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

Input:
Title: CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF THE HYDROXYCINNAMOYL-COA: SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE (HCT) GENE FAMILY IN POPLAR
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall the English in the paper is good, and it was not difficult to read from that perspective, although I saw some grammar and word choice issues with suggested corrections that are noted under general comments.

Article structure is appropriate, although if the journal allows, I'd suggest combining results and discussion section as I think this would be a more effective presentation.

Most figure legend lack sufficient detail (see general comments)
Experimental design: There are two main structural problems I have with this manuscript. It may not be so much an experimental design issue, as much as how the results are presented in the manuscript.

I think it ends up being confusing to refer to the 10 genes as an “HCT gene family” if the authors are defining “HCT” as hydroxycinnamoyl-CoA:SHIKIMATE hydroxycinnamoyl transferases. I think their data suggest that PoptrHCT1 and HCT2 are likely transferases capable of using shikimate as acceptor substrate (and involved in biosynthesis of monolignols), but if I’m interpreting their data correctly it seems likely several/many/most/all of the others are hydroxycinnamoyl-CoA hydroxycinnamoyl transferases that could have different acceptor substrate specificities (e.g. quinate, malate, hydroxyphenylactate). If I am correct here, the title of the paper ends up being inaccurate. There is admittedly some sloppiness in the field in the use of “HCT”, since the most famous, the shikimate transferases implicated in monolignol biosynthesis, are usually just referred to as HCTs, but the term is also used generically for hydroxycinnamoyl-CoA transferases regardless of acceptor substrate. Thus, I think authors need to be very specific in their writing about these enzymes, especially if they might be talking about both enzymes with a specific substrate and implicated function (i.e. the shikimate transferases involved in conversion of p-coumaric acid to caffeic acid) and the class of enzymes in general (i.e. hydroxycinnamoyl-CoA transferases regardless of what acceptor substrate they use).

I am also somewhat confused by the Populus tomentosa data suddenly showing up around line 150. Maybe it is my lack of familiarity with poplar. What is the relationship between Populus trichocarpa and Populus tomentosa and between the HCT genes? It’s sort of strange for the Pto data to suddenly show up in this section without prior mention of it. Has a similar analysis of genes from P. tomentosa already been done, and if so, how does the work here on P. trichocarpa add to knowledge? From context, it seems like there’s a 1 to 1 correspondence between these two species. I think this needs to be better addressed in some way.

The methodology for the biochemical experiment is insufficient. More detail must be provided. See general comments
Validity of the findings: This manuscript details a study using genomics to identify a family of hydroxycinnamoyl-CoA hydroxycinnamoyl transferases from Populus trichocarpa and then use expression analysis to identify those likely involved in lignin biosynthesis. I believe the underlying work is useful and should be publishable, but I think the manuscript needs revision prior to publication. In some cases more detail is needed, and being more focused on some details may help the authors make their case.
Additional comments: Below is the initial review I carried out. I copied and pasted as appropriate into the Reporting, Design, and Validity sections above as appropriate from this review, so some comments below will be redundant. Please note, there are many specific comments below not covered in the other sections.

This manuscript details a study using genomics to identify a family of hydroxycinnamoyl-CoA hydroxycinnamoyl transferases from Populus trichocarpa and then use expression analysis to identify those likely involved in lignin biosynthesis. I believe the underlying work is useful and should be publishable, but I think the manuscript needs revision prior to publication. In some cases more detail is needed, and being more focused on some details may help the authors make their case.

Importantly, I think it ends up being confusing to refer to the 10 genes as an “HCT gene family” if the authors are defining “HCT” as hydroxycinnamoyl-CoA:SHIKIMATE hydroxycinnamoyl transferases. I think their data suggest that PoptrHCT1 and HCT2 are likely transferases capable of using shikimate as acceptor substrate (and involved in biosynthesis of monolignols), but if I’m interpreting their data correctly it seems likely several/many/most/all of the others are hydroxycinnamoyl-CoA hydroxycinnamoyl transferases that could have different acceptor substrate specificities (e.g. quinate, malate, hydroxyphenylactate). If I am correct here, the title of the paper ends up being inaccurate. There is admittedly some sloppiness in the field in the use of “HCT”, since the most famous, the shikimate transferases implicated in monolignol biosynthesis, are usually just referred to as HCTs, but the term is also used generically for hydroxycinnamoyl-CoA transferases regardless of acceptor substrate. Thus, I think authors need to be very specific in their writing about these enzymes, especially if they might be talking about both enzymes with a specific substrate and implicated function (i.e. the shikimate transferases involved in conversion of p-coumaric acid to caffeic acid) and the class of enzymes in general (i.e. hydroxycinnamoyl-CoA transferases regardless of what acceptor substrate they use).

I am also somewhat confused by the Populus tomentosa data suddenly showing up around line 150. Maybe it is my lack of familiarity with poplar. What is the relationship between Populus trichocarpa and Populus tomentosa and between the HCT genes? It’s sort of strange for the Pto data to suddenly show up in this section without prior mention of it. Has a similar analysis of genes from P. tomentosa already been done, and if so, how does the work here on P. trichocarpa add to knowledge? From context, it seems like there’s a 1 to 1 correspondence between these two species. I think this needs to be better addressed in some way.

If allowed by PeerJ, I would suggest combining the Results and Discussion section.

Most of the figure legends lack sufficient detail to be understandable on their own.

Overall the English in the paper is good, and it was not difficult to read from that perspective, although I saw some grammar and word choice issues with suggesrted corrections that are noted below.

Some specific comments:

Line 21: for the sentence starting with “Ten HCT homologs…” I would split it into two sentences and be more conservative. “….. genome.  Both genome duplication and tandem duplication are likely responsible for the expansion…”

Line 36: insert “a” before “vital”

Line 38: replace “with” with “to have”

Lines 44-45: the pathway hasn’t been improved upon, our understand has. Possible rewording “Our understanding of the monolignol biosynthetic pathway has continued to grow, and now 11 enzyme families and 24 metabolites are associated with it.”

Line 47: replace “Combining” with “In conjunction”.

Line 52: replace “revisable” with “reversible”, replace “enzymatic activity” with “in vitro activity”.

Line 55: Perhaps more context could be given for the energy-loss bypass mentioned here. Why does this matter? What’s its relevance here?

Line 57: Perhaps mention that the effect on plant growth is negative.

Line 73: The sentence is awkward. I’d suggest “In this study, we used genome-wide screening to identify 10 HCT homologs...”

Line 97: The phylogenetic tree shown in Figure 2B does not completely correspond with the list of HCTs in Supplementary Table 2. Some things listed in the supplement are not in the tree, and some things in the tree are not in the supplement.

Line 112: replace “addition” with “agent”, replace “with” with “to”

Line 114: insert “supplemented with” after “buffer”.

Line 115: replace “were” with “was”, delete “finally”

Lines 116-120: Authors need to provide more detail here. Some aspects of the experiment here are clearly different than the cited work by Luis et al. For example, detection method used here seems to be MS, but that’s not clear, while Luis et al. used UV-Vis detection (although they were not as specific in their methodology either). It would be helpful for a reader to know more about the reaction conditions used (how much substrate, how much enzyme) here without having to go back to another paper.

Line 128: replace “screened” with “identified”

Line 130: addition of commas would help make this more clear, “… poptrHCT3, 4, and 5, distributed on chromosome V, and PoptrHCT7, 8, and 9, distributed on chromosome XVIII,”

Line 132: For readers not familiar with analysis of genome structure (like me), perhaps some explanation on how to interpret the Ks score would be helpful. See also line 184.

Line 148 and 149: use of the word “genuine” here seems inappropriate. Use more specific language for what you mean. By “genuine” do you mean “uses shikimic acid as an acceptor and has a role in monolignol biosynthesis”? If so, use the more specific language. I think more details about the phylogenetic tree could also be given. Looking at the figure, there’s no mention in the text about “Group I” and “Group II”. What are those groups? On the figure, there’s a scale bar indicating 0.5. This is outside of my area…. What does that value represent? Also, I think some, if not all, of the information from Supplementary Table 2 could be included in the figure legend.

Line 150-160: Mentioned above, this section is somewhat confusing to me. Maybe it is my lack of familiarity with poplar. What is the relationship between Populus trichocarpa and Populus tomentosa and between the HCT genes? It’s sort of strange for the Pto data to suddenly show up in this section without prior mention of it. Has a similar analysis of genes from P. tomentosa already been done, and if so, how does the work here on P. trichocarpa add to knowledge? Additionally, for Figure 4A, what does the numeric scale represent? I don’t find Figure 4B particularly helpful… is this a standard way to show these relationships? It seems like there might be some better way to show that HCT1 and 2 have significant coexpression with monolignol biosynthetic genes, while the others don’t.

Line 163: I would suggest rewriting the sentence as “Recombinant PtoHCT1 protein was expressed in E. coli and purified to examine enzymatic activity.”

Line 165 and 173: I think it is confusing to refer to transfer of caffeic acid from caffeoyl-CoA to shikimic acid as the “reverse reaction”. These BAHD hydroxycinnamoyl-CoA transferases generally can use a variety of hydroxycinnamoyl-CoAs in vitro, including p-coumaroyl-, caffeoyl-, and feruloyl-CoA. I believe transfer of the hydroxycinnamate from the CoA derivative to acceptor (in this case shikimic acid) is viewed as the forward reaction. Reverse reaction is, I think, generally considered reaction of a hydroxycinnamoyl deriviative (here hydroxycinnamoyl-shikimate) with CoA to reform hydroxycinnamoyl-CoA and release free acceptor (here shikimate).

Lines 178-181: I don’t really understand what point the authors are trying to make.

Line 182: delete “finally”.

Line 189-196: do the authors believe that other members of this family, e.g. HCT3-10, might have different acceptor substrate preferences (i.e., something other than shikimic acid)? That might be worth mentioning here.

Line 197: does not need to be included in the paper, but depending on what the authors intend with CSE, chlorogenic acid might be a suitable substrate for some studies of the enzyme, plus it is readily available and inexpensive.

Line 208: “Advice” not “advices”

Figure legends in general need more detail. Figures should be overall understandable without having to refer to text or supplementary material. More specifically:

Figure 1: Please define all enzyme abbreviations in the legend. Should also include 4-coumarate CoA ligase (4CL). Caffeic acid on the right side is missing its hydroxyl.

Figure 2: Legend in 2B could include all the genes shown on the tree. Readers shouldn’t be sent to the supplement to know what genes and species are analyzed in the tree. As mentioned above, please define what the scale bar represents.

Figure 4: As mentioned above, please define what the heat map scale corresponds to. I don’t find 4B very helpful. Is there a better why to visualize the co-expression data. This is somewhat outside my field, but might a “heat map” type of visualization with the Pearson correlations as the value be effective? It could be a matrix of all the HCTs versus several genes implicated in monolignol biosynthesis. HCT1 (and 2) would be hot, HCT 3-10 would be cold (I presume). This is just an idea, but seems to get at the core of what the authors are trying to convey.

Figure 5: Although overall one can see shikimate disappearing and caffeoyl-shikimate appearing, the authors provide too little detail in the methods and in this figure legend to understand what a reader is looking at. I assume this is MS (following LC separation?), but what signal are we looking at in the chromatograms (total negative ion, total positive ion, selected ions?) Are the insets what we should expect? I guess I would not automatically know what to expect those insets to look like (I’ve seen MS detection for these sorts of compounds set up to give a major ion of +1 or -1 from the expected molecular weight, depending on polarity).


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

Input:
Title: CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF THE HYDROXYCINNAMOYL-COA: SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE (HCT) GENE FAMILY IN POPLAR
Review round: 1
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: See attachment.

Specific comments
line 25: please explain what functions of lignin are being considered.
line 28: systematic investigations: in what way? explain.
line45: "improving the pathway" meaning not clear.
line 52: do you mean reversible?
line 59: when using words line a variety, or many. be more precise.
line 72: distinguish more accurately.... please expain.
line 77:further CSE analysis, please explain.
line 86: more details needed on building and validating
line 140: referred HCTs, meaning not clear.
line 143 p- should be in italics in the text and in the figures.
line 173: what does it all mean? What has been learned from the study?
line 179and following. meaning not clear, confusing sentence. Please revise.
figure 1. p should be in italics.
figure 1. Which metabolites give rise to H units in lignin (from p-coumarate)  and C units, G units and S units
figure 1.p-coumaroyl shikimate needs to be corrected.
figure 4. much more can be said about the mybs an other lignin gene associations.
figure 4> "homology genes" should be "homologs"


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

Input:
Title: CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF THE HYDROXYCINNAMOYL-COA: SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE (HCT) GENE FAMILY IN POPLAR
Review round: 2
Reviewer: 1

Output:
Basic reporting: Basic structure of the manuscript is fine. Sufficient background is provided, and it is self contained.

I think the use of the term "HCT" is still problematic. I think better distinction needs to be made between HCT as the shikimate-utilizing transferase implicated in monolignol biosynthesis, and other BAHD hydroxycinnamoyl-CoA transferases that (probably) use other acceptor substrates and have other functions. I personally think saying "HCT-like" or "Bona fide HCT" does not adequately describe to a reader the distinction here. It may end up being a matter of adding additional text to make sure it is clear. I have attempted to do so in the attached copy which has "track changes" correction suggestions and comments

I think it might also be helpful to use the more specific term "orthologs" instead of "homologs" where appropriate (i.e., to refer to the homologs/gene family within the species).

Despite many editing suggestions in the previous version by both myself and the other reviewer, I still found many corrections for English and grammar were needed. Again, see the attached document.
Experimental design: Overall OK. More detail is needed for how the expression construct was made and protein purified. I did not notice this was missing in the last version, but a bit more detail should be provided.
Validity of the findings: OK. I made a few comments where I believe certainty of conclusions was stated too strongly and needed to be softened.
Additional comments: The gist of the paper is 10 HCT orthologs/homologs were identified. Based on phylogeny and structure, two (1 and 2) are likely shikimate-specific for acceptor substrate and associated with monolignol biosynthesis. Two (9 and 10) MAY be involved in dormancy and cold tolerance. The others likely have some other function.


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

Input:
Title: CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF THE HYDROXYCINNAMOYL-COA: SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE (HCT) GENE FAMILY IN POPLAR
Review round: 3
Reviewer: 1

Output:
Basic reporting: The work by Chao et al. describes phylogenetic analysis of the HCT gene family in poplar. Additionally, the authors performed enzyme assays to implicate PtoHCT1 in caffeoyl shikimate biosynthesis suggesting a role in lignin biosynthesis. The HCT gene family is one of the most studied family in poplar, as such most of the conclusions drawn by the authors are confirm what previous studies have reported. This includes characterization of whether family members evolved from the whole genome duplication event or from tandem duplication (Zhang et al. 2018). Additionally, functional specialization into lignin biosynthesis or stress response has also been reported (Zhang et al. 2018).

Specific comments

1.	Authors should consider reconciling their naming nomenclature with existing literature. It appears that they number HCT1-10 by increasing position on chromosomes as well as increasing chromosome number. Previous studies do not follow this pattern and it can be confusing to readers.
2.	Authors should also use the full gene model that is available in phytozome, e.g PT01G04290 should be Potri.001G042900 as found in v3.1 of the P. trichocarpa reference genome.
3.	Previous studies report 9 members of the HCT gene family in poplar. Here, the authors report 10 including PT18G03270 (Potri.018G032700) annotated as a ‘HXXXD-type acyl-transferase-like protein’ in the poplar reference genome. The author should justify and explain its inclusion and relationship to other well established members of the family.

Reference

Zhang, Jin, et al. "Genome‐wide association studies and expression‐based quantitative trait loci analyses reveal roles of HCT 2 in caffeoylquinic acid biosynthesis and its regulation by defense‐responsive transcription factors in Populus." New Phytologist 220.2 (2018): 502-516.
Experimental design: Experimental design was appropriate for the study.
Validity of the findings: Findings confirm what has been previously reported about the HCT gene family with the exception of the number of family members as noted in the review 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: CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF THE HYDROXYCINNAMOYL-COA: SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE (HCT) GENE FAMILY IN POPLAR
Review round: 4
Reviewer: 1

Output:
Basic reporting: I believe the authors have addressed most issues in the manuscript, and it is acceptable for publication pending minor corrections within the manuscript. Many of these I will not detail, but should be able to be corrected by a copy editor.

A few specific issues:

Line 30, “components” instead of  “component”.
Line 45, p in p-coumaroyl should be italicized
Line 55, what is the “t” for?
Line 65, I’d suggest again clarifying what you mean by saying “bona fide HCT utilizing shikimate as an acceptor and involved in monolignol biosynthesis”.
Line 69, there seems to be an extra “.”
Line 81, “… in a previous study are also shown in Supplementary….”
Line 105, “… in a previous study…”
Line 164, instead of “is far from…”, I would again suggest “seem unlikely to be shikimate-specific transferases involved in monolignol biosynthesis”
Line 206, use “utilizes” instead of “catalyzes”
Experimental design: OK
Validity of the findings: OK
Additional comments: See above


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

Input:
Title: GENETIC STRUCTURE AND HISTORICAL AND CONTEMPORARY GENE FLOW OF ASTYANAXMEXICANUS IN THE GULF OF MEXICO SLOPE: A MICROSATELLITE-BASED ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, the manuscript is well written and clear, with good coverage of literature information. The main results are discussed in order to answer the questions proposed by the authors.
Experimental design: The questions proposed by the authors are simple and do not provide any expectations a priori or hypotheses to be tested. I suggest that an improvement should be made in relation to the main question, the expected results, and the hypotheses to be investigated. Nevertheless, the methods applied are quite appropriate to investigate patterns of population structure and gene flow (both contemporary and historical). Despite the low number of genetic markers used (10 microsatellites), the authors compensate with an extensive number of samples.
Validity of the findings: The authors investigated the genetic structure, as well as the current and historic gene flow of the characid fish genus Astyanax spp. by examining the number of genetically differentiated populations existing in the different basins and caves of the Gulf of Mexico slope, as well as the degree of connectivity between such populations. However, since this is a population study with a group that probably has different lineages/species involved in complex processes of isolation, expansion, and hybridization, the discussion is confused in terms of which of these `species' may be involved in the evolutionary events suggested by the authors (more general comments below). In addition, based on contemporary and historical gene flow information, the authors discuss the possibility of different dispersion patterns between surface and caves. I suggest special attention with this interpretation, because historical isolation and the difference in physical connectivity between the two environments (surface and cave) can bias the gene flow pattern in the group, and not be necessarily linked to the dispersion ability of the organism in space. Therefore, to guarantee the best experience for the PeerJ readers, I recommend that these and other minor revisions should be made before considering the present manuscript for publication.
Additional comments: Major revisions:
1# Despite the phylogenetic controversies about the group, the authors investigate the genetic structure of the population in the genus Astyanax spp. I suggest that the authors adopt the most coherent phylogenetic hypothesis available in the literature and indicate a priori which possible lineages (i.e., species) may be involved in the study and in which locations they belong and which genetic clusters they are assigned. That is, for better clarification it would be helpful if these lineages/species are delimited in Figures 1, 2, 3. The lack of hierarchical delimitation of evolutionary units under investigation (i.e., population, lineages, species, etc.) may confuse the reader and hide important biological aspects such as where and which lineages are involved in hybridization and colonization processes.

2# In the section 'Dispersal pattern of Astyanax spp. throughout the Gulf of Mexico slope', Line 394: the authors consider the existence of different dispersion capacities of the species of the genus, concluding higher dispersion capacity for surface populations and low dispersion capacity for cave populations. However, although gene flow (contemporary and historical) is an indirect measure of an organism's ability to disperse (i.e., gametes dispersion) it can be biased by historical factors of landscape evolution and connectivity (e.g., long periods of physical isolation or temporary barriers). Surface basins may have maintained more connecting routes between populations over time than between populations in cave complexes, which indicates less connectivity of environments and not necessarily the low dispersion capacity of organisms. An example of this can be observed in the caves of the 'Sistema de los Sabinos', which presented historical and current gene flow that could be merely the result of the physical connection of these localities (line 431-433).  In addition, isolation by distance often is a result of limited dispersion in continuous environments. Mantel's tests in this study indicated significant correlations (0.696 and 0.633, line 233-239) between genetic and geographical distance for some surface populations. In this case, I believe that determining the different dispersion capabilities across these taxa becomes inconclusive.

Minor revisions:
In the abstract, it is not clear which are the main objectives/issues of the research. Please make it explicit.

Figure 1 - The authors labeled the localities as 'surface and cave populations', which is confusing with the definition of 'population' used in the text to express the observed genetic differentiation. For better clarification, I suggest replacing the term 'population' in Figure 1 by ' sampled localities'.

Figures 2 and 3 - For a better reference of which genetic groups belong to the surface or cave, I suggest the addition of some indicator in the figure that facilitates the rapid distinction by the readers.


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

Input:
Title: GENETIC STRUCTURE AND HISTORICAL AND CONTEMPORARY GENE FLOW OF ASTYANAXMEXICANUS IN THE GULF OF MEXICO SLOPE: A MICROSATELLITE-BASED ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In my opinion the authors need to better describe the morphological and ecological variations within the Astyanax complex. Describing the most important habitats and morphological traits. The author describe this for the caves taxa, but little was said about surface populations.

I also think the authors could better detail the characteristics of the landscape where the study is applied providing a better view on the spatial separation of populations from the caves and the surface. Which ecological factors could influence the genetic variation among populations?

A large part of the discussion is related to the different invasions that caves populations have undergone over time. However, only a few lines in the introduction give the reader a good background on this point.

In the introduction it is also mentioned that several studies of population genetics have already been carried out with this group. Is there any hypothesis to be raised about the direction of gene flow and connection based on these studies?
Experimental design: The research question of the study is well defined, and the methods to address them are ideal. However, a more detailed description of the analysis procedures must be presented.

Below I list some specific observations:

Line 96: I did not understand why the following sentence is cited in the text: "Some of these sites with fewer individuals had previously been studied in Coghill et al. (2014)."

Line 125: DAPC: The authors need to mention that they performed 2 types of analyzes, one with the caves populations and the other without them.

Line 136: AMOVA. I understand the reason for analyzing different groups of populations in AMOVA, but I would like the authors to explain the reason for the specific choice of the models tested. They tested 4 models  (a,b,c and D; Table S2), and they need to be explained in the methods.

Line 145: Since its maximum number of populations is 16, why not test k ranging from 1-16?

Line 159: MIGRATE: The authors should make it  clear which inference they used. Also, the choice of different comparisons between populations must be detailed and justified here in the methods.

General point: I would combine the topics Statistical analyzes and population genetic diversity into one topic, perhaps called genetic diversity
Validity of the findings: The results and Discussion provide an important advance in the study of this group with so many interesting evolutionary characteristics. But some parts of the results also deserve to be better explored.

Below I list some specific observations:

Line 194: The authors could also mention the few comparisons that were not significant.

Line 209: Four or five clusters???

Line 215: AMOVA: I would like the authors to better explore the results of AMOVA,
which means that model D has the least within group variance and the largest between groups?

Line 231: These procedures of including or excluding populations is also not described in the methods

Line 234: The Trans-Mexican Volcanic Belt should be shown in Figure 1

Line 237: Why except Catemaco???

line 240: Again, these different procedures using MIGRATE-N is not explained in the methods.

In fact, this part of the results is very difficult to understand. I didn't quite understand how the comparisons in table 3 were chosen.

-Table 3: It is TR or TRO???

-Why PCH is not included in the caves populations group (Table 3)?

-What are the comparisons called surface & Cave locations? why not use all cave pop???

-How are the genetic clusters found in the present study related to the three possible lineages  (A. mexicanus, A. aeneus and A. hubbsi) mentioned in the Introduction???

- A more in-depth discussion of the genetic structure of populations would be welcome

- All table and figures legends could be improved.

Figure 2: Why not just leave the plots present in parts A´ and B´?
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: GENETIC STRUCTURE AND HISTORICAL AND CONTEMPORARY GENE FLOW OF ASTYANAXMEXICANUS IN THE GULF OF MEXICO SLOPE: A MICROSATELLITE-BASED ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revised manuscript presented a significant improvement in relation to the previous version. The research objectives are clear and the introduction presents a concise context about the investigated issue. Figures and Tables were improved according to the reviewers' suggestions, which made the visual information clearer. In the discussion the authors corrected information that previously seemed ambiguous or out of context, being now more coherent with the hypotheses about the evolutionary processes that determined the observed genetic patterns. Therefore, I believe that after the extensive review the manuscript finds the minimum criteria to be published in PeerJ. This is an important contribution to understanding the geographic and ecological processes that drove the genetic structure and dispersion (with subsequent hybridization) of the three lineages that compose the Astyanax group in the Gulf of Mexico basin.


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

Input:
Title: GENETIC STRUCTURE AND HISTORICAL AND CONTEMPORARY GENE FLOW OF ASTYANAXMEXICANUS IN THE GULF OF MEXICO SLOPE: A MICROSATELLITE-BASED ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors took into account most of my previous comments .
Experimental design: The methodology seems adequate
Validity of the findings: Suggestions were incorporated
Additional comments: The authors took into account most of my previous comments in the Introduction, except the one about the landscape characteristics. I still have doubts about how these characteristics (geographic, ecological, etc.) would affect the connection between populations and I think that a brief description of this would be interesting in the Introduction, but I respect their opinion in thinking that this is not fundamental. The methodology now seems adequate, all my suggestions were incorporated, figures also were improved.

Discussion
Line 417: supported is repeated


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: PHENOTYPE CHARACTERISTICS OF GASTRIC EPITHELIAL MUCUS IN PATIENTS WITH DIFFERENT GASTRIC DISEASES: FROM SUPERFICIAL GASTRITIS TO GASTRIC CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: English language needs a professional editing
Background and context in the field are ok
Structure is ok
Relevant results are self- contained
Experimental design: Everything is ok
Validity of the findings: Everything is Ok
Additional comments: This is an interesting manuscript that clearly shows that variation in mucus composition characterizes different gastric disorders and different grades of gastric mucosa damage.
Beside the need of a clerical language revision, I have few comments:

- all your data are addressed to elucidate the progression from atrophy to gastric cancer, that is perfect to me. However, mucus is important also in drugs absorption process, namely for those that are gastric pH-sensitive (Lahner E, et al. Thyro-entero-gastric autoimmunity: Pathophysiology and implications for patient management. Best Pract Res Clin Endocrinol Metab. 2020;34(1):101373, Sigurdsson HH, et al. Mucus as a barrier to lipophilic drugs. Int J Pharm. 2013;453(1):56-64). Among these drugs oral thyroxine is on the spot due to the large number of treated patients worldwide and the narrow therapeutic index (see for rev Virili C, et al. Gastrointestinal Malabsorption of Thyroxine. Endocr Rev. 2019;40(1):118-136. I believe that this topic should be quoted in the paper.

- Discussion section contains several repetitions of data and should be shortened as well as integrated by the topic suggested above. The role of gastric mucus in different pathophysiologic areas should be highlighted and the related references added.

- Tab 4 is redundant and the data are already in the text.

Minor points
- Intestinal metaplasia and not its acronym (IM) should be cited the first time it appears (line71)
- line 61: alkaline mucin is cited only once. Is this equivalent to the neutral mucin or is a mistake? Please clarify.
- line 156 is GAG acronym correct?


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: PHENOTYPE CHARACTERISTICS OF GASTRIC EPITHELIAL MUCUS IN PATIENTS WITH DIFFERENT GASTRIC DISEASES: FROM SUPERFICIAL GASTRITIS TO GASTRIC CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The introduction section is somewhat vague and reads more like a textbook on the subject. Only 6 references are provided. Authors should provide examples in the literature to support their hypothesis on the putative role of different mucus phenotypes on cancer progression. There are multiple studies evaluating both mucin expression and their glyco-profile and correlation with clinical-pathological features in the gastric context that could be cited.

The histochemical images in the figures should be improved for clarity. Besides the overall view of the lesion, insets with higher-magnification should be provided in these figures.

English revision is advised throughout the manuscript.

Lines 72-74, Introduction – “Exploring the changes of mucus phenotype during the above pathological changes is of great significance to reveal the occurrence and development of gastric diseases”. Could the authors provide evidence in the literature that supports this sentence?

Lines 227-229, Discussion – “From this we can speculate that the disappearance of epithelial mucus phenotype is one of the high-risk factors for the progression of gastric disease.” This is highly speculative without providing some functional prove that there is cause-effect.

Minor points
Line 71, Introduction – define IM abbreviation in full as it is the first time it is mentioned.

Lines 124-126, Results – Authors should provide the exact P value for the different statistical tests, not just the indication that it is over or below 0.05. This applies to all other sections in the manuscript.
Experimental design: An appropriate number of clinical cases is analysed. It is not clear why the authors did not analyse staining in IM cases with the description of the corresponding mucous phenotype characteristics, although these are sparsely mentioned in the main text. Also, Helicobacter pylori infection status is not taken into account in the analysis.
Validity of the findings: The association between loss of differentiation features (in this case presence of mucus-producing cells) and cancer progression is a well-known feature. Overall, as there is progression from pre-neoplasic lesions to increasingly aggressive tumours, differentiation features are lost. In the particular case of gastric cancer, this is well documented, and the expression profile of different mucins has been analysed with antibodies, which is more specific than histochemical methods that only provide information about the nature of the mucous (cf. Reis et al., 1999; Senapati et al., 2008; Namikawa and Hanazaki, 2010; Boltin and Niv, 2013). Although the findings are valid, they are not entirely new. The added benefit to the body of knowledge already published is not clear.
Additional comments: The way the results are reported could be improved for clarity. It is not clear in the end what is the main conclusion of the manuscript. With this type of study (associative in nature) one cannot extrapolate conclusions as to whether the mucous phenotype itself is involved in disease progression or merely a bystander effect, due to shifts in cellular phenotype. This type of conclusion warrants functional assays with relevant study models (cell lines, mice…).


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: PHENOTYPE CHARACTERISTICS OF GASTRIC EPITHELIAL MUCUS IN PATIENTS WITH DIFFERENT GASTRIC DISEASES: FROM SUPERFICIAL GASTRITIS TO GASTRIC CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript by Dong et al describe the changes in gastric mucus characteristics in different lesions. This is a subject addressed in different studies, that here is described is addressed in a very clear manner, using a robust sample of patients. the results are relevant and interesting yet there some issues that need to be addressed:

- designations could be more intuitive: for instance superficial gastritis should be SG and not GS; atrophic gastritis why is CAG?
- in the introduction sentence in lines 76-77 is speculative. Should be toned doen.
- mucins produced in atrophic gastritis are produced by intestinal metaplasia glands. This should be better described.
-The discussion needs to be streamlined and better written.
- some of the images have artifacts and should be improved.


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: PHENOTYPE CHARACTERISTICS OF GASTRIC EPITHELIAL MUCUS IN PATIENTS WITH DIFFERENT GASTRIC DISEASES: FROM SUPERFICIAL GASTRITIS TO GASTRIC CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: Manuscript has been improved and my questions were met. No further comments.
Experimental design: Same as above
Validity of the findings: Same as above
Additional comments: No further 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 DOUBLESEX/MAB-3 DOMAIN TRANSCRIPTION FACTOR DMD-10 REGULATES ASH-DEPENDENT BEHAVIORAL RESPONSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Generally well-written report - see General Comments for specific issues. Appropriate citations. Professional overall structure and figures; however, some figures that include dots to represent individual animals (i.e., figure 3) confusing due to number of animals tested - animal dots looks like lines.
Experimental design: This study is a rational design of a behavioral screen. Some details missing from Methods - see General Comments for details.
Validity of the findings: Study provides a foundation in the behavioral effects of dmd-10 mutation; some interpretations move beyond the data (see General Comments for specifics)
Additional comments: Durbeck et al. investigated the possible role of the doublesex/Mab-3 Domain transcription factor DMD-10 in neuron function in C. elegans by performing a behavioral screen using assays for which specific neural circuitry has previously been identified. From these behavioral studies, Durbeck et al. provide evidence that although dmd-10 is expressed in several neurons types, it has a specific role in glutamate release from the ASH neuron. These studies have been carefully designed, though some required changes remain. As a whole, this study could provide insight for future research into the mechanism and location of action of DMD-10.

Major issues:

1) Despite conducting the egg-laying assay on the same plate across genotypes, and therefore animals experience identical testing conditions, a repeated measures (Within Subjects) statistical analysis is not warranted. At best, this allows for a well-controlled experiment, but as each animal does not go undergo each of the genetic conditions being compared a between-subjects analysis is still required. For this study, the statistics need to be reanalyzed using an ANOVA with appropriate post-hoc analyses (similar to the other tests in this paper).
2) For the GLR-1::GFP imaging studies, the researchers do not provide the precise anatomical location from where this construct was imaged in animals. The anatomical location of these images should be described and or illustrated. As well, authors must confirm that each of the images from every animal was captured from this same location across animals. GLR-1::GFP expression can vary greatly along the ventral nerve cord, with very large aggregates occurring more posteriorly and more punctate expression seen more anteriorly. Once the location of the GLR-1::GFP expression has been identified it should also be followed with a citation supporting the selection of that location in the ventral nerve cord.
3) With regards to the nose touch assay as well as the gentle touch assay, the researchers need to provide details about how much time passes between each touch delivery. Also as the authors are reporting an average of 10 touches, it needs to be noted if the worms are as responsive to the first touch as they are to the last touch. If the responsiveness of worms changes over the course of the 10 nose touches, or the 10 gentle touches, then the average responsiveness (% response) for each touch should be reported in a line graph instead of the average of all touches across animals. If the response from first to last touch is consistent then an average would be justified, however, if this is the case it would suggest that the dmd-10 mutant may have a habituation deficit if it shows no decrement in response over 10 touches (see Giles and Rankin, 2009).
4) For figure 3, two of the dmd-10 mutant strains demonstrate a moderate decrease in percent response to 10 nose touches compared to WT while the third strain shows a dramatic decrease in percent response. Is the dmd-10(gk1125) strain responsiveness level significantly lower than the other two dmd-10 strains? The authors should address what could be the cause of these differing results between mutant strains. This would be more interesting to see if those three strains are showing a difference in responsiveness over the 10 touches, rather than speculating from the average across trials.
5) The interpretation that ASH-mediated odor avoidance is affected by the dmd-10 mutation seems somewhat overstated (figure 5a). It's interesting that this slight decline is significant statistically but it's difficult to suggest that DMD-10 and OSM-10 are equal in their impact on ASH neuron function given the dramatic decline in behavior seen with the osm-10  mutation. Further, in figure 5B showing results of optogenetic activation of ASH, the dmd-10 strain appears to be significant both from wild type and from eat-4, so DMD-10 is likely not necessary for glutamate release from ASH. Given that the largest effect for dmd-10 mutation on behavior is seen with the nose touch reversals, it suggests that perhaps the role of DMD-10 may be in fact more pathway-specific within the ASH neuron. As such, the final sentences of the abstract need to be adjusted to reflect this as DMD-10 is not necessary for all ASH-mediated behaviors. As well, this should be included in the discussion section.

Minor Issues:

1) Line 42 - provide references to support “While some DMD family members have been extensively studied,..”
2) Line 55-re-word “We sought to determine whether DMD-10 is required for normal neuronal signaling in…” as neuronal signaling was not directly investigated in this study. Rather, describe the use of a behavioral screen in order to denote neuron function.
3) Line 60 - provide a reference to support sentence “Behaviors such as egg-laying, chemotaxis, and body bending are associated with signaling at particular synapses.”
4) Line 82-correct the sentence with “encode difference and stimuli”
5) Line 110-give the diameter of the copper ring employed in the egg-laying assay
6) Line 118 - replace with correct statistical analysis, within-subjects design (repeated measures) is not justified for this study (see Major issue #1 above)
7) Nose touch section (lines 130-138) - describe the amount of time that occurs between touches, is it a consistent time period between touches, and what determined the amount of time between touches? Also, report that it was the average across 10 touches that was reported. Ideally, report responsiveness for each individual touch to demonstrate if a plasticity process is at play. (see Major Issue #3 above for more on this)
8) Gentle touch section (lines 141 - 146) – the same as the nose touch section, describe how much time passed between gentle touches and if the averages were reported.
9) For the dry drop assay (lines 149-155) - describe how much time occurs between each dry drop encounter and if the responsiveness changes over time (account for possible adaptation).
10) For Optogenetic Activation of ASH (lines 158 - 168) - provide justification that testing off ATR did does not impact the function of the channelrhodopsin (reference needed). Also, provide measured light intensity that results from the LED setup described. Again, report duration between each of the 10 light stimuli delivered and responsiveness across 10 stimuli. Is there sufficient time between each light presentation to ensure transmitter rundown does not occur? Provide citation to support.
11) Line 177-178, correct wording of sentences “worms that had been laid 3 days prior to the experiment. Worms 178 were twice with M9 buffer and once with water.”
12) Line 178 - Include water type used for the third wash. If this is a typical assay procedure, provide citation.
13) Line 228 begin new paragraph after “...these mutants.”
14) Line 322 – re-word statement “These data suggest that the defect in dmd-10 mutants is not due to a decrease in the sensation of nociceptive stimuli by ASH.” - this was not demonstrated as cell activity of ASH was not measured.
15) Figure 1 caption - Include corrected statistical analysis description.
16) Figure 5A, the moderate decrease in responsiveness to the dry drop assay of the dmd-10 mutant is likely statistically significant from osm-10 outcome (see Major issue #5 above).
For figure 5B, is eat-4 significant from both wild type and dmd-10 mutants? This should be reported and these moderate results discussed.

References:
Giles, A. C., & Rankin, C. H. (2009). Behavioral and genetic characterization of habituation using Caenorhabditis elegans. Neurobiology of learning and memory, 92(2), 139-146.con


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 DOUBLESEX/MAB-3 DOMAIN TRANSCRIPTION FACTOR DMD-10 REGULATES ASH-DEPENDENT BEHAVIORAL RESPONSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors provided sufficient background and the goals of the study were made clear. The data provided including the figures and raw data was well organized and clear. The amount of background or rationale as it relates to the experiment or assay needs to be expanded in certain places, but is mainly clear. I have highlighted where it is not. Please see general comments section.
Experimental design: In their manuscript “The Doublesex/Mab-3 domain transcription factor DMD-10 regulates ASH-dependent behavioral responses “the authors did a thorough investigation of the role of dmd-10 neuronal function in C. elegans using a combination of behavioral assays and optogenetic techniques. The goals of the study align with the scope of the journal and the research proposed and completed fill a gap in the field.
Validity of the findings: The authors determine that dmd-10 gene function is required for ASH mediated behaviors and the experiments assessing GLR-1 expression at the cell surface versus the ventral nerve cord in dmd-10 mutants and wild type animals adds a new level of detail of the role of dmd-10 in neuronal function.  The authors completed many behavioral assays with sufficient replicates with statistical tests to definitely determine the role of the dmd-10 in neuronal function. The raw data provided is well organized and easy to understand and could be replicated.
Additional comments: In their manuscript “The Doublesex/Mab-3 domain transcription factor DMD-10 regulates ASH-dependent behavioral responses “the authors did an investigation of the role of dmd-10 neuronal function in C. elegans using a combination of behavioral and optogenetic techniques. The authors determine that dmd-10 gene function is required for ASH mediated behaviors and the experiments assessing GLR-1 expression at the cell surface versus the ventral nerve cord in dmd-10 mutants and wild type animals adds a new level of detail of the role of dmd-10 in neuronal function.

Overall, the study is well designed, experiments and analysis complete.  The findings are novel and clearly fills a gap in knowledge and establishes a role for dmd-10 in regulating ASH behaviors.

The manuscript is mostly clear, but there are a few areas where the rationale could be better explained or additional information provided to make better connections. I expand and provide examples below.  I also note a few places where there are minor errors or provide general feedback.  Please see my comments below.

Avoid using worm, either use nematode, animal, or C. elegans.

Lines 56-67, the authors mention that they are using behavioral studies to determine where in the nervous system DMD-10 functions. The authors need to provide a better rationale for how using the behavioral assays helps determine where DMD-10 function is required. In other words, make a connection with the genes in the behavioral circuit are in a pathway and that you are using the behavioral assay to determine where loss of dmd-10 functions.

The authors bring up ASH neuron specific behaviors but the transition is a little rough. And the level of background information provided for ASH and regulation of different responses in behavior is extensive and distracting. I suggest simplifying. For example, lines 77-81 the information provided does not further the authors rationale.

Lines 212-213: “We are interested in understanding the role of the Doublesex/Mab domain transcription factor DMD-10.”  In what? A role for neuronal development, neuronal function as it relates to behavior? Be specific

The author needs a better introduction to each of the behavioral assays and why authors are testing certain other mutants associated with the assays for controls. For example, the trashing assay is used to test for defects in motor neuron control. Then the author goes on to mention egl-3 and the egl-3 mutant defects. The author needs to mention how egl-3 is involved with the circuit. Why did you test this mutant? This is the case for all the behavioral assays.

Lines 226-227, the authors need to state what is being compared. For example, no difference compared to wild type.

Lines 228-229, “Along with unaltered behavior driven by motor neurons controlling locomotion, we also found no defect in the motor control of egg laying.” I am not sure what the authors mean by motor control of egg laying. This could be better stated, by saying the neural circuit responsible for egg laying was not defective.  Again, this needs a little more detail. How does this behavioral experiment differ from motor neuron/thrashing experiments? A few lines on the neural circuit for egg laying would help clear things up.

Lines 282-287, the authors make the argument that there was no change in the GLR-1::GFP at the synapses or a better explanation for how the constitutively active GLR variant helps discriminate between altered cell surface GLR-1 and presynaptic GLR-1.

Line 287, functional and properly-localized GLR-1: You are missing a word here or need to remove the semicolon.

In lines 315-320 the authors discuss EAT-4 mutant, but do not mention the phenotype expected.  Make a connection between nose touch and reversal response.

In general, for the discussion, discuss the results in order.  The authors go from discussing working model in figure 6 and discussion of the results to figure 2C. For example, in lines 333-335, the authors discuss figure 2C, before figures 2A-B.

Lines 358-373, In the discussion the writing around total quantification of GLR-1at the ventral cord not altered in dmd-10 mutants and the potential hypotheses for how loss of dmd-10 could be affecting ASH dependent behaviors, is not clear.  I would suggest stating what you found, then present the possible scenarios, followed by how you tested to gain insight into what was happening, and then what you found. I would then explain what this suggests.

Line 366, there is an extra () at the end of the sentence.


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 DOUBLESEX/MAB-3 DOMAIN TRANSCRIPTION FACTOR DMD-10 REGULATES ASH-DEPENDENT BEHAVIORAL RESPONSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The reporting has improved from the previous submission in that the data analysis has been corrected and data display has been improved. There is greater clarity in the figure captions of the result reported in the figures and raw data has been included. Overall writing-style is clear and information has been added to help explain the study approach and the rationale for results.
Experimental design: Experimental design of a behavioral screen followed by a specific receptor expression assay and a neuron activation study are sound approaches to the questions of the study. There is more research to be performed in the future to support the proposed rationale in the discussion.
Validity of the findings: Findings appear valid; however, inclusion of the third dmd-10 mutant strain previously reported is recommended given that the data has been collected, and this appears to be a venue that is amenable to inconclusive results. A rationale should be provided for the different outcomes seen with the third strain though.

As well, how/where strains were procured needs to be provided in the Materials and Methods section. As the dmd-10 mutant strains were originally generated by the Knockout Consortium, this needs to be recognized as per the requirements of using these strains. As well, details of the number of backcrosses performed on each of these strains need to be included.
Additional comments: Overall, the results are clear that there is an effect on behavior in the dmd-10 mutant strains. For clarity, it is necessary to distinguish the statements regarding dmd-10 effects on glutamate release in the Discussion as being speculative as this was not directly tested.  Specific minor comments below:

Line 475 - "The detected defects in ASH-dependent glutamate signaling..." - reword as ASH-dependent glutamate signaling was not directly tested in this study.

FIgure 2C. Missing confidence interval for dmd-10


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 DOUBLESEX/MAB-3 DOMAIN TRANSCRIPTION FACTOR DMD-10 REGULATES ASH-DEPENDENT BEHAVIORAL RESPONSES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Overall, this is a much stronger manuscript and most of the comments, suggestions, and issues that I raised in the original submission were addressed in this resubmission.

The background section was greatly improved with a better introduction of dmd-10 and the overall approach to understand the role of dmd-10 in regulating C. elegans behavior.
Experimental design: The rationale for the experiments was improved by the addition of supporting details and detailed explanation of the experimental design
Validity of the findings: The data and figures provided are statistically sound and well controlled. The figures have been modified and improved. Conclusions are well supported.
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: RESISTANCE TRAINING WITH DIFFERENT REPETITION DURATION TO FAILURE: EFFECT ON HYPERTROPHY, STRENGTH AND MUSCLE ACTIVATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some grammatical corrections are provided.
Experimental design: no comment
Validity of the findings: The statistical analyses should be ran using the absolute values as opposed to the percentage change values. Additionally, I would encourage placing less emphasis on the individual responses because the intervention was only ran once, and there is no time matched control group. This makes it very difficult to understand if we are examining true differences or just random error. Although the typical error is used, this only accounts for measurement error and not random biological variability that occurs over time.
Additional comments: The authors examine differences between repetition durations. Some comments are provided below:

Primary Comments:

1)The fact that the values increased in both groups does not provide a good rationale for using percentage changes instead of the raw values. This method will produce differential results depending on the differences in baseline scores across people (i.e. someone with a smaller pre value will have more weight on the analysis). You also run the risk of losing a normal distribution when using percentage changes (note: to test this you would need to check if the differences in percentage changes are normally distributed). It makes more sense to use the raw values here.
https://bmcmedresmethodol.biomedcentral.com/articles/10.1186/1471-2288-1-6

2) Throughout the discussion and conclusion section I would place much less of an emphasis on the individual-level data because you do not have a time matched control group to compare this to. Even though you have the TE reported this is not the TE performed over the same duration as the intervention groups, and thus it is likely to be artificially lower due to less random biological variability that occurs over time.
https://www.ncbi.nlm.nih.gov/pubmed/25823596

3) The discussion on line 487 needs to be changed: “the 2-s RD protocol showed at least larger than or similar effects in muscle hypertrophy than the 6-s protocol”. There were no significant differences for either muscle group tested, so therefore neither condition is better or worse. Unless you run a test of inferiority you cannot make this statement since this statement can’t be made from just looking at the individual level data.

4) Similar to the comment above, in the conclusion the following sentences are not supported by the data which showed no differences in CSA between conditions: “However, when considering individual data, faster movements may result in larger muscle hypertrophy (mainly for vastus lateralis muscle) compared to slower movements. Thus, we argue that an increased training volume provided by performing faster movements to MF would promote greater muscle hypertrophy when compared to higher TUTs during slower movements.”

5)How did you get the estimated sample size of 10? When entering the values into G*Power it provides a sample size of 19. This is using a paired t test; 2 tailed; effect size dz = 0.68; alpha = 0.05; power = 0.8.

6)Why were females excluded when a within subject design was used? Any influence of the menstrual cycle or effect of sex would be negated by the fact that each individuals is only being compared to themselves. A limitation of the study is therefore, that the findings may not be generalizable to females.

7) In the discussion on line 521: “However, despite the importance of increased TUT, especially in the process of muscle hypertrophy, a higher motor unit recruitment may be expected through a protocol with shorter RD and higher volume.” Would you really expect a difference in motor unit recruitment even given that both exercises were taken to failure? Why would one not recruit all motor units if exercising to failure? By the last repetition I would expect the muscle activation to be similar across protocols. In line with this comment, there were differences in EMG noted, but would you expect these differences to still be present when the individual is reaching volitional failure?

Additional Comments:

Line 556 “In contrast, the individual analyses showed a higher proportion of participants with greater hypertrophy after training with the 2-s RDs compared to the 6-s RDs.” This sentence should be deleted since this is likely to occur by random chance anyway.

Line 81: the word “response” is needed after “hypertrophic”

Line 270: the sentence is not complete

Line 450: add “the” before “6th”

Line 475: add “but” after “last”

Line 575 and 576 should be corrected for grammar: “It has been reported a greater neuromuscular activation and impulse production when performing ballistic movements (Maffiuletti et al., 2016).”


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: RESISTANCE TRAINING WITH DIFFERENT REPETITION DURATION TO FAILURE: EFFECT ON HYPERTROPHY, STRENGTH AND MUSCLE ACTIVATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: I thank the authors for the opportunity to review their work. Overall, I think the introduction/results/discussion need to be much more focused on their actual aim. There was a lot of extra discussion that was interesting but had nothing to do with the primary purpose of the study which was to “investigate the effects of two 14-week resistance training protocols on strength, hypertrophy, EMG, and their force relationships.”  i.e. the only thing that should be of interest is the comparison between groups with each…for example some of your results/discussion are investigating how this changes with session x protocol x knee joint angle…that is not something that was a purpose of the study as reported in the manuscript. Without a non-exercise control condition, it is difficult to say anything about a change across sessions…more appropriate to discuss is this condition different than this other condition. Overall, I request a much more focused introduction/results/discussion.

The individual response discussion should be removed. The issues with this approach has been discussed recently in two review papers by Atkinson and Dankel. With your results, you would want to take the SEM and multiply it by 2.77 (1.96 x sqrt 2) to get the minimal difference…this would then allow you to be confident who exceeded a change from zero…however, this does not necessarily mean that it is different from another score that did not exceed that threshold. This sort of discussion is misleading and it allows for some to conclude that one intervention was better than another when they were in fact not different.
https://physoc.onlinelibrary.wiley.com/doi/abs/10.1113/EP087712
https://link.springer.com/article/10.1007/s40279-019-01147-0

Specific comments below:

Line 44: superior muscle hypertrophy? There were no significant differences between groups.  Probably also worth stating that groups were not different in the strength task that mimicked the way in which they trained (isotonic).

Line 67: how influential is exercise volume for strength? There are a number of studies that show large differences in volume yet similar changes in strength?
https://www.ncbi.nlm.nih.gov/pubmed/28463902
https://www.ncbi.nlm.nih.gov/pubmed/31553889

Line 77: what is meant by “CSA is a well-known estimate of the muscle volume”?

Line 84: I found this paragraph confusing…I was having a difficult time following what the arguments were.

Line 125: testing something that wasn’t trained gives you information about a movement/test that wasn’t performed…gets you around the specificity component…but does that provide information as to adaptations to the exercise they were actually performing?

Line 144: it is not clear to me from the literature or your introduction why you’d expect CSA to be different.

Line 171: your sample size estimation does not appear to be correct. The correct sample size for the numbers you input is n=19.  Remember, you are interested in the difference in changes which is what you’d want to power your study on.

Line 227: in the introduction, you discussed potential differences at each site…but then pool them all together…why?

Line 272: this might explain your lack of difference in the 1RM…you are no longer testing your intervention when you do multiple exposures of the test to each group. This could have been accounted for with a non-exercise control…the lack of this group/condition should be noted in the limitations section.

Line 309: what was the sampling rate of your EMG?

Line 323: please report your results in the absolute values..as opposed to the % change.

Line 332: is there a way to run a Levene’s test for paired data? This is specific to between subject designs…did you run this as between subjet?

Line 334: this effect size appears incorrect…you want to the difference between conditions divided by the SD of that difference…you have paired data

Line 340: see earlier point about removing this

Line 368: what are control variables?

Line 423: your EMG-angle relationship and Force-angle relationship paragraphs were difficult to follow.

Line 486: your point #1 is not supported…it was not larger…it wasn’t different

Line 594: what strength response is important? The one you are repeatedly doing or the adaptations to a movement that wasn’t trained?

Line 635: what about when both arms are trained? Is there still a cross-over? Here is a recent paper that might help support your model
https://www.ncbi.nlm.nih.gov/pubmed/31652423
https://www.ncbi.nlm.nih.gov/pubmed/28177712


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: RESISTANCE TRAINING WITH DIFFERENT REPETITION DURATION TO FAILURE: EFFECT ON HYPERTROPHY, STRENGTH AND MUSCLE ACTIVATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: see below
Additional comments: I thank the authors for the opportunity to review the manuscript again and I appreciate the thoughtful responses to my previous points. However, I still have a number of points of consideration…particularly as it relates to your results and interpretation.

1) You state the importance of repetition duration but I would be very cautious with this interpretation because you found that benefit in only one of your variables (30 degrees). It might be worth stating this as more of an invitation for future work to investigate this, particularly since this seemed counter to your initial hypotheses.

2) The G-power output you provide is interesting because it is counter to what you get with known calculations of the t-distribution. For example, you are interested in the difference in differences which is the interaction….this is equivalent to a paired t-test on the change scores, yet G-power seemingly treats it differently (square the F value gives you the t in this scenario).

3) I have the following points with the statistical analysis
A) there is no need to test baseline differences, the limbs were randomly assigned to a treatment and any difference at baseline would be meaningless for interpretation. The purpose of randomization is random assignment not perfect numerical equality.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310023/

B) how did you run a repeated measures ANCOVA where all factors are within? I don’t think this is necessary based on point A but I am not sure how it was completed in SPSS?

C) the Mann-Whitney Wilcoxin test is a between subject test whereas you have within subject data. An assumption of this test is that here are two independent groups…however your groups have dependency (same person).

4) In the discussion section (line 482) you state that repetition duration is an important variable to be associated with chronic adaptations. This comes after discussing the force-angle relationships with EMG…however, is there evidence that you have differences between conditions across time? Based on your figures it seems like the differences present at beginning of training period are the same differences present at the end of training? In other words, there wasn’t really an adaptation per se?  From what I can tell, the only value that changed differently between conditions was MVC torque at 30 degrees?

Specific comments found below:

5) Line 67: can you rewrite this sentence for clarity.

6) Line 83: a more accurate depiction…compared to what?

7) in the results section on line 379: should this p value be 0.377?

8) it might be better to refer to muscle activation as “EMG amplitude” since that is what  was measured in your study and the Schoenfeld paper you reference.

9) Line 517-535: since this doesn’t appear to differ between protocols, I am not sure it needs a full paragraph of discussion.

10) line 563: please double check Morton paper…I believe it may have been every 3 weeks.

11) in your figures, please connect the individual dots so we know which one came from which person. In other words, connect their Post to their Pre…otherwise the individual plots are not all that informative.


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: RESISTANCE TRAINING WITH DIFFERENT REPETITION DURATION TO FAILURE: EFFECT ON HYPERTROPHY, STRENGTH AND MUSCLE ACTIVATION
Review round: 3
Reviewer: 1

Output:
Basic reporting: The individual plots for the figures are still not correct. You connect the pre test values of one limb with the pre test value of the opposite limb. This is not informative for describing the “response” to the intervention. The pre value for one condition needs to be connected with the post test value of the same condition. This needs to be corrected throughout.
Experimental design: n/a
Validity of the findings: n/a
Additional comments: n/a


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

Input:
Title: A COMMUNITY PERSPECTIVE ON THE CONCEPT OF MARINE HOLOBIONTS: CURRENT STATUS, CHALLENGES, AND FUTURE DIRECTIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Dittami et al. is a product of The HoloMarine working group, a collection of experts with diverse backgrounds and a common interest in understanding how eukaryote-symbiont relationships influence the marine ecosystem. Through a multi-day workshop, this group set out to “discuss critical concepts and opportunities in marine holobiont research and identify key challenges in the field” by highlighting “the potential economic, sociological, and environmental impacts of the holobiont concept in marine biological, evolutionary, and environmental sciences.” This is a bold endeavor. Like many others before it, these exercises are inevitably designed (with rare exception; e.g., McFall-Ngai et al., 2013, PNAS) to manifest into a reiteration of similar communal activities (e.g., Wilkins et al., 2019, PLoS Biology). This manuscript followed that tune by reciting major concepts in holobiont research using common examples and by not providing a novel, forward-thinking synthesis that may serve as a backbone for future research. However, it is only right to attest that the criticisms above reflect personal standards for working groups and this should not hold back or delay this manuscript from publication but more of serve as food-for-though in telling this story. Dittami et al. did provide a well-written manuscript piece that hits on major topics in holobiont research and could serve as a nice resource for those interested in the topic. Provided the manuscript at hand and the revisions it has gone through, I have a few comments that I wish for them to be addressed as part of a minor revision.

First, “The history of the holobiont concept” comes off from the lone perspective that the microbiome is that last missing biological link. While there are components of this that may be true, it does not reflect how the holobiont concept is a spectrum, from some species being heavily reliant on symbionts to barely, if at all. This concept, that was brilliantly addressed by Hammer et al. (2019, FEMS Microbiology Letters) and should be reflected in this, as well.

Second, “Emerging methodologies to approach the complexity of holobiont partnerships” provides the perspective that -omics is the way of the future for this type of research. Recent trends suggest otherwise. While sequencing will, without question, be important, it is simply becoming a component of and not the sole technique for this line of research. This trend should be reflected, as well.

Third, please discuss marine examples of gnotobiotic marine species in  "Emerging methodologies to approach the complexity of holobiont partnerships."

Minor:
Ln 75: Provide an alternate word to “stable” because transient interactions apply, as does the potential for symbioses to fade in or out after a couple generations
Ln 177: I am not sure that I would consider this an emerging concept per se. I'd say it is well-established, especially in the ocean (e.g., corals and sponges) but becoming more widely recognized amongst taxa. Please revise to reflect this.
Ln 239: Perhaps add Burgess et al. (2016, Biological Reviews) here too, as they provide a nice marine and terrestrial perspective on dispersal.
Ln 294: This comes as that Exaiptasia is a coral, which it is not. Please revise to reflect that it is a sea anemone.
Experimental design: See above.
Validity of the findings: See above
Additional comments: See above


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

Input:
Title: A COMMUNITY PERSPECTIVE ON THE CONCEPT OF MARINE HOLOBIONTS: CURRENT STATUS, CHALLENGES, AND FUTURE DIRECTIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This review is the result of a workshop in Roscoff Biological Station who brought together scientists working on marine holobionts from different disciplines and approaches. The review summarizes key concepts of holobiont research applied to the marine system. It discusses specific issues and opportunities related to the research of host-microbe interactions in the ocean. Finally, it emphasizes the need of a link to this research to management strategies. I find this review is valuable for multiple researchers in symbiosis research and marine science field. It is also a valuable resource for teaching! The Authors also mentioned policymakers as a target audience (L183-184). I think that is extremely valuable, but the vocabulary in the current version may not be plain enough or applied enough for that audience. One suggestion is to be more specific on the potential translations for management in the abstract. Why is the holobiont concept important for marine ecosystems and why now? My comments on the structure of the review are reported under Study design.
Experimental design: I much enjoyed the section “the history of the holobiont concept”. I only missed a couple of sentences at the beginning of the first paragraph for a strong statement about why it is the holistic perspective and the holobiont concept important now. I think of the policymakers here again. The paragraph and the whole section develop then this idea nicely, but a strong statement right at the start would help to engage them.
Section “marine holobiont models”. I miss more explanation on the advantages and disadvantages of “environmental” vs “tractable” models. Regarding the paragraph on environmental models, I also do not find well justified the mention of some systems and not others, nor the order they are presented. For example, the paragraph could be structured according to phylogeny, or according to autotrophic vs heterotrophic symbionts, or unicellular then multicellular. I am a bit surprised that sponges, corals and seaweeds are not mentioned or only one species is referred. I find that for environmental models the big taxonomic group can be mentioned, followed by one example of a particular species. Just an idea. The paragraph on tractable models is easier to follow.
Figure 1: I love how it shows all the diversity of marine holobionts, in particular the unicellular hosts, often overlooked in other reviews. But if your target audience includes policymakers, you may consider including a second panel in the figure with bigger picture of keystone holobionts or holobionts of particular interest for ecosystem functioning and services. This is just a suggestion, Authors must decide.
Section “marine holobiont assembly and regulation”. Here I see some problems with the flow of the section. First paragraph focuses on modes of transmission. Second paragraph starts referring to immunity as a mechanism for microbe selection during transmission, and then present dysbiosis as perturbation of microbial transmission and maintenance. There are too many concepts mixed here and not easy to follow. Also, the perturbation of the transmission not necessarily brings the same issues that problems during maintenance. Moreover, the Anna Karenina principle is an example of patterns related to non-deterministic processes and is in the middle of the selection paragraph without detailed explanation. My suggestion is to move the dysbiosis and “Russian dramas” (lines 362-375) to L397. Thus, the second paragraph of this section will focus on selection mechanisms: immunity and chemicals. The third paragraph will discuss other factors shaping the microbiome. The forth paragraph will bring these concepts to the health/disease context, and you end with the conclusive paragraph.
I suggest revising the definition of the Anna Karenina principle. The definition by Zaneveld et al. does not refer to number of parameter but rather to deterministic processes in health vs stochastic process in disease.
Validity of the findings: The conclusion puts together the perspectives on marine holobiont research.
Additional comments: No additional comments


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

Input:
Title: A COMMUNITY PERSPECTIVE ON THE CONCEPT OF MARINE HOLOBIONTS: CURRENT STATUS, CHALLENGES, AND FUTURE DIRECTIONS
Review round: 1
Reviewer: 3

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

Line 269: In this paragraph, I miss a statement to the requirements of marine holobiont model organisms. A table could be helpful summarizing valuable characteristics of mentioned marine holobionts, based on points summarized e.g. in Ruby EG 2008 Nat Rev Microbiol., Box 1.

Line 361/362: The selection of the microbiota by the immune system is an important aspect and should be elaborated. In addition, citations are missing for this claim. (e.g. Franzenburg et al 2013 PNAS). The subsequently mentioned concepts “Anna Karenina principle” and “Rasputin effect” are only of secondary importance for this point.

Line 452. Omics approaches are not the key for functional studies. Similar to large scale ecological studies –omics studies remain correlative and require experimental validation using e.g. CRISPR/cas9 genome editing on the host side or cultivation and transformation on bacterial side. This point needs a deeper discussion in this paragraph.

Line 568: The same is true here. Here, also functional analyses via transgensis (e.g. CRISPR/cas9) is missing.

Line 570: Not clear, what is meant here.

Line 574: What kind of “societal, and economic questions” are meant here? To my mind these kind of questions are not part of the review. Either include a chapter discussing these kind of questions or delete it here.


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

Input:
Title: A COMMUNITY PERSPECTIVE ON THE CONCEPT OF MARINE HOLOBIONTS: CURRENT STATUS, CHALLENGES, AND FUTURE DIRECTIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am happy with the revision by the authors and support publication of this manuscript.
Experimental design: I am happy with the revision by the authors and support publication of this manuscript.
Validity of the findings: I am happy with the revision by the authors and support publication of this manuscript.
Additional comments: I am happy with the revision by the authors and support publication of this manuscript.


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

Input:
Title: A COMMUNITY PERSPECTIVE ON THE CONCEPT OF MARINE HOLOBIONTS: CURRENT STATUS, CHALLENGES, AND FUTURE DIRECTIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The Authors have addressed my comments and those of the others reviewers.


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

Input:
Title: A COMMUNITY PERSPECTIVE ON THE CONCEPT OF MARINE HOLOBIONTS: CURRENT STATUS, CHALLENGES, AND FUTURE DIRECTIONS
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I have no further 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: SENSITIVITY ANALYSIS OF THE INFECTION TRANSMISSIBILITY IN THE UK DURING THE COVID-19 PANDEMIC
Review round: 1
Reviewer: 1

Output:
Basic reporting: Biglarbeigi et al. used existing data to simulate COVID-19 transmission under different reopening scenario with regard to social contact metrics. The prediction can be helpful for policy making if the algorithm is valid.
In fact, social contact is one of the most important factors associated with R0. However, there are still other factors relating to transmissibility, which further affecting R0; for example, universal masking intervention and social distancing. Existing evidence has shown such intervention can reduce transmissibility even though workers keep working in the pandemic.(1,2) Without taking the effects into account, the predictions may not be accurate.
Second, it is inappropriate to report the results along with the discussion. In “4 RESULTS AND DISCUSSION” section, the authors presented the results, but spared little text on discussion. Therefore there is a lack of interpretations regarding whether the findings are an addition to current science, if it is consistent with previous research, what the underlying mechanisms are, and strengths/limitations of the current study, etc. Without in-depth discussion, it is hard for readers to capture the significance of the study.
Ref.:
1.	Wang X, Ferro EG, Zhou G, Hashimoto D, Bhatt DL. Association between universal masking in a health care system and SARS-CoV-2 positivity among health care workers. J Am Med Assoc. 2020;324:703–704.
2.	F-Y Lan, C A Christophi, J Buley, E Iliaki, L A Bruno-Murtha, A J Sayah, S N Kales. Effects of universal masking on Massachusetts healthcare workers’ COVID-19 incidence. Occupational Medicine. 2020; kqaa179.
Experimental design: The authors made several assumptions to build the algorithms, but some of them may not reflect the real situation.
1.	Line 110-115: the authors counted only “health professionals, health and social care associate professionals and those with caring personal service occupations” as essential workers during the lock-down. However, there are workers in other job categories that need to work despite the mitigation policy, such as transportation, public safety, national security, food and other necessary goods, etc.(3) Without including all essential job categories, the contact matrices estimate for the lock-down period would be incorrect.
2.	3.1 Exit strategy scenarios: To my understanding, the BBC Pandemic contact matrices did not calculate contact for each job category, but for working population as a whole. However, contact rate actually varies across occupations. For example, grocery store workers should have much higher contact rates than administrative workers. Therefore, using the BBC Pandemic contact matrices to estimate each scenario may not be appropriate, as the authors listed particular job categories for individual exit strategy.
3.	The paragraph following Line 144, “Accordingly, our calculations show increase in the daily transmissibility due to increase in the number of confirmed COVID-19 inpatients.” I was wondering why the increase in transmissibility was due to “inpatients” but not outpatients or total case numbers. In my opinion, inpatients should be less transmissible than outpatients.
Ref.:
3.	The Lancet. The plight of essential workers during the COVID-19 pandemic. Lancet. 2020;395:1587.
Validity of the findings: The main findings shown in Figure 4 and Figure 6 illustrate the predicted R0 under different scenarios. However, as aforementioned, the contact matrices for each scenario were defined based on the percentage of particular job categories (derived from Office for National Statistics), but not the true contact rates in each occupation, which is unavailable from the BBC Pandemic database. Therefore, I am skeptical about the prediction.

Also, the present algorithm does not take confounding factors into account. These factors include, but not limit to, universal masking and social distancing policies, which have been extensively implemented during the pandemic. Such interventions can actually protect workers even if they have contacts while working, and thus affect R0 in reality.
Additional comments: In sum, I am concerned with applying BBC Pandemic contact matrices to the scenarios comprised of returning particular occupation workers to normal, as well as other policies that can confound the prediction. Because this is a data-driven study, its prediction would be biased if the training database were inadequate to answer the research question.


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: SENSITIVITY ANALYSIS OF THE INFECTION TRANSMISSIBILITY IN THE UK DURING THE COVID-19 PANDEMIC
Review round: 1
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: My major concern is that although the authors propose many detailed methods for the contact matrix and its link to Ro calculator, which as they acknowledged is already existing knowledge, in this results, they simply presented the results directly from Cori et al's approach (by running the 'EpiEstim' R package), which appears less novel.
My minor concern is that how do the authors consider the Rt estimated by using Cori et al's approach as Ro?
As such, I find fewer interests in this manuscript in either theoretical or epidemiological aspects. Thus, unless the authors could justify the novelty of this work, I do not recommend it for publishing.


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: SENSITIVITY ANALYSIS OF THE INFECTION TRANSMISSIBILITY IN THE UK DURING THE COVID-19 PANDEMIC
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have improved basic reporting to my satisfaction.
Experimental design: The authors have added more details on the assumptions for algorithm building, which makes the methodology more clear. However, I am still uncomfortable with the strong assumptions. Most importantly, because this is a data-driven study, if there are essential data unavailable (such as an accurate occupation categorization to define essential workers and the distinct contact matrix for specific occupation instead of the homogeneous contact assumption), the prediction will be limited to be applied to the real world. I acknowledge the authors' novelty on utilizing contact matrix to predict COVID-19 transmission under different scenarios, but it seems to me that the currently available datasets could hardly make precise predictions.
Validity of the findings: Because the quality of available datasets is not satisfactory for the purpose of this study, and the current study is a data-driven study, I am not convinced the findings are valid.
Additional comments: I would suggest the authors to look for more literature before making any strong assumptions. For example, instead of assuming every occupation has the same contact matrix, the authors could define specific weights to individual occupation's contact matrix based on existing literature to better reflect the real world.


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: SENSITIVITY ANALYSIS OF THE INFECTION TRANSMISSIBILITY IN THE UK DURING THE COVID-19 PANDEMIC
Review round: 2
Reviewer: 2

Output:
Basic reporting: I am satisfied with the current version, thanks.
Experimental design: I am satisfied with the current version, thanks.
Validity of the findings: I am satisfied with the current version, thanks.
Additional comments: I am satisfied with the current version, thanks.


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: SENSITIVITY ANALYSIS OF THE INFECTION TRANSMISSIBILITY IN THE UK DURING THE COVID-19 PANDEMIC
Review round: 3
Reviewer: 1

Output:
Basic reporting: The revision is to my satisfaction now, but a few minor corrections should be made before it can be accepted for publication.

1. Line 208-210, "Althogh the results of SC1 suggest that Northern Ireland’s R0 value remains below 1. SC2 suggests R0 values of lower than 1 in all the trusts, however, WHSCT, with R0 = 0.94 shows a higher sensitivity to changes in contact numbers compared to other trusts."
Please correct the spelling 'Althogh' to 'Although', and consider rephrase this sentence for readability.

2. Line 199, "...considering homogeneous social contacts" and Line 228, "...assuming homogeneous social behaviour"
I thought the weights for the average contact numbers were also applied to North Ireland. If that is the case, the assumption should be corrected.
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: SENSITIVITY ANALYSIS OF THE INFECTION TRANSMISSIBILITY IN THE UK DURING THE COVID-19 PANDEMIC
Review round: 4
Reviewer: 1

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