* Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. *

Dear Dr Andrade,

Thank you very much for submitting your Research Article entitled 'Molecular parallelisms between pigmentation in the avian iris and the integument of ectothermic vertebrates' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers.  This is a very well written paper with clear findings that will be of broad interest to the genetics community.  The suggestions made by the reviewers largely concern the presentation of the results in the figures, and should be straightforward to address.  We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.

We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. 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 us at figures@plos.org.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Kelly A. Dyer

Associate Editor

PLOS Genetics

Bret Payseur

Section Editor: Evolution

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: This is a really nice gene study that identifies an interesting gene required for normal pigmentation of the pigeon iris. The avian iris is of considerable interest in the field of pigmentation research as it is thought to harbor evolutionarily ancient chromatophore classes found in ectothermic vertebrates but not elsewhere in endotherms. So any study identifying genes required will iris color makes a substantive contribution to the field. In this particular case, the authors have convincingly shown that SLC2A11B has a premature stop codon in pearl mutant pigeons, which lack yellow pteridines. This particular gene is notable for having been identified several years ago as essential to yellow coloration of xanthophores and leucophores in medaka fish, and so the analyses of Andrade et al. really set the stage for future investigations into conservation of cell lineage and differentiation pathways across a deep phylogenetic divide.

The authors have done a thorough job in their gene identification, using a combination of whole-genome resequencing, mapping by several different metrics, RNA-seq and targeted assessment of downstream gene expression. The study is rigorous and the manuscript is exceptionally clear, making it a real pleasure to read. The only small reservation I had concerned the presentation of candidate pteridine and chromatophore associated gene expression in Figure 4D,E, in which there were some trends but not significant differences. Since these data are not normalized by cell type, but instead represent a complex mixture of cells in the iris, and sample sizes are very small, they really do not contribute substantially. So I would be more inclined to move these panels to a supplementary figure, with the appropriate caveats so they are not over-interpreted by readers.

Although I am very enthusiastic about the paper, I can imagine one might object that the authors have not truly demonstrated a "mechanism" by which SLC2A11B promotes pteridine accumulation, or normal development of the relevant chromatophores. Indeed, it will be exciting to learn these sorts of details. Realistically, however, it seems extremely unlikely that the kinds of analyses that would be required to extract such information could be accomplished in a reasonable time-frame, as they would likely require the authors to find new collaborators or develop new techniques, as well as additional experimental material and perhaps even another model like the original medaka mutant, or a corresponding mutant in zebrafish, either of which would be more amenable to such next steps than pigeon. These would not be trivial undertakings in normal times but are likely to be especially difficult during a pandemic.

Reviewer #2: This is a very convincing and exciting study of pigmentation genomics in pigeon iris coloration. The results were very clear and drawn from a range of data types. I found that the interpretation was fair and, while some could take issue with the sample sizes for the RNA studies, this is logistically challenging and, moreover, are more of supporting data to the clear genomic results. Finally, the paper is exceptionally well-written and makes for a very compelling manuscript—I do not have any specific suggestions for improvement.

My only suggestion would be to add somewhere (likely figure 3?) the intron-exon structure of the SLC2A11B (and any other nearby genes). Basically the results refer to SLC2A11B as being the only gene in this region, however the reader never really gets to 'see' this result directly, as well as any other nearby genes. Thus, including this annotation information visually would be an improvement.

Reviewer #3: Review is uploaded as an attachment

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #3: No

Attachments

Attachment

Submitted filename: andrade_et_al_review.docx

* Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. *

Dear Dr Andrade,

Thank you very much for submitting your Research Article entitled 'Molecular parallelisms between pigmentation in the avian iris and the integument of ectothermic vertebrates' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by one of the previous reviewers. We appreciate your attention to the reviewer's suggestions, and we expect that once you address the one remaining concern about identifying the genetic linkage group for each one of your differential expression hits your article will be accepted. Please see the comments from the Reviewer and their analysis (attached) that addresses this point. Given this is a genetic mapping paper, we agree with the reviewer that this analysis would strengthen the paper.

We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.

We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. 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 us at figures@plos.org.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Kelly A. Dyer

Associate Editor

PLOS Genetics

Bret Payseur

Section Editor: Evolution

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #3: The authors have responded to main point #1 by highlighting the differential expression hits in Supplementary Table 3 that are on the same scaffold as SLC2A11B. This is a step in the right direction. However, this is not what I had originally suggested: I had suggested the authors determine which genetic linkage group each hit belongs to, not which scaffold (indeed the latter is already clear from the table). This is because linkage groups typically span a whole chromosome whereas scaffolds do not. Granted, the scaffold the causative haplotype resides on is a large one (8.6 Mb), and the haplotype is about 1.8 Mb from the scaffold end (so it does seem likely that any putative cis-regulated gene would be located on the scaffold). However, given that A) enhancers can sometimes act at megabase distances, and B) scaffolds are sometimes misassembled, it does not seem unreasonable to be curious whether any of the differential expression hits reside on different scaffolds, which are linked to the one in question. To illustrate what I mean, I have attached the results of a simple analysis, where I downloaded Table S6 from the Holt et al paper, which contains a list of scaffolds and their corresponding linkage groups, and did a simple data join in R to determine the linkage group for each of the genes in Supplementary Table 3. SLC2A11B is on linkage group 20 and indeed the only other hits on that linkage group are on the same scaffold (CHEK2 and TRAFD1). This corresponds to what the authors noted in their response to Main Point #1 (but note the improvement in that we can now make a statement about the entire linkage group). Of the 64 genes, the linkage group for all but 8 could be determined from the Holt et al results. This seems to me to be worth including in the paper. Given that it is such an easy thing to include, I cannot recommend publication until the authors either include it or explain why it should not be included.

Aside from that, I have no further issues to bring up. The matter of the title I will leave to the authors and editors. This was a fun paper to read. I think if the linkage group data is included it will be even better.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #3: Yes

**********

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

Dear Dr Andrade,

We are pleased to inform you that your manuscript entitled "Molecular parallelisms between pigmentation in the avian iris and the integument of ectothermic vertebrates" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

Once your paper is formally accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosgenetics@plos.org.

In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about making your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Kelly A. Dyer

Associate Editor

PLOS Genetics

Bret Payseur

Section Editor: Evolution

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

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Comments from the reviewers (if applicable):

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