Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: PRIORITIZING RIPARIAN CORRIDORS FOR ECOSYSTEM RESTORATION IN URBANIZING WATERSHEDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I appreciate the authors' attention to the comments and review feedback they received. Several changes have been made and the manuscript is in better shape than when it was first reviewed. Regretfully, the manuscript is still not of sufficient writing quality. At a bare minimum, I encourage the authors to revise their abstract, introduction, and conclusions to make sure that all words are necessary and that each sentence is concise and to the point. My attention to the quality of writing is not to punish the authors, but to make sure that their hard work does not go unread, and uncited. Strong verbs and clear adjectives and nouns would greatly improve many passages. I have made a few line edit suggestions, but cannot apply similar changes across the document, as this is not the point of peer review.

By cleaning up the writing, this manuscript will be more attractive to parties looking for an example of a GIS-based floodplain restoration prioritization framework. The references have been improved, but are thin for the length of the manuscript and given the large body of GIS-based floodplain tools that this is similar to.

The conclusions still read more like a methods paper than the "so what?" I would suggest. However, this is at the discretion of the authors.
Experimental design: The design is of sufficient quality, but the writing should be reread and revised as appropriate. There are many ways to skin a cat, and some might disagree with the 20m buffering on each side of the stream based on 30m topography, as well as how riparian/floodplain areas are defined, but I think the recent revisions explain the rationale enough to be useful. The authors performed their due diligence on the NHD request, which is greatly appreciated (in mountainous terrain, the networks can differ substantially, so hopefully it wasn't perceived as a wild goose chase).
Validity of the findings: The findings are valid, although, as cautioned in the first review, the interpretation and applications are what will make this contribution stand out.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A NOVEL TRANSCRIPT OF MEF2D PROMOTES MYOBLAST DIFFERENTIATION AND ITS VARIATIONS ASSOCIATED WITH GROWTH TRAITS IN CHICKEN
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	Comment on languages and grammar issues.
The English language should be improved to ensure that an international audience can clearly understand the text. For example, the authors should avoid putting a coma before an “and” in an enumeration or connecting phrases. Some examples are at lines 37, 40, 59 or 107. I recommend a native English speaker review the manuscript.
•	Figures
There is an error in the order of the figures. Figure S1 appear mentioned in the manuscript at line 188 and Figure S2 at line 134. The authors should change the order of the figures.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: General comments for the author
Lines 84-92. Please, mention in this part the expression of which genes and transcripts are going to be quantified by RT-qPCR
Line 128. Please, add close to the software the reference or URL of the primer software.
Lines 137 – 139. In how many individuals was the MEF2D gene sequenced? In how many individuals were the SNPs genotyped?
Line 150. Please, specify the data analyzed with SPSS instead “other data”.
Line 152. As SEM is an abbreviation, please put the long name and SEM between brackets the first time that appear.
Line 174. It is template instead temple, please change the word.
Lines 177- Line 182. The sizes of the transcripts do not match with the sizes that appear in the electrophoresis gel in Figure 2B.
Lines 195-196. I do not understand what the authors mean when they say that “the expression levels in each tissue are not significantly different”. If they are talking about the expression of the same transcript in different tissues, the expression is significantly different among tissues according to the figure, for example, in figure 3A, the expression is significantly different among liver and cerebellum. Please, clarify this sentence.
Figure 4. Codification letters for significant differences according to the p-value appear in the figure caption. These codification letters are “a” and “b” or “A” and “B”. However, letter “c”, also appear in the figure, please added to the figure caption otherwise, this is confusing. In addition to this, I think there is another error, according with the error bar, the expression in E15 is different to E17, E19 and W7, however, letter A appear in this bar. Please, correct or clarify this error.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A NOVEL TRANSCRIPT OF MEF2D PROMOTES MYOBLAST DIFFERENTIATION AND ITS VARIATIONS ASSOCIATED WITH GROWTH TRAITS IN CHICKEN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The author included a good introduction and discussion. However, authors should considering review the English writing. For example line 191”… two deletion transcripts MEF2D-V1 and MEF2D-V2 were hardly expressed”.
Some data are missing as the cell cycle data. Additionally the Cq values seems to be missing as well in the raw data. Since the data obtained by qPCR are an important for the author’s conclusion Cq values and melting curves are important.
Experimental design: 1. The paper data relied in initial PCR results. However important information is missing to validate the data. In order to ensure the paper reportability it is important to better explain the section 2.3. For example: what was the amount of RNA used to cDNA synthesis? What method was used to calculate the efficiency of the assay to ensure that 2-ΔΔCt was an appropriated quantification method? Which RNA quality checks were used? Please verify all primer sequence, specially 18SrRNA and MYHB1. Please review the MIQE guidelines.
2. There are missing important information in other section as well. Would be important to review materials and methods section. For example: section 2.2 DNA samples the method must be better explained and the information about F2 population should be added to section 2.1 animals. In section 2.5 who many cells were plated and transfected?
3. Which tests were performed to ensure that GAPDH and 18s was indeed appropriated references genes? Specially GAPDH because it was used in a broad of different tissue to compare expression levels. Thus GAPDH expression must be stable and similar in all tissue analyzed in order allow the comparison related to figure 3.
4.Propidium iodide does not stain viable cells. Is missing information at materials and methods section 2.6 or is the data related to viability not cell proliferation? Why a representative data or results values are not shown?
Validity of the findings: The authors bring an intersting data. However it is important to better present/struture the findings.
Additional comments: The paper from Ouyang and collaborator demonstrated the presence of four transcriptor variants of MEF2D gene in chicken tissue. However, the missing of important technical information makes it difficult to analyze some data. The gene variant expression comparison between tissues and embryonic ages are crucial data. The conclusion take from these data need to be better validate.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A NOVEL TRANSCRIPT OF MEF2D PROMOTES MYOBLAST DIFFERENTIATION AND ITS VARIATIONS ASSOCIATED WITH GROWTH TRAITS IN CHICKEN
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No comment.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A NOVEL TRANSCRIPT OF MEF2D PROMOTES MYOBLAST DIFFERENTIATION AND ITS VARIATIONS ASSOCIATED WITH GROWTH TRAITS IN CHICKEN
Review round: 2
Reviewer: 2

Output:
Basic reporting: The language was improve.
Experimental design: 1. How did you check the qPCR efficiency?  It's only possible to used delta delta (Livak and Schmittgen, 2001) if the assay efficiency between reference gene and investigation gene are similar (Pfaffl, 2001). Additionally, are the primers sequence of 18SrRNA and MYHB1 correct? The BLAST search of the sequence listed in the manuscript do not retry the correct products.

2. The cell density should be expressed as cell/cm2 or cell/mL and specify the volume added and the cell area (the cell area is already there as indication of 12-weel plate).

3. There is not such thing is wildly used reference gene. For example, GAPDH expression is influence by glucose, cell cycle, inflammation. Thus, a proper validation must be performed to ensure that the genes used are indeed a good reference gene. Thus, I would like to know if you looked for statistics difference in Cq values between the groups for assume gapdh ou 18s are suitably reference genes. Your most important data is based on qpcr data. We have to make sure that this data was performed correctly. For more information read Kozera and Rapacz, J Appl Genetics (2013) 54:391–406.
Validity of the findings: The manuscript has been improved. However, important information are still missing.
Additional comments: Important information is still missing.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A NOVEL TRANSCRIPT OF MEF2D PROMOTES MYOBLAST DIFFERENTIATION AND ITS VARIATIONS ASSOCIATED WITH GROWTH TRAITS IN CHICKEN
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: I maintained the question numbers.
1. Thank you for the explanation regarding the qPCR efficiency. The primers sequence must be checked. A search in primer blast for the sequence from the manuscript retrieves no product in the 18s primer and another product (NC_006103.5) for MYHB1 primer.
2. Ok, Thank you.
3. It is not clear if the authors tested if the chosen genes were indeed correct reference genes to be used. A simple compare between Cq could help to answer it.
Validity of the findings: no comment
Additional comments: no comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A NOVEL TRANSCRIPT OF MEF2D PROMOTES MYOBLAST DIFFERENTIATION AND ITS VARIATIONS ASSOCIATED WITH GROWTH TRAITS IN CHICKEN
Review round: 4
Reviewer: 1

Output:
Basic reporting: The authors checked the data and corrected the missing information.
Experimental design: no comments.
Validity of the findings: I still have some doubts about the variance expression of the reference genes. However, the manuscript is valid and should be published. I strong suggest inserting the full Cq values of all data as supplementary raw data.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SEA WHIP CORAL LEPTOGORGIA VIRGULATA IN THE MID-ATLANTIC BIGHT: COLONY COMPLEXITY, AGE, AND GROWTH
Review round: 1
Reviewer: 1

Output:
Basic reporting: See edited Word version for comments/revisions using "track changes." (Editing/commenting/revising is much easier in Word than in a PDF, but could not attach Word version to the submission form, so will try to send it to editor separately.)
Experimental design: See edited Word version for comments/revisions using "track changes." (Editing/commenting/revising is much easier in Word than in a PDF, but could not attach Word version to the submission form, so will try to send it to editor separately.)
Validity of the findings: See edited Word version for comments/revisions using "track changes." (Editing/commenting/revising is much easier in Word than in a PDF, but could not attach Word version to the submission form, so will try to send it to editor separately.)
Additional comments: Made my comments/revisions in the Word version of the text. Use "track changes" to see them. (Editing/commenting/revising is much easier in Word than in a PDF, but could not attach Word version to the submission form, so will try to send it to editor separately.)

No major revisions, but one comment I had at the end of the paper is repeated here:

"Good paper! But there’s one thing that needs to be addressed or discussed: might there be a difference, or is there any evidence of a difference (citations?), in growth, colony complexity, age, etc. between colonies inhabiting artificial reefs and those found on natural hard bottoms? I think this is a big gap here. But also, addressing this could lead to all kinds of habitat conservation questions, if one of these habitats is shown to be “better” than the other for the corals. (As you know, these corals are starting to get a lot of attention from both fishers and resource managers.)

Do some digging around and see if this is the case. It’s worth discussing and even speculating about, even if there isn’t a lot of evidence one way or the other out there.

Don’t’ confine your answer to only this species, and don’t necessarily confine it to the exact parameters you studied.

That’s why I was a stickler about adding the word “artificial” when talking about your study sites."


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SEA WHIP CORAL LEPTOGORGIA VIRGULATA IN THE MID-ATLANTIC BIGHT: COLONY COMPLEXITY, AGE, AND GROWTH
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript describes colony complexity, age and growth of four shallow-water populations of Leptogorgia virgulata, an octocoral (sea whip) common in the Mid-Atlantic Bight (Eastern of North America). The ms is well written and organized. Literature is appropriate. Figures are relevant, but I made comments on the map, which should be improved. Other figures are clear and objective. Figure captions need some adjustments. Raw data was supplied, but I suggest that the authors revise names for the excel spreadsheets (not always clear), and a few other details mentioned in the pdf review. Make sure units and variable names are clear in the spreadsheet alone, without the paper.
Experimental design: The ms is a primary research study within the aims and scope of PeerJ. The research question is well defined, and considering the potential impact of the studied populations to anthropogenic activities, it is relevant as baseline data on the biology of the studied objects (corals). Methods are well described and detailed. I have specific comments on the pdf copy.
Validity of the findings: This study adds new information on growth rates and longevity of a common species, which is vulnerable to anthropogenic activities, including fisheries, common in the area. It also contributes to an understanding of basic morphology for the species, which is useful from an ecological and taxonomic point of views. The results are also interesting for comparison with results on the same species in other regions, which was discussed by the authors.
Additional comments: I have included detailed comments in the pdf copy of the manuscript . I have made a few comments that I would like to see addressed. Hence, I'm asking for major revisions to have an opportunity to revise your response. But overall, I think this was a well done project and that your manuscript is worth publication.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: GENETIC ANALYSES REVEAL POPULATION STRUCTURE AND RECENT DECLINE IN LEOPARDS (PANTHERA PARDUS FUSCA) ACROSS THE INDIAN SUBCONTINENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have compiled a large dataset of leopards from 4 landscapes in India to address a number of issues on population structure and bottlenecks. The paper is nicely written, with proper structure and language. I do not see any huge problem in the paper, but provided some comments to help authors to improve their interpretation and applicability.

Also, I believe that authors must consider these four main issues:
1. The paper length is long, particularly in Discussion. Authors are strongly recommended to remove redundant or general parts in Discussion, as they ruin their scope of paper and its exciting findings.
2. Also, I feel that the authors should be a bot careful in interpretation of their findings in conservation. Although the leopard population and range have been reduced, but the heterozygosity of Indian leopards is still high, perhaps highest among Asian leopards, and the extinction rate was calculated as up to 20% for two Indian population, which is far below than many other leopard populations. Thus, in the context of leopard conservation, I think a more careful interpretation is needed.
3. Also, authors are recommended to provide a proper background on genetic studies of leopards, at least in Asia, to provide a comparative baseline for their findings.
4. My final major issue is that more work needed to interpret population structure and combine them with proper ecological data from other literature to rationalize one of the main findings of this paper, which is pop structuring.
Experimental design: Lab experiments have been done properly, field sampling too.
I was curious to know what specific markers were used for species identification (rather than referring to Mondol's paper).
The paper objectives were clear, while I preferred to see all those 4 objectives as some sort of research hypotheses, as what did they expect a priori to find during this survey? Did they expect such a population structure before the analyses, and why?
Validity of the findings: I found findings robust, although some additional tests have been proposed (Ne estimator or PCA) to support their claims.
Additional comments: Abstract: Nicely written, but cannot we consider grey wolf more widespread and highly adaptable than leopards?

Introduction
The introduction is nice and easy to read, but I feel there are redundant sections, particularly for the first two paragraphs with too many citations, many of them really not needed. Although the paper is about the genetic, but the authors have not provided a proper genetic background of leopards, at least within the Asian context, so the reader could learn what we know about leopard genetic and what are the genetic gaps? Based on this background, in following paragraphs, you can narrow your debates down to India.
I recommend you to use these papers for a thorough review of genetic status of leopards in Asia:

Asad, M., Martoni, F., Ross, J.G., Waseem, M. and Paterson, A.M., 2019. Assessing subspecies status of leopards (Panthera pardus) of northern Pakistan using mitochondrial DNA. PeerJ, 7, p.e7243.

Farhadinia, M.S., Farahmand, H., Gavashelishvili, A., Kaboli, M., Karami, M., Khalili, B., Montazamy, S., 2015. Molecular and craniological analysis of leopard, Panthera pardus (Carnivora: Felidae) in Iran: Support for a monophyletic clade in Western Asia. Biol. J. Linn. Soc. 114, 721–736.

Uphyrkina, O., Johnson, W.E., Quigley, H.B., Miquelle, D.G., Marker, L., Bush, M.E., O’Brien, S.J., 2001. Phylogenetics, genome diversity and origin of modern leopard, Panthera pardus. Mol. Ecol. 10, 2617–2633.

Paijmans, J.L., Barlow, A., Förster, D.W., Henneberger, K., Meyer, M., Nickel, B., Nagel, D., Havmøller, R.W., Baryshnikov, G.F., Joger, U. and Rosendahl, W., 2018. Historical biogeography of the leopard (Panthera pardus) and its extinct Eurasian populations. BMC evolutionary biology, 18(1), p.156.

First para: Do you really need all these citations for these generally agreed sentences? I suggest removing non-necessary ones.
Second para: Many of these arguments are not really within the scope of your paper, talking about IUCN status of Amur leopard is not relevant to your work in India. Please consider proper briefing of the whole para.
L110: But Dutta’s work is not at local scale, it is central India, which could be interpreted as regional scale, based on your work.

Methods

Major issues:
Did you use methods of rarefaction that accommodates the effect of small sample size for calculating unbiased allelic richness, i.e. the number of alleles in a sample using HP-RARE 1.0 (Kalinowski, 2005)?
What is Ne of the current population? I suggest you to use the molecular co-ancestry method of Nomura (2008), as implemented in NeEstimator V2 (Do et al., 2014) using the linkage disequilibrium method.
Based on Ne, you then can find published ratios between N/Ne to project the current population size of leopards in India, and compare it to Jhala’s 2015 report of 7000 something. I can suggest you two ratios of 0.11 based on different taxa (Frankham, 1995) and 0.42 calculated from felid case studies (Spong et al., 2000) between census and effective population sizes (N/Ne).
As you believe there are 4 populations, it implies a separation in breeding. I suggest you to calculate the number of migrants successfully entering a population per generation (Nm) as well, as a proof for your claim for populations.
L215: For genetic structure, you have only used STRUCTURE.
I suggest you adding the principal component analysis (PCA) using ‘adegenet’ package (Jombart and Ahmed, 2011) in R (R Development Core Team, 2013).

Also, a main topic in the paper is the reasons behind these population structuring, which I think you need to do additional tests for different hypotheses. For example, another important metircs for population structure is the Mantel test for Matrix Correspondence in GENALEX 6.5, to evaluate isolation by distance. It can tell you more about if isolation between populations is because of distance or because of human barriers (i.e. distance are not large, but human barriers are prominent).

L282: Can you clarify the reason for choosing this grid size?
L302: Citation needed for this method
In occupancy modelling, proper details needed for model building, selection and validation procedures (AIC/over-dispersion etc).

Results
L311 It is a bit strange why no other animal, particularly canids were found in your large dataset, any justification? I expected to see lots of dogs though
L346: A report of private alleles for each population would be great here.
L363-364: It is methods, not results.
L389-394: I suggest reporting psi as below: psi=XX ± SE XX. More details should go to a table, not needed here.
Also, what happened to detection probability and what covariates are associated with that?

Discussion:
It is long, some paragraphs are up-to a page long. Please consider make it shorter, and split long sentences and para into smaller thematic ones.
L402-405: Population, subpopulation and cluster? Please be consistent in using terms across your paper, different terms if used interchangeably will confuse readers.
L408: Mantel test for Isolation by Distance hypothesis will help you to find out better about the reason of separation.
L412: How habitat heterogeneity might cause genetic differences? Not clear. I understand that intraguild competition with tigers can affect pop density of leopards, but how he story ends up in genetic differences?
L414-416: Already mentioned in Results.
L433 and L43 and L437 population OR subpopulation? Please be consistent
L442 Is 0.17 or 0.22 a high extinction rate in current world that many species are suffering higher rates? With preceding debates, I expected higher extinction rate, but it seems that some of Indian leopard populations are doing well.
L454: Citation needed
L454: After such a long para, I should admit that I was lost somewhere half way through, very difficult to keep the track of the narration.
L458: Was T Dutta’s work across a multi PA landscape? I recall it was not within a reserve
L461: Other factors like?
L465-471 This is unjustifiable. To confirm that Indian leopards have decreased, evidence of panda and orangturan and wolves is absolutely irrelevant. Yes, we know many species are declining, but that does not mean a declining trend for another species as an evidence for something else. Please consider reworking or even removing and stick to your data and conclusions
L492: For example (not for e.g.)
L500 ditto
L505 It is not Africa, it is only Tanzania
L506 Did Ilani and Perez reported anything about genetic variability of leopards? I doubt
L506 I recommend to compare your results with another Asian leopard population in west Asia, based on this paper

Rozhnov, V. V, Lukarevskiy, V.S., Sorokin, P.A., 2011. Application of molecular genetic characteristics for reintroduction of the leopard (Panthera pardus) in the Caucasus. Dokl. Biol. Sci. 437, 280–285.

L512 Do you really mean that data on leopard ecology is limited? There are tons of papers out there, making leopards as one of the most studied large carnivores in Asia

Finally, in Discussion, to argue about separation of these 4 populations, I think you need to provide some movement data. I was wondering how long a leopard can disperse or how far are these 4 landscapes that leopards cannot reach from one to the other one?
There are three key papers that you can adopt some movement and dispersal data from:

1. This one shows how far translcoated leopards move (in one case nearly 100 km), tough translocated animals are not usually a proper representative of true dispersal:
Odden, M., Athreya, V., Rattan, S. and Linnell, J.D., 2014. Adaptable neighbours: movement patterns of GPS-collared leopards in human dominated landscapes in India. PLoS One, 9(11), p.e112044.

2. This one shows how far leopards can be translocated to be still within their genetic subpopulation?
Ropiquet, A., Knight, A.T., Born, C., Martins, Q., Balme, G.A., Kirkendall, L., Hunter, L.T.B., Senekal, C., Matthee, C.A., 2015. Implications of spatial genetic patterns for conserving African leopards. C. R. Biol. 338, 728–737.

3. This paper shows dispersal distance of a young leopard, a class in the species with highest rate of dispersal, moving through human landscapes (ca. 80 km), in an area with leopard density similar to many parts of India (4-6 individual/100 km2):
Farhadinia, M.S., Johnson, P.J., Macdonald, D.W. and Hunter, L.T., 2018. Anchoring and adjusting amidst humans: Ranging behavior of Persian leopards along the Iran-Turkmenistan borderland. PloS one, 13(5), p.e0196602.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: GENETIC ANALYSES REVEAL POPULATION STRUCTURE AND RECENT DECLINE IN LEOPARDS (PANTHERA PARDUS FUSCA) ACROSS THE INDIAN SUBCONTINENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	Watch for grammatical errors, e.g., Line 26: “ its’ ” should be “ its ”
2.	Line 31 – include the number of fecal samples used. You indicate 56 unique individuals, 13 loci, 143 leopards so why not include fecal samples used in your work?
3.	Line 63: reword this sentence. I don’t think you mean to say that absence of large carnivores leads to significant changes in trophic cascades but rather significant changes to ecosystem structure and functioning may affect trophic dynamics, which can have cascading effects.
4.	Line 74: insert “The” before “leopard”
5.	Line 105 – here and in all instances change “For e.g.,” to either “For example” or “e.g.,”
a.	E.g., = example given – it is redundant to use both “For example given”
6.	Line 106: insert “by”  “field observations…by…Kranath et al…”
7.	Line 113 – this sentence is wordy and should be rewritten for clarity. Generally, fecal samples are non-invasive, unless you are taking a fecal swab from an immobilized animal. Also minimize the use of the same word (“genetic”) multiple times in the same sentence. Reduce wordiness – e.g., “In this paper, we used fecal samples…”
8.	Lines 128-133 – how were samples collected? For example, were these field surveys intentional for fecal samples or were they opportunistically collected? Foot transects, scat detection dogs, atv/vehicle?
a.	Were field surveys only conducted on established trails? Roadways? If on/surrounding established pathways how far off of the road were samples collected?
b.	Are these details absolutely critical? No, but will they help your reader and any other researchers interested in conducting a similar study understand how you went about obtaining your non-invasive samples? I think so.
9.	Line 142: Remove excess words. Remove “have also” and “leopard”. You’ve established your target species already (leopard) and where possible make your statements active rather than passive.
10.	Line 147: “used the remaining”  Insert “the”
11.	Line 151-152: Reword the last sentence in this paragraph. As written this sentence is illegible.
12.	Line 182: Remove “then” before alleles
13.	Line 186: Replace “was” with “were” as data is the plural of datum
14.	Line 187: Remove “the”
15.	Line 188: Suggest to replace “good quality” with “high quality”
16.	Lines 204-206: I’m confused, this sounds like it should be two separate sentences
17.	Line 213: “we used the program GENECLASS 2.0…” Insert “the”
18.	Line 283: double check the place of commas to separate your divisions. Should this be 3,463,322 km2?
19.	Lines 306-311: You mention 126 fecal samples produced no result – does this mean no result for felid? No result for carnivore? If the latter it is misleading to state you collected 778 large carnivore fecal samples when only 652 were identified as large carnivore. Avoid unintentionally misleading your readers by stating you collected 778 fecal samples and then X% were leopard, Y% were tiger, etc
20.	Line 329: Replace “country-wise” with “countrywide” or “country-wide” but be consistent with the term you use – reference Line 379.
21.	Line 354: “We used microsatellite data” remove “the”
22.	Line 379: “choose either “countrywide” or “country-wide” but be consistent throughout manuscript
23.	Lines 407-411: Can you provide more details on “when” genetic clustering or diversification might have occurred from leopards inhabiting different biogeographic zones? For example, even a rough estimate of whether this is likely to have occurred prior to major human influence on wildlife distribution or after would help place context.
24.	Line 409: For example, an earlier study…” insert “an”
25.	Lines 446-449: This sentence is a bit wordy and awkward. Suggest some mild rewording, and if necessary break into multiple sentences.
26.	Line 455: change to “…loss is a contributing factor of population decline.”
27.	Lines 462-464: Another awkward sentence, having trouble following, particularly “…sampling at subcontinent scale is thus probably indicating the decline patterns at much larger scale.” Suggest rewording.
28.	Lines 447, 478, 480, 500: see comment #5 regarding “For e.g.,”
29.	482 change “increase” to “increases”
30.	Line 498: change “has possibly” to “may have”
31.	Lines 490-491: trim excess words  Change “Apart from sporadic information, we…” to “We”
32.	Line 492: Avoid starting sentence with abbreviations, suggest a change to “For example,…”
33.	Lines 497-501: Long sentence, consider breaking this up to make it easier for the reader to digest.
34.	Line 506: Be very cautious of the data presented in Uphyrkina et al. 2001 for African leopards. Samples were heavily biased towards Southern Africa and not representative of the continent. See Anco et al. 2017 – African leopards were revisited with mtDNA and novel sampling but did not include microsatellite data.
35.	Line 517: Remove “Our results are interesting because”. Start with “We”.
36.	Figure 1: Increase the size (legibility ) of your latitude and longitude markers.
a.	Decrease the size of your symbol for “Individual leopards” to be approximately the same size as is used in the figure to donate sampling locations.
37.	Figure 3: Is there an explanation why the estimated occupancy does not extend to fill the northern extend of the country shapefile you are using?
Experimental design: 1.	The methods you’ve mentioned and describe provide the essential information needed to replicate the study. The only major suggestion I have to increase transparency of your sampling efforts is to detail how/where samples were collected. Refer to comment #8 above. Were fecal samples the intention of field surveys or were they a byproduct of other surveys? Were they collected primarily along/near roads? If so what was the buffer distance? This information would be helpful in the design of future studies. Let’s say for example, most fecal samples were collected opportunistically on/near secondary roads. Having this information and seeing the number of fecal samples your team was able to acquire would be rather useful information in designing a study specifically to search for fecal samples.
2.	The only other comment I have regarding your methods is why bother with microsatellite analyses? They are still informative, yes, but are increasingly being replaced with newer techniques that require less DNA – e.g., RADSeq, Next Gen Sequencing, etc…
Validity of the findings: 1.	You provide a thoughtful interpretation of your results and bring in relevant data and literature to support your speculations. I appreciate you mentioning where your limitations are and to what extend your data support your conclusions without overreaching.
2.	This is an impressive dataset in both samples and geographic coverage and no doubt required the concerted efforts a large and coordinated team. Non-invasive sampling will become increasingly important as populations of threatened species, like the leopard, continue to decline and become more difficult to study using more ‘traditional’ or invasive methods.
3.	If there is one major criticism I have of the paper it is the use of microsatellites. You mention this in Lines 499-501, but I would like to see these conclusions revisited with more novel analyses (RADSeq / Next Gen) as microsatellite analyses are quickly being shelved along with Sanger Sequencing techniques. The information we’re able to gather from these newer techniques can provides exceptional detail when it comes to making inferences about biogeography, anthropogenic impacts, and genetic exchange among populations. We are just beginning to scratch the surface with what we’re capable of learning through genetic techniques and I look forward to what you’ll be able to do with this sample base in future studies.
Additional comments: You have analyzed an impressive number of samples and synthesized that information into a well-put-together manuscript. With some tweaking you should have a solid paper on your hands. A bit confused why Figures 1 and 3 do not share the same spatial extent but that should be easily fixable. Good luck with your revisions!


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: BOVINE TUBERCULOSIS BREAKDOWN DURATION IN CATTLE HERDS: AN INVESTIGATION OF HERD, HOST, PATHOGEN AND WILDLIFE RISK FACTORS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: This is very well written and careful analysis of risk factors associated with the duration of bovine TB restrictions applied to farms in Northern Ireland. Going beyond previous work, the authors explore the association between breakdown duration with badger population estimates and the within-herd diversity of M. bovis isolates.

The risk associated with genotype diversity is striking even given the potential confounding with within herd prevalence (which is carefully quantified and discussed).

The contribution of variables associated with badger population density is less clear and the authors have carefully explored the potential reasons for this and limitations of the data available for the study. However, the most obvious limitation of this data - namely that prevalence within the badger populations could also vary spatially independently of sett density should be acknowledged.

Minor comments:

Line 34, "breakdown" should be defined on first use. Not clear what the distinction being made here is as a breakdown is the terminology used to describe a herd (currently) affected by bTB.

Line 92, "...bTB breakdowns is not not well understood..." ?

Line 115: "(OTF) status." ?

Lin 192: "considered", extra space


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: BOVINE TUBERCULOSIS BREAKDOWN DURATION IN CATTLE HERDS: AN INVESTIGATION OF HERD, HOST, PATHOGEN AND WILDLIFE RISK FACTORS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is extremely well written, in a clear and unambiguous language.
The employed bibliography is adequate and updated.
Figures and tables are relevant for understanding the results. They are not superfluous. Raw data are available.
The work hypothesis are sustained by the undertaken analyses.
Experimental design: The presented work complies with the scope of the journal.
The research question is well defined, relevant and meaningful to the field. The article by Georgina Milne and colleagues is a particularly well written and clearly presented retrospective study using a very rich collection of meticulously selected and organised data from Northern Ireland breakdowns from January 2003 to December 2015.
It is stated how the presented research can help to identiy a knowledge gap.
The aim of the study was to better understand the factors associated with breakdown duration by objectivising well acknowledged bTB factors, using negative binomial and ordinal regression approaches. Different explanatory variables related to herd characteristics, to their residual infection or to local factors were considered. Six variables associated with increasing breakdown length were identified in both models: the herd size, the number of reactors on a first SICCT test, the presence of animals with bTB visible lesions at the abattoir, the local herd-level bTB prevalence, the presence of herds linked via management factors (associated herds) and, very strongly, the genotype (MLVA) richness during the breakdown.
 The investigation is  rigorous.
The methods are described with sufficient detail.
Validity of the findings: The findings are valid. The conclusions are  sound and well supported by the results.
Very interestingly, the contribution of badger’s density (measured using sett metrics) to prolonged breakdowns was not clearly demonstrated in the study.
Additional comments: This a very insightful article that, without doubt, will help to improve bTB management guidelines in Northern Ireland.
Please, add an "is" before "not well understood" at line 92, and a "of" before "the longest breakdowns" at line 303.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: BOVINE TUBERCULOSIS BREAKDOWN DURATION IN CATTLE HERDS: AN INVESTIGATION OF HERD, HOST, PATHOGEN AND WILDLIFE RISK FACTORS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Nothing to add from my previous review
Experimental design: Nothing to add from my previous review
Validity of the findings: Nothing to add from my previous review
Additional comments: Nothing to add from my previous review


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CROSS-CULTURAL ADAPTATION AND VALIDATION OF THE ROMANIAN INTERNATIONAL KNEE DOCUMENTATION COMMITTEE—SUBJECTIVE KNEE FORM
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article structure, figures and tables are all of professional level, however some specific comments for figures and tables that are unnecessary or incorrectly reported are provided below.
Some references should also be added.
The cross-cultural adaptation procedure should be briefly described.
Experimental design: Methods not described with sufficient detail & information to replicate in some respect, and this is pointed out to the authors.
Validity of the findings: All underlying data have been provided; they are robust, however, some departures from statistical soundness are pointed out.
Additional comments: 1.	The authors state they had ‘106 valid sets available for processing’ in all statistical analysis presented n was lower. Apparently there were not 106 complete sets, as attested in the appended Excel file.

2.	The ISPOR criteria for cross-cultural adaptation should be referenced. A reference you could use is the following:
Wild D, Grove A, Martin M, Eremenco S, McElroy S, Verjee-Lorenz A, Erikson P; ISPOR Task Force for Translation and Cultural Adaptation. Principles of good practice for the translation and cultural adaptation process for patient-reported outcomes (PRO) measures: report of the ISPOR Task Force for translation and cultural adaptation. Value Health. 2005;8(2):94-104.
However, a brief description of the cross-cultural adaptation procedure you followed should also be presented.

3.	Lines 100-1: Why was chronicity defined in case symptoms persisted over 4 weeks? Please provide reference.

4.	Line 112: Why was the Spearman’s correlation used instead of Pearson’s? Please provide statistical reasoning and reference(s).

5.	Line 113-4: The statistics for internal consistency and reliability are not clearly stated. Please change. Also, reference 14 is not a primary reference for ICC selection.

6.	Lines 115-6: Test-retest reproducibility should be tested with ICC, not Spearman’s!

7.	Line 143 & 144: ICC is not a statistic for Internal Consistency. Please correct.

8.	Table 1 – page 14: In the table the word Index probably refers to the EuroQuol 5D-5L. Please change. Also, the use of Spearman’s Rho for test-retest reliability is not the appropriate statistic-ICC is.

9.	Figure 1 – page 15: 2 different legends are provided for this figure. Again, the use of Spearman’s Rho for test-retest reliability is not the appropriate statistic-ICC is, as it takes into account a systematic difference that may be present between-ratings. I suggest this figure to be omitted altogether.

10.	Figure 2 &3 – pages 16 & 17: Again, 2 different legends are provided for these figures.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CROSS-CULTURAL ADAPTATION AND VALIDATION OF THE ROMANIAN INTERNATIONAL KNEE DOCUMENTATION COMMITTEE—SUBJECTIVE KNEE FORM
Review round: 1
Reviewer: 2

Output:
Basic reporting: Good English language, with relevant literature references.
Experimental design: The design of the study is respecting all the required steps for validating a functional score.
The statistic method was appropriately performed.
Validity of the findings: It is important to have clinical scores validated in any language, by using those scores we can objectively assess and compare our results.
I consider that this article  is respecting all the requirements for a validation of a clinical score.
Additional comments: The Romanian translation and validation of the IKDC score is a useful assessment tool for all the practitioners in this country .
The study is respecting all the requirements of a validation process.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CROSS-CULTURAL ADAPTATION AND VALIDATION OF THE ROMANIAN INTERNATIONAL KNEE DOCUMENTATION COMMITTEE—SUBJECTIVE KNEE FORM
Review round: 2
Reviewer: 1

Output:
Basic reporting: Reporting is clear and concise. References are now correct.
Experimental design: Up to standard. The methodology for cross-cultural adaptation of patient-reported outcomes is clearly portrayed.
Validity of the findings: All data have been provided, correctly analysed and clearly presented. In table 2 the word 'Index' on the vertical axis can be replaced with Euroqol5d5l for clarity and parity.
Additional comments: Thank you for addressing all comments.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON OF SYMBIODINIACEAE DIVERSITIES IN DIFFERENT MEMBERS OF A PALYTHOA SPECIES COMPLEX (CNIDARIA: ANTHOZOA: ZOANTHARIA)—IMPLICATIONS FOR ECOLOGICAL ADAPTATIONS TO DIFFERENT MICROHABITATS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Very good
Experimental design: The design was relevant for the study
Validity of the findings: Off course, other method could provide more resolution
Additional comments: A good work that provide basis for further studies especially the method that will be able to differentiate between species


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON OF SYMBIODINIACEAE DIVERSITIES IN DIFFERENT MEMBERS OF A PALYTHOA SPECIES COMPLEX (CNIDARIA: ANTHOZOA: ZOANTHARIA)—IMPLICATIONS FOR ECOLOGICAL ADAPTATIONS TO DIFFERENT MICROHABITATS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: The manuscript provide novel and useful information although the purpose is sometimes confusing and not well defined.
Validity of the findings: no comment
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON OF SYMBIODINIACEAE DIVERSITIES IN DIFFERENT MEMBERS OF A PALYTHOA SPECIES COMPLEX (CNIDARIA: ANTHOZOA: ZOANTHARIA)—IMPLICATIONS FOR ECOLOGICAL ADAPTATIONS TO DIFFERENT MICROHABITATS
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Dear Mizuyama et al,

I’ve read your manuscript with interest. You have used nuclear ribosomal DNA and chloroplastic DNA from the psbA to genotype Symbiodiniaceae within a Palythoan species complex. You have statistically shown that the Symbiodiniaceae genotypes are not randomly assorted across the factors of location and host. Rather, you show a host and site driven specificity of the associations.

There is not a lot to go wrong with this dataset and analysis. You have resolved several genotypes with both of the markers and your statistical analyses are sufficient to prove the non-random nature of their distribution.

The writing could improve with regards to the details provided (see specific comments) and in some cases written English. Your analysis of the ITS2 region is far from transparent. You appear to be undertaking editing of the sequences. You also seem to be ignoring additional information in the chromatograms (I cannot comment further because I do not have access to them). You also do not appear to be aware of the amplicon that you are amplifying. You are amplifying the ITS1-5.8s-ITS2 region, not the ITS2 region as this paper is built upon (there may even be 18S and 28S in there, I haven’t checked). This strongly brings into question your analysis of the ITS2 region and the blast results that you provide. I must admit that I find it hard as a reader to trust some of the results with regards to the nrDNA marker. I wonder if better curation of the data would lead to some different results. Unfortunately, I don’t have to time to experiment with this. I would ask that you address some of the specific comments I have raised in the detailed below.

To summarize, your conclusions are largely supported by your results although the nrDNA component of the work should be re-addressed.

I am happy to recommend this work to be published so long as the above issues are thoroughly addressed.

Kind regards,

Ben Hume

Detailed comments
21 The abstract starts with no context of what Symbiodiniaceae are (i.e. zoanthid host-associating algal symbionts), and no context of what “host species” means here (i.e. zoanthid host). The second part of this sentence would be more useful coming first.

44 molecular data, likely due…

81 please provide expected lengths of the amplicons here. You can put what you actually attain in the results.

87 Please provide the chromatogram files as part of the supplementary information so that single peaks can be verified.

87 How and why were the sequences edited?

92 Why are you clustering sequences returned from direct sequencing? This is inappropriate. I am aware of no reason to do this. You should have one sequence per samples and these sequences should be used (after appropriate alignment and cropping for poor quality bp calls) directly for identification of the Symbiodiniaceae. 1bp difference between the most abundant ITS2 sequence represents an enormous difference in terms of phylogenetic and phenotypic distance. As such, the clustering of these sequences will further reduce the resolving power of directly sequenced (i.e. Sanger sequencing) ITS2 amplicon libraries. Perhaps you are undertaking clustering at 100%. Again, without an explanation from you on this, I can’t see what you would do this.

94 above you say that “chromatograms with single peaks were acquired”. Here you talk of how you deal with double peaks. Which is it?

94 The way this is currently written it sounds like you removed only the single nucleotide at which double peaks occurred, or you removed gaps (presumably a gap is from the aligned sequence) in individual sequences rather than removing the entire sequence from the analysis. You cannot modify sequences in this way. You are essentially making up the results by doing this.

96 “each region”. “each marker” would be better.

100 “under default settings other than the clock model being changed to the relaxed log normal model” It would be useful to know why you did this?

112 513-773bp! This is not just the ITS2 region that you are amplifying then. What are you actually amplifying? This will affect the BLAST results too. I’m looking at table 2. It would be useful to see what minimum size match you were using. I.e. if you’re amplicon is 773 bp long, how long are the amplicons that you’re getting matches to. What percentage of the query length are the matches covering?

116 449 sites? Are you saying this is how long the amplicons were? Or rather the number of phylogenetically informative bps?

121 searches

122 Please refrain from using the term “type”. If you mean sequence (which I presume you do), say sequence. Sequence don’t belong to types. Were they exact matches to the C1 sequence? I.e. 0 bp difference? If you mean to refer the the C1 radiation of sequences and species, then say this.

122 “known as generalists in the Indo-Pacific and Atlantic Oceans” This is outdated science. More recent work, including Todd’s work shows that many Symbiodiniaceae are host specific. Modern resolutions also show that the major radiations, i.e. C1, C3, C15, D1, A1 may contains many species, even hundreds (for Cladocopium). See Thornhill 2014 Host specialist lineages dominate the adaptive radiation of reef coral endosymbionts

123 If there were a few substitutions then not all of your samples were matches for the C1 sequence.

124 This is a strange justification; you only need one substitution to make two clades.

125 Genotypes? You’re talking about sequences here right, that represent genotypes.

123 I’ve just looked at your figure 3A. I find it very hard to believe that there was no support for any of these separations. I would like to see the alignment used to make this tree. If there were clear snp differences between the sequences, then these should be seen in the tree.

152 was largest between

171 higher resolution than what?

171 this study is a good example to show that how you analyze the markers is just as important, if not more important than the marker used. If NGS sequencing of the ITS2 had been used and the resulting sequences analyzed to take account of intragenomic variation, it is likely that a resolution equaling that of the psbAncr could have been achieved whilst achieving screening of a wider taxonomic breadth. Smith et al 2017 (Host specificity of Symbiodinium variants revealed by an ITS2 metahaplotype approach), Hume et al 2019 (my own work; SymPortal: a novel analytical framework and platform for coral algal symbiont next‐generation sequencing ITS2 profiling) In fact, it may even be that there is sufficient information in the chromatograms to enable such a resolution. This is currently written with bias towards the psbA marker. COI: I have developed an analytical framework for ITS2; the authors are aware of this from previous reviews.

Fig 3 B and C – why not simply concatenate the fwd and rev psbAncr?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON OF SYMBIODINIACEAE DIVERSITIES IN DIFFERENT MEMBERS OF A PALYTHOA SPECIES COMPLEX (CNIDARIA: ANTHOZOA: ZOANTHARIA)—IMPLICATIONS FOR ECOLOGICAL ADAPTATIONS TO DIFFERENT MICROHABITATS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Although higher number of samples collected in different micro-habitats would be necessary to improved conclusions obtained, authors presented relevant results to future studies.
Experimental design: Aims are well defined compare to the previous version and I think the manuscript has considerably improved.
Validity of the findings: no comment
Additional comments: Authors have addressed all suggestions and comments proposed in the previous version and hence I support it publication in PeerJ


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON OF SYMBIODINIACEAE DIVERSITIES IN DIFFERENT MEMBERS OF A PALYTHOA SPECIES COMPLEX (CNIDARIA: ANTHOZOA: ZOANTHARIA)—IMPLICATIONS FOR ECOLOGICAL ADAPTATIONS TO DIFFERENT MICROHABITATS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Dear Mizuyama et al,

Thank you for taking the time to address the comments made in my review.

You have removed some of the ambiguity in the paper however, there are still some points that could do with improvement (please see below) – some of my points were unaddressed and some are still not clear.

Whilst I believe that the analysis that you have performed could be improved, I see no large issues with the conclusions of the MS as they stand (as I mentioned in my previous review). I would recommend that you have another attempt at addressing the ambiguity and missing information that I point out below, but other than that, I would be happy to recommend this work for publication.

Having taken the time to look through some of your chromatograms I think it is a real shame that you have not taken intragenomic diversity into account when analyzing them. Just from opening up the first three decent chromatograms and looking at them side by side, it is clear that many of the double peaks are non-random (i.e. not sequencing errors; the same double peaks occur in multiple chromatograms). This intragenomic diversity is highly informative! You don’t need NGS to make use it. I’m fully aware that some researchers don’t have access to NGS sequencing, but this is no reason not to make use of intragenomic diversity in your analyses. I have used Sanger sequencing of the full nrDNA region myself (Hume et al.2018: Fine-scale biogeographical boundary delineation and sub-population resolution in the Symbiodinium thermophilum coral symbiont group from the Persian/Arabian Gulf and Gulf of Oman), and my Iranian collaborators who work under exceptionally poorly funded conditions use signals of intragenomic diversity in their chromatograms to identify C. thermophilum and D. trecnchii (e.g. Varaste et al 2018: Symbiodinium thermophilum symbionts in Porites harrisoni and Cyphastrea microphthalma in the northern Persian Gulf, Iran).

I fully appreciate fact that you are pushing to have this paper out in time for a thesis defense and so you are likely uninterested in pursuing this further at this time. However, perhaps, you could mention in your work that this is a viable line of further research that could be taken into account if your analytical approach were to be used again. I think that this would be a good balance to your statements about how lacking the nrDNA marker is in its ability to resolve (in fairness, you appear to have toned down or removed some of these statements now).

I have created an ML tree from your alignment (using iqtree) and indeed I do not see highly supported lineages. Given the ambiguity in your sequence preparation, I would not be willing to say that this is symptomatic of the nrDNA not having the potential to resolve well.

Good luck with the defense!

Kind regards,

Ben

P.s. For future reference, it would be appreciated if you could consistently either mention the line numbers at which you have made your changes (you give them sometimes, but they don’t seem to relate correctly) or include the changes themselves in the rebuttal letter. It’s a little frustrating to have to go through the document looking for the site at which you implemented changes.

101: You are still not being explicit about the region you have amplified. Please use: “partial 18s-ITS1-5.8S-ITS2-partial28S” as you do in your rebuttal letter.

114: Your new text is not helping me understand why you have clustered. I don’t understand how clustering relates to “discriminate taxa of Symbiodiniaceae and obtain representative sequences”. You should not be working with representative sequences; you should be working with the actual sequences. In your rebuttal you say: “We performed clustering to obtain representative sequences (termed as genotypes) to summarize obtained direct sequence data per specimen”. Direct sequence data per specimen is simply the consensus sequence you get from your chromatogram, no? Why would you cluster? In particular you seem to be clustering at 100% identity.

115 You have also not addressed my concerns with regards to what proportion of your sequences you were requiring to be matched when performing your BLASTN. You are using such long sequences and the majority of sequences in the nt database are only the ITS2 region. Are you matching your whole sequence, or only the ITS2, or…?

206. In your rebuttal letter you mention that you are comparing your “higher resolution” to 18S or ITS2 (when direct-sequenced). I don’t see that qualification (direct sequencing) in the new text. You simply flatly compare it to 18S and ITS with no mention of intragenomic variance. In fact now that I look for it, you have not mentioned intragenomic variance once in your entire manuscript despite it being one of the largest complexities in the use of the ITS region.

254. Thank you for including the Hume et al 2019 publication here but it was not my intention to force you to include this. Rather I was hoping to make you more aware of the value of using the intragenomic diversity. This new text currently reads awkwardly. I would suggest you remove the Hume et al reference (which is not the primary literature supporting the point you are trying to make) and replace it with the Thornhill 2014 reference that I suggested in my first review. This paper beautifully points out (using the PsbAncr!) that the ITS2 radiations likely represent multiple (not “various”) species. Importantly, the majority of these species (particularly in Cladocopium) have exactly the same majority ITS2 sequence (i.e. 0bp different). Please readdress this new text accordingly.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EMT-RELATED GENE EXPRESSION IS POSITIVELY CORRELATED WITH IMMUNITY AND MAY BE DERIVED FROM STROMAL CELLS IN OSTEOSARCOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an interesting manuscript that shows the relationship of EMT genes, immune activity, stromal and tumor purity in osteosarcoma. EMT-related gene is positively correlated with immune and stromal activity in osteosarcoma. High immune activity and low EMT were associated with the best OS and RFS in osteosarcoma patients. Although EMT and stromal activity were highly correlated in osteosarcoma, EMT-related genes were more strongly associated with survival than stromal cell genes. The authors claimed stromal cells as a key source of EMT-related gene expression in osteosarcoma patients.
Experimental design: The bioinformatics analysis based on the collected patients data and public datasets are reasonable. The experiment also verified the results of the bioinformatics results.
Validity of the findings: In result 2, why the author claimed the expression of EMT-related gene may result from stromal cells rather than epithelial cancer cells activity in the tumor microenvironment?
Additional comments: This paper provides interesting data confirming the relationship of EMT genes, immune activity, stromal and tumor purity in osteosarcoma.
The following are some minor weakness:
1. The abbreviation “RT-PCR” should be “RT-qPCR”, such as line 31 and 75.
2. The author used 200 EMT related genes obtained from the MSigDB. These genes should be listed in supplementary materials.
3. Inconsistent format P-value, for example, P<0.0001 in line 139, but p<0.0001 in line 143.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EMT-RELATED GENE EXPRESSION IS POSITIVELY CORRELATED WITH IMMUNITY AND MAY BE DERIVED FROM STROMAL CELLS IN OSTEOSARCOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, the study investigated the relationship between EMT-related genes expression, mutation and immune response in osteosarcoma. The article is interesting and compatible with the area of interests of the journal. The experiments are well organized and clear.
Experimental design: The data in this study was derived from three independent microarray datasets GSE36001, GSE39055 and GSE16091. What are the criteria for selecting them?
Validity of the findings: None.
Additional comments: 1. Abbreviations and acronyms are typically defined the first time the term is used within the main text and then used throughout the remainder of the manuscript. Please consider adhering to this convention. such as, OS and RFS in abstract (line 34).
2. Please briefly describe the calculation method of EMT score in the methods (line 86-90), so as not to cause confusion to readers.
3. In legend Fig.4A, bar plot showing the expression of EMT-related and stromal gene signatures in GSE36001 osteosarcoma samples. However, Y-axis show FC. Also, the same in Fig.4C. Did the author reverse the order of 4A and 4C? please verify.
4. The authors find that the expression of EMT genes and immunity are positively correlated, these signatures convey disparate prognostic information. Furthermore, EMT gene expression could derive from stromal rather than epithelial cancer cells. What do these findings specifically reflect in clinical value? What is the innovation of this research? I hope the author can reflect this in the discussion.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EMT-RELATED GENE EXPRESSION IS POSITIVELY CORRELATED WITH IMMUNITY AND MAY BE DERIVED FROM STROMAL CELLS IN OSTEOSARCOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: No Comment.
Experimental design: No Comment.
Validity of the findings: No Comment.
Additional comments: Peng et al. show that there is a strong gene expression correlations among genes related to EMT, immunity, and stromal profile in the context of osteosarcoma using publicly available datasets. Interestingly, these gene expressions are negatively correlated with tumor purity. The authors also stratified patient survival data and found that EMT is more predictive of poor survival (OS, RFS) than immune-related genes and that combining their signatures can provide better prognostic value. They also suggest that EMT gene expression is generally derived from stromal cells rather than epithelial cancer cells.

While the manuscript contain commendable elements (i.e., providing insights of gene activity in osteosarcoma), the current study heavily relied on gene expression profiles and patient survival correlations, which may or may not be directly coupled physiologically. I find the current manuscript lacking of sufficient experimental bolstering. Regardless, the authors can still improve the manuscript. There are several issues I think the authors should address in this regard:

	1. Proper descriptions on the controls (non-cancer) used should be evident in the manuscript, especially in the methods section.
	2. The manuscript also lack the discussion of mechanistic implications of their findings. Reflection on mechanisms should at least be discussed in several parts of the results and discussion sections. However, I should note that this be done cautiously as the authors did not couple their correlations with in vitro or in vivo experiments (which is the main lacking element of the paper).
	3. Conclusions on the EMT lineage formation are presumptuous if the authors are only basing their results on 1 gene expression dataset and patient survival correlations. They also used a very small number of stromal gene markers. More significant stromal markers should be included in this analysis in addition to more cautious reporting of the EMT gene expression lineage formation.
	4. The methods section is not descriptive enough. Please provide more description on how each association/correlation analysis was done.
There are many grammar lapses all throughout the manuscript from the title to the figure legends. I recommend a complete review of grammar, paragraph structures, and paraphrasing knowledge referenced from various sources.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EMT-RELATED GENE EXPRESSION IS POSITIVELY CORRELATED WITH IMMUNITY AND MAY BE DERIVED FROM STROMAL CELLS IN OSTEOSARCOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors answered all my concerns and the article is acceptable.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EMT-RELATED GENE EXPRESSION IS POSITIVELY CORRELATED WITH IMMUNITY AND MAY BE DERIVED FROM STROMAL CELLS IN OSTEOSARCOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EMT-RELATED GENE EXPRESSION IS POSITIVELY CORRELATED WITH IMMUNITY AND MAY BE DERIVED FROM STROMAL CELLS IN OSTEOSARCOMA
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have substantially revised their manuscript, which enhanced the clarity of the
manuscript. I have no further comments to the authors.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EPIDEMIOLOGICAL SCENARIO OF DENGUE IN THE STATE OF MANIPUR DURING THE LAST 3 YEARS
Review round: 1
Reviewer: 1

Output:
Basic reporting: the paper is clear and unambiguous, however still some English errors both spelling and gramma to be improved.
Experimental design: Informed consent was mention when including patients which is not necessary if anonymous. those activities may deducted some important information of those who have better knowledge.
Research question is clear
Method clear
Validity of the findings: Still some issues relate to the representative of data. the refused rate not been reported.
Some redundancy reporting data
I have no idea about the test they compare gender from line 100-106. p value could be enough to report result.
Most of the cases found in 2017 as well as the change in peaking time, it need to have clearer explanation for that time.
In year 2017, most of positive case is confirmed by IgM while NSI rather lower than 2016 and 2018. Any suggestion for that change? any change in method or reagent of test?
Additional comments: Abstract too long compare to the body text. Conclusion also need to be compact and show the main finding for this research. When explain about the epidemic, it is needed to compare with a long period of data, usually, for dengue is 10 years of data to see the seasonality and pattern. Other information about case definition, test used also need to be clarified.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EPIDEMIOLOGICAL SCENARIO OF DENGUE IN THE STATE OF MANIPUR DURING THE LAST 3 YEARS
Review round: 1
Reviewer: 2

Output:
Basic reporting: English language corrections are required throughout the manuscript.

Enough literature survey has not done by the authors.

Repeated representation of data in the form of table and figures.

Article structure is needed to be correct.
Experimental design: Scope of the article is good but lack of proper representation.

Methods: Should provide the information about what kind of statistical analysis has been done for the study and how you have done and which software were used is mandatory.
No description of the study area and study area map.
Validity of the findings: Results:
Table-1 and figure-1 represent the same information hence one should be deleted.
Lines 79-81, 84-85 and 86-87 give the same information and the sentences are repeated too many times.
Line-90: The author did not show any temperature data but compared cases are declining with temperature. Should not state such statements without proper analysis.
Again Table-2 & Figure-2 describes the same information one should delete either table or figure.
All figures and tables describe the same information hence keep either tables only or figures only.
Instead of describing the temporal distribution of dengue by district wise, should have shown spatially for easy understanding.
Is there any specific reason that males are more prone to dengue than females.
Instead of comparing with other authors why this seasonal difference of dengue was observed among the years should discuss briefly. Similarly, why particular the age group is prone for dengue should discuss.
148-150: Need reference for the statement.
Additional comments: Abstract: Line 12-15 is not required in the abstract.
The result is not significant at p < .05.: May be removed from abstract.

Introduction
1sr paragraph: Space after the full stop.

General: English language corrections are required throughout the manuscript.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EPIDEMIOLOGICAL SCENARIO OF DENGUE IN THE STATE OF MANIPUR DURING THE LAST 3 YEARS
Review round: 1
Reviewer: 3

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: The manuscript by Singh et al describes the epidemiology of dengue from one Viral Research and Diagnostic Laboratory from Imphal East. I have the following comments on the manuscript

Introduction
1.	Authors mention that they conducted a study to focussing various epidemiological factors and also for the seroprevalence pattern of dengue virus infection over the last 3 years in Manipur. Have you conducted a sero-prevalence, by testing for IgG antibodies against dengue?
2.	It would be good if you could clearly spell the objective of your analysis

Methods
3.	For the sake of readers, would you elaborate more about the Viral Research and Diagnostic Laboratory Network? Which patients are included in the surveillance? Whether hospitalized or also those attending OPD? What is the case definition followed? Whether all suspected patients are tested or only a sample of patients? What proportion of total suspected patients were in fact tested in each of the three years? Do you have any follow-up data from the patients? Do you have information about severity as well as case fatality of dengue?
4.	You mention that sera are tested for IgM antibodies, NS1 antibodies? Can you please clarify if all sera are tested for both NS1 and IgM? Which kit is used for diagnosis? What is the sensitivity and specificity of these tests?
5.	You mentioned that temporal patterns of dengue cases was explored by plotting monthly incidence for the study period. JNIMS is the tertiary care hospital in Imphal. How correct is your statement that what you calculated is incidence, when in fact all patients in Imphal district (including those attending private sector) are not included in the numerator?

Results
6.	You mention that 1689 suspected patients of dengue (please provide the definition) were referred to VRDL. How many suspected patients of dengue in fact attending during this period?
7.	Fig 1 – Why the number of cases investigated during 2017 was more, as compared to 2016 and 2018? How can you explain such a rise during 2017?
8.	Fig 1 – provide Y axis label
9.	Authors attribute the decrease in the sero-positivity over 2016-18 to the public health issues caused by spread of dengue in Manipur. What exactly do you mean by PH issues? Can you elaborate this point?
10.	I am not sure if you have provided adequate data to support monthly increase/decrease in number of cases by temperate. No analysis has been done to show if the two variables are correlated
11.	Please calculate dengue positivity (#Positive/#Tested) in males and females and then compare the proportion.  The way it is presented in the manuscript,  is less meaningful.
12.	It appears that the virology lab received samples from six neighbouring districts. If the patients/samples from neighbouring districts are included in the analysis, how appropriate is your calculation of incidence, as well as seasonal trend of disease? How sure are you that the referral pattern from different districts did not change over three years?
13.	Did you test 1417 sera which were negative for dengue, for other aetiologies?
14.	What were clinical differences among dengue sero-positive and negative cases?

Discussion
15.	First paragraph  - general statement. Suggest to provide reference
16.	Studies by Atul, Jimmy, Mohan et al? Where were these studies conducted? Are your methods comparable to their studies?
17.	Too superficial.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: PROTEIN EXPRESSION PROFILE CHANGES OF LUNG TISSUE IN PATIENTS WITH PULMONARY HYPERTENSION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors claim they found 74 significantly upregulated and 88 significantly downregulated differentially expressed proteins between control and pulmonary hypertensive lung tissue specimens. At its current state, I think the paper has significant issues that need major reconsideration.
The authors obtained pulmonary tissue from a very small subset and performed cation exchange chromatography and LC/MS MS and analysed data using bioinformatics tools. Given how small the dataset are, the variability in gender between 'control' and experimental and the fact that 'controls' suffered from lung cancer and lobectomy, strongly question the validity of the findings. Variability in gender is significant here since women are more predisposed to pulmonary hypertension than men.
Experimental design: There are lots of variables included in the dataset, such as gender and number. The dataset needs more subjects for a comprehensive bioinformatic analysis. Control subjects should be true controls since lung cancer could have an effect on protein expression as well.
Fig 2- some of the GO terms on the X axis are incomplete
Validity of the findings: Some protein markers found in the study have already been shown to be upregulated in pulmonary hypertension models- intergrin a1 (Umesh et al.) and AMPK (Evans et al). This paper could have corroborated their data had the authors included some experimental data from a larger set of human patients directly implicating the concerned proteins.
Additional comments: Minimal bioinformatics analysis on a small dataset without proper controls and any experimental data to support the hypothesis provides room for a lot of future work.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: PROTEIN EXPRESSION PROFILE CHANGES OF LUNG TISSUE IN PATIENTS WITH PULMONARY HYPERTENSION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please check the article for grammatical or typographic errors, so that the logic is understandable to a general international audience.
I have listed some general comments about the way the manuscript is structured.
Experimental design: I think a one/two line description about the features and/or reach of the GO, KEGG analyses would be helpful for readers who are not experts in the field.
I commend the authors on the vast no. of proteins screened. I felt like it would be easier for the reader if fold changes of some of the major DEPs being looked at were mentioned in the main text. This would make the study more appealing.
Validity of the findings: No comment
Additional comments: The authors have profiled protein expression in lung tissues of patients suffering from pulmonary hypertension. While I commend the authors for identifying potential targets for therapeutic interventions, I think the manuscript is structured in a poor way and it is sometimes hard to follow the logic, especially in the Discussion section. The authors identified genes that are upregulated and downregulated, but then solely concentrated on the ones that were upregulated without providing any justification for the preference. In general, I think the manuscript needs to be revised to be of relevance to a general international audience.
I am curious why there is a significant difference in ages of the test and control populations. Can the authors comment on why the difference is not important.
The authors make extensive use of LC-MS for identifying proteins. It would be helpful to show representative images on how these proteins are identified. Also, it would be helpful to detail a few salient features of the GO, KEGG analyses for the general audience.
Line 183: Can the authors provide example of the specific proteins being looked at, in each of the three different pools?
Line 196: Can the authors be more descriptive of the 10 KEGG signaling pathways? Can the authors comment on interplay between the identified pathways or are they divergent.
Line 220-241: I do not understand the significance of the paragraph. The key finding is that AMPK is upregulated and hence a potential drug target.
In general it would be nicer to have the fold change information for the genes AMPK, ITGA1 etc. in the main text.
Lines 256-274: I do not understand how all of the ancillary functions are related to muscle contraction. It would be helpful if the authors can concisely state the findings. I think in the present form all of the additional description conceals discussion on major findings.
Can the authors comment on what is already known about the field. It seems like some of the key findings of the manuscript have already been highlighted by Evans et al, Umesh et al, Zhang et al etc. I think such prior findings should be discussed in the introduction.
I do not see any new information been provided in Figs. 3 and 4. They seem to be similar to Fig. 2 recast into different axes. It would be nicer to have references added to the pathways in Figs. 7 and 8.
Line 53: familial means the disease is associated with family history. The sentence seems redundant and should be rephrased.
Lines 62, 204, 224 and 229 are hard to follow or redundant.
Line 178: indicates


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: PROTEIN EXPRESSION PROFILE CHANGES OF LUNG TISSUE IN PATIENTS WITH PULMONARY HYPERTENSION
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this paper, the author has analyzed lung tissue specimens from several patients with pulmonary hypertension to study the protein expression profile changes for providing potential new biomarkers. Overall the paper has a present a comprehensive analysis on the protein expression profile with adequate experimental methods. A few things need to be addressed to improve the overall quality of the paper.
Experimental design: See below
Validity of the findings: See below
Additional comments: 1. The English of the paper needs to be examined by a native speaker to avoid some grammar mistakes
2. Some abbreviations, such as GO, KEGG and PPI, didn't have the full descriptions when these terms started to appear, although there were descriptions in later parts.
3. In figure 5, 6 and 7, the author can minimize the pathway maps to the part where has most changes, so that readers can get a more clear understanding


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: PROTEIN EXPRESSION PROFILE CHANGES OF LUNG TISSUE IN PATIENTS WITH PULMONARY HYPERTENSION
Review round: 2
Reviewer: 1

Output:
Basic reporting: In the revised manuscript, the authors have addressed my major concerns
and the manuscript has been improved. I recommend acceptance and publication.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: PROTEIN EXPRESSION PROFILE CHANGES OF LUNG TISSUE IN PATIENTS WITH PULMONARY HYPERTENSION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The quality of the paper has improved a lot and all the points in the feedback have been addressed carefully.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: N/A


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SPATIAL ANALYSIS, LOCAL PEOPLE’S PERCEPTION AND ECONOMIC VALUATION OF WETLAND ECOSYSTEM SERVICES IN THE USUMACINTA FLOODPLAIN, SOUTHERN MEXICO
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is very well-structured, well written, comprehensive,well balance, uses appropriate and current applications of combination data.
Experimental design: The research paper was conducted rigorously and to a high technical standard.  Proper presentation of data for analysis is appreciable.Specific comments are as follows.

1. On line 190-193. The authors suggest that “the more similar the study and policy are, the more accurate the value transfer will be”. This is a commonly held belief (Rosenberger and Phipps, 2007). The stylized fact may be best supported through Johnston and Rosenberger (2010),  Kaul et al. (2013) and Rosenberger (2015).

Rosenberger, R.S. and Phipps, T.T. 2007. Correspondence and convergence in benefit transfer accuracy: Meta-analytic review of the literature. In S. Narvud and R. Ready (eds.), Environmental value transfer: Issues and methods. Dordrecht, Heidelberg, New York, London: Springer. Pp.23-43.

Johnston, R. J. and R. S. Rosenberger. 2010. Methods, Trends and controversies in contemporary benefit transfer. Journal of Economic Surveys 24:479-510.

Kaul, S., Boyle, K. J., Kuminoff, N. V., Parmeter, C. F. & Pope, J. C. (2013), ‘What can we learn from benefit transfer errors? Evidence from 20 years of research on convergent validity’, Journal of Environmental Economics and Management 66(1), 90–104.

Rosenberger, R.S. 2015. Benefit transfer validity and reliability. In Johnstone, R.J., Rolfe, J., Rosenberger, R.S. and Brouwer, R. (eds), Benefit Transfer of Environmental and Resource Values: A Guide for Researchers and Practitioners. Dordrecht, Heidelberg, New York, London, Springer, pp. 307-326.

2. On line 199, authors may need to mention why you choose Meta-dataset of Ghermandi et al. 2010 and Van der Ploeg et al 2010.  Is meta-dataset appropriate to estimate monetary value of Usumacinta ﬂoodplain?.

-	In meta-dataset of Ghermandi et al. (2010),  most primary studies are from developed countries, yet this dataset are applied to estimate wetland in developing countries.  I am wondering why authors do not use    another  dataset (e.g. Camacho-Valdez et al. (2013),  Chaikumbung et al. (2016), Chaikumbung  (2016).    These dataset may suit to estimate the value of  Usumacinta ﬂoodplain   due to greater the similarity of a study and policy site. However, I do not require that authors change to use dataset, only think carefully about this.

Camacho-Valdez, V., A. Ruiz-Luna, A. Ghermandi, and P. A. L. D. Nunes. 2013. Valuation of ecosystem services provided by coastal wetlands in northwest Mexico. Ocean & Coastal Management 78:1-11.

Chaikumbung, M., H. Doucouliagos, and H. Scarborough. 2016. The economic value of wetlands in developing countries: A meta-regression analysis. Ecological Economics 124:164-174.

Chaikumbung, M. 2016. The ecosystem service value of coastal wetlands in developing countries: A meta-regression analysis. International Journal of Applied Business and Economic Research 14:8519-8543

3.	On line 207-212, it would be more helpful for readers if the authors please clearly state the tools and procedure to estimate the money value of wetland ecosystem service within the Usumacinta floodplain.

-	In Appendix A, I find that authors don’t use meta-transfer function to estimate wetland value, yet, using unit value transfer is derived from calculating average value of each ecosystem services from the dataset.  Again, I also note that I don’t need that you change to the method, only think whether estimated value is reliable.

        For example, why the value of riverine ecosystem services for flood control and storm buffering is so high. This value was calculated from wetland valuation studies from France, USA, Laos, Mozambique, Namibia and Botswana which the value ranges from $0.67 to $434,958.

        In my opinion, if data are spread out over a wider range of values, using medium value would be more suitable.   In this case, if using medium value, the value of riverine ecosystem services for flood control and storm buffering is about   5,159.26 US$/ha/year.

4.	In Materials & Methods Section of Local perception of ecosystem services, information about sample selection and a number of respondents should be provided.
Validity of the findings: Findings are easy to understand. However,  the figures in Table 2 are need to be double checked.
-	In the last column, authors should present the figure in average value, not total value.
-	Nevertheless, I believe that money value of ecosystem services in this table should be re-estimated by using medium value.

 Discussion and conclusion  is nicely written. Nevertheless, you may need to add a future research statement.  What are the gaps or limitations with the current data; i.e., a call for directed research?
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SPATIAL ANALYSIS, LOCAL PEOPLE’S PERCEPTION AND ECONOMIC VALUATION OF WETLAND ECOSYSTEM SERVICES IN THE USUMACINTA FLOODPLAIN, SOUTHERN MEXICO
Review round: 1
Reviewer: 2

Output:
Basic reporting: Dear editor,
Now I have concluded revisions for the paper: #39851, entitled "Spatial analysis, local people’s perception and economic valuation of wetland ecosystem services: A case study in the Usumacinta floodplain, Southern Mexico".

In this paper authors considers an integral approach in order to identify specific areas to protect ecosystem services and guarantee the access of communities to natural resources
The authors have done a good job assembling some interesting data, however assembly of data and basic overlap analysis is not sufficient for a significant contribution to the literature. Even though, the topic and the region locally it is very important social and ecologically, authors face the socioecological issues in a very superficial way, as a consequence the approach presented by the authors in lack of novelty.
In general the paper is an informative and interesting work. I understand the local relevance of a research like presented here, however in my opinion the manuscript, such it is here presented, is lacking of relevant ecosystem services and social information.

Since I am not a native English speaker, my opinion about this issue it is not relevant. However, I see a manuscript well structured and easy to follow.
Experimental design: There is not theory or hypothesis that underpins the analysis. The data could be used to answer some novel questions but that has not been done.
In general it not clear how authors deal with the use of different scales of analysis. For example, using maps of bird richness distribution (as a proxy of biodiversity)
Validity of the findings: I considered that if the main goal of the manuscript is showed an integral study or integral approach about ecosystem services issues and the way local people perceive it, authors should consider increased the number local communities in the surveyed. Authors failed the opportunity to have a deeper analysis about these relevant topics. This matters because had only a portion of the total human communities could have a bias perceptions for local people in the region about the value of ecosystem services. Nonetheless, the material presented in this paper retains value. I´m absolutely sure that, if re-framed, resulting information will be remarkable for understanding one of the most cultural and ecological complex region in Mexico.
Additional comments: This is an interesting piece a work that explores an integral approach in order to identify specific areas to protect ecosystem services and guarantee the access of communities to natural resources in Southern Mexico. Along the paper authors tries to make a substantial general point on this important topic. However, author’s methodological approach is unclear and biased. The authors propose to examine these issues using an integral approach which along the the ms it is not clear how authors deal with the use of different scales (e.g birds distribution maps) . Overall, the manuscript is well written and easy to follow.
This article reports on interviews with local people associated with one of the most socio-ecological complex region in Southern Mexico. However, methodologically fails to include a well representative sample of the people´s living in the region, and it  is not clear how authors manage people information.  According to Whyte 1985, who defined perception as "the direct experience of environment... and the indirect information received (by and individual) from other individuals, science, and mass media".

- It is not clear in the manuscript how authors managed the influence of their own-presence in the region on environmental and ecosystem services discourse of local people.

- What are cultural and social aspects (subject at frameworks in decisions-marking) that shape what the group perceives and understands.

- People in the region have an incredible range of perceptions on environmental conditions and hazards, which highlighted the variation among people in this regards, however is not reflected in the manuscript.

- Ecosystem services perceptions. This article reports on contrasting interviews with seven  social different criteria. The task for researchers is to demonstrate both the range of discursive meaning and the social perceptions that reinforce this meaning. However, it not clear, because it not in the ms. how authors deal with. The data collection this paper entails would necessarily be too brief to capture these deeper levels of meaning. The paper would make a very novel contribution to the field by comparing how local people, who nonetheless share similar ideas of ecosystem services, sort themselves out on this issue.

- Finally, the relative weight of people's concern for ecosystem services issues as a strategy for implementing conservation actions.  This is a personal interest of mine, as I work through ideas that conservation fails precisely because so much attention has been paid to economic values (including legislation) and relatively little to public local opinion. As the authors note, public opinion has been reduced to financial matters, as if money were all it took to win over hearts and minds.

Others

It is important to have a deeper literature review. The authors endeavored to maintain a comprehensive approach to the problem but it is hard to see it.
There are many studies in around the world about ecosystem services in particular, and perceptions discourses en general (Indeed, Wilshusen, West, Sundberg, Peet, Watts, among others share in their close attention to conservation discourses).


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SPATIAL ANALYSIS, LOCAL PEOPLE’S PERCEPTION AND ECONOMIC VALUATION OF WETLAND ECOSYSTEM SERVICES IN THE USUMACINTA FLOODPLAIN, SOUTHERN MEXICO
Review round: 2
Reviewer: 1

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: The revised paper of Spatial analysis, local people’s perception and economic valuation of wetland ecosystem services in the Usumacinta ﬂoodplain, Southern Mexico is good and meets the PeerJ criteria.
Thus,  I strongly recommend that PeerJ should publish this paper.