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: ENGINEERING OF CHO CELLS FOR THE PRODUCTION OF VERTEBRATE RECOMBINANT SIALYLTRANSFERASES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The title is not appropriate. What are these diverse environments mentioned in the title?
Quality of the presentation is poor and reported data are not rigorous and somewhat ambiguous. Controls (negative or positive) are missing in most of the proposed experiments.
A number of sections are missing which renders the manuscript difficult to review (no page number). A list of abbreviations and “Materials section” should be provided: pSecTag/FRT/V5-His-TOPO, What are FRT sites line 289 and FRT mechanism line 479? What is pOG44? Where does that come from? SNA-I, RCA-I MAA are from? Which MAA type?

The introduction section gives a very poor description of the context of the study and very limited field background. This section needs to be extensively revised and the research question clearly defined.

Line 56: Explain “SIATs are broadly split”
Lines 61-62: “Generally the stem is less structured and of varied length across SIAT families.” It varies across sialyltransferases themselves see ST6GalNAc I and ST6GalNAc II.
Lines 45-48: as reported recently in the mentioned reference Petit et al. 2018, sialyltransferase sequences are described across eukaryotes, in plants as well as in Bacteria. So “ST6Gal are the oldest” lines 72-73 is not true. In addition, the first ST3Gal enzymes (line75-76) appear much sooner than urochordates since they were already present in sponges (Petit et al. 2015 Mol Biol Evol 906-27).
Lines 69-71: Other crystal structures were obtained recently for the human ST8Sia III (Volkers et al. 2015 Nat Struct Mol Biol 627) and the human ST6GalNAc II (Moremen et al. 2018 Nat Chem Biol ). References should be updated. In this latter study, Moremen and collaborators describe an efficient expression system of glycosylation enzymes for their structural and functional studies. This should be mentioned
Lines 76-77: There are six ST3Gal paralogs in human tissues and eight ST3Gal paralogs in the chicken and zebrafish tissues (Teppa et al. 2016 Int J Mol Sci E1286) and for most of them we know nothing about kinetics and substrate specificities. It is important to describe exactly the sialyltransferases used in this study (sequences, accession numbers, species) and explain why these were chosen and not the others.
Line 95: Explain “specificity to substituted Sia donors”.
Experimental design: The experimental design is also extremely poor. The research question is not well defined. Furthermore, the material section is missing and the method section should clearly describe the research protocol and how measurements were made. There is no description of western blot experiments for instance.
Line 104: What are these selected protein sequences? The alignment need to be shown since it is somewhat difficult to imagine constructs “starting 121 amino acids N-terminal to a Cys that is totally conserved across all animal sialyltransferases (Cys-184 in human)”. In addition, this N-terminal region of vertebrate ST6Gal I is extremely variable (see Petit et al. 2010 JBC). What is the expected molecular weight of these recombinant proteins?
Line 112-114 Explain “Slight variations in ST6Gal1 (zebrafish) ending at LAL, ST6Gal1 (stickleback) ending at AGS and ST3Gal4 (zebrafish) with an additional QKKW are a consequence of an evolving cloning approach”
Line 119-125: It is necessary to describe which sialyltransferase sequence was chosen in which animal species and how molecular cloning was achieved from which tissue?
Line 131 “amplified genes” Line 256 “transfected gene” or cDNAs?
Line 152 It is not clear to me in which medium (with or without?) serum, the recombinant enzymes are produced.
Line 177 crude or purified sialyltransferases?
Line 195 Analysis of proteins on NuPAGE should be shown
Line 216 Explain the rationale using whey permeate as a sialyltransferase acceptor?
Validity of the findings: The results section resembles a methods section although it is obscure which recombinant enzymes are produced. A clear description of the sequences used in the study should be given with accession numbers. Are they soluble in the cell culture medium or still membrane bound (full length enzymes?)? Controls of their efficient production should be provided. It is not clear to me the purpose of using these mutant ST6Gal I proteins? Time course of enzyme activities should be shown and effect of temperatures should be shown. Data are not robust and controlled and conclusion are not well stated
Lines 258-261 “The full length gene has a transmembrane domain that attaches the catalytic domain…” ??...”the stem is cytoplasmic???? Delete this sentence
Line 265 what is human ST6Gal1 B1? Why was the sequence codon optimized?
Line 267 What are the cloned proteins? The multiple sequence alignment should be shown. What is Cys-184??
Line 276-281 It is difficult to understand what these human ST6Gal I sequences are?
Line 281 idem for zebrafish clones (of what?)? H111 K187 rat R105?
Line 287 ST3Gal4i1 what is it?
What about the other transfected cells lectin binding profile?
Explain strong binding of MAA (and not moderate) in figure 1.

Line 305: why asialofetuin? What is the amount used? This is not mentioned in the material and methods? These data are not shown, why?
In figure 2 why incubation reactions are only 30 min? Where is the Wild type human ST6Gal I control?
Line 313 explain the results may be influenced by the choice of acceptor?
Line 313-321: range of protein concentration?? Difficult to understand what is used as an enzyme source? The authors should provide a WB. Another problem is the indirect sialyltransferase assay based on phosphate detection. Controls should be provided since these assays produce high background signals.
Line 336 and 315 exactly the same activities obtained after enzyme purification?
Line 341-346. The samples need to be run on a single gel (figure 3). PNGase F digestion should be shown for all recombinant proteins. Sizes of recombinant enzymes should be equivalent since catalytic domain is almost the same.
Line 363 What are these optimizations?
What about the ST3Gal IV activities?
Line 365-369: Already mentioned.
Figure 5 the WT human sialyltransferase activity should be shown. What are Expt1 and Expt2? Why so much differences between the 2 experiments? What is ST3A1 and ST3A2 and ST3H6
What is the effect of temperature variations on enzyme activities? The human ST6Gal I activity at 37°C should be shown. The activities mentioned in the text are obtained in which conditions? Figure 4 should show all the data obtained for all enzymes at different temperature.
Line 477: low adherence of transfected CHO cells is not mentioned before. Data should be presented. What is the importance of this observation since the recombinant enzyme is produced in the cell culture medium?

Unless otherwise stated, proteins should be noted ST6Gal I and human gene ST6GAL1 and rat, mouse zebrafish genes st6gal1.
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: IN SILICO ANALYSIS REVEALS A SHARED IMMUNE SIGNATURE IN CASP8-MUTATED CARCINOMAS WITH VARYING CORRELATIONS TO PROGNOSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	Some citations are missing.  Line 81, 82, 335.
2.	Line 164, please be specific about what “data” you downloaded from TIMER.  Data is too general.
3.	Line 243-264, the description is quite chaotic here.  The authors compare CASP8-MT to CASP8-WT and HPV-positive, which is unclear at all.  By “CASP8-WT”, I guess you mean CASP8-WT with HPV-negative.  By “HPV-positive”, I guess you mean CASP8-WT with HPV-positive, right?  Please write these very clear so that the reader won’t confuse.  Revise Figure 3a accordingly as well.
4.	Line 292 and 293 should be in the introduction.  Otherwise it also seems confusing why you choose UCEC instead of other cancer types.
5.	Line 294-295 replicates the previous content.  Should be removed.
Experimental design: 1.	It seems a bit mis-match between your title and your content.  I tried very hard to figure out what this “shared immune signature” is, which seems to be the most important meessage in your title.  However, it is not clear at all what this “shared immune signature” is by reading the main manuscript.  Based on the abstract, it seems this immune signature is high neutrophil numbers (but the sixth section of results showed this feature is not shared in UCEC).  Based on the fourth section of results part, it seems this immune signature is these four genes from pathways enriched in CASP8-MT HNSC.  Furthermore, based on the fifth section in results, it seems this immune signature is these enriched categories such as allograft rejection.
2.	A big chunk of differential gene expression analysis results seems to be missing.  In the Materials and Methods section, you describe that the RNA-seq data from CASP8-MT and CASP8-WT are compared through differential gene expression analysis.  However, the results section only present the CASP9-WT who are HPV-negative versus CASP9-MT.  What about the comparison results of the whole group of CASP9-WT versus CASP9-MT regardless of their HPV status?  Why you only exclude HPV-positive patient from this analysis?  I understand that CASP9-MT patients are all HPV-negative, but HPV is just a sub-feature.  CASP9-MT or WT should be the main feature regardless of their HPV status.
3.	In the introduction section, the authors state that among all the genes, “TP53 was the most significant recurrently mutated genes”.  But instead, they decide to focus on another gene “CASP8”.  What is the motivation?  Why they do not want to study TP53 here? What is so special about “CASP8”?
4.	You grouped CASP8 high mutations and mediate mutations to one group.  What if you consider them differently?  Will the patients have more impact with severe mutations?
Validity of the findings: 1.	In the Gene Onotology and GSEA part of Method section, the authors identified 37095 genes.  But as I recall, there are a total of 19000-20000 genes in homo sapiens.  I wonder whether they mapped this correctly.
2.	Again in this GSEA part, they seems to use a set of genes from mouse study.  I am not quite confident in such comparisons.  Unless the authors can provide evidence that this comparison is a common practice, I tend to suggest that the authors should find relevant gene sets from human study.  Related to this, I want to recommend three other GO and pathway tool: EnrichR (http://amp.pharm.mssm.edu/Enrichr/), GREAT (http://great.stanford.edu/public/html/) and DAVID (https://david.ncifcrf.gov/) which offers GO and pathway analysis, even GSEA analysis in one stop.  They may be more reliable than basing on a single gene set.
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: IN SILICO ANALYSIS REVEALS A SHARED IMMUNE SIGNATURE IN CASP8-MUTATED CARCINOMAS WITH VARYING CORRELATIONS TO PROGNOSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper is clearly written and organized. Sufficient introduction and background is provided. Figures are of good quality and addressed the relevant questions. The resources for publicly available data used in this study is clearly referenced.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1. About the motivation of this study, the authors stated that CASP8 in one of the highly mutated genes in HNSC. My question if why chooses to study CASP8 instead of other highly mutated genes? Need more detailed explanations and discussion here.

2. The authors used pre-ranked GSEA instead standard GSEA to interpret gene expression differences between CASP8-WT and CASP8-MT. However, it is well known that pre-ranked GSEA inflates p-values due to the intercorrelation of genes, and it is usually only used when there is no better alternative approaches, for example, RNA-seq data with no or few replicates. But apparently in TCGA, the number of replicates is not an issue. Therefore, my suggestion is to use the standard GSEA instead of pre-ranked GSEA, and to see if still get the similar results.

3. In Figure 4B and Figure S1, the authors compared the effects of immune signature gene expression on survival in CASP8-MT and CASP8-WT samples, and made the interpretation that higher expression of immune signature genes (and CD8+) is only correlated with better survival in CASP8-WT but not in CASP8-MT. My concern is this is a rather strong conclusion, since there is a trend of better survival in CASP8-MT and the p-value is not significant enough probably due to the much small sample size camped to CASP8-WT.

4. The authors did a great job at discussing the potential reasons why CASP8-MT UCECs showed a significantly better survival while the CASP8-MT HNSCs did not. However, the conclusion that this difference might be “attributed to differences in neutrophil infiltration and/or IL33 levels” is way too strong. I would suggest either provide more supporting evidence (which is probably very difficult) or change the conclusions.


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: IN SILICO ANALYSIS REVEALS A SHARED IMMUNE SIGNATURE IN CASP8-MUTATED CARCINOMAS WITH VARYING CORRELATIONS TO PROGNOSIS
Review round: 2
Reviewer: 1

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


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

Input:
Title: IN SILICO ANALYSIS REVEALS A SHARED IMMUNE SIGNATURE IN CASP8-MUTATED CARCINOMAS WITH VARYING CORRELATIONS TO PROGNOSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed my concerns and questions in the revision.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: ELEVATIONAL PATTERNS OF THE PERCENTAGES OF PLANT GENERA WITH TROPICAL AND TEMPERATE AFFINITIES IN NEPAL
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this study, the authors related the percentages of tropical and temperate genera in 100-m elevational bands to several climate variables. They found that the decreasing trends of the percentages of tropical plants with increasing elevation were mainly controlled by mean temperature of the coldest month, and the increasing trends of the percentages of temperate plants with increasing elevation were mainly controlled by mean temperature of the warmest month. It appears worth documenting these patterns in the literature. English in the manuscript needs to be improved. Below are a few specific comments.

Specific comments
Abstract: More details about methods should be mentioned.
Line 91: change “known” to “know”
Line 170: delete one from “one one”
Lines 230 and 237: delete “A”
Line 251: delete though from “though though”


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: ELEVATIONAL PATTERNS OF THE PERCENTAGES OF PLANT GENERA WITH TROPICAL AND TEMPERATE AFFINITIES IN NEPAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: I am regret that I can not recommend this MS for publication, at least in it’s present version. See below.
Experimental design: Not very good.
Validity of the findings: Major weakness in methods.
Additional comments: This manuscript describe the percentages of tropical and temperate plants along an elevational gradient in relation to climatic indices. I am regret that I can not recommend this MS for publication, at least in it’s present version.

The topic itself is interesting. However, the MS did not provide insightful analyses and discussions that can improve our understanding of the altitudinal pattern. The whole MS is mainly talking about two correlations (though some other climate indices were used), and the conclusions are simply based on the correlation analysis in Table 1. The MS reads not very interesting.

There are some clear weakness in methods:
1) The MS conducted only correlation analysis without any multivariate models. However, in Methods and Results, the MS mentioned that they used VIF to diagnose collinearity. VIF is used to avoid collinearity among variables in multivariate models, but there’s no multivariate model in the MS at all.
2) The data used by the MS, the percentage, is well known to suffer to some major statistic problems. Results based on correlation analysis thus may not be reliable, as demonstrated by previous analyses (e.g. some publications by Gaston). The authors may consider some methods based on null model for a more robust analysis for this kind of data.
3) The data for percentages of tropical and temperate plants along elevational gradient is obtained through interpolation of plant occurrence. Consequently, the data will inevitably be affected by the interpolation effect (in some papers named as the mid-domain effect). This effect has strong influence on elevational richness pattern, and can strongly alter the correlations of richness with environmental factors. The percentages used in this MS is calculated from species numbers. Consequently, if the influence of the interpolation effect can not be well addressed, it is not reliable to draw conclusions on the major climate drivers of the percentages based on some simple correlations.
Consequently, I am sorry that I can not recommend this MS to be published in it’s present version.


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: ELEVATIONAL PATTERNS OF THE PERCENTAGES OF PLANT GENERA WITH TROPICAL AND TEMPERATE AFFINITIES IN NEPAL
Review round: 1
Reviewer: 3

Output:
Basic reporting: Using the elevational distributions of over 1300 seed plant genera, the authors estimated the elevational patterns in the percentages of tropical and temperate genera, and explored the determinants of these patterns. The authors tested two different hypotheses respectively explaining the percentages of temperate and tropical genera. The issue addressed in this manuscript is interesting. I have a few comments for the consideration of the authors to improve the manuscript.
Experimental design: As far as I understand, the classification system of biogeographical affinities of seed plant genera developed by Wu is only for Chinese plants, not for global plants. To identify the biogeographical affinities of the genera that are not included in the classification system of Wu, the author used another system developed by Wielgorskaya. Are these two different systems comparable in the definition of biogeographical affinities?

The number of species within genera are different. More importantly, the species numbers of each genus within different elevational belts are also different. The authors only used the number and proportion of genera within each elevational belt. Could the proportions of genera really represent the proportions of different affinities in each elevational belt? If the proportions of species with different affinities were used, would the findings remain the same? Moreover, will the findings reported remain consistent for species with different lifeforms, e.g. woody vs. herbaceous species and annuals vs. perennials?

The authors proposed a new hypothesis named “warming-tolerance hypothesis”, which suggest that species with temperate affinities may could not tolerant the warm climate in low elevations. However, transplanting studies suggest that many species at high elevations could survive the warm climate at lower elevation or at more southern latitudes when they are planted in monoculture in botanical gardens. This observation makes many authors believe that the absence of temperate species from low elevations or latitudes is because temperate species living in warming regions have lower competitive ability than warm-adapted species. I think the authors could try to compare these two alternative hypothesis in explaining the elevational patterns in the percentages of temperate species.
Validity of the findings: no comment
Additional comments: According to the section of “statistical methods”, it seems that the authors conducted multiple regression analysis to select the best model explaining the percentages of tropical and temperate species. however, none of the results on multiple regressions were included in the manuscript or the supplementary materials. I suggest that these results should be included.

The English writing of this manuscript should be improved before publication.

Lines 81 – 84, these two sentences repeated some sentences in the first paragraph. Is it possible to merge the first and second paragraphs into one.
Line 106, insert “along elevational gradients” before and
Line 110, this sentence is not very clear. Consider to rephrase it.
Lines 129, 149, change “around” to “at”
Line 173, change “number” to “numbers”
Line 174, remove the first “and”.
Line 202, change resulting to resulted.
Line 251, remove the first “though”
Line 276, change comma to period.
Line 292, changes “elevational diversity” to “elevational diversity gradient”
Line 297, remove “terms of the”
Figure 1, Why are there white areas in the lowest part of the study area?


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: ELEVATIONAL PATTERNS OF THE PERCENTAGES OF PLANT GENERA WITH TROPICAL AND TEMPERATE AFFINITIES IN NEPAL
Review round: 2
Reviewer: 1

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: The manuscript has been improved. But there are still some weakness in data analyses and writing, before it is accepted.

1) The MS used the MDE model and tried to resolve the problem of interpolation effect and the statistic problem associated with percentage data. If this method is acceptable (which is still controversial), the MS did not clarified their method to calculate the percentage of the tropical and temperate species.
   When using this method, we can use the MDE model to predict the richness for tropical (MDEtrop) and temperate genera (MDEtemp) separately, and for the two group together (MDEtotal). Then we have two choice:
    (1) Percentage of tropical genera = MDEtrop/ MDEtotal
    (2) Percentage of tropical genera = MDEtrop/ (MDEtrop+MDEtemp)
Since the range size frequency distribution of tropical, temperate genera and the two group together should be different, it is possible that the two methods will get different results. If the results are different, which one should the MS use?
The MS did not clarified about this, and only said that “We simulated genus richness over the elevational gradient for all tropical and temperate genera separately.”

2) The MS has made some multivariate analyses in this version, and they made conclusions based on these multivariate analyses. But these results were listed supporting information. This is surprising.

3) The MS proposed the warming-tolerance hypothesis. Is this really a good hypothesis? The cold tolerance hypothesis has sound biological basis, and reflects niche conservatism. However, I am not convinced that fewer temperate species in warmer region was mainly caused by filtering by warmth. Many temperate species can live in gardens (without competition) that are much warmer than their natural distribution regions.


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: ELEVATIONAL PATTERNS OF THE PERCENTAGES OF PLANT GENERA WITH TROPICAL AND TEMPERATE AFFINITIES IN NEPAL
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed most my concerns in the revised manuscript. I think the manuscript has been substantially improved. However, the writing is still not good enough for final publication. Many sentences are not well written. i have listed some below.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Line 24, Abstract: change “Geographical patterns of organisms” to “Geographical patterns of species diversity”
Lines 26-28, "but the  factors or underlying mechanisms controlling the elevational patterns of the percentages of plant genera with tropical and temperate biogeographical affinities have attracted little attention”, could be reworded as “but the factors driving elevational patterns of the percentages of plants with tropical and temperate biogeographical affinities have not been adequately explored”
Line 34, Abstract: “tropical and temperate plant genera” should be “plant genera with tropical and temperate affinities”
Line 35, “percentage of tropical plants” should be “percentage of plant genera with tropical affinity”. Genera with tropical affinity is not the same as tropical genera, because a genus with tropical affinity may contain species that occupy temperate regions. I suggest the authors check this writing throughout the manuscript.
Line 38, Similarly, “temperate plants” should be “plant genera with temperate affinity”
Line 41, “tropical and temperate percentages” is not clear. Consider to reword it.
Line 44, Change “associated controlling factors” to “the factors driving them”.
Lines 43-Abstract, the first sentence of the Discussion section is awkward. Consider to split it into two sentences?
Line 90, “at mid-elevation” should be “along elevational gradients”; line 91, gradient should be gradients.
Line 105, change “controlling factor” to “driver of it”
Line 108, “hump-shaped patterns of elevational diversity” should be “hump-shaped elevational patterns”
Line 112, in their percentage in regional floras
Lines 154 – 169, the consistency between the two data sources for biogeographical affinities should be clearly stated in the text so that readers could judge the reliability of the results.
Line 230, remove “represented by climatic factors”
Lines 225 – 236, because partial regressions analyses were actually not conducted at all, these sentences starting from “Using the predictors ……” should be removed.
Lines 237 – 240, this sentence is not clear. Consider to split it into two as the relationship between the two clauses is hard to understand.
Lines 296 – 297, change it to “no study conducted so far observed different elevational patterns in the percentages of tropical and temperate plants from the trends observed here”
Lines 320 – 325, these sentences repeat previous ones. I suggest that they should either be removed, or integrated with the previous paragraph.
Lines 337 – 349, these sentences also repeats the introduction and the logic between them is hard to follow. I think these sentences could be greatly shortened.


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: ELEVATIONAL PATTERNS OF THE PERCENTAGES OF PLANT GENERA WITH TROPICAL AND TEMPERATE AFFINITIES IN NEPAL
Review round: 3
Reviewer: 1

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


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

Input:
Title: INVASION GENETICS OF THE MUMMICHOG (FUNDULUS HETEROCLITUS): RECENT ANTHROPOGENIC INTRODUCTION IN IBERIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is extremely well written, clear and easy to follow. It has the expected structure, and the sequence data is available at Genbank.
As a general comment, I think that some parts of the paper are excessively long, and with too many citations. This happens in the Introduction, which could be reduced to include much less information about generalities on biological invasions. The information about Fundulus could be more restricted to genetic studies, but it seems to have the aim of reviewing all the published papers on the species instead.
In the discussion there is a similar pattern. Some interesting ideas are discussed, but because it is difficult to reach a conclusion, I would reduce the length of the arguments.
Experimental design: Cyt B (even if it was a larger fragment) was not an ideal option for this analysis. The authors should justify such decision, but the results are valid anyway.
The inclusion of the ecological niche modelling is weird. I am not an expert, so I cannot correct this part, but even if I think that these results should stay in the paper, I have the impression that they should be more connected with the other results, at least in the discussion.
Validity of the findings: The results are robust, even they don't provide much information. No definitive conclusions can be reached, neither on the origin of the Iberian populations (both northern and southern locations of the native range could be the origin), nor in the relationship between the three invaded sites (all 3 have the same haplotype).
I like the ideas in the discussion, but again, I think that they overdiscussed.
Additional comments: I liked the paper. There is an effort in the sampling of the native range, although the results do not reflect it, most likely because cyt B has too low diversity.


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: INVASION GENETICS OF THE MUMMICHOG (FUNDULUS HETEROCLITUS): RECENT ANTHROPOGENIC INTRODUCTION IN IBERIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript "Invasive genetics of the mummichog (Fundulus heteroclitus): recent anthropogenic introduction in Iberia" aims to assess the invasion history of F. heteroclitus in the southern Iberian Peninsula.
The manuscript is well written, well referenced and overall scientifically sound.
Experimental design: The research question is relevant, however the methods used are not the most up-to-date. The authors made use of only a fragment of the mtDNA, for only three locations. These caveats, however, have been addressed in the discussion.

The novelty of the paper resides in the Ecological Niche Modelling approach used. However, this is not explored to its full capacity. I would recommend the authors to change the introduction/results/discussion to increase the focus on ENM.

The methodology is thoroughly described, and easily replicable.
Validity of the findings: The findings of only one major haplotype in the invaded range seem solid, and congruent with a recent invasion, mediated by a small number of individuals. The conclusions that this was done via aquarium trade might be a bit speculative.

I would strongly recommend to increase focus in the ENM.
Additional comments: The manuscript "Invasive genetics of the mummichog (Fundulus heteroclitus): recent anthropogenic introduction in Iberia" aims to assess the invasion history of F. heteroclitus in the southern Iberian Peninsula. The manuscript will make a contribution to the field of invasion biology, and I therefore recommend it to publication pending minor changes.

Minor comments:

line 51: there are reports of over
line 51: of which, 237
line 53: they are recognized as one of
line 75: remove extra paragraph
line 79: remove the between reconstruct and invasive
line 83: remove "all fulfilled for Fundulus heteroclitus" as the species is only introduced in the paragraph below
line 93: Fundulus in full at the beginning of the sentence
line 95: the presence of
line 106, 112, 116: add comma after )
lines 128-132: Although there is some interest in this study using only these aims, it is the ENM approach described in the methods that increase the novelty of this study. Please re-focus the introduction to reflect this angle.
line 156: remove the sequence of the primers, as they are described somewhere else
lines 182-219: This approach is not referred to in the introduction, and is not one of cited aims of the manuscript. Please include it, and the motivation behind itli
line 230: Figure 2 is referenced before Figure 1 - that should be changed
line 232: Figure 1a and 1b are not referenced in the text
lines 250-252: I recommend to expand this section, and elaborate on the findings of the ENM
line 280: This can be explored further. Either via ABC estimates of processes that would lead to maintaining such low genetic diversity. Is the species abundant in the invaded range or is it maintained at low levels? Are these long-term viable populations?
lines 310-311: This suggests that the population is actually not increasing in size
line 352: Which cannot really be discarded by the data used
line 362: Given the caveats of the data here presented, I would be weary to make this claim
lines 377-379: Which again suggests that these populations are not thriving in their invaded range
line 392: remove the
lines 405-413: Please expand this section, keeping in mind that the populations appear to have very limited ability to expand


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: INVASION GENETICS OF THE MUMMICHOG (FUNDULUS HETEROCLITUS): RECENT ANTHROPOGENIC INTRODUCTION IN IBERIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is very well-written. I only found a few minor language-related edits to make, which are indicated below. The level of background information provided was sufficient, and the overall structure of the paper was in accordance with the standards of PeerJ.

Minor edits:

-The title should read “Invasion genetics”, as opposed to “Invasive genetics”.

-Lines 40-44: this is a run-on sentence. It should be revised to read: “We suggest the most probable introduction vector is associated with the aquarium trade. We further discuss the hypothesis of a second human-mediated introduction responsible for the establishment of individuals in the Ebro Delta.”

-lines 44-46 (“Although the species…”) this part can be removed from the abstract. Otherwise, make it clear that it refers to the ecological niche modeling performed (and is not speculation).

-lines 51-52: what does “western European margins” refer to? I assume you mean “East Atlantic coastal areas”. Please revise accordingly.

-line 62: remove “non-native”.

-lines 109-110: according to information detailed below (lines 126-127), and other previous papers, macrolepidotus is the Northern subspecies, and heteroclitus is the Southern subspecies.
Experimental design: The authors emphasize well the originality of their results, and provide sufficient detail on how their work differs from previous publications (lines 128-132). Compared to previous work in this system, they have considerably expanded the number of populations and samples, as well as the length of the mtDNA fragment that was sequenced. The research questions are well defined. Lastly, the methods are described with sufficient detail to allow replication.
Validity of the findings: The data is robust, and the analyses are statistically sound. The conclusions are also well stated. I appreciated that the authors included a paragraph on caveats (lines 264-274), acknowledging the limitations of using only mtDNA information.
Additional comments: no additional comments to make.


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: IMPROVED GENOME OF AGROBACTERIUM RADIOBACTER TYPE STRAIN PROVIDES NEW TAXONOMIC INSIGHT INTO AGROBACTERIUM GENOMOSPECIES 4
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are some suggestions that could help to make the paper more readable:

Abstract
1.	22 –«Obtained from NCPPB» should be deleted here.
2.	31 – The authors should either define genomospecies or write just "species"
3.	32 – Please explain, what is meant by "core genome".

Introduction
1.	31: the authors should define «genomospecies»
2.	46-50 – is this information necessary for this article? Apart from A. tumefaciens and A. radiobacter these species are not mentioned in the manuscript anywhere else. The authors should substitute this part with: «Bacteria of the genus Agrobacterium have been grouped into six species based on the disease phenotype associated, in part, with the resident disease-inducing plasmid (references). Among those six species are A. tumefaciens causing crown gall on dicotyledonous plants, stone fruit and nut trees and A. radiobacter that is not known to cause plant diseases of any kind (Zhang et al., 2014)»
3.	58-59 instead of the first sentence the authors should at least mention here widely used classic approaches to bacterial species definition (see Stackebrandt et al., 2002) as well as novel methods based on the whole-genome analysis (Coutinho et al., 2016), horizontal gene transfer analysis (Bobay and Ochman, 2017) or the core genome analysis, which is used in the present study (Moldovan and Gelfand, 2018).
4.	Line 80: not clear what is meant by «the principle of priority». Probably the authors should explain that and use indirect speech here.
5.	87-88: «has not been reported» probably?
6.	99: not clear what the '4' in «genomospecies 4» stands for. The authors should explain that.
7.	111: not clear by which criterion the evidence is conclusive. Please consider #3 and emphasize which criteria for the species definition are used.

Methods
1.	125-127: Here the authors should mention that no Agrobacterium strains have been sequenced in this laboratory previously.
2.	129-134: The article should be supplemented with the list of commands  employed to construct the genome as well as with Trimmomatic read quality scores.
3.	135-142: Again, the commands or just a list of parameters should be provided as a supplementary material. Here the authors should just list the programmes and expand a little on the meaning of key parameters.
4.	141-142: «Visualisation and annotation of phylogenetic trees was performed with FastTree2 …»
5.	152-156: Please provide references for BLAST and for the image generator.
6.	158-164: The authors should provide a reference for KEGG here.

Results
1.	181: In the Table 1 there is no LM140, only LMG140, which one is it?
2.	207-214:  The authors should include here a reference to a study that employed a 95% identity cutoff, e.g. Coutinho et al., 2016.
3.	Also, In the notation to fig. 1b, there is a 95% protein identity cutoff, the authors should either explain this choice or use a 95% nucleotide identity cutoff and compare CDSs. Along with that, fig. 1B should be mentioned and commented on in the main text.
4.	The authors should indicate on fig. 2 which strains belong to which species and genomospecies; strains mentioned in the text should be highlighted.
5.	On figure 3 it should be indicated, which strains belong to which genomospecies.
6.	On figure 4 there also should be clade annotations: which clade corresponds to which species.
7.	Figure 4B should be explained better. It's not clear what do grey areas and numbers to the right mean.
8.	232: Figure 3 depicts an alignment to Ti plasmid; probably Figure 2 should be referenced here.
9.	245: Probably fig. 5A should be referenced here?
10.	248: fig. 5B?
11.	Objects on figure 5 are too small. I’m afraid the authors have to re-draw it. In the notation a reference for GhostKoala should be included
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors should include a link to the constructed draft genome


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: IMPROVED GENOME OF AGROBACTERIUM RADIOBACTER TYPE STRAIN PROVIDES NEW TAXONOMIC INSIGHT INTO AGROBACTERIUM GENOMOSPECIES 4
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Improved genome of Agrobacterium radiobacter type strain provides new taxonomic insight into Agrobacterium genomospecies 4” reports a comparative genomics analysis of Agrobacterium species including re-sequenced genome of Agrobacterium radiobacter with dramatically improved assembly quality and contiguity. Based on obtained results, authors proposed a reclassification of A. radiobacter/ A. tumefaciens taxonomy.
The topic of research seems interesting and basic reporting is good. The ‘background’ section is clear and sufficient. In the ‘Methods’ and ‘Results’ sections several details were missing that the reader might consider crucial for the logic of the article.
One more moment that appears to be very confusing is numerous formatting inaccuracies, such as incorrect links to figures and Supplements. As an example, Supplement Fig. 1 is mentioned in a text but the Supplement section is absent; at the same time, Fig. 1B is present in the article but not mentioned in the article body. The ‘Declaration’ section with the information regarding the data availability is missing. The list of citations went under the title “Acknowledgements”.  I would strongly suggest a careful proofreading of the article.
Experimental design: L136-139  “Clustering of the predicted proteins was performed with CD-HIT  Identification of unique and shared clusters were done using basic unix commands e.g. csplit, grep, sort and uniq.”
Constructed gene groups should be made available through an open source repository such as GitHub for the reproducibility of the study that I consider to be extremely important in context of the sensitivity of the results to parameters of orthologs clustering.

L206 “phylogenetic tree that was constructed based on the alignment 400 universal single-copy proteins”.
Does it mean that the constructed pan-genome contain exactly 400 universal single-copy genes or any additional filter was applied for the set of core genes?
As one of the main results of the article is based on phyletic pattern of accessory genes, brief description of constructed pangenome such as general statistics of core, accessory and paralogous gene groups in the ‘Results’ section also would be interesting for the reader.

L242. the term ‘core accessory genome’ should be explained. Does it mean ‘the set of genes that are common for a phylogroup but are not common for the species’? As traditional model of pangenome separates all genes on the core, accessory and unique parts, the term requires a definition.

Moreover, it looks like there are methods to achieve a better technical standard. In particular, the methodology of subspecies classification based on phyletic pattern of accesory genes was proposed in (Moldovan and Gelfand 2018).
Validity of the findings: The findings seem reasonable while the description of the methodology should be improved to allow enough support for statements made in the article.
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: IMPROVED GENOME OF AGROBACTERIUM RADIOBACTER TYPE STRAIN PROVIDES NEW TAXONOMIC INSIGHT INTO AGROBACTERIUM GENOMOSPECIES 4
Review round: 2
Reviewer: 1

Output:
Basic reporting: The text and content of the article are better now. Accepted.
Experimental design: Experiments conform to a good research standard. I thank the authors for making scripts and data publicly available
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: IMPROVED GENOME OF AGROBACTERIUM RADIOBACTER TYPE STRAIN PROVIDES NEW TAXONOMIC INSIGHT INTO AGROBACTERIUM GENOMOSPECIES 4
Review round: 2
Reviewer: 2

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


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

Input:
Title: POSITIVE ASSOCIATION BETWEEN EPIPHYTES AND COMPETITIVENESS OF THE BROWN ALGAL GENUS LOBOPHORA AGAINST CORALS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Coral reefs are increasingly being replaced by other functional constituents, including fleshy macroalgae.  However, as noted by the co-authors, the mechanisms of this competitive interaction has rarely been described.  Here they test the competitive nature of Lobophora, as well as turf algae, with and without an epiphytic community.  They demonstrate that: 1) turf algae doesn’t compete with corals despite a significant number of interactions, 2) Lobophora is a competitive “winner”, and 3) that competition is directly related to ephiphytic overgrowth (but not to sedimentation, herbivorous fish biomass, or coral size/species).  The authors should be acknowledged for this interesting set of field-based observations that add to our understanding of algal-coral interactions, and raise many questions for (future) research.

The manuscript is reasonably well written in the context of Peer J style.  The background is relatively inclusive of the existing relevant literature (but see below), and the provided data support the conclusions presented.
Experimental design: The approach is well described and generally analyzed, and the research question is timely and meaningful.  The project seems quite preliminary (i.e., note vs article), but it doesn’t “oversell” the results obtained, and argues for further study.
Validity of the findings: The results are generally robust, and the conclusions are certainly intriguing.  My primary concerns with this study are the lack of detail relative to the central questions/results.  Specifically, the data demonstrate that epiphytes are the primary contributor to competitive dominance in Lobophora-coral interactions.  I am disappointed that there wasn’t any attempt to describe the epiphytic community (ie, micro-algae, fouling inverts, biofilms, etc), and to identify key/any of these taxa using traditional and/or molecular approaches.  Likewise, the authors note numbers of interations between turf and corals, and Lobophora and corals, but “corals” is a broad category in Fiji.  Who specifically?  And did you see more/less fouling on specific genera/species of coral?  These seem like fundamental data for the paper as presented.

Secondarily, and maybe less important for a field study, I assumed that this paper would get to some mechanistic explanation for the results obtained (based on the structure and context of the introduction).  I was therefore disappointed when I got to the end and realized this paper falls short, despite some discussion of chemical defenses, etc.  To that extent, when the authors wrap around to their discussion of allelopathy [line 155: Lobophora can damage corals…], they should include references by Slattery & Lesser (2014), as well as Rasher et al (2010, 2011), that not only demonstrated allelopathy, but identified specific compounds responsible.
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: POSITIVE ASSOCIATION BETWEEN EPIPHYTES AND COMPETITIVENESS OF THE BROWN ALGAL GENUS LOBOPHORA AGAINST CORALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the manuscript is well-written but still has some sentences and paragraphs that could be improved, as pointed throughout this review.

A major problem is that the authors have much more to explore in their data. The presented results are too basic and represent an initial analysis. I would really like to see a more thorough exploration of the data they have collected (available in the supplementary material).

In the introduction: reading of the paragraph within lines 48-52 is disconnected. Part of it may be due to awkward writing, but I would suggest authors to work on a better connection between this paragraph in the intro.

The manuscript also lacks important references of the field of coral-seaweed competition, several of which were conducted in Fiji by Mark Hay´s group. A few examples are provided below:

Rasher, D. B., & Hay, M. E. (2010). Chemically rich seaweeds poison corals when not controlled by herbivores. Proceedings of the National Academy of Sciences, 107(21), 9683-9688.

Rasher, D. B., Stout, E. P., Engel, S., Kubanek, J., & Hay, M. E. (2011). Macroalgal terpenes function as allelopathic agents against reef corals. Proceedings of the National Academy of Sciences, 201108628.

Rasher, D. B., & Hay, M. E. (2014). Competition induces allelopathy but suppresses growth and anti-herbivore defence in a chemically rich seaweed. Proceedings of the Royal Society of London B: Biological Sciences, 281(1777), 20132615.

Rasher, D. B., Hoey, A. S., & Hay, M. E. (2017). Cascading predator effects in a Fijian coral reef ecosystem. Scientific reports, 7(1), 15684.

Clements, C. S., Rasher, D. B., Hoey, A. S., Bonito, V. E., & Hay, M. E. (2018). Spatial and temporal limits of coral-macroalgal competition: the negative impacts of macroalgal density, proximity, and history of contact. Marine Ecology Progress Series, 586, 11-20.

Bonaldo, R. M., & Hay, M. E. (2014). Seaweed-coral interactions: variance in seaweed allelopathy, coral susceptibility, and potential effects on coral resilience. PLoS One, 9(1), e85786.

Dixson, D. L., Abrego, D., & Hay, M. E. (2014). Chemically mediated behavior of recruiting corals and fishes: a tipping point that may limit reef recovery. Science, 345(6199), 892-897.

The authors argue the findings are essentially novel, but I think some of what they present have also been pointed out by the papers below. This does not reduce the importance of the manuscript or their findings, but by putting their results in the context of previous findings, authors will be able to highlight the contribution of their work.

Fong, P., Smith, T. B., & Wartian, M. J. (2006). Epiphytic cyanobacteria maintain shifts to macroalgal dominance on coral reefs following ENSO disturbance. Ecology, 87(5), 1162-1168.

Smith, T. B., Fong, P., Kennison, R., & Smith, J. (2010). Spatial refuges and associational defenses promote harmful blooms of the alga Caulerpa sertularioides onto coral reefs. Oecologia, 164(4), 1039-1048.

Lines 38-40, for example, the authors say: “However, the extent to which algae drive these shifts by outcompeting adult corals and the mechanisms influencing the outcomes of coral-algal competition are still unclear”

This is not true. There is an extensive body of literature on how algae drive these shifts and on the competition mechanisms as well. Therefore, I would suggest the authors to rethink/rewrite this statement.
Lines 48-49: awkward phrasing, please clarify.

Line 54: Nugues & Bak 2008 should probably be:

Nugues, M. M. & Bak, R. P. Differential competitive abilities between Caribbean coral species and a brown alga: a year of experiments and a long-term perspective. Mar. Ecol. Prog. Ser. 315, 75–86 (2006).

Please confirm that.

I could not find specific hypothesis for the influence of sedimentation and herbivore biomass on the frequency and outcomes of contacts. I suggest the authors to include this explicitly by the end of the intro.

Figure 4 seems overcomplicated to me. I think pie charts could do the work better. Also, Figure 4 and 5 could be easily combined into one unique figure to be more informative and straight to the point.
Experimental design: Overall, methods are well-described, but would benefit from some clarifications as described in the comments below:

There is no mention to which coral species the authors investigated or if they did it for all the coral species. There is enough evidence in the literature that the extent and result of seaweed-coral contact is strongly dependent on the identity of both interacting species (coral and seaweed).

Line 82: please replace randomly by haphazardly, which would probably describe your method better. If the authors decide to keep randomly in the text, the I would like to see a better description on how they made sure transects were random indeed.

Lines 83-84: this sentence can be simplified, for example: “we measured the coral perimeter to the nearest centimeter”.

Line 98: please provide more details on the tide conditions and the time frame of the study. For example, all fieldwork was conducted within a month, two months, an year? Please clarify.

Lines 115-116: awkward writing. Please review it. Also, you probably do not need to state that your showing means and standard error at this point of the text.

Line 124: authors provide a X2 result, which is not clearly described in the methods. Please improve the methods section where you describe the statistical analyses.

Lines 131-132: there is a bit of discussion here, please save it to the proper section of the paper.
Validity of the findings: The data is somewhat simple and provide a descriptive overview of coral-seaweed contacts. A major problem of the data is that the authors do not specify the coral species, which would enable a much more refined analysis. This would be of interest to a broader audience. Looking through their raw data, I observed they have this information. Therefore, I strongly suggest the authors to reanalyze the data using coral species, with specific tests for each species. This would strengthen the findings by showing how general the demonstrated outcome is for several coral species. Analysis, findings and conclusions presented so far are too simplistic, which limits the impact of the manuscript. A more profound literature review will also enhance the manuscript.
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: POSITIVE ASSOCIATION BETWEEN EPIPHYTES AND COMPETITIVENESS OF THE BROWN ALGAL GENUS LOBOPHORA AGAINST CORALS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The co-authors have done a reasonable job of addressing my comments (and those of the other reviewer).  In many cases this included adding references and clarifying introductory/discussion points.  On the whole the manuscript is improved, and meets the basic reporting requirements of Peer J.  I will note however, that the added text also has many new typos/grammar issues.  This suggests: 1) a rush job, likely not reviewed by all co-authors, and 2) a lack of respect for the time of reviewers/editors, and ultimately readers.  Both points of which detract from an otherwise interesting story.

For example, line 286 of the track changed revision states: Lobophora to produce RATHER allelochemicals than...  Pretty sure the correct word is OTHER but I can piece together some real sentences with "rather" and a couple of deleted words.  It is not my job, or the readers, to try to figure out what you mean to say.  CORRECT ALL, and all co-authors MUST proof before resubmitting!!!
Experimental design: As noted prior, this paper lacks some fundamental data (i.e. who are the epiphytes), and what is the mechanism of action; but the co-authors have noted that this is part of an MSc thesis from a remote location, implying that data will not be added here.  They have apparently further analyzed some of the specific coral data, and avoided others due to a lack of replication.  Unfortunate that more wasn't available, but there is still a story here that will certainly inform future algal/coral competition research.
Validity of the findings: I think the article provided is an important note that will direct future research efforts.  My major complaint with the current manuscript is that the co-authors don't "own" their short-comings.  Yes, they have made the case to their reviewers, and the editor, that we can't hold this project to the highest standards and/or expect additional data given circumstances.  And, yes, there is still intriguing data here that should be put out to inform future studies.  But it would be great if the next iteration noted these issues in the article.    For example, the co-authors could state something to the effect of: Although this study wasn't able to identify the epiphytes involved (and/or substitute chemistry) for XXX reasons, future studies might approach this topic to better understand algal/coral competitive interactions...
Additional comments: A final revision needs to be carefully edited/proofed, and contextualized if you want this paper to become anything more than a throwawa (i.e., a cited product that defines future research efforts).


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: COVER CROPPING CAN BE A STRONGER DETERMINANT THAN HOST CROP IDENTITY FOR ARBUSCULAR MYCORRHIZAL FUNGAL COMMUNITIES COLONIZING MAIZE AND SOYBEAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please, find my comments on general comments for the author.
Experimental design: Please, find my comments on general comments for the author.
Validity of the findings: Please, find my comments on general comments for the author.
Additional comments: L28 How do you define generalist?
L54 Please, consider a citation to:
RYAN, M. H. & GRAHAM, J. H. (2018). Little evidence that farmers should consider abundance or diversity of arbuscular mycorrhizal fungi when managing crops. New Phytologist early view.
L56 I missed a mention to the changes that agricultural practices may cause on AM fungal taxonomic and functional community composition. Please, consider a citation to:
GARCÍA DE LEÓN, D. CANTERO, J. J. MOORA, M. ÖPIK, M. DAVISON, J. VASAR, M. JAIRUS, T. & ZOBEL, M. (2018). Soybean cultivation supports a diverse arbuscular mycorrhizal fungal community in central Argentina. Applied Soil Ecology 124, 289-297.
OHSOWSKI, B. M. ZAITSOFF, P. D. ÖPIK, M. & HART, M. M. (2014). Where the wild things are: looking for uncultured Glomeromycota. New Phytologist 204(1), 171-179.
L60-L61 Sentence „Thus […] (Njeru et al. 2014)“ does not seem a consequence of previous sentece. Please, consider rephrasing.
L74-LL88 largely repeats the information of previous paragraphs.
L80 Is Noelia Cofré et al 2017 cited correctly? Is Noelia the first name or a surname?
L91 What level is „host identify“ define at? Is it cultivar, plant species, groups of crops?  For instance, should a manuscript regarding wheat focused on wheat „wheat cultivar Arabella 2012“, „wheat“ or „cereals“? How it this for maize and soybean in relation to AM fungi?
L94 What do you mean by „improve the diversity or structure of AM fungal communities“? Improvement implies a goal (i.e. AM fungal community of reference) to achieve? Is this true for Integrated Mycorrhizal Technology? Should the production of food for a growing human population the solely target of agriculture? I personally do not think that agriculture has one unique aim, but some readers may believe food prodution is the only priority for agriculture. As far as I know there are not any „AM fungal community of reference“, which may represent a goal for farmer or scientistist to achieve. If the authors knew of any, I would extremely thankful for them to cite it. If there aren’t, a clarification is needed.
L102-L120 Experiment seems well design.
L117 Perhaps, the repetition of „100 and 100“ is not needed.
L121-L127 Plot scale seems rather small. That does not preclude the interest of study. However, many readers will appreciate a paragragh in the discussion dedicated to how your results might me scale-up.
L147 Could you please summarize the manufacturer’s instructions? Readers from other regions might not be familiar with the kits produced by RIZO, Tsukuba, Japan. For instance, in Europe and North America the DNA extraction market for AM fungi-related purposes is largely dominated by MO BIO Laboratory Inc., Carlsbad, CA, USA.
L150-L168 Nested PCR methods seems sound to me.
L187-L189 Does rarefaction imply that abundance data were used? What would be the effect on the results of using exclusively presence/absence data? There is an open debate that discourage the use of molecular data to estimate the abundance of AM fungi. Please, consider incorporating –and change the methods accordingly, if neccesary- the following citation in the manuscript:
Gamper HA, Young JPW, Jones DL, Hodge A. 2008. Real‐time PCR and microscopy: are the two methods measuring the same unit of arbuscular mycorrhizal fungal abundance?. Fungal Genetics and Biology 45: 581–596.
L192 Perhaps, it is my personally opinion. There are several statistical schools, but I do not particularly like transforming data for fitting a normal distribution. Instead, there are plenty of other statistical tools such as generalized linear models. Could you please explore –and show it to me- how robust your results are to the chosen methodology.
L196-L199 Please, revisit comment on L187-L189.
L200-L215 I agree the methodology described in this paragraph is generally finne. However, I have several concerns: (a) The significance of explanatory variables with function „adonis“ depends on the order they are included in the model in. Could you try and reproduce your results with function „Adonis“ [note capital A], which is robust to this problem?; (b) Rarefaction is a tricky issue. I personally feel rarefaction to the minimum number of sequences waste a lot of information. Perhaps, rarefaction to the median number of sequences might be a good solution. Plus, some readers will appreciate some information about the statistical distribution of the number of sequences (e.g. minimum, Q1, mean, median, Q3, standard deviation, standard error, maximum). It might be shown in the results, but this information is crucial to decide about the rarefaction method. Indeed, you used iNEXT package. iNEXT function from iNEXT package not only interpolates (i.e. rarefaction), but also extrapolates. I find hard to understand how the rarefaction to the miinimum and the use of Chao Extrapolation Index (i.e. INext; see Hsieh et al 2016, Journal of Vegetation Science)
See also my previous criticism about rarefaction on L187-L189 and L196-L199.
L216-L218 Unfortunately, I am not familiar with clamtest function. Many readers might be in the same situation as me. Could you briefly explain what it does?
Final comment: In general, I find the paper extremely interesting. However, it still contains some methodological issues that certainly require the authors’ attentions. Solving these issue could potentially affect some results. Therefore, I decide not comment on results and discussion at this time. I will be very happy to review this manuscript again, once the methodological flaws have been addressed.  At the moment, I cannot trust the results are robust enough to comment on them because some of them might change.


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: COVER CROPPING CAN BE A STRONGER DETERMINANT THAN HOST CROP IDENTITY FOR ARBUSCULAR MYCORRHIZAL FUNGAL COMMUNITIES COLONIZING MAIZE AND SOYBEAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: Literature references are update and according to the topic studied in the manuscript. I have found that some of the references cited in the text are not included in the list of the references and vice versa. Please, check and correct the references in the text and the list of references.
For example, “Clark, 2007” (line 45), “Oehl et al, 2003” (line 355), “Giovannetti et al., 1999” are not in the list of references.
In line 434, is Borrell et al. (2017) or Borrell et al. (2016) as is written in the list of references?
Line 574, where is this reference cited in the text?
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The manuscript titled “Is host crop identity a stronger determinant than cover cropping for arbuscular mycorrhizal fungal communities colonizing maize and soybean roots in a cover crop rotation?” contains novel results that help to understand the effect of the different crops species used in crop rotations on the community structure of arbuscular mycorrhizal fungi.
I think that the title could be reduced without repeating words as cover crops and cover cropping. It will make it clearer.

Introduction:
Some parts of the introduction could be condensed. Some points of the first paragraph are repeated in the third. For example, the information in lines 65-67 is similar to the line 48.

Materials and Methods:
Line 112, you wrote “temperate regions of Japan” and it seems that your experiment is located in different regions around Japan. Maybe you could write “in annual cropping systems in a temperate region of Japan”.
Line 113-119.  Is ryegrass treatment included in these activities (sowing and fertilization)? You should add how you sowed the ryegrass and if it was fertilized or not.
Line 122. In Figure S1, there is a sowing cover crop date in the fallow treatment. This cell should be empty to avoid confusion since it seems that a cover crop was planted in these plots.
Line 133. Did you use an auger to take the samples? Please, specify the apparatus.

Results:
Lines 247-249. Figure 3A shows OTU richness and you are talking about number of OTUs in this sentence. Please, clarify this part.
Lines 263-265. If you are comparing the different cover crops treatments within each cash crop (maize or soybean), the Figure S3A could be clearer if you group the different treatments for maize to the left and the treatment for the soybean to the right as you did in Figure 1.
Line 302, you talk about results from 2014 and 2015 but your experiment started in 2016. Please, clarify this part.

Discussion:
Line 328-329. How did the mustard and fallow affect the AMF colonization? Because ryegrass and vetch treatments also affected the AMF colonization by increasing significantly the colonization compared with fallow and mustard treatments. Please, clarify this part including whether they reduce or increase the colonization compared with which treatment.


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: COVER CROPPING CAN BE A STRONGER DETERMINANT THAN HOST CROP IDENTITY FOR ARBUSCULAR MYCORRHIZAL FUNGAL COMMUNITIES COLONIZING MAIZE AND SOYBEAN
Review round: 2
Reviewer: 1

Output:
Basic reporting: This resubmission has satisfactorily addressed all my previous comments. I do not have any further comments.
Experimental design: See basic reporting.
Validity of the findings: See basic reporting.
Additional comments: See basic reporting.


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: COVER CROPPING CAN BE A STRONGER DETERMINANT THAN HOST CROP IDENTITY FOR ARBUSCULAR MYCORRHIZAL FUNGAL COMMUNITIES COLONIZING MAIZE AND SOYBEAN
Review round: 2
Reviewer: 2

Output:
Basic reporting: Please, find my comments on general comments for the author.
Experimental design: Please, find my comments on general comments for the author.
Validity of the findings: Please, find my comments on general comments for the author.
Additional comments: Introduction:
Lines 51-52. The reference of “García-González et al, 2016” could be more appropriate in line 54. On the other hand, another example of organic matter improvement by cover cropping is García-González et al. 2018 (García-González, I., Hontoria, C., Gabriel, J. L., Alonso-Ayuso, M., & Quemada, M. (2018). Cover crops to mitigate soil degradation and enhance soil functionality in irrigated land. Geoderma, 322, 81-88).
I found large the second paragraph of the introduction. I think that lines from 62 to 66 could be reduce or rewrite with the information of the following sentences.
Line 81: It is not clear for me the new sentence “the level of host plant species in this study”. I do not know if you could use “host species identity”.
Results:
Lines 277-279. This part is confusing for me. Frist, you said there are treatments with higher or lower OUT richness and then in a second sentence you did not find statistical differences among treatments. It is similar in the following lines when you talk about Shannon index on brown mustard. Please, rewrite these sentences and make them clearer because my first impression was that there were statistical differences. In addition, you must check the reference of the number of figures. Figure 3B does not exist.
In lines 347-350 you explain that you performed a PERMANOVA to examine the relative importance of each cover cropping for the AMF communities. And where are these results? Where is the importance of each cover crop on AMF communities?


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

Input:
Title: IMPACT OF INTERCROPPING ON THE COUPLING BETWEEN SOIL MICROBIAL COMMUNITY STRUCTURE, ACTIVITY, AND NUTRIENT-USE EFFICIENCIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, it is an interesting topic to investigate the influence of intercropping on soil microbial communities, microbial activities and nutrient use efficiencies. The introduction is relevant and easy for readers to follow. However, the English language and the quality of scientific writing should be improved. Please recheck the spelling and grammar in the manuscript. Some numbers in the table and figure citations do not match the file provided.
Experimental design: The methods are generally appropriate, but more detail is required and the data analysis should be improved. I suggest that you improve the description of soil respiration and characterization of microbial community composition, and describe all statistical analysis used in this study (e.g. CCA, Spearman correlation were missing).
Validity of the findings: The interpretation of the results is insufficient and the conclusions are not fully justified by the results. For example, L188-190 & 244-245: why higher CUE/NUE ratios were due to higher respiration rates in intercropping treatments? higher respiration would lead to lower CUE as CUE can be calculated as Cgrowth/(Cgrowth+Crespiration) (e.g. Spohn et al. 2016; Keiblinger et al. 2014). are there any evidences that higher respiration could lead to even lower NUE? please elaborate.
Additional comments: L22-23: “the response of” and “in response to” are repetitive, rewrite the sentence.
L25: change “rhizospheric” to “rhizosphere”.
L28: define microbial activity (e.g., soil respiration).
L29: change ”effected” to “affected”.
L68: change “microbial structure” to “microbial community structure”.
L76: is it soil total N or soil total P?
L81: define “sieved soil” (<2mm?). change “considered” to “considered as”.
L87: add one sentence to describe how rhizosphere soil was recovered.
L91: add the company of the pH meter.
L95: remove “determined”.
L102-103: soil respiration rate is one of the key parameters measured in this study, briefly describe how it was measured instead of only referring to the literature.
L107&111: change “ITS1 genes” to “ITS1 region”.
L111: change “on” to “of”.
L117-121: move to “2.6. Statistical analysis”.
L126: Were relative abundances normal distributed here since you used ANOVA? Typically it is not easy to obtain normal distribution of microbial community data.
L134-135: wrong numbers when compared with Table 1.
L143-144: I suggest to calculate soil respiration rates based on soil dry mass or microbial biomass.
L146: wrong figure number, should be “Fig. 1b”.
L156&164: CCA and Spearman correlation were only mentioned in results part, but should be added to section 2.6.
L159-161: r values seem to be not correlated. are those p values?
L164: is Spearman correlation analysis based on mantel test?
L175: replace “such” with “such as”.
L180-181: the decrease of pH doesn’t necessarily indicate it was an important factor governing the microbial community. prove it (e.g. CCA, correlation).
L184-185: how did the SOC change microbial community? and why? please elaborate.
L191: soil total N and DON were not consistently higher in the intercropping treatments based on Table 1.
L199-200: only CUE/NUE ratios were calculated in this study, how could the author tell there was increased CUE? the ratio of fungi: bacteria (Sinsabaugh et al. 2013) rather than fungal abundance might shift soil microbial community CUE.
L214-215: The explanation here is not clear to me. what if dormancy rate of bacteria in soils is higher than that of fungi? then higher abundance of bacteria does not necessarily lead to higher microbial activity.
L217-218: TN and DON don’t seem to play important roles in shaping microbial community structure based on CCA and the results section.
L224-225: based on what kind of analysis? Fig. 4 can’t tell the abundances increased significantly.


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

Input:
Title: IMPACT OF INTERCROPPING ON THE COUPLING BETWEEN SOIL MICROBIAL COMMUNITY STRUCTURE, ACTIVITY, AND NUTRIENT-USE EFFICIENCIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article compared the effect of intercropping and monoculture on soil microbial community composition and activities, the topic of the article is interesting and have values in soil managements. The structure of the article conforms to an acceptable format, and relevant literature references were appropriately provided. However, the statistical analysis should be improved. Some of the discussions are speculative and not supported by the data. Some sentences are just too long and need to be rephrased (such as line 218-220, 192-196).
Experimental design: Research question is not well defined, the objectives of the article are too ambiguous. More detailed hypotheses should be presented in the article, such as the specific difference between monoculture and intercropping on soil microbial communities. In addition, the ultimate goal of this study is to determine whether and how the intercropping management is better than monoculture for soil system, thus a conclusive statement is encouraged to be provided.
The experiment was designed scientifically, and the investigation was conducted to a high technical standard. However, some information was missing in the method section:
1, The procedure of collecting rhizosphere soil should be briefly described, since there are different methods for sampling (Barillot et al., Ann Microbiol, 2013, 471-476).
2, The test of CCA was conducted but it was not mentioned in the statistical analysis.
3, Least significant difference (LSD) was used in ANOVA, but pair-wise comparisons for 4 treatments (sugarcane monoculture; intercropped sugarcane; soybean monoculture; intercropped soybean) were not provided.
Validity of the findings: The article showed the valuable findings that intercropping changed soil physiochemical properties, and subsequently effected microbial structure and microbial activity. I have some questions and suggestions about the discussion and conclusion:
1, The discussion about the ratio of CUE and NUE was based on too much speculation rather than your own data. Please show your results, such as increased CUE or decreased NUE, and explain the results with caution.
2, Several environmental factors were analyzed in CCA, only the factors of pH and SOC were discussed, why?
3, The relative abundance of bacterial and fungal groups at the phylum, genus, and OTU levels were presented, but the analysis on microbial community was unsatisfactory. You specifically discussed some microorganisms, such as Pseudomonas, Fusarium, Chaetomium. Why did you pick these microorganisms? It has limitations to extrapolate to a larger scale by only describing the relationships between certain species and soil managements. To further explore the functional traits of microorganism at community levels in different treatments, I recommend you to use ‘Funguild’ for fungal OTUs to distinguish the pathogens (Nguyen et al., Fungal Ecol, 2015, 1-8).
4. The conclusions should be modified, they should be connected to the original question investigated; in addition, only one reference is published in 2018, please consider citing more latest references.
Additional comments: line22-24 Ambiguous sentence, please rephrase it.
Line 27-32 please show more specific results, with detailed description
line 45 ‘alters’ replace ‘shifts’
line 59 delete ‘were’
line 62-63 Italic for species names
line 65 ‘fungi’ to ‘fungal’
line 66-69 The objectives of this study are too ambiguous, please specify
line 87-88 Please briefly describe the procedure to collect rhizosphere soil
Figure 3 Please add the description for the cluster analysis


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

Input:
Title: IMPACT OF INTERCROPPING ON THE COUPLING BETWEEN SOIL MICROBIAL COMMUNITY STRUCTURE, ACTIVITY, AND NUTRIENT-USE EFFICIENCIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is interesting and well written. Topic and question are relevant and timely. Lian and colleagues analyzed the bacterial and fungi structure and activity in the intercropping and monoculture system. They found that Intercropping changed soil physiochemical properties and microbial community structures. The novelty of this next-generation sequencing survey of soil microbial in the intercropping and monoculture system is the relationships between microbial community structures and microbial activity. As such, it has value.
Experimental design: The research questions are relevant and meaningful. The data analyses are pretty well explained. Below are some more specific comments that I hope are useful as the authors revise the manuscript.
Line 114-115: Did you remove the chimeric sequences from the row data? And how did you do that? Which chimera checking approach available in QIIME did you use?
Line 115-116: Which method did you choose to cluster the sequences into OTU? Such as CDhit, Uclust or some others? Did you standardise sequencing depth of each sample to a fixed value after get the OTUs? Such as 1000, 2000 or some other?
Line 120: please also report richness of microbial, not only Chao1.
Validity of the findings: Lian et al present a study which analyze simultaneously fungal and bacterial community structure in the intercropping and monoculture system. The research questions of the study certainly are current hot topics in microbial ecology. The study is meticulously well executed, the data is robust. The conclusion are well stated, which is linked to original research question and limited to supporting results.
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: IMPACT OF INTERCROPPING ON THE COUPLING BETWEEN SOIL MICROBIAL COMMUNITY STRUCTURE, ACTIVITY, AND NUTRIENT-USE EFFICIENCIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: After one round revision, the manuscript has been improved a lot. However, there are still some concerns need to be addressed. Especially the description of results need to be more precise.
Experimental design: Line102-104: Are you sure SOC, DOC, DON were all measured by TOC analyser? Typically TC,TN were measured differently from DOC and DON measurements.
Line 132: Do you mean rarefy?
Line 137: Was PCoA based on OTU level?
Line 149: change "different on" to "differences of".
Validity of the findings: Some the of explanations are not satisfactory. See the detailed comments.
Additional comments: 1. Change "physiochemical" to "physicochemical" across the manuscript.
2. Line 33: change "significant" to "significantly'.
3. Line 58: change"system have studied" to "systems have been studied".
4. Line 171-172: The description is not precise since bacterial and fungal abundances were only significantly increased for intercropped soybean.
5. Line 175-176: There were no figure captions for all supplementary figures, which makes the description in the MS confusing.
6. Figure 5a needs to be adjusted as one of the error bar was missing.
7.Line 201-202: Description is not precise. Plant pathogen did not increase significantly in I-Sugarcane.
8. Line 243-244: In this case, CUE could only decrease when respiration rates increased strongly than growth increase. Also, it is not clear to me why higher respiration led to lower ratios of carbon and nitrogen use efficiency.
9.Line 246-248: If no statistical significance was detected, I do not think this is a good explanation.
10.Line 258-259: Again since no statistical significance was detected in I-sugarcane, it is better to mention that bacterial and fungal abundances were only significantly higher in I-soybean system.
11. Line 261-263: The explanation is not satisfactory for me. As mentioned no statistical significance of available N was detected.
12. Line 263: "nutrient condition" are not proper words here.
13. Line 282-284: See comments 9 & 11.
14. Line 300: Change "microbial" to "bacterial".
15. Line 301: Change "was" to "as".
16. Line 304: Change "acceleration of" to "accelerate".


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

Input:
Title: IMPACT OF INTERCROPPING ON THE COUPLING BETWEEN SOIL MICROBIAL COMMUNITY STRUCTURE, ACTIVITY, AND NUTRIENT-USE EFFICIENCIES
Review round: 2
Reviewer: 2

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


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

Input:
Title: THE DETAILED DISTRIBUTION OF T CELL SUBPOPULATIONS IN IMMUNE-STABLE RENAL ALLOGRAFT RECIPIENTS: A SINGLE CENTER STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. All figures need legends.

2. Introduction could be imporved with significantly more detail in the description of the cell populations studied. For instance, the relevance of naïve, effector memory, central memory, and effector to the immune state after transplantation is not described.  PD1 is listed as important because it is “an immune regulatory marker,” which does not convey the way in which it is immune regulatory. The statement about CD57 is correct, but lacking in detail. For instance, CD57+ cells tend to have proliferative defects but maintain cytokine production (Immunology. 2011 Sep; 134(1): 17–32. doi:  10.1111/j.1365-2567.2011.03470.x).

3. Clarity could be added by conversion of the lists of mean +/- error bars in the text (i.e. lines 214-218 and lines 248-250) to table format.

4. The figures are relevant to the experimental question and laid out well, but a few features need to be altered to improve clarity. Specifically,
- As noted above, figures should have both titles and legends.
- In Figure 2, on the representative flow plots of CD3 by HLA-DR, either the axes are swapped or the conclusion is misstated.

5. It is believed that English is not the first language for the authors and the paper would benefit from an English editor.  A few specific examples are:
- In the text that describes Figure 1, there is a statement that “there were 4 groups both in CD8+ and CD4+ T cell..” This is confusing because following this statement 6 separate groups are described, and in the figure shows 10.
- Table 1- typo “teat” should be test.
- Lines 268-270 bring in discussion of pre-formed allogeneic cells but the following paragraph does not address this.
- Using the phrase “normal people” to describe the controls implies that transplant recipients are not normal.  “healthy volunteer” may be a better phrase.

6: there are 87 rows of data in T_sub_stat_data and 46 in TCR_stat data but the methods section notes 103 transplant recipients and 44 healthy controls.  Why are there fewer subjects in the supplemental data.

7: Table 1 is too vague– what is meant by generalized infection?  When were patients assessed for HIV, HCV, HBsAg.  It should be mentioned if patients were on immunosuppression other than tacrolimus.

8: Table 3 – should have p values.
Experimental design: 1: The research question is well-defined in the focus on T cell phenotyping after transplantion. However, as presented the experiments are less within the scope of the second portion of the question, about evaluation of immunosuppression. Specifically, the authors demonstrate that the FK506 level is higher in the 1 year post-transplant group than the 5 year post-transplant group, but the immune parameters studied for the most part only show a difference between post-transplant and normal. Thus, the authors have not proven that immunosuppression state is the reason for the difference observed ; correlation but not causation is indicated by the data. Conclusion needs to be toned down.

2: Important details of the analysis are missing from the methods section. Specifically:
- There is no description of compensation controls used in the experiments. The data appear to be compensated, but without a description of the compensation method the quality thereof cannot be evaluated. A notation of cell compensation versus beads would be sufficient.
- The flow cytometer used is not mentioned, so its appropriateness also cannot be evaluated.
- Fluorescence minus one controls are listed as being used to set the gates, but again, in the absence of the data being show these are difficult to evaluate.
- The data in the representative figures of flow cytometry data include instances where there is data adjoining the X axis (Figure 5 top, Figure 6 left), and it is unclear how the gating was completed to factor in the data on the axis. For accurate quantification, the gates must include all events along the axis.

3:  The methods section specifies two different statistical tests used, and in the absence of figure legends it is impossible to know which test was used for each figure and thus whether an appropriate test was used. In general, the Mann-Whitney test appears to be the more appropriate test.

4:  Because of the missing experimental details in the current manuscript, it would be difficult to replicate the study.

5: Ethics appear to be appropriate given the consent form submitted, but a more detailed statement of the ethics safeguards in the methods section as opposed to only in the supplemental materials would be appropriate.

6: No patients received lymphodepletion so any conclusions need to be limited to this group.  Similarly, as this is a single center study of Chinese transplant recipients, conclusions should be limited to this group.
Validity of the findings: 1: CMV serostatus is known to affect the T cell populations described ( for example, Science Reports 2016. 6:26892 doi:  10.1038/srep26892). Thus, some of the differences observed (or lack of differences between 1 yr and 5 yr) could be related to CMV status. Table 3 should include a column of information on proportion of patients who are CMV seropositive vs seronegative, and if there is a mix, the data may need to be divided between the two groups to determine whether there are differences between the time points when controlling for serostatus.

2: The conclusion that T cell populations are different in transplant recipients relative to healthy volunteers is well supported, but the conclusions about differences short- and long-term after transplant and about potential markers of immunosuppression are speculative. Additionally, some of the conclusions about specific parameters require additional justification from the literature:
	- CD8 T cells are listed in the discussion as “suppressive” T cells. Subsets of CD8 T cells can be suppressive, but on the whole these are cytotoxic cells that attack cells that are infected or appear to be foreign.
	- CD57 is listed as being associated with senescence and exhaustion. CD57 is also associated with altered function, but it is unclear whether it actually contributes to senescence (Larbi and Fulop, Cytometry Part A. 85(1)25-35. doi: https://doi.org/10.1002/cyto.a.22351)
	- PD1 is listed as being associated with T cell apoptosis, and the authors suggest that therefore the high PD1 in transplant recipient samples suggests apoptosis. However, PD1 is also associated with reversible exhaustion (see above reference), so it’s unclear why the hypothesis here is that it is associated with apoptosis. Also, PD1 throughout is listed as being associated with immune regulation, without any definition of what immune regulation means in this context.

3: Controls of the data appear to be appropriate, though as discussed above for research question, they are appropriate specifically for the question of differences between healthy and transplant populations, and less so for identifying effects of immunosuppression levels. The majority of the conclusions focus on time post transplant, but some (particularly in abstract summary) suggest that the data provide information about FK506 dose specifically, which is not the case.

4: several of the conclusions are highly speculative and should be more clearly identified as such, or more analysis should b completed to demonstrate their validity.

5: The CD8 T cells analyzed include CD8low cells, which may have had recent TCR stimulation, as indicated by the CD8 downregulation. Thus, this T cell population could vary between the healthy volunteer and patient groups, and some of the other parameters could vary within it.
Additional comments: The manuscript “The detailed distribution of T cell subpopulations in immune-stable renal allograft recipients” discusses the use of T cell phenotyping to describe differences between healthy and stable post-transplant immune states, with the goal of assessing immunosuppressive state.  This research would be of value to publish in PeerJ, but many significant details are missing that would be required for it to be acceptable for publication. However, all of these details should be available to the researchers without further experiments.


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 DETAILED DISTRIBUTION OF T CELL SUBPOPULATIONS IN IMMUNE-STABLE RENAL ALLOGRAFT RECIPIENTS: A SINGLE CENTER STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English grammar usage is very good.

The authors have performed a phenotypic analysis of peripheral blood T cells from normal controls and from kidney transplant patients 1 and 5 years out post-transplant.  Most of the data presented has been previously published for most of the markers analyzed in the current data set.  The authors should do a better job in citing these previous studies; for example Carla Baan and her collaborators have done quite a bit of such phenotypic analysis of T cells from kidney transplant patients.

The structure of the article is professional with no concerns.
Experimental design: No comment.
Validity of the findings: There are a couple of places where further analysis is warranted.

1) For the data in Figure 3, the authors should present the percentage of Vd2 and Vd1 positive cells as a percentage of the CD3+ cells not just as a percentage of the gd+ cells.  If they do this they will see that the Vd2+ cells in the control are approximately 16% of the CD3+ cells whereas for the kidney transplant patients they are less than 2% of the CD3+ cells.

The data in 5 raises questions about gating.  The CD4+ cells in the 5 year post-kidney transplant patients is placed far to the left of the other two groups, particularly for the analysis of CD57.  There is also a problem with the x-axis labeling on the lower left panel.  Why do the authors think that their anti-PD-1 stains the CD8 T cells so distinctly but stains the CD4 T cells as a smear?

Studies from several groups have analyzed the expression of CD28 and other exhaustion markers on non-naive/CD45RO+ cells in transplant and cancer patients.  The authors should also perform and present this analysis to determine percentages of CD28+ and CD28- cells in the CM and EM cell populations.
Additional comments: No additional comments