Kyle, Christopher J

Molecular and genomic tools provide a deeper understanding of the ecology and evolution of species and their capacity to adapt to changing selective pressures, where diversity is presumedly linked to higher fitness and evolutionary potential. Molecular tools can also illustrate how historical processes affect contemporary genetic variation to predict how current population trends may influence future genetic diversity. Genomic investigations increasingly extend beyond variation within host genomes to include diversity of their associated microbiomes, recognized to influence host/environment interactions and adaptation. Muskoxen (Ovibos moschatus) are iconic, Arctic herbivores of ecological, economic, and cultural significance. Demographically, most mainland muskox populations have remained stable or grown over the last century, yet the biggest herds, found on Victoria and Banks Islands (Nunavut and the Northwest Territories, Canada) have experienced recent and drastic population declines. These Arctic island population declines have been associated with warming trends leading to shifting ranges of forage biodiversity, and pathogen expansions directly associated with increased mortality. Genomic investigations have the potential to enhance understanding of these contrasting trends and the adaptive capacities of muskox to cope with rapid ecological change. In this thesis, I assess genetic, genomic, microbiome and diet diversity to better understand the ecology, and evolution of muskoxen. I found extremely low levels of genetic variation associated with population bottlenecks coinciding with major glaciation events and contemporarily low levels of gene flow among populations. Whole genome analyses identified signatures of selection between muskox populations, providing a genetic basis for the divergence of two previously proposed muskox subspecies. Significant differences in diversity, effective population size and inbreeding among subspecies suggests animals from Arctic islands and Greenland are more vulnerable to environmental change. Molecular investigations of diet and microbiome diversity reflected unique capacities of muskoxen to survive on high-fiber forage and exploit shifts in Arctic vegetation that may include continued shrubification. Overall, these data provide insight into the complex relationship between genetic diversity and changing environments, setting a foundation for expanded future investigations of muskox seeking to promote the future viability of this species.

Author Keywords: Genetic Diversity, Genome Assembly, Metabarcoding, Microsatellites, Muskox, Persistence

Heterogeneous environments impose discordant selective pressures on natural populations, where disparate biotic/abiotic factors and variable population connectivity, yield mosaic patterns of genetic variation on the landscape. The ability to maintain or change genetic mosaics of populations becomes key to persistence, as species increasingly need to adapt to rapidly changing environmental and human-associated selective pressures. Specifically, infectious diseases can impose strong and rapid selective pressures on populations, where anthropogenic disruptions of co-evolutionary patterns and altered distributions of hosts and pathogens exacerbate disease risk. Genomic tools provide means to evaluate disease-associated impacts on the genetic landscape of host populations and facilitate implementation of informed conservation efforts. In this thesis, I evaluate disease dynamics in: 1) a long-standing arctic rabies/arctic fox (Vulpes lagopus) system affected by influxes of red fox (V. vulpes), and 2) an invasive bat pathogen system, where the North American introduction of Pseudogymnoascus destructans (Pd) has had variable impacts on bat species and populations. In these systems, signatures of host selection were estimated from temporal and spatial shifts in allelic diversity within genomic regions associated with immune response, highlighting different host mechanisms to enzootic and invasive diseases. In the arctic rabies/fox system, pathogen variants did not influence red fox local disease responses, reflecting more recent expansions of this host to Arctic regions. In contrast, arctic fox revealed genomic patterns consistent with long-term, co-evolutionary processes. In Pd/bat systems, genomic evidence supported the hypothesis that eastern small-footed bats (Myotis leibii) were inherently resistant or tolerant to Pd, the causative agent of white-nose syndrome (WNS). In contrast, WNS-impacted little brown bat (M. lucifugus) populations had varied genomic impacts subsequent to strong selective sweeps from disease. My research illustrates how immunogenetic profiling, in context of demographic processes inferred from neutral genetics, enhances understanding of the varied impacts of changing disease landscapes on host populations/species; insights relevant to other host-pathogen systems. Building on this thesis, future explorations of low coverage genomes, host-imposed reciprocal selection, and impacts on methylation, transcriptomic and proteomic patterns associated with shifts in genetic diversity, would enable more holistic understanding of the geographic mosaics within these disease systems.

Author Keywords: Disease Dynamics, High-throughput Sequencing, Immune System, Natural Selection, Population Genetics, Reduced Representation Sequencing

Rapidly changing landscapes introduce challenges for wildlife management, particularly for large mammal populations with long generation times and extensive spatial requirements. Understanding how these populations interact with heterogeneous landscapes aids in predicting responses to further environmental change. In this thesis, I profile American black bears using microsatellite loci and pooled whole-genome sequencing. These data characterize gene flow directionality and functional genetic variation to understand patterns of dispersal and local adaptation; processes key to understanding vulnerability to environmental change. I show dispersal is positively density-dependent, male biased, and influenced by food productivity gradients suggestive of source-sink dynamics. Genomic comparison of bears inhabiting different climate and forest zones identified variation in genes related to the cellular response to starvation and cold. My thesis demonstrates source-sink dynamics and local adaption in black bears. Population management must balance dispersal to sustain declining populations against the risk of maladaptation under future scenarios of environmental change.

Author Keywords: American black bear, Dispersal, Functional Genetic Variation, Gene Flow Directionality, Genomics, Local Adaptation

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

Identifying population declines and mitigating biodiversity loss require reliable monitoring techniques, but complex life histories and cryptic characteristics of anuran species render conventional monitoring challenging and ineffective. Environmental DNA (eDNA) detection is a highly sensitive and minimally invasive alternative to conventional anuran monitoring. In this study, I conducted a field experiment in 30 natural wetlands to compare efficacy of eDNA detection via qPCR to three conventional methods (visual encounter, breeding call, and larval dipnet surveys) for nine anuran species. eDNA and visual encounter surveys detected the greatest species richness, with eDNA methods requiring the fewest sampling events. However, community composition results differed among methods, indicating that even top performing methods missed species detections. Overall, the most effective detection method varied by species, with some species requiring two to three methods to make all possible detections. Further, eDNA detection rates varied by sampling season for two species (A. americanus and H. versicolor), suggesting that species-specific ecology such as breeding and larval periods play an important role in eDNA presence. These findings suggest that optimized monitoring of complex anuran communities may require two or more monitoring methods selected based on the physiology and biology of all target species.

Author Keywords: amphibian, anuran, conventional monitoring, eDNA, environmental DNA, species richness

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

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

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

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