Genetics

The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are a family of fatal neurodegenerative disorders that primarily affect children. Several subtypes of NCLs have been reported, each being caused by a mutation in a distinct ceroid lipofuscinosis neuronal (CLN) gene; this results in aberrant lysosome function and the accumulation of lipoprotein aggregates (known as ceroid lipofuscin) within cells. Several innate cellular pathways exist to alleviate the stress caused by the buildup of aggregates. The endoplasmic reticulum (ER) is an essential organelle in this process because it is responsible for maintaining cellular homeostasis through protein production, quality control, and regulating several signalling pathways. The unfolded protein response (UPR) consists of several conserved pathways devoted to attenuating ER stress caused by an accumulation of misfolded proteins or aggregates; at the center of this stress response is GRP78, a molecular chaperone that binds to misfolded proteins to facilitate proper folding. The social amoeba Dictyostelium discoideum is an excellent model system for studying NCLs as it encodes more CLN-like proteins when compared to other classical model organisms (e.g., yeast, worm, fruit fly). In this study, D. discoideum was used to elucidate the effects of ER stress and build an understanding of how cells cope with increased stress. Beyond this, ER stress in D. discoideum models for CLN3 disease and CLN5 disease were evaluated. First and foremost, during the induction of ER stress by tunicamycin, there was an increase in intracellular and extracellular amounts of Grp78 accompanied by an increase in stress-related changes to the ER. Furthermore, models of CLN3 disease and CLN5 disease displayed increased amounts of Grp78 as well as a disrupted ER morphology. Interestingly, wildtype D. discoideum, AX3 cells, treated with tunicamycin displayed a similarly disrupted ER when compared to CLN models. Finally, when subjected to tunicamycin-induced ER stress, these NCL models displayed a trend towards increased Grp78 amounts, however, these cells appear to have a reduced sensitivity to tunicamycin-induced stress compared to wild-type cells. In summary, this study highlights D. discoideum as a model for studying ER stress through the conserved role of Grp78 in the stress response and concludes that an aberrant ER stress underlies the pathology of the NCLs.

Author Keywords: Batten disease, Dictyostelium discoideum, ER stress, GRP78, neuronal ceroid lipofuscinoses (NCLs)

Globally, wildlife populations are experiencing increasing rates of range loss, population decline, and extinction. Caribou (Rangifer tarandus) have experienced dramatic declines in both range and population size across Canada over the past century. Boreal caribou (R. t. caribou), one of twelve Designatable Units, have lost approximately half of their historic range in the last 150 years, particularly along the southern edge of their distribution. Despite this northward contraction, some populations have persisted at the trailing range edge, over 150 km south of the boreal continuous range (BCR) in Ontario, along the coast and near-shore islands of Lake Superior. Better understanding the population structure and evolutionary history of caribou in the Lake Superior range (LSR) could help to inform conservation and management actions, such as the delineation of conservation or management units or translocations between populations. In this thesis, I use whole genome sequences from boreal, eastern migratory and barren-ground caribou sampled in Manitoba, Ontario, and Quebec to investigate evolutionary history and population structure. I discovered that the LSR caribou form a distinct group but also some evidence of gene flow with the BCR. Notably, caribou from the LSR demonstrated relatively high levels of inbreeding (measured as Runs of Homozygosity; ROH) and genetic drift, which may contribute to the differentiation observed between caribou occupying the two ranges. Despite inbreeding, the LSR caribou retained Heterozygosity Rich Regions (HRR). I found genomic structure among caribou populations from the LSR and BCR but found these two ranges had similar demographic histories. My analyses indicate that the LSR caribou display distinct genomic characteristics but share ancestry with the BCR, with historical gene flow between these two ranges. Collectively, this dissertation characterizes the population structure and evolutionary history of caribou from the southernmost range in Ontario, providing key insights for the conservation and management of these small and isolated populations.

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

Access to human blood for forensic research and training in bloodstain pattern analysis (BPA) can be difficult due to many ethical, safety and cost concerns. Mammalian blood alternatives can be sourced, especially from local and willing abattoirs, but some concerns remain, and the added difficulties of high variation and species-specific differences in cellular components pose other issues. Therefore, synthetic alternatives to human blood provide practical options for the BPA community. This thesis explores the use of alginate hydrogels as a base material for forensic blood substitute (FBS) development. Hydrogels are first explored as a suitable environment for DNA stability and functionality and compared to other polymer systems. The ability of DNA to remain intact while undergoing electrospray ionization (ESI) is also investigated. The FBS design focuses on mimicking the fluid properties and genetic capabilities of whole human blood – a material not developed in FBSs previously. ESI was used to develop microparticles (MPs) that serve as cellular components of human blood (the red blood cells – RBCs, and white blood cells – WBCs). The microparticles were ionically crosslinked using calcium to provide small MPs (RBCs) or covalently crosslinked with functional DNA to provide larger WBC-like functional particles. The integration of these novel MPs into alginate-based materials is optimized and their use in BPA scenarios is explored. The FBS is tested in BPA scenarios of dripping experiments, impact patterns, and the ability to extract and amplify the contained DNA. In addition, the stability (or shelf-life) of the FBS was also assessed. The FBS exhibited similar spreading ratios to blood and demonstrated feasibility in use for impact angle (a) determination and impact pattern creation. Importantly, the DNA contained within the FBS could be processed with analogous protocols used in DNA evidence processing, enhancing its applicability to BPA research and training.

Author Keywords: Alginate hydrogels, Bloodstain pattern analysis, Electrospray ionization, Forensic blood substitutes, Forensic materials, Synthetic DNA design

Hybridisation is an important evolutionary mechanism with diverse outcomes, including the formation of new lineages, the exchange of alleles between species, or their extinction through genetic swamping. In some cases, hybrids exhibit higher fitness than their parental species, which can lead to hybrid invasions that threaten ecosystems. In North America, hybridisation between the native cattail Typha latifolia and the non-native T. angustifolia produces the hybrid T. × glauca, which is a highly impactful invader in wetlands across large areas of southern Canada and northern USA; contrastingly, in Europe and Asia, where its parental species also co-occur, T. × glauca is rare to non-existent. This thesis examines the evolutionary history of T. latifolia and T. angustifolia, which, in addition to being the parental species of T. × glauca, are two of the most globally widespread Typha species, a genus of plants foundational to wetlands. First, we developed genomic resources, including ~12M nuclear SNPs and plastome assemblies to facilitate genetic research on Typha; we also described a cost-effective library preparation and genotyping protocol that makes population genetic studies of freshwater plants accessible. Then, we applied those genomic resources to investigate the roles of drift, selection, and hybridisation in the divergence of T. angustifolia, T. latifolia, and their sister species, T. domingensis and T. shuttleworthii. We found that speciation in these taxa was driven by drift under allopatry, resulting from historical bottlenecks, and that natural selection has played a minimal role in the divergence of these species; additionally, we observed introgressive hybridisation from T. latifolia into T. angustifolia. Finally, we reconstructed the demographic histories of T. angustifolia and T. latifolia from North America and Europe. We observed that reproductive isolation is strong in Europe, where the two species have potentially been sympatric for ~800,000 years, and weak in North America, where they have been sympatric for only a few centuries. Our results exemplify how the divergence and demographic histories of species can correlate with their strength of reproductive isolation. We emphasise that preventing invasions by hybrids will require limiting the movement of Typha and other historically allopatric species, which likely lack reproductive barriers.

Author Keywords: biological invasions, cattails, demographic histories, evolutionary histories, population genomics, reproductive isolation

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.

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

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

Animal migrations are ubiquitous and one of the most threatened ecological processes globally. Because of the multifaceted nature of migration – seasonal movements between home ranges – it can be difficult to tease apart the underlying mechanisms influencing this behaviour. It is necessary to understand these mechanisms, not only to deepen our fundamental understanding of migration in animals, but also because migrations in many species are vulnerable to environmental change. In Chapter 2, I first systematically identify the broad proximate drivers of migration and offer generalities across vertebrate taxa. I quantitatively reviewed 45 studies and extracted 132 observations of effect sizes for internal and external proximate drivers that influenced migration propensity. Through this meta-analysis, I found that internal and external drivers had a medium and large effect, respectively, on migration propensity. Predator abundance and predation risk had a large effect on migration propensity, as did individual behaviour. Of the studies that examined genetic divergence between migrant and resident populations, 64% found some genetic divergence between groups. In Chapter 3, I explore the genetic basis for migration and identified genes associated with migration direction from pooled genome-wide scans on a population of 233 migrating female mule deer (Odocoileus hemionus) where I identified genomic regions including FITM1, a gene linked to the formation of lipids, and DPPA3, a gene linked to epigenetic modifications of the maternal line. These results are consistent with the underlying genetic basis for a migratory trait which contributes to the additive genetic variance influencing migratory behaviours and can affect the adaptive potential of a species. Finally, in Chapter 4 I used a pedigree-free quantitative genetic approach to estimate heritability and sources of environmental variation in migration distance, timing, and movement rate of the same population of mule deer. I found low heritability for broad patterns of migration timing, and greater variation in heritability for behaviours during migration, with low heritability for distance and duration and high heritability for movement rate along the route. Insights into the genetic and environmental sources of variation for migration are critical both for the eco-evolutionary dynamics of migration behaviour, and for the conservation of species whose migrations may be vulnerable to environmental change. My thesis reveals that broad patterns of migration are driven largely by environmental effects while within these broad patterns, migration behaviour is driven to a measurable degree by genetic variation.

Author Keywords: heritability, migration, Odocoileus hemionus, reduced representation sequencing, whole genome sequencing