Taylor M. Wilcox

Robust detection of rare species using environmental DNA: the importance of primer specificity.

Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probe-based chemistries may represent a particularly powerful tool because of the method’s sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence the specificity and sensitivity of TaqMan MGB assays using co-occurring, closely related brook trout (Salvelinus fontinalis) and bull trout (S. confluentus) as a case study. We found qPCR to be substantially more sensitive than traditional PCR, with a high probability of detection at concentrations as low as 0.5 target copies/µl. We also found that number and placement of base pair mismatches between the Taqman MGB assay and non-target templates was important to target specificity, and that specificity was most influenced by base pair mismatches in the primers, rather than in the probe. We found that insufficient specificity can result in both false positive and false negative results, particularly in the presence of abundant related species. Our results highlight the utility of qPCR as a highly sensitive eDNA tool, and underscore the importance of careful assay design.

Distance, flow and PCR inhibition: eDNA dynamics in two headwater streams.

Environmental DNA (eDNA) detection has emerged as a powerful tool for monitoring aquatic organisms, but much remains unknown about the dynamics of aquatic eDNA over a range of environmental conditions. DNA concentrations in streams and rivers will depend not only on the equilibrium between DNA entering the water and DNA leaving the system through degradation, but also on downstream transport. To improve understanding of the dynamics of eDNA concentration in lotic systems, we introduced caged trout into two fishless headwater streams and took eDNA samples at evenly spaced downstream intervals. This was repeated 18 times from mid‐summer through autumn, over flows ranging from approximately 1–96 L/s. We used quantitative PCR to relate DNA copy number to distance from source. We found that regardless of flow, there were detectable levels of DNA at 239.5 m. The main effect of flow on eDNA counts was in opposite directions in the two streams. At the lowest flows, eDNA counts were highest close to the source and quickly trailed off over distance. At the highest flows, DNA counts were relatively low both near and far from the source. Biomass was positively related to eDNA copy number in both streams. A combination of cell settling, turbulence and dilution effects is probably responsible for our observations. Additionally, during high leaf deposition periods, the presence of inhibitors resulted in no amplification for high copy number samples in the absence of an inhibition‐releasing strategy, demonstrating the necessity to carefully consider inhibition in eDNA analysis.

Sampling large geographic areas for rare species using environmental DNA: a study of bull trout Salvelinus confluentus occupancy in western Montana

This study tested the efficacy of environmental DNA (eDNA) sampling to delineate the distribution of bull trout Salvelinus confluentus in headwater streams in western Montana, U.S.A. Surveys proved fast, reliable and sensitive: 124 samples were collected across five basins by a single crew in c. 8 days. Results were largely consistent with past electrofishing, but, in a basin where S. confluentus were known to be scarce, eDNA samples indicated that S. confluentus were more broadly distributed than previously thought.

The Dual Challenges of Generality and Specificity When Developing Environmental DNA Markers for Species and Subspecies of Oncorhynchus.

Environmental DNA (eDNA) sampling is a powerful tool for detecting invasive and native aquatic species. Often, species of conservation interest co-occur with other, closely related taxa. Here, we developed qPCR (quantitative PCR) markers which distinguish westslope cutthroat trout (Oncorhynchus clarkii lewsi), Yellowstone cutthroat trout (O. clarkii bouvieri), and rainbow trout (O. mykiss), which are of conservation interest both as native species and as invasive species across each other’s native ranges. We found that local polymorphisms within westslope cutthroat trout and rainbow trout posed a challenge to designing assays that are generally applicable across the range of these widely-distributed species. Further, poorly-resolved taxonomies of Yellowstone cutthroat trout and Bonneville cutthroat trout (O. c. utah) prevented design of an assay that distinguishes these recognized taxa. The issues of intraspecific polymorphism and unresolved taxonomy for eDNA assay design addressed in this study are likely to be general problems for closely-related taxa. Prior to field application, we recommend that future studies sample populations and test assays more broadly than has been typical of published eDNA assays to date.

Environmental DNA particle size distribution from Brook Trout (Salvelinus fontinalis)

Environmental DNA (eDNA) sampling has become a widespread approach for detecting aquatic animals with high potential for improving conservation biology. However, little research has been done to determine the size of particles targeted by eDNA surveys. In this study, we conduct particle distribution analysis of eDNA from a captive Brook Trout (Salvelinus fontinalis) in a flow-through mesocosm. Our data suggest that 1.2–10-μm particles are the most common size of eDNA from Brook Trout, which is consistent with our hypotheses that eDNA in the environment is comprised of loosely aggregated smaller particles, resulting in high inter-sample heterogeneity. These findings are similar to those of a study on Common Carp (Cyprinus carpio) in lentic systems.

Climate, Demography, and Zoogeography Predict Introgression Thresholds in Salmonid Hybrid Zones in Rocky Mountain Streams

Among the many threats posed by invasions of nonnative species is introgressive hybridization, which can lead to the genomic extinction of native taxa. This phenomenon is regarded as common and perhaps inevitable among native cutthroat trout and introduced rainbow trout in western North America, despite that these taxa naturally co-occur in some locations. We conducted a synthetic analysis of 13,315 genotyped fish from 558 sites by building logistic regression models using data from geospatial stream databases and from 12 published studies of hybridization to assess whether environmental covariates could explain levels of introgression between westslope cutthroat trout and rainbow trout in the U.S. northern Rocky Mountains. A consensus model performed well (AUC, 0.78–0.86; classification success, 72–82%; 10-fold cross validation, 70–82%) and predicted that rainbow trout introgression was significantly associated with warmer water temperatures, larger streams, proximity to warmer habitats and to recent sources of rainbow trout propagules, presence within the historical range of rainbow trout, and locations further east. Assuming that water temperatures will continue to rise in response to climate change and that levels of introgression outside the historical range of rainbow trout will equilibrate with those inside that range, we applied six scenarios across a 55,234-km stream network that forecast 9.5–74.7% declines in the amount of habitat occupied by westslope cutthroat trout populations of conservation value, but not the wholesale loss of such populations. We conclude that introgression between these taxa is predictably related to environmental conditions, many of which can be manipulated to foster largely genetically intact populations of westslope cutthroat trout and help managers prioritize conservation activities.

A blocking primer increases specificity in environmental DNA detection of bull trout (Salvelinus confluentus)

Environmental DNA (eDNA) is increasingly applied as a highly sensitive way to detect aquatic animals non-invasively. However, distinguishing closely related taxa can be particularly challenging. Previous studies of ancient DNA and genetic diet analysis have used blocking primers to enrich target template in the presence of abundant, non-target DNA. Here we apply a blocking primer to increase the specificity of a TaqMan assay for eDNA detection of rare and endangered bull trout (Salvelinus confluentus) in the presence of the closely related (Salvelinus namaycush). We found that addition of a blocking primer substantially increased assay specificity without compromising sensitivity or quantification ability.

An environmental DNA marker for detecting nonnative brown trout (Salmo trutta)

Brown trout (Salmo trutta) are widely introduced in western North America where their presence has led to declines of several native species. To assist conservation efforts aimed at early detection and eradication of this species, we developed a quantitative PCR marker to detect the presence of brown trout DNA in environmental samples. The marker strongly amplified brown trout eDNA, and produced no amplification of eDNA from 17 other species commonly found in western North America. We field tested this marker and demonstrated positive detections in field samples where brown trout presence was known.

An eDNA assay for river otter detection: a tool for surveying a semi-aquatic mammal

Abstract Environmental DNA (eDNA) is an effective tool for the detection of elusive or low-density aquatic organisms. However, it has infrequently been applied to mammalian species. North American river otters (Lontra canadensis) are both broad ranging and semi-aquatic, making them an ideal candidate for examining the uses of eDNA for detection of mammals. We developed a species-specific assay for detection of North American river otters using eDNA. The assay was tested for specificity against closely-related mustelids native to western North America, and was validated through testing environmental samples.

Capture enrichment of aquatic environmental DNA: A first proof of concept

Environmental DNA (eDNA) sampling—the detection of genetic material in the environment to infer species presence—has rapidly grown as a tool for sampling aquatic animal communities. A potentially powerful feature of environmental sampling is that all taxa within the habitat shed DNA and so may be detectable, creating opportunity for whole‐community assessments. However, animal DNA in the environment tends to be comparatively rare, making it necessary to enrich for genetic targets from focal taxa prior to sequencing. Current metabarcoding approaches for enrichment rely on bulk amplification using conserved primer annealing sites, which can result in skewed relative sequence abundance and failure to detect some taxa because of PCR bias. Here, we test capture enrichment via hybridization as an alternative strategy for target enrichment using a series of experiments on environmental samples and laboratory‐generated, known‐composition DNA mixtures. Capture enrichment resulted in detecting multiple species in both kinds of samples, and postcapture relative sequence abundance accurately reflected initial relative template abundance. However, further optimization is needed to permit reliable species detection at the very low‐DNA quantities typical of environmental samples (<0.1 ng DNA). We estimate that our capture protocols are comparable to, but less sensitive than, current PCR‐based eDNA analyses.