PONE-D-21-19052Introgression and mating patterns between white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) in a natural hybrid zonePLOS ONE

Dear Dr. Matsudaira,

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.

Both of the reviewers agree on that the limited number of genetic markers in this study cannot provide enough resolution to clearly distinguish F1s and backcrossed individuals or to evaluate the degree of gene flow. I have to say that the conclusions are not fully justified by the data in the current manuscript. However, I agree with the reviewer #1 on that this manuscript is publishable if the authors can revise the manuscript and tone down the conclusion. Therefore, my decision is “Major Revision”.

There was a disagreement between the reviewers regarding the limited number of available SNPs or genetic markers. Considering that obtaining larger sample size than the current data is difficult, I am more inclined to agree with the reviewer #1. But as reviewer #2 suggested, please explain the difficulty of obtaining samples and justify the reason why the only the limited sample size and the limited number of genetic markers were used.

There was some unclear points on how the genetic markers were selected and which individuals are included in this study. Please clarify these points on the revised manuscript because these information is essential to make the manuscript technically sound.

Also please address the other points raised by the reviewers than I mentioned below.

Please submit your revised manuscript by Nov 23 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: 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,

Jun Gojobori

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that Figure 1 &  2 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure(s) [#] to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text: “I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only. The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/ The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/ Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/ Landsat: http://landsat.visibleearth.nasa.gov/ USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/# Natural Earth (public domain): http://www.naturalearthdata.com/

[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: Partly

**********

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: Matsudaira and colleges present genetic and phenotypic evidence for hybridisation of white-handed and pileated gibbons in a hybrid zone.

Being unfamiliar with gibbon phenotypic diversity I restrict my comments to the genetic analysis which is within my area of expertise.

The authors present a genetic analysis, using 8 autosomal, MT and Y markers. This appears a novel investigation of a recent and/or ongoing hybridisation of these two species, and a counterpart to the investigation of older introgression described in Matsudaira and Ishida Heredity volume 127, pages312–322 (2021).

As such, I would be pleased if ultimately this paper was published in PLoS One. There is however a question as to how strong the interpretation of the genetic data is.

I certainly would agree with the authors that even a small number of suitably chosen loci can readily differentiate between two diverged lineages, and that these would also provide some information as to introgression/hybrid formation and/or a cline of ancestry. I disagree that there is enough resolution to readily differentiate between F1/F2 or various generations of repeat backcrosses. Given the small number of autosomal markers (less than the number of chromosomes), and the associated uncertainty in estimated ancestry components - even with only K = 2 - many different population processes could result in very similar arrays of ancestry that the authors uncover within the hybrid zone. Thus, I think it is fair to conclude that the authors uncover genetic evidence for recent hybridisation (Recent being relative to the related findings in Matsudaira and Ishida 2021). I do not think that one can say, as the authors do in the Abstract "Four phenotypically white-handed gibbons showed introgressed pileated type mtDNA, but the autosomal SNV admixture was not detected" and therefore ... "some genomic portions have introgressed into the gene pool of the counterpart species". In my opinion, this is too strong an interpretation of their results. I would be much happier if statements of this kind came with a suitable disclaimer. For example: "These gibbons were not only F1 hybrids, but also ones with plural backcross generations" (Lines 405:406). Much better could be, "Our data suggests that these gibbons could be a mixture of F1 hybrids, and perhaps ones with plural backcross generations, but the limited resolution enabled by small number of marker loci cannot rule out other scenarios acting on shorter timescales, such as a mixture of F1/F2 individuals". Of course how the authors want to address this point is up to them. Also, please note this is but one example, and my comment should be interpreted as having the authors rethink all statements on genetic ancestry that are definitive in tone such as Lines 428-429.

Minor points:

Data availability. I searched all three databases and could not find the listed accessions. Can the authors please confirm the availability of data.

Selection of autosomal SNVs: please expand on the relevant methods. How many DNA sequences from GenBank were initially selected?; how many independent fixed differences did you find? How many additional loci came from Perelman et al?; In essence - the reader should have some clarity about how you came to only 8 SNVs.

Lines 70-71: citation for disadvantages of hybridisation.

Reviewer #2: In this manuscript, “Introgression and mating patterns between white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) in a natural hybrid zone”, the authors seek to uncover the degree of introgression and mating patterns between two endangered gibbon species, white-handed and pileated gibbons. They genotyped 88 individuals including 72 individuals from the hybrid zone, 12 purebred white-handed and 4 purebred pileated gibbons, with 8 autosomal SNPs. They also determined 632 bp-sequence on mtDNA and one 2bp-indel on Y chromosome for most of the individuals.

However, as I explained below, I don’t think it’s possible to measure the extent of introgression genetically with this data because of the small number of SNPs and the small number of the purebred samples. Rather, the importance of this study is in proposing potential markers to distinguish the two species and in accumulating genotype information of these endangered species. I recommend to reorganize this manuscript in that way.

1. The major concern is the small number of SNPs analyzed in this manuscript (8 SNPs) and the small number of the purebred individuals (12 white-handed and 4 pileated gibbons). It is hard to say that these SNPs are fixed differences between these species. The authors chose the 8 autosomal SNPs from the previous works of other groups; 5 SNPs from Chan et al. (2013) and 3 SNPs from Perelman et al. (2011). But the numbers of the animals used in the papers are also very small. In Chan et al., 11 white-handed and 4 pileated gibbons were used, and in Perelman et al., only one white-handed gibbon is used. It means, the genotypes of the 8 autosomal SNPs are available only for 13 or 23 white-handed gibbons and 4 or 8 pileated gibbons in total. It is likely that the 8SNPs are not fixed differences between the species but are segregating at high frequency within each species. In that case, it’s not possible to distinguish the two species with these SNPs.

2. In lines 269-270 and in Fig 3., the authors mentioned “the threshold of backcross and pure white-handed gibbons”. How was it determined? Also, how the criteria between F1 and backcross was determined (Fig3)? I guess it came from the identification of species base on vocalization and morphology. If so, it means that in this manuscript, the species identification is not based on genetic data but based simply on the vocalization and morphology. The aim of this manuscript is written as analyzing the samples genetically to evaluate the extent of introgression, but the actual analyses are done the other way round. I think the authors’ true interest is in finding a set of potential markers to distinguish species for future studies and to confirm hybrids which are identified from vocalization and morphology. I do understand the importance of finding the markers for endangered species, for which it’s difficult (/not possible) to get good amount of DNA samples. But the manuscript needs to be modified to make the aim clear.

3. The sample information is not clear enough. The authors collected 129 fecal samples in Khao Yai, 71 fecal samples in Kaeng Krachan and 9 fecal samples on Khao Soi Dao (lines179-181). They also genotyped DNA samples previously collected in Khao Yai (lines 193-194). Then suddenly declared “Overall, 93 DNA samples of 88 individuals were genotyped” without any explanations. Please provide the full sample list with sample ID, sampling location, sex, age category, species identified by vocalization and pelage coloration, and information from genetic data. If the data includes the samples which were used in previous studies, please explain it with the same sample IDs as in the previous works.

4. I think the authors should explain about the endangered status of the two species more, if that is the reason why the amount of genetic data is very limited.

5. The authors wrote “The electropherograms of HVR1 were visualized by MEGA6 " (line 247). Is it a mistake? I couldn’t find electropherograms in this manuscript.

**********

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.

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.

PONE-D-21-19052R1

Introgression and mating patterns between white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) in a natural hybrid zone

PLOS ONE

Dear Dr. Matsudaira,

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.

I generally agree with the two reviewers and I think they made a reasonable suggestions. I ask the authors to address the comments raised by the reviewers before this manuscript is published.

Please submit your revised manuscript by Mar 06 2022 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,

Jun Gojobori

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.

[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 #3: (No Response)

Reviewer #4: (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 #3: Yes

Reviewer #4: Yes

********** 

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

Reviewer #3: N/A

Reviewer #4: 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 #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: Yes

********** 

6. Review Comments to the Author

Reviewer #3: I agree with both previous reviewers that the limited amount of data presented here should not be over-interpreted. In particular, I agree with the statement 1 of reviewer #2, the previously described SNPs might not represent fixed differences with the small number of individuals, especially since ancient admixture events were likely part of their history.

First, the observation in the STRUCTURE analysis could be explained by genetic clines or simply segregating variation rather than recent hybridization. There is one thing I would recommend in terms of analysis, which is a STRUCTURE analysis with higher k (3, 4, 5) to see if the supposed signature of mixing is not just a component restricted to individuals from this area.

Second, the threshold for assigning “hybrid ancestry” seems somewhat low and arbitrary given the small number of loci. Inspecting Fig. 3, there are 5-7 likely hybrids with substantial ancestry from both species (if one assumes these loci are really fixed), while I would be more doubtful about the others. The results on morphology and pelage are lending good support to the hypothesis on observed hybrids, especially in the case of those with clear mixed ancestries. I would like to know if the “five hybrid individuals indistinguishable from typical white-handed gibbons” had a lower assignment of admixed ancestry than the three with atypical patterns (L373).

Third, the discordance of mtDNA nuclear DNA might be pointed out. There are a few individuals with a haplotype assigned to H. pileatus which, in this restricted dataset, have nuclear ancestry only assigned to H. lar. This shows the difficulty in determining ancestries with such few markers. Compare that to humans and Neanderthals, where ancient Neanderthal admixture in modern humans has not been determined by obtaining several mtDNA sequences, nor by retrieving a small amount of nuclear DNA (Green et al., Nature 2006), but only by shallow genome sequencing (Green et al., Science 2010). I agree that it is difficult to obtain information from wild-living primates for this kind of studies, but it is a problem that the confidence in observations must be low when using such a small number of individuals for the reference populations and a very small number of genetic markers. In that sense, I would also add “putative” to any statement about admixture, from the abstract on throughout the paper. I.e. the phrasing should be even more cautious when referring to the genetic results.

Finally, technological innovations may likely offer opportunities to obtain much more information from fecal samples of wild primates, as has been shown in chimpanzees with sequencing after capture (see Fontsere et al., Molecular Ecology 2021). Considering the very fine-grained picture that were obtained in humans and other primate species after sequencing at least substantial parts of the genome, in comparison to single markers or mtDNA sequences, I suggest the authors add a sentence into the discussion about that. This may in the future confirm hypotheses on hybridization gained from the limited data, although of course this also depends on funding.

Some typos:

L191: fecals > fecal

L212: sentence broken

L317/319: who *were* identified

L349: futher > further

L427: not > to

L435: result*ed*

Reviewer #4: This manuscript is about investigating gibbons genetically in the hybrid zones. They make new genetic markers for identifying whether the individual is a “pure-blood” or a “hybrid”. They also discuss on the phenotypic traits for the individuals which were detected as hybrids.

This is a revised manuscript and I was not one of the original reviewers. I will make comments based on the previous reviewers’ comments and the author’s responses.

Overall, the previous comments made by the original reviewers make sense to me and the authors responses are satisfactory. I agree with the previous comments on the limitation of genetic markers. The authors efforts to tone down the conclusion was necessary to make the manuscript more technically sounding.

I have one concern on detecting hybrid using the result of STRUCTURE (e.g. fig 3).

The authors set the criteria for being hybrid as showing contributions of the minor ancestry at a level of more than 0.017. I think it is OK to label the individuals showing “intermediate” level of mixed ancestry as recent hybrids. However, it might be inappropriate to label individuals with marginal value of ancestry (i.e. 0.018 or 0.019) as recent hybrid. This is because the number of markers used is quite limited and even truly “pure-blood” individual may show more than 0.017 of minor ancestry level under this condition.

Then I suggest the author to label the individuals with marginal value of ancestry as “possible hybrids” or so.

********** 

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 #3: No

Reviewer #4: 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.

Introgression and mating patterns between white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) in a natural hybrid zone

PONE-D-21-19052R2

Dear Dr. Matsudaira,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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,

Jun Gojobori

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

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 #3: Yes

Reviewer #4: (No Response)

**********

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

Reviewer #3: Yes

Reviewer #4: (No Response)

**********

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 #3: Yes

Reviewer #4: (No Response)

**********

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 #3: Yes

Reviewer #4: (No Response)

**********

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 #3: (No Response)

Reviewer #4: (No Response)

**********

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 #3: No

Reviewer #4: No