PONE-D-24-52521Are you with me? Co-occurrence tests from community ecology can identify positive and negative epistasis between inversions in Mimulus guttatusPLOS ONE

Dear Dr. Madrigal-Roca,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The reviewers were generally excited about the approch introduced in the manuscript. The main points for improvement are the following.

1. Provide better explanation for the metrics, as they are novel to the PopGen community. Can be done with extended methods, or supplemental methods. In the intro, the network is presented as an alternative approach to the cooccurance stats, but here it is used as a follow up analyses/visualization.
2. Elaborate on strenghts/weaknesses of these methods to detect coupling vs repulsion (suggest using the LD metric vocabulary).
3. Can you compare the preformance of the metircs with established estimators, like linkage disequilibrium (D and D’). We are not ask for full scale benchmark analyses, but comparing the outputs of the different metrics can illustrate the nature of the signals they are best equipped to identify. Mention in disussion that benchmark analyses on simulated data with various properties (missingness, patterns of coupling, repulsion, recombination rates etc) would be an idean next step.
4. Can you synchronize the terminology and labelling of the different methods? Like between Figure 1 and 2, and text for the simulation methods, it is a bit hard to follow which method is which. Perhaps it is just an issue of improving Figure 2 and its legend ? (dont need to state “P-values“ in heading of all 9 panels)
5. Provide a well annotated R script (github ideally) that explains the steps in the analyses, with well described (metadata) dataset. This will increase likelihood of the methods being picked up by other groups.
6. If the cooccurance patterns depend strongly on Cross, then you are reporting higher order interactions. Cite literature on those (rather than organism specific papers), like https://www.sciencedirect.com/science/article/pii/S0168952513000218?via%3Dihub

Intermediate

Think it may be helpful to generate a figure for the data on 336-242, to demonstrate how the numbers look for the 3x3 tables. This may provide a better link to the graphics following! Your call!

Check wording and grammar of entire manuscript, see our minor points!

Please submit your revised manuscript by Feb 12 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Arnar Palsson, Ph.D.

Academic Editor

PLOS ONE

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PLOS24_Inversions

The reviewers were generally excited about the approch introduced in the manuscript. The main points for improvement are the following.

1. Provide better explanation for the metrics, as they are novel to the PopGen community. Can be done with extended methods, or supplemental methods. In the intro, the network is presented as an alternative approach to the cooccurance stats, but here it is used as a follow up analyses/visualization.

2. Elaborate on strenghts/weaknesses of these methods to detect coupling vs repulsion (suggest using the LD metric vocabulary).

3. Can you compare the preformance of the metircs with established estimators, like linkage disequilibrium (D and D’). We are not ask for full scale benchmark analyses, but comparing the outputs of the different metrics can illustrate the nature of the signals they are best equipped to identify. Mention in disussion that benchmark analyses on simulated data with various properties (missingness, patterns of coupling, repulsion, recombination rates etc) would be an idean next step.

4. Can you synchronize the terminology and labelling of the different methods? Like between Figure 1 and 2, and text for the simulation methods, it is a bit hard to follow which method is which. Perhaps it is just an issue of improving Figure 2 and its legend ? (dont need to state “P-values“ in heading of all 9 panels)

5. Provide a well annotated R script (github ideally) that explains the steps in the analyses, with well described (metadata) dataset. This will increase likelihood of the methods being picked up by other groups.

6. If the cooccurance patterns depend strongly on Cross, then you are reporting higher order interactions. Cite literature on those (rather than organism specific papers), like https://www.sciencedirect.com/science/article/pii/S0168952513000218?via%3Dihub

Intermediate

Think it may be helpful to generate a figure for the data on 336-242, to demonstrate how the numbers look for the 3x3 tables. This may provide a better link to the graphics following! Your call!

Minor points.

Line 45

Don’t agree with the last part of sentence “In genetics, non-random relationships between loci, described as linkage disequilibria, are measures of co-occurrence”. The non random relationships are not measures!

Line 49.

Can you reword “Attraction or repulsion of species in their location”

Line 58

Metrics?? “The co-occurrence techniques”

Line 64

Drop “of” “In our study species of Mimulus guttatus”

Line 77

Provide references ”Network analysis is an area of active development in biology.”

Line 79

Singular or plural idea(s)? “The underlaying basic ideas”

Line 104.

Which 10 genomes? “The ten genomes were derived”

Line 130.

“if genotype frequencies” these are all inversion genotypes?

Line 140

Reword, “independence between the pairwise patterns of dosage between inversions” maybe drop “dosage”?

Line 188

Where does this “survival rate” concept come from? Define it above.

Line 205

How many datapoints were in this comparison dataset? “also detect signals derived from linkage.”

Line 211

Used rather than adjusted? “we adjusted robust linear regression instead”

Line 247-251.

Reword this section, introduce the permutation approach earlier in the description.

Line 258.

“in six other crosses where it displays no distortion.” Or state directly, “where both types of homozygotes are seen”?

Line 274

Table 1. Can you annotate the p values in different way? X21 followd by column with its p valunes, and then X22? And a column that has all the same values is useless, i.e. “Selection”

Line 276.

Maybe reword title to “Testing of methods via simulation(s??)”?

Line 286.

Maybe say “counts” were “deflated”, not cell?

Line 309.

“Of the valid contrasts…” sentence is convoluted. Can this be subdivided into 2 or more sentences or reworded?

Line 318

Check refereence to supplement II.

Line 320

Any cases of the opposite?

“but the” instead of “and the”?

Line 366

Was there good or poor congruence? Consider changing the section heading “Congruence between methods” or first sentence “The congruence between the 2x2 table-based methods is depicted in Fig. 3 and Fig. 3S.” to better explain the main result of this section.

Line 371.

“robust”, is this word needed? Anything specificly robust about the regression?

Line 367 and Figure 3.

It is unclear what is shown in the figure (legend missing details, what is above and below diagonal. Labels on figure to small to read, question if inset colour panel is needed for all 9? Make start of Figure 3 legend more transparent “good correspondance was betw…”

Line 384.

Isnt this more of a visualization of the results from previous section??

Heading “Visualization of co-occurance as inversion networks” or some version of this?

Line 390

Quesiton of wording ”there were significant patterns”, maybe “signal”?

Line 391

“Strongest signals were in lines 155”

Line 439

“individuals” rather than “organisms”

Line 442.

Wording, “Also, inversions can be central elements in our understanding about”, maybe drop the “our understanding”?

Line 453.

“interactome” is not the right word here, find another. Also fix in other places in MS.

Line 456

“induced” another verb may be better!

Line 470

Reword “although often not across all crosses” was it not so that this was mainly in few crosses?

Line 479-

Check sentence structure and placement of reference.

Line 482

Strange wording “…where the higher count of plants was found in the homozygous”. Can you rephrase. “more plants were homozygous for inversion 49 …”?

Line 485

“effective population size”?

Line 491

Wording? “Applied to in this study”

Line 507

Add citations to papers about higher order epistasis, see above.

Line 511

“We investigated one such example in detail” rather than “Figure 5 provides a particular example of variability of interactions.” Or some other more descriptive version of this

Line 518-

Reword, in particular klonky text at “In the case of the inversion 32 in homozygous state and the inversion 40 in heterozygous state, it can be seen a significant effect for the heterogeneity”

Line 528

“strong” not “string”?

Line 530

Verb missing…”are?”

Line 533

Maybe we just need more power, or that the dependcies of SDV and inversion interactions are diverse, on case by case basis?

Line 565 and 570

Add references to these sections, so to bring attention to these methods through scholar working on these topics.

Line 579

Or how inversion combinations are depleted or enriched in hybrids between species or subspecies.

Line 584.

Can also be used to study combinations of specific alleles of the genes within inversions, both within one inversion or across inversions.

Line 628

Wording ”in special to” ?

  [Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1:  1. Co-occurrence analyses are usually used to quantify species loss and gain across communities, to investigate patterns of antibiotic cross-resistance, and to identify mechanistic similarities among diseases. Authors need to elaborate the use of co-occurrence analysis for attraction and repulsion between different inversions. This is particularly important as the focal theme of the study is to analyze the epistatic interaction between the inversions. Authors may add a note on the perceived advantage of using Jaccard-Tanimoto index over Sørensen-Dice, and Simpson methods.

2. For Table I, authors have used P-values as suggested by the analysis software, which means raw p-values have been utilized. This is not appropriate, kindly improve the presentation and add scientific P-values < 0.05." This is crucial as the co-occurrence analysis focuses only on the significant positive associations.

3. For the network analysis, some other libraries and approaches can be used to achieve the comprehended results of the Jaccard indexes and taken as a reference for building interaction. You can find alternatives and consider NetworkX in Python instead of igraph in R.

4. The techniques or the extensions authors have used vis a vis R language is not described in the method section. Authors need to emphasize and add a description regarding the usage of R or R Studio for the larger benefit of the readers.

5. Please indicate the type of inversions used for the analysis (paracentric / pericentric). Additionally, a note on the adaptive role or potential of the same need to be added in the discussion section.

6. Authors may consider the following references to incorporate in the revised manuscript:

(i) Chromosome inversions and linkage disequilibrium in Drosophila. Current Science 94:459-64.

(ii) Hundred years of research on inversion polymorphism in Drosophila. Current Science 117:761-775.

Reviewer #2:  Detailed comments are attached in a pdf file! Briefly:

The manuscript investigates epistatic interactions between genome inversions in Mimulus guttatus, focusing on whether these interactions exhibit non-additive effects on fitness. The authors adapt community ecology co-occurrence tests (e.g., chi-square, Jaccard/Tanimoto index J, and affinity score α) to identify deviations in inversion co-occurrence, suggesting epistasis. Network analysis is also employed to assess higher-order interactions.

Key findings:

1. A small subset (<10%) of inversion combinations deviate from additivity, though the direction of these interactions (positive/negative) is inconsistent.

2. Interactions are pairwise without evidence of higher-order effects.

Strengths:

1. A novel methodological framework.

2. Rigorous controls to eliminate confounding effects (e.g., physical linkage, Mendelian inheritance).

Major concerns:

1. [CRITICAL] The manuscript lacks engagement with established frameworks, like linkage disequilibrium (D).

2. Key metrics (e.g., J, α) and statistical methods (e.g., bootstrapping, sequential chi-square) require better explanation and justification.

Overall, the study provides a novel analytical approach to measure epistasis. Further clarifications and methodological comparisons are needed for robustness.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: Yes:  Harshavardhan Thyagarajan

**********

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Attachments

Attachment

Submitted filename: 2024.12.12_PONE-D-24-52521_review.pdf

PONE-D-24-52521R1Are you with me? Co-occurrence tests from community ecology can identify positive and negative epistasis between inversions in Mimulus guttatusPLOS ONE

Dear Dr. Madrigal-Roca,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript is now greatly improved and nearing completion. There are a few editorial issues outstanding, the main one concerns the supplemental methods and LD comparison. Rev 2 did not have access to this, which explains some of the comments, but there are still some issues.

These are the most compelling. The LD addition is an improvement, “We included a simulation-based contrast to illustrate the performance of these two metrics in relation to the LD framework” but I think this should be described in the manuscript (see rev 2). The approach is described in the supplemental section, but I suggest moving it to the main MM. (“Comparison between LD and co-occurrence metrics in an allele-based analysis… fixed association strength of 0.5.”).

And then include a brief summary, like in the RespRev section, to describe the main findings in the manuscript proper. Also, the results of these analyses (points 1-4) in supplemental section, do not include figures or data. Suggest you add that (like 4. “Heatmap plot showing…”). Finally, please define EDF.

Second main thing is language, it needs improvement. We strongly encourage the senior author to read and copy edit the MS thoroughly in this edition. At PLOS it is not in the reviewers or editors role to improve the language.

Please submit your revised manuscript by Mar 31 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Arnar Palsson, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

The manuscript is now greatly improved and nearing completion. There are a few editorial issues outstanding, the main one concerns the supplemental methods and LD comparison. Rev 2 did not have access to this, which explains some of the comments, but there are still some issues.

These are the most compelling. The LD addition is an improvement, “We included a simulation-based contrast to illustrate the performance of these two metrics in relation to the LD framework” but I think this should be described inthe manuscript (see rev 2). The approach is described in the supplemental section, but I suggest moving it to the main MM. (“Comparison between LD and co-occurrence metrics in an allele-based analysis… fixed association strength of 0.5.”).

And then include a brief summary, like in the RespRev section, to describe the main findings in the manuscript proper. Also, the results of these analyses (points 1-4) in supplemental section, do not include figures or data. Suggest you add that (like 4. “Heatmap plot showing…”). Finally, please define EDF.

Second main thing is language, it needs improvement. We strongly encourage the senior author to read and copy edit the MS thoroughly in this edition. At PLOS it is not in the reviewers or editors role to improve the language.

Intermediate

The annotations of columns, terms and p values still varies from table to table, and in tables and the footings. Please carefully check all tables, tidy this up and standardize.

Can you recode large and small numbers, e.g. from 3.29E-30 to more classical annotation 3.29 x 10-30

In text, tables and figures.

Minor points:

Line 17. Maybe insert “unlinked” into the sentence?

Line 56. “Co-occurrence in geneticS…”

Line 68 should this be singular or plural, one method or many?

Line 78 on not of “of 4.5mb on chromosome 6”

Line 83 sentence should end with a citation!

Line 88 pleasre rephrase “Network analysis is an area of active development in biology [20,21].”

Line 203 which metrics ? “between two widely used LD metrics”

Line 206. Figure 2. Is the coupling case – was it deliberate to have one individual of the “fourth type”? In the figure as it “stands”, column 5, line 2.

Legend of table 1, same X2 annotation for both, no explanation of which is which.

Is table 2 justified? Is another representation of the data more helping. This appears a bit like a supplemental table, or is it a list of interesting things?

Figure 3

The scale of X is confusing, can you improve it?

Line 377. “While most inversion pairs show no evidence of interaction, there are interesting exceptions.” Is there maybe more important summary statement at start. Are there different types of patterns to the associations??

Line 387

“The most frequent deviations from independence were observed in heterozygous-homozygous combinations (~58%),” Is this an excess over the expecations. Which fraction of the combinations tested are het-homo? Consider same descriptions for other combinations.

Line 399

Fig missing “as depicted in 3S”?

Line 411

“S trongest signals were more abundant in lines 155, 502, 541, 62, 664, and 909, while in the rest they were scarcer.” Does this rhyme with the JT analyses?

Check title supplemental data file “suplemental”

Also, from supplemental methods file, please cross reference to the manuscript for particulars (methods, tables, figures).

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: I am satisfied that meaningful improvements have been made through revisions in line with the AE’s guidance. While their engagement with LD remains somewhat underdeveloped / could be better articulated, it is an improvement over the previous version. A more direct comparison using LD as a metric—supported by statistical phasing of haplotypes—would have resulted in much stronger understanding. Although not an obstacle to scientific understanding, the language in the manuscript still requires substantial proofreading. I recommend accepting, given some minor revisions, for eg. acknowledging certain caveats. In addition, the extended methods section of the supplement appear to be missing, along with the details of how they carried out the simulation based comparison, and should be included prior to acceptance.

Detailed comments in attached document.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: 2025.02.04_PONE-D-24-52521_review_round2.pdf

Are you with me? Co-occurrence tests from community ecology can identify positive and negative epistasis between inversions in Mimulus guttatus

PONE-D-24-52521R2

Dear Dr. Madrigal-Roca,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Arnar Palsson, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: