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

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

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

Ultrasound imaging identifies life history variation in resident Cutthroat Trout.

Human activities that fragment fish habitat have isolated inland salmonid populations. This isolation is associated with loss of migratory life histories and declines in population density and abundance. Isolated populations exhibiting only resident life histories may be more likely to persist if individuals can increase lifetime reproductive success by maturing at smaller sizes or earlier ages. Therefore, accurate estimates of age and size at maturity across resident salmonid populations would improve estimates of population viability. Commonly used methods for assessing maturity such as dissection, endoscopy and hormone analysis are invasive and may disturb vulnerable populations. Ultrasound imaging is a non-invasive method that has been used to measure reproductive status across fish taxa. However, little research has assessed the accuracy of ultrasound for determining maturation status of small-bodied fish, or reproductive potential early in a species' reproductive cycle. To address these knowledge gaps, we tested whether ultrasound imaging could be used to identify maturing female Westslope Cutthroat Trout (Oncorhynchus clarkii lewisi). Our methods were accurate at identifying maturing females reared in a hatchery setting up to eight months prior to spawning, with error rates ≤ 4.0%; accuracy was greater for larger fish. We also imaged fish in a field setting to examine variation in the size of maturing females among six wild, resident populations of Westslope Cutthroat Trout in western Montana. The median size of maturing females varied significantly across populations. We observed oocyte development in females as small as 109 mm, which is smaller than previously documented for this species. Methods tested in this study will allow researchers and managers to collect information on reproductive status of small-bodied salmonids without disrupting fish during the breeding season. This information can help elucidate life history traits that promote persistence of isolated salmonid populations

List of species used for <i>in vitro</i> screening of the qPCR primers and probe.

<p>Origin refers to the waterbody for Pacific lamprey samples; origin is listed as state for all other samples.</p

Primer sequences for amplification and sequencing of the COI region of the Pacific lamprey mitochondrial genome; and sequences for components of the eDNA assay.

<p>Primer sequences for amplification and sequencing of the COI region of the Pacific lamprey mitochondrial genome; and sequences for components of the eDNA assay.</p

The Dual Challenges of Generality and Specificity When Developing Environmental DNA Markers for Species and Subspecies of <i>Oncorhynchus</i>

<div><p>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 (<i>Oncorhynchus clarkii lewsi</i>), Yellowstone cutthroat trout (<i>O</i>. <i>clarkii bouvieri</i>), and rainbow trout (<i>O</i>. <i>mykiss</i>), 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 (<i>O</i>. <i>c</i>. <i>utah</i>) 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.</p></div