The Value of Support: STEM Intervention Programs Impact Student Persistence and Belonging.

In response to unwaveringly high attrition from STEM pathways, STEM Intervention Programs (SIPs) support STEM students in effort to increase retention. Using mixed methods (survey and focus groups), we studied students at one university who were either supported or unsupported by SIPs to understand how students may differ in experiences believed to contribute to STEM persistence. We evaluated: sense of belonging, scientific self-efficacy, scientific community values, scientific identity, and STEM involvement. The enrollment status of students two and a half years postsurvey was also tracked. SIP students reported significantly higher science identity and sense of belonging and were more involved in STEM-related activities than counterparts unsupported by SIPs. Differences in these measures were correlated with race/ethnicity, college generation status, and age. Notably, SIP students had higher odds of persisting in STEM than students not supported by SIPs. Focus group data provide additional meaning to the measured survey constructs and revealed nuanced qualitative differences between SIP and non-SIP student experiences. Overall, being involved in a SIP at our institution trends positively with theoretical models that explain STEM student persistence. SIPs have the potential to provide and/or facilitate meaningful and critical support, and students without those intentional supports may be left behind.

Evolutionary codependency: insights into the mitonuclear interaction landscape from experimental and wild Caenorhabditis nematodes.

Aided by new technologies, the upsurgence of research into mitochondrial genome biology during the past 15 years suggests that we have misunderstood, and perhaps dramatically underestimated, the ongoing biological and evolutionary significance of our long-time symbiotic partner. While we have begun to scratch the surface of several topics, many questions regarding the nature of mutation and selection in the mitochondrial genome, and the nature of its relationship to the nuclear genome, remain unanswered. Although best known for their contributions to studies of developmental and aging biology, Caenorhabditis nematodes are increasingly recognized as excellent model systems to advance understanding in these areas. We review recent discoveries with relevance to mitonuclear coevolution and conflict and offer several fertile areas for future work.

Mitonuclear Mismatch is Associated With Increased Male Frequency, Outcrossing, and Male Sperm Size in Experimentally-Evolved C. elegans.

We provide a partial test of the mitonuclear sex hypothesis with the first controlled study of how male frequencies and rates of outcrossing evolve in response to mitonuclear mismatch by allowing replicate lineages of C. elegans nematodes containing either mitochondrial or nuclear mutations of electron transport chain (ETC) genes to evolve under three sexual systems: facultatively outcrossing (wildtype), obligately selfing, and obligately outcrossing. Among facultatively outcrossing lines, we found evolution of increased male frequency in at least one replicate line of all four ETC mutant backgrounds tested-nuclear isp-1, mitochondrial cox-1 and ctb-1, and an isp-1 IV; ctb-1M mitonuclear double mutant-and confirmed for a single line set (cox-1) that increased male frequency also resulted in successful outcrossing. We previously found the same result for lines evolved from another nuclear ETC mutant, gas-1. For several lines in the current experiment, however, male frequency declined to wildtype levels (near 0%) in later generations. Male frequency did not change in lines evolved from a wildtype control strain. Additional phenotypic assays of lines evolved from the mitochondrial cox-1 mutant indicated that evolution of high male frequency was accompanied by evolution of increased male sperm size and mating success with tester females, but that it did not translate into increased mating success with coevolved hermaphrodites. Rather, hermaphrodites' self-crossed reproductive fitness increased, consistent with sexually antagonistic coevolution. In accordance with evolutionary theory, males and sexual outcrossing may be most beneficial to populations evolving from a state of low ancestral fitness (gas-1, as previously reported) and less beneficial or deleterious to those evolving from a state of higher ancestral fitness (cox-1). In support of this idea, the obligately outcrossing fog-2 V; cox-1 M lines exhibited no fitness evolution compared to their ancestor, while facultatively outcrossing lines showed slight upward evolution of fitness, and all but one of the obligately selfing xol-1 X; cox-1 M lines evolved substantially increased fitness-even beyond wildtype levels. This work provides a foundation to directly test the effect of reproductive mode on the evolutionary dynamics of mitonuclear genomes, as well as whether compensatory mutations (nuclear or mitochondrial) can rescue populations from mitochondrial dysfunction.

A Caenorhabditis elegans behavioral assay distinguishes early stage prostate cancer patient urine from controls.

Current methods for non-invasive prostate cancer (PrCa) detection have a high false-positive rate and often result in unnecessary biopsies. Previous work has suggested that urinary volatile organic compound (VOC) biomarkers may be able to distinguish PrCa cases from benign disease. The behavior of the nematode Caenorhabditis elegans has been proposed as a tool to take advantage of these potential VOC profiles. To test the ability of C. elegans Bristol N2 to distinguish PrCa cases from controls, we performed chemotaxis assays using human urine samples collected from men screened for PrCa. Behavioral response of nematodes towards diluted urine from PrCa cases was compared to response to samples from cancer-free controls. Overall, we observed a significant attraction of young adult-stage C. elegans nematodes to 1:100 diluted urine from confirmed PrCa cases and repulsion of C. elegans to urine from controls. When C. elegans chemotaxis index was considered alongside prostate-specific antigen levels for distinguishing cancer from cancer-free controls, the accuracy of patient classification was 81%. We also observed behavioral attraction of C. elegans to two previously reported VOCs to be increased in PrCa patient urine. We conclude nematode behavior distinguishes PrCa case urine from controls in a dilution-dependent manner.

Mitochondrial DNA Variation and Selfish Propagation Following Experimental Bottlenecking in Two Distantly Related Caenorhabditis briggsae Isolates.

Understanding mitochondrial DNA (mtDNA) evolution and inheritance has broad implications for animal speciation and human disease models. However, few natural models exist that can simultaneously represent mtDNA transmission bias, mutation, and copy number variation. Certain isolates of the nematode Caenorhabditis briggsae harbor large, naturally-occurring mtDNA deletions of several hundred basepairs affecting the NADH dehydrogenase subunit 5 (nduo-5) gene that can be functionally detrimental. These deletion variants can behave as selfish DNA elements under genetic drift conditions, but whether all of these large deletion variants are transmitted in the same preferential manner remains unclear. In addition, the degree to which transgenerational mtDNA evolution profiles are shared between isolates that differ in their propensity to accumulate the nduo-5 deletion is also unclear. We address these knowledge gaps by experimentally bottlenecking two isolates of C. briggsae with different nduo-5 deletion frequencies for up to 50 generations and performing total DNA sequencing to identify mtDNA variation. We observed multiple mutation profile differences and similarities between C. briggsae isolates, a potentially species-specific pattern of copy number dysregulation, and some evidence for genetic hitchhiking in the deletion-bearing isolate. Our results further support C. briggsae as a practical model for characterizing naturally-occurring mtgenome variation and contribute to the understanding of how mtgenome variation persists in animal populations and how it presents in mitochondrial disease states.

Beyond the Powerhouse: Integrating Mitonuclear Evolution, Physiology, and Theory in Comparative Biology.

Eukaryotes are the outcome of an ancient symbiosis and as such, eukaryotic cells fundamentally possess two genomes. As a consequence, gene products encoded by both nuclear and mitochondrial genomes must interact in an intimate and precise fashion to enable aerobic respiration in eukaryotes. This genomic architecture of eukaryotes is proposed to necessitate perpetual coevolution between the nuclear and mitochondrial genomes to maintain coadaptation, but the presence of two genomes also creates the opportunity for intracellular conflict. In the collection of papers that constitute this symposium volume, scientists working in diverse organismal systems spanning vast biological scales address emerging topics in integrative, comparative biology in light of mitonuclear interactions.

Complex Transmission Patterns and Age-Related Dynamics of a Selfish mtDNA Deletion.

Despite wide-ranging implications of selfish mitochondrial DNA (mtDNA) elements for human disease and topics in evolutionary biology (e.g., speciation), the forces controlling their formation, age-related accumulation, and offspring transmission remain largely unknown. Selfish mtDNA poses a significant challenge to genome integrity, mitochondrial function, and organismal fitness. For instance, numerous human diseases are associated with mtDNA mutations; however, few genetic systems can simultaneously represent pathogenic mitochondrial genome evolution and inheritance. The nematode Caenorhabditis briggsae is one such system. Natural C. briggsae isolates harbor varying levels of a large-scale deletion affecting the mitochondrial nduo-5 gene, termed nad5Δ. A subset of these isolates contains putative compensatory mutations that may reduce the risk of deletion formation. We studied the dynamics of nad5Δ heteroplasmy levels during animal development and transmission from mothers to offspring in genetically diverse C. briggsae natural isolates. Results support previous work demonstrating that nad5Δ is a selfish element and that heteroplasmy levels of this deletion can be quite plastic, exhibiting high degrees of inter-family variability and divergence between generations. The latter is consistent with a mitochondrial bottleneck effect, and contrasts with previous findings from a laboratory-derived model uaDf5 mtDNA deletion in C. elegans. However, we also found evidence for among-isolate differences in the ability to limit nad5Δ accumulation, the pattern of which suggested that forces other than the compensatory mutations are important in protecting individuals and populations from rampant mtDNA deletion expansion over short time scales.

Sex and Mitonuclear Adaptation in Experimental Caenorhabditis elegans Populations.

To reveal phenotypic and functional genomic patterns of mitonuclear adaptation, a laboratory adaptation study with Caenorhabditis elegans nematodes was conducted in which independently evolving lines were initiated from a low-fitness mitochondrial electron transport chain (ETC) mutant, gas-1 Following 60 generations of evolution in large population sizes with competition for food resources, two distinct classes of lines representing different degrees of adaptive response emerged: a low-fitness class that exhibited minimal or no improvement compared to the gas-1 mutant ancestor, and a high-fitness class containing lines that exhibited partial recovery of wild-type fitness. Many lines that achieved higher reproductive and competitive fitness levels were also noted to evolve high frequencies of males during the experiment, consistent with adaptation in these lines having been facilitated by outcrossing. Whole-genome sequencing and analysis revealed an enrichment of mutations in loci that occur in a gas-1-centric region of the C. elegans interactome and could be classified into a small number of functional genomic categories. A highly nonrandom pattern of mitochondrial DNA mutation was observed within high-fitness gas-1 lines, with parallel fixations of nonsynonymous base substitutions within genes encoding NADH dehydrogenase subunits I and VI. These mitochondrial gene products reside within ETC complex I alongside the nuclear-encoded GAS-1 protein, suggesting that rapid adaptation of select gas-1 recovery lines was driven by fixation of compensatory mitochondrial mutations.

Constitutive MAP-kinase activation suppresses germline apoptosis in NTH-1 DNA glycosylase deficient C. elegans.

Oxidation of DNA bases, an inevitable consequence of oxidative stress, requires the base excision repair (BER) pathway for repair. Caenorhabditis elegans is a well-established model to study phenotypic consequences and cellular responses to oxidative stress. To better understand how BER affects phenotypes associated with oxidative stress, we characterised the C. elegans nth-1 mutant, which lack the only DNA glycosylase dedicated to repair of oxidative DNA base damage, the NTH-1 DNA glycosylase. We show that nth-1 mutants have mitochondrial dysfunction characterised by lower mitochondrial DNA copy number, reduced mitochondrial membrane potential, and increased steady-state levels of reactive oxygen species. Consistently, nth-1 mutants express markers of chronic oxidative stress with high basal phosphorylation of MAP-kinases (MAPK) but further activation of MAPK in response to the superoxide generator paraquat is attenuated. Surprisingly, nth-1 mutants also failed to induce apoptosis in response to paraquat. The ability to induce apoptosis in response to paraquat was regained when basal MAPK activation was restored to wild type levels. In conclusion, the failure of nth-1 mutants to induce apoptosis in response to paraquat is not a direct effect of the DNA repair deficiency but an indirect consequence of the compensatory cellular stress response that includes MAPK activation.

Adaptive Evolution under Extreme Genetic Drift in Oxidatively Stressed Caenorhabditis elegans.

A mutation-accumulation (MA) experiment with Caenorhabditis elegans nematodes was conducted in which replicate, independently evolving lines were initiated from a low-fitness mitochondrial electron transport chain mutant, gas-1. The original intent of the study was to assess the effect of electron transport chain dysfunction involving elevated reactive oxygen species production on patterns of spontaneous germline mutation. In contrast to results of standard MA experiments, gas-1 MA lines evolved slightly higher mean fitness alongside reduced among-line genetic variance compared with their ancestor. Likewise, the gas-1 MA lines experienced partial recovery to wildtype reactive oxygen species levels. Whole-genome sequencing and analysis revealed that the molecular spectrum but not the overall rate of nuclear DNA mutation differed from wildtype patterns. Further analysis revealed an enrichment of mutations in loci that occur in a gas-1-centric region of the C. elegans interactome, and could be classified into a small number of functional-genomic categories. Characterization of a backcrossed four-mutation set isolated from one gas-1 MA line revealed this combination to be beneficial on both gas-1 mutant and wildtype genetic backgrounds. Our combined results suggest that selection favoring beneficial mutations can be powerful even under unfavorable population genetic conditions, and agree with fitness landscape theory predicting an inverse relationship between population fitness and the likelihood of adaptation.

Experimental Evolution with Caenorhabditis Nematodes.

The hermaphroditic nematode Caenorhabditis elegans has been one of the primary model systems in biology since the 1970s, but only within the last two decades has this nematode also become a useful model for experimental evolution. Here, we outline the goals and major foci of experimental evolution with C. elegans and related species, such as C. briggsae and C. remanei, by discussing the principles of experimental design, and highlighting the strengths and limitations of Caenorhabditis as model systems. We then review three exemplars of Caenorhabditis experimental evolution studies, underlining representative evolution experiments that have addressed the: (1) maintenance of genetic variation; (2) role of natural selection during transitions from outcrossing to selfing, as well as the maintenance of mixed breeding modes during evolution; and (3) evolution of phenotypic plasticity and its role in adaptation to variable environments, including host-pathogen coevolution. We conclude by suggesting some future directions for which experimental evolution with Caenorhabditis would be particularly informative.

Paternal Mitochondrial Transmission in Intra-Species Caenorhabditis briggsae Hybrids.

To study mitochondrial-nuclear genetic interactions in the nematode Caenorhabditis briggsae, our three laboratories independently created 38 intra-species cytoplasmic-nuclear hybrid (cybrid) lines. Although the cross design combines maternal mitotypes with paternal nuclear genotypes, eight lines (21%) unexpectedly contained paternal mitotypes. All eight share in common ancestry of one of two genetically related strains. This unexpected parallel observation of paternal mitochondrial transmission, undesirable given our intent of creating cybrids, provides a serendipitous experimental model and framework to study the molecular and evolutionary basis of uniparental mitochondrial inheritance.

Paths of Heritable Mitochondrial DNA Mutation and Heteroplasmy in Reference and gas-1 Strains of Caenorhabditis elegans.

Heteroplasmy-the presence of more than one mitochondrial DNA (mtDNA) sequence type in a cell, tissue, or individual-impacts human mitochondrial disease and numerous aging-related syndromes. Understanding the trans-generational dynamics of mtDNA is critical to understanding the underlying mechanisms of mitochondrial disease and evolution. We investigated mtDNA mutation and heteroplasmy using a set of wild-type (N2 strain) and mitochondrial electron transport chain (ETC) mutant (gas-1) mutant Caenorhabditis elegans mutation-accumulation (MA) lines. The N2 MA lines, derived from a previous experiment, were bottlenecked for 250 generations. The gas-1 MA lines were created for this study, and bottlenecked in the laboratory for up to 50 generations. We applied Illumina-MiSeq DNA sequencing to L1 larvae from five gas-1 MA lines and five N2 MA lines to detect and characterize mtDNA mutation and heteroplasmic inheritance patterns evolving under extreme drift. mtDNA copy number increased in both sets of MA lines: three-fold on average among the gas-1 MA lines and five-fold on average among N2 MA lines. Eight heteroplasmic single base substitution polymorphisms were detected in the gas-1 MA lines; only one was observed in the N2 MA lines. Heteroplasmy frequencies ranged broadly in the gas-1 MA lines, from as low as 2.3% to complete fixation (homoplasmy). An initially low-frequency (<5%) heteroplasmy discovered in the gas-1 progenitor was observed to fix in one gas-1 MA line, achieve higher frequency (37.4%) in another, and be lost in the other three lines. A similar low-frequency heteroplasmy was detected in the N2 progenitor, but was lost in all five N2 MA lines. We identified three insertion-deletion (indel) heteroplasmies in gas-1 MA lines and six indel variants in the N2 MA lines, most occurring at homopolymeric nucleotide runs. The observed bias toward accumulation of single nucleotide polymorphisms in gas-1 MA lines is consistent with the idea that impaired mitochondrial activity renders mtDNA more vulnerable to this type of mutation. The consistent increases in mtDNA copy number implies that extreme genetic drift provides a permissive environment for elevated organelle genome copy number in C. elegans reference and gas-1 strains. This study broadens our understanding of the heteroplasmic mitochondrial mutation process in a multicellular model organism.

Selfish Mitochondrial DNA Proliferates and Diversifies in Small, but not Large, Experimental Populations of Caenorhabditis briggsae.

Evolutionary interactions across levels of biological organization contribute to a variety of fundamental processes including genome evolution, reproductive mode transitions, species diversification, and extinction. Evolutionary theory predicts that so-called "selfish" genetic elements will proliferate when the host effective population size (Ne) is small, but direct tests of this prediction remain few. We analyzed the evolutionary dynamics of deletion-containing mitochondrial DNA (ΔmtDNA) molecules, previously characterized as selfish elements, in six different natural strains of the nematode Caenorhabditis briggsae allowed to undergo experimental evolution in a range of population sizes (N = 1, 10, 100, and 1,000) for a maximum of 50 generations. Mitochondrial DNA (mtDNA) was analyzed for replicate lineages at each five-generation time point. Ten different ΔmtDNA molecule types were observed and characterized across generations in the experimental populations. Consistent with predictions from evolutionary theory, lab lines evolved in small-population sizes (e.g., nematode N = 1) were more susceptible to accumulation of high levels of preexisting ΔmtDNA compared with those evolved in larger populations. New ΔmtDNA elements were observed to increase in frequency and persist across time points, but almost exclusively at small population sizes. In some cases, ΔmtDNA levels decreased across generations when population size was large (nematode N = 1,000). Different natural strains of C. briggsae varied in their susceptibilities to ΔmtDNA accumulation, owing in part to preexisting compensatory mtDNA alleles in some strains that prevent deletion formation. This analysis directly demonstrates that the evolutionary trajectories of ΔmtDNA elements depend upon the population-genetic environments and molecular-genetic features of their hosts.

Endogenous ROS levels in C. elegans under exogenous stress support revision of oxidative stress theory of life-history tradeoffs.

BACKGROUND: The oxidative stress theory of life-history tradeoffs states that oxidative stress caused by damaging free radicals directly underpins tradeoffs between reproduction and longevity by altering the allocation of energetic resources between these tasks. We test this theory by characterizing the effects of exogenous oxidative insult and its interaction with thermal stress and diet quality on a suite of life-history traits and correlations in Caenorhabditis elegans nematodes. We also quantify demographic aging rates and endogenous reactive oxygen species (ROS) levels in live animals. RESULTS: Our findings indicate a tradeoff between investment in reproduction and antioxidant defense (somatic maintenance) consistent with theoretical predictions, but correlations between standard life-history traits yield little evidence that oxidative stress generates strict tradeoffs. Increasing oxidative insult, however, shows a strong tendency to uncouple positive phenotypic correlations and, in particular, to reduce the correlation between reproduction and lifespan. We also found that mild oxidative insult results in lower levels of endogenous ROS accompanied by hormetic changes in lifespan, demographic aging, and reproduction that disappear in combined-stress treatments--consistent with the oxidative stress theory of aging. CONCLUSIONS: Our findings demonstrate that oxidative stress is a direct contributor to life-history trait variation and that traditional tradeoffs are not necessary to invoke oxidative stress as a mediator of relationships between life-history traits, supporting previous calls for revisions to theory.

Evolution of a higher intracellular oxidizing environment in Caenorhabditis elegans under relaxed selection.

We explored the relationship between relaxed selection, oxidative stress, and spontaneous mutation in a set of mutation-accumulation (MA) lines of the nematode Caenorhabditis elegans and in their common ancestor. We measured steady-state levels of free radicals and oxidatively damaged guanosine nucleosides in the somatic tissues of five MA lines for which nuclear genome base substitution and GC-TA transversion frequencies are known. The two markers of oxidative stress are highly correlated and are elevated in the MA lines relative to the ancestor; point estimates of the per-generation rate of mutational decay (ΔM) of these measures of oxidative stress are similar to those reported for fitness-related traits. Conversely, there is no significant relationship between either marker of oxidative stress and the per-generation frequencies of base substitution or GC-TA transversion. Although these results provide no direct evidence for a causative relationship between oxidative damage and base substitution mutations, to the extent that oxidative damage may be weakly mutagenic in the germline, the case for condition-dependent mutation is advanced.

Natural variation in Caenorhabditis briggsae mitochondrial form and function suggests a novel model of organelle dynamics.

Mitochondrial functioning and morphology are known to be connected through cycles of organelle fusion and fission that depend upon the mitochondrial membrane potential (ΔΨM); however, we lack an understanding of the features and dynamics of natural mitochondrial populations. Using data from our recent study of univariate mitochondrial phenotypic variation in Caenorhabditis briggsae nematodes, we analyzed patterns of phenotypic correlation for 24 mitochondrial traits. Our findings support a role for ΔΨM in shaping mitochondrial dynamics, but no 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.

In vivo quantification reveals extensive natural variation in mitochondrial form and function in Caenorhabditis briggsae.

We have analyzed natural variation in mitochondrial form and function among a set of Caenorhabditis 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. We performed in vivo quantification of 24 mitochondrial phenotypes including reactive oxygen species level, membrane potential, and aspects of organelle morphology, and observed significant among-isolate variation in 18 traits. Although several mitochondrial phenotypes were non-linearly associated with nad5Δ levels, most of the among-isolate phenotypic variation could be accounted for by phylogeographic clade membership. In particular, isolate-specific mitochondrial membrane potential was an excellent predictor of clade membership. We interpret this result in light of recent evidence for local adaptation to temperature in C. briggsae. Analysis of mitochondrial-nuclear hybrid strains provided support for both mtDNA and nuclear genetic variation as drivers of natural mitochondrial phenotype variation. This study demonstrates that multicellular eukaryotic species are capable of extensive natural variation in organellar phenotypes and highlights the potential of integrating evolutionary and cell biology perspectives.

Selfish little circles: transmission bias and evolution of large deletion-bearing mitochondrial DNA in Caenorhabditis briggsae nematodes.

Selfish DNA poses a significant challenge to genome stability and organismal fitness in diverse eukaryotic lineages. Although selfish mitochondrial DNA (mtDNA) has known associations with cytoplasmic male sterility in numerous gynodioecious plant species and is manifested as petite mutants in experimental yeast lab populations, examples of selfish mtDNA in animals are less common. We analyzed the inheritance and evolution of mitochondrial DNA bearing large heteroplasmic deletions including nad5 gene sequences (nad5Δ mtDNA), in the nematode Caenorhabditis briggsae. The deletion is widespread in C. briggsae natural populations and is associated with deleterious organismal effects. We studied the inheritance patterns of nad5Δ mtDNA using eight sets of C. briggsae mutation-accumulation (MA) lines, each initiated from a different natural strain progenitor and bottlenecked as single hermaphrodites across generations. We observed a consistent and strong drive toward higher levels of deletion-bearing molecules in the heteroplasmic pool of mtDNA after ten generations of bottlenecking. Our results demonstrate a uniform transmission bias whereby nad5Δ mtDNA accumulates to higher levels relative to intact mtDNA in multiple genetically diverse natural strains of C. briggsae. We calculated an average 1% per-generation transmission bias for deletion-bearing mtDNA relative to intact genomes. Our study, coupled with known deleterious phenotypes associated with high deletion levels, shows that nad5Δ mtDNA are selfish genetic elements that have evolved in natural populations of C. briggsae, offering a powerful new system to study selfish mtDNA dynamics in metazoans.

Fitness recovery and compensatory evolution in natural mutant lines of C. elegans.

Deleterious mutation accumulation plays a central role in evolutionary genetics, conservation biology, human health, and evolutionary medicine (e.g., methods of viral attenuation for live vaccines). It is therefore important to understand whether and how quickly populations with accumulated deleterious mutational loads can recover fitness through adaptive evolution. We used laboratory experimental evolution with four long-term mutation-accumulation (MA) lines of Caenorhabditis elegans nematodes to study the dynamics of such fitness evolution. We previously showed that when homozygous mutant populations are evolved in large population sizes, they can rapidly achieve wild-type fitness through the accumulation of new beneficial or compensatory epistatic mutations. Here, we expand this approach to demonstrate that when replicate lineages are initiated from the same mutant genotype, phenotypic evolution is only sometimes repeatable. MA genotypes that recovered ancestral fitness in the previous experiment did not always do so here. Further, the pattern of adaptive evolution in independently evolved replicates was contingent upon the MA genotype and varied among fitness-related traits. Our findings suggest that new beneficial mutations can drive rapid fitness evolution, but that the adaptive process is rendered somewhat unpredictable by its susceptibility to chance events and sensitivity to the evolutionary history of the starting population.