Graduate Theses & Dissertations

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)
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
Enhancing post-mortem interval estimates
The growth of immature insects that develop on human remains can be used to estimate a post-mortem interval (PMI). PMI estimate confidence is negatively affected by: larval killing and preservation methods altering their size, limited morphological parameters to assess larval growth and therefore age, and few available alternate species development data. I compared live specimens to preserved specimens of the same development stages to assess the effects of killing-preservation techniques on morphology, and I introduce a new method that uses digital photography to examine maggot mouthparts for stage grading of Phormia regina. Digital photographic methods enable live insects to be quantified and improve approximations of physiological age. I then use these digital methods to produce a growth-rate model for a beetle commonly found on human remains, Necrodes surinamensis, providing data for PMI estimates that was previously unavailable. Author Keywords: Forensic Entomology, Insect development, Morphometrics, Necrodes surinamensis, Phormia regina, Postmortem interval
Enhancing forensic entomology applications
The purpose of this thesis is to enhance forensic entomology applications through identifications and ecological research with samples collected in collaboration with the OPP and RCMP across Canada. For this, we focus on blow flies (Diptera: Calliphoridae) and present data collected from 2011-2013 from different terrestrial habitats to analyze morphology and species composition. Specifically, these data were used to: 1) enhance and simplify morphological identifications of two commonly caught forensically relevant species; Phormia regina and Protophormia terraenovae, using their frons-width to head-width ratio as an additional identifying feature where we found distinct measurements between species, and 2) to assess habitat specificity for urban and rural landscapes, and the scale of influence on species composition when comparing urban and rural habitats across all locations surveyed where we found an effect of urban habitat on blow fly species composition. These data help refine current forensic entomology applications by adding to the growing knowledge of distinguishing morphological features, and our understanding of habitat use by Canada’s blow fly species which may be used by other researchers or forensic practitioners. Author Keywords: Calliphoridae, Ecology, Forensic Entomology, Forensic Science, Morphology, Urban
Investigating wheat rust virulence evolution through transcriptome analysis of a recently emerged race of Puccinia triticina
Puccinia triticina, wheat leaf rust (WLR), is the most economically damaging fungal rust of wheat on a global scale. This study identified transcriptome changes in a recently emerged race of WLR in Ontario with a new virulence type relative to a possible ancestor race. Also, this study focused on detecting variation in candidate virulence genes and uncovering novel insight into WLR virulence evolution. Various race-by-variety interactions were evaluated using RNA-seq experiments. A list of genes with statistically significant expression changes in each comparison was prepared and predicted effectors were retained for further analysis. Proteins with nonsynonymous substitutions were run through BLASTx to identify potential orthologs. Over 100 candidate effectors with a 2-fold or higher change in transcript level were identified. Seven of these candidate effector genes were recognized to contain single nucleotide polymorphisms (SNPs) which altered the amino acid sequence of the resulting protein. The information gained may aid in targeted breeding programs to combat new WLR races as well as provide the basis for functional analysis of WLR using potential orthologs in a model basidiomycete. Author Keywords: effectors, RNA-seq, rust fungi, SNPs, transcriptome, wheat leaf rust
Exonic Trinucleotide Microsatellites
Trinucleotide repeats (TNRs) are a class of highly polymorphic microsatellites which occur in neutral and non-neutral loci and may provide utility for individual- and population-identification. Exonic trinucleotide motifs, in particular, offer additional advantages for non-human species that typically utilize dinucleotide microsatellite loci. Specifically, the reduction of technical artifacts, greater separation of alleles and greater specificity of amplification products leading to more efficient multiplexing and cross-taxa utilization. This study aims to identify and characterize polymorphic trinucleotide repeats and conserved primer sequences which are conserved across Cervidae (deer) species and their potential for individual identification in forensic wildlife investigations. Chapter one provides a broad introduction to trinucleotide microsatellites, chapter two deals with data-mining TNRs and chapter three applies the identified TNRs as genetic markers for individual identification. Results demonstrate proof-of-concept that exonic TNRs are capable of giving random match probabilities low enough to be employed in individual identification of evidentiary samples. Author Keywords: DNA typing, Exons, Genetic Markers, Individual Identification, Trinucleotide, Wildlife Forensics
Mitogenome characterization of the shortnose sturgeon (Acipenser brevirostrum) for international trade validation of aquaculture-reared caviar
Identifying the population origin of aquaculture-reared caviar is crucial for both conservation and management strategies of farmed fish but could also facilitate international trade of a CITES regulated product. Shortnose sturgeon (Acipenser brevirostrum) is the main source of caviar production in Atlantic Canada, from Breviro Caviar Inc. aquaculture facility. Shortnose sturgeon are also listed as a species-at-risk under the Species At Risk Act. Currently there is no genetic method for delineating wild from aquaculture-reared caviar. By targeting the mitochondrial genome (mitogenome) using novel long-range PCR primers and next-generation sequencing (NGS) methods we have successfully sequenced the full mitogenome of 37 shortnose sturgeon. The purpose of this study was to increase the resolution of diagnostic variation among populations and to validate Canadian aquaculture-reared stock from wild US populations. Results provided a previously unobserved novel control region haplotype in high frequency within both the aquaculture-reared and Saint John River wild sample sets. Similar frequencies were observed with whole mitogenome haplotypes. Diagnostic mitochondrial lineage found in high frequency within the captive Breviro Caviar Inc. population has the potential to allow caviar product from Breviro Caviar Inc. to be distinguished from protected US shortnose sturgeon populations. The application of full mitogenomic characterization provides the potential to further resolve differences between aquaculture and natural Canadian shortnose sturgeon stocks, US/Canadian populations and to contribute to future conservation strategies. Future research identifying signatures of selection on the mitogenome between captive and wild populations and across latitudinal gradients found within the species range. These novel methods have produced a proof-of-concept to provide a "farm-to-fork" validation and ecobrand of Breviro Caviar Inc. product and its aquaculture origin to support importation into US caviar markets. Author Keywords: aquaculture, mitogenome, next-generation sequencing, species-at-risk, sturgeon
SPATIAL AND TEMPORAL GENETIC STRUCTURE OF WOLVERINE POPULATIONS
Habitat loss and fragmentation can disrupt population connectivity, resulting in small, isolated populations and low genetic variability. Understanding connectivity patterns in space and time is critical in conservation and management planning, especially for wide-ranging species in northern latitudes where habitats are becoming increasingly fragmented. Wolverines (Gulo gulo) share similar life history traits observed in large-sized carnivores, and their low resiliency to disturbances limits wolverine persistence in modified or fragmented landscapes - making them a good indicator species for habitat connectivity. In this thesis, I used neutral microsatellite and mitochondrial DNA markers to investigate genetic connectivity patterns of wolverines for different temporal and spatial scales. Population genetic analyses of individuals from North America suggested wolverines west of James Bay in Canada are structured into two contemporary genetic clusters: an extant cluster at the eastern periphery of Manitoba and Ontario, and a northwestern core cluster. Haplotypic composition, however, suggested longstanding differences between the extant eastern periphery and northwestern core clusters. Phylogeographic analyses across the wolverine's Holarctic distribution supported a postglacial expansion from a glacial refugium near Beringia. Although Approximate Bayesian computations suggested a west-to-east stepping-stone divergence pattern across North America, a mismatch distribution indicated a historic bottleneck event approximately 400 generations ago likely influenced present-day patterns of haplotype distribution. I also used an individual-based genetic distance measure to identify landscape features potentially influencing pairwise genetic distances of wolverines in Manitoba and Ontario. Road density and mean spring snow cover were positively associated with genetic distances. Road density was associated with female genetic distance, while spring snow cover variance was associated with male genetic distance. My findings suggest that northward expanding anthropogenic disturbances have the potential to affect genetic connectivity. Overall, my findings suggest that (1) peripheral populations can harbour genetic variants not observed in core populations - increasing species genetic diversity; (2) historic bottlenecks can alter the genetic signature of glacial refugia, resulting in a disjunct distribution of unique genetic variants among contemporary populations; (3) increased temporal resolution of the individual-based genetic distance measure can help identify landscape features associated with genetic connectivity within a population, which may disrupt landscape connectivity. Author Keywords: conservation genetics, Holarctic species, landscape genetics, peripheral population, phylogeography, wolverine
Immunogenetic Responses of Raccoons and Skunks to the Raccoon Rabies Virus
Interactions between hosts and pathogens play a crucial role in their adaptation, evolution and persistence. These interactions have been extensively studied in model organisms, yet it is unclear how well they represent mechanisms of disease response in primary vectors in natural settings. The objective of my thesis was to investigate host-pathogen interactions in natural host populations exposed to raccoon rabies virus (RRV). RRV is endemic to North America, that causes acute encephalopathies in mammals and is commonly regarded as 100% lethal if untreated; however variable immune responses have been noted in natural reservoirs. In order to further understand variable immune responses to RRV, my thesis examined (i) potential immunogenetic associations to RRV using genes intimately associated with an immune response, (ii) the nature of immune responses triggered in the host after infection, and (iii) viral expression and genetic variation, to provide insight into factors that may influence RRV virulence. Immunogenetic variation of RRV vectors was assessed using major histocompatibility complex (MHC) DRB alleles. Associations were found between specific MHC alleles, RRV status, and viral lineages. Further, similarities at functionally relevant polymorphic sites in divergent RRV vector species, raccoons and skunks, suggested that both species recognize and bind a similar suite of peptides, highlighting the adaptive significance of MHC and contemporary selective pressures. To understand mechanisms of disease spread and pathogenesis, I screened for variation and expression of genes indicative of innate immune response and patterns of viral gene expression. RRV activated components of the innate immune system, with transcript levels correlated with the presence of RRV. These data indicate that timing of the immune response is crucial in pathogenesis. Expression patterns of viral genes suggest they are tightly controlled until reaching the central nervous system (CNS), where replication increases significantly. These results suggest previous molecular mechanisms for rabies host response derived from mouse models do not strictly apply to natural vector populations. Overall my research provides a better understanding of the immunological factors that contribute to the pathogenesis of RRV in a natural system. Author Keywords: immune response, major histocompatibility complex, rabies, raccoons, skunks, virus
Understanding Historical and Contemporary Gene Flow Patterns of Ontario Black Bears
Consequences of habitat loss and fragmentation include smaller effective population sizes and decreased genetic diversity, factors that can undermine the long-term viability of large carnivores that were historically continuously distributed. I evaluated the historical and contemporary genetic structure and diversity of American black bears (Ursus americanus) in Ontario, where bear habitat is largely contiguous, except for southern regions that experience strong anthropogenic pressures. My objectives were to understand gene flow patterns in a natural system still largely reflective of pre-European settlement to provide context for the extent of genetic diversity loss in southern populations fragmented by anthropogenic influences. Phylogeographic analyses suggested that Ontario black bears belong to a widespread "continental" genetic group that further divides into 2 subgroups, likely reflecting separate recolonization routes around the Great Lakes following the Last Glacial Maximum. Population genetic analyses based on individual genotypes showed that Ontario black bears are structured into 3 contemporary genetic clusters. Two clusters, located in the Northwest (NW) and Southeast (SE), are geographically vast and genetically diverse. The third cluster is less diverse, and spatially restricted to the Bruce Peninsula (BP). Microsatellite analyses revealed that the NW and SE clusters are weakly differentiated from each other relative to mitochondrial DNA findings, suggesting male-biased dispersal and isolation by distance across the province. I also conducted simulations to assess competing hypotheses that could explain the reduced genetic diversity on the BP, which supported a combination of low migration and recent demographic bottlenecks. I showed that management actions to increase genetic variation in BP black bears could include restoring landscape connectivity between BP and SE; however, the irreversible human footprint in the area makes regular translocations from SE individuals a more practical alternative. Overall, my work suggests that: 1) historical genetic processes in Ontario black bears were likely predominated by isolation by distance, 2) large mammalian carnivores such as black bears can become isolated and experience reduced diversity in only a few generations, and 3) maintaining connectivity in regions under increased anthropogenic pressures could prevent populations from becoming small and geographically and genetically isolated, and should be a priority for conserving healthy populations. Author Keywords: American black bear, carnivore, conservation genetics, Ontario, phylogeography, population genetics
Cytokinin Oxidase/Dehydrogenase (CKX) Gene Family in Soybeans (Glycine max)
Glycine max (soybean) is an economically important plant species that registers a relatively low yield/seed weight compared to other food and oil seed crops due to higher rates of flower and pod abortion. Alleviation of this abortion rate can be achieved by altering the sink strength of the reproductive organs of soybeans. Cytokinin (CK) plays a fundamental role in promoting growth of sink organ (flowers and seeds) by increasing the assimilate demand. Cytokinin oxidase/dehydrogenase (CKX) is an enzyme that catalyses the irreversible breakdown of active CKs and hence reduce the cytokinin content. The current thesis uncovers the members of CKX gene family in soybeans and the natural variations among CKX genes within soybean varieties with different yield characteristics. The identification of null variants of OsCKX2 that resulted in large yield increases by Ashikari et al. (2005) provided a rationale for current thesis. The soybean CKX genes along with the ones from Arabidopsis, Rice and Maize were used to construct a phylogenetic tree. Using comparative phylogeny, protein properties and bioinformatic programs, the potential effect of the identified natural variations on soybean yield was predicted. Five genes among the seventeen soybean CKXs identified, showed polymorphisms. One of the natural variations, A159G, in the gene GmCKX16 occurred close to the active site of the protein and was predicted to affect the activity of enzyme leading to higher accumulation of CKs and hence increased seed weight. Use of such natural variations in marker assisted breeding could lead to the development of higher yielding soybean varieties. Author Keywords: CKX, Cytokinins, Seed weight, Seed Yield, SNPs, Soybeans
Comparative Evaluation of Effective Population Size Genetic Estimation Methods in Wild Brook Trout (Salvelinus fontinalis) Populations
Effective population size (Ne) is a key concept in population genetics, evolutionary biology and conservation biology that describes an important facet of genetic diversity and the capacity of populations to respond to future evolutionary pressures. The importance of Ne in management and conservation of wild populations encouraged the development of numerous genetic estimators which rely on a variety of methods. Despite the number and diversity of available Ne methods, however, tests of estimator performance have largely relied on simulations, with relatively few tests based on empirical data. I used well-studied wild populations of brook trout (Salvelinus fontinalis) in Algonquin Park, Ontario as a model system to assess the comparative performance of multiple Ne estimation methods and programs, comparing the resultant Ne estimates against demographic population size estimates. As a first step, the genetic diversity and ancestry of wild brook trout populations was determined using 14 microsatellite loci. Genetic structure of brook trout populations showed variable contributions from historical supplemental stocking and also identified localized gene pools within and between watersheds, reflecting variable levels of connectivity and gene flow. Once the genetic ancestry and connectivity of populations had been resolved, single sample (point) and two samples (temporal) genetic estimators were used to estimate Ne of populations with pure native ancestry. Values obtained from genetic estimators utilizing both methods were variable within as well as among populations. Single sample (point) estimators were variable within individual populations, but substantially less than was observed among the temporal methods. The ratios of Ne to the estimated demographic population size (N) in small populations were substantially higher than in larger populations. Variation among estimates obtained from the different methods reflects varying assumptions that underlay the estimation algorithms. This research further investigated the effect of sampling effort and number of microsatellite loci used on Ne values obtained using the linkage disequilibrium (LD) estimation method. Ne estimates varied substantially among values generated from subsets of loci and genotyped individuals, highlighting the necessity for proper sampling design for efforts aiming to measure Ne. Despite the variation observed among and within estimation methods, the Ne concept is a valuable for the conservation and management of both exploited and endangered species. Author Keywords: Brook Trout, Effective population size, Genetic Diversity, Genetic Structure

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