Hepatitis E virus (HEV), the causative agent of hepatitis E, is an emerging virus of global distribution. At least four distinct genotypes of HEV exist worldwide: genotype 1 and 2 HEV strains are responsible for waterborne epidemics; genotype 3 and 4 HEV strains are responsible for sporadic occurrences of acute hepatitis E. Genotype 3 and 4 HEVs are zoonotic and have a more expanded host range than genotypes 1 and 2 which are restricted to humans. Genotype 3 and 4 HEV isolates obtained from animal tissues are genetically very similar, or identical in some cases, to human HEV recovered from hepatitis E patients. The objectives of this dissertation research were to assess the zoonotic foodborne transmission of HEV and elucidate the viral determinants of HEV host range. To determine the risk of HEV foodborne transmission, 127 packages of commercial pig liver were tested for HEV RNA. Eleven percent of them were positive for HEV RNA and the contaminating virus remained infectious. We also demonstrated that medium-to-rare cooking condition (56°C) does not completely inactivate HEV, although frying and boiling of the contaminated livers inactivated the virus. To reduce the risk of foodborne HEV transmission, commercial pig livers must be thoroughly cooked for consumption. To determine the host range of genotype 4 HEVs, pigs were inoculated with a genotype 4 human HEV. All pigs developed an active HEV infection indicating that genotype 4 human HEVs can cross species barriers and infect pigs. To identify viral determinant(s) of species tropism, ORF2 alone or in combination with its adjacent 5′ junction region (JR) and 3′ non-coding region (NCR), were swapped between genotypes 1 and 4, 3 and 4, and 1 and 3 to produce 5 chimeric viruses. Chimeric viruses containing ORF2 or JR+ORF2+3' NCR from genotype 4 human HEV in the backbone of genotype 3 swine HEV were viable in vitro and infectious in vivo. Chimeric viruses containing the JR+ORF2+3'NCR of genotypes 3 or 4 HEV in the backbone of genotype 1 human HEV were viable in vitro but non-infectious in pigs, suggesting that ORF1 may also be important for host range. / Ph. D.

Forests provide a number of ecosystem services which sustain and enrich the wildlife, human societies, and the environment. However, many disturbances threaten forest ecosystems, making it necessary to monitor their health for optimal management and conservation. Although there are many indicators of forest health, changes in biogeochemical cycling, loss of species diversity, and invasive plants are particularly useful due to their vulnerability to the effects of climate change and intensive agricultural land use. Thus, this work evaluates the use of imaging spectroscopy to monitor forest nutrient status, species diversity, and plant invasions in the Mid-Atlantic region. The research is divided into four separate studies, each of which evaluated a unique application for imaging spectroscopy data at a different scale within the forest. The first two studies examined loblolly pine nutrient status at the leaf and canopy scales, respectively. The first study determined that loblolly pine foliar macronutrient concentrations can be successfully modeled across the Southeastern US (R2=0.39-0.74). Following on these results, the second study focused on the relationship between physical characteristics, reflectance, and nutrients. Reflectance values and W scattering coefficients produced successful nitrogen models across loblolly pine plots at the canopy scale. Regression models showed similar explanatory power for nitrogen, although W scattering coefficients were significantly correlated with nitrogen at multiple wavelengths and reflectance variables were not. However, the direction of some of the correlations with W and the unusually high directional area scattering factor values indicate a need for further experimentation. The third study found that several imaging spectroscopy algorithms were moderately successful in identifying wavyleaf basketgrass invasions in mixed deciduous forests (overall accuracy=0.35-0.78; kappa=0.41-0.53). Lastly, the fourth study used a novel imaging spectroscopy/lidar fusion to identify canopy gaps and measure species diversity of understory vegetation. The lidar algorithm identified 29 of 34 canopy gaps, and regression models explained 49 percent of the variance in gap species diversity. In conclusion, imaging spectroscopy can be used to evaluate ecosystem health through forest nutrient status, nitrogen models, species diversity estimates, and identification of invasive plant species. / Ph. D.

Since most invasive forest pests first establish in urban areas, detection and containment of these pests within cities is important to the health of all forests. While the emerald ash borer (EAB) (Agrilus planipennis Fairmaire) has proved difficult to contain, efforts continue to mitigate the impacts of its spread. As part of those efforts, the Virginia Department of Forestry (VDOF) initiated its Emerald Ash Borer Treatment Program (EABTP) in 2018, providing financial incentives for insecticidal protection of ash trees. To better understand the role of incentives in promoting urban forest health, I conducted a study of properties, households, and practitioners involved in the program's first year. To examine where EABTP funding helped pay for tree protection, I conducted tree inventories on 16 urban participant properties. Concurrently with tree inventory work, I conducted web and mail surveys to examine homeowner engagement in preservation of threatened trees. Finally, to investigate the role of forest practitioners involved in program implementation, I conducted web surveys of VDOF foresters and Virginia arborists. Results demonstrated that on urban participant properties—typically large and wooded—white ash (Fraxinus americana) was the dominant species. Results from homeowner surveys demonstrated broad support for personal investment in tree preservation, and the significance of attitudinal predictors towards those intentions. Results from practitioner surveys demonstrated substantial, though not unanimous, support for the program as a benefit both to clients and forests. Implications of these findings are discussed in the context of future urban forest health initiatives. / Master of Science / Because most non-native forest pests arrive in cities before spreading to rural areas, detecting and containing these pests within urban forests is important to all forested areas. One non-native pest, the emerald ash borer (EAB), has proved difficult to contain, but there are ongoing efforts to limit the damage it causes as it spreads. As part of those efforts, the Virginia Department of Forestry (VDOF) began its Emerald Ash Borer Treatment Program (EABTP) in 2018, which offered partial reimbursement for the cost of protecting ash trees with insecticide. To better understand how reimbursement payments might help promote the health of urban trees, I studied the properties, households, and practitioners involved in first year of the program. To examine where EABTP funding helped pay for tree protection, I conducted inventories of all trees on 16 participating properties in urban areas. At the same time, I conducted web and mail surveys to examine how homeowners thought about urban tree preservation. Finally, I conducted web surveys of VDOF foresters and Virginia arborists, to investigate roles of these practitioners in implementing the program. Results indicated that on urban participant properties, which were typically large and wooded, white ash was the dominant species. Results from homeowner surveys demonstrated broad support for personal investment in tree preservation, and the significance of attitudes in predicting that support. Results from practitioner surveys demonstrated substantial, though not unanimous, support for the program as a benefit both to clients and forests. These findings are discussed in the context of future urban forest health programs.

Metacommunity ecology explicitly incorporates processes at multiple spatial scales to explain the assembly and dynamics of a community. In a metacommunity, local communities interact with one another through the dispersal of individuals across a region. As such, metacommunities are molded by two sets of processes: local and regional. Local factors are those that directly impact a single local community, such as environmental conditions, competition, and predation. On the other hand, regional factors affect communities across a landscape and include mechanisms such as, immigration and emigration. The potential interactions between local and regional factors make metacommunity dynamics a unique body of theory when compared to classic community theory. However, while the direct influence of dispersal on metacommunity dynamics continues to be a well-researched topic, how dispersal interacts with local factors to shape metacommunity dynamics is a more open topic. In particular, one continuing gap in my knowledge is how dispersal interacts with biotic effect how it may affect metacommunities. One type of local biotic process that can directly affect communities is a strong interactive species, i.e., a species that affects community structure and diversity, and to the best of my knowledge, the interaction between dispersal and strongly interactive species has not been directly addressed experimentally. In the following study, I investigated the interaction of dispersal and a strong interactive species on metacommunity diversity and assembly. I chose Daphnia magna as my strong interacting species due to its biological and physical traits. Dispersal is known to create predictable patterns of diversity as it increases in a metacommunity. We made logical predictions based off of my knowledge of these patterns, and my inclinations regarding how dispersal would interact with a strong interacting species. The following predictions were made in relation to the control: 1) Alpha diversity would be the highest during low dispersal as new species would be introduced and maintained above the extinction threshold. I also predicted beta diversity would decrease with increased dispersal due to the homogenization of communities. 2) In the presence of D. magna, beta diversity would only increase during low dispersal due to possible rescue effects. 3) Temporal variability would decrease for the low dispersal treatment and increase for the high dispersal treatment in the absence of D. magna. 4) Temporal variability would overall increase across all treatments in the presence of D. magna. To carry out the study, I assembled outdoor mesocosms using a 2x3x3x4 factorial design (Daphnia Treatment: no addition of D. magna, addition of D. magna; Dispersal Treatment: no dispersal, low dispersal, high dispersal; three buckets were equivalent to one metacommunity; 4 replicates). There was a significant interaction between D. magna and dispersal. Over time, beta diversity decreased as communities became homogenized; however, the no dispersal treatment homogenized at a slower rate compared to the other treatments. In addition, D. magna appeared to create local selection for certain taxa resulting in the increase of Bosmina and Simocephalus while other taxa decreased, for example Streblocerus. This trend was likely due to the feeding and grazing habits of D. magna which is known to outcompete other large zooplankton for larger phytoplankton taxa. Lastly, D. magna directly influenced temporal variability of metacommunities in the experiment. In particular, the low dispersal treatment increased in temporal variability in the presence of D. magna. Again, this result could likely be attributed to D. magna effects selecting for certain taxa, or by the re-introduction of new or dying species with each dispersal through rescue effects. Overall, the results in my study supported majority of my predictions. It is clear that D. magna had an effect on communities as taxa abundances increased and beta diversity in the no dispersal treatment did not decrease as quickly. This result suggests that the introduction of D. magna as an invasive to non-local waters could pose a threat to local community dynamics. It is important to understand how a strong interactive species can affect communities across a landscape as they can greatly alter diversity and composition. Future studies should focus on expanding the dispersal gradient and incorporating a local strong interactive species and non-local strong interactive species to understand how they may change community dynamics. / Master of Science

Potato (Solanum tuberosum L) is an outcrossing, highly heterozygous cultivated in which the elucidation of the genetic basis of quantitative traits, is more complex than in self-pollinated crops. Both a candidate gene approach and a whole genome SNP genotyping analysis were used to assess allelic variation and to identify loci associated with biosynthesis and accumulation of steroidal glycoalkaloids (SGAs). SGAs are secondary metabolites produced in Solanum species as defense against insects and pathogens. Fragments of genomic DNA coding for regions of five SGA biosynthetic candidate genes were amplified, cloned and sequenced [3-hydroxy-3-methylglutaryl coenzyme A reductase 1 and 2 (HMG1, HMG2); 2,3-squalene epoxidase (SQE); solanidine galactosyltransferase (SGT1); and solanidine glucosyltransferase (SGT2)]. A germplasm panel of six wild potato species [Solanum chacoense (chc 80-1), S. commersonii subsp. commersonii, S. demissum, S. sparsipilum, S. spegazzinii, and S. stoloniferum] and a cultivated clone S. tuberosum Group Phureja (phu DH) was used in an allelic variation analysis. A segregating interspecific F2 population phu DH �" chc 80-1 was screened to assess association with SGAs. Sequence diversity analysis showed a tendency of purifying selection and increased frequency of rare alleles in most of the candidate genes. Genes of primary metabolism (HMG1, HMG2 and SQE) had stronger selection constraints than those in secondary metabolism (SGT1 and SGT2). Sequence polymorphism in HMG2, SQE, SGT1 and SGT2 separated either the phu DH clone which produced no SGAs, or chc 80-1, the greatest SGA accumulator, from other accessions in the panel. Segregation analysis of the F2 population revealed that allelic sequences of HMG2 and SGT2 derived from chc 80-1 were significantly associated with the greatest SGA accumulation. In the whole genome analysis, SNP genotyping and cluster analysis based on putative association with SGA accumulation in the germplasm panel, allowed identification of eight informative SNPs that can be used in future studies. In the segregating F2 population, loci located on five pseudochromosomes were associated with SGA synthesis. Loci on pseudochromosomes 1 and 6 explained segregation ratios of synthesis for α-solanine and α-chaconine, the most common SGAs in most potato species. In addition, loci on seven pseudochromosomes were associated with accumulation. New candidate genes, putatively affecting synthesis and accumulation of SGAs, were identified in adjacent genomic regions of significant SNPs. This research demonstrates how the newly available genome sequence of potato and associated biotechnological tools accelerates the identification of genetic factors underling complex traits in a species with a difficult breeding structure. / Ph. D.

Wildlife biodiversity provides a variety of ecosystem services and is an important indicator of overall ecosystem health. This research investigates the relationship between wildlife biodiversity and landscape characteristics in Virginia. The goal is to produce predictive models of biodiversity within the Commonwealth using environmental characteristics, including fragmentation metrics at the class- and landscape-levels, as well as other environmental variables. The 1248 12-digit watersheds in Virginia are the sampling units for the analyses, with the state stratified into the seven US Environmental Protection Agency's Level III classification. Data on wildlife alpha diversity is based on two sets of species data maintained by the Virginia Dept. of Game & Inland Fisheries (VDGIF). The first chapter provides an introduction to the issue of biodiversity conservation and the background information for this work. The second chapter describes the study using the 2001 National Land Cover Data to calculate class- and landscape-level fragmentation metrics. Best subset regression is used to determine the best predictors for wildlife biodiversity using these metrics. Final selected models range in predictive power from R2 = 0.41 to 0.73 for each of the 7 ecoregions. The third chapter analyzes the relationship between wildlife biodiversity and various environmental variables in order to determine the strength of these factors as drivers for alpha diversity. These variables are then incorporated with the fragmentation metrics in an attempt to improve the biodiversity models. The environmental variable models had R2 = 0.22 to 0.65 across the ecoregions, while R2 = 0.28 to 0.72 when the environmental and fragmentation variables are combined. The last chapter focuses on the conclusions of the studies, the limitations of the data, and the benefits of this work. Overall, our results underline the importance of using fragmentation metrics in Virginia's wildlife models. / Master of Science

Paronychia virginica Spreng. (Caryophyllaceae) is a perennial evergreen herb of exposed, relatively xeric habitats. Approximately 10 mid-Appalachian populations remain in Virginia, West Virginia, and Maryland and are disjunct from populations located primarily in Texas, Oklahoma, and Arkansas. A study was conducted to test the hypothesis that eastern and western populations differ significantly and, therefore, represent at least two distinct taxa. Statistical analyses of 8 qualitative and 24 quantitative morphological characters indicated very highly significant (P < 0.001) variation between eastern and western populations of P. virginica. Characters differing most significantly included sepal pubescence, awn length, awn pubescence, awn curvature, length-width ratio of leaves, and shape of leaf apices. Starch gel electrophoresis was performed and six enzyme systems/nine loci (EST-2, EST-3, LAP, MDH-1, MDH-2, PGI, PGM-1, PGM-2, and SKDH) were identified as being consistently scorable and informative. Although gene flow between populations of P. virginica was shown to be restricted (mean FST = 0.353), populations are maintaining relatively high levels of genetic diversity. Genetic variability was quantified for each population and mean values for number of alleles per locus (A), percent loci polymorphic (P), and expected heterozygosity (HEXP) were found to be 1.95, 47.22%, and 0.204, respectively, exceeding those values reported for seed plants, widespread species, and endemic species. Hierarchical F statistics suggest higher levels of genetic variability within individual populations than among populations, regardless of geographic location. All statistically significant (P < 0.05) deviations from Hardy-Weinberg equilibrium indicated a deficiency in heterozygotes at the respective loci. Considering results from both the morphometric and allozyme analyses, the current author suggests recognizing two distinct subspecies, P. virginica subsp. virginica in the eastern U.S. and P. virginica subsp. scoparia in the south-central U.S. Conservation efforts should be focused on the maintenance of existing populations in both eastern and western regions of the U.S. in order to preserve the genetic and evolutionary potential of these taxa. / Master of Science

As invasive species are so prominent, the influence of wildfire intensity on fuel loading, invasives, species richness, diversity, and evenness were studied at Shenandoah National Park. Most National Parks identify invasive species as the biggest threat to their goal of maintaining native ecosystems. Eight study sites were stratified into three burn classes (high intensity, low intensity, and control), and three transects were randomly located so that nested plots and fuel transects were measured at a distance of 50 ft (15 m), 150 ft (45 m), and 250 ft (75 m) from a road or trail. Field sampling was conducted between May 15, 2004 and June 30, 2004. A subsample of these plots were used to determine specific gravity and quadratic mean diameter for each size class of fuel and to determine the bulk density of the duff and litter layers. High intensity wildfires initially reduced species diversity and evenness in the tree and herbaceous strata, but after 14 years tree species diversity and evenness returned to levels found in unburned areas, while herbaceous strata diversity was not associated with time since burn. Low intensity wildfires resulted in the greatest impacts in the shrub stratum. Presence of invasive species was associated with more even and diverse vegetation in all strata, perhaps because invasive species were relatively sparse. Fuel loadings were reduced initially by high intensity wildfires, but quickly returned to the same level as unburned areas. Although these initial findings indicate that invasive species will not persist after wildfire disturbance, continual monitoring by National Parks would be prudent. / Master of Science

The Clinch Dace Chrosomus sp. cf. saylori is a species of minnow known from only two counties in Virginia. Prior surveys established the species' presence in just eight tributaries to the upper Clinch River. A management plan, which should include both population monitoring and habitat restoration, is still lacking for the species. Population monitoring must balance high detection probability with low risk of injury to captured individuals. I sampled 98 sites in 2014 and 2015 within the putative range of Clinch Dace to refine estimates of its distribution. I sampled 70 of the 98 sites with multiple gears and replication in an occupancy modeling framework. Clinch Dace occupied low-gradient headwater streams with relatively low conductivity in forested watersheds. My surveys uncovered two new tributaries occupied by Clinch Dace, and I was unable to find Clinch Dace in two historically occupied streams. Species detection probability was higher with backpack electrofishing than minnow trapping. N-mixture models suggest that Clinch Dace are more abundant in watersheds with high forest cover although forest cover is highly correlated spatially in the nested stream network. Density estimates from mark-recapture sampling suggest that Clinch Dace occur at low densities in approximately 31.5 km of headwater streams. The mean estimate of global population size was 6,706 individuals. Some populations could be affected by low genetic diversity. I conclude by developing a prioritization framework for restoration and protection of 15 candidate conservation areas. Managers should work with private landowners to implement best management practices in high priority watersheds. / Master of Science

The Reticulated Flatwoods Salamander, Ambystoma bishopi, is an inhabitant of longleaf pine forests in the southeastern United States. Historically distributed across southern Alabama, Georgia, and the Florida panhandle west of the Apalachicola-Flint Rivers, the range of this species has been drastically reduced. It is currently listed as federally endangered under the Endangered Species Act (ESA). Population viability analyses (PVAs) represent a key component of many recovery plans for threatened and endangered species. Here we use 10 years of mark recapture data collected from two breeding populations of A. bishopi to construct a demographic model that can be used to evaluate future extinction risk. In chapter one, we quantify population sizes through time, and estimate the impact of annual variability in numbers on genetic viability. This species exists in small (< 500) breeding populations and exhibit annual fluctuations in abundance characteristic of pond-breeding amphibians. In chapter 2, we adopt a modified version of the von Bertalanffy equation to construct size-at-age curves for A. bishopi that include the metamorphic transition. Individuals exhibit rapid growth in the larval stage such that they emerge as metamorphs at 60% of their final body size. In chapter 3, we employ a Cormack-Jolly-Seber model, modified to include continuous covariates, to generate size-dependent survival curves. Survival of A bishopi exhibits dramatic annual and seasonal variability, but is always positively correlated with body size. Lastly, in chapter 4, we combine the elements of all previous chapters to construct an Integral Projection Model (IPM). Given the prevalence of complete recruitment failure in these populations, and their relatively small size, extinction probabilities under a business-as-usual model were high. Increasing the frequency of successful recruitment drastically reduces extinction risk; however, adult survival exerts the greatest influence on long-term population growth. To assure the recovery of A. bishopi, management must consider all elements of the life-history when allocating resources and effort. More generally, both aquatic and terrestrial habitats must be protected for amphibian conservation to be effective, making them ideal candidates for 'umbrella species' status. Amphibian conservation would also benefit from an increase in systematic, long-term data collection. / Doctor of Philosophy / The southeastern United States is the global salamander hotspot, representing a crucible for diversity. Longleaf pine forests, the predominant ecosystem in the southeast, have been reduced to 3% of their former range, with dire consequences for the animals that inhabit them. The Reticulated Flatwoods Salamander, Ambystoma bishopi, is endemic to the region, and currently listed as federally endangered owing to recent population declines. A recovery plan for the species therefore, is required by law, under the Endangered Species Act (ESA). A salient component of modern recovery plans are population forecasts that evaluate future extinction risk. Such forecasts can then be used to assess alternative management strategies proposed to improve the species' long-term prospects. By studying two of the last remaining populations of A. bishopi from 2010-2019, we were able to collect the data required to construct a demographic model that can be used to run population projections. In some regards, A. bishopi is a typical amphibian, in that their populations show dramatic fluctuations in numbers through time, and they exhibit rapid growth in the aquatic larval stage, achieving 60% of their maximum body size in the first three months of life. Flatwoods salamanders breed in ephemeral wetlands, that often dry before successful metamorphosis can occur. The frequency of pond-drying results in a high probability of extinction for a single population, but survival of breeding individuals was equally important when considering long-term persistence. To assure the recovery of A. bishopi, management must consider all elements of the life-history when allocating resources and effort. More generally, both aquatic and terrestrial habitats must be protected for amphibian conservation to be effective, making them ideal candidates for 'umbrella species' status.