Richard F. Lance

Critical considerations for the application of environmental DNA methods to detect aquatic species

Summary Species detection using environmental DNA (eDNA) has tremendous potential for contributing to the understanding of the ecology and conservation of aquatic species. Detecting species using eDNA methods, rather than directly sampling the organisms, can reduce impacts on sensitive species and increase the power of field surveys for rare and elusive species. The sensitivity of eDNA methods, however, requires a heightened awareness and attention to quality assurance and quality control protocols. Additionally, the interpretation of eDNA data demands careful consideration of multiple factors. As eDNA methods have grown in application, diverse approaches have been implemented to address these issues. With interest in eDNA continuing to expand, supportive guidelines for undertaking eDNA studies are greatly needed. Environmental DNA researchers from around the world have collaborated to produce this set of guidelines and considerations for implementing eDNA methods to detect aquatic macroorganisms. Critical considerations for study design include preventing contamination in the field and the laboratory, choosing appropriate sample analysis methods, validating assays, testing for sample inhibition and following minimum reporting guidelines. Critical considerations for inference include temporal and spatial processes, limits of correlation of eDNA with abundance, uncertainty of positive and negative results, and potential sources of allochthonous DNA. We present a synthesis of knowledge at this stage for application of this new and powerful detection method.

Habitat fragmentation and genetic diversity of an endangered, migratory songbird, the golden‐cheeked warbler (Dendroica chrysoparia)

Landscape genetic approaches offer the promise of increasing our understanding of the influence of habitat features on genetic structure. We assessed the genetic diversity of the endangered golden‐cheeked warbler (Dendroica chrysoparia) across their breeding range in central Texas and evaluated the role of habitat loss and fragmentation in shaping the population structure of the species. We determined genotypes across nine microsatellite loci of 109 individuals from seven sites representing the major breeding concentrations of the species. No evidence of a recent population bottleneck was found. Differences in allele frequencies were highly significant among sites. The sampled sites do not appear to represent isolated lineages requiring protection as separate management units, although the amount of current gene flow is insufficient to prevent genetic differentiation. Measures of genetic differentiation were negatively associated with habitat connectivity and the percentage of forest cover between sites, and positively associated with geographic distance and the percentage of agricultural land between sites. The northernmost site was the most genetically differentiated and was isolated from other sites by agricultural lands. Fragmentation of breeding habitat may represent barriers to dispersal of birds which would pose no barrier to movement during other activities such as migration.

Population structure in an endangered songbird: maintenance of genetic differentiation despite high vagility and significant population recovery

Black‐capped vireos (Vireo atricapilla), an endangered, migratory species dependent upon early successional habitat, have experienced significant recovery since its protection. In light of its vagility and known increase in population size and range, limited genetic differentiation would be expected in the species. Using 15 microsatellite loci and an extensive sampling regime, we detected significant overall genetic differentiation (FST = 0.021) and high interpopulation differentiation compared to other migratory birds. Although proximate sites (separated by < 20 km) tended to be genetically similar, there was no apparent association of either geographical distance or landscape attributes with differentiation between sites. Evidence of a population bottleneck was also detected in a site located near other large concentrations of birds. Although black‐capped vireos are capable of large‐scale movements and the population has experienced a recent expansion, dispersal appears too insufficient to eliminate the genetic differentiation resulting from restricted colonization of ephemeral habitats.

Birds in space and time: genetic changes accompanying anthropogenic habitat fragmentation in the endangered black‐capped vireo (Vireo atricapilla)

Anthropogenic alterations in the natural environment can be a potent evolutionary force. For species that have specific habitat requirements, habitat loss can result in substantial genetic effects, potentially impeding future adaptability and evolution. The endangered black‐capped vireo (Vireo atricapilla) suffered a substantial contraction of breeding habitat and population size during much of the 20th century. In a previous study, we reported significant differentiation between remnant populations, but failed to recover a strong genetic signal of bottlenecks. In this study, we used a combination of historical and contemporary sampling from Oklahoma and Texas to (i) determine whether population structure and genetic diversity have changed over time and (ii) evaluate alternate demographic hypotheses using approximate Bayesian computation (ABC). We found lower genetic diversity and increased differentiation in contemporary samples compared to historical samples, indicating nontrivial impacts of fragmentation. ABC analysis suggests a bottleneck having occurred in the early part of the 20th century, resulting in a magnitude decline in effective population size. Genetic monitoring with temporally spaced samples, such as used in this study, can be highly informative for assessing the genetic impacts of anthropogenic fragmentation on threatened or endangered species, as well as revealing the dynamics of small populations over time.

Crumbling diversity: comparison of historical archived and contemporary natural populations indicate reduced genetic diversity and increasing genetic differentiation in the golden-cheeked warbler

Genetic viability of threatened and endangered species is of increasing concern with habitat loss and fragmentation. Valuable assessments of the genetic status of endangered species are difficult in most cases, where only single sample estimates are available. Using historical and contemporary samples, we assessed the impact of both historical and recent demographic changes on population genetics of the endangered golden-cheeked warbler, (Dendroica chrysoparia). Our study documents a steep decline in genetic diversity in an endangered species over a 100-year period, along with concurrent increase in genetic differentiation, and low contemporary effective sizes for all the populations we evaluated. While adding to the growing body of literature that describes the genetic impacts of habitat fragmentation, our study may also serve as an informative guide to future management of endangered species. Our study underlines the importance of long term population genetic monitoring in understanding the full extent of genetic changes in endangered species.

Mitochondrial Genome Sequencing and Development of Genetic Markers for the Detection of DNA of Invasive Bighead and Silver Carp (Hypophthalmichthys nobilis and H. molitrix) in Environmental Water Samples from the United States

Invasive Asian bighead and silver carp (Hypophthalmichthys nobilis and H. molitrix) pose a substantial threat to North American aquatic ecosystems. Recently, environmental DNA (eDNA), genetic material shed by organisms into their environment that can be detected by non-invasive sampling strategies and genetic assays, has gained recognition as a tool for tracking the invasion front of these species toward the Great Lakes. The goal of this study was to develop new species-specific conventional PCR (cPCR) and quantitative (qPCR) markers for detection of these species in North American surface waters. We first generated complete mitochondrial genome sequences from 33 bighead and 29 silver carp individuals collected throughout their introduced range. These sequences were aligned with those from other common and closely related fish species from the Illinois River watershed to identify and design new species-specific markers for the detection of bighead and silver carp DNA in environmental water samples. We then tested these genetic markers in the laboratory for species-specificity and sensitivity. Newly developed markers performed well in field trials, did not have any false positive detections, and many markers had much higher detection rates and sensitivity compared to the markers currently used in eDNA surveillance programs. We also explored the use of multiple genetic markers to determine whether it would improve detection rates, results of which showed that using multiple highly sensitive markers should maximize detection rates in environmental samples. The new markers developed in this study greatly expand the number of species-specific genetic markers available to track the invasion front of bighead and silver carp and will improve the resolution of these assays. Additionally, the use of the qPCR markers developed in this study may reduce sample processing time and cost of eDNA monitoring for these species.

Effects of a nonnative, invasive lovegrass on Agave palmeri distribution, abundance, and insect pollinator communities

Nonnative Lehmann lovegrass (Eragrostis lehmanniana) has invaded large areas of the Southwestern United States, and its impact on native plants is not fully understood. Palmer’s agave (Agave palmeri), an important resource for many pollinators, is a key native plant potentially threatened by E. lehmanniana. Understanding potential impacts of E. lehmanniana on A. palmeri is critical for anticipating the future of the desert community where they coexist and for addressing management concerns about associated threatened and endangered species. Our study provides strong indications that E. lehmanniana negatively impacts A. palmeri in several ways. Areas of high E. lehmanniana abundance were associated with significantly lower densities and greater relative frequencies of small A. palmeri, suggesting that E. lehmanniana may exclude A. palmeri. There were no significant differences in species richness, abundance, or community composition when comparing flower associates associated with A. palmeri in areas of high and low E. lehmanniana abundance. However, we did find significantly lower connectedness within the pollination network associated with A. palmeri in areas with high E. lehmanniana abundance. Although E. lehmanniana forms thick stands that would presumably increase fire frequency, there was no significant association between E. lehmanniana and fire frequency. Interestingly, medium to high densities of A. palmeri were associated with areas of greater fire frequency. The complex ramifications of E. lehmanniana invasion for the long-lived A. palmeri and interlinked desert community warrant continued study, as these species are likely to continue to be found in close association due to their similar soil preferences.

Genomic investigation of year-long and multigenerational exposures of fathead minnow to the munitions compound RDX.

We assessed the impacts of exposure to an environmentally representative concentration (0.83 mg/L) of the explosive cyclotrimethylenetrinitramine (RDX) on fathead minnows (Pimephales promelas) in one-year and multigenerational bioassays. In the one-year bioassay, impacts were assessed by statistical comparisons of females from breeding groups reared in control or RDX-exposure conditions. The RDX had no significant effect on gonadosomatic index or condition factor assayed at 1 d and at one, three, six, nine, and 12 months. The liver-somatic index was significantly increased versus controls only at the 12-month timepoint. RDX had no significant effect on live-prey capture rates, egg production, or fertilization. RDX caused minimal differential-transcript expression with no consistent discernable effect on gene-functional categories for either brain or liver tissues in the one-year exposure. In the multigenerational assay, the effects of acute (96 h) exposure to RDX were compared in fish reared to the F(2) generation in either control or RDX-exposure conditions. Enrichment of gene functions including neuroexcitatory glutamate metabolism, sensory signaling, and neurological development were observed comparing control-reared and RDX-reared fish. Our results indicated that exposure to RDX at a concentration representing the highest levels observed in the environment (0.83 mg/L) had limited impacts on genomic, individual, and population-level endpoints in fathead minnows in a one-year exposure. However, multigenerational exposures altered transcript expression related to neural development and function. Environ.

Novel microsatellite loci for Agave parryi and cross-amplification in Agave palmeri (Agavaceae)

PREMISE OF THE STUDY To examine the foraging behavior of nectarivorous bats in southeastern Arizona, we developed microsatellite primers in Agave parryi. These markers were also tested for cross-amplification and applicability to assess patterns of genetic diversity and structure in A. palmeri. METHODS AND RESULTS Utilizing DNA sequence data from 454 shotgun sequencing, we identified seven novel polymorphic microsatellite loci in A. parryi and screened them for cross-amplification in A. palmeri. These markers were characterized in two populations of 30 individuals each for each species. In A. parryi, all primers were polymorphic and amplified between three and 12 alleles per population. In A. palmeri, all primers amplified, six were polymorphic, and allelic diversity ranged from one to 16 alleles per population. CONCLUSIONS Our results demonstrate the applicability of these microsatellite primers for population genetics studies in both A. parryi and A. palmeri.

How far is too close? restricted, sex‐biased dispersal in black‐capped vireos

Understanding the interplay of dispersal and how it translates into gene flow is key to understanding population processes, and especially so for endangered species occupying fragmented habitats. In migratory songbirds, there is evidence that long‐distance movement capabilities do not translate well into observed dispersal. Our objectives were to (i) define the fine‐scale spatial genetic structure in endangered black‐capped vireos to characterize dispersal patterns and (ii) to correlate dispersal dynamics to overall population genetic structure using a simulation approach. We sampled 160 individuals over 2 years to (i) describe the fine‐scale genetic structuring and (ii) used this information to model scenarios to compare with actual data on change in population structuring over a 100‐year interval. We found that black‐capped vireos exhibit male philopatry and restricted dispersal distances, relative to females. Our simulations also support a sex‐biased dispersal model. Additionally, we find that fragmentation related changes in rates of dispersal might be a likely cause for increasing levels of population structure over a 100‐year period. We show that restricted sex‐biased dispersal can explain population structuring in this species and that changes in dispersal rates due to fragmentation may be a continuing threat to genetic viability in this species.