The study of adaptive molecular evolution in natural populations has been severely limited by the difficulty of linking genetic variation to phenotypic variation to fitness effects. Most studies connecting genotype, phenotype, and fitness have used reverse genetic approaches to measure the functional effects of specific mutations in the laboratory because this relationship is difficult to measure in natural populations, particularly for complex traits because of the "many-to-one" mapping of genotype to phenotype. Many of the fundamental features of evolving systems, such as evolvability, epistasis, and pleiotropy, however, may be stronger determinants of evolutionary outcomes in natural populations than in the laboratory because artificial selection and breeding schemes are generally more simplistic relative to selection and demographic effects in natural settings. Snake venoms have emerged as a system for the study of the genetics of adaptation in complex, polygenic traits because of their genetic tractability and role in feeding, digestion, and defense, all of which are directly relevant to fitness. Because venom gene expression is tissue-specific (i.e., no pleiotropic constraints) and toxin protein abundance directly influences venom efficacy, venoms are not inherently biased toward a particular mutational pathway, enabling a systematic comparison of the molecular mechanisms underlying adaptive evolution. Venom phenotypes are manifest only upon injection into another animal, and venom functions are directly measurable through various assays, allowing direct tests of adaptive hypotheses in natural prey populations. In this work, we sought to create a genotype-phenotype-fitness map for the venom system of the eastern diamondback rattlesnake (Crotalus adamanteus) and, for the first time, identify the genetic basis of adaptation for a complex, polygenic trait in natural populations. Crotalus adamanteus is the largest species of rattlesnake and exclusively consumes endotherms. Crotalus adamanteus is historically native to seven states in the southeastern United States but has recently been extirpated from Louisiana, is endangered in North Carolina, and is currently under consideration for listing as threatened under the Endangered Species Act. In Chapter 1, we sequenced the venom-gland transcriptome and integrated mass spectrometry data to construct a transcriptome-proteome map for the venom system. We then used this map to identify significant toxin-gene expression differentiation across the range of C. adamanteus, providing candidate-genes for which to test the functional and evolutionary significance of the identified variation. In Chapter 2, we used a similar approach and identified significant ontogenetic differentiation in toxin gene expression; further analyses determined that ontogenetic effects explained more variation in toxin expression than geographic effects, although both juvenile and adult expression patterns varied geographically, and time-series experiments in lab-raised individuals demonstrated that geographic and ontogenetic expression differentiation were not environmentally induced but rather under genetic control. In Chapter 3, we used in vitro functional assays to verify that the expression differences found in the previous two chapters corresponded to differences in venom function. We found that, overall, the statistical differences in toxin expression outlined in the first two chapters equated to functional differences in toxic activities in a predictable, tractable manner, suggesting that the differences identified in the first two chapters were, in fact, biologically relevant. In Chapter 4, we used a target-enrichment approach to sequence the exons of all identified toxins in the venom-gland transcriptome as well as several thousand neutral loci to ascertain the relative roles of expression versus coding-sequence changes in a trait not inherently biased towards either mutational pathway. We found evidence for adaptive changes at both the expression and sequence levels across the entire range, although expression differentiation did appear to be the more frequent molecular mechanism. But, without functional characterizations of the identified sequence and expression evolution, it was difficult to characterize the relative roles demography, selection, and drift played in generating the identified sequence and expression divergence. Although Chapter 3 did link expression variation to functional variation, these assays were not conducted in the actual target of venoms, natural prey. To address these issues, we examined toxin sequence and expression evolution and estimated venom toxicity (i.e., fitness) in sympatric and allopatric natural prey across an island-mainland population pair in Chapter 5 to, for the first time, construct a genotype-phenotype-fitness map for a complex trait in natural populations. We found that expression differentiation was predominantly, or exclusively, the genetic basis of polygenic adaptation, suggesting that over ecological timescales complex traits may preferentially evolve through mutations affecting expression because more molecular mechanisms exist for altering the amount of protein produced than for altering their functions through their primary sequences. In Chapter 1, we found significant expression differentiation in both high- and low-abundance proteins across the range and over 1 million years of divergence, and in Chapter 4, we found both sequence and expression differentiation across the same temporal and spatial scales. In Chapter 5, however, we only identified expression differentiation, and found that this expression differentiation was restricted to low-expression proteins because of physiological and selective constraints on high-expression proteins. These differences in the molecular mechanism underlying adaptive evolution were most likely the result of temporal constraints on generating beneficial variation; because more molecular mechanisms exist for altering protein amounts than protein function, the probability of generating a beneficial expression variant is greater than the probability of generating a beneficial point mutation in the coding-region of a specific protein, and these differences in probability would be most pronounced over extremely short timescales. Given enough time, however, both mutational pathways and proteins expressed at all levels can generate beneficial variation, and these results provide qualitative predictions regarding the process of adaptation for a complex trait. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester 2016. / November 8, 2016. / adaptation, gene flow, protein expression / Includes bibliographical references. / Darin Rokyta, Professor Directing Dissertation; Peter Beerli, University Representative; Greg Erickson, Committee Member; Joseph Travis, Committee Member; Alice Winn, Committee Member.

The Pine Barrens treefrog (Hyla andersonii) is restricted to three isolated (disjunct) regions in the eastern United States: New Jersey, North and South Carolina, and the Florida panhandle and southern Alabama. It is a seepage bog specialist and a species of conservation concern in each state in which it occurs. Previous work showed some differentiation among the three regions; however, these studies had small sample sizes, measured few variables, and/or did not include all three regions. Moreover, the only genetic study of H. andersonii, using allozyme data, did not resolve clear relationships among the three regions. Conservation management of this species requires a clearer understanding of how populations in these three regions may differ from one another and how populations within regions are structured. To extend previous work on this species, I measured differentiation among regions using morphometric, acoustic signal, ecological, and genetic data. I developed 15 microsatellite markers and used targeted sequence capture to collect large-scale nuclear and mitochondrial genomic data to test models of its evolutionary history. It is hypothesized that the species shifted southward during the last glacial maximum (LGM) into one or more refugia, then expanded northward as the glaciers receded. Overall I find a strikingly concordant pattern in which the first axis of variation for each of the data types distinguishes populations along a latitudinal and longitudinal gradient and the second axis distinguishes the set of populations occurring in the Carolinas (CL) from those occurring in the New Jersey (NJ) and Alabama/Florida (AF) regions. I know of no comparable data set that displays such concordance among different types of data across so large a geographic range. The overlap in trait values (i.e. exchangeability) between neighboring regions, however, is substantial in all types of data, except genetic, which supports continued consideration of this taxon as a single species. Using a phylogenetic framework with large-scale genomic data, I found that AF forms a single clade in both the nuclear and mitochondrial trees and that AF is sister to the rest of the Atlantic clade (CL, NJ). Climate models suggest that the distribution of the species has been repeatedly disjunct since at least the last interglacial, but probably even earlier given the genetic divergence time estimates. All three regions also showed little overlap in broad-scale (climate) environmental data, although niche modeling using climate data alone closely matched the known distribution. Fine-scale environmental data (abiotic and biotic) show greater overlap between CL and AF, with some divergence from NJ. I found some association of genetic and morphometric measures with ecological values, mostly for the broad-scale data. Projections for 2050 suggest habitat suitability will be greatly reduced in CL and AF, with only a small area of NJ available. Finally, I found very different patterns of population genetic structure within each region. Little evidence for isolation by distance was found for all regions, suggesting isolation via environment or other factors may be important for connectivity between populations. The results of my research can be applied to the conservation and management of H. andersonii because it is the most comprehensive study of the species using both an ecological and evolutionary perspective across different temporal and spatial scales. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2016. / June 10, 2016. / conservation, disjunct distribution, exchangeability, Hyla andersonii, phylogeography, Pine Barrens Treefrog / Includes bibliographical references. / Emily Lemmon, Professor Co-Directing Dissertation; Joseph Travis, Professor Co-Directing Dissertation; Peter Beerli, University Representative; Scott Steppan, Committee Member; D. Bruce Means, Committee Member.

The family Iguanidae is almost completely restricted to the Western Hemisphere with its main radiations occurring in North and South America. There are also representatives on Fiji, Tonga and the Galapagos Islands, in the Pacific Ocean. Distantly related forms are also found on Madagascar off the Southeastern Coast of Africa. Although the iguanid lizards are familiar to most scientists interested in the tropics, their anatomy and evolution is poorly understood.

Differences in behavior and physiology can arise and change because of variation in life history strategies or differences in discrete polymorphisms. These differences can result from the indirect effects of predation, or responses to erratic, repeated exposures to predators in which the prey individual is not consumed (non-consumptive predation). Behavioral responses in the presence of predators, like changes in mating or antipredator behavior may differ because of life history variation or discrete morphs within populations. There are also physiological responses that occur with behavioral responses. These physiological responses occur through the animals’ stress response. A stress response is an accumulation of endocrine and neuroendocrine responses that return the body back to its normal metabolic and hormonal levels. The least killifish, Heterandria formosa is an excellent system to understand how life history variation influences responses to predation, while, melanic and silver morphs in the Eastern mosquitofish, gambusia holbrooki, are ideal for understanding how variation within populations influence responses to predation. The overarching goals of this dissertation were to evaluate variation in behavior and physiology from predation risk as a result of differences between populations of the least killifish and within a population of the Eastern mosquitofish as well as to review pleiotropic effects that stem from the central melanocortin system and their potential influence on behavior and stress within and between populations. To answer our first questions in the least killifish we created control and predator treatments and measured both populations’ differences in male mating behavior when exposed to a predator. We also analyzed female cortisol levels between the two populations to see if their stress responses varied. We exposed male and female least killifish from both populations to predator and control treatments over 30 days to measure the effect of treatment and population on reproductive output. Results showed that males from the high-predation population were more active overall than males from the low predation population in the control treatment but that males from both populations responded to the presence of predators. Females from the low predator population had higher cortisol levels in predator treatments however cortisol levels from females in high predator population were similar in control and predator treatments. Our results reflect conclusions of another study in which predator presence did not influence reproductive output in another population of H.formosa. In the Eastern mosquitofish we exposed silver and melanic males to predator and control treatments and followed behavior assays with cortisol analyses. We found that melanic males were more active than silver males in their mating behavior and this result is consistent with other studies. All males decreased their mating behaviors in the presence of predators and increased antipredator freezing behavior in predator treatments. Melanic males had higher cortisol levels on average than silver males in control treatments; melanic male cortisol levels were only half as high in the predator treatments as in the control treatments. Differences in morph behaviors and physiology may be mediated by the pleiotropic effects correlated with black coloration and also may help rare eastern mosquitofish morphs persist. Finally, we reviewed the melanocortin system and its pleiotropic effects determined the frequencies of melanic morphs within natural populations and reflected on other pathways that influence black coloration. We found that melanic morphs are rare in polymorphic populations. We also found that populations with discrete polymorphisms mostly consisted of melanic morphs that occurred 33% or less of the time in the population with melanic morphs frequencies higher than 75%. In conclusion, this work is the first to review the frequencies of melanic morphs in populations and assess behavior and stress response in the least killifish and Eastern mosquitofish. We highlight the importance of assessing short-term behavioral responses and also measuring long-term responses through predation exposure in H.formosa. We also provide evidence for the use of short and long-term measures and not solely relying on short-term responses as a means to predict fitness. We also demonstrate how variation within populations, specifically melanic coloration may influence differences in morph behaviors, the propensity to be approached or attacked by predators, and additional behavioral and physiological responses that may arise because of pleiotropy in the melanocortin system. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / 2019 / August 28, 2019. / Behavior, Life History, Melanin, Poeciliids, Predation / Includes bibliographical references. / Joseph Travis, Professor Directing Dissertation; Lisa Eckel, University Representative; Kimberly Hughes, Committee Member; Emily DuVal, Committee Member; Scott Burgess, Committee Member.

Biological hard materials are a remarkable class of materials combining large volumes of mineral with minute organic components into often complex, hierarchical microstructural arrangements. These intricate microstructures offer ideal systems from which form-function relationships can be dissected due to their limited functional demands. They are also of increasing interest to the materials science community due to their high combinations of stiffness and toughness unexpected of ceramic-like materials. Individually, each approach for understanding these materials has suffered from a lack of insight from the other field: the biological perspective has suffered from a lack of analytical rigor while the engineering perspective has been ignorant to the intricacies of evolution as needed to accurately infer the original and current function of these structures. Here I present and execute a unified framework for examining biological hard materials. In order to identify the mechanical import of microstructural changes, this framework tests changes in biologically relevant material properties by measuring mechanical response across the transformation series of microstructures observed in conjunction with ecological shifts. In order to apply this framework, I use mammalian dental enamel as a model system. Dental enamel is the most mineralized tissue in the vertebrate body and is non-repairable and irreplaceable if damaged. Arguably, it has only two functions: transfer masticatory loads to ingesta and resist its own degradation. In mammals, the evolution of a critical tissue constituent--the enamel prism--has resulted in a multitude of enamel microstructural arrangements, some of which have independently evolved consistently in ecologically similar contexts. I sought to characterize changes in the mechanical response of enamel microstructures by providing a survey of elastic modulus and fracture toughness for a diversity of mammals showing a broad array of microstructural forms. Considering the mechanics of damage to mammalian enamel as they pertain to documented microstructural changes within lineages, I then identified three critical functional transitions in enamel microstructures. These functional transitions include: (1) the evolution of the enamel prism, (2) the adaptation to a high wear diet, and (3) the adaptation to a high fracture diet. I investigated potential changes in material response across these transitions. Methodologically, I measured elastic modulus using instrumented nanoindentation across a series of reptilian and mammalian enamels to examine differences in resistance to elastic deformation. I then verified and executed a new method for determining the intrinsic fracture toughness of enamel, crack tip opening displacement, and identified changes in small scale resistance to fracture. I used Vickers microindentation to evaluate differences in resistance to plastic deformation. Lastly, I developed a novel method for quantifying fracture orientation, called Crack Analysis of Propagation Orientation (CAPO). CAPO identifies directions of preferred cracking and provides a proxy of resistance to large-scale fracture effects. These data provide consistent evidence that mammalian enamel microstructures are remarkably consistent in elastic modulus, intrinsic fracture toughness, and hardness. This consistency and their correspondence to values reported in the literature suggests that selection has acted to make enamel microstructures as stiff, hard, and intrinsically tough as possible given the inherent developmental constraints of amelogenesis and material constraints of hydroxyapatite. However, they display marked quantitative and qualitative differences in their resistance to large-scale fracture. Contact with hard particulates in the environment such as plant phytoliths or exogenous grit are expected to result in local indentation damage and the removal of enamel through microcrack growth. Grazing taxa have enamels which include modified radial enamel, a microstructure that channels indentation crack growth into a single direction and suppresses subsurface lateral crack growth. Together, these mechanisms would reduce the removal of enamel pieces by inhibiting microcrack coalescence and offer increased resistance to severe wear. Conversely, contact with large objects such as bone are expected to result in fractures which propagate across the tooth surface. Carnivoran Hunter-Schreger bands qualitatively suppress fracture across bands; this behavior could provide resistance to fatigue crack growth. These results provide evidence that mammalian enamel microstructures are consistent in many of the commonly reported material properties but differ primarily in their large-scale fracture behavior. They further offer avenues for biomimetic ceramic composites with consistent hardness and moduli but with potential damage and fatigue tolerance specific to the loading scenario. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2018. / July 6, 2018. / Biomimetic, Enamel, Mammal, Material Properties, Microstructure, Teeth / Includes bibliographical references. / Gregory Erickson, Professor Directing Dissertation; William Oates, University Representative; Brian Inouye, Committee Member; William Parker, Committee Member; Scott Steppan, Committee Member.

Understanding the maintenance of phenotypic variation within populations has long been a puzzle in evolutionary biology. Many models ignore that fact that animals are not living alone; instead social factors have the potential to alter the development and fitness consequences of alternative phenotypes to promote variation in many systems. Furthermore, while there is empirical evidence that individuals alter phenotypes in response to social cues, it is unclear under what conditions socially-cued plasticity will evolve and be adaptive. My dissertation research combines theory with developmental experiments in sailfin mollies (Poecilia latipinna) to understand if and how individuals alter phenotypes in response to social cues. The first chapter of my dissertation uses an individual-based modelling approach to determine if and when individuals should evolve a strategy that uses social cues during development to alter the expression of alternative phenotypes. We found that socially-cued plasticity evolves under limited conditions where selection acts on survival differences between alternative phenotypes and the expression of socially-cued plasticity is costly. Socially-cued plasticity was not adaptive when selection acted on fecundity. Because costs facilitated the evolution of adaptive socially-cued plasticity, our results suggest that socially-cued plasticity is a special case of plasticity where general models do not hold. Furthermore, we found that socially-cued plasticity is a self-limiting strategy; using social cues to alter phenotypes in adulthood was most likely to evolve when the majority of the population was not using socially-cued plasticity; this scenario allowed social cues to be reliable predictors of environmental conditions. In the second and third chapter of my dissertation, I used sailfin mollies to determine how species with alternative reproductive phenotypes (ARPs) alter their life history and mating behavior in response to social cues during development. Sailfin mollies are a livebearing fish that exhibit extensive variation in body size and correlated traits including age at maturity, morphology and mating behavior, both within and between populations. Together these traits make up a male's ARP. Smalls males mature quickly (50-60 days) and use their disproportionally longer intromittent organ in sneaking behavior. Large males take longer to mature (130-150 days) and use their disproportionally larger dorsal fins in courtship displays to entice female cooperation in mating. Intermediate-sized males, which are intermediate in morphology and time to maturity, switch between courting and thrusting depending on the social context. Previous studies have examined the role of abiotic environmental factors on male ARP in mollies, but found that these factors cannot account for the observed inter- and intra-population variation. Since mollies are gregarious and social environment has been shown to influence adult male behavior, we hypothesize that variability in social conditions can influence the relationships between genotype and phenotype to produce ARP variation. My second chapter describes an experimental study where we examined the relationship between genotype and phenotype by determining how the variation in social environment during development influenced sex-specific differences in life history phenotypes. We found that both variation in the social environment influences life history development in both males and females, but there were sex-specific differences in how social environment modulated the genotype-phenotype relationship. These results suggest that social environment is an important driver of life history differences in sailfin mollies. My final experiment tested the hypothesis that social environment during development affects male alternative mating behaviors. We found that courtship and sneaking behaviors were affected by variation in the social environment, but these effects manifested in complex interactions between experimental treatments. For example, the relationship between body size and courtship displays was affected by a genotype by social environment interaction, and there was a three-way interaction between genotype, developmental stage, and the male’s own body size. In addition,. These results implicated alternative reproductive morph, social environment during development, stage, and body size as non-independent factors in the expression of male ARPs. Results from my dissertation demonstrate that conditions for adaptive evolution of socially-cued plasticity are limited, but despite this, variation in social cues elicited substantial variation in life history and behavior, in ways not accounted for by current life history or sexual selection theory. These seemingly paradoxical results may be resolved by considering the natural history of mollies. Sailfin molly males of different ARPs have differences in survival and therefore results from the modelling chapter suggest mollies may be a system where socially-cued plasticity would evolve. To determine if the patterns observed in this dissertation are adaptive, or are accounted for by gene flow, by exposing animals to social environments they would not typically encounter in nature, or by other non-adaptive processes, future studies should assess mortality in different social environments and reproductive success to determine how social environment affects fitness. Taken together, my dissertation provides a better understanding of how phenotypic plasticity evolves and how social environment affects life history and mating behaviors. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / 2019 / November 7, 2019. / alternative reproductive phenotypes, life history, Poecilia latipinna, social environment, socially-cued anticipatory plasticity, theory / Includes bibliographical references. / Kimberly A. Hughes, Professor Directing Dissertation; Elaine Hull, University Representative; Joseph Travis, Committee Member; Emily DuVal, Committee Member; Don Levitan, Committee Member.

Le but de la recherche menée dans le cadre de cette thèse est de développer et d’appliquer de nouveaux outils de modifications chromosomiques ciblées et d’évolution dirigée in vivo chez Clostridium phytofermentans, une bactérie mésophile et anaérobie qui fermente la biomasse lignocellulosique. L’introduction présente une vue d’ensemble des recherches fondamentales et appliquées dans la biologie et la génétique des Clostridia, incluant les souches pathogènes et environnementales. Une description de l’industrie des biocarburants est détaillée pour montrer les applications possibles de C. phytofermentans dans ce domaine très compétitif. Le premier chapitre présente l’utilisation de la génomique fonctionnelle et de l’inactivation de gènes cibles pour identifier des transporteurs d’hexoses chez C. phytofermentans. Le second chapitre décrit l’application de la technique Genome Editing via Targetron and Recombinases (GETR) pour effectuer de larges délétions chromosomiques chez cette bactérie. Le troisième chapitre présente l’évolution dirigée in vivo chez C. phytofermentans pour améliorer sa résistance aux inhibiteurs issus de la biomasse lignocellulosique, incluant l’analyse des variations transcriptomique et génomiques des souches résistantes. Dans son ensemble, ce travail de thèse souligne les avantages et limites de l’approche ciblé ou de l’approche par évolution in vivo, pour étudier et modifier les Clostridia. / The research aim of this thesis project was to develop and apply new tools for targeted chromosomal changes and in vivo directed evolution of Clostridium phytofermentans, a mesophilic anaerobe that ferments lignocellulosic biomass. The introduction presents an overview of previous basic and applied research in Clostridia biology and genetics, including both pathogenic and environmental strains. A focus on the biofuel industry is reported to describe applications of C. phytofermentans in this competitive industry. Chapter one presents a study using functional genomics and targeted gene inactivation to identify hexose sugar transporters in C. phytofermentans. Chapter two describes the application of Genome Editing via Targetron and Recombinases (GETR) to make large-scale chromosomal deletions in this bacterium. Chapter 3 presents the in vivo directed evolution of C. phytofermentans to enhance its resistance to lignocellulosic inhibitors including analyses of genome-wide transcription patterns and genomic variants that arose in the resistant strains. Together, this thesis work highlights the advantages and limitations of both targeted and evolutional approaches to study and engineer Clostridia.

abstract: Transgenic experiments in Drosophila have proven to be a useful tool aiding in the determination of mammalian protein function. A CNS specific protein, dCORL is a member of the Sno/Ski family. Sno acts as a switch between Dpp/dActivin signaling. dCORL is involved in Dpp and dActivin signaling, but the two homologous mCORL protein functions are unknown. Conducting transgenic experiments in the adult wings, and third instar larval brains using mCORL1, mCORL2 and dCORL are used to provide insight into the function of these proteins. These experiments show mCORL1 has a different function from mCORL2 and dCORL when expressed in Drosophila. mCORL2 and dCORL have functional similarities that are likely conserved. Six amino acid substitutions between mCORL1 and mCORL2/dCORL may be the reason for the functional difference. The evolutionary implications of this research suggest the conservation of a switch between Dpp/dActivin signaling that predates the divergence of arthropods and vertebrates. / Dissertation/Thesis / Masters Thesis Biology 2019

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