DNA assays for genetic discrimination of three Phragmites australis subspecies in the United States.

Premise: To genetically discriminate subspecies of the common reed (Phragmites australis), we developed real-time quantitative (qPCR) assays for identifying P. australis subsp. americanus, P. australis subsp. australis, and P. australis subsp. berlandieri. Methods and Results: Utilizing study-generated chloroplast DNA sequences, we developed three novel qPCR assays. Assays were verified on individuals of each subspecies and against two non-target species, Arundo donax and Phalaris arundinacea. One assay amplifies only P. australis subsp. americanus, one amplifies P. australis subsp. australis and/or P. australis subsp. berlandieri, and one amplifies P. australis subsp. americanus and/or P. australis subsp. australis. This protocol enhances currently available rapid identification methods by providing genetic discrimination of all three subspecies. Conclusions: The newly developed assays were validated using P. australis samples from across the United States. Application of these assays outside of this geographic range should be preceded by additional testing.

Multifaceted DNA metabarcoding of guano to uncover multiple classes of ecological data in two different bat communities.

DNA contained in animal scat provides a wealth of information about the animal, and DNA metabarcoding of scat collections can provide key information about animal populations and communities. Next-generation DNA sequencing technologies and DNA metabarcoding provide an efficient means for obtaining information available in scat samples. We used multifaceted DNA metabarcoding (MDM) of noninvasively collected bat guano pellets from a Myotis lucifugus colony on Fort Drum Military Installation, New York, USA, and from two mixed-species bat roosts on Fort Huachuca Military Installation, Arizona, USA, to identify attributes such as bat species composition, sex ratios, diet, and the presence of pathogens and parasites. We successfully identified bat species for nearly 98% of samples from Fort Drum and 90% of samples from Fort Huachuca, and identified the sex for 84% and 67% of samples from these same locations, respectively. Species and sex identification matched expectations based on prior censuses of bat populations utilizing those roosts, though samples from some species were more or less common than anticipated within Fort Huachuca roosts. Nearly 62% of guano samples from Fort Drum contained DNA from Pseudogymnoascus destructans, where bats with wing damage from White-nose Syndrome were commonly observed. Putative dietary items were detected in a majority of samples from insectivorous bats on Fort Drum (81%) and Fort Huachuca (63%). A minority of guano samples identified as the nectarivorous Leptonycteris yerbabuenae (28%) provided DNA sequences from putative forage plant species. Finally, DNA sequences from both putative ecto- and endoparasite taxa were detected in 35% and 56% of samples from Fort Drum and Fort Huachuca, respectively. This study demonstrates that the combination of noninvasive sampling, DNA metabarcoding, and sample and locus multiplexing provide a wide array of data that are otherwise difficult to obtain.

Multiple stressors in multiple species: Effects of different RDX soil concentrations and differential water-resourcing on RDX fate, plant health, and plant survival.

Response to simultaneous stressors is an important facet of plant ecology and land management. In a greenhouse trial, we studied how eight plant species responded to single and combined effects of three soil concentrations of the phytotoxic munitions constituent RDX and two levels of water-resourcing. In an outdoor trial, we studied the effects of high RDX soil concentration and two levels of water-resourcing in three plant species. Multiple endpoints related to RDX fate, plant health, and plant survival were evaluated in both trials. Starting RDX concentration was the most frequent factor influencing all endpoints. Water-resourcing also had significant impacts, but in fewer cases. For most endpoints, significant interaction effects between RDX concentration and water-resourcing were observed for some species and treatments. Main and interaction effects were typically variable (significant in one treatment, but not in another; associated with increasing endpoint values for one treatment and/or with decreasing endpoint values in another). This complexity has implications for understanding how RDX and water-availability combine to impact plants, as well as for applications like phytoremediation. As an additional product of these greenhouse and outdoor trials, three plants native or naturalized within the southeastern United States were identified as promising species for further study as in situ phytoremediation resources. Plumbago auriculata exhibited relatively strong and markedly consistent among-treatment mean proportional reductions in soil RDX concentrations (112% and 2.5% of the means of corresponding values observed within other species). Likewise, across all treatments, Salvia coccinea exhibited distinctively low variance in mean leaf chlorophyll content index levels (6.5% of the means of corresponding values observed within other species). Both species also exhibited mean wilting and chlorosis levels that were 66% and 35%, and 67% and 84%, of corresponding values observed in all other plants, respectively. Ruellia caroliniensis exhibited at least 43% higher mean survival across all treatments than any other test species in outdoor trials, despite exhibiting similar RDX uptake and bioconcentration levels.

Evaluating the efficacy of sample collection approaches and DNA metabarcoding for identifying the diversity of plants utilized by nectivorous bats.

In this study, we evaluated the efficacy of sample collection approaches and DNA metabarcoding to identify plants utilized by nectivorous bats. Samples included guano collected from beneath bat roosts and pollen-swabs from bat fur, both of which were subjected to DNA metabarcoding and visual identification of pollen (microscopy) to measure plant diversity. Our objectives were to determine whether DNA metabarcoding could detect likely food plants of nectivorous bats, whether sample types would produce different estimates of plant diversity, and to compare results of DNA metabarcoding to visual identification. Visual identification found that 99% of pollen was from Agave, which is thought to be the bats' main food source. The dominant taxon found by metabarcoding was also Agavoideae, but a broader diversity of plant species was also detected, many of which are likely "by-catch" from the broader environment. Metabarcoding outcomes differed between sample types, likely because pollen-swabs measured the plant species visited by bats and guano samples measured all items consumed in the bat's diet, even those that were not pollen or nectar. Overall, metabarcoding is a powerful, high-throughput tool to understand bat ecology and species interactions, but careful analysis of results is necessary to derive accurate ecological conclusions.

Multifaceted DNA metabarcoding: Validation of a noninvasive, next-generation approach to studying bat populations.

As multiple species of bats are currently experiencing dramatic declines in populations due to white-nose syndrome (WNS) and other factors, conservation managers have an urgent need for data on the ecology and overall status of populations of once-common bat species. Standard approaches to obtain data on bat populations often involve capture and handling, requiring extensive expertise and unavoidably resulting in stress to the bats. New methods to rapidly obtain critical data are needed that minimize both the stress on bats and the spread of WNS. Guano provides a noninvasive source of DNA that includes information from the bat, but also dietary items, parasites, and pathogens. DNA metabarcoding is a high-throughput, DNA-based identification technique to assess the biodiversity of environmental or fecal samples. We investigated the use of multifaceted DNA metabarcoding (MDM), a technique combining next-generation DNA sequencing (NGS), DNA barcodes, and bioinformatic analysis, to simultaneously collect data on multiple parameters of interest (bat species composition, individual genotype, sex ratios, diet, parasites, and presence of WNS) from fecal samples using a single NGS run. We tested the accuracy of each MDM assay using samples in which these parameters were previously determined using conventional approaches. We found that assays for bat species identification, insect diet, parasite diversity, and genotype were both sensitive and accurate, the assay to detect WNS was highly sensitive but requires careful sample processing steps to ensure the reliability of results, while assays for nectivorous diet and sex showed lower sensitivity. MDM was able to quantify multiple data classes from fecal samples simultaneously, and results were consistent whether we included assays for a single data class or multiple data classes. Overall, MDM is a useful approach that employs noninvasive sampling and a customizable suite of assays to gain important and largely accurate information on bat ecology and population dynamics.

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.

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 (F(ST) = 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.

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.