Browsing by Author "Rausher, Mark D"

One of the most important questions in evolutionary biology is how new genes and new functions arise and evolve. Among the theories addressing this question, gene duplication is one of the most popular. Previous study has shown that two threonine deaminase (TD) gene copies exist in Solanum lycopersicum, and these two copies have very different functions and low sequence similarities. The primary objective of this study was to widen our understanding of this gene duplication and the subsequent evolutionary processes affecting the duplicate copies by first collecting additional TD sequences from related species, building a gene tree, and inferring the point of gene duplication. The evolutionary processes acting on this gene were then analyzed using the program PAML. Results indicate that 1) The TD duplication probably occurred in before the split of the Solanoideae from the Nicotianoidea; and 2) there is strong evidence for positive selection on one of the TD copies after gene duplication, while for the other TD copy, only weak evidence for positive selection was found; and 3) adaptive improvement of the copy with new function probably spanned a period of at least 25 million years.

Plants and their pathogens coevolve, with pathogen infection and host resistance acting in evolutionary antagonism of each other. Plant-pathogen coevolution has been shown to effect genetic divergence between populations and species, resulting in localized or specialized interactions between hosts and pathogens. Because most of the studies to date investigating plant-pathogen coevolution have been carried out in managed systems and have focused on pairwise interactions, we know little about three aspects of plant pathosystems in natural settings: 1) the role in nature of the gene-for-gene paradigm for genetic determination of resistance; 2) the relationship of host community diversity and structure, and host-pathogen interaction structure, to the coevolutionary process; and 3) the factors which underlie and drive local adaptation and specialization of interactions.

This dissertation constitutes the results of research in which I have begun addressing these aspects in a natural plant-fungus pathosystem comprising three Ipomoea host species and a single rust pathogen, Coleosporium ipomoeae. I have expanded previous characterization of the genetics of plant resistance in one constituent host species in the system by genetic crosses to characterize the basis of resistance in two additional species, finding support for the expectation that the gene-for-gene paradigm of interaction is important in natural systems. I conducted a cross-inoculation experiment designed to assess host and pathogen variation in infectivity and resistance, to investigate patterns of community interaction structure, and the role that antagonistic coevolution may play in structuring the communities which compose pathosystems. In these experiments I found that the coevolutionary interaction in this system leads to genetic divergence and the substantial amount of host and pathogen variation I discovered, but that it tends to preserve one pattern of community interaction structure across communities. I expanded my cross-inoculation experimental design to facilitate analysis of quantitative aspects of pathogenesis by measuring the intensity of infections, to test existing hypotheses concerning local adaptation and specialization in pathosystems. In this analysis I found strong host local adaptation and pathogen local maladaptation for the qualitative interaction trait of infectivity, and I found weak host local maladaptation and pathogen local adaptation for the quantitative interaction trait of aggressiveness. I also found host specialization among pathogens, and specialized resistance among hosts, to be common in this system. In light of these results, I hypothesize that the geographic scale of host-pathogen coevolution in this system is that of the local community, and that differences between host species result in persistent but incomplete host specialization in pathogen races.

Understanding to what extent a particular ecosystem can influence the larger environment is the driving question behind my research. To begin examining this topic, several factors should be considered including 1) the community composition within a certain ecosystem 2) what factors drive community assembly, to predict the composition and organization of other related communities, and 3) the extent of influence of the ecosystem on the surrounding environments.

Chemosynthetic ecosystems are systems that are dependent on chemicals as the base of the food chain, rather than light as in photosynthesis. These systems are frequently biological hotspots within the ocean, where benthic communities can research a higher density than surrounding regions. Methane seeps, where methane and other hydrocarbons migrate through sediments to the seafloor, are one of the major forms of chemosynthetic ecosystems. The known environmental influence of seeps grows stronger as the number of discovered seeps increases, making understanding their breath of impact increasingly relevant.

Data collected in this study utilized two types of underwater research tools, an autonomous underwater vehicle (AUV) and a remotely operated vehicle (ROV). The study begins by examining seep fields about 200 nm off the South Carolina coast, at the Blake Ridge (~2150 m depth) and Cape Fear (~2580 m depth) Diapirs. Geophysical and photographic data were collected during surveys were used to examine the relationship between biomass-dominant invertebrates (mussels, Bathymodiolus heckerae, and clams, Vesicomya cf. venusta) and seafloor physiography. Concentric zonation of mussels and clams at each of the four sites within the seep field suggests the influence of chemical gradients on megafaunal distribution. Distributions of dominant seep features (bivalves, carbonates, bacterial mats) were used to define the active seep site. The relationship between seeps and nearby non-endemic fauna is examined in this study, with a focus on trophic guilds. Geospatial mapping indicated that non-seep-endemic taxa (those not hosting chemoautotrophic endosymbionts) either show positive association (e.g., squat lobsters, cake urchins), negative association (e.g., sea urchins, certain sea cucumbers), or no distributional bias (e.g., sea stars, certain fish) to the presence of a seep.

Further investigation into these faunal relationships may improve understanding of predictive community assembly rules, as well as clarifying the services that seeps provide to the larger ocean ecosystem. Data collection and analytical techniques used here yielded high-resolution habitat maps that can serve as baselines to constrain temporal evolution of seafloor seeps, and to inform ecological niche modeling and resource management.

Another aspect of the study is how seep communities differ when in an extremely low oxygen environment. Methane seeps are typically biological hotspots on the seafloor, with dense faunal communities relative to background (non-chemosynthetic) areas (Carney, 1994). However, some areas of the ocean are extremely low in oxygen, leading to decreases in overall fauna diversity, which can sometimes also affect seep communities. The eastern Pacific contains extensive oxygen minimum zones (OMZs), areas where the dissolved oxygen concentration falls below 22 μM, or 0.5 mg/L (Helly and Levin, 2004; Karstensen et al., 2008), as opposed to averages of 180-270 μM (4-6 mg/L) typically found in the ocean. These zones can intersect the continental slope, affecting benthic organisms, including those found at methane seeps in the region.

The Redondo Knoll Seep (~900 m depth), located just ~30 km off the California coast, shares features of both a seep and an oxygen minimum zone. High-resolution imaging providing a highly detailed photo mosaic and 1 cm resolution bathymetric maps, allowed for a comprehensive site view to further the geologic and microbial examinations. Particularly notable were the extensive microbial mats and, due to its location near the core of a local OMZ (averaging <1 μM oxygen), the lack of endemic seep megafauna or other non-endemic fauna.

While both seeps and OMZs are common by themselves, only a small number of studies have examined them together, such as off the coast of Chile (Sellanes et al., 2010), Pakistan (Fischer et al., 2012; Himmler et al., 2015), and Oregon (Levin et al., 2010). Consequently, there is still much to learn about the ecosystem and organisms at these combination sites.

Overall, this study considers the sphere of influence methane seeps provide the surrounding area through examination of the relationship between endemic fauna, their geologic habitat, and non-endemic fauna, with the intention to use these interactions to better inform seep roles in the environment in the face of anthropogenic disruptions, such as deep-sea mining and climate change, and as well as sites to consider for astrobiological studies.

Floral color polymorphisms are pervasive in nature. Understanding the genetic basis of such polymorphisms allows us to address major evolutionary questions including: what types of genetic changes contribute to the evolution of phenotypic diversification? Have they arisen from new or pre-existing genetic material? And are certain types of genetic changes disproportionately involved? This dissertation address these questions by investigating two polymorphisms involving loss of anthocyanin pigmentation in some of the individuals in the populations. In Chapter 1, I characterized the genetic basis of the “white cup” phenotype in Clarkia gracilis ssp. sonomensis, where no anthocyanins are produced in the basal region of the petal. I demonstrated that the cup pigmentation is controlled by an R2R3-MYB transcription factor using transcriptome analysis, gene expression assays and cosegregation examination. I also found that petal pigmentation requires at least four R2R3-MYB genes, each gene exhibiting a spatiotemporal expression pattern that is different from each other, and each color pattern element is controlled by a different R2R3-MYB gene. In Chapter 2, I examined the evolution of petal pigmentation patterning by analyzing the phylogenetic relationship of these petal R2R3-MYB genes from C. g. ssp. sonomensis, its closely related subspecies, C. g. ssp. albicaulis and their progenitor species, C. amoena ssp. huntiana and C. lassenensis. I also compared the expression domains of these R2R3-MYB genes in these four (sub)species. I found that the R2R3-MYB genes that have region-specific expression patterns are derived from duplication events occurred before polyploidization of C. gracilis. These findings suggest that gene duplication of the R2R3-MYB genes plays an important role in the evolution of petal pigmentation patterning in C. gracilis. In Chapter 3, I identified an R2R3-MYB gene involved in producing purple or yellow anthers in Erythronium umbilicatum using transcriptome analysis, gene expression assays and a likelihood-estimation procedure. This R2R3-MYB gene regulates the expression of three anthocyanin enzyme-coding genes coordinately. However, this R2R3-MYB is present in multiple, highly similar copies. Isolating causal genetic changes from these copies has not been successful, as it would require a better knowledge of the Erythronium genome. In sum, this work shows that R2R3-MYB transcription factors are the primary determinants of floral-color polymorphisms and the R2R3-MYB gene family generated by gene duplication facilitates the evolution of novel characters.

Nectar production is one of several traits that are reduced in flowering plants that display the selfing syndrome, a suite of trait reductions often associated with the transition from outcross-fertilization to self-fertilization. However, the evolutionary mechanisms that contribute to reduced nectar has not been explored. In this dissertation, I use a pair of sister morning glories, Ipomoea lacunosa, a highly selfing species that displays the selfing syndrome, and I. cordatotriloba, a mixed mating species, to address the question: how did reduced nectar evolve in the selfing syndrome? Through a combination of approaches from quantitative genetics, population genomics, and transcriptomics, I describe the genetic architecture of nectar production and attempt to identify candidate genes that could lead to reduced nectar production – nectar volume and nectar sugar concentration – in I. lacunosa. QTL analyses indicate that nectar traits are polygenic and evolved independently from floral size traits, likely due to direct selection. Transcriptomic analyses reveal several sets of genes that are unique to each nectar trait, but both nectar volume and nectar sugar concentration also share some genes in common. Finally, through incorporating population genomic analyses, a short list of candidate genes was obtained that may explain how reduced nectar evolved in I. lacunosa and how nectar traits remain diverged between the two morning glory species even in regions of sympatry.

Accurate species descriptions are critical for understanding evolutionary relationships. Ipomoea “austinii” was proposed to be a new species found in the US, but conflicting evidence suggested that it was instead I. grandifolia. By examining cross-compatibility between these individuals and through genetic analyses, I find no cross-incompatibility and no genetic differentiation between I. “austinii” and I. grandifolia, thus suggesting that I. “austinii” should be reclassified as I. grandifolia.

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.

An unresolved problem in evolutionary biology is the nature of forces that maintain standing variation for quantitative traits. In this study we take advantage of newly developed genomic resources to understand how variation is maintained for flower size and fitness components in a natural population of annual Mimulus guttatus in the Oregon Cascades. Extensive inbreeding depression has been documented in this population for fertility and viability (Willis 1999 a,b), while previous biometric experiments have demonstrated that some of the floral variation in this site is due to common alleles perhaps maintained by balancing selection (Kelly and Willis 2001, Kelly 2003). Detailed comparison of the genetic architecture of these two categories of traits can clarify the relative contributions of mutation versus selection in maintaining trait variation within populations as well as the relevance of standing variation for trait diversification.

We present here the results from a large scale effort to dissect variation for flower size and a suite of genetically correlated traits. In 3 independent F2 mapping populations we mapped QTLs for floral morphology (flower width and length, pistil length, and stamen length), flowering time, and leaf size. We also mapped segregation distortion loci and QTLs for fertility components (pollen viability and seed set) that exhibit inbreeding depression. We compare the genetic architecture of these two sets of traits and find clear differences. Morphological traits and flowering time are polygenic and QTLs are generally additive. In contrast, deleterious QTLs associated with segregation distortion or fertility are partially recessive and include major QTLs. There is also little co-localization between morphological/flowering time and fertility QTLs. The analysis suggests that the genetic basis of segregating variation in morphology is fundamentally different from traits exhibiting inbreeding depression. Further, there is considerable variation in the extant of pleiotropy exhibited by QTLs for morphological traits as well as flowering time and we report that epistasis contributes to the standing variation for these traits. The analysis suggests that the standing variation is relevant for trait diversification and that the variation in floral allometry, plant form, and life history observed in the guttatus species complex could have readily evolved from the standing variation.

Meiotic recombination creates genetic diversity by shuffling combinations of alleles across loci, yet alleles at neighboring loci often remain non-randomly associated. This non-random association is known as linkage-disequilibrium (LD), and it has evolutionarily important effects both within and between species. Nucleotide diversity at a given locus may be reduced by directional selection on the locus, or by selection on neighboring linked loci. Recombination rates and nucleotide diversity are positively correlated across loci within many species, which can be explained by linked selection reducing nucleotide variation disproportionately in regions of low recombination. However, the independent contributions of different types of linked selection are difficult to disentangle. Between species, chromosomal inversions have been proposed to suppress recombination in hybrid inversion heterozygotes and thereby maintain LD and species distinction, but many models of how this happens are overly simplistic—they often ignore non-crossover gene conversion, which reduces LD. Little direct empirical data exist on gene conversion with respect to inversions in hybrids, so despite existing models, inversions may be quite ineffective at keeping hybridizing species distinct. Here, I examine the evolutionary consequences of LD at two levels: nucleotide diversity within species, and recombination-suppression in hybrids between species. I present three investigations driven by this overarching goal of understanding how LD plays into fundamental evolutionary mechanisms. First, I examine nucleotide variation in Drosophila pseudoobscura, and I present a novel test for evidence that particular kinds of selection at linked sites (background selection and/or soft sweeps) may reduce nucleotide variation even in the absence of hard selective sweeps. Second, I show that inversions are permeable to non-crossover gene conversion, which occurs throughout inverted regions in intra- and inter-specific hybrids. I provide a genome-wide empirical analysis of gene conversion rates both within species and in species hybrids, and I estimate that gene conversion occurs at a rate of 1 x 10-5 to 2.5 x 10-5 converted sites per bp per generation in experimental crosses within D. pseudoobscura and between D. pseudoobscura and its naturally-hybridizing sister species D. persimilis. Finally, I use extensive whole-genome sequence data to re-examine patterns of introgression and divergence in the D. pseudoobscura / D. persimilis system. I show how failing to consider variation in evolutionary rate can lead and has led to misinterpretations regarding effects of introgression. Through these genomic examinations, I refine our understanding of how recombination and linkage disequilibrium have shaped the divergence and speciation of Drosophila pseudoobscura and D. persimilis.

Why do species exist where they do? Understanding the forces and processes that shape species’ ranges—and that affect their dispersal and range expansion—have long fascinated biologists. In this work, I focus on understanding diversity and dispersal in Cheilanthes ferns. I first describe a species new to science, Cheilanthes ecuadorensis, from among the understudied South American members of this genus. I then turn to studying the widely distributed, asexual, Australasian species C. distans. Careful review of samples from this species allowed me to find sexual specimens previously unknown to science that exist in a narrow range, as well as to catalogue extensive spore diversity that has gone unrecorded. I find strong evidence for trade-offs related to spore size, with larger spores having higher germination while smaller spores have greater dispersal. Excitingly, I find that spores previously catalogued as abortive are in fact viable, and contribute to the spore size diversity I observe. I then place these findings into phylogenetic context by building a phylogeny for all Australasian Cheilanthes, and use it to explore the relationships of sexual and asexual lineages, of different ploidy levels, and of geographic distributions. These analyses reveal that most dispersal in C. distans occurs over shorter rather than longer distances, in contrast to previous hypotheses posited by fern biologists. I observe that lineages are not limited to particular geographic regions, as well finding that dispersal is asymmetrical and seems to be tracking trade winds. For all my work I rely heavily on herbarium specimens and use them to catalogue morphological variation as well as to obtain DNA sequences that are used for phylogenetic analysis. I implement a variety of statistical and systematic analyses to explore correlations between spore size, reproductive mode, ploidy, germination, and dispersal. While this work expands our knowledge of fern diversity and biogeography, much still remains to be understood, including cataloguing possible novel species, understanding the biology behind spore size determination, and exploring the role of niche in the dispersal and range expansion of C. distans.

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.

The evolutionary transition from outcrossing to self-pollination occurs frequently in flowering plants and has direct and indirect effects on genomics, life history and floral morphology. The life history and floral traits common to selfing plants are collectively called the "selfing syndrome." This dissertation uses the highly selfing morning glory Ipomoea lacunosa to address three major questions in the evolution of highly selfing plants: what are the genomic consequences of selfing, do the morphological changes associated with the transition to self-pollination result from natural selection or genetic drift, and how does the genetic architecture of those morphological traits affect their evolution? In the first chapter, we analyze genetic data from I. lacunosa and its outcrossing sister species I. cordatotriloba to compare the genomic consequences of selfing in I. lacunosa to theoretical predictions. We find that the reduction in genetic diversity is greater than that predicted by theory, suggesting a population bottleneck in I. lacunosa's history. There is also evidence for the relaxation of natural selection. The second chapter combines these genetic data with phenotypic measurements in a Qst-Fst comparison to determine whether natural selection is responsible for life history and floral morphology differences between I. lacunosa and its outcrossing relative I. cordatotriloba. Our analyses reveal that several component traits in the selfing syndrome diverged in response to natural selection. Chapter Three uses a quantitative trait locus (QTL) mapping approach to characterize the genetic architecture of the selfing syndrome and investigate how genetic correlations between traits affected its evolution. We find generally lower levels of genetic correlation between selfing syndrome traits than previous QTL studies of the selfing syndrome. The low level of genetic correlation indicates that independent selection on selfing syndrome traits is responsible for the evolution of the syndrome as a whole.

Color has long served as a model trait for understanding questions at the intersection of genetics, development, and evolution. But recent advances in genomic and gene editing technologies are finally allowing for the in-depth study of the intricate spatial patterning of pigmentation, for example zebra stripes, butterfly wings, and flower spots. The study of plant pigmentation patterning is still in its infancy, but one surprising preliminary result is that patterning, specifically with anthocyanin pigments, always involves subfunctionalized MYB paralogs that each control the initiation of pigmentation in a specific pattern element. These early studies have mostly focused on the mechanism for patterning, though, and few have looked at the evolution and diversification of this complex trait.

Clarkia is a genus of 42 species that display intricate and widely varied pigmentation patterns on their petals while other species in the family have for the most part unpigmented and unpatterned petals, suggesting that pigmentation patterning first evolved at the origin of the Clarkia genus and then has diversified in extant species. In addition, the mechanism for patterning has been attributed to the function of three recently duplicated MYB paralogs in Clarkia gracilis, which makes this genus a tractable system for further understanding the evolution of pigmentation patterning between species. My research aims to answer three fundamental questions about the evolution of pigmentation patterning, using Clarkia as the focal system. 1) Did the duplication of the MYB paralogs coincide with the evolution of pigmentation patterning? 2) Did a patterning ground plan, with each MYB paralog regulating one specific pattern element, evolve in the ancestor of Clarkia and remain generally conserved across the species? 3) Do independent mutations to the MYB paralogs underlie the repeated evolution of similar phenotypes in distantly related species?

I used a combination of phylogenomics, transcriptomics, and classical genetics to answer these three questions. First, I inferred a well-supported species tree for 27 of 32 diploid species in the genus, and then explored a novel method for elucidating the putative parents for the 10 allopolyploid species. Then I used this species tree and whole petal transcriptomes from a majority of Clarkia species and six outgroup species in the Onagraceae family to explore the origin and role of these duplicated MYBs. Presence of these MYB paralogs in the petal transcriptomes of the outgroup species indicate that they duplicated long before the origin of Clarkia, and while all three seem to be integral for patterning across the genus, there is little evidence of a conserved ancestral ground plan to patterning.

I then focused on three distantly related species and used transcriptomics and classical genetics to disentangle the exact role that each paralog plays in patterning in these species, as well as the genetic basis for intraspecies variation in the presence or absence of each pattern element. I find that these paralogs have been repeatedly co-opted for different roles in patterning in the repeated evolution of similar pattern elements in distantly related species. I also find that intraspecies variation in patterning has likely evolved recently and often involves both cis-regulatory and coding mutations to the MYB paralogs. These findings, when combined with previous studies of plant pigmentation patterning, suggests that the modularity of the duplicated R2R3 MYB genes, with specific and less pleiotropically constrained function, may play a central role in pattern diversification in plants

One of the major goals of evolutionary biology is understanding the process of species formation. There is particular interest in how selection can favor species formation through the process of reinforcement. When two diverging taxa produce maladaptive hybrids, selection will favor greater reproductive isolation between the taxa. Reinforcement often results in a pattern of reproductive character displacement, which is defined as two species having greater reproductive isolation in sympatry then in allopatry. Floral-color divergence in the native Texas wildflower, Phlox drummondii, constitutes one of the best documented cases of reinforcement in plants. P. drummondii and a closely related species, P. cuspidata produce similar light-blue flowers throughout the allopatric parts of their ranges. However, in the area of sympatry P. drummondii has dark-red flowers, which has been shown to decrease hybridization between the two species. In the following work, I investigate the causes and consequences of the process of reinforcement and the pattern of character displacement in P. drummondii. First, I identify the genetic basis of the flower color variation as regulatory changes in two genes controlling the type and amount of anthocyanin floral pigments. I then evaluate neutral genetic variation across the range of P. drummondii and conclude there is extensive gene flow between allopatric and sympatric areas of the range, which indicates that selection and not genetic drift is responsible for the flower color variation. By investigating genetic variation at the loci underlying flower color variation I find a molecular signature of a selective sweep at one of the two flower color loci, further indicating that selection is responsible for this flower color variation. Finally, I measure selection on flower color in both sympatry and allopatry. I find no evidence that flower color variation is a response to ecological character displacement or local adaptation in the area of sympatry. I find evidence of pollinator preference for the ancestral allopatric flower color in allopatry, which may explain the persistence of the pattern of character displacement. These investigations of reproductive character displacement and reinforcement address important areas of research in evolutionary biology including the genetic basis of adaptation, the formation of species, and pleiotropy and conflicting selection pressures in species.

The evolution of selfing from outcrossing is one of the most frequent mating system transitions in angiosperms. Plants that are highly selfing typically exhibit a suite of morphological traits termed a "selfing syndrome," including reduced corollas and reproductive structures, loss of corolla pigmentation, little anther-stigma separation, and a low pollen/ovule ratio. The overall consensus among scientist is that the morphological changes that accompany the transition to selfing are adaptive and thus a product of natural selection. Few attempts, however, have been made to determine whether traits of the selfing syndrome are truly an operation of natural selection or if genetic drift could be the acting force. My dissertation examines the roles that natural selection and genetic drift played in the evolution of the selfing syndrome in Ipomoea lacunosa. With the use of field observations, crossing data, and molecular analyses, I show that I. lacunosa has evolved increased selfing ability, decreased anther-stigma distance and smaller, white flowers, compared to its closest relative I. cordatotriloba. Furthermore, using a standard QST - FST comparison, I evaluated the relative importance of selection and drift in the evolution of the selfing syndrome in I. lacunosa. I also identified the genetic basis of flower color divergence between I. lacunosa (white) and I. cordatotriloba (purple) and examined patterns of variation to determine if selection or genetic drift caused the divergence. Analyses revealed that the traits of I. lacunosa characteristic of the selfing syndrome have evolved as a product of natural selection, not genetic drift.

Although much research has been done on how new genes arising from gene duplications has been done, little is still known on how these genes arose and by what mechanisms they evolved their new function. Here I address a case study on how the evolution of a novel defensive gene in the Solanaceae family arose by examining the defensive copy of threonine deaminase (TD2) in tomato (Solanum lycopersicum). This gene evolved by gene duplication from an ancestor involved in biochemical gene synthesis to become an exceptionally stable anti-nutritive defensive gene in insect guts. This stability is hypothesized to have evolved due to three critical ion pairs, ionically bonded amino acids that stabilize proteins. Here, I test whether these three critical ion pairs stabilize the protein and what effects it has the activity of TD2. The removal of the second critical ion pair reduces the Kcat of the enzyme, indicating that it affects activity in a positive manner. Removal of the first and third critical ion pairs increase activity during the temperature and pH stability assays. The results suggest that the interactions and effects of each critical ion pair is complex within the enzyme, but are likely to be stabilizing without sacrificing much activity and in some cases increasing activity as well.

The problem is two fold: how does natural selection operate on systems of interacting genes and how does natural selection operate in natural populations. To address the first problem, I have conducted a theoretical investigation into the evolution of control and the distribution of mutations in a simple system of interacting genes, a linear metabolic pathway. I found that control is distributed unevenly between enzymes, with upstream enzymes possessing the greatest control and accumulating the most beneficial mutations during adaptive evolution. To address the second problem, I investigated the evolution of copper tolerance in the common yellow monkeyflower, Mimulus guttatus. I genetically mapped a major locus controlling copper tolerance, Tol1. A Dobzhansky-Muller incompatibility was hypothesized to also be controlled by Tol1, however, we have demonstrated that it maps to another, tightly linked locus, Nec1. Finally, we investigated the parallel evolution of copper tolerance in multiple new discovered mine populations. We found that copper tolerance has evolved in parallel multiple times via at least two distinct physiological mechanisms. In four mine populations, there was a strong signal of selection at markers linked to Tol1, implying that copper tolerance has evolved via the same genetic mechanisms in these populations.