'' present-day environmental requirements to future climatic landscapes to assess conservation risks.     

Behavior and season affect crayfish detection and density inference using environmental DNA

Although the presence/absence of aquatic invertebrates using environmental DNA (eDNA) has been established for several species, inferring population densities has remained problematic. The invasive American signal crayfish, Pacifastacus leniusculus (Dana), is the leading cause of decline in the UK's only native crayfish species, Austropotamobius pallipes (Lereboullet). Methods to detect species at low abundances offer the opportunity for the early detection, and potential eradication, of P. leniusculus before population densities reach threatening levels in areas occupied by A. pallipes. Using a factorial experimental design with aquaria, we investigated the impacts of biomass, sex ratio, and fighting behavior on the amount of eDNA released by P. leniusculus, with the aim to infer density per aquarium depending on treatments. The amount of target eDNA in water samples from each aquarium was measured using the quantitative Polymerase Chain Reaction. We show that the presence of eggs significantly increases the concentration of crayfish eDNA per unit of mass, and that there is a significant relationship between eDNA concentration and biomass when females are egg‐bearing. However, the relationship between crayfish biomass and eDNA concentration is lost in aquaria without ovigerous females. Female‐specific tanks had significantly higher eDNA concentrations than male‐specific tanks, and the prevention of fighting did not impact the amount of eDNA in the water. These results indicate that detection and estimate of crayfish abundance using eDNA may be more effective while females are ovigerous. This information should guide further research for an accurate estimation of crayfish biomass in the field depending on the season. Our results indicate that detection and quantification of egg‐laying aquatic invertebrate species using eDNA could be most successful during periods when eggs are developing in the water. We recommend that practitioners consider the reproductive cycle of target species when attempting to study or detect aquatic species using eDNA in the field.     

Detecting the movement and spawning activity of bigheaded carps with environmental DNA

Bigheaded carps are invasive fishes threatening to invade the Great Lakes basin and establish spawning populations, and have been monitored using environmental DNA (eDNA). Not only does eDNA hold potential for detecting the presence of species, but may also allow for quantitative comparisons like relative abundance of species across time or space. We examined the relationships among bigheaded carp movement, hydrography, spawning and eDNA on the Wabash River, IN, USA. We found positive relationships between eDNA and movement and eDNA and hydrography. We did not find a relationship between eDNA and spawning activity in the form of drifting eggs. Our first finding demonstrates how eDNA may be used to monitor species abundance, whereas our second finding illustrates the need for additional research into eDNA methodologies. Current applications of eDNA are widespread, but the relatively new technology requires further refinement.     

Predicting community rank‐abundance distributions under current and future climates

Understanding influences of environmental change on biodiversity requires consideration of more than just species richness. Here we present a novel framework for understanding possible changes in species' abundance structures within communities under climate change. We demonstrate this using comprehensive survey and environmental data from 1748 woody plant communities across southeast Queensland, Australia, to model rank‐abundance distributions (RADs) under current and future climates. Under current conditions, the models predicted RADs consistent with the region's dominant vegetation types. We demonstrate that under a business as usual climate scenario, total abundance and richness may decline in subtropical rainforest and shrubby heath, and increase in dry sclerophyll forests. Despite these opposing trends, we predicted evenness in the distribution of abundances between species to increase in all vegetation types. By assessing the information rich, multidimensional RAD, we show that climate‐driven changes to community abundance structures will likely vary depending on the current composition and environmental context.     

Influence of fishes on macroinvertebrate communities and insect emergence production in intermittent stream refuges

1. Drying intermittent stream networks often have permanent water refuges that are important for recolonisation. These habitats may be hotspots for interactions between fishes and invertebrates as they become isolated, but densities and diversity of fishes in these refuges can be highly variable across time and space.
2. Insect emergence from streams provides energy and nutrient subsidies to riparian habitats. The magnitude of such subsidies may be influenced by in-stream predators such as fishes.
3. We examined whether benthic macroinvertebrate communities, emerging adult insects, and algal biomass in permanent grassland stream pools differed among sites with naturally varying densities of fishes. We also manipulated fish densities in a mesocosm experiment to address how fishes might affect colonisation during recovery from hydrologic disturbance.
4. Fish biomass had a negative impact on invertebrate abundance, but not biomass or taxa richness, in natural pools. Total fish biomass was not correlated with total insect emergence in natural pools, but orangethroat darter (Etheostoma spectabile) biomass was inversely correlated with emerging Chironomidae biomass and individual midge body size. The interaction in our models between predatory fish biomass and date suggested that fishes may also delay insect emergence from natural pools, altering the timing of aquatic–terrestrial subsidies.
5. There was an increase over time in algal biomass (chlorophyll-a) in mesocosms, but this did not differ among fish density treatments. Regardless, fish presence in mesocosms reduced the abundance of colonising insects and total invertebrate biomass. Mesocosm invertebrate communities in treatments without fishes were characterised by more Chironomidae, Culicidae, and Corduliidae.
6. Results suggest that fishes influence invertebrates in habitats that represent important refuges during hydrologic disturbance, hot spots for subsidy exports to riparian food webs, and source areas for colonists during recovery from hydrologic disturbance. Fish effects in these systems include decreasing invertebrate abundance, shifting community structure, and altering patterns of invertebrate emergence and colonisation.

     

Creating,curating and evaluating a mitogenomic reference database to improve regional species identification using environmental DNA

Species detection using eDNA is revolutionizing global capacity to monitor biodiversity. However, the lack of regional, vouchered, genomic sequence information—especially sequence information that includes intraspecific variation—creates a bottleneck for management agencies wanting to harness the complete power of eDNA to monitor taxa and implement eDNA analyses. eDNA studies depend upon regional databases of mitogenomic sequence information to evaluate the effectiveness of such data to detect and identify taxa. We created the Oregon Biodiversity Genome Project to create a database of complete, nearly error-free mitogenomic sequences for all of Oregon's fishes. We have successfully assembled the complete mitogenomes of 313 specimens of freshwater, anadromous and estuarine fishes representing 24 families, 55 genera and 129 species and lineages. Comparative analyses of these sequences illustrate that many regions of the mitogenome are taxonomically informative, that the short (~150 bp) mitochondrial ‘barcode’ regions typically used for eDNA assays do not consistently diagnose for species and that complete single or multiple genes of the mitogenome are preferable for identifying Oregon's fishes. This project provides a blueprint for other researchers to follow as they build regional databases, illustrates the taxonomic value and limits of complete mitogenomic sequences and offers clues as to how current eDNA assays and environmental genomics methods of the future can best leverage this information.     

Signals of Climate Change in Butterfly Communities in a Mediterranean Protected Area

The European protected-area network will cease to be efficient for biodiversity conservation, particularly in the Mediterranean region, if species are driven out of protected areas by climate warming. Yet, no empirical evidence of how climate change influences ecological communities in Mediterranean nature reserves really exists. Here, we examine long-term (1998–2011/2012) and short-term (2011–2012) changes in the butterfly fauna of Dadia National Park (Greece) by revisiting 21 and 18 transects in 2011 and 2012 respectively, that were initially surveyed in 1998. We evaluate the temperature trend for the study area for a 22-year-period (1990–2012) in which all three butterfly surveys are included. We also assess changes in community composition and species richness in butterfly communities using information on (a) species’ elevational distributions in Greece and (b) Community Temperature Index (calculated from the average temperature of species'' geographical ranges in Europe, weighted by species'' abundance per transect and year). Despite the protected status of Dadia NP and the subsequent stability of land use regimes, we found a marked change in butterfly community composition over a 13 year period, concomitant with an increase of annual average temperature of 0.95°C. Our analysis gave no evidence of significant year-to-year (2011–2012) variability in butterfly community composition, suggesting that the community composition change we recorded is likely the consequence of long-term environmental change, such as climate warming. We observe an increased abundance of low-elevation species whereas species mainly occurring at higher elevations in the region declined. The Community Temperature Index was found to increase in all habitats except agricultural areas. If equivalent changes occur in other protected areas and taxonomic groups across Mediterranean Europe, new conservation options and approaches for increasing species’ resilience may have to be devised.     

Establishing an environmental DNA method to detect and estimate the biomass of Sakhalin taimen,a critically endangered Asian salmonid

For field ecologists, detecting a target species in the wild is a severe bottleneck to understanding its distribution and population status. Recently, environmental DNA (eDNA) techniques have been developed as a noninvasive monitoring tool for aquatic organisms. While applications of eDNA techniques for biomass estimation have been proposed, little is known about an applicable size range of the organisms, which might affect relationships between biomass and eDNA concentration. Here, we investigated eDNA from Sakhalin taimen (Parahucho perryi), a giant salmonid species of northern Japan. This species is critically endangered and difficult to detect in the wild by conventional sampling methods. Using quantitative real-time PCR, we tested correlations between eDNA concentration and fish density using fish with a wide range of ages and body sizes in aquarium experiments. We found that our new primers and probe were truly species-specific, and that the eDNA concentration was significantly correlated with fish density and body size (p < 0.001). Furthermore, based on our calculation, the eDNA concentrations were rather constant among aquaria with fish in different age and size groups when their total weight was adjusted. These results suggest that eDNA concentrations can be an indicator of biomass of Sakhalin taimen, although further research is needed for its application in natural environments.     

Environmental DNA analysis for estimating the abundance and biomass of stream fish

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Environmental DNA as an emerging tool in botanical research

Over the past quarter century, environmental DNA (eDNA) has been ascendant as a tool to detect, measure, and monitor biodiversity (species and communities), as a means of elucidating biological interaction networks, and as a window into understanding past patterns of biodiversity. However, only recently has the potential of eDNA been realized in the botanical world. Here we synthesize the state of eDNA applications in botanical systems with emphases on aquatic, ancient, contemporary sediment, and airborne systems, and focusing on both single-species approaches and multispecies community metabarcoding. Further, we describe how abiotic and biotic factors, taxonomic resolution, primer choice, spatiotemporal scales, and relative abundance influence the utilization and interpretation of airborne eDNA results. Lastly, we explore several areas and opportunities for further development of eDNA tools for plants, advancing our knowledge and understanding of the efficacy, utility, and cost-effectiveness, and ultimately facilitating increased adoption of eDNA analyses in botanical systems.     

Identifying ‘climate keystone species’ as a tool for conserving ecological communities under climate change

Aim

Climate change affects ecological communities via impacts on species. The community's response to climate change can be represented as the temporal trend in a climate-related functional property that is quantified using a relevant functional trait. Noteworthy, some species influence this response in the community more strongly than others.

Innovation

Leveraging on the concept of keystone species, we propose that species with a strong effect on the community's functional response to climate change beyond their relative abundance can be considered as ‘climate keystone species’. We develop a stepwise tool to determine species' effects on a community's climate response and identify climate keystone species. We quantify the species-specific effect by measuring the difference in the community's climate response with and without the species. Next, we identify climate keystone species as those with a strong residual effect after weighting with their relative abundances in the community.

Main Conclusions

To illustrate the use of the stepwise tool with empirical data, we identify climate keystone species that have a strong effect on the change in the average temperature niche in North American bird communities over time and find the identification tool ecologically relevant. Identification of climate keystone species can serve as an additional conservation method to efficiently protect ecological communities and, in turn, the ecosystem functions they provide.