PONE-D-24-05878Cryptic diploid lineage of Betula ermanii at its southern boundary populations in JapanPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

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

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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

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

Reviewer #1: 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: The research manuscript entitled "Cryptic Diploid Lineages of Betula at its Southern Boundary Populations in Japan" delves into Betula species' previously undiscovered terrain, exploring diploid lineages at the species' southern limits in Japan. The work illuminates Betula populations' hidden diversity and sheds light on this ecologically important genus' evolutionary history.

However, the current work lacks thoroughness and significance in this research due to some flaws. The validity and trustworthiness of the results are questioned due to insufficient sample size. The paper's results are presented in a confusing way, and the interpretations of these results are strongly conjectural. Some of these are mentioned here:

Line 78: Authors have discussed Betula species, but not clear which species authors are talking about.

Line 80: How same species as been named differently in different regions? What is the acceptable botanical name of the species?

Line 87: Authors are talking about which species?

Line 285: Authors stated that B. costata is diploid progenitor of B. ermanii. But, in next lines 286-87, authors are confirming that both are same species. And later on, authors also mentioning inadequate study related to this.

Further, Line 295-297: Authors agreed to the morphological similarity of their diploid specimen to the B. shikokiana, which is confusing, weather both are similar species or different.

Finally, the results are not clear enough to draw any firm conclusions from the study, which casts doubt on the writers' expertise in the field. The data presented in the introduction and discussions appears to be comparable and requires correction.

To summary, this study has had a greater impact on our understanding of cryptic diploid lineages in Betula populations, but manuscript needed to revise with prudence until these core concerns are resolved.

Reviewer #2: Abstract

The abstract provides a concise summary of the study, highlighting the focus on Betula ermanii populations in southern Japan and their morphological and ploidy variations. It effectively outlines the methodology and key findings, setting clear expectations for readers.

Introduction

The introduction provides a thorough background on polyploidy and its role in species diversification, effectively setting the stage for the study. It discusses the relevance of polyploidy to Betula species and outlines the uncertainties in Betula phylogenetics due to cryptic lineages. However, the introduction could be strengthened by providing more context on why Betula ermanii was chosen as the focus of the study and why investigating ploidy levels in this species is important

Materials and Methods

The section on study area and sampling is comprehensive, providing detailed information about the geographic locations of the study populations and the sampling methods employed. The use of common garden populations for comparison enhances the robustness of the study design.

The description of the estimation of ploidy level using flow cytometry is clear and replicable. However, it would be helpful to include more information about the specific protocols followed for DNA extraction and flow cytometry analysis to ensure transparency and reproducibility.

The morphological comparison methodology is well-described, particularly the use of leaf and seed traits for analysis. The inclusion of images (Fig. 2 and Fig. 3) enhances the clarity of the methods section.

Results

The results section presents the findings in a logical sequence, starting with the ploidy level estimation followed by morphological comparisons of leaves and seeds. The use of figures (Fig. 4, Fig. 5, and Fig. 6) aids in visualizing the data and understanding the patterns observed.

The identification of diploid and tetraploid populations based on ploidy level estimation is a significant finding, demonstrating variation within Betula ermanii populations. The morphological comparisons reveal distinct differences between diploid and tetraploid populations, particularly in leaf and seed traits.

Discussion

The discussion section effectively synthesizes the study's findings, contextualizes them within existing research, and offers valuable insights into the genetic and morphological diversity of Betula ermanii populations. With some enhancements to depth and integration, the discussion could further elucidate the ecological and evolutionary significance of the observed patterns and provide more concrete recommendations for future research and conservation efforts.

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Reviewer #1: No

Reviewer #2: Yes: Saleem Wani

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Cryptic diploid lineage of Betula ermanii at its southern boundary populations in Japan

PONE-D-24-05878R1

Dear Dr. Tsumura,

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.

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Kind regards,

Vikas Sharma, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have revised the manuscript as per suggestions of the reviewers. The manuscript can be accepted now.

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 #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 #2: Yes

**********

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

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 #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 #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 #2: the manuscript shows promise in contributing to the understanding of Betula ermanii populations in Japan. By addressing the mentioned points and refining the presentation of the research findings, the study can make a valuable addition to the scientific literature on plant genetics and evolutionary biology.

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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 #2: Yes: Mohd Saleem Wani

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