PONE-D-20-31468

Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region

PLOS ONE

Dear Dr Nguyen, 

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. We received the position of the three reviewers and you can see that the opinions are a little dissonant. Anyway, all the reviewers present very relevant considerations and so I chose to indicate a "Major Review". 

Please submit your revised manuscript by 02/20/2021. 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,

Sergio N. Stampar, Dr.

Academic Editor

PLOS ONE

Additional Editor Comments:

Dear Dr Nguyen,

We received the position of the three reviewers and you can see that the opinions are a little dissonant. Anyway, all the reviewers present very relevant considerations and so I chose to indicate a "Major Review". Please address any doubts and issues raised by the reviewers and I look forward to a new version of the manuscript.

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 Methods section, please provide additional location information of the sampling sites , including geographic coordinates for the data set if available.

3. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the sampling sites  access and, if no permits were required, a brief statement explaining why.

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

"This work was supported by the National Foundation for Science & Technology

Development, Viet Nam, grant code 106.02-2018.313."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"No, the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript"

Additionally, because some of your funding information pertains to [commercial funding//patents], we ask you to provide an updated Competing Interests statement, declaring all sources of commercial funding.

In your Competing Interests statement, please confirm that your commercial funding does not alter your adherence to PLOS ONE Editorial policies and criteria by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” as detailed online in our guide for authors  http://journals.plos.org/plosone/s/competing-interests.  If this statement is not true and your adherence to PLOS policies on sharing data and materials is altered, please explain how.

Please include the updated Competing Interests Statement and Funding Statement in your cover letter. We will change the online submission form on your behalf.

5. We note that Figures 1 and 4 in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

(1) You may seek permission from the original copyright holder of Figures 1 and 4 to publish the content specifically under the CC BY 4.0 license. 

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

(2) If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

6. Please include a caption for figures 5 and 6.

7. 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: Yes

Reviewer #2: Partly

Reviewer #3: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: I Don't Know

**********

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

**********

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

**********

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: Ms. Number. : PONE-D-20-31468

Title : Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region

PLOS ONE

General comments:

The article is very good and very interesting. This article discusses the species diversity of genus Halophila and tries to reveals a high genetic diversity of Halophila major in the Wallacea region using rDNA analysis. This is a good new insight because H. major still has big challenge, especially in Wallacea region. In order to provide a broad impact and strong research and data linkages, it would be better if it could involve cross-country researchers from regional areas, and could be used as a reference for the distribution of Halophila seagrass, especially H. major in ASEAN; I think in Indonesia, this species has just been rediscovered, and I believe it still has a wider range of life, and has a high genetic diversity, not only in the Wallacea region, as you mention in manuscript.

Also, there are several scientific names of species that are not italicized and according to the standard of writing, and should only be the scientific name of the species in italics. And then, there is a lot of writing that needs to be improved, especially related to continuity between sentences and between paragraphs, as well as table and unit. The units should use symbols that have been defined internationally, and are consistent.

Specific comments:

Abstract:

- Clear.

- “The morphological characters show variation, but based on the results of the ITS marker they fom a homogenous population.” Typo ‘fom’?

Introduction:

- Page 3: …“The Halophila section including H. ovalis, H. decipiens Ostenfeld, H. gaudichudii J. Kuo, H. major (Zoll.) Miquel, H. minor (Zollinger) den Hartog, H. nipponica J. Kuo, H. okinawensis J. Kuo and H. stipulacea (Forssk.) Ascherson is known to present as one the most complex challenges plant taxonomy [4,5].” … When biological species that is the first time mentioned in the text, it should attach the authority name and year.

- Page 4: Syringodium isoetifolium, Enhalus acoroides similar comment above.

Material and Methods:

- Page 6: Phylogenetic analysis section….”, including three sequences obtained in this study and 73 sequences of known Halophila sections retrieved from GenBank..”. Total 76 but in Result All sequences were 78.

Results

- Page 7: “Morphological observation and phylogenetic analysis of Halophila major in Vietnamese waters”. Suggestion to reduce, “Halophila major in Vietnamese waters.”

- Page 8: “A total of 78 ITS sequences (including three new sequences from the present study) of H. major collected in five geographic areas: Sunda Shelf (I), Wallacea (II), Sahul Shelf (III), Bay of Bengal (IV) and coast of Japan (V)”.Total sequences are 78 or 76?; this sentence prefer in phylogenetic analysis in method.

- Page 9: in Table 3, please consistent, areas or region is used based on the context.

- Page 10: First paragraph “Area” similar comment above.

Discussion

- Page 11: The number of branching veins for H. ovalis 3-4 from Indonesia samples. Generally, branching cross veins of H. ovalis is 3-4 pairs. How about the branching cross veins of H. ovalis in Vietnam?

- Page 11: “….whereas samples collected in Malaysia, Indonesia and Thailand tend to form a single clade”. Insert Fig. 3 and Why it placed in single clade, How it related with this finding?

- Page 12: Typo. “…..in regions II (Sunda Shelf) and III (Wallacea)”. it means Sunda Shelf (region I) and Wallacea (II)

Figure

- Improve the quality of all Figure (should be min. 350 DPI) follow the journal requirement.

- Figure 1. Suggestion: prefer polygon with different color or pattern to differentiate the region.

- Figure 2. Please insert the scale like 1 cm or 1 mm.

Recommendation: A major revision is required to improve the manuscript.

Reviewer #2: The manuscript entitled "Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea

region" by Nguyen et al. Using only "15" newly collected H. major plants from Viet Nam with other published ITS from GenBank to reveal the phylogentic relationship with other species in the genus Halophila as well the phylogeography of H. major. There are several flaws which are fatal to lead this rejection. First of all, the tile is not falling into their main conclusions(findings), because through out the manuscript, they only discussed about the high genetic diversity in Wallacea with only few sentences in page 12. Second, they seem to be misunderstood the the definition of the term "monophyletic", based on their phylogenetic tree, they mentioned there are 8 subclades in H. major and form nonmonophyletic groups. However, since the sub clades the authors defined in this studied are not well supported at the tips. They is "no" subclade in H. major, and sequences of H. major around the world formed" a "monophyletic clade with well support (supporting values :98/1). This is also supported by the TCS haplotype network which showed no sign of population partition. Third, they use same symbol to code geographic regions as well as 8 clades (I, II, III.....), which is really confusing while reading. Lastly, the resolution of the figures are generally low especially Figure 6.

There are some minor comments as following

Page 5, spell out "DESS"

Figure 1 caption: "where samples were collected change" to "where sequences were obtained from GenBank"

Page 7, "World type" "Red Sea type", you need to explain what are they or simply state previously been defined as "World type"and "Red Sea type" by someone....

There are many sentences developed from the erroneous statement "8 subclades", so the authors may need to reframe the manuscript based on the correct interpretations.

Reviewer #3: The authors analysed rDNA of three H. major collections from Vietnamese islands and similar molecular data from other SE Asia stored in geneBank to demonstrate the origin of this species was located in the Wallacea region. They further estimated the relative divergence times of certain taxa within the section Halophila and also that in section Halophila and section Microhalophila.

Comments:

a. The species Halophila major (H. euphlebia) was ‘re-established’ in 2006, therefore, using the prior to 2006 data from the geneBank to treat as “H. major” could be questionable. For example, the Halophila specimens from Shoalwater Bay, Australia have distinct linear leaf blades with L/W ratio 7-10; cross veins 8-12 (-14), unbranched…Based on the above morphological description, the specimens may not belong to H. major.

b. Abstract stated: “H major was misidentified as H. ovalis in Viet Nam”. However, this statement did not appear in other sections of this manuscript nor any reference was given.

c. Introduction: References [4, 5] did not use section Halophila. Furthermore, H. australis, H. capricorni, and H. sulawesii also include in this section. However, the latest Halophila taxonomy review shows that section Halophila does not include these three species and also not H. stipulacea and H. decipiens (see Kuo 2020).

d. Fig. 3 H. major has eight subclasses. Those from Japan belong to Class III and VIII; Australia belongs to III and VI; while Thailand had IV and VII. Why two different subclasses in the same country (Japan, Australia, Thailand)? Any morphological or environmental difference of the specimens in two different subclasses from the same country?

e. References: should list: Liu et al. (2020) Genetic identification and hybridizationin the seagrass genus Halophila (Hydrocharitaceae) in Sri Lanka waters. PeerJ 2020, 8, e10027

f. Authors estimated relative divergence of H. major and H. ovalis were 6.64 Mya, that was much older than Kim et al.’s estimation of 3.4 Mya. The authors of this manuscript explained these differences were due to ‘technical aspect’ and the length of sequences in the dataset used. They should explain what is ‘technical aspect’.

g. Authors estimated the divergence between section Halophila and section Microhalophila was 32.18 Mya, this estimation implies that the divergence of the genus Halophila would be much earlier than that estimated divergence of genus Halophila in the family Hydrocharitaceae was only 19.41 Mya by Chen et al. The authors of this manuscript should discuss these vast differences.

**********

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

[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-20-31468R1

Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region

PLOS ONE

Dear Dr. Nguyen

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.

==============================

Thank you very much for the new version of the manuscript. Note that one of the reviewers still expects a few changes before his manuscript is accepted for publication. Please make these adjustments so that I can proceed with the revision of the manuscript.

==============================

Please submit your revised manuscript by 04/30/2021. 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,

Sergio N. Stampar, Dr.

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.

Additional Editor Comments (if provided):

Dear authors,

Thank you very much for the new version of the manuscript. Note that one of the reviewers still expects a few changes before his manuscript is accepted for publication. Please make these adjustments so that I can proceed with the revision of the manuscript.

Kind regards

Sergio

[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)

**********

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

**********

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: In the discussion section, suggestions should be made regarding the use of fresh specimens and adding locations, and things that become input and challenges in the future, including collaborative research in order to really get more complete data to better find out its genetic diversity.

Also, there is something that needs to be ascertained from the phylogenetic tree, is H. major in Australia really any specimens based on morphological evidence? If there is, it may be stated that the source of the citation is that H. major is found in Australia in Materials and methods.

Reviewer #2: Although authors have replied most of my comments, there is one exception which related to the symbol (I, II, III etc.) they used in the Figure 1 and in the phylogenetic tree to represent geographic areas and "groups", respectively.

They are several things that I concern while reviewing this revision. Here I list them point by point as follows.

Line 75-76: Delete "In addition, the study....on the IST marker."

Line 88-89: Unclear, had to rephrase.

Line 106-108: This sentence is odd, need to rewrite to clarify what you were trying to say.

Line 109-110:How the genetic partition occur "within" and "among" barrier ? The authors may need to clarify this or simply make this sentence more straight forward.

Line 129: Use "sequences of other regions" instead of "other regions sequences"

Line 140: Use "the" instead of "this".

Line 243-244:I would like the authors to provide the supporting values of each groups they defined. Because technically, if those groups are not support by supporting values they should not be defined as different group. I know the authors already used group instead of clade. One thing the authors can do is be honest with what they found (groups are not well-supported) and add up the information that the grouping of those haplotypes might affiliate with geographic location based on the haplotype network instead of define them as groups without substantial evidence.

Line 328-329:The authors should explain what is the "similar way" they mentioned here? Because the original sentence is not very clear. Additionally, in the tree generated by Waycott et al. 2002, those groups found in H. ovalis samples were not well-supported, therefore, they also over-interpreted their data almost 20 years ago. And I truly believe we should not follow the conclusion which is not correct.

Line 382-383:You only have the morphological data from Viet Nam, therefore, I don't think you can make this conclusion while you mentioned a broad area in previous sentence.

I strongly recommend the authors send their next revision for English editing before submitting back to PloS One, because I did not correct them across the manuscript. I am not a native speaker too, and I always send my manuscript for English editing before submitting, just because it actually help the audiences to read and receive the information that we are trying to deliver.

**********

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: Fery Kurniawan

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.

PONE-D-20-31468R2

Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region

PLOS ONE

Dear Dr. Nguyen,

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 below.

The revised manuscript is well written and reviewer 1 has indicated that their prior comments have all been met. As the stand-in Subject Editor for the manuscript, I appreciate the authors’ and prior reviewers’ efforts to ensure the accuracy and clarity of the manuscript. However, I feel there are several issues that need to be addressed prior to publication.

Principally, results as presented do not clearly support the primary conclusion of significant genetic isolation of Halophila major within the central Indo-Pacific (lines 42-43 and elsewhere). I base this specifically on the lack of phylogenetic support for putative H. major clades and the non-significant PhiCT values presented in the AMOVA table (table 4).

The lack of support for phylogenetic divergence within H. major is acknowledged in the text (eg. lines 381-383) yet elsewhere support for the putative groupings is implied (eg. lines 38-39). At a minimum, this confuses the discussion and should be clarified.  

As described in Excoffier et al. 2010, AMOVA allows the hierarchical partitioning of genetic variation among populations (phiSC), among regions (phiCT), and among individuals within populations (phiST). As presented, table 4 indicates a non-significant phiCT. This may simply be a typo, as suggested by the lack of haplotype sharing among sites (table 2). If, however, this is not a typo, I feel the primary conclusions of the manuscript are unsupported and significant revisions would be needed before publication.

I am therefore recommending major revisions with the caveat that my concerns about project findings may be addressed if AMOVA results in table 4 are not correct as presented.

Below I describe additional recommendations and concerns that should be addressed.

Lines 38-39: Misidentification of Viet Nam H. major was described in a prior study and should not be highlighted here as a new finding.

Line 40: It is unclear who “they” is referencing. Viet Nam samples only?

Lines 42-43: If the AMOVA table is correct as presented this conclusion will need to be revised

Line 52: I’m unfamiliar with using “sections” to describe phylogenetic clades. Is that common in plants? Please correct if not.

Lines 54-59: Author taxonomy citations are inconsistent. My understanding is that presenting the authors last name is standard format. Please confirm and correct as needed.

Line 104-105: Sentence starting with “Oceanic currents…” is unclear. Please revise.

Line 118: Delete “area”

Line 146: Change “Information of the samples…” to “Sample information…”

Methods: Significant methodological details are missing…

• How were pairwise nucleotide differences estimated? What mutational model was used?
• What is a p-distance?
• How were AMOVA groupings determined and what were they?
• Highest Probability Density (BEAST?) is not described.

Lines 231-232: The described H. major groupings are unsupported and should be collapsed into a single branch. Alternatively, the text should be clarified here and elsewhere to avoid any suggestion that there is phylogenetic support for the groups. This does not alone refute the project findings, but it does add some uncertainty.

Table 3: Type “summared”

Lines 281-282: The AMOVA statement will need to be revised if table 4 is correct.

Line 309: Typo “highest density probability”

Please submit your revised manuscript by Sep 11 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,

Jeffrey A. Eble, Ph.D.

Academic Editor

PLOS ONE

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

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

**********

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

Reviewer #1: 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

**********

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

**********

6. Review Comments to the Author

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

Reviewer #1: (No Response)

**********

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

[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-20-31468R3

Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region

PLOS ONE

Dear Dr. Nguyen,

Thank you for submitting your revised manuscript to PLOS ONE. After careful consideration, we feel that you have successfully addressed comments on the previous draft manuscript (revision 2) and no additional changes are requested aside from grammar and wording issues listed below. We invite you to submit a revised version of the manuscript after addressing the issues listed below.

Lines 37-38: "show variation" is unclear. For clarity I recommend indicating among what groups variation was observed. With the addition of this information I as well recommend splitting this statement into two sentences, the first highlighting where variation is observed and the second indicating low support for regional H. major groupings.

Lines 105-106. Sorry for the confusion. My prior concerns about this sentence were not related to the term 'oceanic currents', rather I feel your statement that currents can "act as a gene-exchange line for the mirgration requiring long-distance dispersal abilities" is unclear. I'm pretty sure I understand your point, perhaps something like "Oceanic currents can act to both promote or limit gene-exchange" would be more clear.

Figure 1: The Myanmar  site currently shows a solid circle but instead should be a solid diamond based on the figure 1 legend.

Line 146: "Sample" not "Samples"

Lines 191-193: Suggested revision "The average number of nucleotide differences between sampling locations for the full ITS fragment and per nucleotide was estimated in Mega X (40) using the Kimura 2-parameter model (39)."

Lines 249-250: Which ML and base values are you referring to? I would not consider the values in general to be high given the lack of support for the H. major group. Please revise to avoid giving the false impression that there is significant support for H. major groupings.

Line 251 and elsewhere: Table 2 presents two different estimates of 'evolutionary divergence', one for the full fragment and one standardized estimate of the per nucleotide rate. I recommend changing how these measures are referred to here and elsewhere for clarity. Perhaps something like "Evolutionary divergence as measured by estimated total fragment and per nucleotide differences...".

Table 2 caption: See my note above.

Table 3 caption: "Summarized statistics of haplotypes" is unclear. I recommend something like "Summarized Halophila major sample size, number of haplotypes observed, and estimates of genetic diversity." 

Table 3: I only just noticed that you've included estimates of Tajima's D and Fu's Fs here but they are not mentioned in the results nor discussion. I recommend deleting these measures from table 3 and the methods unless you can find a meaningful way to include the results.

Line 401: Recommend changing "but it trends to form..." to "with unique haplotypes occurring among regions but no or low support for regional groupings."

Please submit your revised manuscript by Oct 07 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,

Jeffrey A. Eble, 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:

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

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

Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region

PONE-D-20-31468R4

Dear Dr. Nguyen,

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,

Jeffrey A. Eble, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: