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Genetic landscapes GIS Toolbox: Tools to map patterns of genetic divergence and diversity.

The Landscape Genetics GIS Toolbox contains tools that run in the Geographic Information System software, ArcGIS ® , to map genetic landscapes and to summarize multiple genetic landscapes as average and variance surfaces. These tools can be used to visualize the distribution of genetic diversity across geographic space and to study associations between patterns of genetic diversity and geographic features or other geo‐referenced environmental data sets. Together, these tools create genetic landscape surfaces directly from tables containing genetic distance or diversity data and sample location coordinates, greatly reducing the complexity of building and analyzing these raster surfaces in a Geographic Information System.

Molecular Ecology Resources

A noninvasive, direct real-time PCR method for sex determination in multiple avian species

Polymerase chain reaction (PCR)-based methods to determine the sex of birds are well established and have seen few modifications since they were first introduced in the 1990s. Although these methods allowed for sex determination in species that were previously difficult to analyse, they were not conducive to high-throughput analysis because of the laboriousness of DNA extraction and gel electrophoresis. We developed a high-throughput real-time PCR-based method for analysis of sex in birds, which uses noninvasive sample collection and avoids DNA extraction and gel electrophoresis.

Molecular Ecology Resources

GONe: Software for estimating effective population size in species with generational overlap

GONe is a user‐friendly, Windows‐based program for estimating effective size (N e ) in populations with overlapping generations. It uses the Jorde–Ryman modification to the temporal method to account for age structure in populations. This method requires estimates of age‐specific survival and birth rate and allele frequencies measured in two or more consecutive cohorts. Allele frequencies are acquired by reading in genotypic data from files formatted for either GENEPOP or TEMPOFS. For each interval between consecutive cohorts, Ne is estimated at each locus and over all loci. Furthermore, Ne estimates are output for three different genetic drift estimators ( F s , F c and F k ). Confidence intervals are derived from a chi‐square distribution with degrees of freedom equal to the number of independent alleles. GONe has been validated over a wide range of Ne values, and for scenarios where survival and birth rates differ between sexes, sex ratios are unequal and reproductive variances differ.

Molecular Ecology Resources

Gene transcription in sea otters (Enhydra lutris); development of a diagnostic tool for sea otter and ecosystem health

Gene transcription analysis for diagnosing or monitoring wildlife health requires the ability to distinguish pathophysiological change from natural variation. Herein, we describe methodology for the development of quantitative real-time polymerase chain reaction (qPCR) assays to measure differential transcript levels of multiple immune function genes in the sea otter ( Enhydra lutris ); sea otter-specific qPCR primer sequences for the genes of interest are defined. We establish a ‘reference’ range of transcripts for each gene in a group of clinically healthy captive and free-ranging sea otters. The 10 genes of interest represent multiple physiological systems that play a role in immuno-modulation, inflammation, cell protection, tumour suppression, cellular stress response, xenobiotic metabolizing enzymes, antioxidant enzymes and cell–cell adhesion. The cycle threshold (C T ) measures for most genes were normally distributed; the complement cytolysis inhibitor was the exception. The relative enumeration of multiple gene transcripts in simple peripheral blood samples expands the diagnostic capability currently available to assess the health of sea otters in situ and provides a better understanding of the state of their environment.

Molecular Ecology Resources

Novel primers for complete mitochondrial cytochrome b genesequencing in mammals

Sequence-based species identification relies on the extent and integrity of sequence data available in online databases such as GenBank. When identifying species from a sample of unknown origin, partial DNA sequences obtained from the sample are aligned against existing sequences in databases. When the sequence from the matching species is not present in the database, high-scoring alignments with closely related sequences might produce unreliable results on species identity. For species identification in mammals, the cytochrome b (cyt b ) gene has been identified to be highly informative; thus, large amounts of reference sequence data from the cyt b gene are much needed. To enhance availability of cyt b gene sequence data on a large number of mammalian species in GenBank and other such publicly accessible online databases, we identified a primer pair for complete cyt b gene sequencing in mammals. Using this primer pair, we successfully PCR amplified and sequenced the complete cyt b gene from 40 of 44 mammalian species representing 10 orders of mammals. We submitted 40 complete, correctly annotated, cyt b protein coding sequences to GenBank. To our knowledge, this is the first single primer pair to amplify the complete cyt b gene in a broad range of mammalian species. This primer pair can be used for the addition of new cyt b gene sequences and to enhance data available on species represented in GenBank. The availability of novel and complete gene sequences as high-quality reference data can improve the reliability of sequence-based species identification.

Molecular Ecology Resources

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These crossspecies microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.

Molecular Ecology Resources

Molecular genetics at the Fort Collins Science Center

The Fort Collins Science Center operates a molecular genetic and systematics research facility (FORT Molecular Ecology Laboratory) that uses molecular genetic tools to provide genetic information needed to inform natural resource management decisions. For many wildlife species, the data generated have become increasingly important in the development of their long-term management strategies, leading to a better understanding of species diversity, population dynamics and ecology, and future conservation and management needs. The Molecular Ecology Lab serves Federal research and resource management agencies by developing scientifically rigorous research programs using nuclear, mitochondrial and chloroplast DNA to help address many of today's conservation biology and natural resource management issues.

Fact Sheet

Genetic tools for wildlife management

Granted interim status in November, 2013, The Wildlife Society’s (TWS) Molecular Ecology Working Group aims to promote scientific advancement by applying molecular techniques to wildlife ecology, management, and conservation. The working group—composed of sci - entists from diverse backgrounds—met for the first time in Pittsburgh at the TWS Annual Conference held in October. Our overarching goal is to enhance awareness of molecular ecology and genetic applica - tions to wildlife biology and act as an informational and networking resource. During the group’s interim status, which runs for three years, we intend to focus on a broad scope of molecular ecology that is applicable to wildlife including genetic and ge - nomic methods, conservation genetics, non-invasive genetic population monitoring, landscape genetics, evolutionary genetics, and molecular forensics

The Wildlife Professional

Successful molecular detection studies require clear communication among diverse research partners

Molecular detection techniques are powerful tools used in ecological applications ranging from diet analyses to pathogen surveillance. Research partnerships that use these tools often involve collaboration among professionals with expertise in field biology, laboratory techniques, quantitative modeling, wildlife disease, and natural resource management. However, in many cases, each of these collaborators lacks specific knowledge about the approaches, decisions, methods, and terminology used by their research partners, which can impede effective communication and act as a barrier to the efficient use of molecular data for ecological inferences and subsequent conservation decision making. We outline a collaborative framework to assist colleagues with diverse types of expertise to effectively translate their scientific and management needs to research partners from other specialties. The molecular techniques used to detect organisms will continue to advance both in sophistication and in the breadth of ecological applications. Our objective is to enable ecologists to harness the full utility of these methods by developing effective collaborative partnerships.

Frontiers in Ecology and the Environment

Microbial ecology of permafrost soils: Populations, processes, and perspectives

Permafrost microbial research has flourished in the past decades, due in part to improvements in sampling and molecular techniques, but also the increased focus on the permafrost greenhouse gas feedback to climate change and other ecological processes in high latitude and alpine permafrost soils. Permafrost microorganisms are adapted to these extreme environments and remain active at low temperatures and when resources are limited. They are also an important component of global elemental cycles as they regulate organic matter turnover and greenhouse gas production, particularly as permafrost thaws. Here we review the permafrost microbiology literature coupled with an exploration of its historical aspects, with a particular focus on a new understanding advanced by molecular biology techniques. We further identify knowledge gaps and ways forward to improve our understanding of microbial contributions to ecosystem biogeochemistry of permafrost-affected systems.

Permafrost and Periglacial Processes

Using biological responses to monitor freshwater post-spill conditions over 3 years in Blacktail Creek, North Dakota, USA

A pipeline carrying unconventional oil and gas (OG) wastewater spilled approximately 11 million liters of wastewater into Blacktail Creek, North Dakota, USA. Flow of the mix of stream water and wastewater down the channel resulted in storage of contaminants in the hyporheic zone and along the banks, providing a long-term source of wastewater constituents to the stream. A multi-level investigation was used to assess the potential effects of oil and brine spills on aquatic life. In this study, we used a combination of experiments using a native fish species, Fathead Minnow ( Pimephales promelas ), field sampling of the microbial community structure, and measures of estrogenicity. The fish investigation included in situ experiments and experiments with collected site water. Estrogenicity was measured in collected site water samples, and microbial community analyses were conducted on collected sediments. During the initial post-spill investigation, February 2015, performing in situ fish bioassays was impossible because of ice conditions. However, microbial community (e.g., the presence of members of the Halomonadaceae, a family that is indicative of elevated salinity) and estrogenicity differences were compared to reference sites and point to early biological effects of the spill. We noted water column effects on in situ fish survival 6 months post-spill during June 2015. At that time, total dissolved ammonium (sum of ammonium and ammonia, TAN) was 4.41 mg NH 4 /L with an associated NH 3 of 1.09 mg/L, a concentration greater than the water quality criteria established to protect aquatic life. Biological measurements in the sediment defined early and long-lasting effects of the spill on aquatic resources. The microbial community structure was affected during all sampling events. Therefore, sediment may act as a sink for constituents spilled and as such provide an indication of continued and cumulative effects post-spill. However, lack of later water column effects may reflect pulse hyporheic flow of ammonia from shallow ground water. Combining fish toxicological, microbial community structure and estrogenicity information provides a complete ecological investigation that defines potential influences of contaminants at organismal, population, and community levels. In general, in situ bioassays have implications for the individual survival and changes at the population level, microbial community structure defines potential changes at the community level, and estrogenicity measurements define changes at the individual and molecular level. By understanding effects at these various levels of biological organization, natural resource managers can interpret how a course of action, especially for remediation/restoration, might affect a larger group of organisms in the system. The current work also reviews potential effects of additional constituents defined during chemistry investigations on aquatic resources.

North Dakota

Coevolution with host fishes shapes parasitic life histories in a group of freshwater mussels (Unionidae: Quadrulini)

Ecological interactions among species often lead to parasitic lineages coevolving with host resources, which is often suggested as the primary driver of parasite diversification. Freshwater mussels are bivalves that possess a parasitic life cycle requiring larval encystment on freshwater vertebrates to complete metamorphosis. The North American freshwater mussel tribe Quadrulini has a suite of life history adaptations including highly specialized patterns of host use, infection strategies, and variable larval morphologies. However, the evolution of life histories has yet to be explored using phylogenetic comparative methods. In this study, we use a holistic approach incorporating biogeographical, ecological, molecular, and morphological datasets to reconstruct the evolution of Quadrulini. Comparative phylogenetic analyses suggested the diversification of Quadrulini has been driven, at least in part, by codiversification with their primary host fishes in Ictaluridae. Major diversification events in both ictalurids and quadrulines were estimated to have occurred in the Mississippi River basin throughout the Miocene. Life history characteristics associated with parasitism were supported to have coevolved with host repertories, supporting the hypothesis that ecological interactions with host fishes have shaped the evolution of highly specialized traits in this group. Our findings demonstrate the importance of ecological interactions with host resources in shaping the evolutionary history of freshwater mussels.

Bulletin of the Society of Systematic Biologists

Transcriptome assembly and differential gene expression of the invasive avian malaria parasite Plasmodium relictum in Hawaiʻi

The malaria parasite Plasmodium relictum (lineage GRW4) was introduced less than a century ago to the native avifauna of Hawaiʻi, where it has since caused major declines of endemic bird populations. One of the native bird species that is frequently infected with GRW4 is the Hawaiʻi ʻamakihi ( Chlorodrepanis virens ). To achieve a better understanding of the transcriptional activities of this virulent parasite, we performed a controlled challenge experiment of 15 ʻamakihi that were infected with GRW4. Blood samples containing malaria parasites were collected at two time points (intermediate and peak infection stages) from host individuals that were either experimentally infected by mosquitoes or inoculated with infected blood. We then used RNA sequencing to assemble a high‐quality blood transcriptome of P. relictum GRW4, allowing us to quantify parasite expression levels inside individual birds. We found few significant differences (one to two transcripts) in GRW4 expression levels between host infection stages and between inoculation methods. However, 36 transcripts showed differential expression levels among all host individuals, indicating a potential presence of host‐specific gene regulation across hosts. To reduce the extinction risk of the remaining native bird species in Hawaiʻi, genetic resources of the local Plasmodium lineage are needed to enable further molecular characterization of this parasite. Our newly built Hawaiian GRW4 transcriptome assembly, together with analyses of the parasite's transcriptional activities inside the blood of Hawaiʻi ʻamakihi, can provide us with important knowledge on how to combat this deadly avian disease in the future.

Hawaii

Trust Species and Habitat Branch: using the innovative approaches of today to conserve biodiversity for tomorrow

Some of the biggest challenges facing wildlife today are changes to their environment from both natural and anthropogenic causes. Natural resource managers, planners, policy makers, industry and private landowners must make informed decisions and policies regarding management, conservation, and restoration of species, habitats, and ecosystem function in response to these changes. Specific needs include (1) a better understanding of population status and trends; (2) understanding of species’ habitat needs and roles in supporting ecosystem functions; (3) the ability to assess species’ responses to environmental changes and predict future responses; and (4) the development of innovative techniques and tools to better understand, minimize or prevent any unintended consequences of environmental change. The Trust Species and Habitats Branch of the Fort Collins Science Center includes a diverse group of scientists encompassing both traditional and specialized expertise in wildlife biology, ecosystem ecology, quantitative ecology, disease ecology, molecular genetics, and stable isotope geochemistry. Using our expertise and collaborating with others around the world, our goal is to provide the information, tools, and technologies that our partners need to support conservation, management, and restoration of terrestrial vertebrate populations, habitats, and ecosystem function in a changing world.

Fact Sheet

A guide to environmental DNA extractions for non-molecular trained biologists, ecologists, and conservation scientists

Ecologists, biologists, and conservation scientists are increasingly interested in the use of environmental DNA (eDNA) data for research and potentially decision-making. While commercial DNA extraction kits are typically user-friendly and accessible, they may fail to deliver the desired results with inherently complex eDNA samples, necessitating protocol optimization or educated selection of alternative approaches. To this end, knowledge of the basic steps and principles of DNA extractions is essential, but traditional education tracks in ecology, conservation, and environmental management typically do not include in-depth training in molecular methods. The primary objective of this paper is to enable scientists with an ecological background and limited molecular training to understand the four key steps of eDNA isolations, and to use this expertise to their advantage. We describe the purpose of commonly used reagents and chemicals, point out alternatives for each key step, explain the impact of certain choices regarding isolation approaches on DNA integrity and purity, and highlight the possibility of a tailor-made “mix and match” approach. We anticipate that this paper will enable field ecologists to develop a deeper understanding of the mechanisms and chemistry underlying eDNA extractions, thus allowing them to make informed decisions regarding the best eDNA extraction method for their research goals. Our intention is not to provide comprehensive, step-by-step protocols, but to offer guiding principles while highlighting alternative solutions. Finally, we hope that this paper will act as a useful resource to support knowledge transfer and teaching.

Environmental DNA

Refinement of eDNA as an early monitoring tool at the landscape-level: Study design considerations

Natural resource managers use data on the spatial range of species to guide management decisions. These data come from survey or monitoring efforts that use a wide variety of tools. Environmental DNA (eDNA) is a surveillance tool that uses genetic markers for detecting species and holds potential as a tool for large-scale monitoring programs. Two challenges of eDNA-based studies are uncertainties created by imperfect capture of eDNA in collection samples (e.g., water field samples) and imperfect detection of eDNA using molecular methods (e.g., quantitative PCR). Occurrence models can be used to address these challenges, thus we use an occurrence model to address two objectives: First, determine how many samples were required to detect species using eDNA; Second, examine when and where to take samples. We collected water samples from three different habitat types in the Upper Mississippi River when both Bighead Carp and Silver Carp were known to be present based on telemetry detections. Each habitat type (backwater, tributary, and impoundment) was sampled during April, May and November. Detections of eDNA for both species varied across sites and months, but were generally low, 0 - 19.3% of samples were positive for eDNA. Overall, we found that eDNA-based sampling holds promise to be a powerful monitoring tool for resource managers, however, limitations of eDNA-based sampling include different biological and ecological characteristics of target species such as seasonal habitat usage patterns as well as aspects of different physical environments that impact the implementation of these methods such as water temperature.

Ecological Applications

Genetically-informed seed transfer zones for Pleuraphis jamesii, Sphaeralcea parvifolia, and Sporobolus cryptandrus across the Colorado Plateau and adjacent regions

(Massatti) Introduction: The majority of native plant materials (NPMs) utilized for restoration purposes are developed for widely distributed species that provide a variety of ecosystem services (Wood et al. 2015; Butterfield et al. 2017). Disturbed ecosystems benefit from the use of appropriate NPMs, which are those that display ecological fitness at the restoration site, are compatible with conspecifics and other members of the plant community, and that do not demonstrate invasive tendencies (Jones 2013). Furthermore, the use of appropriate NPMs can help address specific environmental challenges, rejuvenate ecosystem function, and improve the delivery of ecosystem services (Hughes 2008). While many NPMs have been developed for restoration (e.g., Aubry et al. 2005), there is interest in broadening the diversity of species available and the geographic representation of sources to provide appropriate choices in relation to the characteristics of any restoration site. In addition, researchers are providing guidance to managers and practitioners regarding how best to transfer NPMs across the landscape. For example, guidance on seed transfer has been derived from genecological studies, which utilize common gardens to correlate phenotypic variation to environmental gradients (summarized in Kilkenny 2015), molecular studies, which identify putative adaptive genetic loci and infer environmental drivers of variation (Shryock et al. 2017), and climate modeling studies, which can provide guidance when species-specific data are unavailable (Bower et al. 2014; Doherty et al. 2017). All of these approaches intend to improve the long-term viability of NPMs at restoration sites, thereby improving outcomes and stretching limiting restoration resources (e.g., time and money).

Colorado Plateau

Guidelines for collecting and maintaining archives for genetic monitoring

Rapid advances in molecular genetic techniques and the statistical analysis of genetic data have revolutionized the way that populations of animals, plants and microorganisms can be monitored. Genetic monitoring is the practice of using molecular genetic markers to track changes in the abundance, diversity or distribution of populations, species or ecosystems over time, and to follow adaptive and non-adaptive genetic responses to changing external conditions. In recent years, genetic monitoring has become a valuable tool in conservation management of biological diversity and ecological analysis, helping to illuminate and define cryptic and poorly understood species and populations. Many of the detected biodiversity declines, changes in distribution and hybridization events have helped to drive changes in policy and management. Because a time series of samples is necessary to detect trends of change in genetic diversity and species composition, archiving is a critical component of genetic monitoring. Here we discuss the collection, development, maintenance, and use of archives for genetic monitoring. This includes an overview of the genetic markers that facilitate effective monitoring, describes how tissue and DNA can be stored, and provides guidelines for proper practice.

Conservation Genetics Resources