PONE-D-23-12197Nutritional supplementation, tooth crown size, and trait expression in individuals from Tezonteopan, Mexico.PLOS ONE

Dear Dr. Blankenship-Sefczek,

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 now have two expert reviews of this work. There are significant concerns over the structure of the manuscript, rationale, statistical approach, and discussion of results that may not reflect what the authors were able to demonstrate with the limited data set.

Please submit your revised manuscript by Aug 10 2023 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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Kind regards,

JJ Cray Jr., Ph.D.

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: Partly

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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: 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: Thank you for giving me the opportunity to review this article. In this manuscript, the authors explored the relationship between nutritional stress and tooth size, intercusp spacing, and morphological trait presence and expression. This work is exciting since to date, it is the first to investigate how environmental factors may influence the degree to which expectations under the patterning cascade model of tooth morphogenesis are followed. The authors found no support that nutritional stress results in smaller molars and higher frequencies of Carabelli’s trait. They, however, did find partial support that significant differences exist in intercusp spacing, Carabelli’s cusp trait expression, cusp 5 presence and expression, anterior tooth size and trait expression between nutritionally supplemented and non-supplemented groups. Additionally, the authors found that the spacing between the lingual cusps and buccal cusps of the upper molars appear to be the significant predictors of accessory cusp development. While the results of the research may be tempered by the small sample size, the authors do acknowledge this throughout the manuscript and the value provided by the results of this novel analysis far outweigh sample size concerns. Below, I provide specific comments that I hope will improve the manuscript:

Line 77-80: The way this sentence is currently worded is confusing. Suggested rephrase: The PCM has emerged as a developmental framework to understand how enamel knot spacing and the space and time available for crown formation affect variation in dental morphology.

Line 117-119: The sample of citation 16 was actually conducted on modern humans, I suggest this citation be placed in the appropriate section within the sentence. Additionally, some of the evidence found in the cited studies, especially those that were conducted in fossil hominins and modern humans, provides mixed support for the PCM. I would incorporate this into the sentence to indicate that previous work has not found unequivocal support for this model, especially as it pertains to humans, which is relevant for this study since the current study’s sample is composed of modern humans. This will also bolster this study’s results since this research, similar to previous ones conducted in humans, only found partial support for the PCM.

Line 148: For Hypothesis 2, do the authors expect to find any specific trends with respect to cusp spacing? Would the authors expect to see smaller intercusp distances or larger ones in supplemented vs. non-supplemented groups? The authors provide concrete trends they expect for the other hypotheses based on previous research, whereas in Hypothesis 2, their prediction seems to indicate that they just expect the two groups to exhibit significant differences. If the authors expect specific trends, it would be helpful to clarify here what those would be. If they are unsure about any trends (maybe because no prior study has examined this) that might also be helpful to clarify here. The authors also discuss downstream changes they expect to see in intercusp spacing in hypothesis 3 and 4 as it relates to accessory cusp formation. If these are the specific expectations the authors have for changes in intercusp distance, it might be helpful to reference hypothesis 3 and 4 for the specific trends. For example: “See Hypothesis 3 and 4 for trends expected for intercusp spacing under the PCM”.

Lines 213- 276: The Study Materials section is fairly long. In my opinion, the most important parts are the first and last paragraph of this section. The information provided in the paragraph beginning on line 235 could be reduced and incorporated into the paragraph preceding it. The study by Goodman et al. discussed in the paragraph starting on line 244 would fit well in the discussion around environmental stress and dental phenotypes of the Background section (lines 123-135).

The information provided in the sentences located on lines 257-258, lines 268-270, and lines 270-272 is identical and feels repetitive. This section would read better if some of this overlapping information was reduced.

Lines 278-279: A citation indicating the methods that were followed to obtain the BL and MD measurements would be beneficial here.

Line 322: The authors indicate that trait frequency based on trait presence and absence was compared between supplemented and non-supplemented groups. However, the authors only discuss data collection methodology for raw scores in the previous paragraph (lines 300-303). In that section (line 304), it would be helpful to add a sentence discussing how traits were dichotomized to presence/absence states and citing the system that was followed for dichotomization.

General comment for Tables 3, 4, and 6: The way dental morphological trait frequency is usually presented is by providing trait frequency and the total number of individuals for which the trait was observable. The way they are currently presented is confusing. Are the numbers below “Supplemented” and “Non-supplemented” referring to the number of individuals who were characterized by the respective traits? Are they the % frequency of the trait? I would recommend providing the frequency of the trait (as a proportion) followed by the total number of individuals for whom observations could be made (n), especially since the Wilcoxon test is used to test for difference between the frequencies. If the authors need further clarification on what is meant by the comment here, please refer to Table 3 in Irish, 1997 for how morphological trait frequencies are usually presented, which is a publication cited within this manuscript.

Lines 391-398: The authors identified a significant difference in cusp 5 frequency and expression, but this was only so for M1, not M2. This should be made clear in this section.

Line 396: The authors argue that the results of their analyses indicate that the non-supplemented group had higher grades of cusp 5 expression, but information supporting this is not presented in the manuscript. Table 3, cited to present this information, only indicates results for when dichotomized trait presence is compared between the groups, but not expression. It would be helpful if the authors presented these results in a table, similar to how these results were presented for Carabelli’s trait in Table 4.

Lines 404-408: This section has several statements that do not align with the results presented in Table 5. The authors state that “The supplemented group exhibited significantly larger central incisors (Wilcoxon test: W=2470.5, df=1, p=0.018) and canines (Wilcoxon test: W=1742, df=1, p=0.010), compared to the non-supplemented group”. However, the results attributed to the central incisor in this statement are actually those of the lateral incisor (based on Table 5). While central incisors are larger in the supplemented group, these results are not significant (p=0.404). Conversely, the following sentence discussing the results for the lateral incisor cites Table 6, whereas the results are actually presented in Table 5. Please also correct the sentence in lines 408-410 to refer to the appropriate results as well.

Lines 413-416: While the central incisors and canines of the supplemented group did exhibit significantly higher frequencies of tuberculum dentale than the non-supplemented groups, this was not the case for the lateral incisor, which arguably provides mixed support for prediction 5b, rather than complete support.

Lines 438-439: Authors mixed up the two cusps here. Carabelli’s trait would be a peripheral accessory cusp, while cusp 5 would be a central accessory cusp.

Lines 436-456: To test hypothesis 6, it might be beneficial to run analyses separately for Carabelli’s trait and cusp 5, rather than lumping them together as 5+ cusped molars. This could be presented as an additional table to Table 6 or even as a supplementary table. As the authors point out, Carabelli’s trait is a peripheral accessory cusp, while cusp 5 is a central accessory cusp. Due to differential placement on the crown, some of the intercusp distance expectations would be different for these two cusps. For Carabelli’s all intercusp distances should be smaller to support the potential of accessory cusp development on the periphery (especially that between cusp 1 and 2). On the other hand, to ensure that a centrally placed accessory cusp 5 can develop, we would expect increased distance between cusps 3 and 4 to ensure the fields of inhibition surrounding these cusps allow additional cusp development. This contradicts predictions for Carabelli’s trait. Therefore, I think it would be helpful to show those analyses separately to make sure that considering the two traits at the same time isn’t obscuring the results obtained.

Table 7: As with the previous tables, what is located below the “Supplemented” and “Non-supplemented” columns is very ambiguous. I assume it’s the average intercusp distance within each group? This should be made clear in the labels. For example, instead of simply saying “Supplemented”, I recommend changing the label to “Average ICD (intercusp distance) for supplemented group”.

Table 8: The authors assert that Hypothesis 2 was supported because significant differences between the supplemented and non-supplemented group were found for intercusp spacing. However, these results were only significant for the protocone-metacone distance. Wouldn’t this only lend partial support to Hypothesis 2? Similarly, for Hypotheses 4a and 4b, the results were only significant for M1, but not M2. Wouldn’t this lend partial support to the hypotheses? This is also the case for Hypotheses 5a and 5b as not all results were significant.

Lines 503-504: Again, since these differences are only significant for the protocone-metacone distance, wouldn’t this hypothesis be partially supported?

Lines 534-535: As mentioned previously, differences in central incisor size while larger in the supplemented group, these results are not significant. However, the lateral incisor seems to be significantly larger in the non-supplemented group.

Reviewer #2: Thank you for the opportunity to review this interesting manuscript, “Nutritional supplementation, tooth crown size, and trait expression in individuals from Tezonteopan, Mexico”. This paper employs a dataset collected in Mexico between 1969-1980 to attempt to address questions related to the role of dietary/environmental stress on tooth morphogenesis in humans. The authors focus on the maxillary permanent dentition, and analyze data from dental casts collected during the original study. In the original study, half of participant mothers were given nutritional supplementation and half were not. In the current sample (n=73), n=34 were children of mothers who received nutritional supplementation. Overall, this is an interesting study and fairly unique dataset, but the strength of the data is weak given the attempt to use this particular sample to draw more generalizable conclusions. I would strongly suggest the authors should reframe this as a story about this very unique sample and then speak in more measured terms about the potential applicability to broader concepts such as the PCM.

-This manuscript is quite long. There are two whole pages outlining the hypotheses, after listing research aims. Strongly suggest reducing and synthesizing this section, with perhaps a bulleted list of hypotheses at the end. Or just synthesize and include (brief) hypothesis statements in the results section since these are already briefly reiterated there. There are likely other areas that could be trimmed, particularly in the intro/background.

-The authors engage in multiple testing but not correction of their p-value cut-off. Every hypothesis you add that uses the same underlying sample increases the odds of a false positive. Given this and the small sample size (and thus likely limited power), the authors should be more cautious about not over-interpreting ‘significant’ results. I am concerned about the applicability of their findings to humans more broadly. This is an interesting story about dental morphology in this group, but I am concerned the small sample size and inability to control for confounders may mean these results are not reproducible in other samples.

-The authors have a near-equal number of supported and unsupported hypotheses, and many of their p-values are near the 0.05 cut-off (and would not make a more stringent cut-off correcting for the effects of multiple testing). It is difficult to therefore parse the broader significance of this work. I’m particularly not convinced that the authors have demonstrated “the PCM explains patterns of accessory cusp variation in the entire sample” given that three of the four sub-hypotheses were not supported by the data.

-I would suggest recontextualizing this as a story about this particular group and its relation to the dentition of the local population. What are the frequencies of the cusp traits/crown morphologies studied in this population as a whole? (By population I’m referring to the regional or ethnic group(s) represented in Tezonteopan). Which group (supplemented or unsupplemented) more closely mirrors the broader population in this area of Mexico? Please provide context for whether the supplemented or unsupplemented samples (or neither) is most representative of the predominant cultural group(s) (not specified in the paper) in this region. Is there any other such data from this region? If not, how does it compare to frequencies/tooth sizes for other groups in Mexico and/or the broader global average?

-The authors appear to be discussing the permanent dentition but do not explicitly state this (they do use the capital letter nomenclature used to denote permanent dentition, but please note that this is not commonly used in clinical dentistry). Suggest the authors explicitly state focus on permanent dentition at least once in the beginning of the paper

-If supplementation during pregnancy and weaning was the key aspect of diet, why focus exclusively on the permanent dentition? The relatively lengthy duration of breast feeding (2 years) still does not cover the full period of crown formation for the M2s. Are there dental casts which capture the deciduous dentition?

- I know LEH in the anterior teeth does not correspond to timeline of M1 but what about evidence of LEH in the M2s? It could be informative to note whether those with more LEH were also those who had smaller anterior teeth or were more likely to have accessory cusps

-Similarly, the authors indicate that the un-supplemented group had markers of growth perturbance, but do those who had stunting of their statural growth, for example, have more dental anomalies/smaller teeth? This (and/or LEH data) would help bolster the manuscript’s argument in light of the small sample size and borderline p-values for many of the analyses

-I understand how challenging it is to come by data like this, but given the limitations of the sample, this work would seem to fall more in the realm of hypothesis generation rather than rigorous hypothesis testing. If the authors were to expand their discussion of dental traits in the broader local population (or other regional Mexican populations) they might be able to make interesting observations about what might be similar/different in the presence of nutritional supplementation. This could then serve as a jumping off point for discussing how these results may hint at broader human patterns and indicate areas for future work.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-23-12197R1Nutritional supplementation, tooth crown size, and trait expression in individuals from Tezonteopan, Mexico.PLOS ONE

Dear Dr. Blankenship-Sefczek,

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. There are still concerns over the methodological approach and interpretation of the presented work. Please address these concerns in a revision and a point by point response to these criticisms.

Please submit your revised manuscript by Feb 23 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,

JJ Cray Jr., Ph.D.

Academic Editor

PLOS ONE

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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: (No Response)

Reviewer #2: (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 #2: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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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

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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

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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 revised manuscript has adequately addressed the concerns raised during the previous peer-review process. With the revisions, I have a few minor suggestions to the authors:

Lines 106-117: This paragraph contains information identical to what is present in the Figure 1 legend. For brevity, I would recommend introducing Figure 1 in the preceding paragraph discussing the same publication by Ortiz et al. 2018 (Lines 91-105), and eliminating this paragraph.

Lines 267-300: The authors here reference Figure 2 depicting the measurements taken and Figure 3 visualizing the morphological traits scored, but at the bottom of the manuscript, the figures referenced are reversed. I wanted to bring this to the authors’ attention to make sure the appropriate images will be linked to the appropriate manuscript sections and figure legends.

General comment about Results section: As authors adopt the p-value scheme from Muff et al., it would be helpful to remind the reader about the p-value translation of terminology like “little or no evidence” or “weak evidence” throughout the Results section to facilitate the interpretability of results presented. For example, Line 323-324 would look as follows: “We found little evidence (0.1≤p) that M1…”. This would be a simple change, but it would really ease the reader’s ability to contextualize the strength of the evidence for the various hypotheses.

Lines 339-346: At the end of this paragraph, it would be helpful to explain the implications of the moderate effects uncovered by the sign test for the current study and Hypothesis #2 specifically.

Hypotheses 3 and 4 in the Result section: The way the ordinal logistic regression results are presented (by thresholds) in Tables 4 and 5 is very confusing. What is meant exactly by showing “strong evidence for elevated trait expression for the transition” between ASUDAS grades? It may be more helpful to calculate odds ratios and discuss how the odds of developing more pronounced morphological trait expression changes as a function of supplementation.

Lines 479-481: “With respect to PCM expectations, findings were unclear in the comparison of supplemented and non-supplemented groups”. What exactly is this sentence referring to? Was there any analysis that compared PCM expectations by supplement groups directly? Or is this statement in reference to Hypotheses #1-4?

Reviewer #2: The authors have made important changes to this draft of the manuscript, chief of which were reorganization changes that shortened the background and a change in p-value interpretation. While the utility of a binary or “sharp” p-value cutoff is certainly something being discussed in the literature, the authors’ chosen approach is not without pitfalls or important considerations. I strongly encourage the authors to read “The role of p-values in judging strength of evidence and realistic replication expectations” (Gibson 2021, Statistics in Biopharmaceutical Research) for a discussion of the importance of effect size on p-values. And then also “P-values don’t measure evidence” (Lavine 2024, Communications in Statistics – Theory and Methods) and “Why P values are not measures of evidence” (Lakens 2022, Trends in Ecology and Evolution).

Furthermore, Muff et al. (2022) clearly articulates that p-values treated as a continuous indicator of evidence must be accompanied by effective sizes and confidence intervals (“A minimal requirement is that we report effect sizes, CIs, and (if applicable) Bayes factors”). If the authors choose to stick with this approach, please provide CIs and effect sizes.

I also do not think that a continuous approach to p-values negates the need to consider the role of multiple testing. If the authors have evidence to the contrary, please cite. I appreciate the conundrum of an underpowered sample and the risk of Type II error, but the authors must weigh false positives (and therefore claiming a relationship that doesn’t exist) against simply stating that they do not currently have sufficient evidence to demonstrate a relationship. Including p-values as high as 0.10 as “weak evidence” would seem especially likely to lead to misleading conclusions.

In all, I would strongly caution the authors that application of Muff’s cutoffs might be an erroneous approach (see the Lakens letter). Please at least reference the debate and consider rewording statements such as “strong evidence”, “moderate evidence”, etc on the basis of p-value scores.

I appreciate the authors summarizing their results in Table 10, however, I am confused at some of the result summaries. For example, for Hypothesis 2, they state “small but consistent differences” yet only ICD6 for absolute and relative cusp distances shows p-values less than 0.05. For Hypothesis 3b, the authors state there is “moderate evidence of positive overall effect of non-supplementation on Carabelli cusp scores”, but only 3 of the 6 p-values are below 0.10 and only 1 of those three is below 0.05. I would suggest the authors review this table and be more parsimonious in their interpretations.

The abstract lacks a concluding sentence to summarize for the reader the overarching take-away from this investigation.

I am very enthusiastic about this dataset and appreciate what the authors have attempted to do in this study. However, I remain concerned that the associations are mostly weak and may not hold up to further scrutiny via replication study. If re-contextualizing this as a story about this specific population is not feasible due to lack of comparative data, then I would urge the authors to consider framing this as an exploratory analysis that is providing preliminary data to drive future studies.

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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 #2: No

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[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.

PONE-D-23-12197R2Nutritional supplementation, tooth crown size, and trait expression in individuals from Tezonteopan, Mexico.PLOS ONE

Dear Dr. Blankenship-Sefczek,

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.

There are just a few minor issues that need to be addressed in a revision.

Please submit your revised manuscript by May 10 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:

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We look forward to receiving your revised manuscript.

Kind regards,

JJ Cray Jr., 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.

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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: (No Response)

Reviewer #2: 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

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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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: Lines 42-43: This sentence is confusing. What is meant by “strong evidence of mesiodistal closer approximation”? Please consider rephrasing.

Lines 308-343: I recognize this paragraph was prepared in response to Reviewer 2’s comments, but it is incredibly hard to follow. The authors make the argument that the variables examined in this study are “expected to show developmental integration”, but some of the variation can “result from independent factors”. Isn’t the PCM, which authors rely on this paper, a framework which integrates all of these variables to explain final tooth form? I really struggle to follow the first half of this paragraph discussing effect sizes and intercusp ratios. In my opinion, the second half of the paragraphs is the more relevant discuss that directly addresses Reviewer 2’s comments. I would recommend either revising or eliminating the first half of this paragraph and concentrating on the p-value debate here.

Table 3 and Table 7: Since Tables 3 and 7 only provide statistics, it’s hard to understand how morphological trait expression changes between groups. It would be helpful to add mean ASUDAS scores for each group (supplemented/non-supplemented) and tooth (I1/I2/C/M1/M2), similar to how mean intercusp distances and crown sizes are provided in the other tables. This will help the reader see how mean trait expression changes as a function of supplementation in each tooth.

Lines 603-605: It may be worth discussing the crown size differences as it relates to morphological trait expression here (from Tables 8 and 9).

General comment: The authors indicate that the data utilized in this study are available without restriction, but the raw data does not appear to be attached to the supplement and if it’s housed at an external repository, this isn’t indicated or linked. To comply with PLoS One’s data policy, authors should address this.

Reviewer #2: My thanks again to the authors for this thorough revision. The inclusion of odds ratios, CIs and effect sizes has helped greatly in allowing the reader to evaluate the strength of evidence. I now believe this manuscript is ready for publication.

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Reviewer #1: No

Reviewer #2: No

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Nutritional supplementation, tooth crown size, and trait expression in individuals from Tezonteopan, Mexico.

PONE-D-23-12197R3

Dear Dr. Blankenship-Sefczek,

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,

JJ Cray Jr., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: