PONE-D-24-31160Determining population structure of Atlantic cod (Gadus morhua) found along the south coast of Newfoundland, Canada using next-generation sequencingPLOS ONE

Dear Dr. Babaei,

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 raised some important concerns, especially about the outlier analyses that needs to be addressed in the revision.

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Sven Winter

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: No

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: No

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: Yes

Reviewer #2: Yes

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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: In this manuscript, Babaei et al looked at the population structure of Northern Cod's west Atlantic populations focusing on individuals from the 3Ps NAFO division. The study uses a reduced representation approach and focuses on neutral loci to estimate population structure. Results suggest that cod in the west Atlantic is a metapopulation and the stock from 3Ps is a mix of Northern Cod (2J3KL) and Gulf of St. Lawrence (3Pn4RS) stocks. The paper is clear and the methodology is appropriate, however, there are major points that should be addressed before I can recommend its publication.

- Although, indeed, previous studies haven't looked specifically at 3Ps, there are population genetics studies in cod that include samples from this NAFO division. Although other papers from the same authors have been cited, I am surprised that these studies haven't been mentioned or discussed in the manuscript: Clucas et al., 2019; Kess et al., 2020; Ruzzante et al., 2000; Therkildsen et al., 2013.

- The analyses are appropriate however, regarding detecting outliers, it is recommended to use at least 3 outliers detection methods as it is well known that different methods detect different sets of outlier SNPs. A more conserved approach would be to consider all SNPs found by BayeScan and PCAdapt as outliers, instead of the common SNPs considering the goal is to have a set of neutral markers. In the manuscript, there is no information on the number of outliers each of the two methods detected, so is unknown the proportion of SNPs that weren’t found in common.

- To confirm that all SNPs selected were neutral, a HWE test should be performed during the filtering step which is not shown in the methods.

- The paragraphs explaining the changes in PCAs when removing genetic groups can be confusing. I recommend using hierarchical genetic structure plots.

- I would recommend carefully reading Clucas et al (2019), they too found outliers in chromosomes 18 and 21, which can enrich the discussion section.

- Do the juveniles cluster differently? Also, it is said that other stocks use the area (3Ps) as spawning ground but not all samples from the other NAFO divisions were sampled during spawning season, this should be discussed.

Other recommendations:

- The title is broad, I would advise a more detailed title.

- I recommend being consistent when using location names vs NAFO divisions, for people unfamiliar with the area can be confusing.

- As seasonality is mentioned, it would be good to include in the introduction a brief description of cod’s migratory behavior and spawning time.

-There are two paragraphs about the inversions in the intro, as the focus of the study is the neutral markers, I recommend reducing this information and providing more details about the genetic structure in the region found by different markers: microsatellites and SNPs array.

Minor comments:

Line 28: Southern should be capitalized?

Line 30: Not true! although not the main focus different previous studies have included 3Ps samples.

Line 38: "Which defines population structure"? not sure what this is referring to.

Line 54: What makes it well-suited for population genomic studies? what species wouldn't be well suited?

Line 61: Not clear the Nov-April?

Line 78: Include reference

Line 120: it is important the sampling of juveniles, spawning and non-spawning adults? nothing in the results or discussion about this.

Line 193: were retained? A verb is missing in this sentence.

Line 198: a more conservative approach is to use all outliers. It is well known that different software detects different sets of outliers. How many outliers were detected by each method?

Line 242: Parts of this should be in the methods.

Line 248: Is exactly half? Would be better to include the percentages.

Line 375: "Another explanation", I am not sure I understand the difference with the explanation in the previous sentence.

Line 378: Complete this paragraph with results from Clucas 2019.

Fig1: Include names of places, people outside Canada might not be familiar with the names.

References

Clucas, G. V., Lou, R. N., Therkildsen, N. O., & Kovach, A. I. (2019). Novel signals of adaptive genetic variation in northwestern Atlantic cod revealed by whole‐genome sequencing. Evolutionary Applications, 12(10), 1971–1987. https://doi.org/10.1111/eva.12861

Kess, T., Bentzen, P., Lehnert, S. J., Sylvester, E. V. A., Lien, S., Kent, M. P., Sinclair‐Waters, M., Morris, C., Wringe, B., Fairweather, R., & Bradbury, I. R. (2020). Modular chromosome rearrangements reveal parallel and nonparallel adaptation in a marine fish. Ecology and Evolution, 10(2), 638–653. https://doi.org/10.1002/ece3.5828

Ruzzante, D. E., Taggart, C. T., Lang, S., & Cook, D. (2000). Mixed-Stock Analysis of Atlantic Cod near the Gulf of St. Lawrence Based on Microsatellite DNA. Ecological Applications, 10(4), 1090–1109. https://doi.org/10.2307/2641019

Therkildsen, N. O., Hemmer-Hansen, J., Als, T. D., Swain, D. P., Morgan, M. J., Trippel, E. A., Palumbi, S. R., Meldrup, D., & Nielsen, E. E. (2013). Microevolution in time and space: SNP analysis of historical DNA reveals dynamic signatures of selection in Atlantic cod. Molecular Ecology, 22(9), 2424–2440. https://doi.org/10.1111/mec.12260

Reviewer #2: General comments:

The study presents compelling results, emphasizing the importance of incorporating genetic analyses into stock management. Overall, the research is robust, but given the range of analyses conducted, it is essential to provide access to all scripts used for data analysis and figure generation. Additionally, I recommend reviewing the names of the locations to ensure consistency throughout the text and figures, as inconsistencies can make the study difficult to follow.

Specific comments:

Introduction

Line 108-111: “Individuals tagged within 3Ps were recaptured during spring and summer on the…”. In the previous sentence, you are referring to several studies and, in this sentence, you mentioned individuals tagged within 3Ps were recaptured but did not say to what study you are referring, which makes the whole paragraph very confusing. In addition, I recommend being more specific instead of using “these studies” in line 109, such as citing them at the end of the sentence.

Materials and Methods

Line 132: “Some previously collected samples” How many previously collected samples were included? Please specify this in the text.

Line 138-140: I would include this information in the previous sentence and make it more objective given you are referring to the same set of “previously collected samples”.

Figure 1 legend: Please add the area that 3Ps represent.

Line 160: Please mention what happened with the other 350 samples that were not included in the library prep.

Line 163: From where did you base the use of these enzymes, did you do any kind of in silico simulation using whole genome?

Line 187: Please include the acronym alongside the names of the geographic locations. These locations are a key element of the study, as the research focuses on stock identification. Ensure the naming is clear and concise to avoid any confusion for the reader.

Line 197: Same thing as the comment before, make sure you are concise with the locations name.

Line 215: What multiple methods?

Results

Sequencing and filtering: Please reference here the Supplementary table 2 so the reader can understand how you ended up with the given number of neutral SNPs.

Line 234-236: Between what populations? This is not clear here nor in the methods.

Line 244: As mentioned before, for the readers that are not familiar with these locations, make sure to include the acronym as well for easier visualization using the map you provided.

Figure 3: Given that your study has multiple locations, you need to make sure the reader does not get confused with the names/acronyms. In figure 3A you used the acronyms and figure 3B the names, which makes extremely confusing to understand and compare the genetic clusters. The same issue is present throughout the text.

Table 2: Are these Fst values significant? Please display the p-value somehow.

Line 291-294: Please also provide p-values for a more comprehensive interpretation of your results, specially considering the fine scale genetic structure you are referring here.

Discussion

Line 375-376: Did the study include perhaps any gene flow or effective migration rate analyses? If not, provide a citation in this sentence that support your affirmation.

Line 378-386: This is a very interesting finding. Is there any reference to support such findings? Perhaps in another species with similar characteristics/distribution?

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Reviewer #1: No

Reviewer #2: No

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PONE-D-24-31160R1Genome-wide SNPs reveal novel genetic relationships among Atlantic cod (Gadus morhua) from the south coast of Newfoundland, Canada (subdivision 3Ps), Northern cod stock complex, and Gulf of St Lawrence.PLOS ONE

Dear Dr. Babaei,

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 agree with the reviewer that the manuscript was greatly improved. I also would agree that if a third outlier analysis was performed, it should be added to the methods and results to show that the outliers are agreed on by various methods. I am confident that if the authors add this final info the manuscript will be suitable for publication.  Please submit your revised manuscript by Feb 01 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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

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

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

We look forward to receiving your revised manuscript.

Kind regards,

Sven Winter

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.

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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 #1: 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 #1: Yes

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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: I reviewed the first version of the manuscript. This version is an improved version and it is clear the authors have made an important effort to attend to both reviewer's comments. I just have one recommendation:

I don't see mention of using the 3rd method (Benjamini-Hochberg) for outlier detection in the methods, I think would be important to add a sentence and add the results removing all outliers in the supplementary material as the authors have already done the analysis.

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

Reviewer #1: No

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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.

Genome-wide SNPs reveal novel genetic relationships among Atlantic cod (Gadus morhua) from the south coast of Newfoundland, Canada (subdivision 3Ps), Northern cod stock complex, and Gulf of St Lawrence.

PONE-D-24-31160R2

Dear Dr. Babaei,

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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Kind regards,

Sven Winter

Academic Editor

PLOS ONE

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