PWAT-D-23-00014

Environmental DNA (eDNA) based fish biodiversity assessment of two Himalayan rivers of Nepal reveals diversity differences and highlights new species distribution records

PLOS Water

Dear Dr. Karmacharya,

Thank you for submitting your manuscript to PLOS Water. After careful consideration, we feel that it has merit but does not fully meet PLOS Water'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.

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We look forward to receiving your revised manuscript.

Kind regards,

Bimlesh Kumar

Academic Editor

PLOS Water

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Reviewers' comments:

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Comments to the Author

1. Does this manuscript meet PLOS Water’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn 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 (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

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Reviewer #1: Yes

Reviewer #2: Yes

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

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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: Dear Authors,

I found the manuscript very interesting, this manuscript provides a new insight on the use of eDNA as a tool for assessing biodiversity specially, fish diversity in Nepal. This manuscript has shown that eDNA has a potential, not only for biomonitoring but also for identifying cryptic species and could encourage the conservation efforts. However, I would like to provide some comments and suggestion to improve the manuscript.

1.In Line no. 97 authors should provide sampling point/stretch coordinates, that could be mentioned for better understanding of sampling location.

2.Author should maintain uniformity in mentioning the no. of sites, i.e., “nine sites” and “15 sites” and “seven sites” (Line no. 117 – 120). Also, consistency while using abbreviations.

3.We generally find spatio-temporal variations in diversity of a region, I want a clarification from the author’s side, since the author have done the sampling in different phases for the both KR and TR. What is the rationale behind such sampling decision.

4.In material and method section “Water sample collection for eDNA analysis” authors should provide depth at which water sample has been taken.

5.Author should mention the type of membrane filter (0.45 μm pore size) polymer was used, such as cellulose, nylon etc (line no. 132).

6.In Line no. 146 authors has identified fish species based on their morphology, they should provide or mentioned some keys or manual which they had referred.

7.Line no. 151: Describe COI.

8.In material and method section “Fish diversity comparisons between two river systems” author have mention about principal coordinate analysis (PCoA) plots, however, I didn’t find anything explaining about PCoA in the result section. Further, author should explain Bray-Curtis, Jaccard, Unweighted UniFrac and Weighted UniFrac dissimilarity metrices in detail that is mentioned in figure 6.

9.In line no. 161: I think it should be “After” instead of “And”. Further in line no. 163, author has mention “(S. Korea)”, what does it mean?

10.Rewrite line no. 164 – 166 “For metabarcoding based fish species identification, a ~170 bp fragment of 12S gene was amplified on extracted each eDNA samples using specific MiFish primers (14) with Illumina overhang adaptors”.

11.Author should provide high resolution images and graphs, as figure 1, 2, 4 and 6 are not clear and readable.

Reviewer #2: 1. Interesting and informative work

2. Under material methods, line no 138 - Did you mean five seasons or five sessions? Similarly three sessions or three seasons?

3. In discussion, line 317 management of ecosystems and not management of an ecosystems

4. In discussion, line 318 - resource managers

5. Mention of Black tetra (Black widow tetras - Gymnocorymbus ternetzi) are native to Bolivia, Brazil and Argentina in South America and they are subtropical species. What happens to them during winter when the temperature drops down in these rivers during that time? If it survives then it is worth a mention in the manuscript regarding its adaptation.

6. line 369 - disposal of fish waste. Where are these fishes coming from? caught from lower regions?

7. Identification of non-native fish species in both the rivers could have happened either (a) introduced for fishery purposes or (b) Ornamental aquarium trade. A little insight on this would be useful in the discussion part

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Reviewer #1: No

Reviewer #2: Yes: Deepak Samuel Vijay Kumar

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Environmental DNA (eDNA) based fish biodiversity assessment of two Himalayan rivers of Nepal reveals diversity differences and highlights new species distribution records

PWAT-D-23-00014R1

Dear Dr Karmacharya,

We are pleased to inform you that your manuscript 'Environmental DNA (eDNA) based fish biodiversity assessment of two Himalayan rivers of Nepal reveals diversity differences and highlights new species distribution records' has been provisionally accepted for publication in PLOS Water.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow-up email from a member of our team. 

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Water.

Best regards,

Bimlesh Kumar

Academic Editor

PLOS Water

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Reviewer Comments (if any, and for reference):