PONE-D-20-31568

Extra base hits: widespread empirical support for instantaneous multiple-nucleotide changes.

PLOS ONE

Dear Dr. Lucaci,

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.

As you will see, both reviewers found the writing insufficiently clear and precise. Please take this comment seriously, as it is one of the key criteria of PLOS One, and it is impossible to evaluate well the science if the writing is unclear. In your revision, the key point is thus to improve the writing, but please address all comments by the reviewers.

Please submit your revised manuscript by Jan 14 2021 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:

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We look forward to receiving your revised manuscript.

Kind regards,

Marc Robinson-Rechavi

Academic Editor

PLOS ONE

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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: No

Reviewer #2: No

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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: Lucaci et al present an empirical analysis to assess how widespread single vs multi-nucleotide hits are. On the whole, I find this research conceptually interesting, timely, and reasonable for publication. However, I found this paper quite difficult to review. Far too much of the paper is not written in complete sentences (the ideal number of incomplete sentences is 0), references appear to use the wrong LaTeX command (e.g. [5] should not be used as a sentence subject..), acronyms are not clearly defined when introduced (and from my reading seemed to shift in their definition? or introduced and then called something else later?), and the presentation of the model itself is very confusing. The proposed model itself is NOT that complex, so it is troubling that the presentation is not straightforward to follow.

Further, I have some difficulty clearly interpreting some of figures. For example, the monotone splines in Figure 2 are very misleading especially given that it seems there are wildly different sample sizes across the X-axis. I do not see a clear scientific interpretation of a monotone spline in this context. Fig S3 is ambiguously labeled, the double axes on Fig 4 (with what seem to be semi-outlandish ranges for the left-hand axis?) are virtually impossible to interpret reliably.

Based on my reading, the current manuscript version suffers from an overall lack-of-editing. It was not submitted in an "polished" form. I will be glad to review another version after the text has been significantly cleaned, properly organized, and figures have been designed to yield clear interpretations. Again, I believe the science here is solid, but it is not currently presented clearly at all.

Reviewer #2: Lucaci et al present codon substitution models that allow multiple nucleotide substitution in a codon. They show that such models provide better fit in LRTs. The authors show the results in a sensible flow, but I feel that the picture is quite incomplete.

According to the study, it seems that the majority of datasets and positions across an alignment do have preference for more complex models. It does make sense that more parameter-free models fit the data better, but I was not convinced that they produce better trees. Previous studies show that simpler models lead to more accurate branch-length estimates. I wonder whether these results are biased by the hypothesis testing method. I would suggest to compare the resulting trees of the simulation studies to the starting trees and look at the distances, e.g., branch-length distance. It could be that 3H models are better fitted but generate overestimated branch lengths whereas 1H models are more conservative and produce more accurate trees.

Other than that, I think that the clarity of the text, especially in the Introduction and Methods, could be improved. I had a hard time understanding the details before I reached the Results section. For example, models notations in the first paragraph of the Methods section are only defined in the Results.

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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.

PONE-D-20-31568R1

Extra base hits: widespread empirical support for instantaneous multiple-nucleotide changes.

PLOS ONE

Dear Dr. Lucaci,

Thank you for submitting your manuscript to PLOS ONE. I am sorry for the delay. Reviewer 1 has clarified that they no longer wished to review this manuscript, so I took time to evaluate it myself rather than lose even more time finding another reviewer. 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.

I agree with reviewer 2 that the work is ready for publication in PLOS One, but that the writing should be carefully checked. In addition to the errors noted by the reviewer, I have noticed the following:

"signal due to misaligned" misses a word, probably "misaligned sites".

"for one 384 branch-site tests, [20]" if there is one test it shouldn't have an 's'.

Please submit your revised manuscript by Mar 27 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Marc Robinson-Rechavi

Academic Editor

PLOS ONE

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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 #2: All comments have been addressed

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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 #2: Yes

**********

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

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 #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 #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 #2: The authors have addressed my concerns. The flow of the manuscript was much improved and it is now much easier to follow and understand.

There are several English mistakes in some parts of the text - I suggest that the authors would thoroughly review the writing. Examples: "have have" in line 20; “traction” should be “attraction” in line 45; "these models *have* been unable" in line 44).

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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 #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.

Extra base hits: widespread empirical support for instantaneous multiple-nucleotide changes.

PONE-D-20-31568R2

Dear Dr. Lucaci,

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,

Marc Robinson-Rechavi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: