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: EFFECT OF ISOLATION ON COAT COLOUR POLYMORPHISM OF POLYNESIAN RATS IN ISLAND SOUTHEAST ASIA AND THE PACIFIC
Review round: 2
Reviewer: 1

Output:
Basic reporting: First of all, I would like to thank the author for considering all my suggestions for edits and answering my questions satisfactorily. The author has thoroughly revised the text and figures accordingly and I think that this very interesting and high quality piece of work has greatly improved, concerning content, style, and language. It is much more comprehensive now. However, a couple of new questions and suggestions arose, which I think would be important to consider as well (see below).

Please note that there are still no captions for the supplementary figures.
Experimental design: High quality, see first review
Validity of the findings: High quality, see first review. For exceptions, see general comments below.
Additional comments: My major comments/questions are the following:

(1)	Why do you consider darker colour (melanistic variants) as an aberrant coat colour variation in the context of the ‘island syndrome’ in analogy to the ‘domestication syndrome’? Usually, these ‘syndromes’ are associated with lighter colours only, not darker ones. It would be important to elaborate on this.

(2)	There is another important concept/cause for the observed pattern of aberrant coat colours in island populations, which is – as far as I could see – not discussed (only mentioned briefly in the abstract and aims):  ‘predator release’, i.e., the lack of predator pressure and thus certain selection pressures. There are only few or no predators on most of these islands, as the author shows, and this might lead to a lack of selection against colour patterns that provide less effective camouflage, e.g., light fur, white spots. This, in turn, might lead to a relatively higher occurrence of these coat colour variants in the island populations, compared to the mainland populations, where such aberrant patterns might be selected against. This mechanism has been shown to be a probable cause of white or light colour variants in domestic mammals, including the house mice in the study by Geiger et al. 2018 (besides the selection for tameness).

For this, it would be important to repeat the analysis of a correlation between aberrant coat colour and number of predators including the number of predators on the mainland, which have not been considered for this analysis as far as I could gather from Table 2. (Please correct me if I am wrong.)

In this context – and also for other considerations – it would be important to discuss domestic predators (cats, dogs, ferrets etc.), which also inhabit anthropogenic environments, just as the rats. Are there domesticated predators on these islands? What might their influence be? The absence of all predators, including the domestic ones, is an important prerequisite for the selection for tameness.

(3)	Although you did not find a significant higher occurrence of aberrant coat colour patterns on islands compared to the mainland, you still state in the abstract, discussion, and conclusions, that there is a higher occurrence of such aberrant coat colours on the islands. This is inconsistent and a bit misleading. Could you please include qualifiers such as e.g., “there is a trend towards a higher incidence of aberrant coat colours on the islands compared to the mainland, although not statistically significant”?

(4)	The discussion is still a bit convoluted and therefore hard to grasp. I would therefore suggest summarising certain parts, e.g. the paragraphs about competition and predation could be summarised and discussed together and the genetics part is a bit lengthy and might profit from summarising.

Here are some more minor issues:

- For enumerations, you sometimes use a comma before and/or, and sometimes not. Please be consistent.

- L. 92: The citation by Lomolino needs a page number.

- L. 93: “wolf” should be in lower case.

- L. 94: Falkland wolves also had white on the muzzle and on the lower limbs, not just the tail tip (see Clutton-Brock 1977, Man-made dogs)

- L. 169 ff.: You’ve explained to me that on some islands, lighter specimens are the norm, on others darker ones are the norm: “The ‘normal’ coat colours differ substantially between the regions,... For example, New Zealand specimens are much darker, towards melanism, yet are considered ‘normal’, whereas exactly such a specimen from an area with only light coloured furs would be labelled as melanistic.” Would this mean that the “very dark, toward melanistic” specimens from Polynesia in Table 2 would be considered normal in New Zealand. If this is correct, it would be important to mention here what you explained to me.

Further, although you deleted this part in the newest version, I think it would be an important to mention that you also showed specimens to curators and co-workers. This qualifies your own evaluation. Therefore, I would just write here what you explained to me.

- L. 177 – 188 and 195 - 204: These parts belong to the discussion.

- L. 180 and elsewhere in the text (probably as the result of “track change” in word): Missing space after “.

- L. 215: number of aberrant coat patterns “per biographic area”? Please specify.

- L. 223 – 224: I would exclude the statement about summary statistics in Table S1 from this bracket, as it also concerns information about results not related to the geographic distribution.

- L. 226 & 244: coat “colour” pattern?

- L. 227: Delete “further”. At this point in the manuscript you have technically already done all the analyses.

- L. 233: You could add a reference to Fig. 1 where you mention “a few strands of hair” to illustrate what you mean.

- L. 235: You could add a reference to Fig. 4 here, for comparison.

- L. 236 – 239: You explain that albinism and leucism are two completely different phenomena (to which I agree). So why do you explain leucism on the basis of albinism here?

- L. 238: Which “evenly buff coloured specimens” do you mean here? From which area are they? Please specify.

- L. 239 ff. These specimens lack eyes. So why do you know that they “have black eyes”?

- L. 252: log transformed “island” area?

- L. 267 – 269: This sentence is a bit long and confusing. Consider splitting.

- L. 274 - 278: You mix predation and competition in this sentence. Which one do you mean?

- L. 278 ff.: You are quite negative here, although there is apparently plenty of evidence that these rats are more associated to humans in the introduced areas, due to competition with native rodents. Similar scenarios have also been suggested for commensal house mice in Europe, which are usually outcompeted by other rodent species in non-anthropogenic habitats (e.g., Berry RJ et al. 1982. Colonization by house mice: an experiment. Journal of Zoology 198.3: 329-336.)

- L. 291 – 293: You could refer to Figures 1 and 2 here, for comparison.

- L. 306 – 308: Only the piebald specimens from Hawaii show these coat colour patterns “resembling colour polymorphisms of R. norvegicus”? Please specify what you want to express with this sentence.

- L. 308 ff.: White patches are not only a result of the commensal pathway of domestication, but for domestication in general, i.e., selection for tameness (e.g., domesticated foxes exhibit white spots, although they have been domesticated along the “directed pathway” according to Zeder).

- L. 323 – 327: This part contradicts what you say in the last sentence of the previous paragraph. If there are no predators on Hawaii, why should there be an adaptive advantage of having the same coat colour as the rocks?

- L. 388: “The occurrence of a piebald specimen favours a high degree of isolation.” does not make much sense. Do you mean: The occurrence of a piebald specimen favours “the hypothesis” of a high degree of isolation”?

Conclusions: As described above, this summary does not correspond to your results. According to your statistical tests, there was no higher incidence of coat polymorphisms on the islands. Additionally, you did indeed find a correlation of colour polymorphisms and number of competitors, although you describe here that there was no correlation. Please clarify.

Figures 3 – 5: Not sure if this is necessary, but it might be useful for readers if you shortly note that all these genus and species names on the specimen labels on the photographs are synonymous to R. exulans according to Wilson and Reeder 2005 (?)

Supplementary Data S1 has no captions on top of the columns. Please add those.

Supplementary Table S2: The last header “Relation between island area and aberrant coats” should probably just be “Relation between island area” (because you also test for relations between island area and number of competitors)


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: EFFECT OF ISOLATION ON COAT COLOUR POLYMORPHISM OF POLYNESIAN RATS IN ISLAND SOUTHEAST ASIA AND THE PACIFIC
Review round: 2
Reviewer: 2

Output:
Basic reporting: Refer to comments in my original review.
Experimental design: Refer to comments in my original review.
Validity of the findings: Refer to comments in my original review.
Additional comments: I am largely satisfied with the revisions made by the author, and I found this study to be most interesting. I applaud the author for addressing an interesting topic, compiling an impressive data set, and appropriately revising the original manuscript. I recommend making a few mostly editorial changes.

Line 57: “and dispersal abilities” should be changed to “and reduced dispersal abilities”.
Line 70: “reduction of brain size” should be changed to “reduced brain size”.
Line 71: “change “depigmentation of” to “depigmented”.
Line 98: I think “dog” should follow “feral domestic”.
Beginning of Materials & Methods: collections housed in two museums are mentioned here, but in the new paragraph from lines 210-219, the author points out that there are three collections. In the Acknowledgements, the National Museum of New Zealand is mentioned, so assume that it is the third collection, and it should be added to the beginning of the Materials & Methods. Also, if the new paragraph is correct in stating that there are three collections, then “between” on lines 218 and 219 should be “among”.
Line 150: “Smithsonian Institution” rather than “Smithsonian Institute”.
Line 155: “are as follows per group:” (add the colon :).
Line 169: “faunas” rather than “fauna”.
Lines 228-233: I am now confused about the wording of the statistical tests. An association is a correlation, but a Kruskal-Wallis test is a nonparametric test for differences among medians of more than two samples (analogous to a parametric ANOVA for testing for differences among means). A Mann-Whitney test does not test for a correlation but instead is a nonparametric test for a difference between medians of two samples (analogous to a parametric t-test). I believe some clarification or different wording is in order here. The same comment applies to the results section (lines 265-271) and lines 282-283 in the Discussion. Also, I believe that the test statistics that are associated with the P values should be given in lines 265-271.
Line 279: “amount” should be “number”.
Line 285: Change “This correlation is best explained as coincidental, not causal.” To “This correlation is best explained as coincidental rather than causal.” Also, verify that “correlation” is the correct word here.
Line 306: a comma (,) is needed after “Luzon”.
Line 372: a period (.) is needed after “(Wagner, 1845)”
Line 399: change “population” to “populations”.
Line 439: change “(founding events), thus” to "(founding events) and thus”.


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: DETERMINISTIC PROCESSES DOMINATE SOIL MICROBIAL COMMUNITY ASSEMBLY IN SUBALPINE CONIFEROUS FORESTS ON THE LOESS PLATEAU
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear English used but lot of jargon. The writing style is ambiguous, where many broad level statements are made but not backed up with data or relevant references.
Literature references are not thorough to provide sufficient background information.
Raw data has not been shared, neither code for the models.
There is extensive speculation with the second and third hypotheses.
Experimental design: No justification provided on why the sites were chosen. Neither are replicates highlighted. How were the 23 samples divided between the three sites? How were the replicates handled?
No clear statement on how knowledge gaps were filled.
Methods are not described in sufficient detail, and definitely no information to replicate.
Validity of the findings: Unclear how replication was handled, which means statistical soundness of the data cannot be evaluated. Conclusion is not well stated, and there is broad speculation.
Additional comments: Line Specific Comments:
Line 65: Check format of references
Line 69, Line 75: What does one-sidedness of analytical methods mean? The authors repeatedly use this phrase but have not provided any explanation of what this actually means? What is this sidedness? What is the other-side that needs to be analyzed?
Line 80: What exactly do the authors mean by precisely and robustly disentangle different ecological processes? In general, there are many such sentences throughout the manuscript that do not really explain the point the authors are trying to make.
Line 81: For readers not familiar, what do NRI and NTI actually mean? This should be highlighted here as opposed to Lines 147-148.
Line 86: Aggregation of what processes?
Line 88-89: Measured traits usually represent the functioning of an organism. It is a grey area when the authors write that traits cannot represent the ‘whole’ functioning. What does one mean by whole functioning of an organism? Can the authors provide examples where functional traits have failed to represent the functioning of an organism?
Line 106: How many plots each in WT, LY, and PGQ? Why these broad mountain distinctions? The authors don’t mention this until very later in the manuscript. Why were these sites chosen? How many replicates per plot? Are all results presented downstream of replicate samples, pooled samples? Statistics were performed on what? It is hard to extract this information downstream when the only information provided is 23 samples were studied.
Line 111: 1 g of sieved soil was used for DNA extraction? In that case, the extraction does not represent actual community profile. Soil sieving significantly impacts microbial community structure.
Line 145: Provide reference for Stegen et al.
Line 137: How many times (iterations) was the model run?
Line 158: Show how one arrives at niche breadth from OTU table?
Line 167-168: Where is this functional niche breadth-based dendogram? Is this something the authors created or is this something widely available?
Line 198: Ammonium nitrogen can’t be significantly related to itself.
Lines 203-216: Why should the readers care about this? How are the sites different to begin with? Should a difference be expected?
Line 221: Looks like pH has the longest length
Lines 239-240: It is unclear how the authors arrived at the conclusion that changes of modules represented changes in interspecific relationships. The network analysis figures are impossible to decipher. What do the colors mean? The networks look very tightly linked. What do the modules mean? How many modules were there?
Line 258-260: There is no substantial evidence presented in the manuscript to back this.
Line 261: What one-sidedness?
Line 264-265: Where is the proof that interspecific relationships are driving community assembly? What are these relationships? Are these selection, dispersal ?
Line 280: It will make most sense to regress the environmental variables with results of assembly processes to avoid speculations. If indeed SOC Is the main driving force, this should be visible in the regression with assembly process.
Line 295-302: This should go in the introduction as an added information about the sites.
Line 303-304: Again, this is a stretch to say interspecific relationships (what are these, what is the metric for this?) explained the variation.
Line 321-324: Highly speculative. Litter dependent nutrient accumulation and therefore substrate availability can be tested. Was competition accelerated at one site, across all sites, between sites?
Line 325-340: Applying macroecology principles of deep and shallow phylogeny related historical factors to microecology is questionable. Especially in microbes where horizontal gene transfer is a very active process, the authors have not presented any proof that such extrapolation from macro to micro ecology is valid. This paragraph sheds no extra light, and ends up confusing the reader. What is meant by disjunctive occurrences?
Line 338-340: Did the authors validate that there is no different in paleo and current climate or the difference was not the driving factor?
Lines 341-348: The purpose of this paragraph fails me. What are the authors trying to say?
Line 350-352: The authors should have just stopped at this conclusion. This is the only definitive outcome of this study. Rest is highly speculative and not clearly written, proven, or discussed, and there is use of jargon. For readers less familiar with the field of study, this manuscript does not provide clarity on biogeography of soil bacterial communities, if that is what the authors were aiming for.
Line 361: Where are the source codes for all the modeling? Have these been deposited in any database? If someone attempted to repeat this experiment/analysis, how would they do so? This cannot be repeated.
Lines 366-520: References have to be heavily formatted. Many inconsistencies.


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: DETERMINISTIC PROCESSES DOMINATE SOIL MICROBIAL COMMUNITY ASSEMBLY IN SUBALPINE CONIFEROUS FORESTS ON THE LOESS PLATEAU
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study is about an environmental gradient in a sub-alpine forest in China using 23 plots that they clump in 3 categories based on the mountain ranges. They use 16S tags in illumina and the nutrient content of the soil. The manuscript starts very promising and their hold of community theory is in general sound. However, their review of the recent papers on the issue is not so sound. This is not the first community analysis of soils that has attempted to join several points of view in one analysis (google scholar has14,000 in the last 4 years, many of them with a very deep understanding of the problem).
I particularly like: Delgado-Baquerizo et al., 2016. Microbial diversity drives multifunctionality in terrestrial ecosystems. Nature Communications. http://dx.doi.org/10.1038/ncomms10541. The authors do not know the literature so they try to "discover" new methods, without really going deep into any of them. The authors look for differences between stochastic vs deterministic drivers by looking at the relationship between environmental variables, phylogenetic relatedness and interactions using network analysis (that they do not describe). However, even if they have more than 4000 different OTUs most of the analysis seems to be done at the phyla level or class level losing most of its value, since patterns with such a rough “grain” are unable to disentangle the effect of, precisely, interactions. As an example is the figure 8 where there is nothing to be seen in the “clouds”.
Experimental design: The basic design is good, the number of samples and the sampling is well done, even if always more sequences can be obtained, that is not the point. The theory is the correct one, even if they need to read more and be humble about their uniqueness. The problem here is the analysis, this is why this can be improved if they read and are careful about their statistical approach. They need to explain better at what level is each test, and disentangle the patterns that are jumbled together. The devil is in the details and here there are a lot of details to sharpen. Figure 7 does not make sense, at its presence is the core of the problem, 78% is an unknown…probably due to the resolution of the analysis done, or the lack of the proper question to the analysis.  It seems that somehow, they did all the potential analysis, put them in a blender and got lost in the way. Please rescue the “tread” of the story.
Validity of the findings: The only clear pattern is the relationship with nutrients (RDA analysis). One mountain range is more diverse than the other…but the difference between stochastic vs deterministic drivers that explain such diversity is not clear at all. There is a lot of polish that needs to be done in order to add value to this simple observations.
I recommend that you follow the network analysis done by De Anda et al., 2018 in frontiers, it is an example of a careful network analysis.
Additional comments: I am putting mayor revision given the potential of a good paper if the proper analysis is done. I am adding the pdf with my direct comments, do not pay attention of the highlighting, that was for me to follow the story.


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: DETERMINISTIC PROCESSES DOMINATE SOIL MICROBIAL COMMUNITY ASSEMBLY IN SUBALPINE CONIFEROUS FORESTS ON THE LOESS PLATEAU
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1. No clear hypothesis or justification for conducting the study.
2. What continuum was expected in the sub-alpine ecosystem?
3. The sampling design is flawed (with varying number of samples collected from the sites). Extremely difficult to infer any meaningful conclusion from the statistics.
4. With no conclusive reasoning for the study, there is no understanding of what knowledge gap was identified and addressed.
Experimental design: Flawed. 10 samples were collected from one site, 8 from another, and 5 from the third. No information provided in the methods as to how this was dealt with/how was normalization done. There is inherent heterogeneity within each site as well. Research question in not well defined or meaningful.
Moreover bNTI needs to be tested against SOC matrix using a mantel test.
Validity of the findings: With no hypothesis to test, and irregular sampling strategy, no justification of why samples were studied in sub-alpine system (e.g. were the authors aiming for change in elevation gradient and community assembly patterns?, or if the authors were attempting to study a chrono/climo-sequence, how were their sites classified, or if the authors were attempting to study soil/ecosystem successional stage, what were their soils like?
Additional comments: Line 26: Check sentence structure
Line 33: Abrupt sentence
Line 28-29: What is this continuum? What are the boundaries of this? What was more prevalent at which site?
Line 34: Recommended to perform a mantel test between bNTI and SOC matrices to understand if SOC is really driving the assembly process. The SOC numbers vary considerably within each site, and integrated RDA, MRT, and correlation analysis was not performed on the community assembly results. This is a indirect inference, which can be made stronger and conclusive if the mantel test is done.
Line 35: what are these ‘some’ differences? The abstract needs to be have conclusive results and not general pronouns/adverbs to describe the results.
Line 36-37: The abstract does not provide any information of this continuum, how is the continuum being defined? Did one see stochastic processes at one elevation, which gradually change to deterministic as one moved higher in elevation? Or did the assembly structure change progressively from stochastic to deterministic as SOC increased? The word continuum has been repeated a few times in the abstract, but it is not clear as to what this continuum is.
Line 48-49: Please cite Baas-Becking for the “Everything is everywhere…” sentence.
Line 58: What is the authors’ interpretation of this continuum? Or what is the accepted interpretation of this continuum in the field of microbial ecology?
Line 62: Based on these previous studies where ecological continuum varies based on environmental conditions, can the authors derive hypothesis for their own study?
Line 66-68: What common standard? The studies are ‘limited’ to specific spatial and temporal scales or sampling scales because the question being asked in those studies are probably relevant at those scales. It is unclear what common standard the authors are referring to and trying to prove?
Line 69: How does sampling sub-alpine coniferous forest support the ‘common standard’ that the authors are trying to achieve?
Line 77-78: The sites are not along a transect of elevation.
Line 79: What are the climatic gradients observed for the three sites? Why should a difference be expected between the sites enough to warrant a community assembly study?
Line 83-87: What is the justification for such random sampling (8, 10, 5) from each site? The sites itself are heterogeneous. For the WT site, with such a range in the elevation, where was the sampling done and why? Can’t the authors group their samples according to elevation? There seems to be no logic evident for the sampling strategy. Moreover, if each plot is treated as a replicate for the site (which shouldn’t be the case since the sites are heterogeneous to begin with), then it appears that there were 8 replicates in one site, 10 in another, and 5 in the last one. It is extremely difficult to perform statistics and infer meaningful results with this sampling design.
Line 104: If you are using primers for prokaryotic 16S rRNA gene, there shouldn’t be any eukaryotic sequences to remove.
Line 119: Reference?
Line 121: How was the model controlled for stochastic variation and associated changes in alpha diversity?
Line 163-164: which analysis did you use? Generally people use one or the other.
Line 164-165: Why not spearman correlation analysis between otus and environmental variables?
Line 165: What is PCNM?
Line 166: What are these 23 extracted PCNM variables?
Line 184-186: Define the gradient. These lines finally somewhat depict the reason for conducting community assembly studies in the sites that were selected. This should be reflected in the introduction.
Line 192: Enough for what?
Line 220-221: I don’t think anything can be commented about deterministic vs stochastic responses from partitioning analysis.
Line 225: Network Analysis: PQG site has maximum sampling sites (10). It is not surprising that PQG exhibited more edges, vertices, degree and clustering coefficients. The analysis is biased because the sample size is uneven across the sites. How was this factored for in this study?
Line 227: Fewer modular?
Line 230-231: Nothing can be inferred about keystone species at different sites since the sampling size was different and therefore the results are not comparable.
Line 240: Which parameters of the assembly process?
Line 247: How does one manage microorganisms? There are billions of them in 1 g of soil.
Line 251-252:  This study focused on bacteria. How does a comparison to fungal results make any sense here?
Line 254: Determination?
Line 262: Compared to this literature, does this study actually capture early vs late successional soils? What is the importance of the sites? What gradient was being studied, if any?
Line 289: The irregularity in the number of plots sampled per site makes this analysis very hard to interpret.
Line 300: Makes no sense
Line 307: “some” differences?
Line 307-309: Unfortunately I did not find any extra knowledge from this study, or what this continuum is, and the study contributed to understanding the continuum.
Line 313-320: Check these references, they seem out of place and not arranged alphabetically.
Figure 3a, b, c: How were the individual samples handled in these plots? Were the 8, 10, and 5 samples from the sites averaged per site?
Figure 6a: The legend colors cannot be differentiated. The figure is hard to interpret.
Figure 6b: Write in the text that spearman correlation was performed.
Figure 9: bNTI is a matrix. You cannot do ANOVA to test relation with SOC. You need to do mantel test. Figure 9C and 9D is supposed to show R2 values. Not sure what R is.


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: DETERMINISTIC PROCESSES DOMINATE SOIL MICROBIAL COMMUNITY ASSEMBLY IN SUBALPINE CONIFEROUS FORESTS ON THE LOESS PLATEAU
Review round: 2
Reviewer: 2

Output:
Basic reporting: This is a resubmission of a previously evaluated document, the authors did all the suggestions of the reviewers and I consider that the manuscript is mostly ready for acceptance. The english is much better and the the speculation about the  results have notable been tone down.
This is an interesting study where they survey an altitude gradient for soil microbiome observing that carbon is the main driver of diversity, confirming other studies.
Experimental design: It is a good design with enough replicates and samples for 23 sites
Validity of the findings: It is well done
Additional comments: I believe that this version of the manuscript is good for publication.


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

Input:
Title: DETERMINISTIC PROCESSES DOMINATE SOIL MICROBIAL COMMUNITY ASSEMBLY IN SUBALPINE CONIFEROUS FORESTS ON THE LOESS PLATEAU
Review round: 3
Reviewer: 1

Output:
Basic reporting: Improved version from the last time.
Experimental design: Still a disjoint between what the hypothesis is and how the sampling is suitably set-up to test the hypothesis. Please see line specific comments.
Validity of the findings: Conclusions are not well stated. See line specific comments.
Additional comments: General comments: I still do not see a compelling reasoning for this study or a clear well-defined hypothesis. It is not suffice to say soil A is different from soil B. Given how much heterogeneity exists in soil environments, ofcourse soil A will be different from soil B. The justification for this study is still sending out mixed signals, as if the authors do not have a clear idea of what they are attempting to study and why. Sometimes it reads as if they are studying altitudinal gradient, sometimes it reads that they are studying a climatic gradient and climosequence, and it also reads as if they are studying assembly processes that shift across the lower and upper distribution limit of a subalpine tree species. What is it that is exactly being studied here and why? If the upper and lower limit of distribution define the sampling sites, are the authors studying community assembly in the soils at these zones of distribution? Is that related to the ecology of the trees? Perhaps higher litter at higher distribution areas indicate higher SOC, and therefore a more deterministic force?

Line Specific Comments:
Line 70: Why would the relative importance differ? Were your sites representing successional stages?
Line 73: “different scales” of what?
Line 73-74: I do not see any theoretical support being provided to understand the mechanism. Even after reading the entire manuscript, I do not see a link between the environment that was studied and the results that were obtained. Without a strong hypothesis, the reason for this study is lacking.

Line 82: What is meant by typical soil plots?
Line 84-86: The sentence sounds like these references studied the same site as yours and provided conclusions about microbial community in your sites. This is factually incorrect.

Line 87: Capitalize Cinnamon. This is a soil series.

Line 88-92: The driver gradients are made up of what? Still sending out mixed signals, sometimes it reads as if you are studying altitudinal gradient, sometimes it reads that you are studying a climatic gradient and climosequence, and it also reads as if you are studying assembly processes that shift across the lower and upper distribution limit of a tree. What is it that is exactly being studied here and why? If the upper and lower limit of distribution define the sampling sites, are you studying community assembly in the soils at these zones of distribution? Is that related to the ecology of the trees?

Line 93-98: Which site was at the lower distribution limit and which site was at the upper distribution limit?

Line 104: Sieving significantly alters microbial community composition. Justify why this was done. How are you confident that this is representative of the actual microbial community composition at the site?
Line 117: Incorrect reference
Line 118: Why was archaea removed?
Line 141: Incorrect sentence
Line 241: modular what?
Line 253-254: In your response document you said that bNTI values are not matrix. How did you do mantel test (which you claim to have done) if the bNTI values are not a matrix? Mantel test is for matrices.
Line 258-259: Weird sentence
Line 262-264: Again, you do not have fungal community data here. It makes no logical sense to therefore compare your bacterial community assembly work to someone else’s fungal community work.
Line 271-274: Do the soils in this study fall under any of these category described here?
Line 285-290: Have you considered if SOC is impacted by the upper and lower distribution of your tree species. Litter from the trees will impact SOC, which in turn will impact the community assembly structure, and is perhaps a reason that you see variable selection increase from LY to WT sites.
Line 290-292: Are your sites representative of successional stages?
Line 302-304: Perhaps brought about by transition zones between LY and WT? Why is there greater heterogeneity at PQG?
Line 315-321: Not a strong conclusion. Needs to be refined. What inference can one draw about subalpline coniferous forest density vs soil microbial community assembly processes?
Figure 3: Please write in the caption that these represent average values.
Please check significant figures in Table S1 pH values.
Why is Oikos et al.’s supplementary appendix material being provided? The codes are for that study. How is it relevant to this current study? I am not even sure one can just provide another study’s appendix in one’s supplemental 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: THE MICROBIOME PROFILING OF FUNGIVOROUS BLACK TINDER FUNGUS BEETLE BOLITOPHAGUS RETICULATUS REVEALS THE INSIGHT INTO BACTERIAL COMMUNITIES ASSOCIATED WITH LARVAE AND ADULTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The writing is good overall. Please check the formatting of the references, and confirm that all citations appear in the references section.
Experimental design: The design is good overall, though it seems several replicates for surveying the beetle microbiome were pooled. It perhaps would have been nice to sequence these separately to have true replicates, but overall the samples all looked quite similar.
Validity of the findings: The phylum-level classifications presented throughout the paper and in most of the figures are difficult to interpret given the vast diversity of prokaryotes within each phylum. It would be much more useful to see what bacterial families and genera were most abundant in each of the samples.
Figure 4 in particular would be much more powerful if it included the most abundant genera, rather than phyla. Currently the clustering is uninformative since all of the samples look relatively similar.

Line 36: “for long-term field” -> “for the long-term field”

An important role that bacteria could be playing in these tree associated systems is the degradation of plant defense compounds such as terpenes, which can be toxic to fungi and beetles. Sphingomonads have previously been shown to have versatile biodegradative capacity and could be involved in this process (Aylward et al., Applied and Environmental Microbiology, 2013) and microbial communities associated with pine beetles have also been hypothesized to play this role (Adams et al, Applied and Environmental Microbiology, 2013). I would encourage the authors to emphasize this potential role a bit more, since it also fits with the presence of Burkholderia (as the authors point out in the paragraph starting on line 409).

Line 444: “The bacterial community associated with black tinder fungus beetle likely plays a role in promotion of efficient digestion for extraction of maximum energy from ingested substrates”. Is this really true? Is it possible that the bacterial community is mostly associated with the fungus, and we see it in the beetles simply because of their diet?

I do not think the PiCRUSt analysis adds anything to this study, and it could potentially be omitted to streamline the manuscript. The authors find no significant differences between pathways based on this analysis, and it is hard to interpret findings based on the potential relative abundances of broad functional categories like “membrane transport”. If the authors really wish to keep it, I would recommend just briefly mentioning that it was done and did not show any meaningful differences between samples.

It is unclear to me what the importance of protease inhibitors is in this system. The authors should consider describing the importance of this more in-depth in the introduction if they feel it is an important part of this study.

The discussion is quite long and speculative. I would consider limiting or omitting the PiCRUSt analysis and streamlining the discussion of protease inhibitors. By focusing on the dominant bacterial genera present I think the most important aspects will be conveyed.

Previous work has shown that bacteria of the genera Pseudomonas, Enterobacter, and Rahnella are most abundant in beetle and fungus-associated niches. Is this true here? It’s possible to see this from the supplementary sunburst diagrams, but it would be nice if this was more clearly mentioned in the Discussion.
Additional comments: Overall I think this paper would be suitable for publication if the authors address the comments above and streamline the paper so it is a bit more brief and directed.


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 MICROBIOME PROFILING OF FUNGIVOROUS BLACK TINDER FUNGUS BEETLE BOLITOPHAGUS RETICULATUS REVEALS THE INSIGHT INTO BACTERIAL COMMUNITIES ASSOCIATED WITH LARVAE AND ADULTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The research paper submitted to PeerJ and entitled "Predictive functional profiling and surveying the microbiome of fungivorous black tinder beetle Bolitophagus reticulatus" by Kaczmarczyk-Ziemba et al deals with the metataxonomic analysis of the microbiome associated with the beetle Bolitophagus reticulatus and its predicitive functional profiling.

It almost solely relies on the predictive functional analyses using PiCrust and based on 16S phylogeny. This is too speculative in general and thus I would actually suggest to the authors to formally access the functional potential of microbial communities to perform a shotgun metagenomic analysis. Furthermore, the diversity analyses s quite shallow with only a venn diagram and barplots. This could well be improved.
I would also like to point out that the greengene database that was used to infer taxonomy has not been updated since 2013 so I would suggest the authors to use the Silva one for example.

So while the research question is of interest, the tools used are not completely appropriate.

And, this is why I tend to agree with the last sentence of the conclusion. This study in this form to my point of view is a pilot study and needs to be performed at a wider scale prior to publication.
Experimental design: As indicated above, the choice of approach to infer functional potential is not ideal as well as the database used to assess taxonomical affiliations.

The metagenomic DNA extraction was performed on the whole body of the insects. Could the authors maybe explain why they haven't extracted the gut microbiome, ie dissected the insects and larvaes? That would have been more specific and proper to answer their research question

The authors have performed a 'preliminary biochemical analyses' of sporocarps.
First, I'm unsure what this means (could it be improved? is it incomplete?) and second, why have they not done these analyses on the fruiting bodies collected for this 16S study? To my opinion this approach makes no sens as long as they have not shown that the biochemical analyses of the fruiting bodies studied is constant through time and space...And, why did they specifically choose sporocarps from fallen trees for the biochemical analyses and not for the 16S one?
Validity of the findings: no comment
Additional comments: Generally, I also find that having a 10p discussion for only 4p of result is too long and dilutes the scientific message that the authors are trying to make. I would thus really suggest the authors to shorten the discussion.
I would also recommend to rewrite the introduction significantly as it does not seem linked to the research questions. And it has some vague statements (eg L95-102).

Minor comments:
Often the authors refer to 'previous studIES' and only cite one (eg L70-73 or L91). Also the referencing in the text is not constant (eg L87 "Jonsell, Schroeder & Larson, 2003" should be "Jonsell et al, 2003"). All this should be fixed
Finally, while English is not my mother tongue, I strongly suggest the authors to ask for an English speaker to check their manuscript for minor, but existing, grammatical mistakes.
Scale bars on Figure 1 would be more than appreciated.
Abbreviations should be defined the first time they appear (eg NGS L105)à


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 MICROBIOME PROFILING OF FUNGIVOROUS BLACK TINDER FUNGUS BEETLE BOLITOPHAGUS RETICULATUS REVEALS THE INSIGHT INTO BACTERIAL COMMUNITIES ASSOCIATED WITH LARVAE AND ADULTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Kaczmarczyk-Ziemba and colleagues explore the microbiota of the fungivorous black tinder beetle, Bolitophagus reticulatus. They use 16S rRNA gene amplicon sequencing of 12 samples--3 from each developmental stage (larvae, adult) from each tree type (beech, birch)—to compare the microbiomes. Exploring the microbiota associated with fungivorous insects is interesting and highly warranted, as such studies will help lead towards a broader understanding of how insect microbiota is associated with different diets.

General Comments
•	The grammar/syntax/flow could be improved (some examples included in specific comments below).
•	PICRUSt is not very reliable for 16S amplicon sequencing. 16S only resolves taxonomic placement to the family/genus level, while gene context varies hugely within these genera and families of fungi. Yes, this is commonly published on in the literature, but that doesn’t make it reliable. In my opinion, this work doesn’t add anything to this study regardless, so I would just cut it all out.
•	The breakdown of the bacterial composition of their samples is interesting, although there weren’t many differences. The issue is that the number of samples is rather small for a 16S amplicon study. Further, it is limited by the 16S. I do think the comparison to the work in Aylward et al. 2014 is interesting and a strength of this study; meaning it is interesting to compare beetles that feed on fungus that is cultivated or symbiotic, with beetles that are feeding on ‘free-living’ (with regards to the beetles) fungus.
•	Rf values are stated but no compounds are talked about? They said in their methods the Rf values would be compared to Rf values in a database to distinguish compounds. Were they not able to distinguish any compounds from the Rf values?
•	The statement that they “nearly fully characterize its microbiome” is a completely over the top. 12 samples with 16S amplicon sequencing is just pilot findings in my view.
•	GreenGenes uses incomplete 16S sequences, making it error prone. Consider taxonomic assignment using the Silva database instead.

Specific Comments
•	Lines 67-72: refers to “studies” 4 times
•	73: “gained the raising interest” should be reworded
•	74/76: same paper cited twice in the same sentence.
•	79: One of the most widely distributed beetles?
•	83: It’s not clear that tinder fungus is the same as Fomes fomentarius. I think this should be spelled out.
•	106: varies  vary
•	258: ranking ranging
•	259: what  which
•	278/285/293/392/394: Phyla and class aren’t italicized?
•	337-338: awkward wording
•	355: “these compound affect not only direct insects”  Not only do these compounds directly affect insects
•	421/424: Nevertheless used to start both sentences
•	Discussion of Burkholderia (and other genera) was interesting and linked predicted function to potential function in vivo
•	Protease inhibitors/microbiome sound like two separate projects and aren’t linked together well
-	586: exceed excess?
-	Line 48: “extremely important” seems overstated, and “important” should generally be avoided in scientific writing as the word can come across presumptuous. Try “critical” or “consequential.”
-	Line 50-56 can be shortened to: “Saproxylic beetles can occupy several ecological niches:…”
-	Line 57-58 can be reworded to: “Mycetophilic beetles associate with wood-decaying fungi and use the fruiting body for nourishment and development.”
-	Line 73: considering removing “the raising”
•	Avoid starting a sentence or a clause with the word “it” because “it” is ambiguous in those contexts. Examples: Line 90, Line 332, 372, 507, 509, 517.
-	Line 136: “the inhabiting” can be reworded to “inhabited”
-	Line 308: “allowed to” can be reworded to “allowed us to”
-	Line 480-483: phrasing makes comprehension difficult.
-	Figure 5: y-axis could use a label.
-	Line 167-170 could use brief clarification. How were the samples merged? Were the number of reads of each sample normalized before merging?
-	Lines 79-88 are unclear. It states that B. reticulatus is widely distributed but that it was thought to have limited dispersal until another paper found that the dispersal ability may have been underestimated. It’s not clear if they are trying to link its fungal association with dispersal?
-	Lines 96-102:  They switch from talking about SM of tree being accumulated in fungus to SM produced by fungus. If the “however” in 95 was switched to “additionally” that suggests to me they are interested in investigating both tree SM and fungus SM.
-	Unclear why they investigated protease inhibitors other than “little is known”
-	Mixes adult stage and imago stage. Should either stick with one or make it clear they are the same developmental stage.
-	Figure 3 needs to be labelled better since it was confusing what the difference between the two graphs was. I’m not sure the “zoom in” is necessary and this figure can probably just be the one stacked bar graph.
Experimental design: see above
Validity of the findings: see above
Additional comments: see 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 MICROBIOME PROFILING OF FUNGIVOROUS BLACK TINDER FUNGUS BEETLE BOLITOPHAGUS RETICULATUS REVEALS THE INSIGHT INTO BACTERIAL COMMUNITIES ASSOCIATED WITH LARVAE AND ADULTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Improved
Experimental design: Improved
Validity of the findings: Valid and interesting
Additional comments: I think the authors have done a good job addressing the points raised in the initial round of review. The new manuscript is much easier to read, much more streamlined, and much more effective and making key points. The figures are also easier to follow. I am not the best person to evaluate the results of the biochemical analyses, but it seems as though the authors have been very forthright in their Discussion about possible caveats here, so these seem like valid data to include.
I am recommending "minor revisions" mainly so there is a chance to change the grammatical issues I raise below. I have no further recommendations for changes to the scientific content.

Line 25: “One of the group among them are” -> “among this group are”
Line 30: “the detailed studies focused on microbiome associated” -> “detailed studies focusing on the microbiome associated with”
Line 69: “This beetle belongs to tribe Bolitophagini which represent feeding strategy of dwellers”. I’m not entirely sure what this sentence means, or how the term “dwellers” should be interpreted, but it should be re-worded for clarity. Perhaps, “This beetle belongs to tribe Bolitophagini, which represents the feeding strategy of dwellers, which refers to xxx”
Line 94:  “Know” -> “Known”
I would avoid the term “preliminary” or “pilot” when referring the analyses here. These terms make it sound like the work isn’t ready to be published. If the authors are confident in the results, they can be presented as legitimate analyses along with all the rest. If there are any important problems with these analyses that the authors are aware of, they should just state those outright in the Discussion as potential complicating factors (I think the authors have done a good job of this in the paragraph starting on line 429).
Line 251: “approximately deep” -> “sufficient for microbiome characterization”
Line 289: “allow to identify” => “revealed”
Line 318: “listed as” -> “listed as the”
Line 320: “also those” -> “and also in those”
Line 429: “We are aware that these biochemical analyzes should be treated as preliminary. Fruiting bodies for biochemical analyses were collected earlier than those from which B. reticulatus specimens were collected” -> “The biochemical analyses presented here should be treated with caution since the fruiting bodies sampled for these analyses were collected earlier…”
Line 440: “Additionally, the known endosymbionts has” -> “Additionally, known endosymbionts have”


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 MICROBIOME PROFILING OF FUNGIVOROUS BLACK TINDER FUNGUS BEETLE BOLITOPHAGUS RETICULATUS REVEALS THE INSIGHT INTO BACTERIAL COMMUNITIES ASSOCIATED WITH LARVAE AND ADULTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: The authors addressed my previous 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: CORTICAL MODULATION OF PUPILLARY FUNCTION: SYSTEMATIC REVIEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: After reading this manuscript I am looking for data collected,to be analysed and discussed using Broadmann Areas and white matter tracts especially that contribute directly and indirectly to pupillary function,
The responses differ in the growing cortex from neonates till the elderly brain cortex.This is missing in this review and makes it less informative and citable in the medical world.
Experimental design: The data collected from publications should include
 Broadmann Areas and white matter tracts especially that contribute directly and indirectly to pupillary function,
The responses differ in the growing cortex from neonates till the elderly brain cortex
Validity of the findings: The systemic review should seek these data sets and include them(Broadmann Areas and white matter tracts especially that contribute directly and indirectly to pupillary function.
These responses differ in the growing cortex from neonates till the elderly brain cortex)
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: CORTICAL MODULATION OF PUPILLARY FUNCTION: SYSTEMATIC REVIEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: I enjoyed reading this brief and informative review on cortically driven pupil size changes.

The manuscript is adequately structured, it is written in a clear and coincise way and it covers a significant portion of the relevant literature. The authors set a very ambitious goal, a comprehensive review of the literature on cortical modulations of pupil size. The work goes a long way towards achieving this goal, although I see three areas that could be explored further.

1) there is evidence that attention can enhance the pupillary constriction in response to light - when the light stimulus is at the focus of attention. This effect is qualitatively different from the pupil dilation related to cognitive effort or to “paying more attention”: with a constant level of attention, directing its focus to a more luminous location or feature leads to pupil constriction, while focusing on a darker stimulus leads to pupil dilation (Binda et al., JNeuroscience 2013; JNeurophysiology 2014; JoV 2015; Sci rep 2017; Turi et al., eLife 2018; Mathot et al., 2013; 2014; 2016; 2017; Naber et al., 2013). This body of work has recently been supported by experiments in non-human primates, where stimulation of FEF produces the same pupillary effects as attention shifts: enhanced pupillary light response when the light stimulus is in the stimulated FEF receptive field (Ebitz and Moore 2017).
Thus, dilation is not the only effect of cortical signals on the pupil - although undoubtedly it is the most studied effect in the literature.

2) Naber and Nakayama 2013 is presented as an exception in the literature, reporting pupillary constriction of cortical origin. It is important to state that this work is not isolated. The very same result was reported by Binda et al. 2013 (same issue) using a different approach, and  later replicated (e.g. Sperandio et al., 2018). Also, and perhaps most crucially, Laeng & Endestad 2012 had previously shown that pupil constriction can be elicited by illusory luminance increments.

3) There is growing interest in using pupillometry to track the contents of perception during ambiguous stimualtion (Einhäuser et al., 2008; Naber et al., 2012; Turi et al., 2018) and during imagery (Mathot et al., 2017; Laeng & Sulutvedt 2013) or working memory (Blom et al., 2016), both clearly involving cortical signals.

The studies in my #1, #2 and #3 indicate that turning attention towards a a brighter stimulus (be it a real external stimulus or an internal representation) leads to pupillary constriction. Thus, I do not think it is correct to say that cortical modulations are generally seen as a pupil dilation. There is a group of studies, using a variety of approaches to show that cortical signals can also interact with the pupillary light response and produce pupil constriction. I strongly recommend including these papers, as I feel the review would be more balanced and more useful with these.
Experimental design: The literature search is performed with clearly defined and systematic criteria. My only concern regards the exclusion of any study with less than 15 participants. Of course large Ns are good but why 15? I find it difficult to come up with a justification for picking a specific number (which ends up being different for humans and non human primates, by the way).
Validity of the findings: The conclusions from the literature review are well stated and linked to the original research question. However, I suggest that they are revised after including the three areas of research that I described above (field: Basic Reporting)
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: CORTICAL MODULATION OF PUPILLARY FUNCTION: SYSTEMATIC REVIEW
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: the authors have addressed all my 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: ACCURACY OF A DEEP CONVOLUTIONAL NEURAL NETWORK IN DETECTION OF RETINITIS PIGMENTOSA ON ULTRAWIDE-FIELD IMAGES
Review round: 1
Reviewer: 1

Output:
Basic reporting: RAW data is not provided. Please consider using some sharing services. For example, Figshare is an accademic service and should be free for only 746 original images.
No other comments.
Experimental design: Very old people (>90y) also have RP like fundus. Were they excluded?
No other comments.
Validity of the findings: Authors evaluated deep convolutional neural networks in detection of retinitis pigmentosa on ultrawide-field images. Despite of relatively small size data, the accuracy was very high. However, the real world problem is that the patients with early stage of retinitis pigmentosa (RP) are often overlooked. As the authors said, false positive is OK, but false negative is not good in screening. If the authors present some cases with early RP and their accuracy, the readers will agree to the authors' DNN model is useful for detecting the RP.
No other comments.
Additional comments: In the acknowledgements section, Masyuki may be Masayuki.


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: ACCURACY OF A DEEP CONVOLUTIONAL NEURAL NETWORK IN DETECTION OF RETINITIS PIGMENTOSA ON ULTRAWIDE-FIELD IMAGES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The level of English is below acceptable standards. Composition of sentences is often weird, e.g., second sentence in abstract: “A single-facility, cross-sectional study.” A single-facility study of what/where/who?
In abstract, I don’t think that “patients…. were used”. I suggest that observations and measurements were used to build the model, not parents themselves.
“Training with a convolutional neural network on these learning data objects was conducted and a deep learning model was constructed.” - Convolutional networks technique is a deep learning method by definition. No need to mention it twice.
I don’t think there is a need to include abbreviations to the abstract.
Authors do not refer to supplementary materials in their manuscript (this should be fixed). However, there were some supplementary files and I am not sure why authors attached a copy the manuscript formatted for Journal of Ophthalmology.
It would be helpful to include a database of images as a raw supplementary data.
Experimental design: Description of methods in not sufficient:
It would be useful to describe convolutional network in more details (What was the activation function? What was the stride?, etc.).
Outcomes is a strange subsection that contains only one sentence. This section should be expanded (e.g., by adding basic definition and formulas).
Validity of the findings: Results section: “There were no significant differences in age….” – such claims must be supported statistically.
Discussion is not comprehensive and does not contain any sort of interpretation.
The manuscript lacks “Conclusion” section.
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: ACCURACY OF A DEEP CONVOLUTIONAL NEURAL NETWORK IN DETECTION OF RETINITIS PIGMENTOSA ON ULTRAWIDE-FIELD IMAGES
Review round: 1
Reviewer: 3

Output:
Basic reporting: There are some terms that seem mistranslated
Eg pyramidal cells instead of cones.
I think the manuscript would benefit from a review by an English speaking Japanese ophthalmologist to check there are no other errors like this. Some specific examples are listed below.

In abstract Line 20
“Totally,  373 UWPC…” would be better worded “In total, 373 UWPC…”
In introduction
Line 34 “pyramidal cells” should be “cones”
Line 36 “center” should be “central”
Line 42 It is not clear what “which is spread globally” means
Is it the treatment or the disease spreads globally (throughout the retina)
or the treatment being spread around the world?
Is it the globe of the eye or the globe of the world? (internationally)
Line 91 “sesame salt-like” I think should read as “salt and pepper”
Experimental design: The authors present on the ability of AI/Deep Neural Networks to detect retinitis pigmentosa.
We have seen some major recent papers describing the ability of AI to diagnose common diseases such as diabetic retinopathy, glaucoma and age-related macular degeneration but there is little data in the ability of AI to detect and correctly diagnose rarer retinal diseases.
The authors have a large series of retinal images from a clinic of RP patients.

There is no clear gold standard for RP
Electrophysiology and genetics could be compared to imaging but this is beyond the scope of this research now but will be interesting to see in coming years whether AI can detect early RP

RP is a wide range of diagnoses. A more broad term of inherited retinal diseases would include some disorders with very characteristic and distinct images.
The authors should be more specific about what disorders were excluded from the analysis, eg Stargardts disease, cone dystrophy etc.
Validity of the findings: It is not clear the numbers that were used in the teaching versus the validation part of the experiment.
I think this could be added to table 1

The paragraph beginning with line 209 is not really related to the results of the underlying research presented and might be better in the introduction.

I think a bettter discussion will be on how recognition of RP can be added to other AI being used for other retinal disease so that RP which is rare doesn't get missed or misdiagnosed.
Additional comments: Does the heat map highlight any of the classic ophthalmoscopic triad of RP?
Bone spicule pigmentation, Waxy pallor of the optic disc, Retinal vessel attenuation.
Surprisingly there is little data on the sensitivity and specificity of these signs in RP from the clinical literature.


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: ACCURACY OF A DEEP CONVOLUTIONAL NEURAL NETWORK IN DETECTION OF RETINITIS PIGMENTOSA ON ULTRAWIDE-FIELD IMAGES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors evaluated deep convolutional neural networks in detection of retinitis pigmentosa on ultrawide-field images. Despite of relatively small size data, the accuracy was very high.
Experimental design: Authors should declare that the right and the left images of same patient belong to same group.
Validity of the findings: The uploaded images are useful. Some of images, for example 20130518@150432-R2.tif, is not like RP. If the ERGs were taken precisely, the diagnosis would be true.
Additional comments: In this revised version, the manuscript has been well improved according to the editor and reviewers' 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: ACCURACY OF A DEEP CONVOLUTIONAL NEURAL NETWORK IN DETECTION OF RETINITIS PIGMENTOSA ON ULTRAWIDE-FIELD IMAGES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Authors have address the majority of my comments. Additional suggestions are in the section "General comments for the author".
Experimental design: Authors have address the majority of my comments. Additional suggestions are in the section "General comments for the author".
Validity of the findings: Authors have address the majority of my comments. Additional suggestions are in the section "General comments for the author".
Additional comments: Authors stated that "I already upload the raw images to Figshare." Will these figure be available for readers?
------
/// The manuscript lacks “Conclusion” section. ///
Reply:I am sorry but I can’t what you mean. Our manuscript surely contains the conclusion section.

The so-called "conclusion" contains only one sentence. It is barely a conclusion. Authors must expand this section.


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: OPTIMIZATION OF EXTRACTION CONDITIONS AND DETERMINATION OF PURINE CONTENT IN MARINE FISH DURING BOILING
Review round: 1
Reviewer: 1

Output:
Basic reporting: -Some comments are highlighted in PDF file include text editing and reference details.

-Sample pretreatment (line 97) The authors should state that treatments have been done separately to each edible part of fish samples.

-Extraction rate on axes (figure 2 for example) is defined by (%). I think its is not rate, it is extraction percent since rates literally are related to time.

-In tables (1 and 2); Factors' levels should be presented in actual values instead of coded levels for clarity. also footnotes should be shortened and preferably clarified in text.
Experimental design: -Why 90 degree is used for temperature levels in PCA optimization (line 183) although it showed low extraction% in the prior screening (same for 50 degree level in mixed-acid method). I suggested the levels of this factor should be 70,75 and 80 degrees for PCA and start with 70 degree in mixed-acid to have equal intervals. Also the intermediate level of liquid-solid should be 70:1 (D2) in the same optimization to have a regular intervals between levels. In my opinion, as long as temperature does not greatly affect the extraction % compared to other factors in screening, it should not be included the the design so the number of samples in the design is efficiently reduced.
-Justification of five level design for mixed-acid procedure optimization should be provided.
Validity of the findings: -Is the used HPLC reported before. If yes, reference should be provided and no need for method validation. If the method not reported before, so the author should put some emphases on optimizing the chromatographic conditions especially changes in mobile phase composition as the separated four components (Figure 2) showed four fairly resolved peaks after a waste of running time (5 min). In this case a complete validation according to compendial guid lines should be provided plus a calculated system suitability parameters such as peak resolution, symmetry and column selectivity.

-Line 152, LOD should be calculated based on signal to noise ration or from calibration graph. The authors stated that "LOD was determined using standard solutions". The authors should provide more details about the used concentrations also justify the method used according to official reference.

-Line 156, to determine the % recovery of the method; actually there is nor % recovery for a method, it is for standard or sample solutions. Also how standards were added 0.5, 1 and 2 times the amount found in samples although samples concentration literally are unknown.

-Terms in equation (line 160) should be clarified. I see content added (presumably standard) is the denominator.. assuming that is the recovery for standard added not for the sample.
Additional comments: The manuscript discussed important and interesting topic for purine analysis in marine fish matrix. The manuscript is well written and clearly presented. However some comments are listed that may improve the quality of the work.


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

Input:
Title: OPTIMIZATION OF EXTRACTION CONDITIONS AND DETERMINATION OF PURINE CONTENT IN MARINE FISH DURING BOILING
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: 1. My major concern is regarding the optimization of the purines extraction methods, the authors choose to change one factor while keeping the other factors constant then used an orthogonal design to find the optimum parameters. This design does not allow the study of the interaction between the factors or the quadratic terms. Why the authors did not use a factorial design for screening such as Plackett-Burman designs. Then a central composite design for generating a statistically significant and optimum design that takes into account all the possible terms.
2.  With regards to the HPLC method used for determination of purines, there was not any discussion of how this method was developed for example how the authors choose the mobile phase used during the separation process, what is the pka of the studied purines. Moreover, the authors did not even mention the final pH of the  mobile phase which is a crucial parameter in the ionization and the separation of the studied compounds.
Validity of the findings: The authors should calculate the system suitability parameters regarding the HPLC method, different parameters should be calculated and addressed including, resolution, tailing factor, number of theoretical plates and retention factor. The authors may refer to the USP guidelines for the calculation of these parameters. Moreover, I see no baseline separation between Adenine and Guanine in Fig.1. Finally, the robustness of the method should be assesed
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: EXAMINING THE ASSUMPTIONS OF HETEROGENEITY-BASED MANAGEMENT FOR PROMOTING PLANT DIVERSITY IN A DISTURBANCE-PRONE ECOSYSTEM
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is based on a  thorough analysis of a data set, that is available for re-examination, using state-of-the-art  statistical techniques.  The English expression is of a high quality and the citations are appropriate.
Experimental design: The design of the experiment is appropriate to address the very narrow question of the relative importance of site and disturbance (grazing and fire) ion plant species composition and richness.
Validity of the findings: My concern with this paper is that the study does not tackle the fundamental question in pyrodiversity and pyric herbivory – how spatial scale of burning affects grazing, and if there is a self-reinforcing feedback with post fire grazing through changed fuel arrays.  To do this would require information of the effect of fire patch size on grazing and fire hazard (flammability) of vegetation.

So this paper makes a useful point about the importance of underlying physical gradients in shaping plant species distribution and plant community composition but I do not believe it directly tests the core ideas of pyrodiversity and pyric herbivory.  The Discussion needs, therefore, to be more balanced in light of the very narrow focus of this study objectives and design.
Additional comments: I would scale back the claims of this paper as being a major test of pyric herbivory and pyrodiversity, rather it looks at some facets of these complicated ideas.  The authors should reflect on the caveats with their study and thereby temper their recommendations for managers.  More thought about the biological processes leading to the results would strengthen this paper (i.e. why do some species prosper under some disturbance and edaphic regimes while other species do not).


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: EXAMINING THE ASSUMPTIONS OF HETEROGENEITY-BASED MANAGEMENT FOR PROMOTING PLANT DIVERSITY IN A DISTURBANCE-PRONE ECOSYSTEM
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, well written with scattered minor typos. Meets all the criteria listed by PeerJ.
Experimental design: Seems fine - nice use of long-term vegetation data

Michael Palmer is the go-to guy for multivariate analysis of vegetation data, in particular, and I suspect Daniel McGlinn knows his stuff, as well.
Validity of the findings: See general comments below.
Additional comments: This manuscript describes an analysis of the effectiveness of “pyroherbivory” in maintaining species diversity in grass-grazer ecosystems. The authors test two assumptions: 1) fire and grazing create spatial variability in community structure, and 2) species composition of patches diverge through time in response to these disturbances. Using long-term data from permanent vegetation sampling plots, they find that underlying heterogeneity in soil chemistry, as a proxy for abiotic variability, had a much larger impact on vegetation structure and variability than did patch grazing, as did interannual variation in precipitation. However, patch grazing did have some slight positive effect on species richness. They conclude that “fine tuning” a patch grazing management scheme may provide limited benefits to maintain grassland plant species richness.

This is a very nicely written and easy to follow manuscript that addresses some interesting aspects of the patch-grazing model for rangeland management. This pyroherbivory approach was actually developed and promoted by colleagues of the authors at Oklahoma State, and it has been widely promoted as a way to enhance species diversity without negatively affecting cattle production. The effectiveness of this approach certainly deserves analysis. The patch-grazing model does have strong underlying logic. Traditional grazing approaches promote uniform grazing across a pasture, which logically should result in lower spatial heterogeneity and lower species diversity at the pasture scale. Patch grazing, on the other hand, driven by a rotational burning of subsets of a pasture each year to create different aged patches that are grazed to different degrees will increase heterogeneity and thus pasture scale species diversity. The approach has been tested in many systems on various taxa, and as the authors point out evidence for the success of this approach is mixed.

In the case of the current manuscript the authors find that underlying physical environment has a larger effect than the management scheme, although patch grazing did have a statistically small positive effect. As far as I can tell from the literature and that reviewed in this manuscript, the pyroherbivory approach results in either neutral or positive impacts to some degree, but it never has a negative effect, so in that regard it seems reasonable to suggest the approach has some benefits when implemented, but do the costs of that management scheme justify the benefits? Is the focus on the statistical contribution in your model a valid assessment of whether or not this approach is useful in practice?

Where I am a bit confused by this manuscript is assumption (2) that says patches should diverge in composition over time. Either I don’t understand the pyroherbivory scheme or that assumption is not correct. The authors need to better explain how that assumption works since it is one of the 2 main questions in this manuscript. Here’s my confusion. Again, if a manager burns the entire pasture every year and spreads water and salt licks throughout, then uniform grazing will occur and heterogeneity will be low. Patch burning, for example, would divide the pasture into six units, two of which would be burned each year essentially creating 6 patches that are all burned every three years. Within any given year the treatments would create spatial heterogeneity and fire refugia because 1/3 of the area would be burned that year, 1/3 would be 1 year post burn and 1/3 would be 2 years post burn. Over time all sections would be subjected to the same 3-year fire rotation and grazing pressure. So why should they diverge? Within any given year the three patch types should diverge in community structure to some degree.

The manuscript has scattered typos and a more careful proof reading is needed next time. For example, under methods in the abstract it says “in the grasslands of the preserve” which up to that point neither grassland nor preserve have been mentioned….


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: GENE DRIVES AS A NEW QUALITY IN GMO RELEASES—A COMPARATIVE TECHNOLOGY CHARACTERIZATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors use unambiguous and professional English in their paper. In some cases technical/ juristic terms should be used or defined more clearly. E.g. in line 24 it is stated the experimental field test would be tantamount to the first deliberate release. However the juristic term “deliberate release” applies per definition to any experimental release at least in European legislation.  In line 120 the “precautionary principle” is introduced however it is not referenced or clearly defined what is meant by it (general ethical rule or legally based). Since the “precautionary principle” is used as an important point in later argumentation, this should be more elaborated. It would also help understanding, if central terms like “depth of intervention” clearly defined and would be referenced also by third party literature.

The context and background of the underlying technologies and relevant problems are very good introduced and placed into context. Literature is generally well referenced and relevant. However at some points referencing could be broadened. E.g. in line 105 only literature co/authored by the authors is cited third party literature might strengthen creditability. In line 423 a more general picture of homing efficiencies could be drawn by citing broader literature (e.g. Ganz and Bier2015,  KaramiNejadRanjbar 2017).
Literatur on other available models to analyse gene drives should be provided.

Structure conforms good to PeerJ standards. At some point it is not clear if text  and statements are placed in the right topic. Line 123/124 seems to state a rather a result of a technology characterization than a method or a part thereof. Line 120 to 123 seem to rather state an opinion, or conclusion than a method. If named focus of technology characterization is factual part of the method it should be referenced by literature. In my opinion a scientific characterization should be open ended and does not include conclusions and actions that may be based on its results (“lies on the prevention of..”).
Important parts of the results

In Fig. 3 and Fig. 4 shading oft he colors is not explained despite being seemingly the same graphs, on is named „cross sections“ the other „ cross section overlays“ .
Table 1 lists “Pervasivness”, a term that is not referenced anywere in the the rest paper or other literature.
Fig. 5 it would be reasonable and helpful to include here results from the positive and negative controls displayed in Fig. 3.

Raw data of presented model are provided.
Experimental design: The paper is mostly a literature review supplemented by a basic deterministic inheritance model that visualises gene drive inheritance.
The research question has been well defined and is relevant and the goal to fill a knowledge gap clearly identifiable.
The methods are described with sufficient detail, however central information with respect to the model is to be found in supplemental material.
Validity of the findings: The paper mostly aggregates findings from other papers.
Were the model is concerned the authors might expand on the reasoning for chosing this type of model of other models available in literature.

As positive and negative standards have been introduced to test the model, this results should also be included and put into relation to technologies characterised by the model in the section Discussion and conclusion. E.g. in Fig 5
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: GENE DRIVES AS A NEW QUALITY IN GMO RELEASES—A COMPARATIVE TECHNOLOGY CHARACTERIZATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Very minor comments presented to the authors in the section for General Comments
Experimental design: No problems found
Validity of the findings: Very minor comments presented to the authors in the section for General Comments
Additional comments: It is my opinion that, the manuscript presented by Frie et. al. is of sufficient quality and significance that should be published. It presents a very good assessment of the properties of different Gene Drive systems that should be taken into consideration when performing a risk assessment of GM organisms based on any of these systems.

Minor comments.

1.   Line 17: Compared to previous releases of genetically modified organisms (GMOs) which were primarily plants, gene drives represent a paradigm shift in the handling of GMOs: Current regulation of the release of GMOs assumes that for specific periods of time a certain number of GMOs will be released in a particular region.

This statement does not recognize that, in some instances, the release of a current GMO could lead to the transformation of entire wild populations. If a GMO plant is released in its center of origin/diversity, and there are plenty of sexually compatible relatives, the transgene in principle can spread into the entire wild population -of course depending on the final phenotypes, selective advantage/pressure and other factors. This is important to consider because -albeit with significant differences to the GD issue- the research community working on risk assessment and regulators have already been working with this problem, and methodology including models to simulate the introgression of transgenes into wild populations have also been used.

I believe this issue merits comments and recognition by the authors.

2.  Although it is acknowledged that the use of gene drives could lead to the disappearance of entire populations, that is not always the intention. In Line 64 we read “In agriculture, weeds and crop pests could be eradicated” and indeed this may be the case, however, the prevalent idea is to use GD to revert populations of weeds and insects that have acquired resistance to efficient and safe herbicides and pesticides, to the sensitive phenotype, to again allow the use of those herbicides and pesticides. So at least such applications should also be mentioned even if these are not considered and discussed in detail because that is not the intention of the research reported.

3.  Comment on line 645: In the light of the absence of proven options to a) correct
potential damage or b) just to limit the inherently self-propagating mechanism of gene drives, these functionalities reveal important ‘reasons for very high concern’ with regard to the requirements of the precautionary principle.

I would like to recall what is mentioned in Annex III of the Cartagena Protocol -derived from the Convention of Biodiversity and aligned to the Precautionary principle:

General principles
4. Lack of scientific knowledge or scientific consensus should not necessarily be interpreted as indicating a particular level of risk, an absence of risk, or an acceptable risk.

6. Risk assessment should be carried out on a case-by-case basis. The required information may vary in nature and level of detail from case to case, depending on the living modified organism concerned, its intended use and the likely potential receiving environment.

Methodology
7 (f). Where there is uncertainty regarding the level of risk, it may be addressed by requesting further information on the specific issues of concern or by implementing appropriate risk management strategies and/or monitoring the living modified organism in the receiving environment.

Therefore, I believe that such broad and general statements are not acceptable. I strongly believe that the community of risk assessors/regulators who will read this paper will be aware of the risks associated with the technology -clearly presented by the authors- and will be taken into consideration on any environmental risk assessment of GDO, among many other considerations required. This is in line with the more balanced assessment presented by the authors in their closing paragraph:

“However, as long as this characterization has to rely on theoretical assumptions or initial experimental results in the laboratory scale, the present assessment should
not be used as a basis to choose a “safer” gene drive for release. It rather delivers an early description of the drives specificities that may be a useful contribution for efforts to outline low-risk development paths. There is an urgent demand for further prospective analysis of impact, side effects, countermeasures and the feasibility of low-risk approaches.”


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: CHARACTERIZATION OF INTROGRESSION FROM THE TEOSINTE ZEA MAYS SSP. MEXICANA TO MEXICAN HIGHLAND MAIZE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, this manuscript reads like a Master’s thesis that was re-worked into a manuscript. Unfortunately, the manuscript is overly long making it difficult to distill the key data and interpretation from the manuscript. The manuscript could easily be trimmed to 1/3rd of its length and be stronger.

Page 24 has different line #’s

Throughout manuscript:
‘where’ to ‘were’
‘form’ to ‘from’
Experimental design: I am concerned there are a lot of interpretations from what is really a small number of indivduals. The authors should comment on the power to make interpretations, and the significance of these interpretations, with data from so few indivdiduals.

Line 29: I think it is a misnomer to use the term ‘novel whole-genome sequence data’

Line 154: The authors used v3 of the B73 genome. The current version is v4. I am sure this is because they started the work before v4 was officially available. I am fine with this but the authors should make a note as to why they used v3.

Line 171: The authors refer to Table S17. I did not have the supplemental tables. And these should be cited in order

Line 192-203: The number of markers  for the map seems exceptional low. This is why the QTLs are so large. Can the authors comment on this?

Line 352: It is highly problematic to project SNPs onto a reference genome annotation as shown in Arabidopsis. Thus, the authors should put their SnpEff data in that context. My guess is that a lot of these are artifacts and if they had annotation of their query genomes, then an alternative splice form will alleviate the SNP.
Validity of the findings: While the authors findings are technically sound, the low power of the entire study warrants text to let the reader know this.
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: CHARACTERIZATION OF INTROGRESSION FROM THE TEOSINTE ZEA MAYS SSP. MEXICANA TO MEXICAN HIGHLAND MAIZE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is all quite well done - the writing is clear, references are appropriate, and results are well explained.
Experimental design: There are important and clear research goals, and most of the approaches are well carried out.
My only concern is that the analysis is mainly focused on SNPs called against the reference genome. Additional analysis that would be useful would be to identify any sequences where introgression has lead to copy number variation or (especially) novel genes introgressed. With increasing appreciation for the importance of gene sequences that are missing from the reference genome, I would advocate a "pan genome" style approach to understand this aspect of introgression better.
There are various approaches, but the easiest would simply be to de novo assemble the reads that don't map, to identify genes in the teosinte parent that are missing from the reference. Ideally, the mapping population could be used to place some/all of these sequences onto chromosomes as well, if there are obvious protein-coding or otherwise likely functional genes.
This may be beyond the scope of this current paper, but it should at least be acknowledged that a SNP based approach might be missing something here, and this should be investigated as much as possible here and/or in other papers. At the least, it would be useful to look at copy number variation between the parents based on mapping to the reference genome, and to identify if any regions that were introgressed would lead to gene deletion / duplication, as well as identifying the gene sequences that are novel in the teosente parent even if they can't be placed easily.
Validity of the findings: Findings are well stated and clear. My only additional suggestion is that there are many important traits that were not measured, and it is likely that because of this key phenotypes were missed. Also, as briefly mentioned, there are many traits that could be expressed differently in the highland environment.
It would be ideal to re-do the mapping in the highland environment, obviously for a future paper no this one. IF this is done, I would expect that biotic interactions as well as abiotic factors are important, and lead to higher fitness (yield) in the highlands with certain introgressions. There are likely insects, pathogens, and other organisms that differ substantially between these environments, and could play an important role.
For instance, in sunflower it was found that resistance to local herbivores was driving introgression at key genomic regions during a natural range expansion into texas, leading to the origin of the subspecies H. annuus texanus
Quantitative trait locus mapping identifies candidate alleles involved in adaptive introgression and range expansion in a wild sunflower
KD Whitney et al.
Molecular ecology 24 (9), 2194-2211
Additional comments: This is a nice paper, and adds important new understanding to how Maize and Teosinte have interacted in the areas where they coexist, and how selection may have acted / be acting. The fact that phenotypes associated with this introgression have not yet been identified is not too surprising, since it may be something you didn't measure, as discussed above, or that is only expressed in the highland environment.


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: CHARACTERIZATION OF INTROGRESSION FROM THE TEOSINTE ZEA MAYS SSP. MEXICANA TO MEXICAN HIGHLAND MAIZE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Excellent
Experimental design: Solid,, and with acknowledged challenges that are clearly described.
Validity of the findings: Well done and clear.
Additional comments: I felt like the revisions were done well.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION PROFILE ANALYSIS OF THE HSP20 GENE FAMILY IN BARLEY (HORDEUM VULGARE L.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This manuscript presents identification of barley Hsp20 gene family in Arabidopsis, rice and sorghum based on presence of an ACD domain. Using phylogenetic analysis, authors report four subfamilies of HSP20 multiple gene family. Authors describe features of coding sequence, chromosomal location, intron-exon contain of HSP20 gene family and use RNA-seq data from NCBI-SRA database to reveal up- and down regulation of  barley HSP20 genes upon biotic and abiotic stress. I feel this manuscript will be interest to many researchers in the field. However, there are some issues as follows needs to be addressed.

1) Line 141-147, authors identified 38 Hsp20 gene sequences after removing the repetitive sequences. In my opinion authors should check whether incomplete full-length sequence could be completed using the chromosome sequence information, and amended the amino acid sequence displayed in Table S1.

2) line 151-155, authors introduce a classification of identified HSP20s into C, MT (MTI and MTII), CP, ER, PX and ACD subfamilies. Giving that classification is based in protein location, could it determined by signal peptide or a signature?. In my opinion authors should add information, at some extend, to help audience to distinguish hsp20 into C to ACD subfamilies based in amino acid sequences. Are there reasons to include multiple protein coding gene models for a HSPs in the Fig 1?. For instance, HORVU5Hr1G061160.1, HORVU5Hr1G061160.2, HORVU5Hr1G061160.3 and HORVU5Hr1G061160.4 HSP proteins at the ACD subfamily.

3) Some typos in the manuscript. For instance, line 95 “MATHERIALS”, line 115 “misfits”, line117 “identified”, and line 289 “Hsp20”.

4) The Fig 2 displayed 86 gene models that is less than the 96 reported by authors.

5) In my opinion, it should be useful for the audience if the authors add a little bit on the 10 motifs reported (Fig 3) regarding to ACD domain, N-terminal and C-terminal extension. Do motifs fall into or outside ACD domain?

5) The Fig 4 displayed 35 HSP20 genes, is there a reason for the missing HSP20 gene in the figure?

6) Line 126, Section “Cis-acting regulatory…”, It should be useful to explain to the audience features of the cis-element detection such as strand and matrix similarity for the presence/absence of cis-element.

7) Fig 6, panel B, the scale for the expression levels is missing at “Heat stress in shoots”


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: GENOME-WIDE IDENTIFICATION AND EXPRESSION PROFILE ANALYSIS OF THE HSP20 GENE FAMILY IN BARLEY (HORDEUM VULGARE L.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is clear and used properly in my opinion, the references are sufficient although could be more clear in the information being cited. For example line 66 Liberek citation is mostly about HSP 70 and 100 and although mentions about sHSP the information from this could be obtain from other publications that would focus on sHSP from plants.
The structure is well define, however in the abstract it should be corrected the sentence that mentions that this information is needed to reveal the mechanism.....  Since the data presented in this manuscript is mostly descriptive and it does deal with deciphering any of the mechanism of action of these proteins.
Experimental design: The research is well done and proper, probably syntenic analysis could be made to understand the evolution of the families of HSP20 proteins found.
Validity of the findings: The findings are expected but the information is required to obtain more information about possible target proteins to use for heat stress survival in future experiments. The RNA seq data is acceptable, there are always more conditions to be tried and time points of expression. But for the title of the work it seems appropriate. The conclusions are concrete and accurate with regard to the data and link to the research question from this manuscript
Additional comments: From the phylogeny data it should be taken into account the different genome duplication between the different plants choose, a synteny approach my provide more information about the evolution of this family. The novel signatures for this family that are presented correspond to any other family of proteins? this should be mention.