Environmental and Life Sciences

We studied the physiological and molecular responses of lab-grown Daphnia pulex to shifts in dietary (carbon, nitrogen, phosphorus, and cyanobacteria) and growth media (calcium) nutrient supply using a set of potential biomarkers via qPCR. Each examined nutrient had a strong effect on Daphnia mass-specific growth rate, and we found significant dose-dependent effects of treatment level (medium and low) on the gene expression of selected indicator genes. Furthermore, linear discriminant analysis models using different combinations of treatment levels could separate the animals between nutritional treatments with 86-100% prediction accuracy. This would suggest that Daphnia appear able to respond to nutrient limitation by adjusting their growth rate and associated molecular pathways to deal with an insufficient supply of nutrients. While this study provides valuable information regarding Daphnia's ability to adjust physiological and molecular processes under controlled laboratory conditions, more validation needs to be conducted before applying these potential biomarkers to wild populations to assess the type and intensity of nutritional stress.

Population cycles are characterized by predictable temporal oscillations in population size and are influenced by densities of both predators and prey. These oscillations are influenced by the predator functional response, i.e. the influence of prey density on predator kill rate. The Canada lynx (Lynx canadensis) is a predatory mammal with cyclic northern populations driven by snowshoe hare (Lepus americanus) density. Despite some understanding of the drivers of lynx cycles, we lack understanding of how lynx hunting behaviour, including kill site selection, is influenced by the spatio-temporal distribution of prey. These concepts are explored in chapter one of this thesis.

In chapter two, we (coauthors and I) built on work in Kluane region of the Yukon where lynx and hare populations have been tracked through several population cycles. Over six winters, we deployed GPS collars on >40 individual lynx, some of which were fitted with satellite transmitters, accelerometers, and audio recorders. We validated the use of these technologies for identifying hare kills with an accuracy of >87%. This validation is the foundation for chapter three of this dissertation.

In chapter three, we investigated the drivers of spatial variation in lynx kills. Using snow track transects through four winters of declining hare density, we developed a robust model of habitat-specific hare abundance over time. Using model predictions, in combination with lynx Utilization Distributions derived from GPS locations and related habitat associations, we determined the importance of hare abundance, lynx spatial use, and landscape characteristics such as vegetation density in determining patterns of lynx kills and space use. Lynx kill sites were most strongly predicted by lynx space use, followed by the relative abundance of hares, an index of tree cover density, and elevation. Lynx space use itself was not strongly predicted by hare relative abundance, but rather by a shift to the use of more open habitats when hares were abundant to higher use of denser habitats as hare populations declined; this apparently corresponded to temporal changes in hare distribution. This thesis helps to disentangle the drivers of spatio-temporal variation in predator foraging behaviour, with important implications for understanding predator-prey dynamics.

Author Keywords: animal behaviour, biologging, foraging, habitat selection, Lynx canadensis, predator-prey dynamics

Flavohemoglobins are enzymes primarily implicated in nitrosative stress due to their high nitric oxide (NO) dioxygenase activity and transcriptional upregulation in response to NO. Giardia intestinalis assemblages A, B, and E possess flavohemoglobins (gFlHb) that may function beyond their NO dioxygenase activity, potentially contributing to oxidative stress regulation, as transcriptional profiling revealed that peroxide also induces gFlHb expression. This study investigates gFlHb's NADH oxidase activity in the absence of NO, structural interactions with lipids, and response to reactive oxygen species. Minor differences in NADH oxidase activity among assemblages were observed, and their susceptibilities to inhibition were assessed to evaluate gFlHb as a potential therapeutic target against Giardia infection. Under aerobic conditions, we observed that gFlHb generates hydrogen peroxide, a surprising finding suggesting a self-regulating feedback mechanism involving reactive oxygen species and heme degradation. These findings provide new insight into the role of flavohemoglobins in microaerotolerant parasites like Giardia.

Author Keywords: flavohemoglobin, Giardia intestinalis, heme, hydrogen peroxide, NADH, oxidative stress

DNA methylation (DNAm) is a useful indicator of phenotypic expression and diversity, and can potentially inform adaptations to environmental changes. This thesis uses epigenetic techniques to investigate the mechanisms underlying phenotypic variation in white-tailed deer, black bear, and mountain goat, with a particular focus on age and body size. In the second chapter, we aimed to contribute to wildlife monitoring by developing epigenetic clocks, or predictive models of age, and diagnostic markers of age class and sex. In the third chapter, we aimed to investigate the involvement of DNAm in body size variation of white-tailed deer by developing a model predictive of hindfoot length, and by identifying CpGs and genes that may be involved in hindfoot length variation. My results indicate that DNAm is an effective predictive marker of various phenotypes in these North American large mammals, and that epigenetic methods offer valuable insights for managing human impacts on wildlife.

Author Keywords: Age, Body Size, DNA Methylation, Epigenetics, Phenotypes, Population Genetics

The diversification of mammals has been shaped by climatic fluctuations and geological changes over millions of years. Among them, shrews (Soricidae) stand out as one of the most diverse mammalian lineages. Shrews are found on most continents and have evolved remarkable adaptations at the species and population levels. Although evolutionary studies of shrews have been limited by a lack of genomic resources, this work aimed to address this gap by developing such resources and using various omics approaches to explore adaptation and divergence in shrews, with a particular focus on an isolated population on Bon Portage Island (BPI), Nova Scotia, Canada. BPI shrews exhibit distinctive foraging habits and possess a unique allele for a dietary enzyme, which may represent an adaptation to their specialized diet. My research involved assembling and annotating de novo genomes from three shrew species. In Chapter 2 I conducted a comparative genomic analysis of 20 mammals (including four shrew species) to identify lineage-specific adaptations including accelerated regions, gene family expansions, and positively selected genes. I found shrew-specific variants in genes associated with the nervous, metabolic, and auditory systems, which may underlie some of their unique traits. In Chapter 3, I examined morphological and epigenetic divergence between mainland and island populations of masked shrews (Sorex cinereus), including BPI. Island shrews exhibited smaller body sizes and signs of accelerated biological aging, marked by DNA methylation differences enriched in developmental and digestive pathways. Chapter 4 focused on analyzing genome-wide SNP data to identify regions of differentiation, alongside RNA-seq data to perform a differential gene expression analysis between BPI shrews and other populations. The results from both analyses revealed patterns of differentiation in genes associated with fatty acid metabolism and metabolic regulation that are likely linked to their specialized, largely marine-based diet. Additionally, I reconstructed the demographic history of Nova Scotia masked shrew populations, revealing that the divergence of the BPI population appears to coincide with rising sea levels following the last glacial maximum. These findings shed light on mechanisms of adaptation and divergence, illustrating how ecological pressures, geographic isolation, and dietary specialization shape genomic, epigenomic, and transcriptomic landscapes.

Author Keywords: comparative genomics, epigenetics, genome assembly, island syndrome, population genomics, transcriptomics

Recovery of forest soils from chronic acidification can be enhanced with the use of non-industrial wood ash (NIWA). Non-industrial wood ash is alkaline and contains high concentrations of macronutrients, but trace metal concentrations must be evaluated to limit risk of metal toxicity following application. Additionally, understanding how different forest ecosystem components respond to NIWA is essential to inform current policy regulating its use as a soil amendment. This study evaluated the response of sugar maple (Acer saccharum) sap yield and chemistry, the response of soils beneath maple, American beech (Fagus grandifolia) and mixed species canopies, and maple and beech fine roots, foliage, seedling abundance, and understory vegetation abundance and composition to an application of 6 Mg ha-1 NIWA. Eight 40 x 40 m plots were established in a hardwood stand in Bracebridge, Ontario and were sampled prior-to and up to two years following application of NIWA (n = 4). Non-industrial wood ash significantly increased organic horizon soil pH and macronutrient (Ca, Mg, and K) concentrations with increases in Mg and K extending to the mineral soils. Significantly higher concentrations of some trace metals (Al, Fe, Mn, Cd, Cu, Pb, Zn) were also observed, but these were restricted to the organic horizons. Sugar maple sap, pH, and sweetness were unaffected by NIWA application, and while increases were observed in nutrient and metal concentrations in sap, the differences were small and variable between years, and all concentrations were consistent with those commonly found in maple sap. Fine root biomass of maple and beech trees was not affected by NIWA application, but higher concentrations of K and Mg were observed in the roots of both species, consistent with higher concentrations observed in the mineral soil horizons beneath both species' canopies. Only significant increases were observed in K in sugar maple foliage. Both critical foliar concentrations and diagnosis and recommendation integrated system (DRIS) norms for sugar maple did not indicate mineral nutrient deficiencies at this site; although this site was acidic and nutrient-poor, this may account for the lack of differences observed, particularly between species. Changes observed in understory vegetation were driven by years rather than between treatments. These results suggest that moderate doses of NIWA can provide significant decreases in soil acidity and increase nutrient availability, with limited increases in metal concentrations that are primarily restricted to the organic horizons.

Author Keywords: American beech, metal toxicity, Non-industrial wood ash (NIWA), sap sweetness, sap yield, sugar maple

Microsatellites and microhaplotypes are genetic markers that, through DNA amplicon sequencing, generate genotypes to distinguish between individuals from wildlife populations. Here, these markers were sequenced in caribou (Rangifer tarandus) specimens using Oxford Nanopore's MinION DNA sequencer for the first time. Microsatellite loci previously sequenced with an Illumina MiSeq were compared to MinION Mk1B sequencing data for the same samples/loci, revealing highly consistent microsatellite characterization across platforms. Additionally, a novel panel of caribou microhaplotype loci was developed and sequenced on the MinION Mk1B and Illumina MiSeq. Microhaplotype characterization of the same samples revealed that ambiguous read distributions for the top 3 reads per locus is a key challenge, particularly for the MinION, that hinders concordant haplotype calls across platforms. Potential reasons for this ambiguity include duplicated gene regions and PCR errors. Removing suspected duplicated gene regions and reducing the number of PCR cycles during target DNA amplification may mitigate this problem.

Author Keywords: Conservation Genomics, DNA Profiling, Microhaplotypes, Microsatellites, Nanopore Sequencing, Wildlife Monitoring

Agricultural and urban land uses have been linked to the recent resurgence of eutrophication and salinization issues in the lower Great Lakes. This thesis examined the relationship between watershed land use and stream nitrate-nitrogen (NO3-N), total phosphorus (TP), and chloride (Cl) concentrations across southern Ontario. Using a self-organizing map analysis, the watersheds were classified into eight distinct spatial clusters, representing four agricultural, two urban, and two natural clusters. Agricultural clusters under intensive row crop agriculture exhibited NO3-N and TP concentrations up to twelve and five times higher, respectively, than the most natural-dominated cluster. Urban clusters had Cl concentrations up to nine times greater than the natural-dominated clusters. Three agricultural land use practices, namely continuous corn-soybean rotation, synthetic fertilizer application, and tile drainage, were positively correlated with stream NO3-N concentrations, whereas Cl concentrations increased with urban area and human population density. This thesis also characterized sampling trends of the provincial stream water quality monitoring program and found that sampling frequency has declined since the mid-1990s, while current sites are monitored almost exclusively during the ice-free period. Sampling year-round is critical to capture seasonal variations in NO3-N and Cl, while sampling across a full range of flow conditions is important for describing TP. Exclusion of sampling sites in close proximity of downstream municipal wastewater treatment plants and greenhouses can help isolate and better understand water quality impacts of non-point sources. Although intensive agricultural watersheds in southwestern Ontario draining into Lake Erie remain a priority for research and management, regions experiencing row crop expansion such as along the northern shore of Lake Ontario as well as rapidly urbanizing areas require further attention as these land use shifts will likely increase stream NO3-N and Cl concentrations, placing further pressure on water resources in the lower Great Lakes.

Author Keywords: Chloride, Nitrogen, Phosphorus, Self-organizing map, Southern Ontario, Water quality

Diversified agroecosystems supporting greater genetic, structural, and functional diversity improve soil health and ecosystem function. However, there is limited understanding of how multiple forms of diversification, such as mixing cover crop species and adding arbuscular mycorrhizal fungi (AMF), alter belowground carbon supply to soil. In a controlled environment experiment using rhizoboxes, I investigated the belowgound response of cover crops – red clover (Trifolium pratense) and barley (Hordeum vulgare) – grown in monoculture or mixture, with and without AMF inoculation. Root morphological and mycorrhizal traits that characterize the hypothesized root economics spectrum (RES) were integrated with novel sampling of dissoved organic carbon fluxes and easily extractable glomlin in rhizosphere soil. Results revealed species-specific shifts on the RES suggesting that diversification through species mixing and AMF additions can alter belowground carbon allocation pathways, with potential implacations for plant performance and soil carbon stabilization in agroecosystems.

Author Keywords: Arbuscular mycorrhizal fungi, carbon sequestration, cover crops

Trait variation in wild populations is shaped by the interaction of genetics and the environment. Given these interactions, this thesis explored trait variation in white-tailed deer from two angles: one focused on broad-scale environmental factors, the other on fine-scale genetic mechanisms. The first chapter investigated how climate and habitat variation influence antler size, morphology, and the distribution of record-scoring deer across Ontario, Canada. Our results showed that warmer temperatures and higher percentages of rangeland and forest landcover were linked to larger antlers, while harsher winters with more precipitation had negative effects. The second chapter described the development of novel SNP assays designed to target genetic markers associated with leucism and malocclusions in an isolated island population of white-tailed deer. Together, these results highlight the importance of considering both environmental and genetic factors to understand trait variation in white-tailed deer.

Author Keywords: antlers, Odocoileus virginianus, phenotypic variation, Runs of homozygosity, white-tailed deer