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• 1. [Article] Phenotypic and Mutational Consequences of Mitochondrial ETC Genetic Damage

  Genetic mutation is the ultimate source of new phenotypic variation in populations. The importance of mutation cannot be understated, and constitutes a significant evolutionary force. Although single mutations ...

  Citation × Citation Citation Abstract Copy Title: Phenotypic and Mutational Consequences of Mitochondrial ETC Genetic Damage Author: Lue, Michael James Year: 2015 Genetic mutation is the ultimate source of new phenotypic variation in populations. The importance of mutation cannot be understated, and constitutes a significant evolutionary force. Although single mutations may have little to no impact on organismal performance or fitness, when multiplied across the total number of potential sites within the genome, mutation can have a large impact. Accurate measurement of the rates, molecular mechanisms, and distributions of effects of mutations are critical for many applications of evolutionary theory. Despite the importance of both deleterious and beneficial mutations, their genome-wide patterns and phenotypic consequences are poorly understood when considering the mitochondrial genome. Mitochondria are organelles that are essential for eukaryotic life. They contain their own genome and generate bioenergy (ATP) necessary to sustain life via the electron transport chain (ETC). Because of their role in eukaryotic physiology, understanding how mitochondrial genetic and phenotypic variation can impact populations and evolutionary outcomes is essential. Past studies have implicated DNA-damaging oxidative stress as a source of mutations within somatic tissue, but there is a gap in knowledge regarding its role in heritable damage within the germ line. In this thesis, I aimed to test this possibility by characterizing the phenotypic and mutational consequences of high intracellular ROS levels caused by mitochondrial ETC genetic damage. I performed experiments using Caenorhabditis elegans ETC mutant, gas-1, and mutation-accumulation (MA) lines generated from this ancestral genotype. I quantified organismal fitness (fecundity and longevity), reactive oxygen species (ROS) levels, mitochondrial membrane potential (delta psi m), and ATP levels in these lines, and compared the results to those from a set of wildtype control lines. I begin with a general introduction to the hypothesis and the C. elegans system in Chapter I. In Chapter II, I report the findings from this work. In short, I found that while gas-1 MA lines began the experiment with low lifetime fecundity in comparison to the wildtype strain, their fecundity showed no further decline as expected, and even exhibited higher fecundity levels on days 3-5 of reproduction relative to the gas-1 progenitor. The gas-1 progenitor exhibited higher rates of ROS compared to wildtype, whereas the MA lines reverted back to wildtype levels; a similar pattern was observed for delta psi m, while ATP levels were low in the gas-1 progenitor and remained low in the MA lines. I interpret these findings in light of high-throughput sequencing results from these lines showing that, while nuclear and mitochondrial DNA mutation rates were equal to wildtype in these lines, the genomic pattern of mutation was highly nonrandom and indicative of selection for beneficial or compensatory sequence changes. Because ROS levels declined to wildtype in the evolved (MA), this study was unable to address whether ROS is a major contributor to heritable mutation in this system. I hypothesize that, in addition to their putatively compensatory genetic changes, gas-1 lineages experienced physiological compensation allowing them to survive, and that this was associated with a "slow living" phenotype. In Chapter III, I summarize general conclusions and implications of this study and end by providing suggestions for further study. Title: Phenotypic and Mutational Consequences of Mitochondrial ETC Genetic Damage Author: Lue, Michael James Year: 2015 Genetic mutation is the ultimate source of new phenotypic variation in populations. The importance of mutation cannot be understated, and constitutes a significant evolutionary force. Although single mutations may have little to no impact on organismal performance or fitness, when multiplied across the total number of potential sites within the genome, mutation can have a large impact. Accurate measurement of the rates, molecular mechanisms, and distributions of effects of mutations are critical for many applications of evolutionary theory. Despite the importance of both deleterious and beneficial mutations, their genome-wide patterns and phenotypic consequences are poorly understood when considering the mitochondrial genome. Mitochondria are organelles that are essential for eukaryotic life. They contain their own genome and generate bioenergy (ATP) necessary to sustain life via the electron transport chain (ETC). Because of their role in eukaryotic physiology, understanding how mitochondrial genetic and phenotypic variation can impact populations and evolutionary outcomes is essential. Past studies have implicated DNA-damaging oxidative stress as a source of mutations within somatic tissue, but there is a gap in knowledge regarding its role in heritable damage within the germ line. In this thesis, I aimed to test this possibility by characterizing the phenotypic and mutational consequences of high intracellular ROS levels caused by mitochondrial ETC genetic damage. I performed experiments using Caenorhabditis elegans ETC mutant, gas-1, and mutation-accumulation (MA) lines generated from this ancestral genotype. I quantified organismal fitness (fecundity and longevity), reactive oxygen species (ROS) levels, mitochondrial membrane potential (delta psi m), and ATP levels in these lines, and compared the results to those from a set of wildtype control lines. I begin with a general introduction to the hypothesis and the C. elegans system in Chapter I. In Chapter II, I report the findings from this work. In short, I found that while gas-1 MA lines began the experiment with low lifetime fecundity in comparison to the wildtype strain, their fecundity showed no further decline as expected, and even exhibited higher fecundity levels on days 3-5 of reproduction relative to the gas-1 progenitor. The gas-1 progenitor exhibited higher rates of ROS compared to wildtype, whereas the MA lines reverted back to wildtype levels; a similar pattern was observed for delta psi m, while ATP levels were low in the gas-1 progenitor and remained low in the MA lines. I interpret these findings in light of high-throughput sequencing results from these lines showing that, while nuclear and mitochondrial DNA mutation rates were equal to wildtype in these lines, the genomic pattern of mutation was highly nonrandom and indicative of selection for beneficial or compensatory sequence changes. Because ROS levels declined to wildtype in the evolved (MA), this study was unable to address whether ROS is a major contributor to heritable mutation in this system. I hypothesize that, in addition to their putatively compensatory genetic changes, gas-1 lineages experienced physiological compensation allowing them to survive, and that this was associated with a "slow living" phenotype. In Chapter III, I summarize general conclusions and implications of this study and end by providing suggestions for further study. Close

• 2. [Article] Causes and Consequences of Mitochondrial Variation in Caenorhabditid Nematodes

  Mitochondria are dynamic organelles that harbor their own stream-lined genome and generate much of the ATP necessary to sustain eukaryotic life via an electron transport chain (ETC). Because of the central ...

  Citation × Citation Citation Abstract Copy Title: Causes and Consequences of Mitochondrial Variation in Caenorhabditid Nematodes Author: Hicks, Kiley Ann Year: 2012 Mitochondria are dynamic organelles that harbor their own stream-lined genome and generate much of the ATP necessary to sustain eukaryotic life via an electron transport chain (ETC). Because of the central role for mitochondria in organismal physiology, mitochondrial genetic and phenotypic variation can alter organismal fitness and affect population genetic and evolutionary outcomes. Despite the far-reaching relevance of mitochondria to evolutionary processes and human health, we lack a basic understanding of the causes and consequences of mitochondrial genetic and phenotypic variation. In this thesis, I quantified mitochondrial reactive oxygen species (ROS), membrane potential (δΨM), and mitochondrial morphological traits within Caenorhabditis briggsae natural isolates and mutation-accumulation (MA) lines of both C. briggsae and Caenorhabditis elegans. Substantial natural variation was discovered for most mitochondrial form and function traits measured for a set of C. briggsae isolates known to harbor mitochondrial DNA structural variation in the form of a heteroplasmic nad5 gene deletion (nad5δ) that correlates negatively with organismal fitness. Most among-isolate phenotypic variation could be accounted for by phylogeographic clade membership rather than nad5δ level. Analysis of mitochondrial-nuclear hybrid strains provided support for both mtDNA and nuclear genetic variation as drivers of natural mitochondrial phenotype variation. An MA experimental approach revealed that average levels of both ROS and nad5δ heteroplasmy evolved in remarkably linear ways in C. briggsae maintained under extreme inbreeding. In particular, among C. briggsae isolates prone to acquiring the nad5δ deletion, nad5δ level increased to a plateau of ~50% during successive generations of MA treatment. Conversely, mitochondrial ROS level increased or declined in a strain-specific fashion, which also meant that the relationship between ROS and nad5δ was strain-specific. Further, all lines generated from the isolate with the highest starting level of nad5δ heteroplasmy went extinct prior to generation 20 of MA treatment. Patterns of among-line variance in ROS level were also strain-specific but generally did not conform to the canonical pattern of increasing among-line variance expected for MA experiments. MA lines of C. elegans that had previously been subjected to whole-genome sequencing were found to vary significantly in ROS levels but not in 8-oxo-dG content. Despite a significant positive correlation between 8-oxo-dG and ROS levels, no relationship between oxidative stress measures and base substitution rate or G-to-T transversion rate was revealed. Finally, analysis of patterns of phenotypic correlation for a suite of 24 mitochondrial traits measured in C. briggsae natural isolates support a role for ΔΨM in shaping mitochondrial dynamics, but no such role for mitochondrial ROS. Further, our study suggests a novel model of mitochondrial population dynamics dependent upon cellular environmental context and with implications for mitochondrial genome integrity. This work identifies extensive natural variation and capacity for evolution in organellar traits within multicellular eukaryotic species, with a central role for δΨM in mitochondrial dynamics that may have implications for evolutionary adaptation to thermal niches. Title: Causes and Consequences of Mitochondrial Variation in Caenorhabditid Nematodes Author: Hicks, Kiley Ann Year: 2012 Mitochondria are dynamic organelles that harbor their own stream-lined genome and generate much of the ATP necessary to sustain eukaryotic life via an electron transport chain (ETC). Because of the central role for mitochondria in organismal physiology, mitochondrial genetic and phenotypic variation can alter organismal fitness and affect population genetic and evolutionary outcomes. Despite the far-reaching relevance of mitochondria to evolutionary processes and human health, we lack a basic understanding of the causes and consequences of mitochondrial genetic and phenotypic variation. In this thesis, I quantified mitochondrial reactive oxygen species (ROS), membrane potential (δΨM), and mitochondrial morphological traits within Caenorhabditis briggsae natural isolates and mutation-accumulation (MA) lines of both C. briggsae and Caenorhabditis elegans. Substantial natural variation was discovered for most mitochondrial form and function traits measured for a set of C. briggsae isolates known to harbor mitochondrial DNA structural variation in the form of a heteroplasmic nad5 gene deletion (nad5δ) that correlates negatively with organismal fitness. Most among-isolate phenotypic variation could be accounted for by phylogeographic clade membership rather than nad5δ level. Analysis of mitochondrial-nuclear hybrid strains provided support for both mtDNA and nuclear genetic variation as drivers of natural mitochondrial phenotype variation. An MA experimental approach revealed that average levels of both ROS and nad5δ heteroplasmy evolved in remarkably linear ways in C. briggsae maintained under extreme inbreeding. In particular, among C. briggsae isolates prone to acquiring the nad5δ deletion, nad5δ level increased to a plateau of ~50% during successive generations of MA treatment. Conversely, mitochondrial ROS level increased or declined in a strain-specific fashion, which also meant that the relationship between ROS and nad5δ was strain-specific. Further, all lines generated from the isolate with the highest starting level of nad5δ heteroplasmy went extinct prior to generation 20 of MA treatment. Patterns of among-line variance in ROS level were also strain-specific but generally did not conform to the canonical pattern of increasing among-line variance expected for MA experiments. MA lines of C. elegans that had previously been subjected to whole-genome sequencing were found to vary significantly in ROS levels but not in 8-oxo-dG content. Despite a significant positive correlation between 8-oxo-dG and ROS levels, no relationship between oxidative stress measures and base substitution rate or G-to-T transversion rate was revealed. Finally, analysis of patterns of phenotypic correlation for a suite of 24 mitochondrial traits measured in C. briggsae natural isolates support a role for ΔΨM in shaping mitochondrial dynamics, but no such role for mitochondrial ROS. Further, our study suggests a novel model of mitochondrial population dynamics dependent upon cellular environmental context and with implications for mitochondrial genome integrity. This work identifies extensive natural variation and capacity for evolution in organellar traits within multicellular eukaryotic species, with a central role for δΨM in mitochondrial dynamics that may have implications for evolutionary adaptation to thermal niches. Close