Abstract Mitochondria contain a maternally inherited 16,568bp genome (mtDNA) that encodes for 13 out of more than 70 subunits of complexes of the respiratory chain that produce ATP by oxidative phosphorylation (OXPHOS). As a byproduct of OXPHOS, reactive oxygen species (ROS) are formed, which may play a role in ageing. MtDNA has accumulated numerous polymorphisms during evolution, leading to haplogroups characterized by ancient polymorphisms and defined by letters. MtDNA polymorphisms are thought to be neutral, but some may be slightly deleterious or even advantageous and may influence phenotypes of complex traits. Interestingly, several complex traits such as longevity and maximal aerobic power show maternal inheritance. Associations between mtDNA polymorphisms and longevity have been reported, but no systematic study has been made of the role of mtDNA in longevity. In addition, there are no previous reports on mtDNA haplogroups in elite athletic performance. Associations are demonstrated here between mtDNA haplogroups J, K and U and longevity in Finns. Interestingly, subhaplogroup J2 and haplogroup K, which were found in increased frequency among the 225 very old subjects studied, were not found among the 52 endurance athletes but were present in 11% of the 89 sprint athletes Uncoupling of OXPHOS reduces ATP and ROS production. Thus, a mitochondrial genome with a higher level of uncoupling may promote longevity but may not be favourable in situations that require a high level of ATP production, such as elite endurance performance. A more detailed analysis also showed an association between a combination of three common mtDNA polymorphisms and longevity in both the Finns and the Japanese, providing the first epidemiological support for the assumption that the nature of a mutation is determined by interactions with other mutations in mtDNA. In addition, a systematic approach was applied to study the role of mtDNA in longevity. Association analyses of mtDNA allele combinations in longevity revealed that the mtDNA control region, the tRNA and rRNA genes and the nucleotide repeats in mtDNA may play a role in longevity, since the alleles and allele combinations that showed the strongest associations with longevity, either negative or positive, were among these genes. Differences in overall variation in mtDNA between the very old and their controls were also studied, revealing more differences at synonymous (silent) sites than at non-synonymous (amino acid altering) sites. The findings support previous data suggesting that certain mtDNA haplogroups are associated with longevity. In addition, those haplogroups that increased in frequency among the very old Finns were not found among Finnish endurance athletes. Also, a novel systematic approach was applied to study mtDNA alleles, allele combinations and overall sequence variation in longevity, suggesting that there are interactions between various mtDNA positions and that the tRNA and rRNA genes and short tandem repeats in mtDNA may play a role in longevity.

Knowledge of the distribution of fitness effects of new mutations (DFE) can enable us to quantify the amount of genetic change between species that is driven by natural selection and contributes to adaptive evolution. The primary focus of this thesis is the study of methods to infer the DFE and the study of adaptive evolution in the house mouse subspecies Mus musculus castaneus. Firstly, I extended previous methodology to model the DFE based on polymorphism data. Methods that have previously been used to infer the DFE from polymorphism data have relied on the assumption of a unimodal distribution. I developed new models that can be used to fit DFEs of arbitrary complexity, and found that multimodality can be detected by these models given enough data. I used these new models to analyse polymorphism data from Drosophila melanogaster and M. m. castaneus, and found evidence for a unimodal DFE for D. melanogaster and a bimodal DFE for M. m. castaneus. Secondly, I investigated the contribution of change in coding and non-coding DNA to evolutionary adaptation. I used a polymorphism dataset of ~80 loci from M. m. castaneus sequenced in 15 individuals to investigate selection in protein-coding genes and putatively regulatory DNA close to these genes. I found that, although protein-coding genes are much more selectively constrained than non-coding DNA, they experience similar rates of adaptive substitution. These results suggest that change in functional non-coding DNA sequences might be as important as protein-coding genes to evolutionary adaptation. Thirdly, I used whole genome data from 10 M. m. castaneus individuals to compare the rate of adaptive substitution in autosomal and X-linked genes. I found that, on average, X-linked genes have a 1.8 times faster rate of adaptive substitution than autosomal genes. I also found that faster-X evolution is more pronounced for male-specific genes. I used previously developed theory to show that these observations can be explained if new advantageous mutations are recessive, with an average dominance coefficient less than or equal to 0.25. These results can help to explain the long-studied phenomenon of the large effect of the X chromosome in speciation.

Ranaviruses are large dsDNA viruses that are considered emerging pathogens, and they are known to cause mortality events in amphibian and fish populations. This research utilizes experimental and genomic data to elucidate the mechanisms driving the evolution and spread of ranaviruses, with a focus on host switching within the genus. In Chapter 1, we utilize virus challenge assays to examine potential transfer of ranaviruses between cultured juvenile largemouth bass (M. salmoides) and bullfrog tadpoles (Rana catesbeiana). Additionally, a commonly used antiparasitic treatment containing malachite green and formalin (MGF) was utilized to suppress the immune system of largemouth bass to assess the susceptibility of immunocompromised fish to ranaviruses. The results indicate that tadpoles are not susceptible to Largemouth Bass Virus (LMBV), but that bass are susceptible to ranaviruses isolated from amphibians. Furthermore, immunocompromised fish were more susceptible to both LMBV and FV3 infections than immunocompetent fish. In Chapter 2, we used eight sequenced ranavirus genomes and two selection-detection methods (site-based and branch-based) to identify genes that exhibited signatures of positive selection, potentially due to the selective pressures at play during host switching. We found evidence of positive selection acting on four genes via the site-based method, three of which are newly-acquired genes unique to ranavirus genomes. Our results suggest that the group of newly acquired genes in the ranavirus genome may have undergone recent adaptive changes that have facilitated interspecies and interclass host switching. In Chapter 3, we annotated and analyzed the nearly complete genomic sequence of LMBV to determine its taxonomic classification. The available genomic content and phylogenetic evidence suggests that LMBV is more closely related to amphibian-like ranaviruses (ALRVs) than grouper ranaviruses, and this is further supported by greater genomic collinearity between LMBV and ALRVs. This data suggests that the classification of LMBV as a ranavirus is warranted. The results presented here will help to clarify the taxonomic relationships of ranaviruses, and will also be useful in developing management strategies to limit interspecific and intraspecific viral spread. The information garnered from this research will have far-reaching implications in studies of amphibian conservation, disease evolution, and virology. / text

The genetic mechanisms by which natural populations maintain abundant phenotypic variation and adapt to their local environments remains a controversial topic in evolutionary biology. An intriguing mechanism involving highly mutable microsatellites follows the “tuning knob” model which proposes that stepwise changes in microsatellite allele lengths reciprocally generate phenotypic variation in a stepwise manner. In this study, I explored the predictions of the tuning knob model focusing specifically on transcribed microsatellites within and among natural populations of common sunflower (Helianthus annuus L.) transecting a latitudinal cline. An RNA-Seq experiment was conducted on 95 individuals from Kansas and Oklahoma grown in a common garden. To explore the potential role that microsatellites play in gene expression divergence in common sunflower, enrichment of microsatellites within differentially expressed (DE) genes was assessed. The results showed that A and AG repeat-containing microsatellites are enriched within DE genes and that 83.5% of these microsatellites are located within untranslated regions (UTRs). This finding is consistent with a role for transcribed microsatellites in gene expression divergence. RNA-Seq data were then used to assess microsatellite allele length effects on gene expression. Of all the microsatellites characterized in a reference transcriptome, 3,325 were consistently genotyped. The study identified 479 microsatellites at which allele length significantly correlated with gene expression (eSTRs). When irregular allele sizes were removed from the analysis, the number of eSTRs rose to 2379. eSTRs were most abundant within UTRs (70.4%) which suggests that they are well-positioned as cis-regulatory elements. A population genetic study conducted with 672 individuals across 17 sunflower populations from Saskatchewan to Oklahoma revealed strong signatures of directional selection acting on 13 eSTRs compared to 19 anonymous microsatellites assumed to evolve in a neutral fashion. This demonstrates that longer or shorter alleles may be favored in more extreme environments to that considered in the focal study. A second common garden experiment conducted with populations further north and south of focal populations revealed consistent patterns of correlation between microsatellite allele length and gene expression at some eSTRs. This study provides evidence that a substantial number of transcribed microsatellites function as “tuning knobs” of adaptation in common sunflower by modulating gene expression in a stepwise manner. These findings imply that the genomes of natural populations may include hundreds of active tuning knobs that can facilitate rapid evolution.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22898号 / 農博第2441号 / 新制||農||1083(附属図書館) / 学位論文||R3||N5318(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 松浦 健二, 教授 大門 高明, 教授 寺内 良平 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Understanding the genetic basis of adaptive evolution, and how natural selection has shaped patterns of polymorphism and divergence within and between species are enduring goals of evolutionary genetics. In this thesis, I used whole genome re-sequencing data to characterize the genomic signatures of natural selection along different evolutionary timescales in three Populus species: Populus tremula, P. tremuloides and P. trichocarpa. First, our study shows multiple lines of evidence suggesting that natural selection, due to both positive and purifying selection, has widely shaped patterns of nucleotide polymorphism at linked neutral sites in all three species. Differences in effective population sizes and rates of recombination largely explain the disparate magnitudes and signatures of linked selection that we observe among species. Second, we characterize the evolution of genomic divergence patterns between two recently diverged aspen species: P. tremula and P. tremuloides. Our findings indicate that the two species diverged ~2.2-3.1 million years ago, coinciding with the severing of the Bering land bridge and the onset of dramatic climatic oscillations during the Pleistocene. We further explore different mechanisms that may explain the heterogeneity of genomic divergence, and find that variation in linked selection and recombination likely plays a key role in generating the heterogeneous genomic landscape of differentiation between the two aspen species. Third, we link whole-genome polymorphic data with local environmental variables and phenotypic variation in an adaptive trait to investigate the genomic basis of local adaptation in P. tremula along a latitudinal gradient across Sweden. We find that a majority of single nucleotide polymorphisms (SNPs) (&gt;90%) identified as being involved in local adaptation are tightly clustered in a single genomic region on chromosome 10. The signatures of selection at this region are more consistent with soft rather than hard selective sweeps, where multiple adaptive haplotypes derived from standing genetic variation sweep through the populations simultaneously, and where different haplotypes rise to high frequency in different latitudinal regions. In summary, this thesis uses phylogenetic comparative approaches to elucidate how various evolutionary forces have shaped genome-wide patterns of sequence evolution in Populus. / <p>The research in this thesis was supported by the Swedish research council (to Pär K. Ingvarsson) and the JC Kempe Memorial Scholarship Foundation (to Jing Wang). The PhD study of Jing Wang in Sweden was funded by the State Scholarship from China Scholarship council.</p>

Le genre Mucor appartient au phylum des Mucoromycota, un groupe issu de l’une des lignées ayant divergé très tôt dans l'évolution des espèces fongiques (early diverging lineages). Ces groupes restent encore très peu connus par rapport aux Ascomycètes et Basidiomycètes. Le genre Mucor est un genre d'espèces saprophytes, avec cependant une certaine diversité au niveau du mode de vie. Il existe en effet au sein du genre, des endophytes de plantes (comme M. endophyticus) ou encore des pathogènes opportunistes d'animaux (comme les espèces thermophiles M. circinelloides ou M. indicus). Le genre est ubiquiste mais il existe des associations à certains habitats qui semblent dénoter une certaine spécialisation. L’objectif de cette thèse était de mieux connaître les potentialités génétiques du genre Mucor lui permettant ce mode de vie ubiquiste, son potentiel d'adaptation mais également de mieux comprendre l'existence au sein du genre d'espèces semblant s'être spécialisées en colonisant préférentiellement ou exclusivement certains habitats comme le fromage. Afin d'atteindre cet objectif des études transcriptomiques et génomiques comparées ont été menées dans le cadre de cette thèse, afin de déterminer les principales caractéristiques des génomes de Mucor aussi bien structurelles que fonctionnelles, identifier les similitudes au niveau des espèces étudiées et aussi leur spécificités et en fonction des modes de vie/habitats et déterminer s'il existe chez les espèces fréquemment rencontrées dans les fromages (et notamment pour celles considérées comme technologiques) des traces d'adaptation voire de domestication. / The genus Mucor belongs to the phylum Mucoromycota; a group that derived from the lineages that diverged early in the evolution of fungal species (early diverging lineages). These groups have been less well studied and are less well understood in comparison to Ascomycetes and Basidiomycetes. The genus Mucor is composed of saprophytic species, but also encompasses species with diverse lifestyles.For example, it includes plant endophytes (such as M. endophyticus) or opportunistic animal pathogens (such as the thermophilic species M. circinelloides or M. indicus). The genus is ubiquitous but there are some associations with specific habitats which seem to indicate specialisation. The aim of this thesis is to better understand the genetic potential of the genus Mucor in particular, to decipher how it maintains this ubiquitous lifestyle, its capacity to adapt to diverses habitats and to better understand the existence within the genus of species that may have undergone specialization allowing them to preferentially or exclusively colonise certain habitats, such as cheese. In order to achieve this, we have performed comparative transcriptomic and genomic studies in order to determine the main structural and functional characteristics of the Mucor genomes, identify similarities among the species studied and also assess whether there exist specific genetic associations with lifestyle/habitat and determine whether the species frequently found in cheese (in particular those species considered as technological) harbour imprints of adaptation or even domestication.

É crescente a busca por fontes de energia renováveis em substituição a o uso dos combustíveis fósseis, devido a grande preocupação mundial com o aquecimento global e as mudanças climáticas. O Brasil é considerado o detentor do processo de produção de etanol mais economicamente viável. Estimativas apontam para o fato de que a produção de etanol de primeira geração não será suficiente para atender a futura demanda global pelo biocombustível. Diante disto, a produção de etanol a partir da biomassa lignocelulósica se mostra como uma potencial solução. No entanto, durante o pré-tratamento e a hidrólise da biomassa, há formação de vários compostos tóxicos tais como o furfural, HMF, ácido fracos, e compostos fenólicos, os quais exercem efeitos inibitórios sobre as leveduras, tendo como consequência queda no rendimento fermentativo. Além das vantagens tecnológicas que o processo industrial brasileiro apresenta quanto à incorporação da produção de etanol 2G nas plantas já existentes, soma-se a abundância de matéria prima proveniente da própria indústria sucroalcooleira. No entanto, é de extrema necessidade o desenvolvimento de leveduras capazes de resistir as diversas condições inibitórias provenientes do novo substrato, as quais são potencializadas pelo reciclo celular. Neste sentido, o presente trabalho objetivou o desenvolvimento de novas linhagens de leveduras através das técnicas de hibridação e evolução adaptativa/seleção. Para isto, foi realizado o cruzamento massal envolvendo 5 linhagens de Saccharomyces cerevisiae, previamente selecionadas por demonstrar alta tolerância em fermentações em mosto misto a base de hidrolisado lignocelulósico e melaço de cana-de-açúcar. Inicialmente estudos foram realizados com a intenção de se obter altas taxas de esporulação, a fim de se propiciar uma grande quantidade de cruzamentos aleatórios para consequente geração de uma ampla biodiversidade, aumentando assim a possibilidade de se obter indivíduos com fenótipos melhorados. A cultura resultante do cruzamento massal foi seguida de evolução adaptativa/seleção, buscando, após cerca de 51 gerações, um enriquecimento da cultura com as linhagens mais tolerantes. Por meio de avaliação de crescimento em microplacas (DO 600nm), foram selecionadas 10 isolados evoluídos, os quais foram submetidos a ensaio de fermentação em bancada, simulando tanto quanto possível as condições industriais. Ao final, foi possível destacar uma linhagem por apresentar teor de reserva de trealose significativamente maior que as demais linhagens avaliadas, demonstrando assim a geração de um fenótipo melhorado. / Searching for renewable energy sources to substitute the fossil fuels use is growing, due to a great concern worldwide for global warming and climate change. Brazil is considered the holder of the most economically viable process of ethanol production. Estimates indicate that ethanol production of first generation will not be enough to supply future global demand for biofuel. Therefore, an ethanol production from the lignocellulosic biomass show up as a potential solution; however, during biomass pretreatment and hydrolysis, several toxic compounds such as furfural, HMF, weak acid, and phenolic compounds are formed, which exert inhibitory effects on yeasts, resulting in a fermentative yield decrease. Besides the technological advantages, presents in Brazilian industrial processes to incorporation of 2G ethanol production in existing factories, add up the abundance of feedstock comes from the own sugar and alcohol industry. However, the development of yeasts strains, resisting to inhibitory conditions from the new substrate which are potentiated by the cellular recycle, is extremely necessary. In this sense, the present work aimed the development of new yeasts strains by hybridization and adaptive evolution techniques. Mass mating was carried out involving 5 strains of Saccharomyces cerevisiae, previously selected by demonstrating high tolerance to fermentation from mixed-must composed by lignocellulosic hydrolyzate and sugarcane molasses. Previous studies were carried out to get high rates of sporulation that promote random crosses and broad biodiversity, in order to obtain individuals with improved phenotypes. The culture resulting from the mass mating was followed by an adaptation/selection, during 51 generations, generating enrichment of more tolerant strains. By means of microplate growth evaluation (DO 600nm), 10 evoluted isolates were selected, which were submitted to lab scale fermentation, simulating as much as possible as industrial conditions. At the end, it was possible to highlight a lineage demonstrating significantly higher trehalose reserve content than the other lineages evaluated, thus demonstrating a generation of an improved phenotype.

The gene regulatory network (GRN) controls the expression of genes providing phenotypic traits in living organisms. In particular, transcriptional regulation is essential to life, as it governs all levels of gene products that enable cell survival and numerous cellular functions. However, there is still poor understanding of how shifts in gene regulation alter the underlying evolutionary dynamics and consequently generate evolutionary innovations. By employing Wagner's GRN model, this dissertation investigates how the interplay of simple evolutionary forces (mutation and recombination) with natural selection acting on gene regulatory dynamics can generate major evolutionary innovations. In this dissertation, firstly, I review all currently available research papers using Wagner's GRN model, which is also employed as the computational model used extensively in the remaining chapters. I then describe how Wagner's GRN model and its variants are implemented. Finally, network properties such as stability, robustness and path length in initial populations are investigated. In the first study, I explore the characteristics of compensatory mutation in the context of genetic networks. Specifically, I find that 1) compensatory mutations are relatively insensitive to the size and connectivity of the network, 2) compensatory mutations are more likely to occur in genes at or adjacent to the site of a previous deleterious mutation and 3) compensatory mutations are more likely to be driven by mutations with a relatively large regulatory impact. In the second study, I further investigate the evolutionary consequences of the properties of compensatory mutation discovered previously. Specifically, I find that 1) compensatory mutations can occur regardless of patterns of selection, 2) networks with compensatory mutations exhibit proportionately higher robustness when compensatory mutations interact closely with deleterious mutations or have large effects on gene regulation, and 3) regulatory complexity can arise as a consequence of the propensity for co-localised and large-effect compensatory mutations. In the third study, I provide a mechanistic understanding of how recombination benefits sexual lineages. Specifically, I find that 1) recombination together with selection for developmental stability can drive populations towards the optimum, 2) recombination does not frequently disrupt well-adapted lineages as conventionally expected, and 3) recombination facilitates finding good genetic combinations which are robust to disruption, although it also rapidly purges weaker configurations. In the final study, I show that the selection pressure acting on rewiring gene regulation is critical to increasing benefits for sexual lineages whilst mitigating costs of sex and recombination. Specifically, I find that 1) strong selection strength can greatly benefit low-fitness sexual lineages, especially at the early stage, 2) recombination is initially costly, but it can rapidly evolve to compensate for costs of sex and recombination, and 3) sexual lineages with low levels of sex and recombination can outcompete strictly asexual populations under higher selection pressure and lower mutation rates. The results presented for all of the studies are important for mechanistically understanding evolutionary innovations through altering transcriptional regulatory dynamics. These innovations include 1) facilitating alternative pathway evolution, 2) driving regulatory complexity, 3) benefiting sexual reproduction, and 4) resisting invasion against asexual lineages.

É crescente a busca por fontes de energia renováveis em substituição a o uso dos combustíveis fósseis, devido a grande preocupação mundial com o aquecimento global e as mudanças climáticas. O Brasil é considerado o detentor do processo de produção de etanol mais economicamente viável. Estimativas apontam para o fato de que a produção de etanol de primeira geração não será suficiente para atender a futura demanda global pelo biocombustível. Diante disto, a produção de etanol a partir da biomassa lignocelulósica se mostra como uma potencial solução. No entanto, durante o pré-tratamento e a hidrólise da biomassa, há formação de vários compostos tóxicos tais como o furfural, HMF, ácido fracos, e compostos fenólicos, os quais exercem efeitos inibitórios sobre as leveduras, tendo como consequência queda no rendimento fermentativo. Além das vantagens tecnológicas que o processo industrial brasileiro apresenta quanto à incorporação da produção de etanol 2G nas plantas já existentes, soma-se a abundância de matéria prima proveniente da própria indústria sucroalcooleira. No entanto, é de extrema necessidade o desenvolvimento de leveduras capazes de resistir as diversas condições inibitórias provenientes do novo substrato, as quais são potencializadas pelo reciclo celular. Neste sentido, o presente trabalho objetivou o desenvolvimento de novas linhagens de leveduras através das técnicas de hibridação e evolução adaptativa/seleção. Para isto, foi realizado o cruzamento massal envolvendo 5 linhagens de Saccharomyces cerevisiae, previamente selecionadas por demonstrar alta tolerância em fermentações em mosto misto a base de hidrolisado lignocelulósico e melaço de cana-de-açúcar. Inicialmente estudos foram realizados com a intenção de se obter altas taxas de esporulação, a fim de se propiciar uma grande quantidade de cruzamentos aleatórios para consequente geração de uma ampla biodiversidade, aumentando assim a possibilidade de se obter indivíduos com fenótipos melhorados. A cultura resultante do cruzamento massal foi seguida de evolução adaptativa/seleção, buscando, após cerca de 51 gerações, um enriquecimento da cultura com as linhagens mais tolerantes. Por meio de avaliação de crescimento em microplacas (DO 600nm), foram selecionadas 10 isolados evoluídos, os quais foram submetidos a ensaio de fermentação em bancada, simulando tanto quanto possível as condições industriais. Ao final, foi possível destacar uma linhagem por apresentar teor de reserva de trealose significativamente maior que as demais linhagens avaliadas, demonstrando assim a geração de um fenótipo melhorado. / Searching for renewable energy sources to substitute the fossil fuels use is growing, due to a great concern worldwide for global warming and climate change. Brazil is considered the holder of the most economically viable process of ethanol production. Estimates indicate that ethanol production of first generation will not be enough to supply future global demand for biofuel. Therefore, an ethanol production from the lignocellulosic biomass show up as a potential solution; however, during biomass pretreatment and hydrolysis, several toxic compounds such as furfural, HMF, weak acid, and phenolic compounds are formed, which exert inhibitory effects on yeasts, resulting in a fermentative yield decrease. Besides the technological advantages, presents in Brazilian industrial processes to incorporation of 2G ethanol production in existing factories, add up the abundance of feedstock comes from the own sugar and alcohol industry. However, the development of yeasts strains, resisting to inhibitory conditions from the new substrate which are potentiated by the cellular recycle, is extremely necessary. In this sense, the present work aimed the development of new yeasts strains by hybridization and adaptive evolution techniques. Mass mating was carried out involving 5 strains of Saccharomyces cerevisiae, previously selected by demonstrating high tolerance to fermentation from mixed-must composed by lignocellulosic hydrolyzate and sugarcane molasses. Previous studies were carried out to get high rates of sporulation that promote random crosses and broad biodiversity, in order to obtain individuals with improved phenotypes. The culture resulting from the mass mating was followed by an adaptation/selection, during 51 generations, generating enrichment of more tolerant strains. By means of microplate growth evaluation (DO 600nm), 10 evoluted isolates were selected, which were submitted to lab scale fermentation, simulating as much as possible as industrial conditions. At the end, it was possible to highlight a lineage demonstrating significantly higher trehalose reserve content than the other lineages evaluated, thus demonstrating a generation of an improved phenotype.