PONE-D-21-08538

DNA Barcoding of Brackish and Marine Water Fishes and Shellfishes of Sundarbans, the World’s Largest Mangrove Ecosystem and UNESCO Natural Heritage Site in Bangladesh

PLOS ONE

Dear Dr. Habib,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jul 04 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,

Bi-Song Yue, 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.We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.  

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services.  If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

• The name of the colleague or the details of the professional service that edited your manuscript
• A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)
• A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

4. Please include a separate caption for each figure in your manuscript.

5.We note that Figure(s) 1 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:

a)   You may seek permission from the original copyright holder of Figure(s) 1 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].”

b)   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/

[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: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

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

Reviewer #1: Samples of fish, crabs, shrimp some mollusks were CO1 barcoded and some Mitrochondrial 16s ribosomal DNA was all sequenced for some. The data for fish seem good, crabs and shrimp ok and mollusks poor. the data are valuable for knowing what fish and crustaceans are present. A lot of data are presented (GC content and transitions vs transversions for example) but why they are presented is not well explained. What was actually done is hard to figure out because there has been no filtering. For example 3 samples were theoretically analyzed for each morphological species , but then it turns out one, two 3 or more individual individuals were collected and there is no accounting for how many out of the number sampled were successfully sequenced. The crab/shrimp data show 11 Scylla olivivacea were sequenced out of the 35 total sequences. there are data for 4 species of mollusks. the mollusk data are presented and not discussed.

Overall, the paper does not stay on track. The existing introductory sections need to be shortened and the general audience need to be introduced to additional topics (GC content - Tranversions vs transitions-divergence) be explained. The authors address CO! vs 16s in the introduction, but then do not return to it in the discussion.

Having data from this region is important and the data should be presented in a clear concise fashion. I didn't suggest reject because the data are important. The paper should be totally redone.

DNA barcoding

Line comment

1-3 suggest Title: Barcoding of Fish and Shellfish of the worlds largest mangrove ecosystem

absrtract

24-aquatic fauna , be specific looks like fish, bivalvees and crabs?

44 delete wild animals

43-63 reduce text by 40%

64-78 reduce 40%

79-98 reduce by 40% is 16s CO1 an issue of debate for your groups?

99-107 move up with story on mangroves and reduce 40%

185 188 numbers do not add up

189 to 190—you said 3 of each kind earlier—need to show data on numbers of samples somewhere.

Reviewer #2: PLOS ONE

PONE-D-21-08538

DNA Barcoding of Brackish and Marine Water Fishes and Shellfishes of Sundarbans, the World’s Largest Mangrove Ecosystem and UNESCO Natural Heritage Site in Bangladesh

The authors produced an ambitious inventory of DNA barcodes of aquatic life in the Sundarbans mangrove ecosystem. They also compared the performance of COI and 16S barcodes. The data will be useful for future biodiversity studies, development of environmental DNA assays, and taxonomy studies. The paper is well-written overall. I recommend publication with no required major revisions. The following suggestions should be considered by the authors:

Throughout, use past tense when writing about the results of the study.

Throughout, please check the significant digits in reported results. I expect most results reported as percentages should have 3 significant digits, not 4.

Abstract, line 22: Change “apply DNA barcoding tool” to “apply a DNA barcoding tool”.

Abstract, line 25: Change “sequences of 16S rRNA gene” to “sequences of the 16S rRNA gene”

Abstract, line 26: The reference to “3 aquatic taxa” is not very precise. I’d suggest changing “3 aquatic taxa (fish, crustaceans and mollusk)” to “aquatic fish, crustaceans, and molluscs”

Abstract: Give the standard deviations for all the average K2P distances reported.

Abstract, lines 36-37: Change “misidentification of mud crab species of Sundarbans” to “misidentification of a mud crab species of the Sundarbans”.

Abstract, line 37: Change “In case of molluscs” to “In the case of molluscs”

Abstract, lines 38-40: Change last sentence to “The present study describes the development of a molecular and morphometric cross-referenced inventory of the fish and shellfish of the Sundarbans. This inventory will be useful in future biodiversity studies and in informing future conservation plans.

Introduction, line 56: I think instead of “exclusively adapted” you mean “uniquely adapted”.

Introduction, line 64: Change “The Sundarbans is the world’s single largest 64 continuous mangrove forest lies…” to “The Sundarbans, the world’s single largest 64 continuous mangrove forest, lies…”

Introduction, line 76: Change “All of these factors educe urgent need…” to “All of these factors highlight the urgent need…”

Introduction, line 80: Change “under the term’s DNA barcoding or DNA” to “under the terms DNA barcoding or DNA taxonomy”

Materials and Methods, lines 151-153: Please give final concentrations of PCR components rather than volumes.

Materials and Methods, line 157: Do you mean a 1.0 % agarose gel?

Materials and Methods, line 168: Was there any reciprocal BLAST or other error checking of the database matches?

Results, line 222: Delete “respective”, it is not needed here.

Results, lines 268-269: Were the mollusk samples tested for PCR inhibition? If the samples were inhibited, a second DNA extraction, or 10-fold dilution of the DNA sample might improve the sequencing results.

Discussion, line 283: Delete “among entities within species”

Discussion, line 284: Change “runs” to “provides”

**********

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

[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.

DNA barcoding of brackish and marine water fishes and shellfishes of Sundarbans, the world’s largest mangrove ecosystem

PONE-D-21-08538R1

Dear Dr. Habib,

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,

Bi-Song Yue, Ph.D

Academic Editor

PLOS ONE