PONE-D-23-38093Enhanced dietary reconstruction of Korean prehistoric populations by combining d 13 C and d 15 N amino acids of bone collagenPLOS ONE

Dear Dr. Choy,

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 Feb 22 2024 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,

John P. Hart, 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. In your manuscript, please provide additional information regarding the specimens used in your study. Ensure that you have reported human remain specimen numbers and complete repository information, including museum name and geographic location. 

If permits were required, please ensure that you have provided details for all permits that were obtained, including the full name of the issuing authority, and add the following statement:

'All necessary permits were obtained for the described study, which complied with all relevant regulations.'

If no permits were required, please include the following statement:

'No permits were required for the described study, which complied with all relevant regulations.'

For more information on PLOS ONE's requirements for paleontology and archeology research, see https://journals.plos.org/plosone/s/submission-guidelines#loc-paleontology-and-archaeology-research.

3. Thank you for stating the following financial disclosure: 

"This work was supported by Research Funds for Young Faculty Club at Hanyang University (HY-2018-00000001315) and also supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2022S1A5A2A03051382)."

Please state what role the funders took in the study.  If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" 

If this statement is not correct you must amend it as needed. 

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

4. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.

Additional Editor Comments:

Both reviewers are positive about your work. However, both indicate the need for revision to clarify various sampling, methodological, statistical, and interpretive issues. Please address all of the comments and suggestions while making your revisions.

[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: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Reviewer comments to the authors

General comments

Dear authors, your article presents highly valuable data for the dietary reconstruction of Korean prehistoric populations. The use of Compound-Specific Stable Isotope Analysis from Amino Acids (CSIA-AA) distinctly strengthens this study and contributes significant insights to the published pool of CSIA-AA data in archaeology. This, in turn, optimises and enriches prospects for future research in the field of dietary reconstructions.

I extend my congratulations to the authors for conducting such an exemplary study. Additionally, I would like to suggest minor revisions that could potentially further enhance the work for its publication.

Furthermore, I encourage the authors to report the scripts for the PCA and the MixSIAR models. Sharing this information would significantly contribute to advancing the use of CSIA-AA in this field and optimise its interpretation.

Minor revisions

- Page 3 (lines 64-66), to the comment “Amino acids (AAs) can be divided into two groups on the basis of their synthesis and function: essential amino acid (EAA) and non-essential amino acid (NEAA)”:

I would like to suggest to make clear that this distinction is related to the carbon pathways and that it's different for nitrogen, for which they should rather differentiate between source and trophic amino acids (see e.g. O'Connell (2017) on the nitrogen metabolic pool).

O'Connell TC. (2017) 'Trophic' and 'source' amino acids in trophic estimation: a likely metabolic explanation. Oecologia. 184(2):317-326. doi: 10.1007/s00442-017-3881-9.

- Page 8 (line 216), about derivatisation method(s?):

It's important to note that the derivatisation process applies to the actual amino acid, not the isotope value itself. However, I'm unclear on whether you employed two distinct derivatisation approaches—one for measuring nitrogen isotope values and another for carbon. Your description suggests this might be the case, but it's not entirely clear, especially regarding the method used for carbon isotope measurements. Could you please provide clarification on this aspect?

- Page 10 (lines 273 - 274), to the sentence: “Standard deviations of carbon isotope measurements were generally ±0.4‰ as determined by repeated injections of the 10 AA standard mixture”:

Have you considered propagating the error due to inclusion of exogenous carbon from your solvents? I believe that's something important to do. See Docherty et al (2001)

Docherty G, Jones V, Evershed RP. Practical and theoretical considerations in the gas chromatography/combustion/isotope ratio mass spectrometry delta(13)C analysis of small polyfunctional compounds. Rapid Commun Mass Spectrom. 2001;15(9):730-8. doi: 10.1002/rcm.270. PMID: 11319796.

- Page 11 (line 312), regarding: “four essential (Ile, Leu, Phe, Val)”:

Please, be cautious regarding leucine, isoleucine, and valine, as they do not exhibit typical behaviour as 'essential' for nitrogen. Previous observations have noted fractionation in these amino acids (indicating their role as trophic amino acids). In all three cases, the diet-to-consumer offset was observed to be quite high, approximately 5 permille, with a significant level of uncertainty. For more insights, refer to experimental studies such as McMahon and McCarthy (2016).

McMahon, K.W. and McCarthy, M.D., 2016. Embracing variability in amino acid δ15N fractionation: mechanisms, implications, and applications for trophic ecology. Ecosphere, 7(12), p.e01511.

- Page 11 (lines 315-317), regarding: “In order to assess quality control (QC), we used the relationship between proline and hydroxyproline since the hydroxylation of proline to form hydroxyproline does not involve exchange of nitrogen atoms [56]”.

You should be careful here, as you only consider the quality of proline and hydroxyproline. Ideally, you should estimate the d13C and d15N bulk stable isotope values from the amino acid values and compare them with your measured bulk SIA values. See e.g. Fontanals-Coll, et al. (2023), but also older research by Styring and Howland. I consider this really important to assess the quality of your research, please try to address.

Fontanals-Coll M, Soncin S, Talbot HM, von Tersch M, Gibaja JF, Colonese AC, Craig OE. Stable isotope analyses of amino acids reveal the importance of aquatic resources to Mediterranean coastal hunter-gatherers. Proc Biol Sci. 2023 Feb 22;290(1993):20221330. doi: 10.1098/rspb.2022.1330.

- Page 13 (lines 379 - 380), regarding “Previous studies have shown that the d13CGly-Phe values are an effective proxy for discriminating between terrestrial and marine sources [6, 7, 11, 58]”.

The figure does not provide an explanation of how the shaded regions, within which terrestrial and aquatic values are expected to fall, were created. I assume you utilised data from these studies? If so, please make this explicit, perhaps in the figure's caption, to enhance clarity.

- Page 13 (lines 385-386), about: “Our d13CVal-Phe results show that d13CVal-Phe can separate terrestrial from aquatic sources.”

Figure? or Figure 4B could be: axis x: Gly-Phe, axis y: Val-Phe rather than bulk d15N as it has been previously done.

- Page 14 (lines 394-396), regarding: “To assess differences in food groups, we carried out principal component analyses (PCA) with five d13C (Phe, Leu, Val, Ile, Thr) and four d15N (Val, Leu, Ile, Phe) values of essential amino acids.”

Consistency is crucial here. Although you decided to run the PCA with essential amino acids, you included trophic amino acids. You should exclude Val, Leu, and Ile for their stable nitrogen isotope values for this. I suggest to re-run the PCA and decide between two different options: a) Repeat the analysis but excluding d15N for Ile, Leu, and Val, or b) Consider the inclusion of all (essential, non-essential, trophic) amino acids. Both approaches will provide different insights; however, the first option (a), while not altering the results significantly, ensures consistency for readers and researchers in the field.

- Page 15 (lines 419-420), regarding: “A MixSIAR model was conducted with five d13CAA values (Leu, Val, Ile, Thr, Phe) and one d15NAA value (Phe).”

I would like to once again encourage the authors to publish the scripts. It's not entirely clear how you constructed your MixSIAR model. Did you consistently take into account the calorific or protein contribution of different foodstuffs? For instance, did you consider the protein contribution of cereals, assuming 70% carbohydrates and 30% proteins? If so, I am satisfied with the model. However, I believe that, like me, other researchers would appreciate having this information available. This isn't just for the purpose of assessing your work but also for the benefit of learning and improving their own methodologies and interpretations , especially considering that CSIA-AA is still cutting-edge.

Reviewer #2: This research paper presents bulk collagen and amino acid stable isotope data (carbon and nitrogen) from bone material (human and animal) of four sites in South Korea. The four sites are Prehistoric cemeteries and represents different chronocultural periods. Four human bones and 4 animal bones are spread on three sites of the Mumum period (Bonze Age culture for this geographical area) and 28 human bones along with 11 animal remains from different species (diversified and representing the resources potentially consumed) are from a site with 1600 burials of the Proto-Three kingdom period (Imdang, more recent than Mumum). The archaeological material is enhanced with modern local domesticated plants (n=9, C3 and C4 cereals) to create a local isotopic baseline. Bulk and CSIA-AA data are discussed thanks to statistical analyses (PCA) and Bayesian models. Results indicate (1) different dietary interpretations when comparing bulk and AA data and (2) show how isotopic data improve the knowledges about Prehistoric Korea subsistence strategies, still underdeveloped in the scientific community. The paper deserves publication for both aspects of methodological considerations and archaeological information provided.

Specific comments are proposed below

P 3, l 72 and 73: provide the trophic step for both Glu and Phe

P4, l102-104: late in the manuscript one can understand the real interest to use isotopic analysis to study diet, but this sentence deserves more information about the archaeological issue in this specific context

P4, l113: did you correct the C isotopic data (Suess effect)? if yes mention it in the paper, if not explain why.

P5 l114-116: better explain the archaeological issue of this study

P5: Mumum material: explain potential difficulty to get material and the exact anatomical part sampled. Why only 4 remains on 3 sites? how it can reflect a pattern or if not explain better how it improves the previous knowledge (to add also in the conclusion part).

The same for Imdang. Explain it is coming from scarified humans and how they can reflect a pattern and if not how it improves the previous knowledge (to add also in the conclusion part).

L134-136: push forward this issue and also highlight it in the conclusion.

P8-9: all the technical part should go in a Supplementary Material file

P11, l306: P>0.05?

P14, l405-409: it seems that it is twice the same sentence; check and rewrite what is necessary

P20 conclusion: add sentences about the initial archaeological issues (cf previous comments) and more explain the archaeological issues about agriculture and game in these two different periods. Explain why these two periods are interesting to compare and explain how despite the very small sample size and the specific origin of the material, the significance of the isotopic data (both bulk collagen and AA).

**********

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: Yes: Maria Fontanals-Coll

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: PLOS ONE Review - M Fontanals-Coll.pdf

Enhanced dietary reconstruction of Korean prehistoric populations by combining d 13 C and d 15 N amino acids of bone collagen

PONE-D-23-38093R1

Dear Dr. Choy,

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,

John P. Hart, Ph.D.

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 #1: All comments have been addressed

Reviewer #2: 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

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: 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

Reviewer #2: 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

Reviewer #2: 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: Dear authors,

Thank you for the good work in addressing my comments. In my opinion, it is a fantastic piece of work worthy of publication, and this has been my suggestion to the journal.

Good luck in your future endeavours.

Kind regards,

Reviewer 1

Reviewer #2: (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 #1: Yes: Maria Fontanals-Coll

Reviewer #2: No

**********