Protein-coding sequences have long been assumed to evolve under selection, but the quantification of the process at the nucleotide sequence level only started when a simple null model, the neutral theory of molecular evolution, was formulated by Kimura. Several methods were developed, which were based on the assumption that synonymous sites (nucleotides at third codon positions which do not change the encoded amino acid) evolve close to neutrally, and could be used as local neutral standards. Most of our current knowledge on the direction and strength of selection still depends on this simple assumption. One method, notably the non-synonymous to synonymous substitution rate ratio (dN/dS) has gained prevalence and is still widely used, in spite of the growing body of evidence that synonymous sites evolve under selection. In this thesis, I quantify the strength of selection in different sequence compartments of mammalian genomes, in order to obtain estimates of their functional importance from comparative genomics analyses. I quantify the fraction of mutations that have been selectively eliminated since the divergence of the species pairs examined, the so called genome wide selective constraint. This in turn is used to approximate the genomic deleterious mutation rate, which is an important parameter for several evolutionary problems. As estimates of selection depend on a large extent on the chosen neutral standard, here I use orthologous transposable elements, so called ancestral repeats, as these have been found to be evolving at a largely neutral fashion, and contain the least number of constrained sites in mammalian genomes. This enables me to quantify the level of selection even at synonymous sites, and the results suggest that these sites indeed evolve under constraint, the consequences of which I discuss. The selective constraint estimates enable me to test some simple hypotheses, such as Ohta's nearly neutral theory of molecular evolution, which suggests that selection is more efficient in species with larger effective population sizes. Beside the choice of neutral standards, there are several additional factors which are known to affect the selective constraint estimates. Here I also test the consequences of one of these, notably when sequences are not at compositional equilibrium (i.e. their GC content is away from the equilibrium GC content), which predicts that sequences with different GC content should evolve with different rates. This can cause bias in the estimates of level of selection or can even imitate selection in sequences which evolve completely neutrally. This effect is quantified here, and a simple correction is discussed.

<p>The emergence of multidrug resistant bacteria world wide is a serious problem, and very few new drugs are under development. The selection of resistant bacteria is affected by factors such as mutation rate, biological fitness cost and the rate of fitness compensation. This thesis is focused on how mutation rate affects resistance to fluoroquinolones and on exploring a dosing strategy that might slow resistance development. </p><p>In a set of urinary tract <i>Escherichia coli</i> isolates MIC values above the breakpoint for the fluoroquinolones norfloxacin and ciprofloxacin carried at least three resistance-associated mutations. In these isolates the number of resistance mutations correlated with the mutation rate. During step-wise selection for decreased susceptibility to fluoroquinolones, the accumulation of mutations in <i>E. coli</i> was associated with an increasing biological cost both <i>in vitro</i> and <i>in vivo</i>. However, in some lineages an additional selection step for resistance was associated with a partial restoration of fitness. During step-wise selections we found, as expected, that reduced ciprofloxacin susceptibility frequently hitchhiked with a strong mutator phenotype. More surprisingly, we also found that reduced susceptibility was frequently associated with the emergence of rifampicin-resistant populations. We hypothesise that this correlation reflects selection for fitness-compensating mutations in RNA polymerase.</p><p>Mutant prevention concentration (MPC) dosing has been proposed as a strategy to reduce the selection of resistant bacterial populations. Based on limited data it had been thought that MPC might be a simple multiple of MIC, which can easily be determined. However, we showed for a collection of susceptible urinary tract <i>E. coli </i>that MPC could not be predicted from MIC and must be measured directly for relevant populations. Using an <i>in vitro</i> kinetic model we also showed that the pharmacodynamic index that best predicted prevention of resistance development in wild type <i>E. coli</i> was an AUC/MPC of > 22 for ciprofloxacin.</p>

The emergence of multidrug resistant bacteria world wide is a serious problem, and very few new drugs are under development. The selection of resistant bacteria is affected by factors such as mutation rate, biological fitness cost and the rate of fitness compensation. This thesis is focused on how mutation rate affects resistance to fluoroquinolones and on exploring a dosing strategy that might slow resistance development. In a set of urinary tract Escherichia coli isolates MIC values above the breakpoint for the fluoroquinolones norfloxacin and ciprofloxacin carried at least three resistance-associated mutations. In these isolates the number of resistance mutations correlated with the mutation rate. During step-wise selection for decreased susceptibility to fluoroquinolones, the accumulation of mutations in E. coli was associated with an increasing biological cost both in vitro and in vivo. However, in some lineages an additional selection step for resistance was associated with a partial restoration of fitness. During step-wise selections we found, as expected, that reduced ciprofloxacin susceptibility frequently hitchhiked with a strong mutator phenotype. More surprisingly, we also found that reduced susceptibility was frequently associated with the emergence of rifampicin-resistant populations. We hypothesise that this correlation reflects selection for fitness-compensating mutations in RNA polymerase. Mutant prevention concentration (MPC) dosing has been proposed as a strategy to reduce the selection of resistant bacterial populations. Based on limited data it had been thought that MPC might be a simple multiple of MIC, which can easily be determined. However, we showed for a collection of susceptible urinary tract E. coli that MPC could not be predicted from MIC and must be measured directly for relevant populations. Using an in vitro kinetic model we also showed that the pharmacodynamic index that best predicted prevention of resistance development in wild type E. coli was an AUC/MPC of &gt; 22 for ciprofloxacin.

Most clonal plants exhibit a modular structure at multiple levels. At the level of the organs, they are characterized by functional modules, such as, internodes, leaves, branches. At the level of the genetic individual (clone or genet), they possess independent evolutionary and physiological units (ramets). These evolutionary units arise through the widespread phenomenon of clonal reproduction, achieved in a variety of ways including rhizomes, stolons, bulbils, or lateral roots. The focus of this study was Populus tremuloides, trembling aspen, a dioecious tree that reproduces sexually by seed and asexually through lateral roots. Local forest patches in western populations of Populus tremuloides consisted largely of multiple genotypes. Multi-clonal patches were dominated by a single genotype, and in one population (Riske Creek) we found several patches (five out of 17) consisting of a single genotype. A second consequence of modularity is that during the repeated cycle of ramet birth, development and death, somatic mutations have the opportunity to occur. Eventually, the clone becomes a mosaic of mutant and non-mutant cell lineages. We found that neutral somatic mutations accumulated across 14 microsatellite loci at a rate of between 10^-6 and 10^-5 per locus per year. We suggest that neutral genetic divergence, under a star phylogeny model of clonal growth, is an alternative way to estimate clone age. Previous estimates of clone age couple the mean growth rate per year of shoots with the area covered by the clone. This assumes a positive linear relationship between clone age and clone size. We found, however, no repeatable pattern across our populations in terms of the relationship of either shape or size to the number of somatic changes. A final consequence of modularity is that during clonal growth, natural selection is relaxed for traits involving sexual function. This means that mutations deleterious to sexual function can accumulate, reducing the overall sexual fitness of a clone. We coupled neutral genetic divergence within clones with pollen fitness data to infer the rate and effect of mildly deleterious mutations. Mutations reduced relative sexual fitness in clonal aspen populations by about 0.12x10^-3 to 1.01x10^-3 per year. Furthermore, the decline in sexual function with clone age is evidence that clonal organisms are vulnerable to the effects of senescence.

Most clonal plants exhibit a modular structure at multiple levels. At the level of the organs, they are characterized by functional modules, such as, internodes, leaves, branches. At the level of the genetic individual (clone or genet), they possess independent evolutionary and physiological units (ramets). These evolutionary units arise through the widespread phenomenon of clonal reproduction, achieved in a variety of ways including rhizomes, stolons, bulbils, or lateral roots. The focus of this study was Populus tremuloides, trembling aspen, a dioecious tree that reproduces sexually by seed and asexually through lateral roots. Local forest patches in western populations of Populus tremuloides consisted largely of multiple genotypes. Multi-clonal patches were dominated by a single genotype, and in one population (Riske Creek) we found several patches (five out of 17) consisting of a single genotype. A second consequence of modularity is that during the repeated cycle of ramet birth, development and death, somatic mutations have the opportunity to occur. Eventually, the clone becomes a mosaic of mutant and non-mutant cell lineages. We found that neutral somatic mutations accumulated across 14 microsatellite loci at a rate of between 10^-6 and 10^-5 per locus per year. We suggest that neutral genetic divergence, under a star phylogeny model of clonal growth, is an alternative way to estimate clone age. Previous estimates of clone age couple the mean growth rate per year of shoots with the area covered by the clone. This assumes a positive linear relationship between clone age and clone size. We found, however, no repeatable pattern across our populations in terms of the relationship of either shape or size to the number of somatic changes. A final consequence of modularity is that during clonal growth, natural selection is relaxed for traits involving sexual function. This means that mutations deleterious to sexual function can accumulate, reducing the overall sexual fitness of a clone. We coupled neutral genetic divergence within clones with pollen fitness data to infer the rate and effect of mildly deleterious mutations. Mutations reduced relative sexual fitness in clonal aspen populations by about 0.12x10^-3 to 1.01x10^-3 per year. Furthermore, the decline in sexual function with clone age is evidence that clonal organisms are vulnerable to the effects of senescence.

The rates and patterns at which nucleotide substitutions occur vary significantly across the genome sequence of vertebrates. A prominent example is the difference in the rate of evolution of functional sequences versus nonfunctional (neutrally evolving) sequences, which is explained by the influence of natural selection on functional sequences. However, even within neutrally evolving sequences there is striking variation in the rates and patterns of nucleotide substitutions. Unraveling the underlying processes that induce this variation is necessary to understand the basic principles of variation in neutral substitution profiles, which in turn is crucial for the identification of regions in the genome where natural selection acts. This research question builds the main focus of the present thesis. I have studied the causes and consequences of variation in different patterns of nucleotide substitutions. In particular, I have investigated substitutional strand asymmetries in mammalian genes and could show that they result from the asymmetric nature of DNA replication and transcription. Comparative analysis of substitutional asymmetries then suggested that the organization of DNA replication and the level of transcription are conserved among mammals. Further, I have examined the variation in CpG mutation rate among human genes and could show that beside DNA methylation also GC content plays a decisive role in CpG mutability. In addition, I have studied the signatures of GC-biased gene conversion and its impact on the evolution of the GC isochore structure in chicken. By comparison of the results in chicken to previous results in human I found evidence that karyotype stability is critical for the evolution of GC isochores. Finally, beside the empirical studies, I have performed theoretical investigations of substitution rates in functional sequences. More precisely, I have explored the temporal dynamics of estimates of the ratio of non-synonymous to synonymous substitution rates dN/dS in a phylogentic-population genetic framework.

Most clonal plants exhibit a modular structure at multiple levels. At the level of the organs, they are characterized by functional modules, such as, internodes, leaves, branches. At the level of the genetic individual (clone or genet), they possess independent evolutionary and physiological units (ramets). These evolutionary units arise through the widespread phenomenon of clonal reproduction, achieved in a variety of ways including rhizomes, stolons, bulbils, or lateral roots. The focus of this study was Populus tremuloides, trembling aspen, a dioecious tree that reproduces sexually by seed and asexually through lateral roots. Local forest patches in western populations of Populus tremuloides consisted largely of multiple genotypes. Multi-clonal patches were dominated by a single genotype, and in one population (Riske Creek) we found several patches (five out of 17) consisting of a single genotype. A second consequence of modularity is that during the repeated cycle of ramet birth, development and death, somatic mutations have the opportunity to occur. Eventually, the clone becomes a mosaic of mutant and non-mutant cell lineages. We found that neutral somatic mutations accumulated across 14 microsatellite loci at a rate of between 10^-6 and 10^-5 per locus per year. We suggest that neutral genetic divergence, under a star phylogeny model of clonal growth, is an alternative way to estimate clone age. Previous estimates of clone age couple the mean growth rate per year of shoots with the area covered by the clone. This assumes a positive linear relationship between clone age and clone size. We found, however, no repeatable pattern across our populations in terms of the relationship of either shape or size to the number of somatic changes. A final consequence of modularity is that during clonal growth, natural selection is relaxed for traits involving sexual function. This means that mutations deleterious to sexual function can accumulate, reducing the overall sexual fitness of a clone. We coupled neutral genetic divergence within clones with pollen fitness data to infer the rate and effect of mildly deleterious mutations. Mutations reduced relative sexual fitness in clonal aspen populations by about 0.12x10^-3 to 1.01x10^-3 per year. Furthermore, the decline in sexual function with clone age is evidence that clonal organisms are vulnerable to the effects of senescence. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Small bacterial genomes are believed to be evolutionarily derived from larger genomes through massive loss of genes and are usually associated with symbiotic or pathogenic lifestyles. It is therefore intriguing that a similar phenomenon of genome reduction has been reported within a group of free-living phototrophic marine cyanobacteria Prochlorococcus. Here I have investigated the roles of natural selection and mutation rate in the process of Prochlorococcus genome size reduction. Using a data set of complete cyanobacterial genomes including 12 Prochlorococcus and a sister group of 5 marine Synechococcus, I first reconstructed the steps leading to Prochlorococcus genome reduction in a phylogenetic context. The result reveals that small genome sizes within Prochlorococcus were largely determined by massive gene loss shortly after the split of Prochlorococcus and Synechococcus (a process we refer to as early genome reduction). A maximum likelihood approach was then used to estimate changes in both selection effect and mutation rate in the evolutionary history of Prochlorococcus. I also examined the effect of selection and functional importance of a subset of ancestor-derived genes those are lost in Prochlorococcus but are still retained in the genomes of its sister Synechococcus group. It appears that purifying selection was strongest when a large number of small effect genes were deleted from nearly all functional categories. And during this period, mutation rate also accelerated. Based on these results, I propose that shortly after Prochlorococcus diverged from its common ancestor with marine Synechococcus, its population size increased quickly and thus the efficacy of selection became very high. Due to limited nutrients and relatively constant environment, selection favored a streamlined genome for maximum economies in material and energy, causing subsequent reduction in genome size and possibly also contributing to the observed higher mutation rate.

No description available.

Unexpected high mutations detected in new emerging variants of concern (VOCs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially in the case of omicron, raises concerns and efforts to understand their evolutionary trajectory. Several hypotheses have been discussed in literature to conceptualize the source of their emergence, including intra-host viral evolution in immunocompromised patients. These patients grant opportunities for the emergence of new variants through a persisting virus winning against host immunity, and selection for viral mutations driven by treatment interventions. VOCs have in common high mutation rate exceeding the average rate of 1-2 mutations per month. Not many studies have investigated the evolutionary rate of SARS-CoV-2 in immunocompromised candidates. Therefore, the purpose of this study is to reveal potential mechanisms underlying the emergence of VOCs by exploring substitution rate of SARS-CoV-2 genomes from surveyed COVID-19 immunocompromised patient’s studies. First, SARS-CoV-2 genome sequences were collected at sequential time series throughout host infection, which were reported in the previous studies. Filtration criteria was applied to reanalyze patients with prolonged infection documented for ≥ 2 months, and comprehensive sequenced samples for ≥ 6 time points. Then, phylogenetic analysis was conducted using Nextclade (https://clades.nextstrain.org/), followed by mutation rate analysis using two substantial similar approaches to calculate the rate in i) substitutions per month and ii) substitutions per site (per year). The mutation tendency of SARS-CoV-2 in immunocompromised hosts was compared to reported VOCs, particularly to omicron. The highest observed mutation rate accounted for approximately 2.2 mutations per month, which is higher than the average rate. High mutation rate was due to prolonged infection and selection pressure by treatment interventions (i.e., convalescent plasma and antibodies). Here, higher rate of intra-host viral evolution in immunocompromised patients is detected, potentially leading to the emergence of VOC. Hence, this research highlights the need for sequencing efforts in high-risk individuals, updating treatment strategies along with further analysis on adaptive mutants pronounced due to intra-host evolution. Together, such findings provide an ultimate synergy for future public health guidelines and infection control measures.