Environmental DNA for conservation

Biodiversity must be documented before it can be conserved. However, it may be difficult to document species with few individuals (Thompson, 2013; Goldberg et al., 2016), thus it requires a multitude of tools to detect species that occur in low numbers or are elusive (see the various chapters in this volume). One tool that has become useful for conservation efforts utilizes environmental DNA, which is DNA shed into the environment by organisms (eDNA; Taberlet et al., 2018). Typically this involves taking environmental samples such as soil, water, air, or using biological surrogates for sampling biodiversity (e.g. leeches, sponges, carrion flies, etc.; Schnell et al., 2012; Calvignac-Spencer et al., 2013; Lynggaard et al., 2019; Mariani et al., 2019) and using laboratory approaches to concentrate, isolate, and test for target DNA through polymerase chain reaction (PCR) amplification (Taberlet et al., 2018). The utilization of eDNA for species detection is part of a larger field of non-invasive DNA sampling, which more broadly includes collecting DNA passively from wildlife, through collection of faeces, saliva, feathers, hair, or other methods of sampling shed DNA. Environmental DNA has been used to document presence/absence of a target species (Ficetola et al., 2008a, 2008b; Himter et al., 2017) or to quantify relative abundance for biodiversity from varied environments such as the arctic (e.g. Leduc et al., 2019; Von Duyke et al., 2019), marine (e.g. Port et al., 2016; Jo et al, 2017; Stoeckle et al., 2018), freshwater (e.g. Lacoursi^re-Roussel et al., 2016; Doi et al., 2017), and tropical (e.g. Schnell et al., 2012; Gogarten et al, 2020) ecosystems. The application of this technology includes the detection of invasive species, pathogens (including DNA and RNA), species of conservation concern, and biodiversity (Acevedo-Whitehouse et al., 2010; Rees et al., 2014; Sakai et al, 2019). In this world -of \u27fast-paced technological advances, not all new methods prove useful in an applied context. Although eDNA has not been used regularly in biodiversity conservation for more than a decade, it has proven to be an extremely practical and informative tool. The utility of eDNA is supported by ongoing advancements and development of novel applications. There is no easy way to standardize the application or methods of eDNA as the conservation question, and the target system must drive the selection of a range of options at every step. However, guidelines now exist for the best practices of optimizing a sampling scheme and sample processing for eDNA applications (Goldberg et al., 2016; Jeunen et al., 2019; Klymus et al., 2020; The eDNA Society, 2019; Shu et al, 2020). Further, the ranks of experienced eDNA practitioners have expanded globally; thus, it is fairly easy to find expert consultation. Therefore, it is now practical and prudent to adopt eDNA in the service of biodiversity conservation efforts

Assessing the potential impacts of a changing climate on the distribution of a rabies virus vector

Common vampire bats (Desmodus rotundus) occur throughout much of South America to northern MeÂxico. Vampire bats have not been documented in recent history in the United States, but have been documented within about 50 km of the U.S. state of Texas. Vampire bats feed regularly on the blood of mammals and can transmit rabies virus to native species and livestock, causing impacts on the health of prey. Thus cattle producers, wildlife management agencies, and other stakeholders have expressed concerns about whether vampire bats might spread into the southern United States. On the other hand, concerns about vampire- borne rabies can also result in wanton destruction at bat roosts in areas occupied by vampire bats, but also in areas not known to be occupied by this species. This can in turn negatively affect some bat roosts, populations, and species that are of conservation concern, including vampire bats. To better understand the current and possible future distribution of vampire bats in North America and help mitigate future cattle management problems, we used 7,094 vampire bat occurrence records from North America and species distribution modeling (SDM) to map the potential distribution of vampire bats in North America under current and future climate change scenarios. We analysed and mapped the potential distribution of this species using 5 approaches to species distribution modeling: logistic regression, multivariate adaptive regression splines, boosted regression trees, random forest, and maximum entropy. We then projected these models into 17 ªworst-caseº future climate scenarios for year 2070 to generate hypotheses about how the vampire bat distribution in North America might change in the future. Of the variables used in this analysis, minimum temperature of the coldest month had the highest variable importance using all 5 SDM approaches. These results suggest two potential near-future routes of vampire bat dispersal into the U. S., one via southern Texas, and a second into southern Florida. Some of our SDM models support the hypothesis that suitable habitat for vampire bats may currently exist in parts of the Mexico-U.S. borderlands, including extreme southern portions of Texas, as well as in southern Florida. However, this analysis also suggests that extensive expansion into the south-eastern and south-western U.S. over the coming ~60 years appears unlikely

No filters, no fridges: a method for preservation of water samples for eDNA analysis

Background: Advancements in the detection of environmental DNA (eDNA) for detecting species of interest will likely allow for expanded use of these techniques in the field. One obstacle that continues to hinder applications in the field is the requirement of a cold chain of storage for water samples containing eDNA. While eDNA has been successfully preserved using Longmire’s lysis buffer applied to filters, it has yet to be tried with freshwater samples collected for eDNA detection of an invasive species. We tested the utility of Longmire’s solution (100 mM Tris, 100 mM EDTA, 10 mM NaCl, 0.5 % SDS, 0.2 % sodium azide) as an additive to freshwater samples for preservation of eDNA. Results: Environmental DNA was effectively preserved in 15 mL water samples with Longmire’s solution added; eDNA positive detection was comparable to freezing the samples at −80 °C and occurred out to 56 days at the highest concentration (5 mL Longmire’s solution: 15 mL sample water). Medium and low concentrations of Longmire’s solution added to 15 mL of sample water generally preserved eDNA out to 56 days but not as well as did freezing or application of the highest concentration of Longmire’s lysis buffer. Treatment and degradation time had a significant effect on average DNA concentration of samples, although not the interaction of treatment and time. Perfect detection occurred out to 56 days with the high Longmire’s treatment group but DNA concentration was significantly lower at this time point compared to 28 days. Conclusion: We conclude that Longmire’s lysis buffer is a viable alternative to cold chain storage that can simplify the collection of eDNA by eliminating the need for filtering and allow more time for sample collection when added at our highest concentration (1 part Longmire’s:3 parts water sample), which could translate to an increase in the chances of detecting a rare or elusive species

PHYLOGENETIC AND POPULATION GENETIC ASSESSMENT OF RAFINESQUE’S BIG-EARED BAT (\u3ci\u3eCORYNORHINUS RAFINESQUII\u3c/i\u3e)

Rafinesque’s big-eared bat (Corynorhinus rafinesquii) is distributed across the Southeastern United States. Due to habitat loss and low population numbers, this species is a Federal species of concern and protected by every State within its range. Effective management of any species of concern is dependent on an unambiguous understanding of taxonomic relationships. However, for this species, there are discordant inferences about subspecific designations from previous studies. Further, there have been no assessments of population genetic status for this species. Such assessments could provide information on genetic diversity and population connectivity and increase our understanding of the need for management and conservation of this species. Therefore, our goals were to assess population level genetic diversity and connectivity among 5 colonies in Arkansas (139 individuals) and to infer the evolutionary relationships of these bats to C. rafinesquii collected across its distribution (additional 216 individuals). We used mitochondrial DNA control region sequences and 11 microsatellite loci to infer genetic relationships, estimate levels of genetic diversity, and examine population connectivity among 5 colonies in Arkansas. Although we identified two phylogenetically divergent mitochondrial DNA lineages, these correspond to neither current subspecific designation nor nonoverlapping geographical groups. Genetic diversity and population connectivity estimated from mitochondrial DNA was high in Arkansas populations probably due to occurrence of both evolutionary lineages within each colony. However, estimates from microsatellite DNA of genetic diversity, population connectivity, and effective population sizes in these populations were low. Further, our results suggested a weak signal of population bottleneck in Arkansas colonies and low genetic connectivity. Current conservation efforts should continue to focus on protection of roosts and improvement of habitat corridors to connect populations

Using DNA to Identify the Source of Invasive Mongooses, \u3ci\u3eHerpestes auropunctatus\u3c/i\u3e (Carnivora: Herpestidae) Captured on Kaua‘i, Hawaiian Islands

Two small Indian mongooses (Herpestes auropunctatus) were live-captured in 2012 at separate locations on the Hawaiian Island of Kaua\u27i, which was previously considered to be free of this invasive species. Genotypes from these two individuals were compared to genotypes of H. auropunctatus from the islands of Hawai\u27i (n =39), O\u27ahu (n =91), Maui (n = 39), and Moloka\u27i (n = 19) to determine the island of origin of the Kaua\u27i individuals. Genotypes were generated from each individual using five microsatellite loci. Genetic clustering was estimated by Bayesian inference of spatial clustering of individuals and clustering of groups of individuals. Both analyses separated the samples into three distinct genetic clusters (K = 3). Kaua\u27i individuals consistently formed a single cluster with individuals from O\u27ahu, whereas Hawai\u27i and Maui formed a second cluster, and Moloka\u27i was the third cluster. Thus, we conclude that the origin of two H. auropunctatus captured on Kaua\u27I was O\u27ahu. All three genetic clusters showed evidence of transportation of mongooses between islands, indicating that sampled islands in the archipelago are capable of acting as both donors and receivers of mongooses

Exploring the value of a global gene drive project registry

Recent calls to establish a global project registry before releasing any gene-drive-modified organisms (GDOs) have suggested a registry could be valuable to coordinate research, collect data to monitor and evaluate potential ecological impacts, and facilitate transparent communication with community stakeholders and the general public. Here, we report the results of a multidisciplinary expert workshop on GDO registries convened on 8–9 December 2020 involving 70 participants from 14 countries. Participants had expertise in gene drive design, conservation and population modeling, social science, stakeholder engagement, governance and regulation, international policy, and vector control; they represented 45 organizations, spanning national and local governmental agencies, international organizations, nonprofit organizations, universities, and district offices overseeing local vector control. The workshop aimed to gather perspectives on a central question: “In what ways could a gene-drive project registry both contribute to and detract from the fair development, testing and use of GDOs?” We specifically queried the perceived purpose of a registry, the information that would need to be included, and the perceived value of a registry. Three primary findings emerged from the discussion: first, many participants agreed a registry could serve a coordinating function for multidisciplinary and multisector work activities; second, doing so may require different design elements, depending on the target end-user group and intended purpose for that group; and third, these different information requirements lead to concerns about information sharing via a registry, suggesting potential obstacles to achieving transparency through such a mechanism. We conclude that any development of a gene-drive project registry requires careful and inclusive deliberation, including with potential end-users, to ensure that registry design is optimal

Using Genetics to Assess Differentiation Among Raccoons in an Area with Variable Rabies Status in Alabama

The western spread of raccoon rabies in Alabama has been slow and even appears to regress eastward periodically. While the disease has been present in the state for over 30 years, areas in northwest Alabama are devoid of raccoon rabies. This variation resulting in an enzootic area of raccoon rabies primarily in southeastern Alabama may be due to landscape features that hinder the movement of raccoons (i.e., gene flow) among different locations. We used 11 raccoon-specific microsatellite markers to obtain individual genotypes to examine gene flow among areas that were rabies free, enzootic with rabies, or had only sporadic reports of the disease. Samples from 70 individuals were collected from 5 sampling localities in 3 counties. The landscape feature data were collected from geographic information system (GIS) data. We inferred gene flow by estimating FST and by using Bayesian tests to identify genetic clusters. Estimates of pairwise FST indicated genetic differentiation and restricted gene flow between some sites, and an uneven distribution of genetic clusters was observed. Of the landscape features examined (i.e., land cover, elevation, slope, roads, and hydrology), only land cover had an association with genetic differentiation, suggesting this landscape variable may affect gene flow among raccoon populations and thus the spread of raccoon variant of rabies in Alabama

Picky eaters are rare: DNA-based blood meal analysis of \u3ci\u3eCulicoides\u3c/i\u3e (Diptera: Ceratopogonidae) species from the United States

Background: Biting midges in the genus Culicoides (Diptera; Ceratopogonidae) have been implicated in the transmission of a number of parasites and highly pathogenic viruses. In North America, the complete transmission cycles of many of these pathogens need further elucidation. One way to increase our knowledge about the evolution and ecology of Culicoides species and the pathogens they transmit is to document the diversity of vertebrate hosts that Culicoides feed upon. Our objective was to identify the diversity of Culicoides hosts in the United States. Results: We sequenced two vertebrate mitochondrial genes (cytochrome c oxidase subunit 1 and cytochrome b) from blood-engorged Culicoides to identify Culicoides species and their blood meals. We detected the mitochondrial DNA of 12 host species from seven different Culicoides species from three states. The majority of the identified blood meals were from the C. variipennis species complex in California. The hosts included both mammals and birds. We documented new host records for some of the Culicoides species collected. The majority of the mammalian hosts were large ungulate species but we also detected a lagomorph and a carnivore. The bird species that were detected included house finch and emu; the latter is evidence that the species in the C. variipennis species complex are not strictly mammalophilic. Conclusions: These results demonstrate that Culicoides will feed on multiple classes of vertebrates and may be more opportunistic in regards to host choice than previously thought. This knowledge can help with identification of susceptible host species, pathogen reservoirs, and new vector species which, in turn, will improve disease outbreak risk assessments

A molecular analysis to assess codling moth \u3ci\u3eCydia pomonella\u3c/i\u3e L. (Lepidoptera: Tortricidae) predation by orchard birds

The codling moth Cydia pomonella L. (Lepidoptera: Tortricidae) is a major economic pest in organic apple orchards. Observational methods, prey removal experiments and correlative experiments with exclosures or nest boxes have demonstrated that birds contribute to the removal of this insect pest. However, the majority of research conducted in the last several decades has taken place outside of the United States and methods for studying biological pest control have advanced dramatically and now include molecular techniques. We conducted a proof-of-concept study to test a DNA-based approach to detect C. pomonella prey in the diets of birds occupying organic apple orchards. We tested published Tortricidae primers, polymerase chain reaction (PCR) and sequencing for detection of C. pomonella in avian feces. We also tested the quality of DNA isolated and purified from fecal samples using two DNA extraction kits. Finally, we field-tested this tool to identify the presence or absence of C. pomonella in the laboratory and from field samples. C. pomonella DNA was amplified in less than 1% of field samples and was successfully sequenced in 0.5%. A single species, the brown-headed cowbird Molothrus ater (Boddaert), had fecal samples positive for C. pomonella DNA. While our results do not provide evidence that birds play a strong role in the control of C. pomonella in western Colorado organic apple orchards, the approach we present is a new tool for understanding bird-mediated ecosystem services, avian feeding ecology, and supporting management decisions for sustainable agricultural practices and farmland biodiversity

Rooting Out Genetic Structure of Invasive Wild Pigs in Texas

Invasive wild pigs (Sus scrofa), also called feral swine or wild hogs, are recognized as among the most destructive invasive species in the world. Throughout the United States, invasive wild pigs have expanded rapidly over the past 40 years with populations now established in 38 states. Of the estimated 6.9 million wild pigs distributed throughout the United States, Texas supports approximately 40% of the population and similarly bears disproportionate ecological and economic costs. Genetic analyses are an effective tool for understanding invasion pathways and tracking dispersal of invasive species such as wild pigs and have been used recently in California and Florida, USA, which have similarly long-established populations and high densities of wild pigs. Our goals were to use molecular approaches to elucidate invasion and migration processes shaping wild pig populations throughout Texas, compare our results with patterns of genetic structure observed in California and Florida, and provide insights for effective management of this invasive species. We used a high-density single nucleotide polymorphism (SNP) array to evaluate population genetic structure. Genetic clusters of wild pigs throughout Texas demonstrate 2 distinct patterns: weakly resolved, spatially dispersed clusters and well-resolved, spatially localized clusters. The disparity in patterns of genetic structure suggests disparate processes are differentially shaping wild pig populations in various localities throughout the state. Our results differed from the patterns of genetic structure observed in California and Florida, which were characterized by localized genetic clusters. These differences suggest distinct biological and perhaps anthropogenic processes are shaping genetic structure in Texas. Further, these disparities demonstrate the need for location-specific management strategies for controlling wild pig populations and mitigating associated ecological and economic costs