Doctor of Philosophy in Forestry (PhD) 
Genomic and phenotypic architecture of a spruce hybrid zone (Picea sitchensis x P. glauca)
North Dakota State University
The emergence of locally unfamiliar climates due to anthropogenic global warming is compelling scientists and resource managers to seek ecological data and management strategies from non-local climates, known as climate analogs. In this context, novel climates—emerging conditions with no analog in the observational record—represent widening gaps in the ecological knowledge base. Identification of novel climates is essential to climate change adaptation. However, methods to detect novel climates have not kept pace with this necessity. The goal of this dissertation is to advance methods for detection of novel climates in the context of ecology and forest management. I develop a multivariate metric for climatic novelty, sigma dissimilarity, that uses the local historical range of interannual climatic variability as a scale for measuring the ecological significance of climatic differences. I apply this metric at three scales—continental, jurisdictional, and local—each of which offers a distinct perspective on the implications of climatic novelty. At the continental scale, I assess the emergence of novel climates in North America, where they are an important source of extrapolation error in ecological modeling. I demonstrate the potential for novel climates to emerge throughout the continent, particularly at low topographic positions. At the jurisdictional scale, I assess the emergence of novel climates that are not represented in a structured knowledge system for forest management—the Biogeoclimatic Ecosystem Classification for British Columbia. A parallel novelty assessment using sigma dissimilarity and random forest classification indicates a robust pattern of novel climates in BC, for which analogs from outside BC must be identified. At the local scale, I demonstrate that dependencies among climate variables can produce larger and earlier departures from natural variability than is detectable in individual variables. This multivariate departure intensification effect—evident in distinct regions of the planet in global climate models—indicates adaptive challenges for ecological and human communities as their local climates become unfamiliar. The identification of locally unfamiliar and regionally novel climates is an important step in anticipating and adapting to climate change. Further, the challenges presented by novel climates are yet another basis to advocate for global emissions reductions.
Anthropogenic climate change is shifting species ranges and exerting high selection pressures on populations of all taxa, including trees. Temperate tree species of the northern hemisphere share a history of large-scale postglacial colonization during the Quaternary, providing a natural laboratory for the study of evolutionary responses to climate fluctuations. This dissertation aims at improving our understanding of the mutual influences of demography and evolutionary patterns during range expansions in trees using Picea sitchensis (Sitka spruce) as a focal species.I first focused on the most recent P. sitchensis expansion event in south-central Alaska to study the interplay between demography and population genetics by combining neutral genetic markers and tree ring data. This multidisciplinary approach allowed me to assess the pace of neutral evolution across five centuries of colonization. Allelic richness was efficiently recovered in the colonizing population by early, open-grown colonizers on the Kodiak Archipelago during a long phase of low population growth. However, heterozygosity remains low compared with the nearest mainland populations. These results highlight the long-term importance of early colonizing genotypes in genetics of populations and the influence of pollen dispersal in maintaining standing genetic variation during forest expansion.Local hybridization of P. sitchensis colonizers with foreign pollen from white spruce (Picea glauca) populations occurred repeatedly during the early colonization period. However, introgression was suppressed in subsequent generations growing under a closed canopy. As the two species occupy separate climatic niches, selection against hybrids, intensified by competition, might explain this pattern. Spring precipitation tended to affect hybrid growth more negatively than pure P. sitchensis genotypes, but this effect was nonsignificant.I finally assessed the extent to which demographic parameters of range expansion can be estimated from genomic data through simulations using the approximate Bayesian computation framework. Simple 3-parameter models could be successfully estimated with genetic markers developed from reduced-representation methods currently available for nonmodel species. Models of higher complexity presented challenges, especially when ongoing migration after expansion was considered, and the accuracy of results depended on the time of expansion. The demic expansion models examined here were inadequate to infer the colonization history of P. sitchensis.
Climate change is disrupting local adaptation in temperate and boreal tree species. As climates shift, tree breeding zones are becoming dissociated from their historical climatic optima and no longer represent optimal seed deployment zones. Assisted gene flow (AGF) policies that match reforestation seedlots with future climates require accurate knowledge of genetic variation in climatically adaptive traits in breeding populations. In this thesis I evaluate the effects of selective breeding on climatic adaptation in the two most planted species in western Canada, lodgepole pine (Pinus contorta) and interior spruce (Picea glauca, P. engelmanii and their hybrids), to inform provincial AGF prescriptions. I compared natural stand seedlots (n = 105 pine, 154 spruce) with selectively bred seedlots (n = 20 pine, 18 spruce) from across Alberta and British Columbia in common garden experiments. Phenotypic variation among breeding zones was assessed for growth, phenology and cold hardiness in relation to climate. For both species, phenotypic differences between natural and selected seedlings in growth traits were substantial. Height gains resulted from increased growth rate and delayed growth cessation, but autumn cold hardiness was not substantially reduced. Seedlings were also genotyped for ~30,000 candidate single nucleotide polymorphisms for growth and adaptive traits. Selection for growth has shifted interior spruce hybrid ancestry in some breeding populations, but these effects are not consistent across zones. A genome-wide association study of pine identified many trait-associated SNPs. Positive effect allele frequencies among pine breeding zones were strongly associated with climatic variation. Selection has resulted in small increases in the frequency of positive effect alleles in breeding populations. Associations among cold hardiness phenotypes, genotypes and climate dominated signals of local adaptation were preserved in breeding populations. Selection, breeding and progeny testing combined have produced taller pine and spruce seedlings without compromising climatic adaptation. Strong phenotype-genotype-climate associations suggest AGF will be necessary to match breeding populations with future climates, but selectively bred and natural seedlots can be safely redeployed using the same AGF prescriptions. Multi-locus genomic profiles of adaptive traits associated with climate provide an accurate, rapid method to assess climatic adaptation that is independent from long-term provenance trials.
Natural hybrid zones provide a great opportunity to study the evolutionary relationshipsbetween closely related species. I have combined ten microsatellites (SSR) and 311 singlenucleotide polymorphism (SNP) markers with quantitative data to investigate the geneticstructure, interspecific gene flow and adaptation of the economically and ecologically importantPicea glauca (white spruce) x P. engelmannii (Engelmann spruce) hybrid zone in westernNorth America. Climate modelling and paleoclimate analysis was used to study the historicalevolutionary relationships between hybridizing species; and to predict future patterns of geneticvariation in the zone. This modelling suggests these species may have been in contact for aslong as 21,000 years. Current levels of admixture and introgression are extensive, assuggested by both the SSR and SNP analyses, with populations showing elevational andlatitudinal unimodal clines in admixture. Hybrids occupy intermediate environments in the zoneand show a higher genetic contribution from Engelmann spruce than from white spruce onaverage. Despite a long history of interspecific gene flow, pure species and hybrids areadapted to different environments. Results of the quantitative analysis based on long-term dataon growth and survival, as well as bud phenology and cold hardiness, indicate that the white xEngelmann spruce hybrid zone is maintained by adaptation to the length of growing seasonsand the persistence of the snowpack (exogenous selection), in which hybrids are fitter thanpure species in intermediate environments, fitting the "Bounded hybrid superiority" model of hybrid zone maintenance. I identified 12 outlier SNPs among the 311 SNPs; these were genesresponsible for carbohydrate metabolism, signal transduction and transcription factors. Theseresults have significant implications for forest management and breeding of spruce species inBritish Columbia, where this species complex is managed as one species without consideringthe complexity in population structure and adaptive differences between pure species andhybrids.
Natural hybrid zones may be viewed as important biological systems for examining the role of selection in creating and maintaining species differences. Where ecological differences exist between hybridizing species, these zones may provide useful insight into the genetic architecture of important traits involved in adaptation. I have evaluated the genomic and phenotypic architecture of the economically and ecologically important Picea sitchensis (Sitka spruce) x P. glauca (white spruce) hybrid zone along the Nass and Skeena river valleys in northwestern British Columbia using chloroplast and mitochondrial markers, twelve microsatellite loci (SSRs), and 268 single nucleotide polymorphisms (SNPs), in combination with morphological variation, and phenotypic data from a common garden. Maternally- and paternally-inherited organelle markers, in combination with bi-parentally inherited nuclear markers, were used to estimate both the historic and contemporary direction and extent of gene flow within the hybrid zone. Sitka spruce mitotype ‘capture’ throughout the introgression zone point towards asymmetric gene flow, congruent with microsatellites and SNPs, indicating extensive long-term introgression and widespread recombination with more Sitka spruce than white spruce ancestry in hybrid populations. Significant clinal variation was observed for marker-based hybrid indices and morphological traits associated with climate and geography, while growth and cold hardiness traits evaluated in a common garden exhibited weak to non-significant clines. These results indicate extrinsic selection appears to play a strong role in the distribution and structure of this hybrid zone, which fits expectations for the environmentally-determined bounded hybrid superiority model of hybrid zone maintenance. However, intrinsic mechanisms of hybrid zone maintenance could not be ruled out. Finally, broad-scale patterns of variation, combined with fine-scale analysis of candidate SNP-specific patterns of introgression revealed a suite of candidate loci that may be targets of extrinsic or intrinsic selection. These loci may be involved in either adaptation to climate across the zone, particularly precipitation gradients, or involved in the maintenance of species barriers. These results have important implications for genetic conservation of adaptive variation, selection of seed sources for current reforestation within this ecologically transitional area, and appropriate scale and direction of seed transfer relating current genotype-climate associations to future climate predictions for this region.
Climate change will affect the regeneration, growth, survival and distribution of trees. Here, I use common gardens to empirically test establishment, growth and the potential for persistence, adaptation and migration for two iconic North American trees, whitebark pine (Pinus albicaulis) and lodgepole pine (Pinus contorta ssp. latifolia). Whitebark pine is of conservation concern due to range-wide diebacks, while lodgepole pine is critical to forest productivity and carbon sequestration. Whitebark seeds were planted north of the current range in areas predicted to be climatically suitable through the 2050s; these germinated and survived in varying proportions at all locations. Establishment and growth were positively affected by moderate snow-cover durations, heavier seed weights, and warmer provenance temperatures. Whitebark pine seedlings grown from seeds sown in growth chambers spanning current and predicted-future temperatures demonstrated positive responses to warmer growing seasons. Lodgepole pine seedlings in the same chambers outgrew the whitebark pine seedlings at all but the coldest temperatures. Together, these results suggest that whitebark pine may lose its competitive advantage to other species within its narrow alpine-treeline niche as the climate warms, but that it is capable of establishing in climatically-suitable areas north of its current range. Using tree-ring data from long-term lodgepole pine common garden trials, I built universal growth-trend response functions to forecast future growth trends relative to genetics, climate and tree age. The models predict growth reductions for all populations by the end of the 21st century based on middle-of-the-road climate models, except in far northern areas near and within Yukon, Canada. Analogous models built using summer and winter climate indices indicate that the growth declines are primarily caused by warmer summers, and may be offset by growth increases resulting from warmer winters. I found that populations are most sensitive to annual temperatures and summer aridity, but that sensitivity to climate varies due to local adaptation. Overall, my research will help forest professionals and conservationists forecast changes in forest productivity and species growth and survival under warming temperatures.
Genecological studies in widely distributed tree species have revealed steep genetic clines along environmental gradients for traits related to adaptation to local climate. In the face of a changing climate, the ecological and economic importance of conifers necessitates an appraisal of how molecular genetic variation shapes quantitative trait variation. I have combined transcript profiling with association mapping to better understand the genomic architecture of adaptation to local climate in conifers, using Sitka spruce (Picea sitchensis) as a model. A microarray study during the fall hardening period revealed wholesale remodeling of the transcriptome within a population originating in the centre of the species range, and substantial variation in the autumn transcriptome was observed when populations from the northern and southern limits of the range were compared. Based on these data, a suite of candidate genes was selected and screened for single nucleotide polymorphisms (SNPs) in a panel of 24 individuals. A diverse array of biological processes were represented among the candidate genes, including stress response, carbohydrate, lipid and phenylpropanoid metabolism, light signal transduction, and transcriptional and posttranscriptional regulation. Nucleotide diversity in Sitka spruce was approximately average for a conifer (π = 3.49 x 10⁻³), and linkage disequilibrium decayed rapidly. Tests of selective neutrality suggest widespread purifying selection within these candidate genes, though evidence for positive selection was detected within a few. In addition, I observed evidence for diversifying selection in 8% of the studied genes, which exhibited high population differentiation relative to the genome-wide average FST of 0.12. To identify genetic determinants of phenotypic variation in locally adaptive traits, an Illumina GoldenGate assay was used to genotype 768 SNPs in a mapping population comprised of 410 individuals from 12 geographical populations collected from across the species range. After correcting for population structure and relative kinship, associations were detected in 28 of the candidate genes, which cumulatively explained 28% and 34% of the phenotypic variance in cold hardiness and budset, respectively. Most notable among these associations were five genes putatively involved in light signal transduction, the key pathway regulating autumn growth cessation in perennials. This study represents a significant step toward the goal of characterizing the genomic underpinnings of adaptation to local climate in conifers, and provides a substantial resource for breeding and conservation genetics in a changing climate.
Common garden experiments in widely distributed tree species have demonstrated that phenotypic traits timing of bud set exhibit clinal variation across provenance climatic and geographic gradients, emphasizing the importance of these traits in local adaptation. With rapid advances in molecular techniques, spatial patterns of genomic variation underlying these traits can also be studied. Here I assess whether 17 putatively adaptive single nucleotide polymorphisms (SNPs) previously shown to be statistically associated with cold adaptation phenotypes vary clinally along a temperature gradient in natural, mature populations of Sitka spruce (Picea sitchensis). I also test the hypothesis that clinal strength is stronger in mature spruce populations than in seedling populations due to selection. Regressions were run for each of the 17 SNPs with logit-transformed major allele frequency as the dependent variable and provenance mean annual temperature (MAT) as the independent variable. Next, differences in strength of clines between mature and seedling populations were estimated for each SNP separately and for the 17 SNPs as a group. Finally, I ran two alternate analyses – a full analysis that included all seedling populations and a truncated analysis that limited the range of MAT observed in seedling populations to match that of mature populations. My results vary between the full and truncated analyses. In seedlings, the full analysis revealed clines in 11 SNPs (65%) compared to six SNPs (35%) in the truncated analysis. Mature populations had significant clines for five SNPs (29%). For the full analysis, the group test supported the one-sided hypothesis that mature populations have significantly steeper clines than seedlings across SNPs (P=0.027).Parallel clines in seedling and mature populations were observed for a subset of the SNPs, which strengthens their importance for local adaptation. However, low power limited my ability to make conclusive statements about differences in clinal strength between mature and seedling populations. While most SNPs were present in most populations, I also observed that the northern, disjunct population of Kodiak Island, AK was fixed for the highest proportion of SNPs (59%). This suggests that this recently founded population may lack adaptive diversity to respond to rapid climate change in the future.