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 GUIDELINE FOR REPORTING EXPERIMENTAL PROTOCOLS IN LIFE SCIENCES
Review round: 2
Reviewer: 2

Output:
Basic reporting: * basic reporting
** Clear, unambiguous, professional English language used throughout.
   No, I do not consider the language acceptable. It needs a thorough review.
   This has not been addressed.
   A subset of things that I spotted.
   1. line 17: I do not think 'Life Sciences' is supposed to be capitalized.
   2. line 29: 'when produced repeatedly' should read 'when executed repeatedly'? Not sure what the intent was here.
   3. line 30: 'the descriptions' -> 'their descriptions'
   4. line 37: 'what' -> 'What'
   5. line 89: oxford comma 'reviewers, and editors' (there are other cases like this)
   6. Spelling error on legend in figure 8.
   7. multiple cases of 'equipments' -> 'equipment'
** Intro & background to show context.
   1. Despite the authors claim to have rewritten the introduction to discuss the differences between methods sections and protocols I cannot find any evidence of this. lines 570-578 from the discussion should probably be in the introduction
   2. There is still no discussion of why the authors have chosen to take a checklist approach. For example, checklists are widely used in safety critical situations such as in airplane cockpits and operation rooms and there are many studies demonstrating their effectiveness. Did this fact or maybe some other fact about checklists have any bearing on the author's choice of format?
   3. How is the required subset of the SP Checklist any different from the requirements of minimal information models? If this is an adaptable checklist (line 61) then it should be possible to rank all 51 elements. Furthermore it should be possible to provide advice about the circumstances under which that ranking should change. Regardless of whether the authors call their required subset a minimal information model, it acts as one in the absence of an explanation of how to identify situations where certain elements would not be required.
   4. lines 61-65: I agree with the sentiment of these sentences completely, but I think this belongs in the discussion (where it is indeed already discussed). As it stands the authors do not provide citations to support what are very strong claims.
** Literature well referenced & relevant.
    1. The authors still do not discuss other approaches to improving the communication of protocols. The authors are describing a tool that can be used to improve protocol reporting. There are other tools that also claim to do this. The point was not that PSL or KeFED in particular 'support' the current work, but rather that the current work needs to be situated in the larger context of other work on protocols. This touches on the fact that the authors have not addressed the question about why they have taken checklist approach from our previous comments.
** Structure conforms to PeerJ standards, discipline norm, or improved for clarity.
   Yes.
** Figures are relevant, high quality, well labelled & described.
   1. Figure 8 is useful but I think it needs more explanation. I also think that it is probably appropriate to put this in the introduction or as Figure 1 in the results. I can grok it because I maintain an ontology, but I imagine that most readers are going to be hopelessly lost. The crossing black lines are very hard to parse, though I'm not sure anything can be done about it. At the very least a listing of what the curie (qname) prefixes mean is needed. Tangentially related: the ontology seem to be using both an old and new versions of RO and BFO?
   2. The simplified figures are much easier to understand. My original comment was a reaction to the information overload evoked by the original figures 2-8.
   3. The reduced number of tables is more manageable. I think I may be reacting to the way the current tables are typeset to be wider than the text which breaks up the flow and makes it hard to follow the structure of the sections. Table 15 is great, and I might even put it as Table 1 as context for the rest of the results.
   5. Some of the figure captions need more detail. In addition, even if abbreviations are defined once for tables (eg NC, O, R, S) they should probably still be provided again for each figure that uses the abbreviation so that readers do not have to go hunting.
** Raw data supplied (see PeerJ policy).
   1. The reasoning about Zenodo is the same reasoning that we used for shortened links. Similarly the author's own rational for including DOIs and not urls from the response to editor comment 2 applies here as well. GitHub is a commercial organization and it does not archive your code, github links are not persistent. GitHub's urls do not have persistence guarantees. Using Zenodo does not duplicate GitHub. It provides a persistent citable link (as a DOI) to a specific version of the repository as it existed at the time of publication so that it is clear to any readers what version of the repo the reviewers saw. Zenodo will also redirect anyone to the live version of the repository and there is no confusion about what the living artifact is (i.e. GitHub). See also: the software citation principles https://doi.org/10.7717/peerj-cs.86. If the authors do not want to use zenodo to provide a persistent citable version of their repository then they should at least link to the commit hash version of the tree so that the provenance can be preserved. For example https://github.com/oxgiraldo/SMART-Protocols/tree/010eba87813652e492461bf8bd14418dc72193b8. Regardless of whether this is in compliance with peerj requirements I as a reviwer cannot claim to have reviewed a GitHub repository because it can change after review. I can claim to have reviewed a GitHub repository at a specific commit.
   2. It is fine for the CIAT protocols not to be included, but it needs to be made clear that they are not available as part of the data for this paper since is not clear that that is what 'unpublished' implies in this case. Maybe something to the effect of "75 are unpublished and thus not available in the dataset for this paper" on line 141 would help.
Experimental design: * experimental design
** Original primary research within Scope of the journal.
   Yes.
** Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap.
   OK.
** Rigorous investigation performed to a high technical & ethical standard.
   OK.
** Methods described with sufficient detail & information to replicate.
   1. The methods are clearer but I still think there is a fundamental disconnect between the review of 500+ protocols, the findings as a result that review, and the checklist that the authors propose.

      Briefly, all I am asking for is an articulation of the principles that were employed to determine what elements were in the 51 total and what were in the 17 required.

      I think that the findings from the review are interesting and important on their own, and I realize that it is out of scope to request the authors to demonstrate that the checklist is effective.
      However, the "analysis" mentioned on line 165 is completely opaque.
      The methodology for the development of the ontology is well articulated in https://doi.org/10.1186/s13326-017-0160-y. If the current elements for the checklist were derived entirely from the ontology, then simply say so and cite the ontology paper in the methods section. If the elements were all initially derived from the ontology, then it would be nice to know which elements were not considered for inclusion in the checklist. If the answer is that all 16, 17 (the number of R's I count in table 15), 18, or 51, elements were the ones that the experts said should be included that is fine, but I cannot tell if that is the process. How do versions 0.1, 0.2, and 1.0 differ? What elements were added or removed between versions? What was the criteria?

      This is partially covered on line 153 "Our checklist includes the data elements that are common across these resources." Are there any elements on the checklist that were not present in all of those resources? If so, what resources were they present in? A version of Table 15 that has one more column which lists the inclusion criteria would be sufficient, even if the criteria was "our experts agreed that this was important to include."

      Another concrete example of the problem: why did the number of elements drop from 18 to 16 (17?) between revisions?

      Also to this point. I have reviewed a number of protocols myself, though not as many as the authors. I have come to a different set of conclusions about what is required and what is optional. I would very much like to be able to reconcile our choice of elements, but the methodology for inclusion, and the decision to mark as required or suggested is not described.

      To give some examples.

      For samples and organisms, why is the repository or vendor that the sample procured from not even on the list? Was it considered? This is a serious question, because not only which vendor, but which vendor site animals are procured from is know to have experimentally meaningful effects. (NOTE: organism is missing in the text see Data point 3.) I need to know the starting set of all terms considered for inclusion in order to be able to determine whether the resulting set of elements I am seeing is the result of a decision or because something was simply missed out the outset. This could go in an extended version of table 15 where column one is extended to include all the elements originally considered.

      Why is the version of software required if I am using Adobe Photoshop? Does the version have an impact on the outcome of my experiments? Why is version, which in this case has no effect, required when vendor, which is known to have an effect, not even listed for organisms? For Photoshop it does not, but for FreeSurfer it does https://doi.org/10.1371/journal.pone.0038234. "users are discouraged to update to a new major release of either FreeSurfer or operating system without repeating the analysis ... in view of the large and significant cross-version differences ..." Should a biologist cropping an image of a gel have to conform to the needs of the neuroimaging community?

      For reagents why are identifiers not required (where they can be obtained)? (Full disclosure: I maintain a tool for crawling RRIDs out of the literature.)

      For kits why are the vendor and catalog number not required? They seem like they would be a more reliable way to make sure you have the same "QuickPCR kit."

      My point is not that the authors should have done any of the things that I mention above.
      My point is that I do not know why they did what they did so I cannot assess why my results are different from theirs.
      We are working in different domains, so one major contributing factor might be that the input protocols are somehow different, but I test that unless I know how the authors made their determination.

      The inclusion of a particular element is going to depend on (among other things) the criteria that are used to determine whether it is experimentally relevant to the results. The authors seem to prefer human readability over identifiability, but they have not explicitly articulated their criteria, so I do not know for sure.

      I am not saying that the authors should promote the use of RRIDs or that they should prefer identifiers over names, but I want to know how they made the decision, otherwise I cannot reconcile my results with theirs.

   2. With regard to the number of elements. The point was not that 10 is better than any other number or that the authors should be writing a certain kind of paper. The point is that the authors should be able to provide a ranked list of their elements from most important to least important. In context, the question was posed as a journal who was interested in adopting this checklist but only has room for 10 elements (or 5, or 6, or 12 elements, etc.). If the authors can only tell the journal "well you have to use all 18" then that is a lost opportunity.
Validity of the findings: * validity of findings
   1. A note on our intention with asking for an explanation of why this checklist is useful. First, that question came from Maryann, so I can only speak as a proxy here. That said, it is not that we have anything against checklists, or any other tool in particular. It is more that there are so many tools out there already and we still haven't solved the problem. Is the issue that we don't have the right tool? Or is the issue that the tools we have, have not been supported/promulgated after release/publication? This is also the reason for the later question about promulgation.

      I cannot speak to the question about mismatch between experts and protocols since that one came from Maryann. In the event that readers might raise the same question, maybe a list of the domains in which the experts were expert like the one provided in the rebuttal would help?

      I can however, state that the lack of cross validation in other domains is still a concern. My question about why vendor is not listed as a required element for organisms is an example of this. In some domains the protocol will need to be that specific, in others it will not.

      We also very much agree that more work and effort on a variety of solutions for protocols is needed. The reason we brought up those questions is because we personally have received them from experimentalists. We were suggesting that it is important to make the value of the checklists explicit in the paper as the authors have done in their response to our comments.
** Data is robust, statistically sound, & controlled.
   1. Stats OK.
   2. The suggestions from table 15 for organism/sample do not match what is in the text. All 3 required elements organism, organism part, and Sample/organism identifier, are not explicated in the text starting on line 319.
   3. I don't know whether this is an issue for the authors, but table 14 should come before the bullets starting on line 480 so as not to break up the list.

** Conclusions are well stated, linked to original research question & limited to supporting results.
   1. With regard to licensing. If you have reviewed the author instructions for numerous journals and they do not say anything about how the protocol will be licensed then that is an important finding.
   2. We concur with the authors in that we have been unable to find any work directly addressing the relationship between protocol quality and reproducibility beyond the ones that they cite.
   3. I would still like the sentence starting on line 569 to be explicitly qualified because I think that it can be misread to imply that protocols are routinely published. This is clearly not what the authors intend. Something to the effect of "Currently, when protocols are published, they are treated like any other scientific publication, as if they are just text." The additional implication being that methods sections certainly are not FAIR and that even when published independently protocols are still not FAIR, as the authors discuss elsewhere.
   4. We are not asking for a continuation of the study, we are asking for a sentence or two describing plans for continuation if there are any.
   5. We do not consider it useless to publish checklists. We are asking the authors to consider that there are many people that have reached 'new tool fatigue' and who are likely to dismiss efforts like this out of hand, especially if the authors do not articulate how their approach is any different. How does this make the lives of experimenters easier? I will echo the question about wanting evidence for the sentence on line 583-584, and having that connected to how this approach is different. We are not discouraging the work at all, we are asking the authors to articulate why they themselves are not discouraged given the repeated failures of efforts that on the surface appear to be similar to theirs. For example, checklists could be easier to adopt than ontologies or minimal information models. There is no expectation that the authors solve the promulgation problem, which is one of the most fundamental problems period. There is however, a desire to know whether the authors have considered anything beyond the publication of this paper.
** Speculation is welcome, but should be identified as such.
   1. line 516: 'can be used' -> 'could be used'. The authors provide no evidence about the efficacy of their checklist.
Additional comments: I think that this paper is really two papers. This first is about the findings you have made about the current practices for reporting protocols in the literature and among domain experts. You have lots of data here that is sufficient for a single publication. The second is about the checklist. Currently you have no data about the checklist or whether or not it is effective. That is fine if you articulate the criteria used to select the elements of the checklist, but it is still hard to defend. Having more evidence about the efficacy of the checklist will make it much easier to publish the checklist itself.


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: DO LATITUDINAL GRADIENTS EXIST IN NEW ZEALAND STREAM INVERTEBRATE METACOMMUNITIES?
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is clear and precise and written with close to flawless English. The structure follows the journal’s standards as well a good academic writing and findings are stated in concise manner. In general, the figures and tables are relevant, clear and well structured and raw material is provided. The scope of the study is well stated in the introduction and relevant references are cited.
Experimental design: The study is applicable to the scope of the journal. Both the biological and environmental data sets are large enough to study the topic. The design supports the research questions. The methods are described thoroughly .The chosen statistical methods are applicable to the research questions and the results are reported in a meaningful way.
Validity of the findings: The authors address a highly important topic of the factors affecting freshwater metacommunity structure. There is an ongoing need to understand how communities of dynamic freshwater environments are structured in order to conserve freshwater organisms. The authors discuss the importance of temporal factors affecting metacommunities in fluvial systems, which I consider to be one of the key factors to take into account in studies focusing on these environments. The authors also discuss about the uniqueness of catchments within region, which is another important thing to acknowledge in my view.
Additional comments: Specific comments

Introduction

page 3, line: ‘island size’ comes up somewhat out of nowhere here. Maybe change the order of this and next sentence.
page 3, line 45: please change ‘drives’ with ‘drive’
page 3, line 54: I was very interested to read what Jocque et al think are the reasons for this. Maybe add a sentence of this.
page 3, line 58: How does this unpredictability affect the examination of latitudinal factors on communities? Authors do address this quite well in the discussion but maybe some of it should also be discussed here.
page 4, lines 72-74: Could the authors explain here how latitudinal diversity gradient is linked to dispersal ability.
page 4, line 75: word ‘this’ seems redundant

Material and methods

page 5, lines 101-102: Why were sites with higher human impact not included and will the results found in the study apply to such environments? Including such environments might have broadened the range of the measured environmental variables, which might have resulted in stronger species sorting. What is the authors’ view on this?
page 7, line 158: This could be included as last sentence instead of placing it here as a separate sentence.
page 5, lines 109-110: Please consider changing the structure of the sentence. Maybe: “Benthic macroinvertebrate sampling was performed between February and April 2006 using...”
page 5, line 168: Please consider changing the word ‘completeness’ to something else. Surely one cannot sample everything from a site.
page 8, line 193: Couldn’t there be spatially structured environmental variables also in the pure spatial effects? Were the environmental variables spatially autocorrelated and was this tested?
page 8, line 202: Were the sampling sites within a region connected with same river network?
page 9, line 215: I am surprised that the stream order did not correlate with any of the measured environmental variables. Was this really the case?
page 9, lines 229-230: This paragraph contains only one sentence. Please consider joining with some other paragraph
page 11, line 284: Please change ‘neseted’ to ‘nested’.
page 12, lines 289-290: Yes, but also on the range of the measured environmental variables.

Results

page 12, 306-307: I like the figure as it presents a clear difference in the communities. However, through the figure one cannot clearly state that this is because of latitude and not some other factor. Please consider revising the sentence.
page 12, lines 308-309: Is the reason for this discussed?

Discussion

pages 14-15, lines 366-368: Can this be understood so that studying the effect of climatic factors on communities in such environments may not be reasonable? Does this finding reflect the concept of each stream harboring unique conditions which may question the categorization of streams based purely on latitude?

page 16, lines 412-413: If samples per region would have been larger, would similar results have been observed? What is the authors’ view on this?
Table 1: Maybe the table would be better located in the supplementary material.


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: DO LATITUDINAL GRADIENTS EXIST IN NEW ZEALAND STREAM INVERTEBRATE METACOMMUNITIES?
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper focuses on metacommunity processes as driver of latitudinal diversity patterns. I find this study a relevant contribution to debate on latitudinal gradients of diversity as most previous literature approach it from community level. In addition, most of those studies did it in the north hemisphere, the southern hemisphere remaining poor studied. This is a point that authors should remark in the introduction section in order to increase the interest of the manuscript. It is overall well written and the English writing is correct. However, I suggest them to take a last quite reading to solve some minor mistakes. I consider the background is somewhat incomplete and authors should mainly improve it on latitudinal patterns of diversity (and provide new references). The part concerning metacommunity issues is good enough but given it is framed on latitudinal patterns authors must improve the background for this part. The structure of the article matches the standard sections and figures are of high quality, relevant to the content of the article and appropriately labelled. I find the addressed results are relevant to the hypotheses stated. However, I have some concerns which are detailed in the "General comments for the author".
Experimental design: Research questions are well defined and are relevant enough to get publication. I think authors should better highlight the knowledge gap they are investigating as it is explicitly stated in first sentence of the abstract. I find this piece of research to be rigorously conducted with high technical standard. Methods are well described and information on them is good enough. In fact, I find this section too extensive (introduction + results + discussion take 7 pages while methods by itself takes 7,5 pages), and this the reading of the paper a little tedious. In order to solve this point, given that EMS analysis is used as an additional exploratory analysis and their results do not provide clear patterns and relevant results, I suggest authors to briefly mention this point in terms of methods, results and discussion and extend on it in one appendix. In this way, they can significantly reduce the methods section, as this point takes 2,5 pages of it.
Validity of the findings: In my opinion, the impact and novelty of the paper should be more clearly stated.  Although I consider data are robust enough and statistically sound, I encourage authors to share data and R code. At least, a table with taxa list and another with local, metacommunity and regional richness should be provided. Conclusions are well stated and adequately linked to the research questions.
Additional comments: Abstract

I find three strong statements in the abstract which should be better supported along the manuscript: i) unpredictability of New Zealand’s dynamic stream ecosystems (please, provide data on environmental features of the study area); ii) fauna that has evolved to cope with these conditions (please, provide information on proportion of endemics-biogeographic traits-range size); iii) while local community structure turned over along this latitudinal gradient, metacommunity structure was highly context dependent and dispersal traits did not elucidate patterns (please, show in a clear way the inverse latitudinal pattern at community level).

Introduction

Please, provide references and background about latitudinal patterns and nuances for other groups/regions. Overall, I miss a paragraph briefly reviewing the extensive literature on latitudinal patterns, and specifying that although a general pattern can be found, there are many exceptions mainly depending on the origin and adaptations of the targeted group of organisms (see general reviews by Hillebrand 2004 in AmNat; Heino 2011 in Freshwater Biol; book “Patterns and processes in macroecology” by Gaston & Balckburn 2000; as well as concrete studies as for example Dobrovolski et al 2011 in GlobalEcolBiogeogr; Hortal et al 2011 in EcolLett; Hof et al 2008 in GlobalEcolBiogeogr; Boyero et al 2012 in GlobalEcolBiogeogr; etc.). Authors should also mention the role of random factors driving latitudinal patterns as the main domain effect (Colwell & Lees 2000 in TrendsEcolEvol). Are there studies for latitudinal patterns in New Zealand? If yes, please provide some references. If not, it is worth you show the latitudinal pattern for regional and local richness with your data. It is interesting for comparson with the metacommunity latitudinal pattern. Authors should also mention that most studies on latitudinal patterns have been performed in North America and Europe, their main findings pointing to dispersal ability in combination with climatic legacies from Pleistocene glaciations as important drivers of latitudinal patterns (again, please mention some references as those I provided above).

Line 40: Please, provide some reference to support you statement about the influence of seasonality and predictability on latitude-biodiversity relationship.

Line 40-41: I feel authors should first briefly lists the main described mechanisms driving the latitudinal patterns. In the last two decades there has been an intense debate about drivers of latitude-biodiversity relationships and how they interact, and multiple hypothesis have been put forward as possible explanations. Most prevalent among them are those related to energy-water availabitiliy, area size, biological interactions, past environments and effective evolutionary time (e.g. Ricklefs 2004 in EcolLett; Pianka 1966 in AmNat; Willig et al. 2003 in AnnuRevEcolEvolSyst; Mittelbach et al 2007 in EcolLett).

Line 43: I feel “similarly placed” is a little ambiguous. Does it mean very close in terms of geographical distance, same latitudinal range, both? Please, concrete.

Lines 62-65: Although you provide some references, I think it is a key point to understand the context of the study hypotheses and findings, so I recommend to extend a little on geology, biogeography and climatic history particularities of New Zealand as well as on the general features of its stream fauna. What main groups of organisms form stream benthic fauna in New Zealand? Provide some basic data about its origin. Are they mainly widespread species arriving from the continent? Have they evolved to new local species resulting in a high proportion of endemics adapted to the special features of New Zealand streams? Very little is known about latitudinal patterns in New Zealand, so I think some context about these points would help to increase the understanding on the relevance of this study by making it more comparable with the extended literature dealing with latitudinal patterns in North America and Europe.

Methods

Lines 99-100: “Site selection followed a series of criteria to minimise differences between regions”. Do authors refer to the points stated in the following sentence? I guess so, but for me it is not clear enough as it is currently written.

Data for analyses: although authors mentioned it for the nMDS analysis, I think they should state more clearly if, overall, all analyses are based on presence-absence or abundance data. Please, specify.

Line 126: “previously-identified important local habitat variables”. How was it done? Is it taken from previous references or authors performed some analysis? Please, specify.

Line 220: It should be “by Blanchet et al., (2008)” instead of “by (Blanchet et al., 2008).

Line 221-223: I find this sentence a little confusing. Please, rewrite.

Line 247: Please, specify the number of permutations you used.

Lines 248-249: I find this sentence a little confusing. Please, rewrite.

Lines 298-300: Good point. I find this approach very interesting. In terms of community ecology, I am not sure if it is correct to remove species based only in geographic rarity, i.e. rarity of occupancy (as authors did), or should be this species removed only when, in addition to geographic rarity, they also show demographic rarity (rarity of individuals within areas or density rarity). I mean, if one species is only found in one place but display high abundance or even adults and larvae are found in the same place, this species can be totally considered as a member of the community. What about community structure? Does it depend on the total number of sites forming the metacommunity? I think this sentence could be extended a little on this point.

Results

Line 302: Are data on spatial extents of metacommunities given in any place of the manuscript?

Line 305: I think authors should provide information about the variables that mainly summarise these first to axes.

Line 307: I suggest to complement results of PERMANOVA with a table/plot showing the latitudinal trend of local and regional richness. Showing the latitudinal pattern of local and regional richness will also help to interpret the results at metacommunity level. As authors have community data and there is a biogeographic regionalization of New Zealand, it would be very easy to perform it and I am sure it will provide a very clear and visual insights of latitudinal diversity patterns of the study area. I consider this point relevant to researchers working on latitudinal patterns and could help your paper be mentioned in further research on this topic.

Line 308: Does author mean that the latitudinal pattern for the whole study area is broken and divide into two similar patterns for each single island? I think this is an interesting point on which author should go in depth in the discussion section.

Lines 309-311: Authors should provide data on percentage of completeness for each accumulation curve. How they support this statement? How could readers know if the regional pool is well sampled or not? They should also provide some reference on what could be considered adequate inventories for invertebrates (see for instance Sánchez-Fernández et al. 2008 in DiversDistributions).

Lines 311-312: I think this is an interesting point that could be related to the strong-weak dispersal ratio. I suggest authors to write on it in the discussion section. For strong dispersers, regional curves are expected to reach the asymptote faster than for weak dispersers. As it matches the results, authors could relate dispersal with range size and biogeography of species and therefore with metacommunity structure and dynamics. It could also encourage authors to introduce some sentence dealing with the relevance of their findings in terms of conservation as this is a point that I missed in the paper and could increase its relevance.

Lines 318-319: Very relevant finding. Authors should definitely discuss on it as most of the studies on latitudinal patterns in Europe and North America find poor dispersers to be accumulated in lower latitudes whereas strong dispersers predominate in high latitude areas. Please, provide also references.

Discussion

Lines 375-377. In order to increase the interest of the paper for a general audience, authors should provide some relevant information (maybe in the introduction section) on the features of the stream studied, mainly in terms of temporal dynamics (flow regimes, temporality or intermittency, influence of droughts and floods, etc). In the current state of the manuscript, it is only stated in a very sligh way.

Lines 405-406: As previous studies have found nuances in the latitudinal patterns depending on the targeted taxonomic group, authors should write a little about particularities of stream invertebrate fauna from New Zealand (at least at order level, they work on the whole community). This can provide very useful and relevant information for researchers working on this topic what could be mentioned in next studies.

Lines 442-443. Authors should provide information on climate patterns of New Zealand in some place along the manuscript (maybe in the methods section), because when a reader arrives to this point it is not clear yet if there is a clear climate gradient and how much can it influence the nature of New Zealand streams (temporality, intermittence, etc.). This is a relevant point to understand the idiosyncrasies of benthic stream faunas. Maybe a map on this point would be great (if not, at least some reference).

Conclusion

Lines 453-454. In order to better understand this point, as I mentioned above, it should be very interesting that authors provide some key aspects about biogeographic traits of species (at least, how is the proportion of endemics vs widespread species?).

Figures

Figure 2. I suggest to include the boundaries of the five biogeographic regions in the map and also change the symbols of sampling sites for each metacommunity (manly to get a better readability of PCA and nMDS plots). Maybe it is better to insert the letters for each metacommunity in the map. Please, provide % of completeness for accumulation curves. I guess letters for each metacommunity in several plots follow a latitudinal order from low to high latitude, but maybe it should be specified in any place (alternatively they can be ordered according to their latitudinal centroids).


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: DO LATITUDINAL GRADIENTS EXIST IN NEW ZEALAND STREAM INVERTEBRATE METACOMMUNITIES?
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have answered my questions adequately and made all relevant corrections. I have no more comments for this manuscript.
Experimental design: No additional comments needed
Validity of the findings: No additional comments needed
Additional comments: I congratulate the authors for a very interesting paper.


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: DO LATITUDINAL GRADIENTS EXIST IN NEW ZEALAND STREAM INVERTEBRATE METACOMMUNITIES?
Review round: 2
Reviewer: 2

Output:
Basic reporting: I have now had the opportunity of reviewing the new version of the manuscript. Overall, I feel happy about the effort made by authors to modify the manuscript based on the suggestions made by the other reviewer and myself and to argue when these suggestions were not taking into account. Please, if they want to include my name in the Acknowledgements section, substitute "Felix Picazo Mota" by "Félix Picazo".
Experimental design: I have no additional suggestions on this point.
Validity of the findings: I have no additional suggestions on this point.
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: ALLOCATION PATTERN AND ACCUMULATION POTENTIAL OF CARBON STOCK IN NATURAL SPRUCE FORESTS IN NORTHWEST CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no commen
Experimental design: no commen
Validity of the findings: no commen
Additional comments: This study has investigated the allocation pattern and accumulation potential of carbon stock in natural spruce forests in northwest China. The authors have comprehensively estimated the C partitioning in all ecosystem components (trees, shrubs, herbs deadwood, litter, and soil) across age classes in natural spruce forests in northwest China based on 39 plots. As we all know, the ecosystem components may play different role in the C stock and cycling of forest ecosystems. However, forest inventory is a hard work that needs to cost much time, labors, and also money. These results are helpful us to more accurately understand the carbon stock dynamics and sequestration potential in these natural forests. Overall, the methodology, data analyses, and scientific presentation are in a reasonable status. I recommend publication of this work after a minor revision.

1.	The biomass equations of some other tree species (2. Populus davidiana, 3. Salix caprea and S. rehderiana, 4. Betula utilis) were also given in table 2; however, there is not any information in materials and methods part. So how are about the distribution proportion of these tree species in the Spruce forest?
2.	In the manuscript, the soil coring technique was used to estimate fine root biomass. However, how the authors separated the the root of tree, shrub and herb when they estimated the fine biomass of each vegetation types?
3.	In Line 175, Equation (2), the forest biomass C stock was calculated as the sum of biomass of tree component, shrub component, herb component, deadwood, fine root and litter. However, root biomass had been calculated in tree component, shrub component, and herb component. Thus, is there any double counting of root biomass in calculating the forest biomass C stock?
4.	In the conclusion, the author concluded stand age is not a good predictor of soil C stock, and Fig. 2 also showed the obviously difference of SOC among different stand age classes. However, in a recent study, Wang et al (2016) reported the ecosystem carbon stock showed an initial decrease and then increase trend with the soil sampling depth of 0–100 cm in natural forest succession in Chines Loess Plateau. I suggest that the authors might discuss the present study with the previous works.

Wang et al. 2016. Dynamics of ecosystem carbon stocks during vegetation restoration on the Loess Plateau of China. Journal of arid land, 8(2), 207-220


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: ALLOCATION PATTERN AND ACCUMULATION POTENTIAL OF CARBON STOCK IN NATURAL SPRUCE FORESTS IN NORTHWEST CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Mixed of unprofessional sentence
A lot of grammar error.
Literature and reference were very old , > 5 years

Citation formatting of more than 2 citation for one sentence

Raw data and figures shared
White space after figure. Kindly check the upload instruction.
The author tends to write all the findings in the graph and data into words with minimal discussion. This style of writing although acceptable, it creates overlap explanation.
The acronym is, C for carbon, SOC and etc were used a lot and introduced incorrectly
Paragraph is not justifiable
Experimental design: Data analysis and review article type.
Only 2 types of trees were used. What are the % of the chosen tree types compared to the population?
Mean and standard error, do you have any indicator that which level is acceptable?
Validity of the findings: Some information in the article were assumption that if you collect this much of data it should represent the rest of the data.

The sample size determination should be explained

How does your findings make any different in terms of policy with the current understanding of carbon stock in China sequestration

Benefits of having your data versus the current High Carbon Stock (HCS) approach.
Additional comments: Difficult to read the article when too many acronym is used.

Biomas usually associated with energy. Stick with carbon stock.


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: ALLOCATION PATTERN AND ACCUMULATION POTENTIAL OF CARBON STOCK IN NATURAL SPRUCE FORESTS IN NORTHWEST CHINA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript “Allocation pattern and accumulation potential of carbon stock in natural spruce forests in northwest China” by Yue et al. reports a comprehensive assessments of C storage in spruce forests characterized by stand age. The data used here were basically acquired as a part of large national project (NFP) that has been operating for last 20 years. Estimation and control of forest C stock is a very timely topic related to contemporary climate change and forest management issues. Therefore the research theme, presented in the article is well suited for PeerJ. However the paper has some basic weaknesses which must be overcome before considering for publication.
1.	English language needs thorough revision. Some sentence structures are poor and not qualified as professional English. For example, Line 22-23, 51-53, 59, 63-65,268,320-321.
2.	The arguments to justify the research as mentioned in Introduction (line 66-69 and 78-80) is not correct. All these components are generally included in stock assessment protocols (See IPCC Guidelines for National Greenhouse Gas Inventories).
3.	The importance of fine root and dead wood can be moved to introduction section. Discussions should contain the critical analysis of the findings. From Line 283-291.
4.	Most of the discussions consists of repetition of same things under two sections (4.2 Allocation pattern and 4.3 Stock dynamics), my suggestion is try to combine the two sections and precisely discuss the each components.
5.	Figure 1 please explain what does the 15 dot points in sketch map indicate .
6.	Figure 2 Please Split it or convert to Table.
7.	Figure 3 Caption of the figure should be self-explanatory. Please mention what is CSP, CSR, age classes (i, ii.... & i-ii, ii-iii etc.), add error bar and statistics.
8.	Table 1 H (m) should be Height (m), unit of basal area cm2?
9.	Table 2 please mention only those equations that have been used in the current study.
10.	Table 4 Table caption should be More detailed (explain age class and statistics).
Experimental design: 1.	The location of the study areas are not completely described. Location map showed that all 39 sampling plots were spread over large area of 4 locations, nearly 300-700 km apart from each other. It’s not clear how the 4 plots of 20mX50m (age class IV, for example) were distributed over those 4 locations and how these are comparable if not in same geographic location.
2.	No information about systemetic sampling/selecting plots, I mean the protocol for selection of 20 mX 50 m plot in a large forest area. For example in a similar study (Cui et al  PLoS ONE 10(9): e0137452) in Shaanxi province,  28.28 m×28.28 m plots were established in each 4km×8 km grid along a transect line.
3.	By the universal definition, all particles less than 2mm are generally considered as soil (fine earth). So sieving the air dried soil samples by a 0.25mm sieve (Line 166) before chemical analysis is clearly a wrong procedure with the possibilities of an erroneous estimation. Because small aggregates, particulate organic matter can be lost due to use of fine screen. (Please see the sieve size in Global Change Biology (2014) 20, 2644–2662, CATENA (2016) 137: 651-659).
4.	The Decomposition class and specific density of dead trees need detailed procedures as the referred literature in not available in English (Line 133-136).
5.	No sampling time (year, month) for soil or root sampling was mentioned.
6.	Please mention how fine roots were sorted (hand picking/wet or dry sieving)(Line 156).
7.	The method of estimating C sequestration potential (CSP) using the equation-3 needs reference (Line 186).
Validity of the findings: Minor revisions.
1.	Not clear (Line144)
2.	Not clear (Line 206)
3.	No regression analysis was performed (in section 2.5 Statistical analysis) (Line 215).
4.	At each soil depth? (Line 219)
5.	No Regression analysis ? (Line 239)
6.	Line 331 (In this study-----) and Line 333 (Such differences---) is contradictory.
7.	Line 336 -338 not clear.
8.	Line 359 Please explain how soil formation correlate with forest development in the present study sites. Need reference.
9.	L397 Age class iv is less than 70%.
Additional comments: The authors investigated ecosystem C accumulation pattern and potential in spruce forests in China. The subject-matter is interesting and fit with the scope of PeerJ. The Authors correctly identified and addressed the major components of ecosystem C stock. But as the study location covered a large geographical area, a systematic survey-protocol should be followed during the plot selection and/or sampling, with the description of forest, landscape, soil and climate of each sampling sites. In addition, methodological robustness in sampling-preparing-analyzing is crucial, where I found a lacking in soil sample processing (pass through 0.25mm screen instead of standard 2mm sieve) which might have influence on objectives of the study.


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: ALLOCATION PATTERN AND ACCUMULATION POTENTIAL OF CARBON STOCK IN NATURAL SPRUCE FORESTS IN NORTHWEST CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors responsed all the commentary carefully in the revised manuscript, i recommend the manuscript to be accepted after this revision.
Experimental design: The authors responsed all the commentary carefully in the revised manuscript, i recommend the manuscript to be accepted after this revision.
Validity of the findings: The authors responsed all the commentary carefully in the revised manuscript, i recommend the manuscript to be accepted after this revision.
Additional comments: The authors responsed all the commentary carefully in the revised manuscript, i recommend the manuscript to be accepted after this revision.


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: ALLOCATION PATTERN AND ACCUMULATION POTENTIAL OF CARBON STOCK IN NATURAL SPRUCE FORESTS IN NORTHWEST CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: Better writing style then previous version

Format in 455 : reference
paragraphs were missalign
Experimental design: Within the journal scope.
Author had provided explanation in rebuttal
Validity of the findings: The result is valid
Additional comments: The impact of the study may justify the government program, but thats all.


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: ALLOCATION PATTERN AND ACCUMULATION POTENTIAL OF CARBON STOCK IN NATURAL SPRUCE FORESTS IN NORTHWEST CHINA
Review round: 2
Reviewer: 3

Output:
Basic reporting: Authors' actions are satisfactory
Experimental design: Authors' actions are satisfactory
Validity of the findings: Authors' actions are satisfactory
Additional comments: The manuscript has been improved satisfactorily.  Just three minor comments:
1.	In line 197 (pdf version) ……in the soil layer m (totally five sampling layers in this study)- not totally- should be total five….
2.	 In different Tables  ‘Stand age classes’ were presented as I, II, III etc. which were explained only in Table 1. I think you can use either   I (<40 yr), II (40-60 yr)….. this style in all Tables or mention this in Table captions.
3.	In Table 6 footnote  please pest  ‘Data with different lowercase ……………..’


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: PHYSICAL FITNESS PREDICTS TECHNICAL-TACTICAL AND TIME-MOTION PROFILE IN SIMULATED JUDO AND BRAZILIAN JIU-JITSU MATCHES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Review the cites throughout the paper
-	After of et al “.,” (i.e.  Sacripanti et al., 2008)
-	When there are several references separated with “;” (change “,” x “;”)
-	Change: “and” X “&”
-	When the author is directly cited the year goes between (). For example, line 209, by Sacripanti et al. (Sacripanti 2008) x Sacripanti et al. (2008).
Experimental design: More details should be added to the inclusion criteria of the participants (lines 107 a 110). For example, years of practice, frequency of weekly training and competitive level reached. This to guide the practical implications of the readers.
Validity of the findings: This job that presents interesting contributions towards the field of combat sports, especially for the specialties of grappling.
Additional comments: Good work. just ask them to review the following:

Review the cites throughout the paper
Line 71: Gi, describe it the first time, thinking about readers who are not Martial Arts and combat sports.
Line 81: change “inestigations” x “investigations”
Line 189-191: cite and reference the site of the rules of the International Judo Federation and International Brazilian Jiu-Jitsu Federation.
Line 219 a 233 Time motion analysis. Because the intensity of the efforts is assessed subjectively, it is necessary to indicate who valued it. For example, high-ranking professor of Judo and BBJ or researchers with a high level of specialization in the disciplines, etc.
In Table 2, add the number of athletes: BBJ (n = 24); Judo (n = 20).
Line 347, “in belt and jacket combat sports” change for “in the specialties of grappling in combat sports”
Line 426 about limitations. I see a limitation that should be pointed out in the discussion that corresponds to the selection of the sample.


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: PHYSICAL FITNESS PREDICTS TECHNICAL-TACTICAL AND TIME-MOTION PROFILE IN SIMULATED JUDO AND BRAZILIAN JIU-JITSU MATCHES
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: must include the reference "sit and reach test"
must incorporate the reference of Power of the lower limbs (horizontal jump)
must incorporate the reference of Upper Limbs Power (Medicine Ball Throw)
must incorporate the Upper Limbs Endurance reference (flexion test)
must incorporate the abdominal resistance reference (sitting test)
must incorporate the reference for the calculation and classification of the "effect size"
must review the citations and references format
Validity of the findings: no comment
Additional comments: Should review aspects of the format in general, mainly citations and references.
All tests must be cited and referenced.
Rhe "effect size" must be cited and referenced.


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: PHYSICAL FITNESS PREDICTS TECHNICAL-TACTICAL AND TIME-MOTION PROFILE IN SIMULATED JUDO AND BRAZILIAN JIU-JITSU MATCHES
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. Review line 200. The word analysis is repeated.

2.Reading lines 86-88, where the phrase "in this context, the present investigation aimed to study the relationship between physical fitness variables and competitive success, through technical-tactical analysis in judo and bjj athletes"appears. It seems to me that relate the variables related to physical fitness with sports success was not the objective of the study. Given that, this relationship is analyzed with simulated combat and not with a tactical technical analysis in real competition conditions, and the competition is much more complex than a simulated condition in a laboratory. What I recommend is to leave that sentence like as:"In this context, the present investigation aimed at studying the relationship between physical fitness variables and technical-tactical analysis in judo and bjj athletes”.

3.Regarding the revision of the data table (Excel) there is a difference with the text. In the Excel file appears n = 24 BJJ athletes and n = 16 Judo athletes, while in the text appears n = 24 BJJ athletes and n = 20 Judo athletes, check and correct that.
Experimental design: 1. In the subjects section, in the paragraph of lines 103 to 106, I suggested to provide more information about the subjects, such as frequency of training, weekly hours of training and years of experience. This would be useful for the practical application of the study. In this way, future readers will be able to understand in a better way the level of the selected subjects, and understand if they were competitive or recreational athletes.
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: PHYSICAL FITNESS PREDICTS TECHNICAL-TACTICAL AND TIME-MOTION PROFILE IN SIMULATED JUDO AND BRAZILIAN JIU-JITSU MATCHES
Review round: 2
Reviewer: 1

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


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: PHYSICAL FITNESS PREDICTS TECHNICAL-TACTICAL AND TIME-MOTION PROFILE IN SIMULATED JUDO AND BRAZILIAN JIU-JITSU MATCHES
Review round: 2
Reviewer: 2

Output:
Basic reporting: All modifications were made.
Experimental design: All modifications were made.
Validity of the findings: All modifications were made.
Additional comments: All modifications were made. The work is of high quality and a contribution to the knowledge of combat sports training


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 SURVEY ON SLEEP ASSESSMENT METHODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: From my point of view, authors could follow the Preferred Reporting Items
for Systematic reviews and Meta-Analyses (PRISMA)
guidelines (Liberati et al., 2009).
Validity of the findings: As a general advice, authors could add some tables summarizing the findings of previous studies on each method reviewed in their work. This could give a clearer idea to the reader on the validity of each method.
Additional comments: Manuscript ID: 23959v1

Journal: PeerJ

Title: A survey on sleep assessment methods

Abstract

1) Lines 44-45. Authors wrote that “Objective methods such as actigraphy present a sensibility higher than 90%.” Here they could also state that specificity of actigraphy is lower compared to sensibility, being one of the limitation of such technology.

2) Line 46. Authors wrote “such as the patient’s perception of his sleep”. Please add “her” before or after “his”.

Introduction

1) Lines 56-57. Authors wrote that “such, most health care systems invest a lot of money in the creation of sleep centres and sleep units in hospitals”. Is there any reference supporting such sentence?

2) Lines 67-69. Authors wrote that “The main motivation of this survey is to produce a comprehensive and unbiased literature review from which we can extract a complete classification of sleep assessment methods (including new technologies such as mobile apps).” I suggest that authors explicit the main features of examined populations, e.g., children, adolescents, adults, healthy controls, patients (if patients, which kind of disorder).

3) Line 98. I suggest that authors follow the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines (Liberati et al., 2009).

4) Line 98. Authors could show the flow-chart of the bibliographic search for each database, according to the PRISMA guidelines.

Classification of sleep detection methods

1) Line 156. Authors should specify here that PSG is the gold standard for the sleep assessment.

2) Lines 180-182. The same sentence also applies to sleep diaries.

Medical Assistance Methods

1) As a general advice, authors could add some tables summarizing the findings of previous studies on each method reviewed in their work. This could give a clearer idea to the reader on the validity of each method.

Self-assessment methods

1) Line 316. Authors should add “her” before or after “his”.

Hardware devices

1) Line 406. Zeo acquires a single electrical signal composed by brain activity, muscle tone and eye movement. Therefore, Zeo cannot be considered an actigraph.

Tables

1) Table 1. The definition of total time out of bed should be the interval between FOB and IIB.

2) Table 2. Authors could revise the works on the Mini Sleep Questionnaire (e.g., Falavigna et al., 2011; Natale et al., 2014), a self-assessment tool of both sleep and wake originally proposed by the research group coordinated by Professor Peretz Lavie (Zoomer et al., 1985), aiming to assess if it fulfils their inclusion criteria.

Figures

1) Figure 1. From my point of view, actigraphy should be considered as a technology requiring medical assistance, because actigraph is a medical device.

2) Figure 1. In the legend, authors should specify the meaning of acronyms reported in such figure.


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 SURVEY ON SLEEP ASSESSMENT METHODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The review is interesting, informative and coherent with the aims of the journal, but several major revisions are needed.

Major points
-	The main problem concerns the originality of the paper. Authors recently published a paper on the journal “Sleep Medicine” in which they reviewed the current methods to assess sleep based on questionnaires and diaries (cit. Ibanez V., Silva J., Cauli O. A survey on sleep questionnaires and diaries. Sleep Medicine, 2018; 42:90-96). Albeit the present paper is a more extensive and general survey on sleep assessment methods, I see an overlap with the “Sleep Medicine” review. Furthermore, such paper is not even cited. Clearly, a partial overlap is unavoidable, but I think that it should be reduced. The “Sleep Medicine” paper should be cited, and the originality of the dissertation on questionnaires and diaries should be improved. Moreover, Table 2, 3 and 4 contain information that are largely reported in the (very similar) tables in Ibanez et al., 2018, so they should be eliminated or strongly differentiated by the old ones (e.g. improving their informative value).
-	I suggest a complete revision of the English across the text (it is often wrong, unclear or repetitive).

Abstract
-	The “results” section of the abstract actually does not report results, but methods.

Medical Assistance methods
-	for each described method a) the principal studies should be cited (at present, I see references only for PSG, and they are not exhaustive); b) the main sleep disorders/problems that can be assessed should be reported.
-	Lines 272-273: describe what sensors are used and how the device detects sleep stages.

Hardware devices:
-	For both contactless and contact devices, a table reporting the devices specifics (the name of the devices, ranking/review, variables measured by the devices) could be useful.

Contact hardware devices:
-	Lines 406-407: It seems that Zeo Personal Sleep Manager is presented as an actigraph, while it detects simplified EEG measures.

A critical discussion about accuracy and validation
-	lines 429-430: authors should cite the studies at the basis of their ranking

Figures
- Fig. 2: the figure should be improved: a) it should be referred to a diagnostic classification system; b) for each disorder, authors should report at least one relevant study that use PSG for the assessment of that disorder. Moreover, the present figure is incomplete (e.g. insomnia is missing) and peculiar (medical-psychiatric sleep disorders are reported as parasomnias?).

Minor points
-	line 205: the brief summary for each method is redundant, since the cited methods are described later. I suggest the removal of the summary and the integration of the information that it contains in the main description of the methods.
-	line 305: “polysomnogram” should be abbreviated
-	Fig. 1: abbreviations should be reported in the figure legend
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: A SURVEY ON SLEEP ASSESSMENT METHODS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Please refer to the general comments for the author.
Experimental design: Please refer to the general comments for the author.
Validity of the findings: Please refer to the general comments for the author.
Additional comments: Manuscript ID: 23959v2

Journal: PeerJ

Title: A survey on sleep assessment methods

The authors have adequately addressed my previous concerns. I do not have any further suggestions.


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 SURVEY ON SLEEP ASSESSMENT METHODS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors made all the required changes.
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: THE GLOBAL BURDEN OF HIV-1 DRUG RESISTANCE IN THE PAST 20 YEARS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors find out that the drug resistance with non-B subtypes and recombinants has been growing and they pointed out that transmitted drug resistance seems to be stable over time. They not only verify an increase in drug resistance along with antiretroviral therapy (ART) being accessible in resource constrained areas, but also disclose the plateau effect on decreasing drug resistance in areas covered by the latest drug regimens.
Experimental design: This paper studies the development of HIV-1 drug resistance in the past 20 years and estimates the global burden of drug resistance with data from two HIV sequence repositories Los Alamos and Stanford HIVdb. The formal one is used to derive the worldwide prevalence, time trends and geodemographic predictors of HIV drug resistance while the latter is used for sensitivity analysis. Three available algorithms ANRS, Rega and HIV db are applied to figure out resistance to reverse transcriptase as well as protease inhibitors (NRTIs, NNTRIs and PIs). Moreover, country-level sociodemographic and health indicators and the list of surveillance drug resistance mutations are obtained from World Bank and WHO, respectively, for the analyses of country-specific resistance and transmitted drug resistance.
Validity of the findings: However, two major limitations of the study are the lack of information on resistance to integrase inhibitors or rare recombinants and the inconsistency of data from different sources, which may affect the validity the of the results.

Aside from the above issues, I also have some minor remarks on the manuscript for further consideration.

- Line 166 (…has been decreasing by 3%...): I checked the row of Calendar Year in Table 2 and could not find out how the authors got the lower bound 3%.

- Line 178-179 (…the kappa agreement between B subtype and antiretroviral-naïve was -0.06.): It would be more readable if the authors mention how the get the value -0.06.

- Line 184-185 (All results are provided in the supplementary materials.): The authors should have specified the corresponding data set for results in the current paragraph. If those results are concluded from Supplementary Table S2, they had better point out the data attributes for reference, say, the sex M is compared with sex F and the B subtype with non-B subtype, as they did for Table 2 in the manuscript.

- Line 188-189 (In relation to latest generation PI/NNRTI drugs, specifically the PI darunavir (DRV) and the NNRTIs etravirine (ETR) and rilpivirine (RPV).): the sentence appears to be incomplete.

- Line 201 (…higher literacy rates, and public health expenditure.): From Figure 3, it seems that another factor “adjusted net national income” should be added, which is also related to a higher prevalence of B subtype (correlation value 0.63).
Additional comments: I would like the authors to revise and resubmit their manuscript according to my review given above.


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: THE GLOBAL BURDEN OF HIV-1 DRUG RESISTANCE IN THE PAST 20 YEARS
Review round: 1
Reviewer: 2

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: The present manuscript derives trends and patterns of antiretroviral resistance from published nucleotide sequences extracted from the Los Alamos and Stanford databases. These analyses consider either all available sequences or sequences from treatment naive patients. Based on this, the authors  derive time trends for different subtypes, levels of resistance for different countries, and  consider associations between resistance and demographic/socio-economic characteristics (of different countries). Overall this is a well written paper presenting an interesting analysis on an important subject.

I have several comments that need to be addressed :

* I think that the limitations of the study approach and their impact on the results should be discussed and emphasised  more. This applies in particular to sampling bias, i.e. the fact that different types of patients are sampled for different dates and countries (which could result in some of the differences presented here) and also the fact that some countries are strongly overrepresented in these databases.This concern applies in particular to sequences from treatment experienced patients (for which treatment history plays a key role in the observed resistance patterns) but to a lesser extent also for treatment naive sequences. I guess for many sequences no information on prior treatment is available, which can result in an additional bias. Generally, it would be very useful if the authors could provide a detailed discussion of the challenges associated with interpreting sequence data for which no or only very limited demographic and clinical data is available. As stated above, I think that this work is  despite these limitations  a valuable analysis, but the limitations should be emphasised more than in the current version (in lines 265-267).

* On a related note,  it would be useful if the authors could provide a discussion of the pros and cons of the present approach compared to the more traditional systematic-review approach (e.g. Rhee et al. Plos Med 2015).

* It is unclear what the correlations summarised in lines 200-205 mean: For example the correlation of subtype with sanitation, physicians, public health expenditure, etc. just reflects the known fact that subtype B is the predominant subtype in the US and western Europe. Similarly, it is unclear to what extent confounding was taken into account in these analyses.

*Given that this work is based on publicly available sequences, the accession numbers of the analysed sequences (by country and category[naive, all] )  should be provided to ensure the reproducibility of the results.


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: RAPID EVOLUTION OF THE HELICOBACTER PYLORI ALPA ADHESIN IN A HIGH GASTRIC CANCER RISK REGION FROM COLOMBIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper submitted Gutierrez-Escobar et., al. is a comprehensive evolutionary analysis of the AlpA gene in Colombian populations.
In general terms I find the paper well written. The state of the art and background of the paper is well supported, as well as the paper’s objective. However there are several gramatical errors that the authors must address. The ones that I’ve found are listed as follows:
37. I think is singular: “adhere”.
59. It progresses.
59. ulcers, not ulcera.
99,115: keep consistent nomenclature when referring to the DNA vs protein.
217: Sure is ten to the minus one? If that is the case use 0.2525 instead of the expression with exponent. That value is not significant and as a result the values of the GENCONV algorithm would not be significant as well. Please verify this.
Please show the lowest and the highest p-value.

In general, resolution of figures must be improved. Text in the phylogenetic trees and in figure 4 looks pixellated.
Experimental design: Rationale, experimental design, methodology and results are clear and within the scope of the journal.
Please include the original sequences used, the alignment used for this paper and sequence accession numbers if available. Please make sure all software include versions (i.e. muscle).
Validity of the findings: The nature of this paper is descriptive, there are no wet lab experiments carried out. Similar analyses have been done for other adhesins in H. pilory, however it is probably the first paper looking at Colombian data for this particular marker in this bacterium.
Its conclusions are within the reach of the experimental design. However, it is not clear to me why the authors mention gene duplication as a conclusión when apparently they didn’t use paralogous genes? AlpA belongs to a locus where other gene is located: AlpB which is very similar to AlpA. Both genes are paralogous to one another and the result of a duplication. Since the authors didn’t use any sequence belonging to AlpB no conclusión can be drawn with respect to gene duplication. All the analysis was done with AlpA, conclusions must be circunscribed within the reach of the experimental design and the data as well.
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: RAPID EVOLUTION OF THE HELICOBACTER PYLORI ALPA ADHESIN IN A HIGH GASTRIC CANCER RISK REGION FROM COLOMBIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: It’s better for the manuscript to be checked by a native English speaker or professional English editing service.
Experimental design: See comments below
Validity of the findings: See comments below
Additional comments: It’s better for the manuscript to be checked by a native English speaker or professional English editing service.  For example, there are grammatical errors
L132: to identified -> to identify
L140: The HBK, Snn and chi squared were performed -> The HBK, Snn and chi squared tests were performed
L202: “The Tajima test was negative” -> “Tajima’s D was negative” or “Tajima’s D was negative”
L222: “Functional divergence … was performed” -> “Functional divergence analysis … was performed”?
L258: that -> than

L118, L131: reference of MEGA 7 should be
Kumar S Stecher G, Tamura K . 2016 . MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger data sets. Mol Biol Evol . 33(7):1870–1874.

Figure 5 legend: p > 0.01 -> p < 0.01

Table 3: p > 0.05 -> p < 0.05?  There is no explanation about the asterisks.

Table 1: There is no explanation what the Rm (table title) or RM (legend) is.

Figure 1 and 2 legends: remove “significant” because it is not defined in the manuscript and “significant consensus tree” is not a correct scientific term

L203-204: There is no explanation how Fisher’s exact test was conducted, and Table 2 doesn’t seem to show the result.

L206 and Table 3: There is no explanation what the Nm value is and how the values in Table 3 indicate a constant genetic flow.

L213-220: The number of recombination tracks depends on time to the most recent common ancestor of the Colombia strains.  At least, it’s better to show when each strain was isolated if the authors have the data.

Table 4: There is no definition about the “Significant values”.

Overall, the authors don’t pay attention to multiple testing corrections when they repeat statistical tests and show p-values.

L131-133: Was the sliding windows analysis conducted using Mega 7?  There is no result of the analysis shown in the manuscript.

2.5 and L222-233: The methods to infer positive selection used in the manuscript don’t assume recombination, and there is no theoretical justification to use them.  A proper alternative is omegaMap.

Title: There is no explanation in the main text about “a high gastric cancer risk region from Colombia”.  Where is it, and how is the cancer risk in the region?


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: SELECTIVE CONSTRAINT ACTING ON TLR2 AND TLR4 GENES OF JAPANESE RANA FROGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript describes the characterization of Toll-like receptors from three species of Japanese frogs. The manuscript is clearly written and organized. The authors did a very thorough job of presenting their data in figures and tables (both in the main body and supplemental data). Their DNA data is accessible and their use of vertebrate animals was approved by an ethical committee.

I have a few minor suggestions for improvement (listed in order of appearance in manuscript, not by priority).

Line 14 - Change to "Toll like receptors (TLRs) are an important component of innate immunity, and the first line of pathogen defence because they can recognise pathogen-associated molecular patterns.”  I do not think it is necessary to provide the acronym because you do not use it in the abstract and give it again in the main body of the text.

Abstract – Nothing about expression level analysis is included and would be interesting to note. Also, it could be interesting to mention that some the of PSS were located in the more variable LLR domain.

Line 21 - I think it would be better to end the abstract in a more positive way. I suggest something like: "We found that both of these genes are evolutionarily conserved, with overall evidence supporting purifying selection. In contrast, site-by-site analysis of selection identified several TLR2 and TLR4 codons under positive selection. Although it remains unclear whether the Bd fungus is a selective force acting on TLRs of Japanese frogs, our results indicate that certain sites in TLRs of these species may have experienced pathogen-mediated selection."

Line 47 – Wording is not clear. Do the authors mean that TLR4, in ADDITION to 11 other TLR genes were isolated from a Bombina maxima transcriptome? Or, it was one of the 11?

Line 51 – Change to "An overall signature of purifying selection was identified in TLRs of urodele amphibians, although a few individual codons were found to be evolving under positive selection."

Line 61 – Would transition better from the previous paragraph if re-written to "Chytridiomycosis is a disease in amphibians caused by the fungal pathogen Batrachochytridium dendrobatidis (Bd)."

Line 72 – I think the goal of this paper could be better described something like this (since you are not really characterizing the dynamics between infection and TLR alleles): “Here we characterized two candidate TLR genes in three Japanese Rana species, in order to better understand the immune gene complements of Bd-tolerant species."

Table 3 – I would add “loci from these three species” to the very end of the table legend

Table S1 - I recommending move this information "E– raised in captivity from eggs, T- raised in captivity from tadpoles, A- adults collected in field.” from the legend to a footnote below the table because it is not primary to table’s description.

Table S2 - The names of primers in Table S2 do not match those in Fig. S1 and Fig. S2

Fig. S1 legend - I recommend modifying legend to read “Nucleotide alignment of representative TLR2 transcripts” since not all alleles were included in the figure.

Fig. S2 – the vertical text “exon boundary” is very small and hard to read. I recommend making it a little larger so that the reader does not have to zoom in so much to see it.

Fig. S5 - Amino acid numbering is very small and hard to read. I recommend making it more like the size of the numbering in Fig. S4.
Experimental design: Line 83 – There is space missing after the period

Line 92 – Change to “…unique in that the majority of their coding sequence is located within a single exon.”

Line 122 – When looking at Table 3, it appears that you combined the MHC data from all three species for the dn/ds analysis. I recommend you make that clear here in the methods.

Line 143 – Could you explain a little more about how to interpret the TMM-normalized values presented in Table 4? It would be helpful for the reader to have something to reference in the methods when looking at the table.
Validity of the findings: Line 170 – Since you discuss your results by gene here, rather than by species, it would be helpful to reorganize the table so that it reflects the same organization (and matches the other tables).
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: SELECTIVE CONSTRAINT ACTING ON TLR2 AND TLR4 GENES OF JAPANESE RANA FROGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The context of the paper is generally clearly stated and references are relevant. However what is not clear to me is the resistance of Japanese frogs to Bd pathogen. In line 63-65 of Introduction, the authors write that there is ”no evidence of Bd-related declines in East Asian frogs”. This is very interesting, but it is unclear if this is due to resistance of frogs or perhaps frogs for some reason do not come into contact with the pathogen? Are there any studies investigating resistance of East Asian frogs to Bd? If yes, please provide references. If there are no such studies, please specify that frog resistance/tolerance is a only a possibility. I also suggest to moderate the statements in the abstract (line 19: “frogs from East Asia appear to be tolerant”) and the conclusion (line 224-225: “our study adds to the growing body of literature related to genes that impact Bd resistance” ).
2. Some accession numbers are not accessible (line 151). Also the text files with genbank submissions are not readable. The authors mention transcriptome data of outgroup species Odorrana sp, which is not available online. Please make sure all data is available to the public.
3. The paper is clearly written, and I find the language correct. One suggestion concerns usage of word “extrapolation” in line 218. Do the authors mean “extract” here?
4. Table 1. Statistic “k” is not mentioned in the Methods.
Experimental design: 1. Please explain in more detail the aims of the study at the end of the Introduction, specifically what the authors mean by "characterization" of two candidate TLR genes (line 72).
2. Although the authors refrain from any statistical tests comparing expression data, some statements suggest this analysis was done. I would avoid statements about comparison of expression levels as no firm conclusions can be made from presented results (eg. line 141).
Validity of the findings: no comment
Additional comments: The paper “Selective constraint acting on TLR2 and TLR4 genes of Japanese Rana frogs” describes genetic variation and selection in two Toll-like receptor genes in three species of frogs. The topic of molecular basis and evolution of innate and adaptive immunity in amphibians is timely, due to potential threats caused by fungal pathogens, thus it may be of interest to a wide public. The paper is clearly written and with no methodological flaws. With minor comments to this article, I recommend this manuscript for publication in PeerJ.


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

Input:
Title: SELECTIVE CONSTRAINT ACTING ON TLR2 AND TLR4 GENES OF JAPANESE RANA FROGS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors provide a good, concise introduction to the topic. I particularly like lines 27 - 30 which introduce TLRs. The literature cited throughout seems to be appropriate to the topic at hand.

Lines 72 - 73 represent the entire extent of any mention of the rationale for the study and the introduction includes no explanation as to what was done overall and how it was achieved. I believe that this detracts some the cohesion of the paper and makes it feel disjointed and messy in places.

This constitutes my primary concern with the paper and requires addressing in order for it to be published.

Please provide sufficient detail in at the end of the introduction in order to give the reader a whole overview of the project. i.e. "Here we characterise the genetic diversity and selection patterns of TLRs in 3 Japanese frogs, we also investigated the expression TLRs using transcriptomics"

You should also clearly define your hypotheses. i.e. "In order to test the hypothesis that a shared resistance to Bd in Japanese frog species may be the result of selection on TLRs, here we ...." or similar.

The structure of the article conforms to what is expected by the journal and, overall, the article is well written however in my opinion there are a few incidences where it could be improved.

ABSTRACT -

Lines 14 - 15: This sentence does not flow well in my opinion. I would consider splitting this into two.

Lines 17 - 19: Again, this sentence does not read particularly smoothly. Consider a restructure so that it reads "Evidence from other vertebrates shows that TLR2...." and being a new sentence at the point "Such genes therefore..." I believe that this would improve the flow but would also remove the problem of having an abbreviation as the first word of a sentence, which I don't like.

Lines 19 - 20: I think it would be helpful to more explicitly state that you are interested in the genetic diversity that underlies these TLRs rather than any structural or functional aspect of the TLRs themselves. This is true also in the introduction.

INTRODUCTION -

Lines 30 - 33: There are a couple of terms in this sentence that would benefit from brief explanation, particularly LRR. Explaining complex terms will greatly improve the accessibility of this paper for interested non-experts.

METHODS -

Lines 76 - 86: This section would benefit from restructuring and condensing. In my opinion the sentence about ethical approval for lethal sampling (which is repeated twice) should be made more explicit and come before everything else. I would also like to know how long these animals were house in the laboratory and how long the samples were stored prior DNA extraction.

Line 83: Missing space between 2. and Animals.

Lines 111 - 114: The reason for cloning the sequences may not be clear from some readers and should therefore be expressly stated and explained. It may also be helpful to provide more detailed but concise cloning methodologies regarding the media used for incubation phases etc.

Lines 141 - 146: The incorporation of expression data into the study is interesting. However at present I feel that it is so badly woven into the article as a whole that is detrimental to the quality of the paper. I think the primary issue is that the introduction contains very little in the way of foreshadowing the authors intentions or hypotheses. Therefore this section about expression data appears from no where and it potentially difficult to rationalise at first. Please better outline the intentions and predictions of this study in the introduction and provide a qualifying statement in the methods section i.e. "In order to investigate baseline expression of our candidate TLRs we examined ....." I think it would be helpful to provide more information regarding the structure of the transcriptome data being analysed so that this work stands on its own. At present this portion feels like an extension of the study of Law et al. 2017 which in my opinion undermines this work as a whole.

RESULTS -

Lines 155 -156: Discussion possibly creeping into results here, consider moving to the discussion section.

Lines 170 - 174: As per my previous comment regarding better explanation of the expression analysis, this section is currently almost non-sensical given the information contained within this article as a stand alone piece and would greatly benefit from further details in the methods section.

DISCUSSION -

Lines 178 - 180: I would like to see the articles which generated these data cited here so that due credit is given.

Line 182: I believe this should read "domain structure TO that of other frogs".

Lines 191: I believe this should read " as well as THOSE of over 3000 transcribed..."

TABLES and FIGURES -

Table 1: Caption states that no values for D are below 0.01 however the value for R. ornativentris is 0.008. This is probably a rounding issue that should be addressed or acknowledged.

Figure 1: The drab colour scheme of Figure 1 is underwhelming. While this is not a problem per se, I believe the overall look of the article would be greatly enhanced by a more eye catching figure. Consider a difference colour scheme.

ARCHIVING -

Data accession numbers and fasta files are provided with the texts however I found it impossible to locate the data of GenBank with the accession numbers provided in the text. This may be a problem my end but should be checked.
Experimental design: This article constitutes original research that is within the scope of the journal.

There are several apparent issues regarding experimental design. However, I believe that most are due to how the work has been reported as per my comments in the report section.

For example:

More clearly defining the research questions and primary methodological approach in the introductory sections will greatly enhance the quality and readability of this manuscript.

I have no-concerns over the rigour or ethics that underly this work.

Certain areas of the methodologies should be reported in greater detail in order to enhance repeatability. Specifically the sections referring to molecular cloning and the generation and analysis of expression data should be revised as per my comments in the reporting section.

My main comment regarding the methods of this study is that if the aims of this study were really to investigate the link between selection at TLRs and resistance to chytrid in Japanese frogs then the investigators should also have characterised the TLR diversity of several susceptible species by way as a comparison group. As it stands the study tells us little about the role that chytridiomycosis plays in the selection apparently acting on the TLR loci in question.
Validity of the findings: The findings of signatures of purifying selection of TLR loci in Japanese frogs is interesting in their own right, as is the evidence provided for the existence of particular TLR types in amphibians.

However, given my concern regarding a lack of a Bd susceptible comparison group discussed in the experimental design section, I believe that this paper does not truly investigate any link between Bd and selection at the TLR and evidence for such is almost entirely inferred from other sources.

The authors correctly point out the need for further research to establish whether observed selection at the TLR loci is indeed due pressure exerted by Bd or another pathogen. However, given that Bd is integral to the story that the authors attempt to build into this paper, it begs the question as to why some of this research was not conducted for this paper. If the authors cannot provide a stronger case for a link between the observed selection and Bd then I would like to see the amphibian disease angle of the paper toned down throughout.

Lines 224 - 225: Whilst the potential for Bd to exert such selective pressures is irrefutable, I think it would be more appropriate at this stage to say that this study "adds to the growing literature related to gene that potentially impact Bd resistance"
Additional comments: The findings of this work are interesting and I enjoyed reading the manuscript. I particularly thought the background presented in the introduction was strong.

However, I believe that there are several issues which can be addressed which will greatly improve the quality of this article. Please see my specific comments in the relevant sections of the review form however I will provide a summary of the major points below.

1. You should provide a more comprehensive introduction to the project in the introduction section. Outline the exact rationale behind the work. State your hypotheses and provide an overview of the methodologies that were used to test them. This will greatly improve the cohesion of the paper, which at present feels like two distinct smaller pieces of work bolted together (TLR diversity / selection vs Expression).

2. Whilst the potential for such a relationship is clear, your experimental design does not really provide any insight into whether or not Bd is causative behind the TLR selection that you observe. Whilst you correctly acknowledge the need for further research, Bd is still one of the central columns of the paper. Unless you can provide a stronger case for a potential correlation between Bd resistance and TLR selection, I would suggest shifting the focus of the paper away from Bd slightly once the need for the research has been established in the introduction.

Aside from those issues my remaining points are relatively minor.

I look forward to reading your revised manuscript.

All the best,

Lewis Campbell


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: SELECTIVE CONSTRAINT ACTING ON TLR2 AND TLR4 GENES OF JAPANESE RANA FROGS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Figure 1, S1, S2, S3 and S4 legends. Species name should Lithobates catesbeianus.

Be consistent with the shorthand name you use for Batrachochytrium dendrobatidis (Bd vs. the Bd fungus).
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: No additional comments


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

Input:
Title: SELECTIVE CONSTRAINT ACTING ON TLR2 AND TLR4 GENES OF JAPANESE RANA FROGS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed all the points given in the review. I am satisfied with the answers and changes to the manuscript. I recommend the manuscript in its current form for the publication in PeerJ.
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: SELECTIVE CONSTRAINT ACTING ON TLR2 AND TLR4 GENES OF JAPANESE RANA FROGS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have greatly improved the reporting of this manuscript in this revised version.

I have only a few minor comments.

The newly added background section regarding expression of TLRS (lines 52 - 57 in pdf) feels out of place. Consider moving this to before the section regarding Bd (70 -  77) or incorporating it into the section 78 - 87.

I welcome the addition of a hypothesis statement (lines 84 -85) as per my last round of comments. I do however, feel like the current hypothesis feels very specific given that we know how the results come out. I would personally prefer to see something along the lines of " Given the potential immunological importance of TLRs against fungal pathogens, we hypothesized that TLRs would be subjected to purifying selection in species which show marked resistance to Bd.)

Line 86 - Since the transcriptomics was actually for another study I would prefer to see the word transcriptomics replaced with "published transcriptional data".

Line 94 -95 - "For a minimum of five weeks"

Line 95 - "And thus considered healthy"

Line 100 - I would again like to see "the transcriptome data set" replaced with "published transcriptomic data".

Line 161 - Reference the original Gosner stage paper here  (Gosner 1960).

Slightly contrary to my comments in the previous round of review, I think it might improve the paper to briefly mention the limitations which prevent you from being conclusive about a link to Bd in the discussion.

I.e. (On line 222) all three species studied. **As we did not examine the patterns of selection in species which are susceptible to Bd any link is currently speculative, nevertheless, we cannot rule out the possibility of adaptive evolution, potentially .... and so on **

I like the new figure 1 very much.
Experimental design: no comment

My previous concerns have been addressed by more extensive reporting.
Validity of the findings: no comment
Additional comments: Well done on improving this manuscript. My remaining comments are minor and I hope will improve it further still.

The flow of the manuscript is much better! However, I still find the results section a difficult read. This is not a fault per se so I have not suggested edits above. However if you are so inclined I think the readability of the results section could be improved.

Your new figure one looks great and will definitely go a long way to making for a good looking type set manuscript.

Congrats.