Bioinformatics

Since the mid-Pleistocene, evolutionary histories of North American species were shaped by extreme climatic oscillations involving major range and habitat shifts at a rapid pace. The peopling of the continent and the subsequent human colonisation waves created further pressures affecting North American faunal and plant populations. Cervidae (deer family) are a diverse group which arrived in North America approximately 5 million years ago, and is represented on the continent by several extant species. The overarching aim of my thesis is to understand how North America's dynamic history shaped the evolutionary trajectory of the region's Cervidae species. In Chapter 2, I investigated the speciation and hybridisation history of the sister species white-tailed (Odocoileus virginianus) and mule deer (Odocoileus hemionus) using whole genome data of individuals from across their ranges, including zones of sympatry and allopatry. I found negligible patterns of ancient gene-flow suggesting white-tailed and mule deer divergence occurred via drift and their hybridisation is the result of secondary contact. In Chapter 3, I furthered our understanding of the Odocoileus species pair evolutionary history through explicit demographic inference and selection analyses. I used deer samples from across North America and found strong signals of climate- and human-induced population declines. Particularly, this work highlights the impact of European settlers and patterns of conservation concerns for mule deer. For Chapter 4, I clarified the phylogenetic relationship of a contentious taxon, Torontoceros hypogaeus, that went extinct during the late Pleistocene extinction event. I analysed 11k years old DNA of the single specimen representing the species, performed phylogenetic and divergence analyses, and found it belongs to the Odocoileus genus as Odocoileus (Torontoceros) hypogaeus which should be included in North America's late Pleistocene extinction list. Finally, in Chapter 5, I investigated genetic diversity over time in five North American Cervidae using contemporary and ancient DNA. I found patterns of change in genetic diversity that are consistent with known dispersal and demographic histories of our target species. Altogether, my thesis provides valuable insight into the evolutionary history of Northern American Cervidae, and on how they have been impacted by the continent's dynamic past.

Author Keywords: Cervidae, Demographic history, Extinction, Human impact, Hybridisation, Speciation genomics

Mutations in the CLN7 (MFSD8) gene, causes CLN7 disease, a subtype of neuronal ceroid lipofuscinosis. MFSD8 is a lysosomal transmembrane protein that transports chloride across membranes. Experimentation regarding Dictyostelium discoideum revealed that mfsd8 deficiency altered lysosomal enzyme activity. During starvation, the aggregation of mfsd8¬¬- cells was delayed, and cells formed more mounds that were smaller in size, phenotypes that were attributed to reduced cell-substrate adhesion and altered lysosomal enzymatic activities. This study examines the possible transcriptomic and secretomic basis for these phenotypes. This work generated new datasets for examining the effect of mfsd8 loss on the transcriptome and secretome. The validity of these datasets was supported by use of western blotting and RT-PCR along with a set of assays probing relevant biological processes. Together these results elucidate the biological mechanisms behind the observed phenotypes and lay the foundation for future studies to further study the cellular role of MFSD8.

Author Keywords: Battens disease, CLN7, Dictyostelium discoideum, MFSD8, Secretome, Transcriptome

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

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

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

Genetic variation is the raw material and basis for evolutionary changes in nature. The loss of genetic diversity is a challenge many species are facing, with genomics being a potential tool to inform and prioritize decision making. Whole genome analysis can be an asset to conservation biology and the management of species through the generation of more precise and novel metrics. This thesis uses whole genome re-sequencing to characterize the demographic history and quantify genomic metrics relevant to conservation of caribou (Rangifer tarandus) in Québec, Canada. We calculated the ancestral and contemporary patterns of genomic diversity of five representative caribou populations and applied a comparative population genomics framework to assess the interplay between demographic events and genomic diversity. When compared to the census size, NC, the endangered Gaspésie Mountain caribou population had the highest ancestral Ne:NC ratio which is consistent with recent work suggesting high ancestral Ne:NC is of conservation concern. These ratios were highly correlated with genomic signatures (i.e. Tajima's D) of recent population declines and explicit demographic model parameters. Values of contemporary Ne, estimated from linkage-disequilibrium showed Gaspêsie having among the highest contemporary Ne:NC ratio. Importantly, classic conservation genetics theory would predict this population to be of less concern based off this metric alone. Inbreeding measures suggested nuanced patterns of inbreeding and correlated to the demographic models. This study suggests that while the Québec populations are all under decline, they harbour enough ancestral genetic variation to replenish any lost diversity, if conservation decisions are made in favour of these populations, specifically supporting NC.

As the generation of genetic data accelerates, Big Data has an increasing impact on the way bioinformatics software is used. The experiments become larger and more complex than originally envisioned by software designers. One way to deal with this problem is to use parallel computing.

Using the program Structure as a case study, we investigate ways in which to counteract the challenges created by the growing datasets. We propose an OpenMP and an OpenMP-MPI hybrid parallelization of the MCMC steps, and analyse the performance in various scenarios.

The results indicate that the parallelizations produce significant speedups over the serial version in all scenarios tested. This allows for using the available hardware more efficiently, by adapting the program to the parallel architecture. This is important because not only does it reduce the time required to perform existing analyses, but it also opens the door to new analyses, which were previously impractical.

Author Keywords: Big Data, HPC, MCMC, parallelization, speedup, Structure

Giardia intestinalis is an anaerobic protozoan that lacks common eukaryotic heme-dependent respiratory complexes and does not encode any proteins involved in heme biosynthesis. Nevertheless, the parasite encodes several hemeproteins, including three members of the Type II cytochrome b5 sub-group of electron transport proteins found in anaerobic protist and amitochondriate organisms. Unlike the more well-characterized cytochrome b5s of animals, no function has been ascribed to any of the Type II proteins. To explore the functions of these Giardia cytochromes (gCYTB5s), I used bioinformatics, immunofluorescence microscopy (IFM) and co-immunoprecipitation assays. The protein-protein interaction in silico prediction tool, STRING, failed to identify relevant interacting partners for any of the Type II cytochromes b5 from Giardia or other organisms. Differential cellular localization of the gCYTB5s was detected by IFM: gCYTB5-I in the perinuclear space; gCYTB5-II in the cytoplasm with a staining pattern similar to peripheral vacuole-associated protein; and gCYTB5-III in the nucleus. Co-immunoprecipitation with the gCYTB5s as bait identified potential interacting proteins for each isotype. The most promising candidate is the uncharacterized protein GL50803_9861, which was identified in the immunoprecipitate of both gCYTB5-I and II, and which co-localizes with both. Structural analysis of GL50803_9861 using Swiss Model, Phyre2, I-TASSER and RaptorX predicts the presence of a nucleotide-binding domain, which is consistent with a potential redox role involving nicotinamide or flavin-containing cofactors. Finally, the protein GL50803_7204 which contains a RNA/DNA binding domain was identified a potential partner of gCYTB5-III. These findings represent the first steps in the discovery of the roles played by these proteins in Giardia.

Author Keywords: Cytochrome b5, Giardia intestinalis, Heme, Interactome, Protein structure prediction