PONE-D-21-04061

A 3D basicranial shape-based assessment of local and continental northwest European ancestry among 5th to 9th century CE Anglo-Saxons

PLOS ONE

Dear Dr. Plomp,

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.

All reviewers found this work to be of great interest. However, there was a consistent concern over statistical approach and interpretation of those results. There are other minor issues to be addressed as well.

Please submit your revised manuscript by May 08 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

JJ Cray Jr., 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 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 archaeology research, see https://journals.plos.org/plosone/s/submission-guidelines#loc-paleontology-and-archaeology-research.

3. Please include a copy of Table 1 which you refer to in your methods section.

4. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Partly

**********

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: 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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

5. Review Comments to the Author

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

Reviewer #1: The manuscript by Plomp and colleagues explores the morphological affinities of Anglo-Saxon skeletal collections, with the goal of contributing to the discussion about the impact of continental populations in the formation of Anglo-Saxon biological diversity. The question presented is of interest and relevance, given that it has been discussed by many authors from different disciplines. As such it could be of relevance for an inter-disciplinary audience and therefore could be suited for PLoS One.

However, there are a series of aspects of the methods adopted that should be clarified or revised before I can recommend the manuscript for publication. I list below my questions and comments, which I hope will help the authors to revise the manuscript.

1. My main concern with the current version of the manuscript relates to the use and interpretation of the results from the classification of the individuals based on the LDA, as I believe the authors are not properly taking into account the inherent limitations of using modern human morphology to infer ancestry.

a. The first aspect of these limitations to consider is that, even for the anatomical regions that best discriminate modern human populations, there is considerable overlap among them. Because of that, it would be important to either demonstrate that the source populations can be successfully and reliably discriminated and/or take into account the uncertainty in the classification per se. The authors demonstrate through MANOVAs that the source populations are different, but this is based on PCs and not on the CVAs. So, my first suggestion would be to test how often the Danish individuals classify as pre-Medieval English, and vice-versa. If only a few of the individuals from each source population classify incorrectly, this supports the notion that these populations have morphological differences that allow for reliable discrimination. If that is not case, however (which would be expected for modern human populations), the results of the classification of the earlier Anglo-Saxons must be presented more cautiously (see my next point).

b. The second approach to consider is to incorporate better the uncertainty of the classification results. Because the classification is usually done based on the smallest distance to the centroids of the reference populations, in situations where there is a lot of overlap in the variance of the series, individuals who belong to a population can be frequently misclassified. It is not clear in M&M (see my next point) exactly how the classification was done, but it is important that if it is based on posterior probabilities or typicalities that the authors consider the probabilities of belonging to each source population. Having a higher probability of belonging to group A does not necessarily reject the hypothesis that it could belong to group B (which is ultimately the hypothesis being tested in this study). If the source populations are not that distinct, which is a possibility here, it is particularly important to be more explicit about how the authors are considering false-positive affiliations.

2. The Material and Methods section should be more explicit about how the analyses were conducted. In particular for the classification, it is hard to understand how it was done. For example, it is not clear to me what the authors mean by: “We repeated this process 100 times for each Anglo-Saxon individual and then calculated the average attribution percentages.” Why was the analysis repeated a 100 times? Was the source data permutated somehow? Why not use more traditional classification analyses and cross-validation through posterior probabilities? I am not saying the authors should change the method, just that I do not understand what the method is. Therefore, more justification is required here.

3. Similarly, I do not understand why the authors chose 55% as the cut value of membership to each population. It seems unreasonable that this is the cut-value, especially since the source populations may be very similar to start with. This value must be justified better, but I strongly recommend the authors adopt a more conservative value here to avoid false positives.

4. While it is a nice visual exploratory analysis, I do not see how the CVA plots are informing the conclusions of the manuscript in any meaningful way. I suggest the authors try to integrate the patterns observed from the CVA plots more explicit in their interpretations.

5. It would be helpful if the MANOVAS results between pairs of series were presented in a table, to facilitate the visualization of the results.

6. Finally, I think the discussion is also not giving sufficient attention to the limitations of the morphological analyses. I agree with the authors that isotopic analyses are limited and that their results can be biased. But it must also be considered that the reason of the discrepancy may be due to the limitations of morphological analyses. At least, it would be important for the authors to argue explicitly why we should consider their results more reliable than other sources of information. Once again, I don’t say this because I think morphological data is not reliable, which it is, but it is important to address its limitations more explicitly, especially when writing for a journal that is targeting a broader inter-disciplinary audience.

Reviewer #2: This manuscript provides important data on the population history of Anglo-Saxon British populations and demonstrates that Early Medieval Britain was heterogeneous in terms of genetic ancestry. The authors use well accepted geometric morphometrics and linear discriminant function analyses (LDA) to analyze individuals from Anglo-Saxon, pre-Medieval, and Iron Age/Viking sites. In so doing, they demonstrate that Early Medieval populations were comprised of both local and non-local (from mainland Europe) groups, which both contradicts and supports previous research that involves historical, stable isotope, and genetic data. Additionally, the population structures change through time. Overall, the manuscript is well written and distills the information down in a very understandable way while contributing to an important dialog around the peopling of Britain.

The comments and suggestions are primarily minor in nature and are documented in the attached PDF. I'll briefly highlight the main ones here:

The authors indicate that their sample size is 237; however, I came up with 236.

If possible, I'd like to see some more discussion regarding the sex estimations of the individuals in their sample, since this is a key foundation of their study. The authors indicate that they based the sex estimations on the pelvis and skull. It does not look like there were any indeterminate/ambiguous skeletons, which is usually rare. Were all individuals placed into female or male categories based on the pelvis and skull, or was the skull or pelvis used? What if the skull and pelvis "disagreed"?

A paragraph in the Discussion that addresses the results when pooling females and males reads, to me, more like it belongs in the Results. I suggest it be moved to Results and a brief statement added in the Methods.

I do not see Table 1 that is referenced in the narrative (only Table 2 and Table S1).

I am curious whether the LDAs were cross-validated. Cross-validation can ensure that the individual under analysis was not included in the method development and is a more accurate reflection of an unknown individual.

The last sentence of the Abstract and Conclusion state that "identity in Early Medieval Britain was similar to what it is today--inclusive and dynamic." While this may be true, this study does not examine social identity of past or present peoples (nor the bioarchaeological literature on identity). Ultimately, we do not know how people may have identified (or identified others) and whether such social identities map onto the genetic/ancestral data. I concur that the population was heterogeneous and dynamic in terms of ancestry, but not necessarily in identity. Also, heterogeneity doesn't necessarily equate to "inclusivity." It may be that there were invasions or forced migrations, which arguable isn't inclusion (making space for others). It may also be argued that modern Britain, like anywhere, isn't inclusive. I suggest limiting this conclusion to the heterogeneous and dynamic nature of the ancestries/genetics and avoiding reference to identity altogether.

I look forward to seeing this revised and published!

Reviewer #3: This paper provides a novel approach to address longstanding questions regarding human migration patterns. I believe it should be published with minor revisions, as discussed below:

In the Materials and Methods section, it is stated that the sample is summarized in Table 1, but there is no Table 1 included in the text. This table should be added to clarify the demographics of the sample.

It would be helpful to include date ranges for the Danish sites, as is done with the other sites.

Each anatomical landmark that was collected should be named and/or described, either within the text, in a new table, or as part of Figure 2.

In Figures 3 and 4, it would be helpful to include the percentage of variance explained along each axis in each of the axis labels.

Would it be possible to describe the variance found along the different CV axes in anatomical terms? What anatomical patterns are described along each axis?

The second paragraph of the Discussion section is repetitive, using the phrase “our decision to keep the female and male specimens separate did not skew our results” twice. This should be reworded to avoid this repetition.

I appreciate the authors’ thoughtful comparison of their results to previously published historical, isotope, and genetic evidence.

In the seventh paragraph of the Discussion section, there is an inappropriate use of a semicolon, “The historical texts do not mention one; nor do the isotope and DNA studies.” This semicolon should be replaced with a comma.

It is assumed in the study that variations between the Early and Middle Anglo-Saxon samples are caused by differing patterns of ancestry, but is it possible that there are other factors influencing these differences? Does basicranial morphology change over time in the Danish or pre-Medieval samples?

In the Supplementary Document, there are two tables both labeled as Supplementary Table 1. Change the title of one of these tables to Supplementary Table 2. Also, the titles of these tables should be changed to “female” and “male” rather than “females” and “males” since the term “each” is used.

Reviewer #4: 1. Summary of the research and your overall impression

The authors have designed a novel study to identify the ancestral origins of Anglo-Saxon peoples in Medieval Europe and trace the path of migration across time. Previous research has used historical, archeological, or genetic evidence to track the migration of mainland European peoples to the British Isles. However, these data are incongruent and tell conflicting stories. The current study importantly adds a new line of evidence to paint a clearer picture of this very complex human phenomenon to see how mainland European and local populations interacted with one another. The authors employed cutting-edge technology to acquire biological data from the skeletons of historic archeological populations. Then, geometric morphometric techniques were used to analyze the data. Specifically, three-dimensional coordinate landmark data were abstracted from digital photogrammetric reconstructions of a relatively large sample of skulls. Using statistical methods borrowed from paleontology, the basicranial shape of three major groups separated by time and space were compared to identify morphological similarities; thus, a proxy for migration and ancestry is given. The authors found that Early Anglo-Saxons were mostly derived from continental Europe populations whereas Middle Anglo-Saxons were mostly derived from local populations. The proposals for future research give readers possibilities to explore this topic further and enhance the accuracy of the data.

The manuscript is easy to follow and sensibly organized. The introduction proceeds from general to specific ideas and flows into the methodology. The results are clearly presented and are supported by the data. The conclusions can be traced back through the results and answer the original question. The primary strength is the relatively large sample size of 237 individuals. Studies involving skeletal collections are typically abhorrently small due to access or preservation issues. This sample size allows for the authors to make more generalizable inferences about ancestral origins. Additionally, the use of landmark morphometric techniques to collect and analyze data, in conjunction with contextual evidence, is relatively novel for bioarcheological studies such as this. The authors do well at plainly explaining their methodology without getting caught up with the theoretical and mathematical underpinnings. However, there are a couple details that were left out of the methodology to make the study reproducible.

I recommend that this study be accepted for publication after minor revisions.

2. Evidence and examples

Major Issues

1. No major issues noted.

Minor Issues

1. Table 1 referenced in the first line of the Material and Methods section is absent in the manuscript. If a table is not provided showing sub-sample sizes, including the number of males and females for each group, along with totals, a description of the sample breakdown should be given in the text.

2. A list of the 34 landmarks used is not given. Page 6, last paragraph refers to landmarks used by Havarti and Weaver (2006) and a figure is referenced in the current study. Once could infer the names of said landmarks, but without labels the reader does not know precisely which landmarks were used. Thus, the study could not be repeated. Include a table listing these landmarks and label the points in Figure 2 with numbers cross-referenced to the new table. A brief summary of how many landmarks are midline and how many are bilateral would be a helpful, as commonly seen in similar studies of bilaterally symmetric structures.

3. The observer error sub-study described on page 7 first full paragraph does not indicate whether there was a single or multiple observers. I inferred a single observer and was able to confirm this in the “Authors’ Contributions” section. A mention of a single observer in the text would mitigate expectations for inter-observer error test results.

4. The computer program MorphoJ is incorrectly referenced. Dr. Klingenberg’s lab website (https://morphometrics.uk/MorphoJ_page.html) indicates the preferred citation to be Klingenberg, C. P. 2011. MorphoJ: an integrated software package for geometric morphometrics. Molecular Ecology Resources 11: 353-357.

5. The authors use an uncommon or possibly incorrect symbol for Wilk’s lambda used in reporting MANOVA test results on page 12. Wilk’s lambda is usually abbreviated with the upper-case lambda symbol (Λ) or less commonly a lower-case lambda (λ). The barred lambda given in the manuscript may be a symbol formatting error.

6. The presentation of results is very clear and easy to follow. The authors do not include the effect size for MANOVA results on page 7. Including the partial eta-squared value will help assess the magnitude of the difference in basicranial shape between the groups. Following Cohen J (1992). A power primer. Psych Bull, 112, 155-159, once can interpret partial eta-squared effect sizes as 0.01 = small, 0.06 = medium, and 0.14 = large. A p-value can only tell you if there is a difference or not, but the effect size indicates how large or small that difference is. This may help to tease out subtle differences between groups as statistically significant, but practically insignificant.

7. The two other possibilities for future results on page 19 are well considered. For the first suggestion, the authors should also contemplate including an outgroup in future work to act as a control. Discriminant analysis creates a function that maximizes the differences between groups, no matter how small or practically insignificant they may be. The incorporation of another population removed by geography and/or time can help show that the methodology used is working as it should.

8. Figures 3 and 4 are well laid out but have axis titles and labels that are difficult to read. The numbers are generally referenced in the text, but the figures are difficult to interpret. Also, the captions for Figures 3 and 4 do not give titles for a or b. ‘a’ is CV1 versus CV2 and ‘b’ is CV2 versus CV3.

9. The authors interchangeably use the terms “individuals” in place of “specimens” when referring to once living human beings. The connotation of “individual” maintains the humanity of these persons while they provide us with information about our past. Using these terms will help overcome anthropology’s contentious history of digging up the dead without permission.

Reviewer #5: The paper investigates craniometric variation of pre-medieval and medieval populations from the British Isles and Denmark in an effort to further reconstruct population history of the "Anglo-Saxons". It focuses on the basicranium as proxy for relatedness, from which it infers ancestry. Based on multivariate statistics (Manova, LDA) the authors conclude that the continental ancestry of Anglo-Saxons decreased from the early to a later phase.

The paper is generally well written, the methods are those that are commonly used when dealing with morphometric variation, the samples are not huge, but probably sufficient to address the general question the study is aimed at. Some methodological aspects require clarification and revision prior to publication.

Major

The first CVA computed on the Procrustes residuals of males and females separately probably does not yield reliable results. By my account the number of variables (p*d=102) is 3times larger than any of the groups, which is not in line with general recommendations (e.g. Bookstein & Mitteroecker, 2011).

The summary of the Manova is confusing: it reports significant differences for pairwise-comparisons and a different value for Wilks' lambda for each of these comparisons. Normally, one would expect one Wilks' lambda for the female Manova and one for the male, so it is not clear what is being reported here and how p-values for pairwise comparisons were obtained.

The key results are obtained by LDA, to classify the Anglo-Saxon samples as either pre-Medieval British or as Danish. The authors find a shift from high to low Danish classification rates between early and Middle Anglo-Saxons. I am a bit skeptical about the female result (only 13 Middle_Anglo-Saxons): It is consistent with the larger male sample and suggests that continental ancestry shifted similarly in males and females. The authors repeated the LDA on a pooled sample to ascertain that the results from the sex-specific analyses are not biased. However, the pooled middle Anglo-Saxon sample remains predominantly male, so some uncertainty remains, as does the possibility of differential ancestry in males and females. Considering that some genetic data suggest high male continental ancestry, it would be worth pursuing this question, though I reckon this would require additional samples from Middle Anglo-Saxon females.

Minor

Table 1 lists only the Anglo-Saxon samples, omitting info for sex of the pre-Medieval and Danish samples. It provides results of the LDA individually, which is ok for a supplementary document. I would suggest including a more synthetic version (e.g. sample size by sex/chronology/geography) in the text, since this information is important for assessing the scope of the study.

**********

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

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

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

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: 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: PONE-D-21-04061_reviewer (peer review).pdf

PONE-D-21-04061R1

A 3D basicranial shape-based assessment of local and continental northwest European ancestry among 5th to 9th century CE Anglo-Saxons

PLOS ONE

Dear Dr. Plomp,

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 several minor comments that should be addressed before final acceptance thus I am sending it back to you as a minor revision. Please address the reviewer comments. Note only an editorial review will be conducted upon resubmission.

Please submit your revised manuscript by Jun 19 2021 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: http://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

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.

[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 #2: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

Reviewer #5: (No Response)

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: 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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: 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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: 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 appreciate the effort the authors made in clarifying the methodological questions I had, as well as the addition of the new analyses. They have successfully addressed all my concerns, and this article will be a meaningful addition to the literature. My only remaining suggestions is to move the discussion of the supplementary analyses to the Results section, instead of the Discussion. I know that one of the other reviewers asked for this and the authors decided not to make these changes, but I'll echo that reviewer's opinion that it does not seems to fit well in the discussion, as it is not truly contributing to the contextualization of the data analyzed.

Reviewer #2: The authors sufficiently incorporated the more substantial issues brought up in the peer review; however, I am unsure if they saw or responded to the PDF'd document I originally uploaded with the first review. I don't see responses to some of the points brought up therein. In particular, I suggest changing "colonization" in the keywords to "ancestry" since the paper doesn't actually discuss colonization, but instead ancestry and population heterogeneity (ln 31); change "men" to "males" since we can't comment on gender as skeletal biologists, and the two terms shouldn't be conflated (ln 62); change "determined" to "estimated" when discussing sex since as skeletal biologists, we cannot determine the sex of someone--we can only estimate their assigned sex (ln 90). There are other minor comments in the PDF that can help with phrasing and clarification.

Overall, the manuscript is improved.

Reviewer #3: Thank you for addressing my concerns and those of the other reviewers.

I have noticed one remaining typo - In line 167, the word "difference" should be used rather than "different."

Reviewer #4: (No Response)

Reviewer #5: The authors have addressed key concerns, thereby clarifying the study design and results, with one exception.

My original comment:

"The authors repeated the LDA on a pooled sample to ascertain that the results from the sex-specific analyses are not biased. However, the pooled middle Anglo-Saxon sample remains predominantly male, so some uncertainty remains, as does the possibility of differential ancestry in males and females. Considering that some genetic data suggest high male continental ancestry, it would be worth pursuing this question, though I reckon this would require additional samples from Middle Anglo-Saxon females."

The authors' response:

"We agree that further research with a larger sample size would be beneficial, especially if combined with aDNA and isotope data. We hope to pursue this further in the coming years. As for the current analyses, the CVP PCs were calculated based on balanced samples, which should minimize any error due to sample size differences. This has now been added to Line 159"

This is not obvious to me: As clearly summarized in the discussion, the pooled LDA was carried out on the entire sample, not on a sex-balanced subsample, so my point remains unaddressed, or something needs to be clarified here. Note the acronym CVP is nowhere defined, I can only guess it refers to the pooled LDA. The reference to Line 159 seems erroneous.

In light of this, I still think that subsample sizes for female Danish and Middle Anglo-Saxons and male early Anglo-Saxons are small relative to the number of variables (PCs retained) and induce uncertainty for the classification results. I am not saying this uncertainty invalidates the interpretation, but it should be acknowledged in the discussion.

**********

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

Reviewer #3: No

Reviewer #4: No

Reviewer #5: 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.

A 3D basicranial shape-based assessment of local and continental northwest European ancestry among 5th to 9th century CE Anglo-Saxons

PONE-D-21-04061R2

Dear Dr. Plomp,

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,

JJ Cray Jr., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: