Shafer, Aaron

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

Hybridization contributes to the genetic diversity and can impact speciation. This study investigates the genetic evidence of recent hybridization under climate change in sympatric populations of northern and southern flying squirrels in Ontario. Using low-coverage whole-genome sequences, my research examines the existing population structure and measures the genomic variation of the Glaucomys species. The global estimates of FST (0.308) and DXY (0.141) are indicative of substantial differentiation between the species. Measures of genetic diversity (π), differentiation (FST), and divergence (DXY) across the genome reveal insights into the divergent selection driving speciation. Results indicate an absence of contemporary hybridization or introgression at a site with longstanding sympatry. Across both species' genomes, signatures of selection align with four different scenarios for the formation of genomic landscapes of differentiation, shedding light on the complex speciation history of these flying squirrels. These findings enhance understanding of evolutionary dynamics, adaptation, speciation, and genetic differentiation.

Author Keywords: Genomic differentiation, Glaucomys, northern flying squirrel, southern flying squirrel, speciation

The neuronal ceroid lipofuscinoses (NCLs), collectively referred to as Batten disease, are a group of neurodegenerative diseases that affect all ages, primarily children. Batten disease is caused by mutations in 1 of the 13 ceroid lipofuscinosis neuronal (CLN) genes (CLN1-CLN8, CLN10-CLN14), each of which causes an NCL subtype when mutated. One of the NCL subtypes, CLN5 disease, is caused by mutations in the CLN5 gene. CLN5 is a soluble lysosomal protein that localizes to the endoplasmic reticulum (ER), the Golgi complex, the cytoplasm, and extracellularly. CLN5 has four putative molecular functions, including as a ceramide synthase, glycoside hydrolase, depalmitoylase, and bis(monoacylglycerol)phosphate synthase. CLN5 plays various roles within the cell, such as lipid metabolism, autophagy, and proteasome degradation. However, the function and the exact pathway in which CLN5 is involved are unclear. In addition, CLN5 is a secreted protein that, as shown via bioinformatics analysis, contains a signal peptide sequence. Furthermore, there are currently 70 CLN5 disease-causing mutations reported in the NCL mutation database. 12 CLN5 disease-causing mutations have been studied thus far in terms of their cellular impact, as well as the release of CLN5 to a certain extent. However, there is a lack of research into the functionality of the signal peptide in CLN5 and an in-depth analysis of the molecular impact of mutations in CLN5 disease. Consequently, this Ph.D. thesis focused on using comparative transcriptomics to reveal biological pathways affected by cln5-deficiency, revealing mechanisms that regulate the secretion of Cln5 and CtsD, and using Dictyostelium to gain insights into the molecular effects of mutations in CLN5 disease. Comparative transcriptomics reveal many differentially expressed genes that are linked to phenotypes observed in cln5-deficient cells and identified pathways affected in other CLN5 disease models, such as autophagy. Furthermore, novel findings, like affected expression of lysosomal enzymes and pathways, including secretion, are identified within the comparative transcriptomics analysis. Subsequently, this research also shows the secretory role of the signal peptide in Cln5 and CtsD. Finally, this Ph.D. thesis revealed that mutations in CLN5 disease affect the lysosomal biology and secretion of Cln5 and other lysosomal enzymes.

Author Keywords: Batten disease, CLN5, Dictyostelium discoideum, Enzymes, Lysosome, Secretion

Wildlife translocation programs are widely employed as a strategy to reintroduce extirpated species into regions they once inhabited but no longer do. Reintroduction programs can be successful at re-establishing extirpated populations and also provide unique opportunities to study post-reintroduction population dynamics and behavioural ecology. The wild turkey (Meleagris gallopavo) is a forest generalist species that, prior to European colonization, inhabited much of the Carolinian zone in Ontario. This species was hunted to extirpation in the early 1900's and reintroduced in the mid-1980's through a series of wildlife trade agreements and coordinated trap and transfer efforts. Ontario's contemporary populations are seemingly thriving, with wild turkey harvest permitted in many regions of the province. However, given this species history of extirpation, understanding the size, distribution, and behavioural ecology of Ontario's reintroduced population of wild turkeys is essential to their long-term persistence in the province. We captured and radio-tagged 77 wild turkeys over four years in Peterborough, Ontario and studied their movement, sociality, and habitat preferences. My findings indicate that Ontario may contain relatively high densities of this species when compared with other parts of their range. My analyses also elucidated interesting aspects of this species habitat selection patterns within an anthropogenic landscape, in addition to novel findings surrounding wild turkey sociality and genetic structure.

Author Keywords: behaviour, genetics, Ontario, reintroduction, wild turkey, wildlife management

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

Hybridization contributes to the genetic diversity and can impact speciation. This study investigates the genetic evidence of recent hybridization under climate change in sympatric populations of northern and southern flying squirrels in Ontario. Using low-coverage whole-genome sequences, my research examines the existing population structure and measures the genomic variation of the Glaucomys species. The global estimates of FST (0.308) and DXY (0.141) are indicative of substantial differentiation between the species. Measures of genetic diversity (π), differentiation (FST), and divergence (DXY) across the genome reveal insights into the divergent selection driving speciation. Results indicate an absence of contemporary hybridization or introgression at a site with longstanding sympatry. Across both species' genomes, signatures of selection align with four different scenarios for the formation of genomic landscapes of differentiation, shedding light on the complex speciation history of these flying squirrels. These findings enhance understanding of evolutionary dynamics, adaptation, speciation, and genetic differentiation.

Author Keywords: Genomic differentiation, Glaucomys, northern flying squirrel, southern flying squirrel, speciation

The cost of reproduction refers to the use of resources for the production of offspring that decreases the availability of resources for future reproductive events and other biological processes. Models of sex-allocation provide insights into optimal patterns of resource investment in male and female sex functions and have been extended to include other components of the life history, enabling assessment of the costs of reproduction. These models have shown that, in general, costs of reproduction through female function should usually exceed costs through male function. However, those previous models only considered allocations from a single pool of shared resources. Recent studies have indicated that the type of resource currency can differ for female and male sex functions, and that this might affect costs of reproduction via effects on other components of the life history. Using multiple invasibility analysis, this study examined resource allocation to male and female sex functions, while simultaneously considering allocations to survival and growth. Allocation patterns were modelled using both shared and separate resource pools. Under shared resources, allocation patterns to male and female sex function followed the results of earlier models. When resource pools were separate, however, allocations to male function often exceeded allocations to female function, even if fitness gains increased less strongly with investment in male function than with investment in female function. These results demonstrate that the costs of reproduction are affected by (1) the types of resources needed for reproduction via female or male function and (2) via trade-offs with other components of the life history. Future studies of the costs of reproduction should examine whether allocations to reproduction via female versus male function usually entail the use of different types of resources.

Author Keywords: Cost of Reproduction, Gain Curve, Life History, Resource Allocation Patterns, Resource Currencies

In endotherms, physiological functioning is optimized within a narrow range of tissue temperatures, meaning that the capacity to dissipate body heat is an important parameter for thermoregulation and organismal performance. Yet, experimental research has found mixed support for the importance of heat dissipation capacity as a constraint on reproductive performance. To investigate the effects of heat dissipation capacity on organismal performance, I experimentally manipulated heat dissipation capacity in free-living tree swallows, Tachycineta bicolor, by trimming feathers overlying the brood patch, and monitored parental provisioning performance, body temperature, and offspring growth. I found that individuals with an enhanced capacity to dissipate body heat (i.e., trimmed treatment) provisioned their offspring more frequently, and reared larger offspring that fledged more consistently. Although control birds typically reduced their nestling provisioning rate at the highest ambient temperatures to avoid overheating, at times they became hyperthermic. Additionally, I examined inter-individual variation in body temperature within each treatment, and discovered that body temperature is variable among all individuals. This variability is also consistent over time (i.e., is repeatable), irrespective of treatment. Further, I found that individuals consistently differed in how they adjusted their body temperature across ambient temperature, demonstrating that body temperature is a flexible and repeatable physiological trait. Finally, I used a bacterial endotoxin (lipopolysaccharide) to examine the regulation of body temperature of captive zebra finches (Taeniopygia guttata) during an immune challenge. Exposure to lipopolysaccharide induces sickness behaviours, and results in a fever, hypothermia, or a combination of the two, depending on species and dosage. I asked what the relative role of different regions of the body (bill, eye region, and leg) is in heat dissipation/retention during the sickness-induced body temperature response. I found that immune-challenged individuals modulated their subcutaneous temperature primarily through alterations in peripheral blood flow, particularly in the legs and feet, detectable as a drop in surface temperature. These results demonstrate that the importance of regional differences in regulating body temperature in different contexts. Taken together, my thesis demonstrates that heat dissipation capacity can affect performance and reproductive success in birds.

Author Keywords: body temperature, heat dissipation, tree swallow, zebra finch

Warming climates have had various consequences on terrestrial and aquatic food webs that are expected to persist. There is evidence suggesting that certain organisms are better equipped to handle changing climates compared to others. Therefore, the purpose of my thesis was to study the adaptability of Daphnia under temperature stress and nutrient limitation. First, to examine the effects of dietary phosphorus limitation and temperature on daphniid life-history and population growth, a series of experiments were conducted in the laboratory. In general, I found that Daphnia body growth rates and life-history traits to food carbon to phosphorus (C:P) ratios change with temperature. Next, I identified a protocol to limit the genomic DNA (gDNA) from ribonucleic acid (RNA) extractions. I found that using a modified phenol-chloroform extraction protocol was the most effective way to remove gDNA from extracted Daphnia RNA samples. Overall, results from this study show that temperature and food quality interactions are more complicated than previously thought. Furthermore, the RNA extraction protocol developed will be useful in future studies examining gene expression responses in Daphnia.

Author Keywords: ecological stoichiometry, gene expression, life-history, nutrient limitation, RNA puritiy, temperature

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