The Genes Among Us: Molecular Methods for Species Monitoring and Biodiversity Surveys in Aquatic and Terrestrial Ecosystems

Molecular tools offer opportunities to non-invasively monitor species of conservation concern by identifying the DNA left in the environment (eDNA) through deposition of skin cells, mucus, gametes, excrement, or scat. Species detections using molecular genetic techniques (qPCR, ddPCR, sequencing, high-throughput sequencing) can be linked to a specific geographic region, a temporal window, or a specific population. These techniques are of particular interest for rare, threatened, or invasive species, which are difficult or costly to detect through traditional means. The first generation of this science has shown that DNA can be extracted from many different sources, including marine, freshwater, and terrestrial habitats, including scat. Attention can be focused on one focal species or on multiple species. This proposed symposium will focus on the use of molecular tools to aid in the detection of aquatic and terrestrial biodiversity. We propose a series of speakers whose research tackles a range of conservation questions using aqueous, terrestrial, and scat sampling. Our collection will highlight the diverse systems and questions that can be tackled with indirect, passive sampling techniques of the genetic material left behind. Our proposed symposium will provide information for environmental managers by quantitatively assessing population dynamics, gene flow, population connectivity, population distributions, and biodiversity. Several speakers will discuss the assumptions inherent in passive sampling of genetic material and the scope of inference allowable with lab and field studies. Our proposed list of presenters represent academic institutions, federal agencies, and institutes from the western United States, and cover a range of taxonomic expertise, including cetacean, amphibian, fish, invertebrate, and disease pathogen studies. The speakers will be grouped by the techniques they use and the systems they work in. Our symposium will attract attendees from both The Wildlife Society and American Fisheries Society due to the breadth of ecosystems and species covered.

8:00AM Oregon on the Cutting Edge: Creating the Foundation for 21st Century Molecular Monitoring
  Emily Dziedzic, Taal Levi, Richard Cronn, Brooke Penaluna, Jamie Anthony, Brian Sidlauskas
Rapid technological advancements and decreasing sequencing costs have sparked a genomics revolution enabling the development of molecular applications to monitor biodiversity and assess management policies. The Oregon Biodiversity Genome Project (OBGP) was established to develop genetic tools to appraise the distribution and ecology of regional taxa in order to better understand how ecosystems function and respond to management intervention. Such tools allow the rapid evaluation of policy impacts on surveilled species. Traditional methods to measure biodiversity cannot keep pace with biodiversity loss, and strides have been made to enhance our ability to monitor wildlife using molecular detection and diet analysis tools with environmental DNA. To develop these tools we need extensive genomic data, and OBGP has developed a pipeline to create a comprehensive georeferenced reference sequence database including full mitogenomic data for Oregon’s freshwater species and invertebrate pollinators. Preliminary results suggest that full mitochondrial genomes provide better taxonomic resolution for identifying species using molecular methods. These data are being made publicly available to facilitate molecular tool development for inland aquatic species and insect pollinators. Our tool development pipeline puts Oregon on the leading edge of molecular monitoring and can provide a template for other regions to expand their management capabilities.
8:20AM Casting a Broader Net: Using Microfluidic Metagenomics to Capture Aquatic Biodiversity Data from Diverse Taxonomic Targets
  Kevin Weitemier, Laura Hauck, Brooke Penaluna, Tiffany Garcia, Richard Cronn
Environmental DNA (eDNA) assays for single- and multi-species detection show promise for providing standardized assessment methods for diverse taxa, but techniques for evaluating multiple taxonomically-divergent assemblages are in their infancy. We evaluated whether microfluidic multiplex metabarcoding and high-throughput sequencing could identify diverse aquatic and riparian assemblages from 48 taxon-general and taxon-specific metabarcode primers. eDNA screening was paired with electrofishing along a stream continuum to evaluate congruence between methods. Microfluidic metabarcoding detected all 13 species observed by electrofishing, with overall accuracy of 86%. Taxon-specific barcoding primers were more successful than taxon-general universal metabarcoding primers at classifying sequences to species. Both types of markers detected a transition from downstream sites dominated by multiple fish species, to upstream sites dominated by a single species; however, we failed to detect a transition in amphibian population structure. Overall, we identified 878 predicted taxa, with most sequences (49.8%) derived from fish (Actinopteri, Petromyzontidae), Oomycetes (21.4%), Arthropoda (classes Insecta, Decapoda; 16.6%), and Apicomplexan parasites (3.83%). Our work shows that microfluidic metabarcoding can survey multiple phyla per assay, providing fine discrimination required to resolve closely-related species, and enable data-driven prioritization for multiple forest health objectives.
8:40AM Aquatic Biodiversity from a Bottle
  Brooke Penaluna
Aquatic biodiversity has long-been a proxy for assessing environmental change. Environmental DNA is revolutionizing how we can survey biodiversity in streams by offering a rapid and accurate assessment of multiple aquatic species from various taxa. We evaluate the detection of multiple aquatic species of fish, amphibians, crayfishes, mussels, macroinvertebrates, and pathogens using eDNA in neighboring streams and compare that with electrofishing. eDNA suggests more lineages of sculpins than expected by electrofishing from cryptic lineages across streams. eDNA can detect individuals to species before those individuals are developed enough for field identification (young-of-year O. mykiss). Pacific trout are detected further upstream with eDNA than electrofishing and freshwater mussels are found in streams collectively suggesting eDNA can detect rarer sequences associated with sparsely-distributed species. Amphibians are not detected consistently across primers within or across streams. Our approach of using multiple primer sets allows us to focus on different subsets of taxa and provides built-in redundancy of species detections, which are warranted to sample an aquatic community in a reasonably comprehensive way.
9:00AM Early Detection of Aquatic Invasive in Big Water Using Microfluidic Metagenomic Edna Sequencing
  Rebecca Flitcroft, Brooke Penaluna, James Capurso, Bruce Hansen, Richard Cronn
Public land management in the western U.S. targets ecological sustainability and the well-being of local communities. An important part of this mandate for range and forest managers is the conservation of aquatic habitats that support native species. One element of this work is vigilance in early detection and eradication of aquatic invasive species. To promote management of moving waters under federal jurisdiction, the Pacific Northwest Region (6) of the USDA Forest Service developed an Aquatic Invasive Species Monitoring Program in big water environments (i.e. lakes, reservoirs, navigable rivers). Microfluidic metagenomic eDNA sequencing is the tool chosen to accomplish the goals of this project. Questions regarding the utility of this method and its application in big water environments were explored to determine whether this tool would be useful in accomplishing the projects goals. Three specific objectives were explored: 1) Sequences and voucher specimens of focal invasive species in the Pacific Northwest were collected from the field and museums to develop taxon-specific primers. 2) Field experiments using varying amounts of water and pumping tools were completed in the field at places of known invasive species occurrence. 3) Sampling was completed throughout the summer field season to identify whether species detections changed temporally.
9:20AM Harnessing Genetics to Evaluate Stream Capture As a Contributor to Freshwater Biodiversity
  Karen Bobier, Byron J. Freeman, John Wares
The world’s freshwater habitats occupy less than 1% of the Earth’s surface, yet they are home to ~10% of all known species including 1/3 of vertebrate species. This over-representation of diversity in freshwater habitats has been generated by many biotic and abiotic mechanisms. Here, I am investigating the contribution to aquatic biodiversity by the geologic process known as stream capture or river piracy. Stream capture, usually the result of erosion and uplift, diverts the path of a stream from one river system to another. This process is a potential mechanism of dispersal and vicariance for freshwater organisms and may act as a driver of biodiversity. In this study I am focusing on the stream capture event that transferred the headwaters of the Chattahoochee River to the Savannah River system. By comparing genetic data for populations of multiple species that occur in both the Chattahoochee and Savannah Rivers, I can test for simultaneous divergence and assess the role of stream capture as a mechanism of dispersal and vicariance.
09:40AM Break
1:10PM A New Method for More Affordable and Effective Noninvasive Genetic Sampling Using High-Throughput SNP Genotyping
  Charlotte Eriksson, Joel Ruprecht, Taal Levi
Noninvasive genetic samples are particularly valuable for the study of rare or elusive species where other methods pose ethical and/or logistical concerns, however, the utility of these samples is challenged by low quantity and quality of DNA. Genotyping techniques using Single Nucleotide Polymorphisms (SNPs) have emerged as a promising alternative to traditional microsatellites by producing higher genotyping success and lower error rates. SNPs allow for amplification of shorter DNA fragments making them more suitable for genotyping degraded DNA. In addition, the bi-allelic nature of SNPs facilitates automated allele scoring and high-throughput genotyping, with results that are easily shared among research groups. We present a genotyping method based on next-generation sequencing of highly multiplexed PCR products targeting previously identified SNPs. The use of short and highly multiplexed amplicons combined with powerful sequencing systems such as the Illumina HiSeq facilitates cost-effective genotyping with high success rates using only standard laboratory equipment. We demonstrate the powerful utility of this method for individual identification by genotyping noninvasive samples and comparing to tissue samples from four different carnivores in Eastern Oregon. Our approach holds potential to address the critical need for an accurate, and affordable method of non-invasive genetic monitoring of elusive or wide-ranging species.
1:30PM Noninvasive Genetics of Aardvarks to Illuminate Landcape Connectivity and Phylogeographic History
  Clinton Epps, Rachel Crowhurst, Robert Spaan, Hannah Tavalire, Matthew Weldy
The aardvark (Orycteropus afer) occurs across much of sub-Saharan Africa and is the only extant member of Order Tubulidentata. Due to their cryptic behavior, space use is poorly understood, and dispersal, genetic structure, and phylogeographic history have not been evaluated. Here, we developed field and laboratory methods for non-invasive study of this keystone species. We used bioinformatics to identify prospective markers and optimized primers for 19 variable nuclear microsatellites and mitochondrial DNA sequence. We developed tracking methods to locate buried aardvark feces, enabling efficient non-invasive genetic sampling. We collected 274 genetic samples at 15 sites in eastern and southern Africa from 2016-2018; large reserves were sampled intensively. Nuclear genetic differentiation saturated at ~1000 km. Populations in eastern South Africa & Swaziland were strongly genetically differentiated from Kenya and populations in western South Africa, but even within western South Africa, multiple genetic clusters were identified. Individual re-detections were separated by <3.2 km. We used aardvark activity to develop a simple habitat model in one region, and evaluated whether that model predicted fine scale genetic structure. Although preliminary, our study is the first evaluation of genetic structure and gene flow for this species, and paves the way for a range-wide phylogeographic investigation.
1:50PM Biodiversity of Cottus in Western North America: A Molecular Perspective
  Michael Young, Rebecca Smith, Kristine Pilgrim, Kevin McKelvey, Michael K. Schwartz, Daniel Isaak, Sharon Parkes
The taxonomy of sculpins (Cottus, Cottidae) remains one of the last major unresolved puzzles in the systematics of North American freshwater fishes. Therefore, we began an effort to clarify the evolutionary history and taxonomy of sculpins from across western North America using molecular tools. First, we crowd-sourced collection of specimens (n = 7,673 and counting) via outreach to biologists in the western U.S. and Canada, and compiled the results of those collections on a webpage ( Second, we sequenced mitochondrial and nuclear genes of specimens from most basins in the West, and applied standard phylogenetic techniques to assess patterns of diversity. Those analyses revealed five discrete species complexes of inland sculpins, each composed of an array of lineages of which only some were accorded taxonomic recognition despite relatively deep phylogenetic divergence among most or all groups. Most lineages were specific to particular basins and allopatric with respect to other members of their species complex, but were commonly sympatric with members of other complexes. Coastal forms harbored much less diversity and tended to exhibit less divergence among lineages, although this was also evident in the most widely distributed of the inland forms.
2:10PM Identification and Screening of New Diagnostic SNPs for Detecting Hybridization and Introgression between Rainbow Trout and Yellowstone Cutthroat Trout
  Matthew Campbell, Thomas Delomas
Interspecies hybridization from non-native species can threaten the integrity and survival of native fish populations. If hybrids are fertile and comparable in fitness to the pure species, introgression can eliminate pure populations. Even when hybrids are sterile, hybridization can waste reproductive effort and increase competition for resources. These potential negative outcomes necessitate monitoring hybridization and introgression in native fish populations with the potential for interspecies hybridization. Detection of hybrid individuals can rapidly and accurately be performed through genetic analysis if diagnostic genetic markers can be identified. Single nucleotide polymorphisms (SNPs) are currently the marker of choice for high-throughput genotyping, and are increasingly being assayed through genotyping-by-sequencing of amplicon panels. We developed a bioinformatic pipeline that identifies new SNPs in existing amplicon panels and used it to identify diagnostic SNPs for assessing hybridization and introgression. From an existing rainbow trout Oncorhynchus mykiss panel containing 379 amplicons, we identified 28 SNPs diagnostic between rainbow trout and Yellowstone cutthroat trout O. clarkii bouvieri. We report on the screening of several Yellowstone cutthroat trout populations of particular management and conservation importance. We also describe how the bioinformatics pipeline we developed can be used cheaply and efficiently used to identify diagnostic SNPs in other taxa.
2:30PM Monitoring Anadromous Fish Population Dynamics Using Environmental DNA
  Taal Levi, Jennifer Allen, Meredith Pochardt, Ted Hart
The first generation of eDNA science has shown that DNA from organisms can be extracted from many different sources and identified taxonomically. The next generation will use this information for environmental management. We tested the ability of eDNA to produce useful quantitative information for monitoring spawning eulachon and Pacific salmon returning adults and outmigrating smolts. We quantified eDNA concentrations and stream flow contemporaneously with two years of salmon weir counts in Auke Creek, near Juneau, Alaska, and with five years of eulachon mark-recapture population estimates from the Chilkoot River near Haines, Alaska. We show that the daily flow-corrected eDNA rate (eDNA concentration x stream flow) closely tracks daily numbers of both returning and outmigrating salmon. Similarly, the peak and area-under-the-curve of the flow-corrected eDNA rate respectively explained over 90% of the variance in eulachon population estimates. eDNA thus promises accurate and efficient enumeration, but delivering the most robust numbers will need high-resolution stream-flow data, at-least-daily sampling, and a focus on species with simple life histories. Given adequate calibration, eDNA-based methods could be used on large spatial or long temporal scales to monitor fish populations at a fraction of the cost of traditional methods, particularly as eDNA sampling becomes automated.
2:50PM Refreshment Break
3:20PM Experimental Evaluation of Abundance Estimates Using Environmental DNA across Flow Regimes and Aquatic Taxonomic Groups.
  Christopher Cousins, Justin Sanders, Taal Levi, Jennifer Allen, Tiffany Garcia
The management and conservation applications of environmental DNA (eDNA) include confirming species occupancy and monitoring invasive or rare/endangered species. Recently, the technology has been extended to estimate population abundance, establishing a relationship between DNA concentration and counts of individuals in the system. Studies using eDNA for abundance estimates have found evidence that eDNA is more predictive of species abundance than biomass, suggesting that eDNA may be used to estimate animal counts. These studies have largely been done using mesocosms, which may not accurately represent results in lotic systems. To test these assumptions, and the effect of flow on eDNA abundance estimates, we designed two controlled laboratory experiments. The first tested the assumption that quantifiable eDNA amounts are more closely tied to numbers of individuals, using multiple size classes of American bullfrogs (Lithobates catesbeianus) held in static control water. The second tested the effects of flow rate on quantifiable eDNA concentrations, using varying numbers of Zebrafish (Danio rerio) across different flow regimes. Both studies highlight the complexities of estimating biomass using eDNA methodologies and help clarify the limitations of estimated small densities and high flow rates. These experiments test assumptions inherent in abundance estimates using eDNA, and how flow alters estimates.
3:40PM Designing Environmental DNA Surveys in Complex Lentic Systems: Backpack Sampling for Rare Amphibians in Sierra Nevada Meadows.
  Caren Goldberg, Karen Pope, Nicolette Nelson, Jonah Piovia-Scott
Environmental DNA (eDNA) detection of rare species is challenging in complex aquatic systems. In the summer of 2017, we conducted visual encounter surveys (VES) and used a backpack sampler (ANDe; Smith-Root) to collect concurrent eDNA water filter samples from 53 sites in 12 Sierra Nevada meadows. Sample volumes of 0.21-4 L took 1-17 minutes to collect. We analyzed samples using species-specific qPCR assays for Rana sierrae, R. cascadae, R. catesbeiana, and Pacifastacus leniusculus. A Bayesian multi-scale occupancy model estimated the probability of detecting R. sierrae or R. cascadae at meadows with VES detections to be 0.91. Within meadows, detection ranged from 0.47 when no frogs were seen nearby to 0.86 when at least one frog was seen in the vicinity. We detected R. catesbeiana and P. leniusculus at the one sampling site where each was expected to occur. There was some evidence that number of individuals detected using VES was related to target species eDNA quantity (p=0.070) but the explanatory power of this relationship was low (r2=0.071). Our results indicate that this method of eDNA sampling can be effective for detection of rare species in complex aquatic systems, outperforming VES at the foot of meadows and in off-channel pools.
4:00PM Using Environmental DNA to Validate the Occurrence and Habitat Associations of Torrent Salamanders to Inform ESA-Listing Decisions
  Lindsey Thurman, Deanna Olson, Christopher Cousins, Tiffany Garcia, Brooke Penaluna
We lack fundamental ecological information on many amphibian species, which is needed to more effectively identify the drivers of losses and ideally reduce the velocity of their declines. Torrent salamanders (Rhyacotriton sp.) are Northwest-US endemic, headwater-stream associated amphibian species that have a dearth of basic ecological information, but are thought to be highly vulnerable to habitat changes. This taxon currently has two candidates for listing by the Endangered Species Act: the Cascade torrent salamander (Rhyacotriton cascadae) and the Columbia torrent salamander (R. kezeri). Herein we address U.S. Fish & Wildlife Service pre-listing information needs for these species using a three-tiered approach. First, we developed a habitat suitability model for both species by compiling existing information on known occurrences (historical and contemporary) and habitat associations. Second, we field-validated and improved these habitat suitability models by using visual encounter surveys and environmental DNA (eDNA) inventory methods to evaluate occupancy and abundance across each species range. From this information, we will provide a more comprehensive projection of current and future distributions for these torrent salamanders given projections of environmental and climatic changes.
4:20PM Adding Early-Detection Surveillance for Invasive Mussels to USGS Stream Gaging Networks
  Adam J. Sepulveda, Christian Schmidt, Jon J. Amberg, Patrick Hutchins, Christian Stratton, Chris Mebane, Matthew Laramie, David Pilliod
A critical tactic in minimizing invasive species costs is the development of effective, early-detection systems. To this end, we evaluated the efficacy of adding environmental (e)DNA surveillance to the U.S. Geological Survey (USGS) streamgage network, which consists of >10,000 streamgages systemically visited by hydrological technicians. Incorporating eDNA sample collection during routine streamgage visits could provide early detection of invasive species with minimal additional cost. Hydrological technicians collected monthly eDNA water samples from streamgages downstream of Columbia River Basin reservoirs thought to be vulnerable to invasive dreissenid mussels. We tested water samples for dreissenid mussel DNA and also for kokanee and yellow perch DNA, which allowed us to assess if streamgages are adequately located to provide eDNA surveillance of taxa known to be present in upstream reservoirs. Two streamgage locations yielded unverified, positive detections for dreissenid DNA. We also detected kokanee and yellow perch DNA at streamgages downstream of reservoirs where these fish occur. Field collection, lab analyses, and personnel time required for collection of three eDNA samples at a streamgage cost US$400-$500. Given these results, incorporating eDNA biosurveillance into routine streamgage visits might decrease costs associated with an invasion since early detection maximizes the potential for eradication, control, and mitigation.
4:40PM Developing an Environmental DNA (eDNA) Filter That Is Self-Preserving and Biodegradable
  Austen Thomas, Phong L. Nguyen, Jesse Howard, Caren Goldberg
Environmental DNA studies often rely on water filtration in the field and immediate sample preservation to prevent DNA degradation during sample transport. However, filter membrane transfer steps for preservation can increase risk of sample contamination, and the reliance on single-use filter housings produces significant plastic waste. We therefore created an eDNA filter housing (compatible with any suction pump) comprised of a biodegradable, hydrophilic plastic that functions to automatically preserve captured eDNA via desiccation – no filter membrane transfer steps, and no chemical or cold storage required. We tested the self-preservation capabilities of the filters in a mesocosm study with New Zealand mudsnails and compared with ethanol preservation. Quantitative PCR results indicated no significant difference in eDNA quantity between the two preservation treatments over a 2-month storage period (F1,26 = 1.878, p= 0.182). The average eDNA quantity (SQ) for self-preserved filters was slightly higher (319 copies) than ethanol-preserved samples (290 copies). These data indicate that self-preserving eDNA filter housings are a viable alternative to standard ethanol preservation methods. The new filter housings also help to reduce the risk of sample contamination, minimizes protocol steps, and result in less plastic waste.

Organizers: Brooke Penaluna, Tiffany Garcia

Location: Reno-Sparks CC Date: September 30, 2019 Time: 8:00 am - 5:20 pm