PONE-D-21-38977Analysis of the earliest complete mtDNA genome of a Caribbean colonial horse (Equus caballus) from 16th century Haiti.PLOS ONE

Dear Dr. Delsol,

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. Please submit your revised manuscript by Apr 15 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.

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PLOS ONE

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5. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This research was funded by the National Science Foundation to Emery and Delsol (DDIG 1930628), the Fulbright program (to Delsol), and additional support from the Florida Museum Ancient DNA Laboratory (from Guralnick). Permission for excavation and export of the faunal materials for curation at the Florida Museum was provided to Deagan by the government of Haiti, and permission for analysis was provided by the curators of the Historical Archaeology (Cobb) and Environmental Archaeology (Emery) programs of the Florida Museum.”

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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

Reviewer #3: Partly

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

Reviewer #4: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

Reviewer #4: 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

Reviewer #3: Yes

Reviewer #4: 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: It is an interesting research. Author briefed an interesting history about horses in the Americas and take us an overview about the origin of early translocated individual and its phylogenetic relationship with modern breeds in the Americas.

Reviewer #2: The manuscript reads very well and presents a nice story that fits with the historical records for the introduction of the horse into the Americas in the 16th century. The only minor issue with the results presented is that it is only based on a single specimen.

While the authors indicate that the sequence is publicly available at the NCBI, no accession number is provided. The accession number needs to be added before the paper can be published.

For the rest, I only have a few minor comments:

Line 73: "By the 1650s century" delete "century"

Lines 167-175. It would have been nice if the authors would have added a map of the archeological site in the supplementary material.

Lines 232-237: I assume the medium-joining network mentioned in this paragraph is the one shown in figure 3?

Line 259: "Figure S1" should be "Figure S2"

Reviewer #3: The paper reports assembly of the whole mt genome of a single horse dating back to 16th century. It is well written, and although only one sample was analyzed I think it adds to current knowledge.

However, I have several suggestions to improve the paper before publication on PLOS ONE, mainly related to reproducibility and data availability.

In particular, authors declared that they made the data publicly available, but I wasn't able to find any link pointing to them.

I list my concerns below regarding Data availability and Methods (and reproducibility)

Data availability issues:

Raw reads should be deposited on SRA. The assembled genome should be deposited in GenBank. Authors declared that the data are available, but they are not.

Methods and reproducibility issues:

First of all, I think authors performed all the analysis (mapping, assembling, phylogenetic tree construction) using the Geneious commercial software. This should be clearly declared.

Most importantly, for each analysis step, all the parameters and options used should be clarified so that a user buying Genious and having access to the data would be able to repeat the experiment. I think we are still far from this. I give some suggestions below, but I invite the authors to carefully check the methods and identify more missing details.

Line 210: “The 3’ and 5’ reads were imported, paired, and deduplicated in Geneious (https://www.geneious.com/).” This description should be more detailed. 1) What does it mean that read were “imported, paired”? Reads are already provided as paired by the sequencing instrument. Did the authors mean that the tried to merge paired reads in a single read? 2) What does it mean that reads were “deduplicated”? Did authors remove duplicates? 3) I assume 3’ and 5’ reads are the so-called forward and reverse reads. I think it is more common to name them forward and reverse, but this is not an issue.

Line 211: “The sequence was attributed to a horse after a BLAST search on the reads.” Did the authors use the blast online service? If so, they should provide the link and/or a reference to BLAST publications. Did the authors use default parameters? Did the authors blast against nt or specifically against the horse genome? Which threshold did they set in order to accept the hits as horse?

Lines 211-213: Which software was used for mapping? Which parameters were used to ensure that the maximum proportion of mismatches was 5%?

Lines 213-214: It is not clear how authors went from mapping reads to assemble the mitochondrial genome. This should be described.

Lines 216-218: Which software used for building the consensus? How were contigs generated? Which requisites were required for a contig to be used in the assembly? (e.g. contig length, contig coverage). Which overlap between contigs was required to assemble them into a scaffold? “We built the consensus sequence of the whole mitochondrion of the Puerto Real horse with a threshold of 75% and a quality set on “Highest”. The consensus was generated out of contig pileups that represented a mean coverage of 5622.9X of the horse reference mitochondrion.”

Lines 229-230: “The mitochondrial data was partitioned into coding, non-coding, and RNA regions to produce a phylogeny using different model estimates for each of these regions.” What are the differences between the model estimates? Are they implemented in Geneious? What are their names?

Reviewer #4: This study focused on a single ancient horse sample from 16th century /Caribbean colonial horse/. Complete mitochondrial sequencing of this single sample was performed. According to the authors, this mitochondrial genotype is associated with group A, and is the most closely related to a modern specimen of the American Chincoteague pony breed from North America (JN398377). I did not find a deposit of your genomic sequence and Genbank. Why?

The article is well discussed historically, but in contrast, relatively genetic research in this region is lacking. It would be good to make a comparative analysis of the available genetic data on the biodiversity of mitochondrial lines (modern and ancient) for America and the Iberian Peninsula. I would recommend adding data on modern local horses from the study region.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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PONE-D-21-38977R1Analysis of the earliest complete mtDNA genome of a Caribbean colonial horse (Equus caballus) from 16th-century Haiti.PLOS ONE

Dear Dr. Delsol,

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.

Please submit your revised manuscript by Jul 01 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,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

[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

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

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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

Reviewer #3: Yes

Reviewer #4: Partly

**********

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

Reviewer #1: Yes

Reviewer #3: N/A

Reviewer #4: No

**********

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

Reviewer #3: Yes

Reviewer #4: No

**********

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

Reviewer #3: Yes

Reviewer #4: 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: (No Response)

Reviewer #3: All my comments and concerns have been addressed, and I think that the paper is now suitable for publication. Authors provided the required details for reproducing the work using Geneious and the other software they used. Authors also provided accession numbers for the raw reads and the genome assembly. The data will be made available after publication.

Reviewer #4: The authors of the article did not comment on my two remarks.

- I did not find a deposit of your genomic sequence and Genbank?

- It would be good to make a comparative analysis of the available

genetic data on the biodiversity of mitochondrial lines (modern and ancient) for

America and the Iberian Peninsula. I would recommend adding data on modern local

horses from the study region.

In addition, the article is not written according to the requirements of PlosOne. As an example, too short a description of the results and the discussion. The abstract section is not formatted according to the requirements of the journal.

The Materials and Methods section describes the isolation of ancient DNA that does not conform to the protocols of good practice for working with ancient DNA. Тhere are no control reactions for DNA contamination as example.

I cannot believe that DNA isolation is possible in the described way to dissolve 500 mg of dry bone material in 1 ml of buffer.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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

Reviewer #1: No

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.

Analysis of the earliest complete mtDNA genome of a Caribbean colonial horse (Equus caballus) from 16th-century Haiti.

PONE-D-21-38977R2

Dear Dr. Delsol,

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,

Tzen-Yuh Chiang

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

**********

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

**********

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

Reviewer #3: 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

**********

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

**********

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)

**********

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

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