Herbivory is thought to be nutritionally inefficient relative to carnivory and omnivory. But, herbivory evolved from carnivory in many lineages, suggesting that there are advantages to eating plants. To understand the adaptive significance of the transition from carnivory to herbivory, I proposed five hypotheses for the adaptive evolution of herbivory and reviewed the current freshwater literature to identify conditions where eating plants might be adaptive over eating animals. I tested three of these ideas (Suboptimal Habitat, Heterotroph Facilitation, and Lipid Allocation) using the herbivorous Sailfin Molly (Poecilia latipinna)and identified each as a potential mechanism for the evolution of herbivory. To understand the origins of herbivory in Sailfin Mollies, I reconstructed ancestral habitats and dietsacross a phylogeny of the genus Poeciliaand then used phylogenetically independent contrasts to identify patterns of diet evolution. I found that the degree of herbivory increases with increasing salinity affiliation, suggesting that in this genus, herbivory evolved as an adaptation for invading less productive saline habitats from freshwaters. This result is consistent with the Suboptimal Habitat hypothesis, which states that herbivory allows organisms to invade and persist in ‘suboptimal’ habitats. To understand how herbivory is maintained in extant populations, I raised juvenile Sailfin Mollies in mesocosms and enclosure cages placed in the Everglades to document that dietary autotrophic lipids play a role in early life history by supporting rapid growth (Lipid Allocation). However, dietary bacterial fatty acids promoted fish survival, consistent with the Heterotroph Facilitation hypothesis, which states that indirect detritivory supplements the herbivorous diet. Finally, I quantified periphyton quality/availability and consumer density across the Everglades landscape to examine the correlates of trophic dynamics in nature. Results revealed that herbivores can persist in diverse habitats and survive on varying resources when habitats are unfavorable, supporting the Suboptimal Habitat hypothesis.

Global climate change is a real-time problem that presents threats to many species. Climate change can alter ecosystems and may lead to species extinction. Species can respond to climate change by moving to a better environment or adapting. Therefore, it is necessary to rely on several approaches and perspectives to anticipate ecological impacts of climate change. A common strategy uses models to understand how populations respond to different climate scenarios. Ecological models have helped usunderstand population persistence, but they often ignore how populations adapt to environmental stress. Adaptive evolution has been ignored because it was assumed that evolution was too slow to have any effect on ecology. Current research has shown that some populations are able to rapidly adapt to novel environments and this is essential for population persistence. We used a population genomics approach to understand how different precipitation regimes affect the perennial bunchgrass, Pseudoroegneria spicata, in the eastern Idaho sagebrush steppe. Our objective was to determine how genetic diversity changes under manipulated precipitation regimes and whether these changes were consistent with patterns of genetic diversity under natural precipitation regimes. The manipulated precipitation regimes consist of three precipitation treatments: control, drought with 50% ambient precipitation, and irrigation with 150% ambient precipitation. The natural precipitation regimes consist of two treatments: low elevation (drier than the experimental site) and high elevation (wetter). We collected plant tissue to isolate plant DNA and then used sequenced DNA for analyses. We used a hierarchical Bayesian model to estimate genotypes and allele frequencies across all loci. We found that there were low levels of genetic variation across all experimental precipitation treatments. When examining genetic differentiation, we found there was stronger differentiation in the natural precipitation regimes. Our study focuses on the short-term responses to climate to understand how environmental stress influences genetic diversity.

We explored using de-oiled algal biomass (algal cake) as a low-value substrate for production of lactic acid in fermentations with Lactobacillus casei, and strategies for increasing lactic acid production at low pH. L. casei 12A algal cake (AC) fermentations showed carbohydrate and amino acid availability limit growth and lactic acid production. These nutritional requirements were effectively addressed with enzymatic hydrolysis of the AC using α-amylase, cellulase, and pepsin. Producing 0.075 g lactic acid per g AC from AC digested with all three enzymes. We explored heterologous expression of the cellulase gene (celE) from Clostridium thermocellum and the α-amylase gene (amyA) from Streptococcus bovis in L. casei 12A. Functional activity of CelE was not detected, but low-level activity of AmyA was achieved, and increased > 1.5-fold using a previously designed synthetic promoter. Nonetheless, the improvement was insufficient to significantly increase lactic acid production. Thus, substantial optimization of amyA and celE expression in L. casei 12A would be needed to achieve activities needed to increase lactic acid production from AC. We explored transient inactivation of MutS as a method for inducing hypermutability and increasing adaptability of L. casei 12A and ATCC 334 to lactic acid at low pH. The wild type cells and their ΔmutS derivatives were subject to a 100-day adaptive evolution experiment, followed by repair of the ΔmutS lesion in representative isolates. Growth studies at pH 4.0 revealed that all four adapted strains grew more rapidly, to higher cell densities, and produced significantly more lactic acid than untreated wild-type cells. The greatest increases were observed from the adapted ΔmutS derivatives. Further examination of the 12A adapted ΔmutS derivative identified morphological changes, and increased survival at pH 2.5. Genome sequence analysis confirmed transient MutS inactivation decreased DNA replication fidelity, and identified potential genotypic changes in 12A that might contribute to increased acid lactic acid resistance. Targeted inactivation of three genes identified in the adapted 12A ΔmutS derivative revealed that a NADH dehydrogenase (ndh), phosphate transport ATP-binding protein PstB (pstB), and two-component signal transduction system (TCS) quorum-sensing histidine kinase (hpk) contribute to increased acid resistance in 12A.

Determining the properties of mutations is fundamental to understanding the mechanisms of adaptive evolution. The major goal of this thesis is to investigate the mechanisms of bacterial adaptation to new environments using experimental evolution. Different types of mutations were under investigations with a particular focus on genome rearrangements. Adaptive evolution experiments were focused on the development of bacterial resistance to antibiotics. In paper I, we performed stochastic simulations to examine the role of gene amplification in promoting the establishment of new gene functions. The results show that gene amplification can contribute to creation of new gene functions in nature. In paper II, the evolution of β-lactam resistance was studied by evolving S. typhimurium carrying a β-lactamase gene towards increased resistance against cephalosporins. Our results suggest that gene amplification is likely to provide an immediate solution at the early stage of adaptive evolution and subsequently facilitate further stable adaptation. In paper III, we isolated spontaneous deletion mutants with increased competitive fitness, which indicated that genome reduction could be driven by selection. To test this hypothesis, independent lineages of wild type S. typhimurium were serially passaged for 1000 generations and we observed fixation of deletions that significantly increased bacterial fitness when reconstructed in wild type genetic background. In paper IV, we developed a new strategy combining 454 pyrosequencing technology and a ‘split mapping’ computational method to identify unique junction sequences formed by spontaneous genome rearrangements. A high steady-state frequency of rearrangements in unselected bacterial populations was suggested from our results. In paper V, the rates, mechanisms and fitness effects of colistin resistance in S. typhimurium were determined. The high mutation rate and low fitness costs suggest that colistin resistance could develop in clinical settings. In paper VI, a novel Metallo-β-lactamase (MBL) with low resistance against β-lactam antibiotics was employed as the ancestral protein in a directed evolution experiment to examine how an enzyme evolves towards increased resistance. For most isolated mutants, in spite of their significantly increased resistance, both mRNA and protein levels were decreased as compared with the parental protein, suggesting that the catalytic activity had increased.

Evidence indicates seed oil melting point is likely an adaptive quantitative trait in many flowering plant species. An adaptive hypothesis suggests selection has changed the melting point of seed oils to covary with germination temperatures because of a trade-off between total energy stores and the rate of energy acquisition during germination under competition. The predicted differences in relative fitness under different temperatures have not yet been tested and little is known about the genetic basis of differences in oil composition. I used Arabidopsis thaliana to: (1) assess the fitness consequences of high and low melting point seeds germinating at different temperatures, (2) assess what genes underlie natural variation in seed oil composition, and (3) consider how these genes may be used to create oils with particular characteristics. To assess the effects of seed oil melting point on timing of seedling emergence and fitness, I competed high and low melting point lines of A. thaliana under cold and warm temperatures. Emergence timing between these lines was not significantly different at either temperature, which comported with warm temperature predictions but not cold temperature predictions. Under all conditions, plants competing against high melting point lines had lower fitness relative to those against low melting point lines, which matched expectations for undifferentiated emergence times. To assess the genetic basis of naturally occurring variation in seed oil melting point, the seed oil compositions of 391 accessions of A. thaliana were used in a genome-wide association study. Twelve genes were tightly linked with SNPs significantly associated with seed oil melting point variation. Seven encoded lipid synthesis enzymes or regulatory products. The remaining 5 encoded products with no clear relation to seed oil melting point. Results suggest selection can alter quantitative trait variation in response to local conditions through a small set of genes. 268 seed-expressed, candidate genes were linked to 103 SNPs associated with A. thaliana seed oil fatty acids. Eight genes were involved in lipid metabolism, and thirty-four encoded regulatory products. I discuss how knowledge of these genes can be used to breed and engineer desirable oil compositions for industry and nutrition. / text

Reproductive traits evolve rapidly at the morphological, physiological and molecular levels, a taxonomically robust pattern that is thought to arise from sexual selection. In internally fertilizing organisms, female promiscuity results in competition between multiple male ejaculates for fertilizations in the same female reproductive tract, extending sexual selection past courtship and copulation. In this post-copulatory arena, biochemical interaction between male ejaculates and female reproductive tracts form a dynamic molecular interface that modulates female post-mating responses essential to reproductive fitness. Consistent with the hypothesis that these interactions are subject to sexual selection, sperm and seminal proteins are known to evolve rapidly in a broad range of taxa. The female role in this process, however, in terms of both molecular mechanisms and evolutionary dynamics, remains unclear.The presented dissertation research examines the biochemical nature and evolutionary consequences of post-copulatory sexual selection in two sister-species of cactophilic Drosophila, D. mojavensis and D. arizonae. I first present data that female post-mating response in crosses between these to species is perturbed, severely reducing the reproductive output of heterospecific crosses. A breakdown of reproductive tract interactions in matings between divergent lineages suggests that male and female contributions to reproductive outcomes are coadapted. Next, I use a combination of bioinformatic analyses, comparative sequence analyses, and biochemical assays to elucidate candidate female reproductive tract proteins that may be involved in ejaculate-female dynamics. 241 candidate female reproductive proteins are identified, the most intriguing of which are recently-duplicated secreted proteases. Finally, I explore the evolutionary history of two families of secreted proteases within geographically isolated populations of D. mojavensis. I show that both families evolve rapidly through a complex process involving gene duplication, gene conversion, pseudogenation and positive selection, a unique pattern never before documented in reproductive proteins.Collectively, my dissertation research suggests that females are active participants in the evolution of reproductive tract interactions. Further exploration of how sexual reproduction coevolves between males and females, both in terms of interacting biomolecules, and dynamic evolutionary histories, remains an important challenge for future research.

Microbial-based industrial production has experienced a revolutionary development in the last decades as chemical industry has shifted its focus towards more sustain- able production of fuels, building blocks for materials, polymers, chemicals, etc. The strain engineering and optimization programs for industrially relevant phenotypes tackle three challenges for increased production: optimization of titer, productivity, and yield. The yield of production is function of the robustness of the microbe, generally associated with complex phenotypes. The poor understanding of complex phenotypes associated with increased production poses a challenge for the rational design of strains of more robust microbial producers. Laboratory adaptive evolution is a strain engineering technique used to provide fundamental biological insight through observation of the evolutionary process, in order to uncover molecular determinants associated with the desired phenotype. In this dissertation, the development of different methodologies to study complex phenotypes in microbial systems using laboratory adaptive evolution is described. Several limitations imposed for the nature of the technique were discussed and tackled. Three different cases were studied. Initially, the n-butanol tolerance in Escherichia coli was studied in order to illustrate the effect of clonal interference in microbial systems propagated under selective pressure of an individual stressor. The methodology called Visualizing Evolution in Real Time (VERT) was developed, to aid in mapping out the adaptive landscape of n-butanol tolerance, allowing the uncovering of divergent mechanisms of tolerance. A second case involves the study of clonal interference of microbial systems propagated under several stressors. Using VERT, Saccharomyces cerevisiae was evolved in presence of hydrolysates of lignocellulosic biomass. Isolated mutants showed differential fitness advantage to individual inhibitors present in the hydrolysates; however, some mutants exhibited increased tolerance to hydrolysates, but not to individ- ual stressors. Finally, dealing with the problem of using adaptive evolution to increase production of secondary metabolites, an evolutionary strategy was successfully designed and applied in S. cerevisiae, to increase the production of carotenoids in a short-term experiment. Molecular mechanisms for increased carotenoids production in isolates were identified.

Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2017-04-05T18:01:32Z No. of bitstreams: 1 DissLPMoc.pdf: 5018835 bytes, checksum: 6e118e7b00ba50c95eaca8d0df293f3a (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-04-19T13:35:49Z (GMT) No. of bitstreams: 1 DissLPMoc.pdf: 5018835 bytes, checksum: 6e118e7b00ba50c95eaca8d0df293f3a (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-04-19T13:36:00Z (GMT) No. of bitstreams: 1 DissLPMoc.pdf: 5018835 bytes, checksum: 6e118e7b00ba50c95eaca8d0df293f3a (MD5) / Made available in DSpace on 2017-04-19T13:42:20Z (GMT). No. of bitstreams: 1 DissLPMoc.pdf: 5018835 bytes, checksum: 6e118e7b00ba50c95eaca8d0df293f3a (MD5) Previous issue date: 2016-03-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Biodiesel is a renewable fuel and its production generate raw glycerol (RG) as main byproduct. The use of RG as carbon source in microorganism cultivations poses as an alternative to add value and reduce the environmental impact of this residue. However, RG impurities (salts, esters, alcohol and soap) can inhibit cell growth. Techniques that aims adapting microorganisms to environments containing contaminants by adaptive evolution have been employed to overcome inhibition problems. Adaptation strategies allows imposing a certain selective pressure upon the population, favoring the appearance of mutants and selection of most beneficial mutations, which will make the cell more suited to develop itself in a hostile environment. This work employed Adaptive Evolution methodology to obtain an E. coli K12 strain adapted to RG concentrated by rotary evaporation (RGRota). Cultivations were carried out in plates (E. coli – USP strain) incubated at 37 ºC, as well as shaken flasks (E. coli – UMinho strain), kept at 37 ºC and 300 rpm, involving transfers to defined media gradually enriched with RGRota. Obtained evolved strain as well as the wild-type strain E. coli – UMinho were characterized in cultivations using 2 L, bench-scale bioreactor, equipped with monitoring and control system. During shaken flask experiments, growth was followed by optical density (OD) readings. In bioreactor cultures, samples were withdrawal to analyze cell concentration of the suspension (OD and dry cell weight), concentrations of glycerol, ethanol and organic acids (liquid chromatography), concentration of viable cells (colony forming units counting) and morphology. Cultures characterization were carried out with E. coli – USP in shaken flasks, the values of maximum specific growth rate (μmax) remained between 0.40 e 0.45 h-1 and they showed little influence of strain or media composition. These results suggest that the selected strain did not have differentiated characteristics from the wild-type strain. For E. coli – UMinho, two adaptation strategies were evaluated: successive transfer during exponential growth phase (OD = ~2.5) and during stationary growth phase (OD = ~10). In both cases cells evolved, showing increased μmax values, with more homogeneous populations being observed for adaptation conducted under the first strategy. After 26 days of adaptation, corresponding to 534 generations, an evolved strain, exhibiting μmax of 0.60 h-1 and capable of growing in medium containing 29 g/L of glycerol from RGRota was selected by the methodology of successive transfers in exponential phase. This growth rate was 27.6 % superior to that achieved by the wild-type strain (0.47 h-1). Evolved and wild-type strains were cultivated in bioreactor, containing defined medium prepared with GBRota to have 40 g/L of glycerol. The evolved one maintained μmáx of 0.61 h-1. Acetate formation was observed, with yield of 0.19 g acetate/g glycerol, which caused growth inhibition and limited biomass yield to 0.26 gbiomass/gglycerol. When the wild-type strain was cultivated in bioreactor, exponential growth started after 24 h of lag phase and it presented μmax of 0.28 h-1, biomass yield of 0,39 gbiomass/gglycerol and acetate yield of 0.19 gacetate/gglycerol. The evolved strain obtained, capable of growing in the biodiesel production residue, showed a μmax value similar to the best results reported in the literature for E. coli adaptation in pure glycerol (0.7 h-1), what demonstrates the successful application of the adaptive evolution methodology. Acetate accumulation can be reduced by Genetic Engineering techniques to manipulate metabolic pathways and this will lead to development of an industrial strain which can be employed as a platform of high value products using unrefined glycerol as substrate. / O biodiesel é um combustível renovável cuja produção gera o glicerol bruto (GB) como principal subproduto. O aproveitamento de GB como fonte de carbono em cultivos de microrganismos se apresenta como uma alternativa para agregar valor e reduzir o impacto ambiental deste resíduo. Contudo, as impurezas do GB (sais, ésteres, álcool e sabão) podem inibir o crescimento das células. Técnicas que visam adaptar os microrganismos via evolução adaptativa a ambientes contendo contaminantes vêm sendo empregadas para contornar problemas de inibição. As estratégias de adaptação permitem impor uma certa pressão seletiva sobre a população, favorecendo o aparecimento de mutantes e a seleção de mutações benéficas, que tornam a célula mais apta a se desenvolver em um ambiente hostil. O trabalho empregou a metodologia de Evolução Adaptativa para obter uma linhagem de E. coli K12 adaptada ao GB concentrado por rotaevaporação (GBRota). Os cultivos foram realizados tanto em placas (linhagem E. coli – USP) incubadas a 37ºC, como em frascos agitados (linhagem E. coli – UMinho), mantidos a 37ºC e 300 rpm, envolvendo transferências para meios definidos gradualmente enriquecidos com GBRota. A linhagem evoluída obtida assim como a linhagem selvagem E. coli – UMinho foram caracterizadas em cultivos em biorreator de bancada de 2 L, dotado de sistema de monitoramento e controle. Durante os experimentos em frascos agitados, o crescimento foi acompanhado por leitura de densidade ótica (DO). Nos cultivos em biorreator, amostras foram coletadas para análises de concentração celular da suspensão (DO e massa seca), da concentração de glicerol, etanol e ácidos orgânicos (por cromatografia líquida), da concentração de células viáveis (por contagem de unidades formadoras de colônia) e de morfologia. Para os cultivos de caracterização da E. coli – USP realizados em frascos agitados, os valores da velocidade máxima específica de crescimento (max) permaneceram entre 0,40 e 0,45 h-1, sendo pouco influenciados pela linhagem ou pela composição dos meios, sugerindo que a metodologia adotada para adaptação em placa não foi eficiente, já que a linhagem selecionada não possuía características diferenciadas em relação à linhagem selvagem. Para a E. coli – UMinho foram avaliadas duas estratégias de adaptação: transferências sucessivas na fase exponencial do cultivo (DO = ~2,5) e na fase estacionária (DO = ~10). Em ambos os casos, as células evoluíram, apresentando aumento nos valores de max., sendo que populações mais homogêneas foram observadas na adaptação realizada pela primeira estratégia. Após 26 dias de adaptação, correspondendo a 534 gerações, foi selecionada pela metodologia de transferências sucessivas na fase exponencial, uma linhagem evoluída apresentando velocidade máxima específica de 0,60 h-1, resultado superior em 27,6% ao da linhagem selvagem (0,47h-1), capaz de crescer em meio contendo ~30 g/L de glicerol proveniente do GBRota. As linhagens selvagem e evoluída foram cultivadas em biorreator contendo meio preparado com GBRota na concentração de 40 g/L de glicerol. A linhagem evoluída manteve o μmáx de 0,61 h-1. Foi observada formação de acetato, com rendimento de 0,19 gacetato/gglicerol, o que causou inibição do crescimento e limitou o rendimento em biomassa a 0,26 gbiomassa/gglicerol. Enquanto que, para a linhagem selvagem o cultivo em biorreator apresentou uma fase lag de 24 h, um max de 0,28 h-1, rendimento em biomassa de 0,39 gacetato/gglicerol e rendimento em acetato 0,19 gacetato/gglicerol. A linhagem evoluída obtida no presente trabalho, capaz de crescer no resíduo da produção de biodiesel, apresenta max semelhante aos melhores resultados relatados na literatura para adaptação de E. coli em glicerol puro (0,7 h-1), demonstrando o sucesso da aplicação da metodologia de evolução adaptativa. O acúmulo de acetato pode ser amenizado utilizando técnicas de Engenharia Genética para manipulação das vias metabólicas e permitindo o desenvolvimento de uma linhagem industrial que poderá ser empregada como plataforma para obtenção de produtos de alto valor agregado usando o glicerol não refinado como substrato.

The study of biological invasions is not only essential to regulate their vast potential for ecological and economical harm, they offer a unique opportunity to study adaptive evolution in the context of recent range expansions into novel environments. The North American invasion of the gypsy moth, Lymantria dispar L., since its introduction in 1869 to Massachusetts, has expanded westward to Minnesota, northward to Canada, and southward to North Carolina. Fluctuating range dynamics at the southern invasive edge are heavily influenced by heat exposure over their optimal (supraoptimal) during the larval stage of development. We coupled genomic sequencing with reciprocal transplant and laboratory-rearing experiments to examine the interactions of phenotypic, genetic, and environmental variation under selective supraoptimal regimes. We demonstrate that while there is no evidence to support local adaptation in the fitness-related physiological traits we measured, there are clear genomic patterns of adaptation due to differential survival in higher temperatures. Mapping of loci identified as contributing to local adaptation in a selective environment and those associated with phenotypic variation highlighted that variation in larval development time is partly driven by pleiotropic loci also affecting survival. Overall, I highlight the necessity and inferential power gained through replicating environmental conditions using both phenotypic and genome-wide analyses.

Antibiotic resistance is a worldwide health threat, as bacteria continue to evade antibiotic treatment. In order to survive, bacteria utilize a number of resistance mechanisms, including efflux pumps, which efficiently export antibiotics outside of the cell to reduce intracellular damage. While such mechanisms are well known, there remains a significant gap in knowledge regarding how different environmental dynamics, such as the rate of antibiotic introduction or the diversity within a microbial community, play a role in resistance. In this work, we used the AcrAB-TolC efflux pump as a case study to explore how such complex dynamics promote antibiotic resistance and its evolution. First, through a combined effort using experiments and mathematical modeling, we discovered that the rate of antibiotic introduction impacts the fraction of resistant bacteria in a population. We then explored the impact of mixed populations on survival following antibiotic treatment. In mixed microcolonies, we found that resistant cells can harm their susceptible neighbors by exporting antibiotics to increase the local concentrations of these drugs. Next, we aimed to understand how these environmental effects may impact longer-term survival of an antibiotic treatment, focusing on the evolution of resistance over ~72 hours. Through a series of adaptive evolution experiments, we identified that near-MIC treatments were the most likely to promote antibiotic resistance, regardless of whether the strains contained the AcrAB-TolC pump at wild type or overexpressed levels, or whether the strains lacked the pump altogether. In studying antibiotic introduction rates on evolution, we found that slower introduction rates facilitated the evolution of high levels of resistance with a minimal fitness cost. Meanwhile, mixed populations demonstrated limited evolvability after rapid antibiotic introductions. This work provides important insights into the impacts of environmental factors, such as the rate of antibiotic introduction and the homogeneity of populations, on the promotion and evolution of antibiotic resistance. These lessons may help inform future policies on antibiotic use and mitigate the continued pattern of resistance evolution.