Genetic determination and JARID2 over-expression in a thermal incubation experiment in Casque-Headed Lizard

Gabriel Suárez-Varón; Eva Mendoza-Cruz; Armando Acosta; Maricela Villagrán-Santa Cruz; Diego Cortez; Oswaldo Hernández-Gallegos

doi:10.1371/journal.pone.0263804

Peer Review History

Original SubmissionJanuary 25, 2022
28 Feb 2022 Decision Letter - Patrick R Stephens, Editor PONE-D-22-02482A study of thermally-induced sex reversal in casque-headed lizardsPLOS ONE Dear Dr. Cortez, 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 reviewers were enthusiastic about the topic and the potential importance of the findings. However both authors found issues with the text. James Walker volunteered to be identified, and made a number of editorial suggestions. You can find a document in the PLOS One editorial system with his suggest edits (the final version that he sent to me is labeled JWalker edits). These are only meant to be taken as suggestions, and I leave it to your discretion which of them you wish to implement. However, many of his suggestions seem sound to me so please do carefully consider his feedback. The other reviewer identified more substantial issues, particularly areas where the clarity of the manuscript could be improved. Please do respond directly to each of the issues raised by this reviewer in your rebuttal letter. Please submit your revised manuscript by Apr 14 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, Patrick R Stephens, Ph.D. 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. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: The authors present an interesting paper bringing new insight into the sex determination system of the brown basilisk. The paper is clearly written and well presented. In an ideal world, the sample sizes would be larger, but I appreciate the difficulties associated with using wild caught animals. Due to the small sample sizes and limited temperature range the available eggs could be incubated at, this study was unable to definitively conclude whether or not the species has sex reversal. As such, I strongly suggest that the title be changed to more accurately reflect the findings of the study. It currently reads like sex reversal was shown to occur in this species, and the short title "Lack of sex reversal in casque-headed lizards" is actually what was found. I think that it is likely that this species does has sex reversal, and I would be keen to see this expanded on in the discussion. Currently only two other reptiles have been definitively shown to posses sex reversal, and only one of these has an XY system. Both are Australian lizards, so from an evolutionary perspective, sex reversal occurring in a New World species is very interesting. I noticed that 82 eggs were incubated, but only 48 across the three incubation temperatures reached the target developmental stages. That seems like quite a high mortality rate - are the authors able to comment on this? Was the mortality observed at a particular temperature, were perhaps some of the eggs not actually viable when they were initially incubated? The temperatures aren't particularly extreme, so I would be surprised to see temperature specific mortality. If sex specific mortality has occured this could affect the results presented in the manuscript. My major concern with the manscript is the tissue used for the RNA-seq analysis. As the methods are currently written I am not entirely clear what tissue was used. Under the "laboratory conditions" section of the methods the posterior part of the embryo was used for histology, so was clearly the part of the embryo containing the gonads. Then the rest of the embryo was preserved for further genetic analysis. This is quite a large part of the embryo containing a wide variety of different tissue types. Then in the "RNA extraction and RNA-seq analysis" section samples were obtained from whole embryos. Were they sub sampled from the other half of the embryo, or were these whole embryos that weren't also used for histology? Regardless, what was the actual tissue type that was sequenced? Or was a large part of the embryo homogenised? This information is important not only for the methodological clarity of this manuscript, but also because the RNA-seq data has been deposited publicly on SRA, so any other researchers looking to use this data need to have a clear understanding of what the actual sample was. An additional concern is if the same tissue type wasnt used, this significantly affects the validity of the differential gene expression analysis. Based on the low number of differentially expressed genes, I assume that the tissues were consistent between samples, but this does need to be explictly stated. The discussion feels a little unfinished, and would benefit from a short concluding paragraph, particularly highlighting future research directions, or how it might be possible to definivitely show whether or not the species has sex reversal. Overall, it is a well presented manuscript, and would be suitable for publication in PLoS One if the concerns outlined above are addressed. ****** 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 [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: Suarez-Varon.et.al.fullMS.2022.docx Attachment Submitted filename: Suarez-Varon.et.al.fullMS.2022 Jwalker edits.docx https://doi.org/10.1371/journal.pone.0263804.r001
Revision 1
28 Mar 2022 Author Response 10 March 2022 Patrick R Stephens, Ph.D. Academic Editor PLOS ONE Dear Dr. Stephens, The authors would like to thank you and the reviewers for their thoughtful insights into our manuscript. These comments and suggestions have largely improved the quality of our work. We have addressed all of the comments in the manuscript (of Reviewer#1 and edits of J. Walker). A detailed list of the corrections is shown below (comments of Reviewer#1). The newly revised version of the manuscript includes the editor and reviewers’ comments. Throughout the entire manuscript, several paragraphs were rewritten for better understanding, and the discussion was expanded. Academic Editor’s comments: Both reviewers were enthusiastic about the topic and the potential importance of the findings. However, both authors found issues with the text. James Walker volunteered to be identified and made a number of editorial suggestions. You can find a document in the PLOS One editorial system with his suggest edits (the final version that he sent to me is labeled JWalker edits). These are only meant to be taken as suggestions, and I leave it to your discretion which of them you wish to implement. However, many of his suggestions seem sound to me so please do carefully consider his feedback. The other reviewer identified more substantial issues, particularly areas where the clarity of the manuscript could be improved. Please do respond directly to each of the issues raised by this reviewer in your rebuttal letter. Answer: We thank the Academic Editor for these comments. We have now carefully studied the suggestions made by Dr. Walker. We have accepted most of these suggestions as they clearly improved the text. We have also clarified the questions raised by Dr. Walker regarding the use of some words, the meaning of some sentences, and the common names or families of reptiles we described. Additionally, we have addressed the comments raised by Reviewer#1 regarding the lack of important information in the Methods and the Discussion. Please find below our point-by-point answer to Reviewer#1 comments. We addressed Dr. Walker suggestion’s in the marked-up copy of your manuscript. Reviewer#1’s comments: 1) Reviewer#1: Due to the small sample sizes and limited temperature range the available eggs could be incubated at, this study was unable to definitively conclude whether or not the species has sex reversal. As such, I strongly suggest that the title be changed to more accurately reflect the findings of the study. It currently reads like sex reversal was shown to occur in this species, and the short title "Lack of sex reversal in casque-headed lizards" is actually what was found. Answer: We thank Reviewer#1 for the detailed revision of our work. Following her/his advice, we have changed the title of the study to reflect the main findings. The new title is: “Genetic determination and JARID2 over-expression in a thermal incubation experiment in Casque Headed Lizard”. Page 1 in the revised version of the manuscript. 2) Reviewer#1: I noticed that 82 eggs were incubated, but only 48 across the three incubation temperatures reached the target developmental stages. That seems like quite a high mortality rate - are the authors able to comment on this? Was the mortality observed at a particular temperature, were perhaps some of the eggs not actually viable when they were initially incubated? The temperatures aren't particularly extreme, so I would be surprised to see temperature specific mortality. If sex specific mortality has occurred this could affect the results presented in the manuscript. Answer: We thank Reviewer#1 for this comment. We agree that the number of eggs that were incubated and the number of eggs reported for the histological/genotypic analyses do not match. We have now corrected this mistake. We would like to clarify that of the 22 females, a total of 130 eggs were obtained. These eggs were randomly assigned to three incubation temperatures (26, 29 and 32°C). Of these, 42 eggs (32%) became contaminated with fungal infection and the embryos died with no effect of temperature on mortality rates (X2 > 0.05). Of the 88 eggs that successfully reached the target developmental stages, 40 were used in a parallel study that aimed to establish the effect of incubation temperatures (26, 29 and 32°C) on the development of the embryos: Suárez-Varón. etal. (2021). REVISTA MEXICANA DE BIODIVERSIDAD. 92:923795 (http://rev.mex.biodivers.unam.mx/index.php/es/variacion-del-estadio-embrionario/). Thus, we used 48 eggs to study the association between incubation temperature, genotype, and the histology of gonads (present study). We have added more information to the Methods. Page 5, first paragraph. 3) Reviewer#1: My major concern with the manuscript is the tissue used for the RNA-seq analysis. As the methods are currently written I am not entirely clear what tissue was used. Under the "laboratory conditions" section of the methods the posterior part of the embryo was used for histology, so was clearly the part of the embryo containing the gonads. Then the rest of the embryo was preserved for further genetic analysis. This is quite a large part of the embryo containing a wide variety of different tissue types. Then in the "RNA extraction and RNA-seq analysis" section samples were obtained from whole embryos. Were they sub sampled from the other half of the embryo, or were these whole embryos that weren't also used for histology? Regardless, what was the actual tissue type that was sequenced? Or was a large part of the embryo homogenised? Answer: We thank Reviewer#1 for this comment. We agree that the Methods are not clear about the tissues used for the analyses. Embryos that reached the target developmental stages were divided into two segments. An upper segment that comprised the head and eyes. And a lower segment where the gonads were located. Each individual was assigned a number so we could match the genetic material versus the histology of the gonads. The upper (head/eyes) segment was then divided longitudinally into two fragments of equal size. One that was homogenized and from which we extracted DNA for the genotypic verification of the Y chromosome, and a second fragment that was also homogenized and from which we attempted to purify RNA; RNA was degraded in many samples though. We have added more information to the Methods. Page 5, first and last paragraphs; Page 6, second paragraph. 4) Reviewer#1 comment: This information is important not only for the methodological clarity of this manuscript, but also because the RNA-seq data has been deposited publicly on SRA, so any other researchers looking to use this data need to have a clear understanding of what the actual sample was. An additional concern is if the same tissue type wasnt used, this significantly affects the validity of the differential gene expression analysis. Based on the low number of differentially expressed genes, I assume that the tissues were consistent between samples, but this does need to be explictly stated. Answer: Reviewer#1 is correct. Using different tissues to conduct differential gene expression analysis may have led to potentially odd results. Besides, detailed information about the samples used is important to replicate our results. As explained above, we used half of the upper segment (head/eyew) of the embryos for DNA/RNA extractions. Tissues were homogenized prior to the purification of the genetic material. We have added more information to the Methods. Page 5, first and last paragraphs; Page 6, second paragraph. 5) Reviewer#1 comment: The discussion feels a little unfinished, and would benefit from a short concluding paragraph, particularly highlighting future research directions, or how it might be possible to definivitely show whether or not the species has sex reversal. Answer: We thank Reviewer#1 for this comment. We have added a new paragraph at the end of the Discussion where we describe the limitations of the work and future research directions. Page 12, second paragraph. Please do not hesitate to contact us if you need any further information or clarification. Diego Cortez, PhD Centro de Ciencias Genómicas UNAM Ave. Universidad s/n. CP-62210. Cuernavaca. México email: dcortez@ccg.unam.mx Oswaldo Hernández, PhD Laboratorio de Herpetología Universidad Autónoma del Estado de México Instituto Literario #100 Centro. CP-50000. Toluca. México email: ohg@uaemex.mx Attachments Attachment Submitted filename: Suarez-Varon.et.al.Rebuttal_Letter.pdf https://doi.org/10.1371/journal.pone.0263804.r002
20 May 2022 Decision Letter - Patrick R Stephens, Editor PONE-D-22-02482R1Genetic determination and JARID2 over-expression in a thermal incubation experiment in Casque-Headed LizardPLOS ONE Dear Dr. Cortez, 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 needs a few additional minor revisions for polish and readability, but overall this is a really solid piece of work. This should be ready to go after a final small revision. Great work. Please submit your revised manuscript by Jul 04 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, Patrick R Stephens, 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 (if provided): My apologies for how long this decision took. Since we were unable to obtain a second review in a timely fashion I have acted as the second reviewer for this revision. I think there are a few minor revisions you still need to make, but overall this is a really solid work. Once you make a few small changes to enhance readability, I believe this will be ready to go. Here are my additional suggestions: Line 58: Change “their sex determination mechanisms remain poorly understood” To “the factors that drive variation in sex determination mechanisms among species remain poorly understood” Your next sentence demonstrates that we know a lot about how sex determination works in squamates in general, making the first sentence seem like an exaggeration as written. Line 76: Delete “however” Should read: . . .chromosomes, non-avian reptiles have . . . Methods: PLOS is pretty flexible about formatting. However, for readability the formatting should be internally consistent. I suggest that you follow the formatting you use in the Results for headings and subheading throughout. Line 112: bold the heading like in the discussion Line 121: there should be a line of space in between 121 and 122, bold the heading Line 141: bold the heading Line 148: bold the heading Line 165: bold the heading Line 261: By “top-second gene” do you mean the gene with highest expression difference between 26°C and 32°C? If that is the case, I would delete the phrase “top-second” as I find it really unclear. I’m also not sure what’s “remarkable” about this result from the intro. In this context “remarkably” implies a result running counter to what most people would expect or that is unusual or unexpected in some other way. However, from what we have read up to this point it’s not clear why you think the result is remarkable. I would change this sentence to “JARID2 was the gene with the highest expression difference between 26°C and 32°C . . .” In the discussion, where you immediately explain what’s remarkable about the result, I think it’s fine to use this phrasing. Line 322: I would delete “top-second” here as well. I find the phrase really unclear. Change the sentence to: “Remarkably, we observed that JARID2 was the gene with the highest expression difference between low and high incubation temperatures.” [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 ******** 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: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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: 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: 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: The ammendments the authors have made to the manuscript have addressed my questions, and the manuscript is acceptable for publication in PLoS One ****** 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 [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. https://doi.org/10.1371/journal.pone.0263804.r003
Revision 2
14 Jun 2022 Author Response My apologies for how long this decision took. Since we were unable to obtain a second review in a timely fashion I have acted as the second reviewer for this revision. I think there are a few minor revisions you still need to make, but overall this is a really solid work. Once you make a few small changes to enhance readability, I believe this will be ready to go. Here are my additional suggestions: Line 58: Change “their sex determination mechanisms remain poorly understood” To “the factors that drive variation in sex determination mechanisms among species remain poorly understood” Your next sentence demonstrates that we know a lot about how sex determination works in squamates in general, making the first sentence seem like an exaggeration as written. A. Done, manuscript was modified to attend this suggestion (line 58). Line 76: Delete “however” Should read: . . .chromosomes, non-avian reptiles have . . . A. Done, manuscript was modified to attend this suggestion (line 77). Methods: PLOS is pretty flexible about formatting. However, for readability the formatting should be internally consistent. I suggest that you follow the formatting you use in the Results for headings and subheading throughout. Line 112: bold the heading like in the discussion A. Done, manuscript was modified to attend this suggestion (line 113). Line 121: there should be a line of space in between 121 and 122, bold the heading A. Done, manuscript was modified to attend this suggestion (line 124). Line 141: bold the heading A. Done, manuscript was modified to attend this suggestion (line 143). Line 148: bold the heading A. Done, manuscript was modified to attend this suggestion (line 150). Line 165: bold the heading A. Done, manuscript was modified to attend this suggestion (line 167). Line 261: By “top-second gene” do you mean the gene with highest expression difference between 26°C and 32°C? If that is the case, I would delete the phrase “top-second” as I find it really unclear. A. Done, we delete the phrase “top-second” (line 263). I’m also not sure what’s “remarkable” about this result from the intro. In this context “remarkably” implies a result running counter to what most people would expect or that is unusual or unexpected in some other way. However, from what we have read up to this point it’s not clear why you think the result is remarkable. I would change this sentence to “JARID2 was the gene with the highest expression difference between 26°C and 32°C . . .” A. Done, manuscript was modified to attend this suggestion (line 262). In the discussion, where you immediately explain what’s remarkable about the result, I think it’s fine to use this phrasing. A. Thanks. Line 322: I would delete “top-second” here as well. I find the phrase really unclear. Change the sentence to: “Remarkably, we observed that JARID2 was the gene with the highest expression difference between low and high incubation temperatures.” A. Done, manuscript was modified to attend this suggestion (line 324). Attachments Attachment Submitted filename: Suarez-Varon.et.al.Response_to_Reviewers.docx https://doi.org/10.1371/journal.pone.0263804.r004
17 Jun 2022 Decision Letter - Patrick R Stephens, Editor Genetic determination and JARID2 over-expression in a thermal incubation experiment in Casque-Headed Lizard PONE-D-22-02482R2 Dear Dr. Cortez, 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, Patrick R Stephens, Ph.D. Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0263804.r005
Formally Accepted
28 Jun 2022 Acceptance Letter - Patrick R Stephens, Editor PONE-D-22-02482R2 Genetic determination and JARID2 over-expression in a thermal incubation experiment in Casque-Headed Lizard Dear Dr. Cortez: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Patrick R Stephens Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0263804.r006

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