Genetics

There are three cattail (Typha) taxa in Canada: T. latifolia (native), T. angustifolia (introduced), and their hybrid T. x glauca. The latter is invasive in regions around the Laurentian Great Lakes, and I investigated the potential role that commercial suppliers may be playing in the introduction of non-native Typha by comparing genotypes of North American, European, and commercially available plants. I found that Ontario garden centres are importing both hybrids and non-native lineages of T. angustifolia into Canada, but was unable to identify the provenance of T. latifolia. I also investigated the possibility that the hybrid cattail leaf litter shade and leachate influences germination and early growth of the parental species of the hybrids. Using three common garden experiments, I found that T. x glauca leaf litter suppresses germination rates of the three taxa. In the early seedling growth experiment, plant performance varied by taxa, and for the competition experiment there were no intra- or interspecific competition or treatment effects on the performance of plants. Overall, my research identified a potential mechanism allowing T. x glauca to dominate wetlands, and also shows that non-native lineages are being introduced into Canada through commercial trade

Author Keywords: Competition, Germination, Non-native lineages, Plant nurseries, Seedling Growth, Typha spp.

Identifying the population origin of aquaculture-reared caviar is crucial for both conservation and management strategies of farmed fish but could also facilitate international trade of a CITES regulated product. Shortnose sturgeon (Acipenser brevirostrum) is the main source of caviar production in Atlantic Canada, from Breviro Caviar Inc. aquaculture facility. Shortnose sturgeon are also listed as a species-at-risk under the Species At Risk Act. Currently there is no genetic method for delineating wild from aquaculture-reared caviar. By targeting the mitochondrial genome (mitogenome) using novel long-range PCR primers and next-generation sequencing (NGS) methods we have successfully sequenced the full mitogenome of 37 shortnose sturgeon. The purpose of this study was to increase the resolution of diagnostic variation among populations and to validate Canadian aquaculture-reared stock from wild US populations.

Results provided a previously unobserved novel control region haplotype in high frequency within both the aquaculture-reared and Saint John River wild sample sets. Similar frequencies were observed with whole mitogenome haplotypes. Diagnostic mitochondrial lineage found in high frequency within the captive Breviro Caviar Inc. population has the potential to allow caviar product from Breviro Caviar Inc. to be distinguished from protected US shortnose sturgeon populations. The application of full mitogenomic characterization provides the potential to further resolve differences between aquaculture and natural Canadian shortnose sturgeon stocks, US/Canadian populations and to contribute to future conservation strategies. Future research identifying signatures of selection on the mitogenome between captive and wild populations and across latitudinal gradients found within the species range. These novel methods have produced a proof-of-concept to provide a "farm-to-fork" validation and ecobrand of Breviro Caviar Inc. product and its aquaculture origin to support importation into US caviar markets.

Author Keywords: aquaculture, mitogenome, next-generation sequencing, species-at-risk, sturgeon

The endangered North Atlantic right whale (Eubalaena glacialis) has been internationally protected from whaling since 1935 but recovery has been slow compared to the southern right whale (Eubalaena australis) due to anthropogenic mortalities and poor reproduction. Prey availability, genetic variability, and alleles of genes associated with reproductive dysfunction have been hypothesized to contribute to low calf production. The North Atlantic Right Whale DNA Bank and Database contains 1168 samples from 603 individuals. I added 115 new genetic profiles to the database which now contains profiles for 81% of individuals alive since 1980. Paternity assignments using these profiles resulted in 62% of sampled calves being assigned a father and only 38% of candidate males being assigned a paternity. This may suggest false exclusion due to genotyping errors or the existence of an unknown group of males. The use of the DNA database allowed for the identification of 10 deceased individuals which has implications for identifying cause of death and reducing mortalities. However, genetic identification is dependent on the time of post-mortem sample collection which influences DNA quantity and quality. An assessment for variations in methylenetetrahydrofolate reductase, a candidate gene associated with reproductive dysfunction, revealed six females heterozygous for a synonymous A/T variant in exon four which may influence reproductive success through changes in enzyme production, conformation or activity.

Author Keywords: Eubalaena glacialis, Forensic Identification, Genetic Profiling, North Atlantic Right Whale, Paternity, Reproductive Dysfunction

The amitochondrial protozoan, Giardia intestinalis, encodes four members of the cytochrome b5 (CYTB5) family of heme proteins of unknown function. While homology models can predict the likely fold of these proteins, supporting experimental evidence is lacking. The small size of the cytochromes (~16 kDa) makes them attractive targets for structural analysis by Electron Paramagnetic Resonance spectroscopy (EPR) and Nuclear Magnetic Resonance spectroscopy (NMR). EPR measurements are particularly useful in defining the geometry of the coordination environment of the heme iron; such measurements indicated that the planar imidazole rings of the invariant histidine axial ligands are nearly perpendicular to each other, rather than in the coplanar orientation observed within mammalian CYTB5s. This may be due to geometrical constraints imposed by a one-residue shorter spacing between the ligand pair in the Giardia cytochromes b5 (gCYTB5s). Following optimization of sample and instrument conditions for NMR experiments, a comparison of the 1D 1H-NMR spectra of gCYTB5 isotype I to those of three of its heme-pocket mutants (Tyr51→Phe, Tyr61→F, and Cys84→Ala) were used to tentatively assign the heme methyl and vinyl protons. Mutant Tyr61→F had the greatest effect on the wild-type spectrum due to maximum through-space contacts with the heme macrocycle and its proximity to the His63 axial ligand. These experiments are a prelude to further NMR experiments that can lead to solving the complete structures of these proteins.

Author Keywords: cytochrome b5, heme b, mutant protein, paramagnetic iron, resonant spectroscopy, sequence homology

Ustilago maydis is a basidiomycete smut fungus and the causal agent of common smut of corn. Disease progression and fungal development in this pathogen occur in planta, terminating in the production of dormant teliospores. Dormant spores of many fungi are characterized by reduced metabolic activity, which is restored during spore germination. The transition out of dormancy requires the rapid translation of stored mRNAs, which may be stabilized through natural antisense transcript (NAT)-mediated mechanisms. Transcript analysis revealed that as-ssm1, a NAT to the mitochondrial seryl-tRNA synthetase (ssm1), is detected in the dormant teliospore and absent in haploid cells. Disruption of ssm1 leads to cell lysis, indicating it is essential for cellular viability. Presented data supports the hypothesis that as-ssm1 has a role in facilitating teliospore dormancy through stabilizing ssm1 transcripts, which reduces mitochondrial function. as-ssm1 expression during in planta development begins 10 days post-infection, coinciding with the first appearance of dormant teliospores. To assess the impact of as-ssm1 expression on cell division, virulence and mitochondrial function, as-ssm1 was ectopically expressed in haploid cells, leading to increased ssm1 transcript levels and the formation of double-stranded RNA. These expression mutants are characterized by attenuated growth rate, virulence, mitochondrial membrane potential and oxygen consumption. Together, these findings support a role for NATs in moderating mitochondrial function during the onset of teliospore dormancy.

Author Keywords: Dormant teliospore, Mitochondria, mRNA stability, Natural antisense transcripts, Non-coding RNA, Ustilago maydis

Sex-specific genetic structure is a commonly observed pattern among vertebrate species. Facing differential selective pressures, individuals may adopt sex-specific life historical traits that ultimately shape genetic variation among populations. Although differential dispersal dynamics are commonly detected in the literature, few studies have investigated the potential effect of sex-specific functional connectivity on genetic structure. The recent uses of Graph Theory in landscape genetics have demonstrated network capacities to describe complex system behaviors where network topology intuitively represents genetic interaction among sub-units. By implementing a sex-specific network approach, our results suggest that Sex-Specific Graphs (SSG) are sensitive to differential male and female dispersal dynamics of a fisher (Martes pennanti) metapopulation in southern Ontario. Our analyses based on SSG topologies supported the hypothesis of male-biased dispersal. Furthermore, we demonstrated that the effect of the landscape, identified at the population-level, could be partitioned among sex-specific strata. We found that female connectivity was negatively affected by snow depth, while being neutral for males. Our findings underlined the potential of conducting sex-specific analysis by identifying landscape elements that promotes or impedes functional connectivity of wildlife populations, which sometimes remains cryptic when studied at the population level. We propose that SSG approach would be applicable to other vagile species where differential sex-specific processes are expected to occur.

Author Keywords: genetic structure, Landscape Genetics, Martes pennanti, Population Graph, sex-biased dispersal, Sex-Specific Graphs

Migratory and sedentary ecotypes are phenotypic distinctions of woodland caribou. I explored whether I could distinguish between these ecotypes in Manitoba and Ontario using genetic signatures of adaptive differentiation. I anticipated that signatures of selection would indicate genetic structure and permit ecotype assignment of individuals. Cytochrome-b, a functional portion of the mitochondrial genome, was tested for evidence of adaptation using Tajima's D and by comparing variations in protein physiology. Woodland caribou ecotypes were compared for evidence of contemporary adaptive differentiation in relation to mitochondrial lineages. Trinucleotide repeats were also tested for differential selection between ecotypes and used to assign individuals to genetic clusters. Evidence of adaptive variation in the mitochondrial genome suggests woodland caribou ecotypes of Manitoba and Ontario corresponded with an abundance of functional variation. Woodland caribou ecotypes coincide with genetic clusters, and there is evidence of adaptive differentiation between migratory caribou and certain sedentary populations. Previous studies have not described adaptive variation in caribou using the methods applied in this study. Adaptive differences between caribou ecotypes suggest selection may contribute to the persistence of ecotypes and provides new genetic tools for population assessment.

Author Keywords: Adaptation, Cytochrome-B, Ecotype, RANGIFER TARANDUS CARIBOU, Selection, TRINUCLEOTIDE REPEAT

The recent northward expansion of the white-footed mouse (Peromyscus leucopus) in response to climatic changes provides a natural experiment to explore potential adaptive genetic variation within the clock gene Per1 in Peromyscus undergoing latitudinal shifts, as well as, the possibility of hybridization and introgression related to novel secondary contact with its sister species the deer mouse (Peromyscus maniculatus). Because clock genes influence the timing of behaviors critical for survival, variations in genotype may reflect an organism's ability to persist in different environments. Hybridization followed by introgression may increase the adaptive potential of a species by quickly generating adaptive variation through novel genetic recombination or by the transfer of species-specific alleles that have evolved in response to certain environments. In chapter 2, I used microsatellite and mtDNA markers to test for hybridization and introgression between P. maniculatus and P. leucopus and found that interbreeding is occurring at a low frequency (<1%). In chapter 3, I tested for a latitudinal cline in a polyglycine repeat located within the Per1 gene of Peromyscus and discovered a putative cline in the Per1-142 and Per1-157 allele of P. leucopus and P. maniculatus, respectively. Chapter 4, further expands upon these findings, limitations, and the lack of evidence supporting introgression at the Per1 locus. Despite this lack of evidence, it is possible that novel hybridization has or could lead to adaptive introgression of other genes, allowing for the exchange of adaptive alleles or traits that could be advantageous for range expansion and adaption to future environmental changes.

Author Keywords: Clock genes, Hybridization, Latitudinal gradient, Per1, Peromyscus, Range Expansion

Moose are an iconic species, known for their large size and impressive antlers. Eight subspecies are classified in circumpolar regions of the planet - four in North America. Two subspecies are similar in shape and size, the north-western moose (Alces alces andersoni) and the eastern moose (Alces alces americana). It was previously believed that these two subspecies meet in northern Ontario. Earlier genetic population studies used a small number of samples from Ontario, primarily in broad studies covering all of North America.

A comprehensive genetic study of moose populations in Ontario has not previously been conducted. We examined the genetic diversity and population structure at 10 polymorphic loci using 776 samples from Ontario, as well as outgroups from representative populations – Manitoba/Cape Breton, representing A. a. andersoni, and New Brunswick/Nova Scotia, representing A. a. americana. Results indicated three genetic populations in the province, in north-western Ontario, north-eastern Ontario and south-central Ontario. RST values, compared against both FST and Jost's D values for phylogenetic analyses, indicated no phylogenetic pattern which suggests no subspeciation present in the province.

Population movement patterns in Ontario were studied. Gene flow was estimated using genetic and spatial data. Isolation by distance was only seen within the first distance class of 100 kilometres and then not seen again at further distances, indicating that moose display philopatry. There were very few migrants travelling across the province, with a greater number moving gradually north and west, towards better habitat and food sources.

A forensic database in the form of an allele frequency table was created. Three loci showed very low levels of heterozygosity across all three populations. Probability of identity was calculated for the three populations and quantified. Samples with known geographic origins were run against the database to test for sensitivity, with identification of origin occurring at an accuracy level between 87 and 100%.

Within Ontario, there are not two different subspecies, as previously believed, but two different populations of the same subspecies meeting in northern Ontario. The genetic data does not support previous research performed in Ontario. The sample sizes in our research also provide a more comprehensive view of the entire province not seen in any previous studies. The comprehensive research enabled the building of a reliable forensic database that can be used for both management and forensic purposes for the entire province.

Author Keywords: Alces alces, Genetic Diversity, Moose, Ontario, Phylogeography, Subspecies

Magnolia acuminata (Cucumber tree) is the only native Magnolia in Canada, where it is both federally and provincially listed as endangered.Magnolia acuminata in Canada can be found inhabiting pockets of Carolinian forest within Norfolk and Niagara regions of southwestern Ontario. Using a combination of nuclear and chloroplast markers, this study assessed the genetic diversity and differentiation of M. acuminata in Canada, compared to samples from the core distribution of this species across the United States. Analyses revealed evidence of barriers to dispersal and gene flow among Ontario populations, although genetic diversity remains high and is in fact comparable to levels of diversity estimated across the much broader range of M. acuminata in the USA. When examining temporal differences in genetic diversity, our study found that seedlings were far fewer than mature trees in Ontario, and in one site in particular, diversity was lower in seedlings than that of the adult trees. This study raises concern regarding the future viability of M. acuminata in Ontario, and conservation managers should factor in the need to maintain genetic diversity in young trees for the long-term sustainability of M. acuminata in Ontario.

Author Keywords: conservation genetics, cpDNA, forest fragmentation, Magnolia acuminata, microsatellites, population genetic structure