<p>Examining the genetic basis across repeated origins of the same phenotypic adaptation allows us to address several questions pertaining to the genetic basis of adaptation. First, whether the genes and types of mutations that are involved in adaptation are predictable. Second, whether the underlying genetic changes can constrain future evolutionary trajectories. Here, I have focused on the genetics of blue to red flower color shifts, an adaptive shift that has repeatedly occurred across angiosperms. First, I review the literature and determine the relative contribution of functional vs. regulatory mutations to the evolution of red flowers can be predicted both on the mutational target size of each type of mutation and the degree of their associated deleterious pleiotropy. Chapter 2 characterizes the genetic basis of red flowers in Penstemon barbatus using a combination of gene expression and protein function assays. I demonstrated that multiple inactivating mutations to one anthocyanin pathway enzyme, F3'5'h, have occurred, but no mutations to any other component of the anthocyanin pathway have contributed to the evolution of red flowers. This suggests that F3'5'h may be a particularly favorable target for selection and also that evolutionary reversal to blue flowers would be highly unlikely. Chapter 3 investigates the genetic basis of an additional 12 origins of red flowers within Penstemon. Again, using a combination of gene expression and enzyme function assays, I found the genetic basis of these additional origins red flowers in Penstemon is highly predictable, involving redundant inactivating mutations to F3'5'h, and tissue-specific regulatory mutations to a second gene F3'h. Thus, the genetics of red flowers in Penstemon often involves inactivation of a non-pleiotropic gene, F3'5'h, but tissue-specific regulatory mutations to the pleiotropic gene F3'h. Furthermore, the presence of redundant inactivating mutations in many red-flowered Penstemon species indicates that the evolutionary reversal to blue flowers would be unlikely.</p> / Dissertation

<p>Determining the genetic basis of adaptation has become a central focus of evolutionary biology, and the incorporation of increasingly sophisticated analytical tools from molecular biology has made identifying causal genes a practical reality. The work presented herein addresses the effects of pleiotropic constraint on evolutionary change at the level of individual genes and genetic networks. In the first chapter, I combine molecular phylogenetic analyses and direct assays of enzymatic function to determine the evolutionary processes following a gene duplication in the anthocyanin pathway. My results show that, prior to duplication, the DFR gene was constrained from functional improvement by its multiple enzymatic roles. Following duplication, this constraint was released and adaptive evolution proceeded along both paralog lineages. In the second chapter, I determine the molecular genetic basis of a flower color transition that is associated with change in pollinator attraction in morning glories. A regulatory change in a branching gene in the flavonoid biosynthetic pathway restricted flux down the cyanidin-producing branch, conferring nearly exclusive production of red pelargonidin pigment in flowers. I further demonstrate that this regulatory change was restricted to floral tissue, and that ancestral pathway flux predominates in vegetative tissues. I propose that deleterious pleiotropic effects prevented evolutionary change via enzymatic changes in the pathway due to the numerous essential products downstream of this branching point. Together, these two results show that evolutionary change may be constrained by the molecular genetic context in which prospective adaptive mutations occur.</p> / Dissertation

Inspired by Gould, who claimed life would be arriving at a different outcome each time it were allowed to run from the same beginning, I have attempted to determine the repeatability of the adaptive course of one Pseudomonas fluorescens lineage. In addition, my study aimed to establish whether the likelihood of parallel evolution of the two synonymous single-nucleotide substitutions was contingent upon a prior motility-impairing deletion or a prior increase in fitness. Further, the study was designed to provide empirical data addressing the long-standing question of the effect of starting fitness on the ensuing rate of adaptation. Although no exact replay of the initial evolutionary trajectory was observed, I have demonstrated that gtsB, but not gtsC gene, is likely to be a mutational hotspot under the low glucose with a recovery of two undescribed mutations in gtsB. My data are consistent with a notion that substitutions in gtsB may be contingent upon Δ35kB(fliJ-PFLU4466) motility-impairing deletion, but not the fitness increase associated with it. Finally, the features of the adaptive landscape of P. fluorescens in the minimal glucose provide languid support for Fisher’s hypothesis of a decrease in adaptation rate with the rise in the starting fitness. Taken together, these original results reinforce the non-negligible role of history in shaping the outcomes of biological evolution and call for caution in attempting a formulation of rigid predictive models of evolutionary change. Inspiré par les travaux de Stephen J. Gould qui affirmait que la vie sur terre arriverait à une forme différente si elle repartait à zéro, je présente ici mes travaux où je teste la reproductibilité du cours adaptatif d’une lignée expérimentale de Pseudomonas fluorescens. L’objectif de cette étude était de déterminer si la probabilité que deux mutations synonymes évoluent en parallèle est affectée par la présence d’une délétion affectant la motilité de la bactérie ou de l’augmentation de la valeur sélective de celle-ci. De plus, le design expérimental de cette étude permet de tester si la valeur sélective initiale d’une population affecte le taux d’adaptation de cette même population. Bien d’une reproductibilité exacte du cours adaptatif initial ne fut pas observée, je démontre que le gène gtsB est probablement un « hotspot »mutationnel permettant l’adaptation à de bas niveau de glucose, ayant trouvé deux mutations dans ce site; alors que le gène gtsC ne l’est pas. Mes données sont également conséquentes avec le fait que les mutation dans le gène gtsB dépendent de l’effet de la délétion Δ35kB(fliJ-PFLU4466) affectant la motilité de la bactérie, mais non de l’augmentation de la valeur sélective qui y est associée. Finalement, la forme du plateau adaptative associé à de bas niveaux de glucose chez P. fluorescens supporte l’hypothèse émise par Fisher qui stipule que le taux d’adaptation d’un organisme diminue avec la valeur sélective initiale qui y est associée. L’ensemble de ces résultats supporte le rôle non-négligeable de l’histoire de vie d’une population en ce qui attrait à l’évolution future de cette même population. Aussi, ces résultats appelle à la prudence quand vient le temps de formuler des modèles prédictifs des changements évolutifs d’une population.

Inspired by Gould, who claimed life would be arriving at a different outcome each time it were allowed to run from the same beginning, I have attempted to determine the repeatability of the adaptive course of one Pseudomonas fluorescens lineage. In addition, my study aimed to establish whether the likelihood of parallel evolution of the two synonymous single-nucleotide substitutions was contingent upon a prior motility-impairing deletion or a prior increase in fitness. Further, the study was designed to provide empirical data addressing the long-standing question of the effect of starting fitness on the ensuing rate of adaptation. Although no exact replay of the initial evolutionary trajectory was observed, I have demonstrated that gtsB, but not gtsC gene, is likely to be a mutational hotspot under the low glucose with a recovery of two undescribed mutations in gtsB. My data are consistent with a notion that substitutions in gtsB may be contingent upon Δ35kB(fliJ-PFLU4466) motility-impairing deletion, but not the fitness increase associated with it. Finally, the features of the adaptive landscape of P. fluorescens in the minimal glucose provide languid support for Fisher’s hypothesis of a decrease in adaptation rate with the rise in the starting fitness. Taken together, these original results reinforce the non-negligible role of history in shaping the outcomes of biological evolution and call for caution in attempting a formulation of rigid predictive models of evolutionary change. Inspiré par les travaux de Stephen J. Gould qui affirmait que la vie sur terre arriverait à une forme différente si elle repartait à zéro, je présente ici mes travaux où je teste la reproductibilité du cours adaptatif d’une lignée expérimentale de Pseudomonas fluorescens. L’objectif de cette étude était de déterminer si la probabilité que deux mutations synonymes évoluent en parallèle est affectée par la présence d’une délétion affectant la motilité de la bactérie ou de l’augmentation de la valeur sélective de celle-ci. De plus, le design expérimental de cette étude permet de tester si la valeur sélective initiale d’une population affecte le taux d’adaptation de cette même population. Bien d’une reproductibilité exacte du cours adaptatif initial ne fut pas observée, je démontre que le gène gtsB est probablement un « hotspot »mutationnel permettant l’adaptation à de bas niveau de glucose, ayant trouvé deux mutations dans ce site; alors que le gène gtsC ne l’est pas. Mes données sont également conséquentes avec le fait que les mutation dans le gène gtsB dépendent de l’effet de la délétion Δ35kB(fliJ-PFLU4466) affectant la motilité de la bactérie, mais non de l’augmentation de la valeur sélective qui y est associée. Finalement, la forme du plateau adaptative associé à de bas niveaux de glucose chez P. fluorescens supporte l’hypothèse émise par Fisher qui stipule que le taux d’adaptation d’un organisme diminue avec la valeur sélective initiale qui y est associée. L’ensemble de ces résultats supporte le rôle non-négligeable de l’histoire de vie d’une population en ce qui attrait à l’évolution future de cette même population. Aussi, ces résultats appelle à la prudence quand vient le temps de formuler des modèles prédictifs des changements évolutifs d’une population.