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: GRAFT SURVIVAL OF PINUS ENGELMANNII CARR. IN RELATION TO TWO GRAFTING TECHNIQUES WITH DORMANT AND SPROUTING BUDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment. I have listed the review results in "General comments for the author".
Experimental design: no comment. I have listed the review results in "General comments for the author".
Validity of the findings: no comment. I have listed the review results in "General comments for the author".
Additional comments: In my opinion, this article is a very excellent study that used statistical methods to validate the effectiveness of grafting as one of the methods selected to propagate Pinus engelmannii. Therefore, I hope that this article will be published and will be seen by many readers. I have read this article many times, but I do not have many comments on it. If I have any comments, they are the following two points. These are minor comments.
1.	In Tables 3 and 6, the authors examine the model selection of several factors. It is quite understandable that GT and PSB have a significant impact, but is HR or DS an essential factor in estimating graft survival? If the authors have a clear idea, please put it in the "Discussion" section.
2.	The main purpose of this paper is to examine the factors that should be considered for grafting. However, statistical method selected is one of the important parts that reinforce the originality of this article. Therefore, I feel that the "Discussion" section should contain more information on the selection of statistical models. For example, the part corresponding to lines 319-330 in the "Results" section could be included in the "Discussion" section. Could the authors please reconsider this?


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: GRAFT SURVIVAL OF PINUS ENGELMANNII CARR. IN RELATION TO TWO GRAFTING TECHNIQUES WITH DORMANT AND SPROUTING BUDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors addressed all of the questions and made all of the necessary changes.
Experimental design: The experimental design is appropriate.
Validity of the findings: In the perfect form.
Additional comments: Dear respected author,

Thank you for your efforts in revising the manuscript in an appropriate manner. Annotated pdf file contains only minor writing errors in the results and discussion sections. Please fix these mistakes.

Sincerely,


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: IMPACT OF URBANIZATION ON FUNCTIONAL DIVERSITY IN MACROMYCETE COMMUNITIES ALONG AN URBAN ECOSYSTEM IN SOUTHWEST MEXICO
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some areas in need of improved English.  Some additional references encouraged. Mostly professional structure, figures and tables, but see comments. Needs a clearly stated hypothesis in the Introduction.
Experimental design: Research seems to be original and a question/objective defined that is relevant and meaningful. Gap addressed could be made more universal. Investigation appears rigorous though methods need improved clarity and additional information.
Validity of the findings: Replication in terms of multiple urbanization gradients tested would be an improvement.  As is, the study only examines one gradient.  Each location along the single gradient has multiple plots (i.e. pseudureplicates). Underlying data not available, unsure of where they can be found. Conclusions are mostly clear and linked to original questions.
Additional comments: The authors characterize the “macromycete” fungal communities across an urbanization gradient.  The key results show that soil temperature and air humidity are driving factors in species richness and that the urbanization gradient (which combines street/road amounts, building density) is negative for functional richness but positive for functional divergence (the latter I am still not sure I fully understand). Overall, this is a reasonable study but I am unsure of how interesting it might be beyond a small set of readers.  The case could be made more convincingly that this is a global and not a solely Mexican pattern. Study needs revision, many small items to attend to and several larger considerations.

One area to expand and reconsider:  Look at the patterns of mycorrhizal, saprotroph and parasite groups separately...do they do different things across the gradient?  See the Gomez-Hernandez et al 2019 Fungal Ecology paper cited

Figure 4 and 5 (maybe even Fig 1) and Table 2 could go to the supplement without harm.

Line (number given followed by “:” and suggested correction or comment) by line comments:

28: mutualists
28: pathogens, mutualists and decomposers…
32: I think the functional aspect is more about the specific ways in which the organisms processes energy and matter and less to do with evolution.
35: in order to infer
41: with increased level of
42: sites which appear to be
51: But in line 44-45 it says FDiv increased with urbanization.  This will be confusing until readers further get into the paper.  It probably should be stated as FRic not “functional diversity”
75: Do not use the word “Nowadays” here and elsewhere. Can start the sentence at the following word.
82: Consider changing "Macromycetes" to "Fungi" at least here in the introduction.  Then, in the methods, clearly state that this study only focuses on "macromycetes" and then explain what criteria the study used to determine and survey only macromycetes.
98: Important instead of relevant
114: See Avis et al Restoration Ecology https://doi.org/10.1111/rec.12397  for an example of using functional diversity in the context of ecological restoration including some sites of urban settings.
120: It would be preferred if the authors propose specific hypotheses about the relationship between urban gradients and macromycetes.
156-158: The sentence seems more like it belongs in the Results section.
162: Was there a size or diameter minimum that was considered to be "macromycete" at least according to this study?  this needs to be clarified so that we understand how only "macros" were considered.  Where is the boundary between macro and micro in this study?
163: How or why was this determined? At a minimum, state references that support this contention and point out limitations of this choice.  There is very good evidence that some "macromycete" individuals are extremely large while others are quite small -- and I would surmise that population genetic tests were not conducted on all of these species to determine individual genet size. So, this study's criteria, as written, has not accounted for this.  Since this is an unknown, it would be better to state this as “observations” and not “individuals”.  Then in the methods describing the diversity metrics make it very clear that this assumption is not met and that the study uses “observations” instead of true measures of individuals.
177: What’s the rationale for three groups?  What were “all traits”?  Why not just look at trophic group?
187: For each functional index, explain how the index was calculated.  For example, was FRic the total number of functional types (including all three trait groups)?  How did this handle repeats of a trait group? These need to be clearly defined FDiv and FEve as well.
188: remove “convex hull”
197: Were data normal and identically distributed? In the results ,there is no mention of this and/or any subsequent transformations conducted to deal with data and residual issues.
210: What is “nutritional”?  Trophic groups?  Not mentioned or described previously.
215: Better stated as "collections" or "observations" rather than individuals.
224: Given that just more than half of the communities should we expect the answer to change?  This issue is not at all dealt with in the Discussion section and should be. Or, can the authors survey for another year? If they could, this study would be much stronger.
243: species, no “of”
244: Jaccard not mentioned in Metods
245: similarity
277: Can a table comparing the different models used and their respective AIC be shown?  This will help the reader see the different models considered.
278: indices
295: What is “true”?  Explain.
323: should read "...have shown that..."
324: The global relationship is probably hump-shaped but under tropical conditions, such fungal communities are at the downward slope of the hump (and conversely, in boreal-arctic regions the relationship is at the start, the upslope of the hump).  This might be worth clarifying if the readers are globally focused.  Urbanization happens globally.
350: should read "...appear to affect..."
386: Since the text that follows is a list that explains the statement prior, use a ":" instead of ";"  Alternatively, and my preference, would be to just make these all shorter sentences.
399: stop the sentence after "...2013).  The increasing..."
404: more urban communities
405: Not sure I understand this point. Could it be elaborated?  How does this show the center of the trait range?
420: Should say "...study suggests that urbanization..."  Correlation is not causation and this study just looks at the relationship.  It does not experimentally test the effect of urbanization which is what would be needed to state it in the way the authors wrote this.
430: Again, worth referencing Avis et al Restoration Ecology 2016
433: Reference here e.g. Peay et al New Phytologist (2010) 185: 529–542 and Branco and Ree (2010) PlosOne https://doi.org/10.1371/journal.pone.0011757
504: Mycorrhizal


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: IMPACT OF URBANIZATION ON FUNCTIONAL DIVERSITY IN MACROMYCETE COMMUNITIES ALONG AN URBAN ECOSYSTEM IN SOUTHWEST MEXICO
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: To the Authors,

I have now reviewed manuscript ID: #59452, entitled ‘Impact of urbanization on functional diversity within macromycete communities along an urban ecosystem in Southwest Mexico’. This study sought to determine the effects of an urbanization gradient on macromycete communities within an urban ecosystem of Southwest Mexico. The authors have measured various macromycete functional and trait diversity metrics, along with community composition, and explanatory variables of the biotic metrics above.

Major concerns:
The overall writing style, tone, language, and clarity need improving to properly reflect the study conducted. This will improve the overall comprehension as a reader of the manuscript. Various parts of the writing style throughout the manuscript need to be toned down to keep within the aim, scope, and limitations of the study. I suggest the authors consult with an editing service or colleague to help proofread and revise this manuscript. Methods need to be more extensive to ensure the repeatability of the study; some parts remain unclear; a few questions pertaining to methods are asked and provided in the general comments section below.

Weaknesses: Writing style needs major revision to convey the aim, scope, limitations and justifications in a comprehensible manner to improve overall clarity. More explanation should be provided in the Introduction to help guide the reader along as to the differences between ‘macromycetes’ and broadly based fungal groups, and more information about the important roles of fungi and their intimate connection with their surround environment, as PeerJ is not a microbial ecology-based journal.

Strengths: Important to understand fungal community patterns in urban ecosystems, and even more so for sub-tropical/tropical ecosystems. Hand-sampling fungi is not easy; consider DNA metabarcoding approach in the future, to assess fungal community patterns from environmental samples collected in this region—food for thought.

General comments and concerns:

Title
The title is a bit unclear and suggest revising to improve comprehension and clarity in order to accurately convey the study being presented. For example, replacing “macromycete communities” with “fungal communities”—this may help engage the reader more. Moreover, these fungal communities were examined along an urbanization gradient within an urban ecosystem of Southwest Mexico; would suggest rephrasing the title to

Abstract
The Abstract could be shortened to keep within the aims, scope, and limitations of the study (e.g., this is a case study; please tone down language and writing style). Moreover, is there a justification in using the term ‘macromycetes’ as opposed to ‘fungi’? The difference between macromyete-fungi and fungi, should be clear to the reader in the Abstract, as PeerJ is not a microbial ecology-based journal. Abstract needs to be revised such that the reader is guided along a story as to what the problem is with urbanization and macromycetes; knowledge gap for macromycetes in this area; why are macromycetes important.

Please keep any interpretations to the Discussion section.

Ln 25-27: This sentence lacks context and should be revised or deleted. For example, “abundance of individuals” does not provide the reader as to what type of individual is being referred to (i.e., this study only examines macromycetes); likewise, “loss of species which play different roles in ecological communities” is vague and shrouds the overall cohesiveness and clarity of the message being conveyed.

Ln 27-30: This sentence lacks context and should also be reorganized to improve comprehension and clarity.

Ln 30: Please specify the species organisms you are referring to (i.e., macromycetes); and be consistent throughout the manuscript to remind the reader.

Ln 30-32: What is meant by “they incorporate an evolutionary background”? This sentence should be revised; unclear.

Ln 35: “to infer the effect of urbanization on macromycete communities” can be omitted. Would indicate/incorporate this earlier in the sentence (i.e., aim of the study was to assess patterns of macromycete species richness, functional diversity, and community composition along an urbanization gradient in an urban ecosystem of SW Mexico).

Ln 36-38: This sentence can be shortened and/or condensed with previous sentence (not necessary to put in the dimensions of the plots in the abstract).

Ln 43-44: Interpretations should be kept to the Discussion section.

Ln 44-46: This sentence would be more appropriate as Discussion material, not Abstract material.

Ln 46-48: Keep interpretations to Discussion section.

Ln 50-52: This sentence is speculative as “ecosystem productivity” and “stability” were not measured. Please refrain from using this type of language throughout the manuscript.

Ln 48-52: These sentences need to be revised to keep within the aims and scope of the study, and what was actually measured and what the data and results show.

Introduction

Justification could be built upon by focusing on knowledge gaps in the research and the critical roles that fungal communities play in not only non-urban ecosystems, but urban ecosystems as well.

Can condense paragraphs 1 and 2 to shorten the introduction and expand on various topics to make the introduction more relevant, cohesive, and comprehensible for readers that are not familiar with microbial groups such as fungi (e.g., Macromycetes).

Ln 59-60: How many decades? Also, this sentence could use an in-text citation.

Ln 70-72: Which research? Please reference. Moreover, this sentence needs more context as this study is only focusing on fungal groups, and in saying “exploring biotic communities” does not provide the reader with context to comprehend more specifically the message being conveyed. Same with “declines in species richness” and “abundance of individuals”. The end of this sentence—“there is a decreasing continuum of human influence from city centres out toward wild areas” is a bit vague and seems to be a bit of a non-sequitur. Please consider revising this sentence.

Ln 72-74: It is not clear which kind of biotic communities the authors are referring to—in addition, is this the case for all biotic communities? Please be consistent in using the organismal group being studied (i.e., macromycetes), if this is true for this group.

Ln 77: Please cite this reference here, then provide the results of this study in the following sentence starting with “The results showed…”

Ln 77-81: This sentence is a bit lengthy, consider revising.

Ln 82: This sentence needs a reference.

Ln 82-88: Unclear the difference between Macromycetes and fungi—this needs to be explained more in the Introduction for those readers that are unfamiliar with fungi.

Ln 85-90: Do Macromycetes fall within these categories of fungi? (i.e., saprotrophic, wood-decaying, and mycorrhizal fungi). This is unclear in these sentences.

Ln 96-99: This sentence could be rephrased to provide more context (i.e., evaluating variation in Macromycete species diversity and community distribution patterns…..).

Ln 99-101: What is meant by this? What kind of species? Do other species have different responses? This sentence is vague and lacks context that is pertinent to the study—should be revised.

Ln 101: Would be a good idea to provide an example of a specific Macromycete functional trait.

Ln 117-120: The aims and research questions could be made more clear.

Methods

Ln 129-134: Is there evidence to suggest elevation could be a contributing factor?

Ln 132-134: What is the justification for these dimensions? (i.e., why 2 large plots for each site, and 5 within each plot?)

Ln 136-144: These methods should be made more clear such that the study is repeatable.  How were these explanatory variables measured? Proper instrumentation should be given as well where appropriate.

Ln 145-158: Is this a standard method in identifying urbanization gradients? References would be useful here.

Ln 160-167: This section needs a more extensive description of sampling methods and applicable references (i.e., are these standard procedures for sampling macromycetes?; Were any sterile techniques applied?; Are sterile techniques necessary?; Is there a standard procedure or reference in which to identify micro- and macro-morphological characteristics?).

Ln 172: Change “was” to “were”.

Ln 173: Justification for using Jaccard similarity when the data has been presented as abundance, as opposed to presence/absence?

Ln 172-174: These methodological descriptions need to be more extensive.

Ln 176-178: Reference?

Ln 178-181: Reference?

Ln 176-193: The functional and trait diversity methods need to be made clearer here and referenced appropriately.

Ln 195-198: These sentences are unclear—please consider revising.

Ln 195-203: Statistical analyses need to be made clearer and explicit, as well as properly referenced analyses. Moreover, were the biotic data standardized prior to analyses? Were the abiotic data normalized prior to analyses? This is not clear.

Ln 199-211: These sentences need to be revised and made more extensive to guide the reader along as to what analyses were used for which specific research questions.

Results

Ln 223-224: What is meant by “complete”? This is unclear.

Ln 225-228: Please revise these sentences—not comprehensible.

Ln 229: “Within the latter group of sample”….—easier to refer to the samples as opposed to this language, to improve readability.

Ln 229-232: These sentences are unclear—please revise.

Ln 233-242: Please consider providing these data results in a table of the manuscript.

Ln 233-242: Please be consistent in p-value significant figures.

Ln 243: Typos

Ln 244-246: This is unclear.

Ln 251: The Monte Carlo methods are not described in this manuscript.

Ln 277-278: This is not clear

Ln 285-286: What does this mean?

Discussion
The Discussion section is dense with in-text citations and suggest removing where appropriate. This section also needs to be shortened in length. Moreover, some of the information provided in this section may be more fitting as Introduction material. Some pieces of information are difficult to interpret, and it is unclear in various areas throughout this section as to how the authors have arrived at their conclusions, supported by their data/results and why; there does not seem to be a clear link of their results and the discussion material provided. Please omit from using strong speculative language when discussing the interpretations of the results, and please keep within the aims, scope, and limitations of the study.

Ln 301-308: More appropriate as Introduction material.

Ln 301-307: Lengthy sentence—please consider shortening.

Ln 309-314: More appropriate as Introduction material.

Ln 315-318: Please interpret and discuss the results; the discussion section should not be a repeat of the results.

Ln 318-321: This sentence is unclear; how is this related to an urbanization gradient? Please consider revising.

Ln 327-336: These sentences can be condensed.

Ln 361-366: How is this related to the results?

Ln 369-371: The message being conveyed in this sentence is not clear. Please revise.

Ln 378: The amount of niche space is not described well in the introduction.

Ln 378-383: These sentences are unclear, and it is not clear to the reader how the authors arrived at this conclusion. Please revise.

Ln 385-390: Please shortened—too long.

Ln 394: This study did not test for ecosystem productivity; please keep within the aim and scope of study.

Ln 401-403: This is not explained well—please revise.


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: IMPACT OF URBANIZATION ON FUNCTIONAL DIVERSITY IN MACROMYCETE COMMUNITIES ALONG AN URBAN ECOSYSTEM IN SOUTHWEST MEXICO
Review round: 2
Reviewer: 1

Output:
Basic reporting: Yes, sufficient.
Experimental design: There remains concern in how the interpretations of "functional" are portrayed. See below.
Validity of the findings: See "additional comments".
Additional comments: This manuscript is improved and several areas are much better than the previous version.  I appreciate that the authors have considered the previous comments seriously and also that they have pushed me to study deeper the current state of functional trait ecology. However, several key areas still need to be addressed in order to make this manuscript acceptable for publication.

The interpretation provided for “functional” and the extent to which the authors extend interpretations and discussions beyond the limits of the evidence provided remain a serious point of concern.  In essence, they have measured five “morphology-related” (line 427) traits of the macrofungi surveyed in the forests of different sites along the urbanization gradient. From these data, they have extrapolated in the discussion about how the responses measured illustrate a “reduction in ecosystem productivity” (line 429) and other processes.

Given only the morphological evidence, it is not at all clear how these could be connected to ecosystem productivity or other processes. The five traits selected have no clear (at least as defined by the authors to this point) and direct relationship to ecosystem productivity, function or service -- unless they are presuming that pileus size and spore production are elements of ecosystem productivity substantial enough to compare to primary productivity by autotrophs in the forests.  By analogy, this is akin to stating that the size of bird nests and number of eggs in the bird nests is a significant portion of ecosystem production -- this isn’t to say that bird nests and eggs, nor macrofungal reproductive structures are unimportant. But the evidence of how these measures are connected to ecosystem processes needs to be there, and in this document, as written, we only have a set of five reproduction-related morphological traits of these species.

Rather, their measures of “functional” traits are adequate for what they have defined (lines 105-109) as “response” traits (“quantifiable ...reproductive characteristics...related to fitness” -- the latter term I take to refer to the evolutionary type of fitness connected to reproductive output) -- mushroom cap size, size and shape of spores are all aspects of macrofungal reproduction.  As far as I know, it would be a stretch to say that larger pilei and larger spores are reliable measures of ecosystem productivity.

In its current state the manuscript provides no direct measure or evidence of “effect” traits (“how species contribute to ecosystem functionality”) which, again, is typically considered as having roles in primary productivity, nutrient cycling, carbon storage, etc.  As written, the manuscript does not adequately address how these data are related to these “effect” traits and only gives us the “response” of a few traits of macrofungal reproduction.  Nor has the manuscript sufficiently explained how measuring only these five morphological traits we can get to a “functional” understanding of forests and the impact of urbanization on these forests by way of fungal responses. Perhaps there is some way to explain how these five traits connect to the functional view, but as is, the manuscript does not do so convincingly.

To address these concerns, I make two suggestions.  First, throughout the manuscript including the title, consider using the term “morphology-related trait” instead of “functional” to more clearly show what was measured.  The authors could still make the contention that this trait set is an element of “functional diversity” but repeated use of what was actually measured would consistently remind readers that only morphology and not some ecosystem process (e.g. decomposition or carbon sequestration) was considered by this study. Given this, they should minimize their extrapolation of these data to speaking to ecosystem processes.

Second, it is not out of the question to include some actual “effect” trait data in this manuscript that could be related to the urbanization gradient.  Although I suggested this in the previous comments, the rebuttal to this comment was not clear [How is classifying a species of fungi as ectomycorrhizal or saprotrophic “subjective”?] and this new manuscript has not yet adequately addressed the point of concern.  How would it not be a part of this study to address “effect” type functional traits, especially if it wouldn’t take much more effort (and no collection of additional data) to do so? If I am missing something here, then it needs to be clarified in the manuscript as readers will share my confusion.

One way to do this with the data already collected and shared (see Supplemental File S4) is to use the simple model provided in Avis et al (2016, Restoration Ecology, see below) and calculate an “effect” type functional value trait index for each of their sites.  To do this, each taxon listed in S4 can be scored as ectomycorrhizal, saprotrophic or parasitic, and then by using the formula below, an “effect” type functional value can be calculated for each plot.  These data can then be compared and statistically analyzed across the urbanization gradient. This really wouldn’t be much more work and from the sounds of it in the reply to this comment, probably related to your species richness patterns.

The Simple Model of Avis et al 2016 (Restoration Ecology)
(1) Simple Model Site Simple = [ FEcM × ln(#speciesEcM + 1 ] +[ FSap × ln(#speciesSap + 1 ] +[ FPar × ln(#speciesPar + 1 ] Where F is functional value as defined in Table S3b, EcM is EcM fungi, Sap is saprotrophic fungi, and Par is parasitic fungi.

With the suggested approach above, the authors can easily test their contention in their rebuttal [“We think that using trophic groups for studies on functional diversity is not appropriate due to very dissimilar species are grouped together based only on one trait (trophic strategy) and a lot of information is missing.’] In doing so, they can greatly expand the relevance of their work.

Additional line by line comments.

Line 71: Change “decreasing continuum” to “decrease of human influence…”
Line 119 and through the manuscript:  Should “true” be “taxonomic”? If not, then other diversity, for example, “functional diversity” would be “false”.
Lines 176-177:  I’m not sure what “practical purposes” would prevent changing “individual” to “collection unit” throughout the paper
Lines 183-185: This sentence can be removed as it goes without saying.
Line 187:  State the index used in relationship to the variables measured, i.e. what Jost formula was used and what were the input variables, species observed?  This is particularly important to do since no true knowledge or evidence exists as to how many actual genetic “individuals” were observed in this study.  How a “Jost” approach might incorporate that consideration into “true diversity” should be considered as well.


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: IMPACT OF URBANIZATION ON FUNCTIONAL DIVERSITY IN MACROMYCETE COMMUNITIES ALONG AN URBAN ECOSYSTEM IN SOUTHWEST MEXICO
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No 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: MULTIOMICS-BASED ANALYSES OF KPNA2 HIGHLIGHT ITS MULTIPLE POTENTIALS IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Zhang et.al used multi-omics approaches to make the prediction of HCC outcome and presented valuable information regarding the change of KPNA2 variants during HCC. The paper is generally well written and structured. However, the paper has some shortcomings regarding to several data analyses and text:

1). The font size is too small for most of the figures.

2). Figure 5A, Are there any sub-clusters in your PPI analysis? What are your clustering coefficient and the minimum required interaction score you used? Could you indicate in the figure legend what the colors of each dot represent?

3). The finding of the connection of KPNA2 and immune cells is very interesting. Can you find more data to indicate the association of KPNA2 and immune cells in different disease stages? It will be helpful if the authors can provide some experimental information on it.

4). Can you do the co-expressed analysis of KPNA2 to see the gene expression pattern related to HCC? And find out the top positive and negative correlated genes with KPAN2 in the HCC?

5). Figure 8, please explain why using paired samples test?

6).  Figure 11. Can the authors also look at the relationship between different KPNA2 transcripts with some HCC’s serum markers, like AFP?
Experimental design: no comment
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: MULTIOMICS-BASED ANALYSES OF KPNA2 HIGHLIGHT ITS MULTIPLE POTENTIALS IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the current study, the authors provide a relatively comprehensive analysis of molecular features of KPNA2 in hepatocellular carcinoma (HCC) by using multi-omics-based analyses. This suggests a potential association between KPNA2 and the possibility of using molecular features of KPNA2 as diagnostic and prognostic prediction tools for HCC.

The whole study was well conducted and the manuscript is well written. However, there are still some questions/problems that are needed to be resolved before final acceptance.

1. What are the potential molecular mechanisms that KPNA2 may regulate the genesis and development of HCC? The authors should provide a more detailed introduction about this in the Introduction session based on current knowledge.

2. Please move the data in the supplementary figure 1 to figure 1 because it is critical to the main story. The same action should be performed for the data in figure S4.

3. In the discussion, the authors also describe that “KPNA2 could affect carcinogenesis by mistaken translocation of cancer-associated cargo proteins such as c-Myc and PLAG1, which could be found in the liver-specific KPNA2 PPI network.” Please provide reference to support that KPNA2 could affect carcinogenesis by mistakenly translocating c-myc.

4. Please add the reference for the sentence “Liver is a central immunomodulator that ensures organ and systemic protection while maintaining immunotolerance.”

5. Please re-check the whole manuscript to resolve the grammatical problems. If possible, please rephrase some of the long sentences into short ones, which can make the description easier for the readers to understand.
Experimental design: 1. Based on the current study, KPNA2 seems to play its role in the hepatic cells by transporting a variety of proteins between the cell nucleus and cytoplasm. However, at the last of the study, the authors examined KPNA2 in the plasma. Are there any studies suggesting plasmic KPNA2 primarily comes from the liver? Is it possible that the plasma KPNA2 may be released by other cells, e.g. immune cells or from other organs? If yes, this data may not be suitable to indicate the alteration of HCC or the change of KPNA2 in HCC.
Validity of the findings: 1. In the discussion, the authors say that “the constitutive risk models involving CpG sites methylation are presumably more predictive for HCC prognosis than KPNA2 expression level alone.” Please provide the evidence to support this conclusion.

2. Interestingly, the authors discussed that KPNA2 could affect carcinogenesis by mistakenly translocating cancer-associated cargo proteins, while a truncated KPNA2 may inhibit the nuclear import of p53. Are there any studies showing how KPNA2 distinctly regulates the transport of oncogenes and tumor suppressor genes?
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: MULTIOMICS-BASED ANALYSES OF KPNA2 HIGHLIGHT ITS MULTIPLE POTENTIALS IN HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: I would like to thank the authors for addressing my initial comments. The authors have provided a nicely detailed and thorough response to the comments from the previous review and have addressed my major concerns. The manuscript now reads with greater focus and clarity. I would like to recommend this manuscript to be published on the journal.
Experimental design: None
Validity of the findings: None
Additional comments: None


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: MULTIOMICS-BASED ANALYSES OF KPNA2 HIGHLIGHT ITS MULTIPLE POTENTIALS IN HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: I appreciate the authors for providing a thorough rebuttal letter. Based on the replies, I have two further questions.

1. Based on the current study, KPNA2 seems to play its role in the hepatic cells by transporting a variety of proteins between the cell nucleus and cytoplasm. However, at the last of the study, the authors examined KPNA2 in the plasma. Are there any studies suggesting plasmic KPNA2 primarily comes from the liver? Is it possible that the plasma KPNA2 may be released by other cells, e.g. immune cells or from other organs? If yes, this data may not be suitable to indicate the alteration of HCC or the change of KPNA2 in HCC.

Authors’ reply: -Thank you for your reasonable and thoughtful suggestion. KPNA2 is indeed expressed in most normal tissues, up-regulated in a variety of organ originated cancers, and is a plasma protein. We examined the plasma KPNA2 level in HCC patients and normal individuals given that their most other tissues and organs were comparable except the difference of livers between the two groups. It has to be admitted that if other tissues and organs weigh a lot in KPNA2 release, the plasma KPNA2 could not accurately reflect the change of KPNA2 in HCC. It needs to be further verified in future. Thus, we include it as another potential explanation for the ELISA outcome in the Discussion section. (Line: 452-456)

Q: Can the authors provide evidence to support that “most other tissues and organs were comparable except the difference of livers between the two groups”?

2. Interestingly, the authors discussed that KPNA2 could affect carcinogenesis by mistakenly translocating cancer-associated cargo proteins, while a truncated KPNA2 may inhibit the nuclear import of p53. Are there any studies showing how KPNA2 distinctly regulates the transport of oncogenes and tumor suppressor genes?

Authors’ reply: -Most studies focused on identifying the cargo proteins of which KPNA2 mediated nuclear localization. Both oncoproteins and tumor suppressor proteins are nuclear translocated with KPNA2 such as E2F1, OCT4, c-Myc, p53, p27, MDC1, FGF1/2, LEF-1, CHK1, BRCA1, S100A2, S100A6, RECQL, RAC1, p65, JNK1, STAT3, c-Jun, NBS1, TBP-2 (Han & Wang 2020; Martinez-Olivera et al. 2018; Zhou et al. 2021). Then, their downstream signaling including PI3K/AKT, P53, MAPK, cell cycle, Wnt/β-catenin, and EMT-related pathways affect the growth, invasion, and metastasis of tumors (Han & Wang 2020). Given that KPNA2 binds to oncoproteins and tumor suppressor proteins indistinctively, it might depend on oncogenic or tumor suppressive signaling to distinctively regulate the transport of oncoproteins and tumor suppressor proteins. For example, KPNA2 was one of candidate target genes of p53 which inhibits KPNA2 in liver cancer to suppress tumor cell growth. Moreover, lncRNAs, microRNAs, and transcriptional factors such as E2F1 (Drucker et al. 2019) have been reported to regulate KPNA2 expression.

Q: Could the authors add their reply for this question into the discussion part?
Experimental design: N/A
Validity of the findings: N/A
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: MULTIOMICS-BASED ANALYSES OF KPNA2 HIGHLIGHT ITS MULTIPLE POTENTIALS IN HEPATOCELLULAR CARCINOMA
Review round: 3
Reviewer: 1

Output:
Basic reporting: All my questions were satisfactorily answered by the authors.
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: KEY AUXIN RESPONSE FACTOR (ARF) GENES CONSTRAINING WHEAT TILLERING OF MUTANT DMC
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript identified 67 ARF genes encoding auxin response factor in wheat, putatively involved in IAA signaling to control wheat tillering. This study provided valuable clues for functional characterization of ARFs in wheat. However, some data must be re-organized and re-analyzed. There are also too much grammar errors in the manuscript, which must be improved in language.

Some comments:

1: In line 52, the “Oryza sativa L.” should be added between rice and (Wang et al., 2007) as the same as maize, millet and so on.

2: Statistical analyses are not described in the materials and methods. A section should be added to describe these. Subsequently, significant differences should be examined between WT and dmc in Figure2, Figure 10 and Figure 11.

3: I am confused why the author chose 10 μM IAA to spray the wheat seedlings (Figure 3)? Did the authors perform assay that would generate curve at the different concentrations?

4: Quantitative real-time PCR experiments normalisation is usually conducted with at least two validated reference genes. Please provide new experimental data with a second control gene to show stable expression of the first reference gene under all experimental conditions.


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: KEY AUXIN RESPONSE FACTOR (ARF) GENES CONSTRAINING WHEAT TILLERING OF MUTANT DMC
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors conducted a systematic and comprehensive analysis of wheat ARF family genes, providing valuable clues for functional characterization of ARFs in wheat. Analysis of wheat ARF family genes suggest that TaARFs and IAA signaling were involved in controlling wheat tillering, and the abnormal expressions of TaARF11 and TaARF14 were major causes constraining the tillering of dmc mutants. This study provides a necessary foundation for wheat tillering regulation mechanism and for breeding by increasing tillers to increase yield.
1. Please add statistical results for qRT-PCR data in Figure 10 and Figure 11. For readers’ convenience, the legends should be more detailed, and key genes should be emphasized in order to get a clear conclusion directly from your figure.
2. Due to the mechanism of auxin in regulating tillering has been reported in Arabidopsis and rice, more discussion should be given about the function of auxin in tillering.
3. Please reconsider if it is necessary to emphasize dmc mutant in the title.


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: CORTINARIUS SUBSALOR AND C. TIBETICISALOR SPP. NOV., TWO NEW SPECIES FROM THE SECTION DELIBUTI FROM CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the present manuscript, Cortinarius subsalor and C. tibeticisalor belonging to the section Delibuti, are described from China as new to science, which is clear and unambiguous.However, 1) the English need to polish by a native speaker; 2) the latest literature references need to update; 3) in instruction part, the author need to add a paragraph according to the molecular systematics of this genus or section;
Experimental design: OK
Validity of the findings: 1) Lack of the treebase datasets; 2) need add the MP analysis to this research;
Additional comments: The molecular data also support C.subsalor and C.tibeticisalor as new species. The phylogenetic analyses and biogeography of sect. Delibuti are discussed and a key to the species of this section currently known in the world is provided, which is good for supporting two new species. This research is significative to push the diversity of Chinese fungi. However, some commens also give to communicate:

1) there are many latest references missing about this genus  in instruction part, the author have to add them;

2) please add the paragraph in instruction partabout the phylogeny of this genus;

3)  add the BLAST result for top 10 taxa, which is easily get the identified accuracy, including the related 5 parameters;

4) this basidiospores data is ave, please add the holotype specimen dataset single in here, which is easily to get for reader.

5) this epithet need to change, in which both new species are similar to C. salor in same paper, which is more   far-fetched;

6) add a distribution diagram of this group in China;

7) mark which is type specimen in this tree;

8) add the drawing picture for hyphae and basidiospores at least in Figs. 2 and 3;

other comments see the revised PDF.


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: CORTINARIUS SUBSALOR AND C. TIBETICISALOR SPP. NOV., TWO NEW SPECIES FROM THE SECTION DELIBUTI FROM CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: A few English sentences (or words) need to be modified. Please refer to the comments in the attachment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The MS could be published after minor revisions.

The authors are suggested to to make the following revisions:
1)A few English sentences (or words) need to be modified. Please refer to the comments in the attachment.
2)Please make some checks and improvements to the "Key to species of sect. Delibuti".  For example, the treatment of  species "C. largodelibutus" seems not so good.
3)For other suggestions for improving the MS, please refer to the notes in the attachment.


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: CORTINARIUS SUBSALOR AND C. TIBETICISALOR SPP. NOV., TWO NEW SPECIES FROM THE SECTION DELIBUTI FROM CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: In the present manuscript, two fungal species, Cortinarius subsalor and C. tibeticisalor belonging to the section Delibuti, are described from China as new to science. The comments on areas where the article have meet my standards with suggested improvements.
Experimental design: Good
Validity of the findings: ok
Additional comments: 1) The drawing picture for hyphae and basidiospores in Figs. 2 and 3 are good.
2) It is better to add basidiospores data set of both holotype and AVE, but only holotype data.


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 COMPREHENSIVE REVIEW AND CALL FOR STUDIES ON FIREFLY LARVAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors submitted a review on lampyrid larvae and I would expect that a review means to set a topic into a wide perspective which was not clearly present in original studies dealing with specific questions. The authors did not try to do anything like that.
I expected an approach based on relationships, but the review is a repetition of isolated findings.
Any comparison with other groups, i.e., a wider perspective, is missing.

Concerning PeerJ requirements the manuscript fulfils the criterion for acceptance.
It contributes to the volume of scholarly literature, although it does not contribute to knowledge.
Experimental design: Simple design - the list of literature with findings rewritten.
Validity of the findings: Some 'unpublished' info reported without any hard information and experiment design
Additional comments: I made numerous comments into the manuscript. Hopefully, you find them useful.


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 COMPREHENSIVE REVIEW AND CALL FOR STUDIES ON FIREFLY LARVAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript introduces an interesting topic about the larvae of fireflies (Coleoptera: Lampyridae). It is a literature review covering  231 publications. Authors identified main important topics related to the firefly larvae and tried to find out the geographical and topical biases and main gaps in the firefly larval research.

Generally, I like the manuscript and topic it presents very much. I think this has a great potential to be a landmark study which can be highly cited by all subsequent researchers on firefly ecology, morphology, biology, behavior, and other aspects. It fits to the scope of PeerJ, too.

The text is generally rather clearly written but I think some additional proof reading by English native speaker would be beneficial (a few sentences hard to read, plural vs singular, etc).

Introduction provides the most important background of the field, however, I think that the last part with results and call for future work does not belong there, as it is more appropriate for Discussion or Conclusions. After removing that last part, Introduction would be quite short, and could be definitely improved by adding information about the geographic distribution, classification, species richness, etc. of Lampyridae to provide the readers a bigger picture.

I do not know exactly the policy of PeerJ about using taxonomic names in manuscripts, but usually author plus year or at least author should be mentioned for each species and genus-name when it appears first time in the text. I keep this on editor whether or not this is needed.

Regarding figures, I really do like figures 1 and 2, which show the morphology of firefly larvae and are really well-drawn. This may attract attention definitely. However, the legends could be improved in my opinion. For example, in Figure 1B-E, some examples of genera or species could be added in order reader better understands where to find these variations in lantern disposition. The same for all drawings in Figure 2. What is more, the legend of figure 2 has some introductory story which should probably go into main text. This might be, however, some format requirement of PeerJ (?) because I see similar explanatory sentences also in Figures 3-5.
Results given in Figures 3-5 can be more user-friendly when they are put in the Table. Alternatively, it would be also interesting to merge these three aspects together, in order readers can see how many papers for example focused on the Luciolinae (aspect 1) behavior (aspect 2) in Asia (aspect 3), or Photurinae life cycle in North America, etc. This would allow better discussion and better presentation of results, and conclusions.

Regarding the organization of main text, there is twice a conclusions part called "Conclusions" (in between Methods and Discussion, which is weird to me), and "Concluding remarks" (at place where it is supposed to be, i.e. at the end of the manuscript, after discussion). These should be probably merged into one.

There are some minor issues in References; they should be double-checked for the required format of PeerJ.
Experimental design: This is to me probably the most problematic part of the presented study. Authors divided World into eight regions and tried to show which regions are understudied regarding the larval fireflies. However, they did not use any of the official realms/regions classifications defined by Wallace and used for ages (i.e, Oriental, Neotropical, Palaearctic, etc.) or still quite recently by Holt et al. (2013). Instead, they divided whole World into eight regions, from which four (half!) is America (South America, North America, Central America, and the Caribbean). On the other hand, for example, whole Asia (i.e., from Turkey and Saudi Arabia through Mongolia to Indonesia, covering many completely different areas in terms of geography, aridity, temperature, flora, etc.) is one region only!
Authors should definitely either provide very good explanation for their division of the World, or change this completely and hence also results, conclusions and respective figure. It seems to me really inappropriate to compare the Caribbean with whole Asia...

Further, I miss for example Liew and Schilthuizen (2014) PeerJ in this review, and there are also some citations therein (which I have not examined, however).
Validity of the findings: There are quite a number of "hypotheses yet to be tested" mentioned in the discussion, these can be probably summarized in the Conclusions part at the end of the main text.

Also, see my remarks on the problematic division of the World into eight proportionally totally different regions by authors, which affected the final conclusions.
Additional comments: Some changes are required in order the manuscript is improved to the higher quality, and conclusions can be better supported by data (i.e. geographic aspect problem).
However, I want to make clear that I definitely support publishing this manuscript (but improved version) in PeerJ!

Major issues are reported in previous sections, and most of my comments/suggestions are embedded directly into the PDF attached.


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 COMPREHENSIVE REVIEW AND CALL FOR STUDIES ON FIREFLY LARVAE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall, the review manuscript uses clear and professional English throughout, although some contexts can be improved. It contains references to the most up-to-date work on firefly larva but may not be comprehensive enough. It provides sufficient background which fits into the broader field of knowledge about firefly larva's biology aspects with appropriate references.

However, the review manuscript structure does not fully conform to an acceptable format of PeerJ's 'standard sections'. See line numbers 101-105. The Conclusion heading should be after Survey Methodology and before Acknowledgement sections. In addition to that, there is another conclusion section (under subheading). See line numbers 388-408. The headings/sections should be the topic of interest, not the "Discussion".

Figures are relevant to the article's content but not appropriately labelled, particularly category title of axis X and Y of Figure 3-5.

This review is essential and timely because studies on firefly larva are scarce compared to studies published on adult fireflies. The authors made a good introduction to fill the gaps in larval biology knowledge based on published literature.
Experimental design: The Survey Methodology needs a bit more clarity, and from the current form of writing, the sources of literature are not yet exhaustive. A comprehensive review should also include extensive searching, which means that the authors should demonstrate that their reviews are not biased or skewed toward specific browsers or databases when possible. Instead of browsers, one would prefer to start first with the most comprehensive and well-organised databases such as Scopus and Web of Science (although pay-walled). There would be discrepancies, but there would also overlapping results among these databases. The cutoff dates of review (year of publications) are not stated.

The choice of string search or keywords is essential, and usually, we would make it more variable not to be too restrictive. For instance, using Web of Science, "Lampyridae" AND "larva" will return 97 results, but if you use "firefl AND larva" (using asterisk to denote "fireflies or firefly", "larva or larvae"), you will get even more papers. In another instance, using the exact string searches, Scopus returned 109 and 208 results, respectively. If not the entire WOS, the authors might want to look into the Zoological Records database (partly owned by WOS). Also, authors might want to dig deeper into classic studies in Biodiversity Heritage Library (BHL) regarding taxonomy and behaviour studies and not just rely on FIN's website, which is merely a list of references. There are cases where literature is inaccessible because of a pay-wall. It is worth mentioning if some literature came from author's personal copies. From the accumulation of literature, perhaps it would be great to map the critical points in the study of Lampyridae by chronological order: e.g. when the study on larva sparked the interest, and then pinpoint where the gaps start appearing.

The review needs more improvement in its organisation. Please check again the formatting and see examples from other literature review published in PeerJ.
Validity of the findings: This manuscript aimed to review and indicate key gaps in the biology of firefly larvae based on available literature. The authors reviewed 231 papers and categorised the findings based on geographic region, subfamilies and five major themes. Although they have demonstrated an excellent reference to literature, especially on the morphology section, the argument they presented is not well-developed to meet the goal set out in the Introduction.  In line 87, "only papers.." it is unclear whether the authors referred to the 231 papers or were the papers examined a subset of 231 papers?

I wasn't sure whether the review is about trends in research of firefly larva or is it about gaps of knowledge in larval biology? It's somewhat in between.

The Conclusions are too vague. As they call for more studies, they did not explicitly outline what gaps we can learn from this review and identify what future directions we could take. The questions in the Conclusions are too general, and the authors already pointed some of them out in the texts. For instance, on what firefly larvae eat, authors have already addressed a few studies that showed evidence of diet type of larvae. However, what is lacking (gap) is that firefly larva is a diet specialist; hence different firefly species would require different prey species. Prey species availability would also depend on the habitat where firefly species thrive. The future studies are challenging and probably still skewed to the well-studied region or subfamilies (which is common)- this is where the authors would provide where best to tackle this issue.
Additional comments: I commend the authors for their excellent effort to come up with a review on firefly larva. Although the manuscript can be considered as a topic of high interest, the current form of writing is not yet ready for publication and it needs major improvement before it can be accepted for 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: A COMPREHENSIVE REVIEW AND CALL FOR STUDIES ON FIREFLY LARVAE
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is much better than previous version. Authors made a really good job to improve the manuscript.
Experimental design: This is much better than previous version. Authors made a really good job to improve the manuscript.
Validity of the findings: This is much better than previous version. Authors made a really good job to improve the manuscript.
Additional comments: I am happy to see the improved version of this manuscript. Now it is really worth to be published almost as it is. There are only a few minor issues in the manuscript:

line 27: Results: "only 139 species in 47 genera" - when reader does not know how many species and genera are in Lampyridae, it is difficult to understand if 139/47 is a small or high number...

line 73: Methodology: "lampirid" really lampirid or lampyrid?

line 165: bioluminescence in Elateroidea - authors cite only Bocakova et al. (2007), however, to cover also more lineages and new findings I would suggest citing also e.g., Bi et al. (2019) ZooKeys and Fallon et al. (2018) eLife.

line 275 - "emit bioluminescence" better: emit light?

line 283 - Linnaeus, 1758 should not be italicized

line 293 - "larval bioluminescent" should be probably "larval bioluminescene".

line 376 - "))" instead of ")" after Rosa et al. 2020

line 444 - e.g. - a dot is missing

line 454 - lampyrid fireflies sounds odd to me...just fireflies would be enough

line 455 - lampyroid should always be within quotation marks as this is not the real superfamily but only an informal name for the clade consisting of fireflies and relatives


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 COMPREHENSIVE REVIEW AND CALL FOR STUDIES ON FIREFLY LARVAE
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript has improved substantially and most of the reviewers' questions and concerns have been addressed. However, there are a few points the authors may need to double-check before the manuscript can be published:

73 Please check if this is a typo. "Lampiri" or "Lampyri"?

155 heavily hardened sclerites; no, not in the aquatics

156 they describe shape as if it is a box with top bottom and sides but that is not the shape it is more like a top and bottom, the bottom including the lateral elements

217 NOT pleurae. Singular is pleuron, pleural is pleura

222 NOT Pyrophanes. A Pyrophanes larva having gills is totally unsubstantiated

224 Use currently accepted name. Sclerotia substriata (Gorham, 1880)

228 not Aquatica it is Sclerotia

336 Aquatica larvae can move, not sure if they swim, across the bottom of ponds

337 Sclerotia larvae actually swim upside down just below the water surface and parallel to it with just the tip of the abdomen and thus the terminal spiracles piercing the water surface. They are back swimmers as they swim on their backs but they still swim towards their head end

360 Typo: "void". Change it "to avoid"

369 Check Fu & Ballantyne 2008 where they noted Pygoluciola qingyu will attack ants head on

Figure 1 Genera names should be italicised
Figure 2 Same comment as the above. Scientific names should be italicised


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: SARS-COV-2 SEROPREVALENCE IN A HIGH-ALTITUDE SETTING IN PERU: ADULT POPULATION-BASED CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: OK
Experimental design: OK
Validity of the findings: OK. Just attend to some concerns.
Additional comments: The authors analyzed the prevalence of antibodies to SARS-CoV-2 in Cusco, after the first epidemic wave. They found a prevalence of antibodies in all the populations groups over 30%, with positive correlation of antibody presence with household positivity and negative correlation with wearing mask all times. The information is relevant and interesting. Some minor concerns should be addressed for publication of this manuscript.

Major comments:
1.	The authors described in Discussion differences in prevalence between countries and inside Peru, In addition to comparing the reported sensitivity of the tests used in each study, it would be interesting to mention the antigens used in each of these tests, and the limitations of comparing prevalence between different studies because of this heterogeneity.
2.	In addition, although the companies argues for very high sensitivity and specificity, did the authors perform any quality control test to validate the performance of the test used: for example, sensitivity with sera from patents with known previous COVID-19 and specificity with sera collected before the pandemic?


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: SARS-COV-2 SEROPREVALENCE IN A HIGH-ALTITUDE SETTING IN PERU: ADULT POPULATION-BASED CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Huamani et al. assessed the seroprevalence of SARS-COV-2 in a high-altitude setting in Peru. Overall, it is well written and the subject is interesting. I enjoyed reading it. The method section is clear and the results are significant. The discussion and conclusion are in accordance with the results.
Experimental design: The study is well designed and methods and analyses are clear.
Validity of the findings: The results are significant and presented properly. There is no comment.
Additional comments: In the introduction, it would be good that some information about the number of death and confirmed cases for Peru be added.


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: SARS-COV-2 SEROPREVALENCE IN A HIGH-ALTITUDE SETTING IN PERU: ADULT POPULATION-BASED CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is an interesting study, following the trends of regional studies of SARS-CoV-2 seroprevalence in Peru.
Their findings highlight the importance of such information.
Experimental design: The experimental design is appropriate to address the research question.
Validity of the findings: Conclusions are well stated, linked to original research question.
Additional comments: This is an interesting study, following the trends of regional studies of SARS-CoV-2 seroprevalence in Peru.
Their findings highlight the importance of such information.
I only recommend to extend the Discussion regarding the implications of the findings coupled with the ongoing vaccination plan. Secondly, Figure 1, please add a relative map showing South America and Peru, in order to international audience have more clear the location of the study.
Please include the recent publications, even on on Peer J, of other similar studies in Peru, e.g. in Chiclayo or Lambayeque, at Lima, the capital in EClinical Medicine, among others.


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: SARS-COV-2 SEROPREVALENCE IN A HIGH-ALTITUDE SETTING IN PERU: ADULT POPULATION-BASED CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 4

Output:
Basic reporting: In this study, Huamaní et al. estimated the seroprevalence of COVID-19 (?) in three regions of Cusco, Peru.
I found this study interesting and novel but there are many flaws that need to be addressed.
Experimental design: Please see below.
Validity of the findings: Please see below.
Additional comments: ABSTRACT:

It should indicate that the participants were ≥18 years old. The title should also indicate that the study included an adult population.

The statement “Little evidence exists about the prevalence of COVID-19 infection at high altitude.” is not accurate. The authors have not performed an exhaustive search of the literature. There are numerous studies that have estimated the prevalence of SARS-CoV-2 in high-altitude cities. This also applies to INTRODUCTION.

Based on the aim of the study (the estimation of the seroprevalence of COVID-19 in Cusco), this reviewer assumed that this study used a representative population of Cusco, but that does not seem to be the case. The aim of the study was not specifically addressed. Therefore, the conclusions of the study are misleading. Did the authors estimate the seroprevalence of COVID-19 and SARS-CoV-2? How was COVID-19 diagnosed?

Since the analysis of antibodies did not discriminate between IgM and IgG, then the authors should indicate that the diagnosis of SARS-CoV-2 infection was based on the identification of anti-SARS-CoV-2 total antibodies (IgM or IgG). More importantly, is there a valid justification for not performing a separate analysis for IgM and IgG? If data is available, this analysis should be performed and included in the study.

MATERIALS AND METHODS

It was mentioned that the cross-sectional study was part of a longitudinal cohort study. Which longitudinal study are the authors referring to? Has this longitudinal study already been published? If not, then further details of the main (longitudinal) study have to be provided.

At what temperature were the samples stored?

It was mentioned that up to 1800 participants were enrolled. However, Results show that 1924 participants were analyzed. This needs clarification.

Was there any difference in ethnicity across regions?

How was obesity diagnosed? Was it self-reported?

RESULTS:

The study aimed to estimate the seroprevalence of SARS-CoV-2 antibodies in Cusco, with emphasis on a high-altitude population. Since data were shown for 3 regions (one at low altitude), it is natural to expect a deeper analysis of the ORs for seroprevalence by altitude. Please provide the analysis of the OR for seroprevalence at Cusco City and periphery of Cusco vs. Quillabamba (reference), adjusted for age, gender, comorbidities, and population density.

Table 1: Are the values expressed as the media? Did you mean median or average? What do you mean by “Previous COVID-19 test”? Do data show only positive results? What kind of test? The statement “Asymptomatic during pandemic” is meaningless. Please specify the period/duration relative to the serological test performed in the study. Please change “Personal history” for “Self-reported medical history” or “Self-reported comorbidities”. Why information on other important comorbidities were not collected? In “personal history”, did you mean “obesity”? To be consistent, Tables 1 and 3 should show data for all comorbidities. Was type 2 diabetes actually discriminated from type 1? What were the diagnosis criteria used? Was information on gender (orientation) collected? Were the participants asked about their biological sex?

Table 2: Why does the title of the table refer to the prevalence of serum antibodies to SARS-CoV-2 in the general population in a high-altitude setting in Peru” if Quillabamba (~1000 m elevation) was included? Was not altitude defined (in the study) as an elevation over 2500 m? The comparison of prevalence across regions is interesting. Quillabamba had the lowest prevalence. This must be further discussed and contrasted with the findings from numerous previous studies (Quevedo-Ramirez, Respir Physiol Neurobiol 2020; Accinelli, Arch Bronconeumol 2020; Intimayta-Escalante, High Alt Med Biol 2020; Woolcott, High Alt Med Biol 2020; Seclen, Diabetes Res Clin Pract 2020, Perone Sci Total Environ 2021; Cano-Pérez, Am J Trop Med Hyg 2020.). These references should be included in Introduction. Please correct: ”Models adjusted by” (adjusted for…”). What do the authors mean by “adjusted for study setting”? Data cannot be adjusted for the same variable. This should be clarified in the table footnotes.  Table should show the n size for each variable/category. Data show that only 50% of all who died had positive antibodies. Is this correct? This deserves extensive discussion. How was BMI measured?

The authors presented some of their data as adjusted odds ratios, however, all odds ratios were interpreted as probabilities. That is inappropriate and must be corrected.

Correct grammar and spelling throughout the manuscript. I would suggest the manuscript be revised by a native English speaker.

Table 3: “DEVELOPED SOME OF THESE SYMPTOMS IN THE LAST 3 MONTHS”. Three months prior to what date? HBP: does this acronym stand for high blood pressure? Did you mean hypertension? How was Cardiac disease and Renal disease defined? Could you explain in Methods? Could you use the proper terms for “Smell / taste alteration”? What do you mean by “Always wear…”. This does not sound accurate. I doubt the subjects used protective gear at home. Please clarify.

Figures for review should be provided using a common format. Not all have the applications to open EPS files. I could not see Figure 1.

DISCUSSION:

The fact that the first case in Cusco was reported in March 2020 and the blood samples collected in September 2020 may have introduced an important bias in the estimates of the prevalence considering that IgG and IgM were not separately analyzed. This is a major limitation of the study that should be discussed.

The comparison of prevalence across regions is interesting. Quillabamba had the lowest prevalence. This must be further discussed and contrasted with the findings from numerous previous studies (Quevedo-Ramirez, Respir Physiol Neurobiol 2020; Accinelli, Arch Bronconeumol 2020; Intimayta-Escalante, High Alt Med Biol 2020; Woolcott, High Alt Med Biol 2020; Seclen, Diabetes Res Clin Pract 2020, Perone Sci Total Environ 2021; Cano-Pérez, Am J Trop Med Hyg 2020.).

This study has numerous limitations, but I do not see any effort at self-criticism. It has been referred that this study is not representative of the whole population of Cusco. However, nothing is mentioned about the people who are hospitalized. This population may represent a large proportion of cases with seropositivity. Also, it should be discussed the rate of acceptance or rejection to be surveyed. Was there any difference on the participation among and between families across regions?

CONCLUSIONS:

The conclusion that “the altitude seems not to influence estimates” is not accurate. Your data suggest the opposite. I would like to see the unadjusted ORs for all your estimates in Table 2. In addition, as I suggested in my previous comment, the ORs estimates of seroprevalence for Cusco and peripheral regions vs Quillabamba should also be adjusted for comorbidities and population density, in addition to age and sex. Based on the additional findings from the analyses requested, discussion should be modified 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: SARS-COV-2 SEROPREVALENCE IN A HIGH-ALTITUDE SETTING IN PERU: ADULT POPULATION-BASED CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Satisfactory
Experimental design: Ok
Validity of the findings: Valid
Additional comments: The authors addressed adequately the concerns of the reviewers.


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: SARS-COV-2 SEROPREVALENCE IN A HIGH-ALTITUDE SETTING IN PERU: ADULT POPULATION-BASED CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Thank you for addressing most of my comments. Still, some points have not been properly addressed.
Experimental design: The work needs further revision.
Validity of the findings: The work needs further revision.
Additional comments: Given the limitations of the study acknowledged by the authors, and the presence of underadjustment (for instance, prevalence ratios were not adjusted for comorbidities) in a population-based study, the Conclusions of the study (Line 337) need revision. The statement "altitude seems not to influence estimates as the only variable, as done by other factors such as population density or population size" is not supported by the findings. The conclusions stated in the Abstract are supported by the main findings of the study. Thus, the Conclusions at the end of the manuscript should be consistent with those stated in the Abstract.

A significant part of the study focuses on the factors associated with SARS-CoV-2 seropositivity. The authors attempted to explain this association using regression analysis adjusting only for age, sex, and study setting. Thus, the limitation of undeardjustment must be acknowledged in the Discussion, more so when the authors argument  to exclude many variables from their final model was based on the lack of significant correlation between the excluded variables and the dependent variable.

The authors are reluctant to discuss their findings in relation to those from numerous “ecological” studies published in the literature. These uncited studies have examined the prevalence of SARS-CoV-2 or COVID-19 in regions located at high altitude (Quevedo-Ramirez, Respir Physiol Neurobiol 2020; Accinelli, Arch Bronconeumol 2020; Intimayta-Escalante, High Alt Med Biol 2020; Woolcott, High Alt Med Biol 2020; Seclen, Diabetes Res Clin Pract 2020; Perone, Sci Total Environ 2021; Cano-Pérez, Am J Trop Med Hyg 2020). This is surprising and disappointing considering that their analysis included populations from three regions in Cusco, two located at high altitude and one at low altitude. The rationale for the analysis of these three Cusco regions only must be stated in Methods.


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: SARS-COV-2 SEROPREVALENCE IN A HIGH-ALTITUDE SETTING IN PERU: ADULT POPULATION-BASED CROSS-SECTIONAL STUDY
Review round: 3
Reviewer: 1

Output:
Basic reporting: Thank you for addressing all my comments.
Experimental design: no comment
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: CAUTION AHEAD: REASSESSING THE FUNCTIONAL MORPHOLOGY OF THE RESPIRATORY ORGANS IN AMPHIBIOUS SNAILS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a very straightforward paper criticising the findings/interpretations in another paper. The arguments are well presented and appropriately phrased, even though it is evident that the author is not happy at all about the approach taken in the criticized paper.
Experimental design: Not primary research (even though the arguments are partly based on primary research in which the author was involved), so I will not comment on the experimental design.
Validity of the findings: I am no expert on lung/gill morphology of gastropods, so I have to take the arguments at face value. Given that the author has pointed out in much detail where he finds fault with the conclusions of the criticised paper by Mueck et al. and gives a balanced view acknowledging uncertainties, I am left with the impression that the Mueck et al paper does indeed have quite a few flaws. In the very least, this will inspire readers to draw their own conclusions in light of the arguments made in this MS, so I think it is worthy of publication even if there should be scope for discussion about some of the points made. The only thing I am wondering about is why this is not published in the same journal as the criticised paper - has this been tried? Surely an editor must take a criticism of this scope into account (and offer a reply to the authors of the criticised paper).
Additional comments: Line 162: I think "do not show" is missing before "any"? The sentence would make more sense then.


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: CAUTION AHEAD: REASSESSING THE FUNCTIONAL MORPHOLOGY OF THE RESPIRATORY ORGANS IN AMPHIBIOUS SNAILS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an unusual paper in that it is mainly a critique of (two) recent publications (Mueck, Deaton & Lee 2020a,b: J Shellfish Res 39(1): 125-132, 133-141) rather than an original contribution. It is obvious (and I agree with the details) that the criticized papers do have a number of shortcomings (confusions and misinterpretations), nevertheless:

(1)	This critique should preferably be published in the original journal of the criticized papers (Journal of Shellfish Research) and

(2)	In any case the criticized authors (Mueck, Deaton & Lee) should be given the possibility to answer the critique, and their response should be published side by side with this critique in order to be fair.
Experimental design: no comment
Validity of the findings: see above under basics
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: CAUTION AHEAD: REASSESSING THE FUNCTIONAL MORPHOLOGY OF THE RESPIRATORY ORGANS IN AMPHIBIOUS SNAILS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a well written and justified critique of Mueck et al. 2020. The author does a nice job of pointing out major errors and misinterpretation in Mueck et al. 2020 and the implications such issues will create for interpretation and understanding of the biology, evolution, and functional morphology of ampullariids. Given the large number of errors and the ease at which they could have been caught in peer review, this should never have made it to press. Unfortunately, this happens, which in turn necessitates corrective reviews such as this one. Hopefully Mueck et al. 2020 and the editors of the editors of the Journal of Shellfish Research will provide a link to this paper once it is published so others less familiar with ampullariid anatomy won't be misled and the errors compounded.

I only noted a few minor spelling and grammatical errors, but nothing of substance that would necessitate revisions.
Experimental design: There were no experiments, as this was a critique of a previously published study. The author does a nice job of laying out the issues and references them very well.
Validity of the findings: The underlying premise of the critique is robust and fully supported with clear examples and solid citations. The conclusions are wells stated and limited to the points raised throughout.
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: EFFECTIVENESS, TOLERABILITY AND SAFETY OF DIRECT ACTING ANTIVIRALS IN MEXICAN INDIVIDUALS WITH HEPATITIS C VIRUS GENOTYPE-1 AND PREVIOUS PEGYLATED INTERFERON AND RIBAVIRIN THERAPY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Although the manuscript is decent enough in clarity, it needs to be revised accordingly to further enhance the clarity and unambiguous. Also, I strongly believe that the English language should be improved to ensure that an international audience can clearly understand your text. Thus, I suggest the authors have a colleague who is proficient in English and familiar with the subject matter review the manuscript, or contact a professional editing service.

At times, authors have used the old citations (around 10 years old) where a lot of advances and improvements have taken place within the literature.

Table and figure titles and legends have to be revised according to the context being reported/illustrated (especially look for grammar and comprehension)
Experimental design: This is original research within the aims and scope of the journal, where the research question was well-defined that fills an identified knowledge gap. Rigorous investigation has been performed to a technical and ethical standard. Although methods were described sufficiently, I suggest authors specifically mention the type of the study design in both abstract and the main text. Also, I am wondering about the gap (in years) between two sub-cohorts i.e., what about years 2015 and 2016 that are missing from both the sub-groups.
Validity of the findings: Meaningful replication encouraged where rationale & benefit to literature was clearly stated.  All underlying data have been provided, which are robust. The data on which the conclusions were based are made available. However, I believe the conclusions should be well-stated further based upon the results.
Additional comments: All the general comments can be found as annotations or comments in the manuscript pdf file itself. I recommend the authors to consider the comments provided while revising 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: EFFECTIVENESS, TOLERABILITY AND SAFETY OF DIRECT ACTING ANTIVIRALS IN MEXICAN INDIVIDUALS WITH HEPATITIS C VIRUS GENOTYPE-1 AND PREVIOUS PEGYLATED INTERFERON AND RIBAVIRIN THERAPY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Melendez Mena et al. report the effect of Hepatitis C treatment with Direct Acting Antivirals (DDAs) in a cohort of 83 Mexican patients who had not responded to IFN/ribavirin treatments. The study focuses on patients with genotype 1 strains.
The results show that while IFN/ribavirin treatment had failed in more that 60% of cases, DDAs resulted in sustained virological response in 97% of patients, with marked improvement of liver function and inflammation parameters.
This study is well conducted and the manuscript is well written, although the only novelty compared to the existing literature is the Mexican context.
I only have minor comments, mostly regarding the presentation of the data:
1) Line 56. Why “CTP”? Perhaps the authors mean “CPS”
2) Line 57. Why “despite”? All parameters improve and so it is expected that transaminases also decrease.
3) Line 62. CHC is not defined. Also, many acronyms are defined only in the abstract and that may confuse the uninitiated reader, since this is a very acronym-heavy manuscript. I suggest that the authors make sure all the acronyms are defined in the main text and, perhaps, that they are used more sparingly.
4) Sagnelli et al., 2013 is not the right reference here, since it does not mention anything about worldwide mortality.
5) Line 213-214 – It is not clear here how fibrosis was measured and I cannot see the results of these measurement in table 5 or anywhere else in the manuscript. Yet, lack of change in fibrosis is mentioned in the abstract and throughout the manuscript.  The author should explain this point more clearly.
6) Line 219. Could elevated bilirubinemia due to ribavirin treatment or does it occur also in –RBV patients? Could the author comment on this?
7) Line 221. Here and throughout the manuscript, the authors describe the treatment groups as +/-RBV, but they never explain how the cohort was divided for +RBV and –RBV treatment, why, and how many f patients were in each group. The authors should describe the experimental design in more details in M&M and clearly define the number of patient in each group.
8) The first part of the discussion, especially lines 241 and 247 is too detailed and the “data not shown” should be presented and described in Results.
9) Line 391. “Installation” does not seem to be the right word. Perhaps “onset” or “development”?
10) Fig. 1 is not referenced in the text and not described anywhere. It looks like the data of Fig. 1 are also shown in the various Tables, and I would suggest to remove it.
11) I suggest to move Supp table 1,2 and 4 to the main text, because these are very relevant data for the study.
Experimental design: The experimental design is adequate and the research is meaningful. Please see some of my minor comments under Basic Reporting asking for clarification of some aspects of the experimental design.
Validity of the findings: The finding are valid and relevant and the conclusions are well supported by the data.
Additional comments: None


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: EFFECTIVENESS, TOLERABILITY AND SAFETY OF DIRECT ACTING ANTIVIRALS IN MEXICAN INDIVIDUALS WITH HEPATITIS C VIRUS GENOTYPE-1 AND PREVIOUS PEGYLATED INTERFERON AND RIBAVIRIN THERAPY
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: No 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: EFFECTIVENESS, TOLERABILITY AND SAFETY OF DIRECT ACTING ANTIVIRALS IN MEXICAN INDIVIDUALS WITH HEPATITIS C VIRUS GENOTYPE-1 AND PREVIOUS PEGYLATED INTERFERON AND RIBAVIRIN THERAPY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Melendez Mena et al. report the effect of Hepatitis C treatment with Direct Acting Antivirals (DDAs) in a cohort of 83 Mexican patients who had not responded to IFN/ribavirin treatments.
This is the first revision of a manuscript that I have previously reviewed. The authors have addressed satisfactorily all my previous suggestions and I don’t have any further comment.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: No 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 CROSS-SECTIONAL STUDY OF THE NASAL AND FECAL MICROBIOTA OF SOWS FROM DIFFERENT HEALTH STATUS WITHIN SIX COMMERCIAL SWINE FARMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language is mostly clear, just some small editing required:
- Line 27: check sentence structure
- 30: clarify: each sample from an individual animal?
- 32 and elsewhere: 16S rRNA _gene amplicon_ sequencing (you didn't sequence RNA!)
- 130: /hr = ?
- 163: "separately" = unclear
- 166: diveristy = typo
- 182 and elsewhere: 0-1 decimals is sufficient and more adequate
- 196: belongs rathet to the next chapter?
- Fig 3 caption: the caption says FECAL microbiota in 3b, is this a typo?
- 222: very unclear. Do you mean individual OTUs or orders or what?
- 223: plyLum = typo
- 226 and elsewhere: please use tables rather than long lists of taxa.
- 266: unclear sentence, rewrite
- 321: was this really at the SPECIES level?
- 341: explain more clearly!

Tables:
- Table 3: this does NOT look like relative abundance data. What is the unit? Absolute counts can't be compared like this.
- Report ALL observed families / genera as a supplementary table.
- Report the parities etc. PER FARM (table!).

Figures:
- Fig 2: show P values in the fig.
- Fig 4: legends too small!
- Fig 4a: why are there several missing pools here?

Data:
- The data provided as attachment is NOT the raw sequencing data! The BIOM taxonomy table is also nice to have, though.
Experimental design: This is original primary research with a relevant and well defined research question. However, there are some major problematic aspects in the experimental design and several missing data which should be reported.

- Negative controls are not reported at all. The nasal mucosa is a low-abundance microbiota, and proper negative controls are absolutely essential to ensure validity of results. Please perform DNA extraction and sequencing from unused sampling instruments. This of course should have been done together with actual samples, but better late than never.
- The pooling of the samples is unfortunate, as it drastically reduces the statistical power and resolution of the data. Considering the low costs of 16S sequencing currently, I wonder if this is really absolutely necessary. Could you consider re-doing the sequencing individually?
- Some potentially significant background information is missing. Report details of feeding, and of culling protocols in each farm.
- The production stage of the healthy sows very likely strongly influences the microbiota. This must be more adequately considered (another reason to perform sequencing individually).
- Report how and when the samples were frozen.
- Report if the PureLink protocol includes mechanical lysis. This is critical.
- An old version of QIIME and an EXTREMELY old version of the Greengenes database were used. The bioinformatics need to be re-run using up-to-date version (preferably something else than Greengenes).
- Report read counts per sample type. Apparently these were very small per sample.
- Why was OTU based cutoff used, rather than read count based? The nasal microbiota samples could very well really be low diversity. This kind of cut-off may bias the results.
- Fig 3: the clustering doesn't seem very clear. Perform PERMDISP to check if it's actually just about dispersion differences.
- 269: Can't compare absolute read counts! Use relative abundances.
- 283: Impossible to compare OTU counts between studies! Alpha diversities perhaps a bit better, but should probably re-analyze the sequencing data from beginning.
- 295: Please analyze WHAT was were the differences revealed by PCoA.
- 316 and elsewhere: should also consider effects of feed!
- 325: Family level too broad for any meaningful comments.
Validity of the findings: This study provides useful baseline data on sow microbiotas, if the shortcomings are corrected.

Data: please see part 2.
- Negative controls are missing and the pooling drastically weakens the study.
- Much of essential metadata is missing.
- The sequencing data is not provided.

Conclusions: these are well in line with the reported results and not exaggerated or overly speculative. After complementing the study and reporting as suggested here, the authors may be able to interpret the results a bit further.
Additional comments: Studies like this are important and very welcome. I do hope that you are able to improve the work as suggested and would like to see the work published in the improved form.