Evolution of haploid and diploid populations reveals common, strong, and variable pleiotropic effects in non-home environments

  1. Vivian Chen
  2. Milo S Johnson
  3. Lucas Hérissant
  4. Parris T Humphrey
  5. David C Yuan
  6. Yuping Li
  7. Atish Agarwala
  8. Samuel B Hoelscher
  9. Dmitri A Petrov
  10. Michael M Desai
  11. Gavin Sherlock

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Abstract

Adaptation is driven by the selection for beneficial mutations that provide a fitness advantage in the specific environment in which a population is evolving. However, environments are rarely constant or predictable. When an organism well adapted to one environment finds itself in another, pleiotropic effects of mutations that made it well adapted to its former environment will affect its success. To better understand such pleiotropic effects, we evolved both haploid and diploid barcoded budding yeast populations in multiple environments, isolated adaptive clones, and then determined the fitness effects of adaptive mutations in ‘non-home’ environments in which they were not selected. We find that pleiotropy is common, with most adaptive evolved lineages showing fitness effects in non-home environments. Consistent with other studies, we find that these pleiotropic effects are unpredictable: they are beneficial in some environments and deleterious in others. However, we do find that lineages with adaptive mutations in the same genes tend to show similar pleiotropic effects. We also find that ploidy influences the observed adaptive mutational spectra in a condition-specific fashion. In some conditions, haploids and diploids are selected with adaptive mutations in identical genes, while in others they accumulate mutations in almost completely disjoint sets of genes.

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  1. Version published to 10.7554/elife.92899 on eLife
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    Reply to the reviewers

    Manuscript number: RC-2023-01901

    Corresponding author(s): Gavin, Sherlock

    We thank the reviewers for their comments and their generally positive reviews – the reviews were constructive, and we have revised the manuscript to deal with all the requested changes and suggestions. We believe the manuscript is improved as a result, and hope that the reviewers agree that it is now suitable for publication. Below with provide a point-by-point reply that explains what revisions we have made. Reviewers’ comments are italicized, while our responses are highlighted.

    Reviewer #1:

    *It would be interesting have an idea of the global mutation rates and spectra in the diploid and haploid lineages across the conditions as well. The S. cerevisiae mutational spectrum has been shown to be dependent on the environment and genetic background to an extent but not ploidy. Ploidies differ in terms of just the frequency. How similar/ dissimilar are the overall mutational spectra here? Were there any homozygous mutations in the diploids? *

    • We have plotted the mutation types in the diploid and haploid lineages across the conditions to compare the frequencies of each type of mutation between ploidies, which is now presented as Supplemental Figure 3. The mutation types between ploidies for each of the conditions look similar. Homozygous diploids are indicated in Table 2.

    *Fitness gains and losses can happen without trade-offs if neutral home mutations are non-neutral in non-home conditions. Can the authors comment on that in this context. Physico-chemically, how different are the home/ non-home environments? How do the fitness effects correlate across the environments in the absence of these adaptive mutations? It would also be useful to know the extent of fitness variance of the populations in the home and away environments, this would aid the reader better grasp the significance of fitness gains/loss. *

    • We agree that trade-offs could occur as a result of mutations that are neutral in the home condition showing trade-offs in the non-home conditions. However, in the newly added Supplemental Table 1, it is clear that most lineages have several passenger mutations, yet for lineages carrying the mutations in the same candidate beneficial mutation, they have largely similar pleiotropic profiles, suggesting that the influence of neutral mutations that arise in the home environment do not play a large role in determining fitness in other environments, at least for those tested. We have not generated strains that only contain the passenger mutations – while that would empirically test the fitness effects of the passenger mutations, it would be extremely time consuming to generate such strains, and the results would be unlikely to change our claims in the paper.

    *A summary table of the pleiotropic effects would be very useful as in Bakerlee et al. 2021. *

    • We have added a summary table (Supplemental Table 3) of the pleiotropic effects as suggested. Reviewer #2

    *The major conclusion of the manuscript state that "mutations in the same genes tend to produce similar pleiotropic effects", suggesting that a number of times this does not occur. For instance, the authors comment on the case of PDR3, which does not always produce 'cost-free' adaptation across environments. I believe that, to strengthen and better define their conclusion, the authors should develop a quantitative analysis of the reproducibility of pleiotropic profiles (that considers how many times genes have been found mutated). The heatmaps provided are compelling, but make it hard to generalize on how often, and to what extent, the gene mutated can predict pleiotropy across various environments. *

    • We have calculated pairwise correlations between pleiotropic profiles for mutations that arose in the same environment either in the same gene, or in different genes, and added this Supplemental Figure 10. These data show that by and large, correlations between mutations in the same gene are higher than those for different genes.

    *In the concluding sentence of the discussion, it is unclear whether the authors are speculating about a role of the strength of selection in determining pleiotropy based on their results, or if that only represents a suggested hypothesis to test in future studies. *

    • We have modified the concluding sentence to clarify its meaning (it was a suggested hypothesis)

    *The method used to identify putative adaptive mutations should be described in more detail. For instance, I seem to understand that only one mutation per lineage is considered 'adaptive'. However, many lineages seem to have more than one mutation. Based on what reported in the method section, the adaptive mutations have been hand-picked based on previous knowledge of selection in the environments of choice ("the list of genes was curated based on those genes' interactions with other identified genes or pathways known to be involved in the adaptation of that specific condition from previous work"). If this assumption is correct, the criteria for such a curation should be specified in more detail. *

    • We have further clarified our criteria in the text; note, there was not a requirement for there to be only a single beneficial mutation per lineage, though very few lineages had two candidate beneficial mutations.

    *The term 'Pareto front' is technical and left undefined. *

    • We have clarified the meaning of Pareto front

    *The section ' adaptation can be cost-free' only refer to figure 4, (with adaptive mutant lineages from populations evolved in fluconazole), while it comments extensively on mutation isolated in clotrimazole (reported in Sup. Fig10, not mentioned in the section). *

    • We thank the reviewer for noting our oversight – we have also now referenced the supplementary figure too (now Supplementary Figure 11). Reviewer #3

    *It would be helpful if the authors could clearly provide information on the zygosity of the evolved mutations, as the presence of mutations in homozygous or heterozygous states can impact the results of the study. *

    • We have added zygosity information to the genotypes in the text and in Table 2, Summary of Adaptive Mutations

    *Do any of the evolved lineages have multiple adaptive mutations or other potentially adaptive mutations? If so, it would be great if the authors could provide a table listing these lineages and mutations. *

    • We have added Supplemental File 1, which enumerates the adaptive and passenger mutations found in each lineage. Candidate adaptive mutations are in highlighted in red. Of the ~200 adaptive lineages, 4 have two candidate adaptive mutations, while the rest have only one.

    *In the Pooling of the Isolated Clones section of the Methods, the ancestor and subject pools were mixed in different ratios for different types of pools. While not strictly necessary, it would be helpful to provide a brief explanation for this. *

    • We have added a brief explanation

    *The conditions listed in Table 1 and Supplemental Figure 2 do not seem to match perfectly. *

    • We have corrected Supplemental Figure 2 such that it matches Table 1

    Supplementary Figure 6 demonstrates reproducible fitness estimates across lineages with the same mutations but distinct barcodes, supporting the authors' inference of adaptive mutations. However, it also appears to show no evidence of interactions among these mutations. Can the authors clarify if this is due to the absence of lineages with multiple mutations or if no observable interactions were found?

    • See response above – there are very few lineages with more than one candidate beneficial mutation. The remaining passenger mutations are thus likely neutral.

    *In the Pleiotropy is common, strong and variable section of the results, all three conditions were noted to have their evolved lineages tested in other conditions and presented in Supplementary Figure 5. However, due to the rapid dominance of lineages evolved in clotrimazole, there is no comparison data for them in Supplementary Figure 5. *

    • Unfortunately, we were not able to generate robust fitness remeasurements in the clotrimazole condition, due to the rapid takeover by lineages that were evolved in that condition

    *In the Results section on cost-free adaptation, it would be beneficial to include any compositional differences, such as pH, between the two drugs used that could have contributed to the fitness effects of the evolved lineages in pH 7.3. *

    • We are not aware of any such differences – we did not pH any of the media other than the media with a specific pH.

    *Results - Adaptation can be cost-free: While the authors did state "at least across the conditions in which we remeasured fitness" at the end of the paragraph, it may be prudent to exercise caution when stating "cost-free adaptation" as only a few conditions were tested. For instance, an all-beneficial or all-deleterious result can sometimes be obtained solely based on the chosen conditions. *

    • We have added additional caution in the text based on the reviewer’s suggestion.

    *Colormaps in Figure 4, Supplemental Figure 6, 10, and 11: The colors for values below -0.2 are uniform, whereas the heatmaps exhibit darker blues. *

    • We have edited the color scales on Figure 4 and Supplemental Figures 6, 10, and 11 (now Supplemental Figures 6, 11, and 12) such that the scales are uniform.

    *Results - Pleiotropy varies according to the mutated gene: "For example, haploid lineages adapted in glycerol/ethanol with mutations in IRA1 show the same pattern of fitness effects across conditions (Supplemental Figure 6)." I believe the authors are referring to Supplemental Figure 11. *

    • The reviewer is correct – we have fixed this reference to what is now Supplemental Figure 12

    *On the topic of IRA1, IRA2, and GPB2 in the section "Pleiotropy varies according to mutated gene" in the Results: Although IRA1 mutants exhibit highly similar patterns, it is challenging to ascertain which of the two genes, GPB2 or IRA2, has a more similar pattern. *

    • We have create a new supplemental figure showing the correlation between mutations in the same gene and mutations in different genes for lineages evolved in the same condition – see response to Reviewer #1 above.

    Results - Pleiotropy varies according to mutated gene: From "If lineages isolated from the same home environment have similar pleiotropic profiles..." to the end of that paragraph. While it is true that "pleiotropy varies according to target genes and not environment alone," it may be premature to suggest that the environment is the "main driving force" of pleiotropy without some form of statistical analysis.

    • We did not intend to suggest that environment is the main driving force - that section was somewhat poorly worded. We have modified the wording to make that clearer.

    *Discussion - line 5, paragraph 2: "For example, in glycerol/ethanol, the haploid adapted lineages have a trade off at 37{degree sign}C but the diploid adapted lineages do not (Supplemental Figure 11)." I believe the authors are referring to Supplemental Figure 6. *

    • We thank the reviewer for spotting this and have fixed the figure reference.
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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    The authors present an intriguing study on the pleiotropic effects of adaptive mutations in yeast populations evolving in different environments. The study used haploid and diploid barcoded budding yeast populations to understand the pleiotropic effects of adaptive mutations in "non-home" environments where they were not selected. The findings indicate that pleiotropy is common, and most adaptive evolved lineages show fitness effects in non-home environments, which can be beneficial or deleterious. The results also highlight how ploidy influences the observed adaptive mutational spectra in different conditions. The methodology involved whole-genome sequencing and pooled fitness remeasurement assays in 12 environments with various perturbations. The study concludes that pleiotropic effects are unpredictable, but lineages with adaptive mutations in the same genes tend to show similar effects. Overall, the study provides insights into the dynamics of adaptation and the impact of pleiotropy in different environments.

    Major comments

    1. It would be helpful if the authors could clearly provide information on the zygosity of the evolved mutations, as the presence of mutations in homozygous or heterozygous states can impact the results of the study.
    2. Does any of the evolved lineages have multiple adaptive mutations or other potentially adaptive mutations? If so, it would be great if the authors could provide a table listing these lineages and mutations.

    Minor comments

    1. In the Pooling of the Isolated Clones section of the Methods, the ancestor and subject pools were mixed in different ratios for different types of pools. While not strictly necessary, it would be helpful to provide a brief explanation for this.
    2. The conditions listed in Table 1 and Supplemental Figure 2 do not seem to match perfectly.
    3. Supplementary Figure 6 demonstrates reproducible fitness estimates across lineages with the same mutations but distinct barcodes, supporting the authors' inference of adaptive mutations. However, it also appears to show no evidence of interactions among these mutations. Can the authors clarify if this is due to the absence of lineages with multiple mutations or if no observable interactions were found?
    4. In the Pleiotropy is common, strong and variable section of the results, all three conditions were noted to have their evolved lineages tested in other conditions and presented in Supplementary Figure 5. However, due to the rapid dominance of lineages evolved in clotrimazole, there is no comparison data for them in Supplementary Figure 5.
    5. In the Results section on cost-free adaptation, it would be beneficial to include any compositional differences, such as pH, between the two drugs used that could have contributed to the fitness effects of the evolved lineages in pH 7.3.
    6. Results - Adaptation can be cost-free: While the authors did state "at least across the conditions in which we remeasured fitness" at the end of the paragraph, it may be prudent to exercise caution when stating "cost-free adaptation" as only a few conditions were tested. For instance, an all-beneficial or all-deleterious result can sometimes be obtained solely based on the chosen conditions.
    7. Colormaps in Figure 4, Supplemental Figure 6, 10, and 11: The colors for values below -0.2 are uniform, whereas the heatmaps exhibit darker blues.
    8. Results - Pleiotropy varies according to the mutated gene: "For example, haploid lineages adapted in glycerol/ethanol with mutations in IRA1 show the same pattern of fitness effects across conditions (Supplemental Figure 6)." I believe the authors are referring to Supplemental Figure 11.
    9. On the topic of IRA1, IRA2, and GPB2 in the section "Pleiotropy varies according to mutated gene" in the Results: Although IRA1 mutants exhibit highly similar patterns, it is challenging to ascertain which of the two genes, GPB2 or IRA2, has a more similar pattern.
    10. Results - Pleiotropy varies according to mutated gene: From "If lineages isolated from the same home environment have similar pleiotropic profiles..." to the end of that paragraph. While it is true that "pleiotropy varies according to target genes and not environment alone," it may be premature to suggest that the environment is the "main driving force" of pleiotropy without some form of statistical analysis.
    11. Discussion - line 5, paragraph 2: "For example, in glycerol/ethanol, the haploid adapted lineages have a trade off at 37{degree sign}C but the diploid adapted lineages do not (Supplemental Figure 11)." I believe the authors are referring to Supplemental Figure 6.

    Significance

    General assessment:

    The research is well-conducted, utilizing both haploid and diploid barcoded yeast populations, and isolating adaptive clones to determine fitness effects in non-home environments. The double-barcoding system allowed the authors to perform pooled fitness measurements of a large number of lineages coming from different home-environments in a plethora of conditions accurately and efficiently. The inclusion of a multiple evolution conditions followed by fitness measurements in a broader range of conditions allowed the authors to study the effect of environment to pleiotropy.

    The low number of generations used in this study, however, could hamper the discovery of more adaptive mutations, particularly those with smaller effects, and also make the study underpowered for studying epistasis among the evolved mutations. Furthermore, while the definition of pleiotropy in this study is reasonable and practical, it also makes most, if not all, generalist mutations pleiotropic and hence it's not surprising to see pleiotropy to be so common in this study.

    Advance:

    This study is an extension of a few recent publications. Jerison et al. (2020) evolved 20 haploid founder replicates in 11 environments for about 700 generations and measured the fitness of evolved clones - one clone from each replicate - across these conditions. This study provides in-depth analyses of how environments affect pleiotropy and a certain level of analyses for the underlying mutations. Bakerlee et al. (2021) evolved several hundred barcoded haploid and diploid populations in a few environments for 1000 generations and traced not only the fitness changes but also the dynamics of pleiotropy longitudinally. The environments used in this study are similar to each other, and so were the results, with the exception of environments with high (37˚C) and low (21˚C) temperatures. The current study utilized their double-barcoding system to allow for testing both haploids and diploids in a broader range of conditions. Although only three of the starting environments were chosen for further analyses, these environments are more dissimilar, and the putative underlying adaptive mutations in the evolved clones were identified and more thoroughly analyzed.

    The study's findings offer valuable insights into the intricate relationship between adaptation, pleiotropy, and environmental dynamics. While the complexity of pleiotropy and the multitude of factors that influence it make it challenging to comprehensively address all aspects in a single study, the results presented here contribute significantly to our understanding of this phenomenon. Nevertheless, further research of this nature is crucial to deepen our knowledge of the underlying mechanisms and to identify overarching patterns that can be applied across diverse systems. Overall, this study represents a promising step towards advancing our understanding of pleiotropy and its role in adaptive evolution.

    Audience:

    While the current study focuses on yeast as a model organism for evolutionary experiments, the implications of pleiotropy extend far beyond basic research. An understanding of the pleiotropic effects of mutations is crucial for comprehending the mechanisms of evolution and developing effective clinical interventions. As pleiotropy can affect disease outcomes and drug responses, the insights gained from this study can have far-reaching implications in the fields of biology and medicine. Thus, this study contributes not only to our understanding of yeast genetics but also to broader areas of research and application.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, the authors address the question of pleiotropy (the multiple effects of a single mutation on different traits) of adaptive mutations occurred during evolutionary processes. To do this, they evolve populations of S.cerevisiae strains in 12 environments, they identify the major adaptive mutation occurring in a subset of them, and they use a barcoding system to address their effect on fitness in environments where they were not evolved. The study confirm a number of conclusions from previous studies, such as the frequency of positive and negative pleiotropy of evolved lines when tested in other environments. The major novelty of this work is represented by the focus on single adaptive alleles and the conclusion that mutations in the same genes tend to produce similar pleiotropic effects.

    Major comments:

    1. The major conclusion of the manuscript state that "mutations in the same genes tend to produce similar pleiotropic effects", suggesting that a number of times this does not occur. For instance, the authors comment on the case of PDR3, which does not always produce 'cost-free' adaptation across environments. I believe that, to strengthen and better define their conclusion, the authors should develop a quantitative analysis of the reproducibility of pleiotropic profiles (that considers how many times genes have been found mutated). The heatmaps provided are compelling, but make it hard to generalize on how often, and to what extent, the gene mutated can predict pleiotropy across various environments.
    2. In the concluding sentence of the discussion, it is unclear whether the authors are speculating about a role of the strength of selection in determining pleiotropy based on their results, or if that only represents a suggested hypothesis to test in future studies.
    3. The method used to identify putative adaptive mutations should be described in more detail. For instance, I seem to understand that only one mutation per lineage is considered 'adaptive'. However, many lineages seem to have more than one mutation. Based on what reported in the method section, the adaptive mutations have been hand-picked based on previous knowledge of selection in the environments of choice ("the list of genes was curated based on those genes' interactions with other identified genes or pathways known to be involved in the adaptation of that specific condition from previous work"). If this assumption is correct, the criteria for such a curation should be specified in more detail.

    Minor comments:

    1. The term 'Pareto front' is technical and left undefined.
    2. The section ' adaptation can be cost-free' only refer to figure 4, (with adaptive mutant lineages from populations evolved in fluconazole), while it comments extensively on mutation isolated in clotrimazonle (reported in Sup. Fig10, not mentioned in the section).

    Referees cross-commenting

    I tend to agree with reviewers 1 and 3 that, given the focus on individual mutations of this manuscript, more information about their nature is important. On top of the zygosity, I would be curious to know whether mutations predicted to inactivate the gene (frameshifts, stop codon), have different pleiotropic profiles than AA substitutions.

    To answer the reviewer's 2 second major point, my understanding is that most of the lines have accumulated other mutations (marked with a '+' sign in Fig4 and FigS6,S10,S11). I suspect, however, that none of these mutations have been considered adaptive given the criteria described in the 'identifying adaptive mutations' session (e.g. mutations in coding regions appearing in more than one clone in a given condition, and with median fitness in the original home greater than 0).

    Significance

    The manuscript address a question (the emerge of pleiotropy during evolutionary adaptation) which has been extensively studied. However, it does it in a more comprehensive way that previously achieved, including many environments, both haploid and diploid organisms, and by focusing on single adaptive mutations. Most of the conclusions match the ones of previous studies. Perhaps the only exception is represented by the conclusion that individual genes, more than home environments, are proposed to dictate the pleiotropy profiles. However, the fact that mutations affecting the same genes often produce similar pleiotropy profiles is not necessarily unexpected. Overall, the paper is clearly written and can represent a valuable resource for a rather specialized community interested in the origin of pleiotropy during evolutionary adaptation.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    The objective of the study was to investigate the impact of specific adaptive mutations on trade-offs and pleiotropic effects in haploid and diploid Saccharomyces cerevisiae populations. The authors identified adaptive mutations from strains evolved in three specific home conditions and then conducted fitness assays in up to 12 environments (home/non-home). They highlight that ploidy level plays a condition-specific role in shaping the adaptive mutation spectra. Adaptive mutations showed fitness effects in both home and non-home environments, which were beneficial in some cases and detrimental in others.

    Major comments

    It would be interesting have an idea of the global mutation rates and spectra in the diploid and haploid lineages across the conditions as well. The S. cerevisiae mutational spectra has been shown to be dependent on the environment and genetic background to an extent but not ploidy. Ploidies differ in terms of just the frequency. How similar/ dissimilar are the overall mutational spectra here? Were there any homozygous mutations in the diploids?

    Fitness gains and losses can happen without trade-offs if neutral home mutations are non-neutral in non-home conditions. Can the authors comment on that in this context. Physico-chemically, how different are the home/ non-home environments? How do the fitness effects correlate across the environments in the absence of these adaptive mutations? It would also be useful to know the extent of fitness variance of the populations in the home and away environments, this would aid the reader better grasp the significance of fitness gains/loss.

    Minor comments

    A summary table of the pleiotropic effects would be very useful as in Bakerlee et al. 2021. Citation errors e.g. Consistent with prior work (Jerison, et al., 2021)... is either incorrect or not referenced.

    Significance

    The paper is well written highlighting an interesting question, the take home message is incremental at best in the context of the overall literature. In general, my suggestion would be to tone down the conclusions a bit, as the evidence isn't very clear cut .

  10. Version published to 10.1101/2023.02.28.530341v1 on bioRxiv