Genotype-environment interaction and the maintenance of genetic variation: an empirical study of Lobelia inflata (Campanulaceae)

High levels of genetic variation are often observed in natural populations, suggesting the action of processes such as frequency-dependent selection, heterozygote advantage and variable selection. However, the maintenance of genetic variation in fitness-related traits remains incompletely explained. The extent of genetic variation in obligately self-fertilizing populations of Lobelia inflata (Campanulaceae L.) strongly implies balancing selection. Lobelia inflata thus offers an exceptional opportunity for an empirical test of genotype-environment interaction (G × E) as a variance-maintaining mechanism under fluctuating selection: L. inflata is monocarpic and reproduces only by seed, facilitating assessment of lifetime fitness; genome-wide homozygosity precludes some mechanisms of balancing selection, and microsatellites are, in effect, genotypic lineage markers. Here, we find support for the temporal G × E hypothesis using a manipulated space-for-time approach across four environments: a field environment, an outdoor experimental plot and two differing growth-chamber environments. High genetic variance was confirmed: 83 field-collected individuals consisted of 45 distinct microsatellite lineages with, on average, 4.5 alleles per locus. Rank-order fitness, measured as lifetime fruit production in 16 replicated multilocus genotypes, changed significantly across environments. Phenotypic differences among microsatellite lineages were detected. Results thus support the G × E hypothesis in principle. However, the evaluation of the effect size of this mechanism and fitness effects of life-history traits will require a long-term study of fluctuating selection on labelled genotypes in the field.


Comments to the Author(s)
Thank you for the work done in clarifying this manuscript. Unfortunately, I still do not think that the conclusions the authors draw match with their results. I do not think that the GxE shown in this study is sufficient to support temporal GxE as a mechanism for maintaining variation in this system. There is some GxE across these manipulated environments, but this in itself not particularly surprising, and does little to support the main hypothesis of the paper.
Studying these genotypes in more clearly stressed conditions in growth chambers -such as drought or cold stress would provide more interesting results across genotypes and better address the hypothesis than the treatments used in this study.

Review form: Reviewer 3 (Melis Akman)
Is the manuscript scientifically sound in its present form? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation? Accept with minor revision (please list in comments)
Comments to the Author(s) I think the authors addressed my major comments well in the manuscript. I understand the limitation of sample size for the proposed analyses for this dataset. Please see my specific comments below: Abstract: 24: meaning of "levels" is not very clear. Do you mean variation is continuous, which could be as a result of several mechanisms such as f-dependent selection, heterozygote advantage and/or variable selection? 28: Before L. inflata you need a connection word, such as "Thus". 39: Before "Although", the abstract can use a strong statement about the finding of the study. I think the 2nd and 3rd paragraphs in Discussion read more like Results, and would be better suited for that section. I am sorry this is a vague comment but I think the Discussion needs more work with references to earlier studies. Using the framework constructed in the Intro can guide to talk about why this study agrees/conflicts with some other studies or species would add to the Discussion.
Decision letter (RSOS-191720.R0) 04-Nov-2019 Dear Dr Simons, The editors assigned to your paper ("Genotype-environment interaction and the maintenance of genetic variation: an empirical study of Lobelia inflata (Campanulaceae)") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. The reviewers have provided a range of comments and suggestions for further improvements. The most problematic comments (from our perspective, and by implication yours) come from Reviewer 2, who expresses concerns regarding the study conclusions that drawn from your data. Please seriously consider how to address these concerns -you need to persuade the Editors and this reviewer that you've made further sincere efforts to improve the manuscript and delineate changes made both in a point-by-point response and tracked changes manuscript.
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Comments to Author:
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) I appreciate the response of the authors to my previous review/comments. Overall I think the manuscript is improved. However, there are a few instances where the additions the authors made to previous comments come across as out of place, taking away from the flow of the paper. I think a little more effort to incorporate those changes into the manuscript would improve the readability, strengthening the manuscript. 1) Lines 49-54: Specific, detailed, example of overdominance in a system in the opening paragraph seems a bit random and out of place. 2) Lines 94-95: While I appreciate the authors adding this in response to my comment, it reads awkwardly and comes across as a fairly big caveat. I don't think this needs to be the case. Do the authors have any reason to think that pathogens are important in this system? Perhaps rewording this sentence to say something more like, "Frequency dependent selection is likely to be less important in selfing systems than outcrossing ones, as no selection acts on mating systems components … etc". This way it makes it clear you are not completely negating a potential role for frequency dependent selection, but explaining why it may be less important in selfing systems. Then you could just drop the throwaway pathogen line. 3) Line 362: If I am following this argument correctly, time to first flower and time to senescence likely do not contribute to GxE for fitness (as opposed to "thus may contribute to GxE in fitness" as stated in the manuscript), as these two traits "showed strong and relatively fixed genotypic effects". Is this correct? Whereas height at first flower is more likely to contribute to GxE in fitness? Is this is not correct, I am not following the argument made in the paragraph completely, and need further clarification. 4) Lines 372-373: Again, I appreciate the authors addressing a comment of mine regarding the potential influence of a seed bank, however, presented at this point in the paragraph/discussion it comes across as a throw away sentence, out of place and not contributing much to the paper. I would either further develop this argument a bit, or get rid of it (or move it).

Reviewer: 2
Comments to the Author(s) Thank you for the work done in clarifying this manuscript. Unfortunately, I still do not think that the conclusions the authors draw match with their results. I do not think that the GxE shown in this study is sufficient to support temporal GxE as a mechanism for maintaining variation in this system. There is some GxE across these manipulated environments, but this in itself not particularly surprising, and does little to support the main hypothesis of the paper.
Studying these genotypes in more clearly stressed conditions in growth chambers -such as drought or cold stress would provide more interesting results across genotypes and better address the hypothesis than the treatments used in this study.

Reviewer: 3
Comments to the Author(s) I think the authors addressed my major comments well in the manuscript. I understand the limitation of sample size for the proposed analyses for this dataset. Please see my specific comments below: Abstract: 24: meaning of "levels" is not very clear. Do you mean variation is continuous, which could be as a result of several mechanisms such as f-dependent selection, heterozygote advantage and/or variable selection? 28: Before L. inflata you need a connection word, such as "Thus". 39: Before "Although", the abstract can use a strong statement about the finding of the study.

Intro:
I like the new Intro a lot. I think the authors addressed my suggestions, thanks for that! 128: I do not think you need the sentences after "Each …". Instead give a brief explanation of the results to engage the readers. I think it is ok to discuss the limitations in Discussion only and not mention them in the Intro.
Results and Discussion: I think the 2nd and 3rd paragraphs in Discussion read more like Results, and would be better suited for that section. I am sorry this is a vague comment but I think the Discussion needs more work with references to earlier studies. Using the framework constructed in the Intro can guide to talk about why this study agrees/conflicts with some other studies or species would add to the Discussion.
Author's Response to Decision Letter for (RSOS-191720.R0) See Appendix A.

RSOS-191720.R1 (Revision)
Review form: Reviewer 1 Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Recommendation? Accept as is
Comments to the Author(s) I am happy with the edits made by the authors and think the manuscript is acceptable for publication. The only final point I would suggest is that the authors add a sentence to the first paragraph in the discussion clearly stating the main finding of their paper. This would make the opening of the discussion much more compelling.

Review form: Reviewer 4
Is the manuscript scientifically sound in its present form? No

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? Yes

Recommendation?
Major revision is needed (please make suggestions in comments) Comments to the Author(s) I think that the research explore an important issue in understanding the maintaining of genetic variation by considering GxE (E means environment change in space or time) interaction as a process of natural diversification. However, the analysis of the data is not clear enough, and it was done in an inefficient manner in order to answer the questions. Conclusions are not clearly justified by a non-efficient analysis.
I think that there are lack of results and more explanation in data analysis. For example, many results presented in the text can be presented more clearly in tables. The authors discussed about the rank of the genotypes but this information is not presented and it is so difficult to read from the figure 1. Model including the region of origin is not described. It is not clear why the authors decided to adjust p-value by FDR sometimes yes and sometimes no. They are not considering that using FDR, they are increasing the rate of false negatives and they use the FDR to argue that genotype effect is not significant. In this specific case, a large probability of error type II is worse than a large probability of error type I. I think that it is necessary to include more tables and a better description of the analysis.
The authors said that they used mixed-effect models, and the best method to estimate variance components is restricted maximum likelihood (REML), however, results in Table 1 match with the results of using minimum least square because they include mean squares. Using REML method allows analyzing all the data together, even with the present unbalance, and it avoids analyzing separately pieces of data that make more difficult to understand the results. Assuming that Geno and GxE are random, the test for genotypic effect has to be built dividing the MS_genotypes by MS_GxE in the case (3,11), the F value should be 0.137/0.909=0.1507, in the others three cases values are not exactly the same but they match better.
When analyzing GxE it is possible to separate two components, one due to cross over interaction (COI) and the other one to non-cross over interaction. COI is related with changes in the rank of genotypes and non-COI is related just with changes in the variances. The first kind of interaction is more important than the non-COI, because it explains changes in the behavior of genotypes while the non-COI explains differences in magnitude only. To know how much of both components are in the GxE would be very nice in order to interpret better the results.
As the author mentioned, Figure 1 is not easy to understand because the different data sets analyzed, an analysis using REML and all the data together could clarify the interpretation of results. However, I have to mention that a mixed model will not solve all the problems of unbalance. GxE will always be estimated with available data. X-axis in figure 1 shows the different environments and they can be sorted in many different ways with different effect on the reader. I think that it is better if one graph for each analysis could be less confuse. Others options are: i) to substitute the environment by the average relative fitness of the environment, or ii) to do two-by-two graphs. Graphing by pairs of environments will show more clearly the kind of interaction between the two environments.
When analyzing statistical interaction, we need to consider two environments and two genotypes to measure the level of interaction, then, we cannot talk about the interaction of one genotype or one environment if it is not relative to another set of genotypes and environments.
In summary, I recommend to reanalyze the data, to include more tables and more results and to present them in a better way.

11-Feb-2020
Dear Dr Simons: On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-191720.R1 entitled "Genotype-environment interaction and the maintenance of genetic variation: an empirical study of Lobelia inflata (Campanulaceae)" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
The reviewers and Subject Editor have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your manuscript.
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• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data has been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that has been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-191720.R1 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Associate Editor Comments to Author: Please ensure that you address the final point suggested by the Reviewer 1: "The only final point I would suggest is that the authors add a sentence to the first paragraph in the discussion clearly stating the main finding of their paper. This would make the opening of the discussion much more compelling." Reviewer 4's comments were solicited to take into account that the more critical reviewer in the previous round of review was unavailable to assess the revision -we apologise this has taken longer than usual. We're of the view that, given earlier reviewers were broadly in favour of publication, we can move toward that goal -though we'd recommend you take the Reviewer 4 comments on board (as far as practical), as they offer a number of potentially useful suggestions for clarifications.
Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) I am happy with the edits made by the authors and think the manuscript is acceptable for publication. The only final point I would suggest is that the authors add a sentence to the first paragraph in the discussion clearly stating the main finding of their paper. This would make the opening of the discussion much more compelling.

Reviewer: 4
Comments to the Author(s) I think that the research explore an important issue in understanding the maintaining of genetic variation by considering GxE (E means environment change in space or time) interaction as a process of natural diversification. However, the analysis of the data is not clear enough, and it was done in an inefficient manner in order to answer the questions. Conclusions are not clearly justified by a non-efficient analysis.
I think that there are lack of results and more explanation in data analysis. For example, many results presented in the text can be presented more clearly in tables. The authors discussed about the rank of the genotypes but this information is not presented and it is so difficult to read from the figure 1. Model including the region of origin is not described. It is not clear why the authors decided to adjust p-value by FDR sometimes yes and sometimes no. They are not considering that using FDR, they are increasing the rate of false negatives and they use the FDR to argue that genotype effect is not significant. In this specific case, a large probability of error type II is worse than a large probability of error type I. I think that it is necessary to include more tables and a better description of the analysis.
The authors said that they used mixed-effect models, and the best method to estimate variance components is restricted maximum likelihood (REML), however, results in Table 1 match with the results of using minimum least square because they include mean squares. Using REML method allows analyzing all the data together, even with the present unbalance, and it avoids analyzing separately pieces of data that make more difficult to understand the results. Assuming that Geno and GxE are random, the test for genotypic effect has to be built dividing the MS_genotypes by MS_GxE in the case (3,11), the F value should be 0.137/0.909=0.1507, in the others three cases values are not exactly the same but they match better. When analyzing GxE it is possible to separate two components, one due to cross over interaction (COI) and the other one to non-cross over interaction. COI is related with changes in the rank of genotypes and non-COI is related just with changes in the variances. The first kind of interaction is more important than the non-COI, because it explains changes in the behavior of genotypes while the non-COI explains differences in magnitude only. To know how much of both components are in the GxE would be very nice in order to interpret better the results.
As the author mentioned, Figure 1 is not easy to understand because the different data sets analyzed, an analysis using REML and all the data together could clarify the interpretation of results. However, I have to mention that a mixed model will not solve all the problems of unbalance. GxE will always be estimated with available data. X-axis in figure 1 shows the different environments and they can be sorted in many different ways with different effect on the reader. I think that it is better if one graph for each analysis could be less confuse. Others options are: i) to substitute the environment by the average relative fitness of the environment, or ii) to do two-by-two graphs. Graphing by pairs of environments will show more clearly the kind of interaction between the two environments.
When analyzing statistical interaction, we need to consider two environments and two genotypes to measure the level of interaction, then, we cannot talk about the interaction of one genotype or one environment if it is not relative to another set of genotypes and environments.
In summary, I recommend to reanalyze the data, to include more tables and more results and to present them in a better way.

See Appendix B.
Decision letter (RSOS-191720.R2) 19-Feb-2020 Dear Dr Simons, It is a pleasure to accept your manuscript entitled "Genotype-environment interaction and the maintenance of genetic variation: an empirical study of Lobelia inflata (Campanulaceae)" in its current form for publication in Royal Society Open Science.
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal. Thank you for the opportunity to respond the reviewers' comments. I include all detailed responses in-line below, in blue font and prefixed by "AMS:" (retaining the reviewers' comments intact) and include a document with tracked changes. Revisions in response to reviewers #1 and #3 were straightforward. As you point out, the most problematic comments are those of Reviewer #2, who clearly remains sceptical about "the study conclusions drawn" from the results, rather than about the statistical validity our results themselves (significant genotype-environment interactions [GxE] caused by variable slopes). Specifically, the issue is about inferences from observed GxE to maintenance of genetic variation. I completely agree in principle with the criticism. However, I wish to point out that the manuscript explicitly makes a claim that is not as strong as the claim being criticized, and is one that is supported by the results. I have therefore made an earnest effort to respond to this criticism by clarifying hypotheses under test, and by fully acknowledging and explaining limits on inference.
I hope the manuscript revisions are to your satisfaction. Sincerely,

Andrew M. Simons
Author response to reviewer comments: Reviewer: 1 Comments to the Author(s) I appreciate the response of the authors to my previous review/comments. Overall I think the manuscript is improved. However, there are a few instances where the additions the authors made to previous comments come across as out of place, taking away from the flow of the paper. I think a little more effort to incorporate those changes into the manuscript would improve the readability, strengthening the manuscript. 1) Lines 49-54: Specific, detailed, example of overdominance in a system in the opening paragraph seems a bit random and out of place.
AMS: I agree, and details have been removed. We retain only simple mention of the example to bridge from discrete polymorphism to quantitative genetic variation.
2) Lines 94-95: While I appreciate the authors adding this in response to my comment, it reads awkwardly and comes across as a fairly big caveat. I don't think this needs to be the case. Do the authors have any reason to think that pathogens are important in this system? Perhaps rewording this sentence to say something more like, "Frequency dependent selection is likely to be less important in selfing systems than outcrossing ones, as no selection acts on mating systems components … etc". This way it makes it clear you are not completely negating a potential role for frequency dependent selection, but explaining why it may be less important in selfing systems. Then you could just drop the throwaway pathogen line.

AMS:
We have re-worded, as requested, so as not to imply that frequency-dependent selection is eliminated as a possibility. Note that support for fluctuating selection as a mechanism does not require elimination of other mechanisms.
3) Line 362: If I am following this argument correctly, time to first flower and time to senescence likely do not contribute to GxE for fitness (as opposed to "thus may contribute to GxE in fitness" as stated in the manuscript), as these two traits "showed strong and relatively fixed genotypic effects". Is this correct? Whereas height at first flower is more likely to contribute to GxE in fitness? Is this is not correct, I am not following the argument made in the paragraph completely, and need further clarification.
AMS: This was confusing because the distinction between fitness components (time to first flower etc.) and fitness itself (fruit number) as not made clearly enough. In these interpretations in the Discussion (that we were asked to include), we hypothesize that components that are inflexible across environments (i.e. with strong genotypic effects) might be an underlying cause of rank-order differences in fitness itself, following the logic of, for example Sara Via's (1991) classic work on GxE and specialization, which we now cite as well. We clarify, too, that these ideas are introduced as hypotheses for future test. 4) Lines 372-373: Again, I appreciate the authors addressing a comment of mine regarding the potential influence of a seed bank, however, presented at this point in the paragraph/discussion it comes across as a throw away sentence, out of place and not contributing much to the paper. I would either further develop this argument a bit, or get rid of it (or move it).
AMS: I can see how this appears as a "throw-away", but the original point about seed dormancy raised here by the reviewer is a valid one, so I have revised to integrate the point into the general context of how life-history traits may interact in complex ways, thus making inference about underlying causal relationships with fitness difficult.

Reviewer: 2
Comments to the Author(s) Thank you for the work done in clarifying this manuscript. Unfortunately, I still do not think that the conclusions the authors draw match with their results. I do not think that the GxE shown in this study is sufficient to support temporal GxE as a mechanism for maintaining variation in this system. There is some GxE across these manipulated environments, but this in itself not particularly surprising, and does little to support the main hypothesis of the paper.