eDNAssay: A machine learning tool that accurately predicts qPCR cross-amplification.

Environmental DNA (eDNA) sampling is a highly sensitive and cost-effective technique for wildlife monitoring, notably through the use of qPCR assays. However, it can be difficult to ensure assay specificity when many closely related species co-occur. In theory, specificity may be assessed in silico by determining whether assay oligonucleotides have enough base-pair mismatches with nontarget sequences to preclude amplification. However, the mismatch qualities required are poorly understood, making in silico assessments difficult and often necessitating extensive in vitro testing-typically the greatest bottleneck in assay development. Increasing the accuracy of in silico assessments would therefore streamline the assay development process. In this study, we paired 10 qPCR assays with 82 synthetic gene fragments for 530 specificity tests using SYBR Green intercalating dye (n = 262) and TaqMan hydrolysis probes (n = 268). Test results were used to train random forest classifiers to predict amplification. The primer-only model (SYBR Green results) and full-assay model (TaqMan probe-based results) were 99.6% and 100% accurate, respectively, in cross-validation. We further assessed model performance using six independent assays not used in model training. In these tests the primer-only model was 92.4% accurate (n = 119) and the full-assay model was 96.5% accurate (n = 144). The high performance achieved by these models makes it possible for eDNA practitioners to more quickly and confidently develop assays specific to the intended target. Practitioners can access the full-assay model online via eDNAssay (https://NationalGenomicsCenter.shinyapps.io/eDNAssay), a user-friendly tool for predicting qPCR cross-amplification.

Do metapopulations and management matter for relict headwater bull trout populations in a warming climate?

Mountain headwater streams have emerged as important climate refuges for native cold-water species due to their slow climate velocities and extreme physical conditions that inhibit non-native invasions. Species persisting in refuges often do so as fragmented, relict populations from broader historical distributions that are subject to ongoing habitat reductions and increasing isolation as climate change progresses. Key for conservation planning is determining where remaining populations will persist and how habitat restoration strategies can improve biological resilience to enhance the long-term prospects for species of concern. Studying bull trout, a headwater species in the northwestern USA, we developed habitat occupancy models using a data set of population occurrence in 991 natal habitat patches with a suite of novel geospatial covariates derived from high-resolution hydroclimatic scenarios and other sources representing watershed and instream habitat conditions, patch geometry, disturbance, and biological interactions. The best model correctly predicted bull trout occupancy status in 82.6% of the patches and included effects for: patch size estimated as habitat volume, extent of within-patch reaches <9°C mean August temperature, distance to nearest occupied patch, road density, invasive brook trout prevalence, patch slope, and frequency of high winter flows. The model was used to assess 16 scenarios of bull trout occurrence within the study streams that represented a range of restoration strategies under three climatic conditions (baseline, moderate change, and extreme change). Results suggested that regional improvements in bull trout status were difficult to achieve in realistic restoration strategies due to the pervasive nature of climate change and the limited extent of restoration actions given their high costs. However, occurrence probabilities in a subset of patches were highly responsive to restoration actions, suggesting that targeted investments to improve the resilience of some populations may be contextually beneficial. A possible strategy, therefore, is focusing effort on responsive populations near more robust population strongholds, thereby contributing to local enclaves where dispersal among populations further enhances resilience. Equally important, strongholds constituted a small numerical percentage of patches (5%-21%), yet encompassed the large majority of occupied habitat by volume (72%-89%) and their protection could have significant conservation benefits for bull trout.

Occupancy modeling and resampling overcomes low test sensitivity to produce accurate SARS-CoV-2 prevalence estimates.

BACKGROUND: We evaluated whether occupancy modeling, an approach developed for detecting rare wildlife species, could overcome inherent accuracy limitations associated with rapid disease tests to generate fast, accurate, and affordable SARS-CoV-2 prevalence estimates. Occupancy modeling uses repeated sampling to estimate probability of false negative results, like those linked to rapid tests, for generating unbiased prevalence estimates. METHODS: We developed a simulation study to estimate SARS-CoV-2 prevalence using rapid, low-sensitivity, low-cost tests and slower, high-sensitivity, higher cost tests across a range of disease prevalence and sampling strategies. RESULTS: Occupancy modeling overcame the low sensitivity of rapid tests to generate prevalence estimates comparable to more accurate, slower tests. Moreover, minimal repeated sampling was required to offset low test sensitivity at low disease prevalence (0.1%), when rapid testing is most critical for informing disease management. CONCLUSIONS: Occupancy modeling enables the use of rapid tests to provide accurate, affordable, real-time estimates of the prevalence of emerging infectious diseases like SARS-CoV-2.

Using bear rub data and spatial capture-recapture models to estimate trend in a brown bear population.

Trends in population abundance can be challenging to quantify during range expansion and contraction, when there is spatial variation in trend, or the conservation area is large. We used genetic detection data from natural bear rubbing sites and spatial capture-recapture (SCR) modeling to estimate local density and population growth rates in a grizzly bear population in northwestern Montana, USA. We visited bear rubs to collect hair in 2004, 2009-2012 (3,579-4,802 rubs) and detected 249-355 individual bears each year. We estimated the finite annual population rate of change 2004-2012 was 1.043 (95% CI = 1.017-1.069). Population density shifted from being concentrated in the north in 2004 to a more even distribution across the ecosystem by 2012. Our genetic detection sampling approach coupled with SCR modeling allowed us to estimate spatially variable growth rates of an expanding grizzly bear population and provided insight into how those patterns developed. The ability of SCR to utilize unstructured data and produce spatially explicit maps that indicate where population change is occurring promises to facilitate the monitoring of difficult-to-study species across large spatial areas.

An improved environmental DNA assay for bull trout (Salvelinus confluentus) based on the ribosomal internal transcribed spacer I.

The majority of environmental DNA (eDNA) assays for vertebrate species are based on commonly analyzed regions of the mitochondrial genome. However, the high degree of mitochondrial similarity between two species of charr (Salvelinus spp.), southern Dolly Varden (S. malma lordii) and bull trout (Salvelinus confluentus), precludes the development of a mitochondrial eDNA assay to distinguish them. Presented here is an eDNA assay to detect bull trout based on the first ribosomal internal transcribed spacer (ITSI), a nuclear marker. This assay successfully detects bull trout and avoids detecting Dolly Varden as well as brook trout (S. fontinalis), Arctic char (S. alpinus), and lake trout (S. namaycush). In addition, this assay was compared with an extensively used mitochondrial bull trout assay and it was found that the ITSI-based assay produced higher detectability. Our results suggest this assay should out-perform the published mtDNA assay across the range of bull trout, while the added specificity allows reliable bull trout detection in areas where bull trout co-occur with other charr such as Dolly Varden. While clearly a superior assay in this instance, basing assays on ITSI is not without problems. For vertebrates, there are fewer ITSI sequences available than commonly sequenced regions of the mitochondrial genome. Thus, the initial in silico screening of candidate assays must be preceded by much more extensive sampling and sequencing of sympatric or closely related taxa. Further, all copies of the internal transcribed spacers within an individual may not be identical, which can lead to complications. Lastly, the copy number for ITSI varies widely across taxa; the greater detectability associated with this assay cannot be assumed for other species.

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.

Repurposing environmental DNA samples-detecting the western pearlshell (Margaritifera falcata) as a proof of concept.

Information on the distribution of multiple species in a common landscape is fundamental to effective conservation and management. However, distribution data are expensive to obtain and often limited to high-profile species in a system. A recently developed technique, environmental DNA (eDNA) sampling, has been shown to be more sensitive than traditional detection methods for many aquatic species. A second and perhaps underappreciated benefit of eDNA sampling is that a sample originally collected to determine the presence of one species can be re-analyzed to detect additional taxa without additional field effort. We developed an eDNA assay for the western pearlshell mussel (Margaritifera falcata) and evaluated its effectiveness by analyzing previously collected eDNA samples that were annotated with information including sample location and deposited in a central repository. The eDNA samples were initially collected to determine habitat occupancy by nonbenthic fish species at sites that were in the vicinity of locations recently occupied by western pearlshell. These repurposed eDNA samples produced results congruent with historical western pearlshell surveys and permitted a more precise delineation of the extent of local populations. That a sampling protocol designed to detect fish was also successful for detecting a freshwater mussel suggests that rapidly accumulating collections of eDNA samples can be repurposed to enhance the efficiency and cost-effectiveness of aquatic biodiversity monitoring.

qPCR detection of Sturgeon chub (Macrhybopsis gelida) DNA in environmental samples.

The Sturgeon chub (Macrhybopsis gelida) is a cyprinid fish native to the Missouri and Mississippi River basins of the U.S. Suspected long-term declines in the size of its distribution have prompted a review of its conservation status by the U.S. Fish and Wildlife Service, a process which depends on reliable methods to delineate the distribution and status of extant populations. To facilitate monitoring of Sturgeon chub populations, we developed a quantitative PCR assay to detect Sturgeon chub DNA in environmental samples. The assay consistently detected Sturgeon chub DNA in concentrations as low as 2 copies per reaction, and did not amplify DNA from non-target fish species that are sympatric in the upper Missouri River basin. Field tests of this assay with environmental samples successfully detected Sturgeon chub from sites known to be occupied. This assay offers an extremely sensitive methodology that can be applied to determine the range of Sturgeon chub, regardless of variation in habitat characteristics.

Ecological segregation moderates a climactic conclusion to trout hybridization.

For decades, it has been assumed that introgressive hybridization between introduced rainbow trout and native cutthroat trout in western North America will lead to genomic extinction of the latter. A broad-scale re-examination of their interaction indicates that ecological differences between these species and demographic processes are dictating the location and extent of their hybrid zones, and that runaway introgression between these taxa is unlikely.

The importance of data quality for generating reliable distribution models for rare, elusive, and cryptic species.

The availability of spatially referenced environmental data and species occurrence records in online databases enable practitioners to easily generate species distribution models (SDMs) for a broad array of taxa. Such databases often include occurrence records of unknown reliability, yet little information is available on the influence of data quality on SDMs generated for rare, elusive, and cryptic species that are prone to misidentification in the field. We investigated this question for the fisher (Pekania pennanti), a forest carnivore of conservation concern in the Pacific States that is often confused with the more common Pacific marten (Martes caurina). Fisher occurrence records supported by physical evidence (verifiable records) were available from a limited area, whereas occurrence records of unknown quality (unscreened records) were available from throughout the fisher's historical range. We reserved 20% of the verifiable records to use as a test sample for both models and generated SDMs with each dataset using Maxent. The verifiable model performed substantially better than the unscreened model based on multiple metrics including AUCtest values (0.78 and 0.62, respectively), evaluation of training and test gains, and statistical tests of how well each model predicted test localities. In addition, the verifiable model was consistent with our knowledge of the fisher's habitat relations and potential distribution, whereas the unscreened model indicated a much broader area of high-quality habitat (indices > 0.5) that included large expanses of high-elevation habitat that fishers do not occupy. Because Pacific martens remain relatively common in upper elevation habitats in the Cascade Range and Sierra Nevada, the SDM based on unscreened records likely reflects primarily a conflation of marten and fisher habitat. Consequently, accurate identifications are far more important than the spatial extent of occurrence records for generating reliable SDMs for the fisher in this region. We strongly recommend that practitioners avoid using anecdotal occurrence records to build SDMs but, if such data are used, the validity of resulting models should be tested with verifiable occurrence records.

Environmental DNA assays for the sister taxa sauger (Sander canadensis) and walleye (Sander vitreus).

Sauger (Sander canadensis) and walleye (S. vitreus) are percid fishes that naturally co-occur throughout much of the eastern United States. The native range of sauger extends into the upper Missouri River drainage where walleye did not historically occur, but have been stocked as a sport fish. Sauger populations have been declining due to habitat loss, fragmentation, and competition with non-native species, such as walleye. To effectively manage sauger populations, it is necessary to identify areas where sauger occur, and particularly where they co-occur with walleye. We developed quantitative PCR assays that can detect sauger and walleye DNA in filtered water samples. Each assay efficiently detected low quantities of target DNA and failed to detect DNA of non-target species with which they commonly co-occur.

A Noninvasive Tool to Assess the Distribution of Pacific Lamprey (Entosphenus tridentatus) in the Columbia River Basin.

The Pacific lamprey (Entosphenus tridentatus) is an anadromous fish once abundant throughout coastal basins of western North America that has suffered dramatic declines in the last century due primarily to human activities. Here, we describe the development of an environmental DNA (eDNA) assay to detect Pacific lamprey in the Columbia River basin. The eDNA assay successfully amplified tissue derived DNA of Pacific lamprey collected from 12 locations throughout the Columbia River basin. The assay amplifies DNA from other Entosphenus species found outside of the Columbia River basin, but is species-specific within this basin. As a result, the assay presented here may be useful for detecting Entosphenus spp. in geographic range beyond the Columbia River Basin. The assay did not amplify tissue or synthetically derived DNA of 14 commonly sympatric non-target species, including lampreys of the genus Lampetra, which are morphologically similar to Pacific lamprey in the freshwater larval stage.

Trade-offs and efficiencies in optimal budget-constrained multispecies corridor networks.

Conservation biologists recognize that a system of isolated protected areas will be necessary but insufficient to meet biodiversity objectives. Current approaches to connecting core conservation areas through corridors consider optimal corridor placement based on a single optimization goal: commonly, maximizing the movement for a target species across a network of protected areas. We show that designing corridors for single species based on purely ecological criteria leads to extremely expensive linkages that are suboptimal for multispecies connectivity objectives. Similarly, acquiring the least-expensive linkages leads to ecologically poor solutions. We developed algorithms for optimizing corridors for multispecies use given a specific budget. We applied our approach in western Montana to demonstrate how the solutions may be used to evaluate trade-offs in connectivity for 2 species with different habitat requirements, different core areas, and different conservation values under different budgets. We evaluated corridors that were optimal for each species individually and for both species jointly. Incorporating a budget constraint and jointly optimizing for both species resulted in corridors that were close to the individual species movement-potential optima but with substantial cost savings. Our approach produced corridors that were within 14% and 11% of the best possible corridor connectivity for grizzly bears (Ursus arctos) and wolverines (Gulo gulo), respectively, and saved 75% of the cost. Similarly, joint optimization under a combined budget resulted in improved connectivity for both species relative to splitting the budget in 2 to optimize for each species individually. Our results demonstrate economies of scale and complementarities conservation planners can achieve by optimizing corridor designs for financial costs and for multiple species connectivity jointly. We believe that our approach will facilitate corridor conservation by reducing acquisition costs and by allowing derived corridors to more closely reflect conservation priorities.

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.

Quantitative PCR Assays for Detecting Loach Minnow (Rhinichthys cobitis) and Spikedace (Meda fulgida) in the Southwestern United States.

Loach minnow (Rhinichthys cobitis) and spikedace (Meda fulgida) are legally protected with the status of Endangered under the U.S. Endangered Species Act and are endemic to the Gila River basin of Arizona and New Mexico. Efficient and sensitive methods for monitoring these species' distributions are critical for prioritizing conservation efforts. We developed quantitative PCR assays for detecting loach minnow and spikedace DNA in environmental samples. Each assay reliably detected low concentrations of target DNA without detection of non-target species, including other cyprinid fishes with which they co-occur.

Patterns of hybridization among cutthroat trout and rainbow trout in northern Rocky Mountain streams.

Introgressive hybridization between native and introduced species is a growing conservation concern. For native cutthroat trout and introduced rainbow trout in western North America, this process is thought to lead to the formation of hybrid swarms and the loss of monophyletic evolutionary lineages. Previous studies of this phenomenon, however, indicated that hybrid swarms were rare except when native and introduced forms of cutthroat trout co-occurred. We used a panel of 86 diagnostic, single nucleotide polymorphisms to evaluate the genetic composition of 3865 fish captured in 188 locations on 129 streams distributed across western Montana and northern Idaho. Although introgression was common and only 37% of the sites were occupied solely by parental westslope cutthroat trout, levels of hybridization were generally low. Of the 188 sites sampled, 73% contained ≤5% rainbow trout alleles and 58% had ≤1% rainbow trout alleles. Overall, 72% of specimens were nonadmixed westslope cutthroat trout, and an additional 3.5% were nonadmixed rainbow trout. Samples from seven sites met our criteria for hybrid swarms, that is, an absence of nonadmixed individuals and a random distribution of alleles within the sample; most (6/7) were associated with introgression by Yellowstone cutthroat trout. In streams with multiple sites, upstream locations exhibited less introgression than downstream locations. We conclude that although the widespread introduction of nonnative trout within the historical range of westslope cutthroat trout has increased the incidence of introgression, sites containing nonadmixed populations of this taxon are common and broadly distributed.

Capturing subregional variability in regional-scale climate change vulnerability assessments of natural resources.

Natural resource vulnerability to climate change can depend on the climatology and ecological conditions at a particular site. Here we present a conceptual framework for incorporating spatial variability in natural resource vulnerability to climate change in a regional-scale assessment. The framework was implemented in the first regional-scale vulnerability assessment conducted by the US Forest Service. During this assessment, five subregional workshops were held to capture variability in vulnerability and to develop adaptation tactics. At each workshop, participants answered a questionnaire to: 1) identify species, resources, or other information missing from the regional assessment, and 2) describe subregional vulnerability to climate change. Workshop participants divided into six resource groups; here we focus on wildlife resources. Participants identified information missing from the regional assessment and multiple instances of subregional variability in climate change vulnerability. We provide recommendations for improving the process of capturing subregional variability in a regional vulnerability assessment. We propose a revised conceptual framework structured around pathways of climate influence, each with separate rankings for exposure, sensitivity, and adaptive capacity. These revisions allow for a quantitative ranking of species, pathways, exposure, sensitivity, and adaptive capacity across subregions. Rankings can be used to direct the development and implementation of future regional research and monitoring programs. The revised conceptual framework is equally applicable as a stand-alone model for assessing climate change vulnerability and as a nested model within a regional assessment for capturing subregional variability in vulnerability.

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.

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.

Cottus schitsuumsh, a new species of sculpin (Scorpaeniformes: Cottidae) in the Columbia River basin, Idaho-Montana, USA.

Fishes of the genus Cottus have long been taxonomically challenging because of morphological similarities among species and their tendency to hybridize, and a number of undescribed species may remain in this genus. We used a combination of genetic and morphological methods to delineate and describe Cottus schitsuumsh, Cedar Sculpin, a new species, from the upper Columbia River basin, Idaho-Montana, USA. Although historically confused with the Shorthead Sculpin (C. confusus), the genetic distance between C. schitsuumsh and C. confusus (4.84-6.29%) suggests these species are distant relatives. Moreover, the two species can be differentiated on the basis of lateral-line pores on the caudal peduncle, head width, and interpelvic width. Cottus schitsuumsh is also distinct from all other Cottus in this region in having a single small, skin-covered, preopercular spine. Haplotypes of mtDNA cytochrome oxidase c subunit 1 of C. schitsuumsh differed from all other members of the genus at three positions, had interspecific genetic distances typical for congeneric fishes (1.61-2.74% to nearest neighbors), and were monophyletic in maximum-likelihood trees. Microsatellite analyses confirmed these taxonomic groupings for species potentially sympatric with C. schitsuumsh and that fish used in morphological comparisons were unlikely to be introgressed. Its irregular distribution, in the Spokane River basin in Idaho and portions of the Clark Fork River basin in Montana, may have resulted from human-assisted translocation.