Graduate Theses & Dissertations


Population Genetics and Gut Microbiome Composition Reveal Subdivisions and Space Use in a Generalist and Specialist Ungulate
Natural populations are often difficult and costly to study, due to the plethora of confounding processes and variables present. This is of particular importance when dealing with managed species. Ungulates, for example, act as both consumers and prey sources; they also provide economic benefit through harvest, and as such, are of high ecological and economic value. I addressed conservation and management concerns by quantifying subdivision in wild populations and combined movement with non-invasive sampling to provide novel insight on the physiological drivers of space use in multiple species. This thesis explored biological patterns in ungulates using two distinct approaches: the first used molecular genetics to quantify gene flow, while the second examined the relationship between movement and the gut microbiome using high-throughput sequencing and GPS tracking. The goal of the first chapter was to quantify gene flow and assess the population structure of mountain goats (Oreamnos americanus) in northern British Columbia (BC) to inform management. I used microsatellites to generate genotype data and used a landscape genetics framework to evaluate the possible drivers behind genetic differentiation. The same analyses were performed at both a broad and fine scale, assessing genetic differentiation between populations in all of northern BC and in a case management study area northeast of Smithers BC. The results indicated panmixia among mountain goats regardless of scale, suggesting distance and landscape resistance were minimally inhibiting gene flow. Therefore, management at local scales can continue with little need for genetically informed boundaries, but regulations should be tailored to specific regions incorporating data on local access and harvest pressure. My second chapter aimed to determine the extent to which the gut microbiome drives space-use patterns in a specialist (mountain goat) and generalist (white-tailed deer, Odocoileus virginianus) ungulate. Using fecal samples, we generated genomic data using 16S rRNA high-throughput sequencing to evaluate gut diversity and gut microbiome characteristics. Additionally, individuals were fitted with GPS collars so that we could gain insight into movement patterns. Gut microbiome metrics were stronger predictors of space use and movement patterns with respect to home range size, whereas they were weaker predictors of habitat use. Notably, factors of both the gut microbiome and age of a given species were correlated with changes in space use and habitat use. Ultimately, this research linked high-throughput sequencing and GPS data to better understand ecological processes in wild ungulates. Author Keywords: gene flow, genomics, gut microbiome, home range, population genetic structure, ungulates
Assessment of Potential Threats to Eastern Flowering Dogwood (Cornus florida) in Southern Ontario
In Canada, eastern flowering dogwood (Cornus florida L.) is an endangered tree that occurs only in the Carolinian forest of southern Ontario. Threats to this species include habitat fragmentation and the fungal pathogen dogwood anthracnose (Discula destructiva). I conducted a population genetic analysis using seven nuclear microsatellite markers to determine if fragmented populations are genetically isolated from one another and have low levels of genetic diversity. Genetic comparisons suggest on-going dispersal among sites and relatively high genetic diversity within most sites; however, smaller populations and younger trees were less genetically diverse. I also used linear mixed effects models to assess potential relationships between several ecological variables and the prevalence of dogwood anthracnose. Disease severity was higher in trees on shallow slopes and in larger trees; the latter also had higher likelihood of infection. Insights from this study will be important to incorporate into future management strategies. Author Keywords: Cornus florida, Discula destructiva, dogwood anthracnose, Eastern flowering dogwood, endangered, population genetics
Characterizing the demographic history and prion protein gene variation to infer susceptibility to chronic wasting disease in a naïve population of white-tailed deer (Odocoileus virginianus)
Assessments of the adaptive potential of natural populations are essential for understanding and predicting responses to environmental stressors like climate change and infectious disease. The range of stressors species face in a human-dominated landscape, often have contrasting effects. White-tailed deer (Odocoileus virginianus, deer) are expanding in the northern part of their range following decreasing winter severity and increasing forage availability, caused by climate change. Chronic wasting disease (CWD), a prion disease affecting cervids, is likewise expanding and represents a major threat to deer and other cervids We obtained tissue samples from free-ranging deer across their native range in Ontario, Canada which has yet to detect CWD in wild populations of cervids. High throughput sequencing was used to assess neutral genomic variation and variation in the gene responsible for the protein that misfolds into prions when deer contract CWD, known as the PRNP gene. Neutral variation revealed a high number of rare alleles and no population structure, consistent with an expanding population of deer. Functional genetic variation revealed that the frequencies of variants associated to CWD susceptibility and disease progression were evenly distributed across the landscape and the frequencies were consistent with deer populations not infected with CWD. These findings suggest that an observable shift in PRNP allele frequencies likely coincides with the start of a novel CWD epidemic. Sustained surveillance of genomic and genetic variation can be a useful tool for CWD-free regions where deer are managed for ecological and economic benefits. Author Keywords: Canadian wildlife, population genetics, prion, PRNP, RADseq, ungulate
Range dynamics of two closely related felids
Species ranges are changing and the rate at which the climate is warming is faster than anything previously seen in the past, consequently species will need to adapt quickly, track the climate or perish. Cold adapted terrestrial species are the most vulnerable, because they are limited by the availability of land at the cold edge of their range. This means that many alpine, boreal and polar species essentially have nowhere to go as the climate warms. Habitat generalists are widely distributed across the globe and are highly adaptable to anthropogenic change. Our future biodiversity may only consist of several habitat generalists. The Canada lynx (Lynx canadensis) is a boreal species that has limited range expansion potential at the cold end of its range and its range has already contracted by 40%. The lynx has nowhere to go as climate warming progresses in this current century. Therefore, understanding the causes of its range contraction could enlighten us on conservation and management strategies that we might undertake as climate warms. My analyses indicated that the Canada lynx seems to have tracked the habitat that it is adapted to in more northern homogenous boreal forests and the bobcat (Lynx rufus), a habitat generalist, has simply replaced it in the south. Author Keywords: Anthropogenic Change, Competition, Connectivity, Lynx canadensis, Lynx rufus, Range change
Investigating the regional variation in frequencies of the invasive hybrid cattail, Typha × glauca
Interspecific hybridization rates can vary depending on genomic compatibilities between progenitors, while subsequent hybrid spread can vary depending on hybrid performance and habitat availability for hybrid establishment and persistence. As a result, hybridization rates and hybrid frequencies can vary across regions of parental sympatry. In areas around the Laurentian Great Lakes, Typha × glauca is an invasive plant hybrid of native Typha latifolia and introduced Typha angustifolia. In areas of parental sympatry in Atlantic Canada and outside of North America, T. × glauca has been reported as either rare or non-existent. I investigated whether the low frequencies of hybrids documented in Nova Scotia, Atlantic Canada, are influenced by reproductive barriers that prevent hybrid formation or environmental factors (salinity) that reduce hybrid performance. I identified an abundance of hybrids in the Annapolis Valley (inland) and a scarcity of hybrids in coastal wetlands through preliminary site surveys throughout Nova Scotia. In Annapolis Valley populations, flowering times of progenitor species overlapped, indicating that asynchronous flowering times do not limit hybrid formation in this region. Viable progeny were created from interspecific crosses of T. latifolia and T. angustifolia from Nova Scotia, indicating that there are no genomic barriers to fertilization and germination of hybrid seeds. Typha × glauca germination in high salinity was significantly lower than that of T. latifolia, but there was no difference at lower salinities. Therefore, while germination of hybrid seeds may be impeded in the coastal wetlands where salinity is high, inland sites have lower salinity and thus an environment conducive to hybrid germination. However, I found that once established as seedlings, hybrids appear to have greater performance over T. latifolia across all salinities through higher ramet production. Moreover, I found that T. latifolia sourced from Ontario had reduced germination and lower survivorship in high salinities compared to T. latifolia sourced from Nova Scotia, which could indicate local adaptation by T. latifolia to increased salinity. These findings underline that interactions between environment and local progenitor lineages can influence the viability and the consequent distribution and abundance of hybrids. This, in turn, can help explain why hybrids demonstrate invasiveness in some areas of parental sympatry but remain largely absent from other areas. Author Keywords: flowering phenology, Hybridization, invasive species, physiology, pollen compatibility, salinity tolerance
Genomic architecture of artificially and sexually selected traits in white-tailed deer (Odocoileus virginianus)
Understanding the complex genomic architecture underlying quantitative traits can provide valuable insight for the conservation and management of wildlife. Despite improvements in sequencing technologies, few empirical studies have identified quantitative trait loci (QTL) via whole genome sequencing in free-ranging mammal populations outside a few well-studied systems. This thesis uses high-depth whole genome pooled re-sequencing to characterize the molecular basis of the natural variation observed in two sexually selected, heritable traits in white-tailed deer (Odocoileus virginianus, WTD). Specifically, sampled individuals representing the phenotypic extremes from an island population of WTD for antler and body size traits. Our results showed a largely homogenous genome between extreme phenotypes for each trait, with many highly differentiated regions throughout the genome, indicative of a quantitative model for polygenic traits. We identified and validated several potential QTL of putatively small-to-moderate effect for each trait, and discuss the potential for real-world application to conservation and management. Author Keywords: evolution, extreme phenotypes, genetics, genomics, quantitative traits, sexual selection
Do birds of a feather flock together
Populations have long been delineated by physical barriers that appear to limit reproduction, yet increasingly genetic analysis reveal these delineations to be inaccurate. The eastern and mid-continent populations of sandhill cranes are expanding ranges which is leading to convergence and warrants investigation of the genetic structure between the two populations. Obtaining blood or tissue samples for population genetics analysis can be costly, logistically challenging, and may require permits as well as potential risk to the study species. Non-invasively collected genetic samples overcome these challenges, but present challenges in terms of obtaining high quality DNA for analysis. Therefore, methods that optimize the quality of non-invasive samples are necessary. In the following thesis, I examined factors affecting DNA quality and quantity obtained from shed feathers and examined population differentiation between eastern and mid-continent sandhill cranes. I found shed feathers are robust to environmental factors, but feather size should be prioritized to increase DNA quantity and quality. Further, I found little differentiation between eastern and mid-continent populations with evidence of high migration and isolation-by-distance. Thus, the two populations are not genetically discrete. I recommend future population models incorporate migration between populations to enhance our ability to successfully manage and reach conservation objectives. Author Keywords: feathers, genetic differentiation, non-invasive DNA, population genetics, population management, sandhill crane (Antigone canadensis)
Assessing Molecular and Ecological Differentiation in Wild Carnivores
Wild populations are notoriously difficult to study due to confounding stochastic variables. This thesis tackles two components of investigating wild populations. The first examines the use of niche modeling to quantify macro-scale predator-prey relationships in canid populations across eastern North America, while the second examines range-wide molecular structure in Canada lynx. The goal of the first chapter is to quantify niche characteristics in a Canis hybrid zone of C. lupus, C. lycaon, and C. latrans to better understand the ecological differentiation of these species, and to assess the impacts of incorporating biotic interactions into species distribution models. The goal of the second chapter is to determine if DNA methylation, an epigenetic marker that modifies the structure of DNA, can be used to differentiate populations, and might be a signature of local adaptation. Our results indicated that canids across the hybrid zone in eastern North America exhibit low levels of genetic and ecological differentiation, and that the importance of biotic interactions are largely lost at large spatial scales. We also identified cryptic structure in methylation patterns in Canada lynx populations, which suggest signatures of local adaptation, and indicate the utility of DNA methylation as a marker for investigating adaptive divergence. Author Keywords: Ecological Epigenetics, Ecological Genetics, SDM
Evaluation of silver nanoparticles (AgNPs) and anti-GD2-AgNP antibody-drug conjugates as novel neuroblastoma therapies
Neuroblastoma (NB) has one of the highest mortality rates in pediatric oncology due to relapsed and refractory disease. Current aggressive multi-modal treatments are inhibited by dose-limiting toxicities and are associated with late-effects and secondary malignancies, emphasizing the necessity for novel therapeutics. Uniquely, most NB cells highly express disialoganglioside (GD2) a cell surface glycolipid that can provide a target for tumour-specific delivery. This study demonstrates a comprehensive evaluation of silver nanoparticles (AgNPs) and the first preliminary evaluation of anti-GD2-AgNP antibody-drug conjugates (ADCs) against NB in vitro. This present study validates the potential for AgNPs as an anti-cancer agent against NB as AgNPs demonstrated preferential toxicity towards NB cells through metabolic inhibition and indicative morphological alterations, while a less tumorigenic cell line demonstrated resistance to AgNP treatment. Therefore, this work identified an AgNP cell-type-dependent cytotoxicity effect. Low conjugation efficiency of the anti-GD2 monoclonal antibody, 14.G2a, to NHS-activated AgNPs failed to exert greater toxicity than the AgNPs alone. Collectively, this thesis provides novel information regarding the anti-cancer effects of AgNPs against NB with recommendations for anti-GD2-AgNP ADCs. Author Keywords: ADC, Chemotherapy, GD2, Neuroblastoma, Silver nanoparticles
Frog Virus 3
Understanding the maintenance and spread of invasive diseases is critical in evaluating threats to biodiversity and how to best minimize their impact, which can by done by monitoring disease occurrences across time and space. I sought to apply existing and upcoming molecular tools to assess fluctuations in both presence and strain variation of frog virus 3 (FV3), a species of Ranavirus, across Canadian waterbodies. I explored the temporal patterns and spatial distribution of ranavirus presence across multiple months and seasons using environmental DNA techniques. Results indicate that ranavirus was present in approximately 72.5% of waterbodies sampled on a fine geographical scale (<10km between sites, 7,150 km2), with higher detection rates in later summer months than earlier. I then explored the sequence variability at the major capsid protein gene (MCP) and putative virulence gene (vIF-2α) of FV3 samples from Ontario, Alberta, and the Northwest Territories, with the premise of understanding pathogen movement across the landscape. However, a lack of genetic diversity was found across regions, likely due to a lack of informative variation at the chosen genetic markers or lack of mutation. Instead, I found a novel FV3-like ranavirus and evidence for a recombinant between FV3 and a ranavirus of another lineage. This thesis provides a deeper understanding into the spatio-temporal distribution of FV3, with an idea of how widespread and threatening ranaviruses are to amphibian diversity. Keywords: ranavirus, frog virus 3, amphibians, environmental DNA, phylogenetics, wildlife disease, disease surveillance, major capsid protein, vIF-2α Author Keywords: amphibians, environmental DNA, frog virus 3, phylogenetics, ranavirus, wildlife disease
Using environmental DNA (eDNA) metabarcoding to assess aquatic plant communities
Environmental DNA (eDNA) metabarcoding targets sequences with interspecific variation that can be amplified using universal primers allowing simultaneous detection of multiple species from environmental samples. I developed novel primers for three barcodes commonly used to identify plant species, and compared amplification success for aquatic plant DNA against pre-existing primers. Control eDNA samples of 45 plant species showed that species-level identification was highest for novel matK and preexisting ITS2 primers (42% each); remaining primers each identified between 24% and 33% of species. Novel matK, rbcL, and pre-existing ITS2 primers combined identified 88% of aquatic species. The novel matK primers identified the largest number of species from eDNA collected from the Black River, Ontario; 21 aquatic plant species were identified using all primers. This study showed that eDNA metabarcoding allows for simultaneous detection of aquatic plants including invasive species and species-at-risk, thereby providing a biodiversity assessment tool with a variety of applications. Author Keywords: aquatic plants, biodiversity, bioinformatics, environmental DNA (eDNA), high-throughput sequencing, metabarcoding
De novo transcriptome assembly, functional annotation, and SNP discovery in North American flying squirrels (genus Glaucomys)
Introgressive hybridization between northern (Glaucomys sabrinus) and southern flying squirrels (G. volans) has been observed in some areas of Canada and the USA. However, existing molecular markers lack the resolution to discriminate late-generation introgressants and describe the extent to which hybridization influences the Glaucomys gene pool. I report the first North American flying squirrel (genus Glaucomys) functionally annotated de novo transcriptome assembly with a set of 146,621 high-quality, annotated putative species-diagnostic SNP markers. RNA-sequences were obtained from two northern flying squirrels and two southern flying squirrels sampled from Ontario, Canada. I reconstructed 702,228 Glaucomys transcripts using 193,323,120 sequence read-pairs, and captured sequence homologies, protein domains, and gene function classifications. These genomic resources can be used to increase the resolution of molecular techniques used to examine the dynamics of the Glaucomys hybrid zone. Author Keywords: annotation, de novo transcriptome, flying squirrels, high-throughput sequencing, hybridization, single nucleotide polymorphisms


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