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: TRANSCRIPTOMIC ANALYSIS REVEALS ETHYLENE SIGNAL TRANSDUCTION GENES INVOLVED IN PISTIL DEVELOPMENT OF PUMPKIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: See below.
Experimental design: See below.
Validity of the findings: See below.
Additional comments: The manuscript by Li and colleagues presents comprehensive transcriptomic and candidate gene analysis associated with pistil development of pumpkin plants. The authors used RNA sequencing (RNA-Seq) technologies to identify the potential key genes involved in the development of pistils. The experimental design is a typical comparison, and the aborted and normal pistils of pumpkin were used in this analysis. Several DEGs were validated using qRT-PCR. The authors also investigated the expression levels of ethylene candidate genes in different flower tissues and highlighted ERDBF3 and ERTF10 genes. Thus, one of the main findings is related to ethylene signal transduction genes as important players in the pistil developmental processes. The work may be technically sound, but I feel that the treatment of data is little bit shallow and do not go beyond descriptive statistical analyses. The study is too focused on the technology/RNA-Seq approaches and not enough on biology. I also found many problems in this manuscript and pointed them as follows.

Major concerns:
(1)	Poor figures and legends
As a whole, the authors should improve the quality of the illustrations (Figures). In addition, the legends were too poor. For example, Figure 1: Enlarging the font size in x-y axes and labels is needed. It is difficult to check the scientific quality. The authors should describe the significance levels in Figures 1B and1 C.
Figure 2: The current legend was confused with that of Figure 3. You should explain software to generate this figure. What is RichFactor?
Figure 3: The font size is too small. The authors should use magenta/cyan instead of red/green. It is important from the viewpoint of "Colorblind Barrier Free".
Figure 4: The figure legends need more information about the bar plots. What are significant differential expressions? What do the bars define? Average, median, outliers, distribution, error bars? Did you perform any statistical tests? The authors should explain all the gene abbreviation.
Figure 5: See my comments to Figure 4.

(2)	Identification of DEGs and functional enrichment analysis
I was not able to find any statistical tests (e.g., DESeq) to identify 3817 DEGs. Also you mentioned in L152-143: ”The |log2 (fold change)| > 1 and FDR<0.05 were used as significance cut-offs for the expression differences.” This is too arbitrary without any statistical tests. The correction method of p-values is not clear.
Please describe the parameters for transcriptome mapping, functional annotation, and enrichment analysis, such as TopHat2 and GOseq R. In the GO- and KEGG pathway enrichment analysis, did you use FDR correction for multiple testing problems?

(3)	Biological significance and discussion
Table 2: Changes in most of candidate PAL genes are small but significant. Is this study, the authors mentioned about the role of PAL and ethylene candidate genes. It is absolutely wrong to put such argue that these genes are relevant for the biosynthesis and signal transduction pathways without any experimental evidence. It is highly important to conduct a silencing/mutant study of either any one of the mentioned genes to support your conclusion. Without experimental evidence and support, the authors cannot claim their role in the biological processes.

(4)	Raw and processed data
The raw transcriptome data were deposited in the NCBI SRA under BioProject number PRJNA554766. This is great. However, the authors should add all the processed data such as the results including functional annotations and FPKM values of all transcripts as supplementary data (e.g. MS Excel file).


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: TRANSCRIPTOMIC ANALYSIS REVEALS ETHYLENE SIGNAL TRANSDUCTION GENES INVOLVED IN PISTIL DEVELOPMENT OF PUMPKIN
Review round: 1
Reviewer: 3

Output:
Basic reporting: The ms by Qingfei Li et al. describes the results of a transcriptomic analysis on pumpkin pistils and the expression of some selected genes in different parts of the flowers in such species. The authors claim the importance the ethylene response and phenilpropanoid biosynthesis during pistil development.  In general, the manuscript is well written, although it could be improved changing the structure of some sentences.
The introduction of the work indicates properly what is known about sex determination of flowers in cucurbits, which is a very early stage of flower development. As the topic of the work is pistil development the introduction should be illustrative about what is known for this in cucurbits and other species and how hormones impact on it.

-The main experiment of the work, the RNA seq, is not accessible and raw data have not been disposed in any database.
Experimental design: The main experiment of the work is an RNAseq. The authors have tested only two biological replicates in the experiment, aborted vs. “normal pistil”.  They should describe in more detail what are aborted and normal pistils, and of course, indicate the developmental stage of the flowers at moment of the collection of the samples. I mean, for example flowers at anthesis? Before?... What I observe in the Figure1 A is a mix of pistils in different developmental stages. It could be also beneficial for unfamiliar readers to include the normal pistil for comparisons, and pictures of the flowers at the moment of collection. In addition, the origin of the abortion must be indicated in the text. Is due to the absence of ovules? defects in the cell division? Could this cause be responsible of part of the DEG observed? All this points should be addressed.
Validity of the findings: An important concern that I have is about how well annotated is the Cucurbita moschata genome, and how this information could be used in ontology analysis. In the list presented by the authors in supplementary figure 2, I see that they found a lot of ERF, but it make reference to a transcription factor family, and not to a function!
This point make the conclusions derived very weak.
For example AP2 and ANT genes (included in the list) belongs to this family but no relation with ethylene has been reported.
In addition, the authors named the different genes that they tested by Q-PCR with Arabidopsis thaliana names, as for example  NPR1 or ERDBF3, without indicating the Cm locus. Are they sure that the genes at least are orthologous?
As a suggestion, I will reanalyse all this data carefully, and separating the up-regulated genes from the repressed genes.
Additional comments: No additional comments


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

Input:
Title: TRANSCRIPTOMIC ANALYSIS REVEALS ETHYLENE SIGNAL TRANSDUCTION GENES INVOLVED IN PISTIL DEVELOPMENT OF PUMPKIN
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors addressed all my comments.


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

Input:
Title: TRANSCRIPTOMIC ANALYSIS REVEALS ETHYLENE SIGNAL TRANSDUCTION GENES INVOLVED IN PISTIL DEVELOPMENT OF PUMPKIN
Review round: 2
Reviewer: 2

Output:
Basic reporting: See below.
Experimental design: See below.
Validity of the findings: See below.
Additional comments: I appreciate the authors' efforts to address the comments expressed during the previous round of reviews. The revised manuscript has much improved. However, I still had a minor comment about data reproducibility and transparency. The authors did not answer the following question in the previous review.
> I was not able to find any statistical tests (e.g., DESeq) to identify 3817 DEGs.


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: TRANSCRIPTOMIC ANALYSIS REVEALS ETHYLENE SIGNAL TRANSDUCTION GENES INVOLVED IN PISTIL DEVELOPMENT OF PUMPKIN
Review round: 2
Reviewer: 3

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: I'll put my comments following the answers of the authors:

Experimental design
The main experiment of the work is an RNAseq. The authors have tested only two biological replicates in the experiment, aborted vs. “normal pistil”. They should describe in more detail what are aborted and normal pistils, and of course, indicate the developmental stage of the flowers at moment of the collection of the samples. I mean, for example flowers at anthesis? Before?... What I observe in the Figure1 A is a mix of pistils in different developmental stages. It could be also beneficial for unfamiliar readers to include the normal pistil for comparisons, and pictures of the flowers at the moment of collection. In addition, the origin of the abortion must be indicated in the text. Is due to the absence of ovules? defects in the cell division? Could this cause be responsible of part of the DEG observed? All this points should be addressed.

Agreed. According to the reviewer’s suggestion, we added more detail description of the aborted and normal pistils and indicated the developmental stage of the flowers at moment of the collection of the samples in the new manuscript.

---- I still see a problem using only two biological replicates in RNAseq experiments. The authors have added additional information about the nature of the sampling for the RNAseq, explaining that samples are collected from 5mm buds, and they refer to the fig1A. As I commented in my first report, samples in fig1A seems a mix of different developmental stages of pistils. Which one represents the pistil of a 5mm bud? 5mm bud are buds where pollination has not yet occurred? Is there any "normal" pistil to compare? Are there macroscopic differences between the two kind of pistils analyzed? The authors also indicated that one difference between aborted and normal pistil is the absence of ovules. Again, I wonder if part of the DEG observed could be related to this fact instead of proper pistil development. These points should be addressed and, at least, considered in the discussion.-------

Validity of the findings
An important concern that I have is about how well annotated is the Cucurbita moschata genome, and how this information could be used in ontology analysis. In the list presented by the authors in supplementary figure 2, I see that they found a lot of ERF, but it make reference to a transcription factor family, and not to a function! This point make the conclusions derived very weak. For example AP2 and ANT genes (included in the list) belongs to this family but no relation with ethylene has been reported.
In addition, the authors named the different genes that they tested by Q-PCR with Arabidopsis thaliana names, as for example NPR1 or ERDBF3, without indicating the Cm locus. Are they sure that the genes at least are orthologous?
As a suggestion, I will reanalyse all this data carefully, and separating the up-regulated genes from the repressed genes.

The Cucurbita moschata genome is annotated well. The ERF, AP2 and ANT in the new supplementary Table 4 were annotated as ethylene-responsive transcription factor in the NCBI blast and Cucurbita moschata genome. So, they have relation with ethylene.
According to the reviewer’s suggestion, the Cm locus of genes that were tested by Q-PCR have been added in the new manuscript. They are orthologous. Thanks for your suggestion.

Again I'm not agree with the consideration that all genes in the list are "ethylene signal transduction genes"or "ethylene-responsive factors". At least in Arabidopsis, AP2/ERF transcriptions factors are a huge family, that are involved in multiple developmental processes and respond to many abiotic a biotic stresses as well as multiple hormones, but this doesn't mean that all them respond to ethylene. If authors consider that genes included in the list are clearly ethylene response genes, please include a reference where this is stated.
Other point that I suggested but not considered by the authors was to perform the GO analysis separating Up-regulated and down-regulated genes. Many times these analysis make results clearer.


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: BEHAVIOURAL VARIABILITY AMONG CAPTIVE AFRICAN ELEPHANTS IN THE USE OF THE TRUNK WHILE FEEDING
Review round: 1
Reviewer: 1

Output:
Basic reporting: This section is fine.
Experimental design: This also is fine.
Validity of the findings: Findings are accurate but see longer comments to author.
Additional comments: This paper offers detailed observations on styles of eating among a couple small groups of elephants at a zoo. To begin, the paper's title really needs to be more specific and informative, something like, Variability among a zoo's African elephants in use of the trunk while feeding. (It doesn't seem like HIGH behavioral variability, and POPULATION is too sizable a term for this study, plus it pertains only specifically to use of the trunk while feeding. Some variability is to be expected, e.g., individual elephants vary in their interactions with humans, Rossman et al.)

The study is well-executed with extensive detailed observations. It first provides a very nice comparative review on manipulation of food by primates and elephants with uses of hands, fingers, and the elephant's trunk. Then it sets forth the goal of examining how the trunk is used in feeding, with a couple of hypotheses. Elephants were provided items of food each day, the largest being branches 2-3 meters long.

Elephants living in the wild or in naturalistic environments are surrounded by living plants, ranging from grasses available on the ground to huge trees. Using their trunk, tusks, and feet, they are well-equipped with a range of strategies for processing these growing items into food for themselves. A limitation of this study is that it takes place in a stripped-down environment that offers no opportunities for elephants to exhibit their typical approaches for acquiring and preparing food items for consumption.

There is no mention of elephants using the foot to assist in feeding during these observations, perhaps because, with food being delivered in elephant-bite-sized pieces, there was no need to use the range of special skills elephants employ to prepare their food. Using personal communications, I have verified that both wild and domesticated Asian elephants in India use their feet to assist in feeding, not only to break branches into more manageable pieces, but also to knock dirt off of grass clods that are held by the trunk (personal communication, L. Hart, May 12, 2020). These are routine behaviors performed by elephants as they are feeding. And in South Africa, captive African elephants in a spacious naturalistic environment show similar behavior with grass clods, and also use the foot to break up items such as a melon into manageable pieces (personal communication, Z. Rossman, May 10, 2020).  The zoo environment in this study provided only limited avenues for manipulating items, or at least there was little need to use a wider range of strategies with the food and environmental items that were provided.

It seems the study revealed the stringent limitations of the zoo environment, resulting in the elephants exhibiting much less of their behavioral repertoire than would be seen in a more naturalistic environment where they are surrounded by growing vegetation. This limitation bears discussion in the paper.

Nonetheless the paper provides some interesting data and i support publishing it.

Several typos are highlighted. Many of these are incorrect singular/plurals, or apostrophes.  Also, the format is inconsistent in the references regarding upper and lower case for the titles.


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: BEHAVIOURAL VARIABILITY AMONG CAPTIVE AFRICAN ELEPHANTS IN THE USE OF THE TRUNK WHILE FEEDING
Review round: 1
Reviewer: 2

Output:
Basic reporting: Abstract
Whilst the opening line of the abstract sounds good, it’s not very accessible to a general audience, or a lay one, and it’s not factually correct as it’s the order Artiodactyla that contains the largest mammals on Earth; the whales. I would suggest explaining which animals you are referring to, for example, “The Proboscideans, an order of mammals including elephants, are the largest of the Earths land animals”.

Introduction
Line 26: change ‘able’ to ‘ability’
Line 32: remove ‘also’
Line 37: add ‘elephants’ before ‘trunk’
Line 43: change ‘individuals’ to ‘elephants’, and merge with the following sentence to read: “…1.5-2 meters long in adult elephants, and is mainly composed…” (I suggest this because grammatically, the second sentence isn’t complete since it starts with ‘it’).
Line 47: a sentence shouldn’t really start with ‘it’.
Line 64: perhaps add something like ‘in trunk use’ at the end. I won’t comment any further on sentence structure, or English language, as that’s not what reviewing is for.

Methods
Line 76: Add the year.
Please make it clear that you did live observations, perhaps in line 117 where you discuss proximity to the elephants. Also, please make clear what this proximity was in some sort of distance estimate.

Behavioural observation: please describe fully how you recorded the behaviour, i.e. I am assuming from what you’ve written that you scored feeding behaviour continuously during each 15-minute focal sample, as you appear to have durations for feeding as a state behaviour, but this hasn’t explicitly been stated nor has a definition of ‘feeding’ been given. I see you have an ethogram with definitions of each type of manipulation behaviour you recorded, but this should be referenced here not in Data Analysis. You also need to state how you recorded these; i.e. were they events counted as frequencies, which you then converted to percentages for each individual per food item? These methods need to be described in enough detail to be replicated.

Line 159: was branch diameter measured before the branches were put in or estimated by eye?

Line 163: number of occurrences of what? This isn’t clear.

Line 169: what is a ‘general score’?

Discussion
The discussion is good, well done. I have a few minor comments.

Line 265 onwards: It is not clear here if you are still referring to the Hart study or back to your own data again.

Line 324: ‘may be more important’ instead of ‘are more important’

Line 395: delete ‘not’ in ‘not to be’
Line 397: capital G needed on gripping
Line 401: a sentence should not begin with ‘and’, just continue this from the previous one.
Experimental design: The main ‘design’ elements in this study are 1) the behavioural sampling, of which I can’t judge because not enough detail has been provided, and 2) the statistical analysis. The analysis is complex hard to follow. I am not convinced that so many tests for significant differences were required for the purpose of this study. Whether the p-value is significant or not shouldn’t really matter, especially here, as this is a descriptive study documenting the types of manipulation strategies used by feeding elephants. The aim, to work out if there is more or less individual variation when elephants manipulate larger more ‘simple’ objects versus performing complex delicate tasks doesn’t surely need all these tests to tell us that?

It’s very difficult to judge statistics when you can’t see the data, and you’re new to the study, so I won’t try to tell you what should have been done, but it is hard to follow what was done and why here, and leaves me feeling there must have been a simpler way to analyse the data.

Please also use the guidelines set out by Budaev (https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1439-0310.2010.01758.x) when describing what type of PCA was done and please report the relevant statistics (KMO & Bartlett’s test as well as a factor loading table).

The PCA and the PCA figures make sense, but you could help the reader and tie into your text by circling the three different groups from Figure 2 in different colours. Figure three is good also. However, figure 4 seems to me like it would contain all of the same information if the significance testing were removed. This would also allow you to include the rare behaviours, to show if they were performed most my certain individuals. I’m also not convinced that the two groups need to be treated separately, unless you are expecting group-effects.

Lines 200-206: in relation to my previous comments, I don’t feel like these differences needed to be tested for significance, it’s quite clear from Figure 2 what the relationship is between the different items. Keep this in if you wish to, but I’m not convinced it adds much.

Line 215: this sentence doesn’t make sense ‘whereas in group A elephants in the frequency of using it’

For the inter-individual differences section: I don’t see why the elephants were analysed according to group A and B, since you’re looking at individuals, they can all be compared to each other. And again, I am not convinced the significant differences really add anything; p-values are fairly meaningless in this context. If you disagree, this is fine, it’s not necessary to change it.
Validity of the findings: The findings all appear to be valid, but it should be acknowledged that no male elephants were included in the study, nor were any tusked elephants, which may impact handling strategies.
Additional comments: This manuscript is well written, but it could be improved by using an English Language service.


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: BEHAVIOURAL VARIABILITY AMONG CAPTIVE AFRICAN ELEPHANTS IN THE USE OF THE TRUNK WHILE FEEDING
Review round: 1
Reviewer: 3

Output:
Basic reporting: Thank you for submitting this manuscript. I really enjoyed reading it. My only substitutive comment is on the English language.

I found your interesting findings a little bit difficult to follow in places, which leads me to the recommendation that your English language should be improved to ensure that an international audience can clearly understand your text. This is partly due to some grammatical issues including the incorrect use of plurals (e.g. abstract lines 4 and 11 and main text lines 68, 95, 111, 212, 312) and tense changes both within sentences and within paragraphs (e.g. lines 100, 212-219, 238, 281, 368). For example: “The absence of bones in the trunk allows substantial degree of freedom for the movements in all directions” should be “The absence of bones in the trunk allows a substantial degree of freedom for movement in all directions”. These grammatical errors are extremely common errors for non-native speakers so you not alone in making these errors. It will also help the clarity and flow of your work if you try to stick to one tense, such as the past tense, when describing your results (e.g. lines 212-219). I also found some of your sentence structures a little convoluted in places and some of the discussion section in particular was a little long. This could be improved by writing in an active and precise scientific style. I’ve given a few examples here and below, but you do need to thoroughly check the whole manuscript. I am confident that you can improve this.

Minor comments:
-	Your current title is a little bit vague as you just say elephant behaviour varies and not what type of behaviour. It would be great if you could be more specific as it will help your audience locate your article.
-	Proboscideans (or even Elephantinae/ Elephants) are the largest terrestrial mammals - the blue whale is the largest mammal.
-	Abstract: “impact of item properties” should be “food item” for clarity
-	I am not entirely sure about your use of the word "personal" in your abstract as it is a slight anthropomorphism of your study subjects. Even in the field of animal personality, researchers tend to talk about inter-individual differences.
-	Line 32: Use either "in addition" or "also" not both
-	Line 64: "Evocated" - evaluated?
-	Lines 37-64. I recommend you slightly re-structure this paragraph (or paragraphs - I'm not sure if there is a break in there) so that the paragraph is in a more logical order that creates a clear narrative. For example, you give specific details about the size of the trunk on lines 43-44 but the species these trunks belong to are not introduced with their latin names until lines 51-52. On lines 51-52, you say that African elephants "pinch" more than Asian elephants, but do not describe what that behaviour is until the following line. Providing the information required to interpret sentences before the current sentence will help your readers.
-	Line 68: "expected" - hypothesised?
-	Line 68: “the more simple is the task” – the simpler the task
-	Line 80: Delete "are"
-	Lines 82 & 83: "to" Beauval, should be "at"
-	Lines 113-114: "soil" - do you mean flooring?
-	Line 143: "this" should be "which"
-	Line 274: "od" – of
-	Lines 396-401: Please check these sentences as there seems to be a capital letter missing and odd placement of full stops.
-	Figures 3 & 4: Please display the full error bar (not just the top half) as it will aid in interpretation.
Experimental design: The research questions and the methods, including Table 1, are generally well described, although the English needs some work as detailed above. I commend the authors on their detailed collation of trunk behaviours in this study.

Minor comments:
-	Line 76: In what year?
-	Table 1: Shake - "stir" implies a circular movement - is that what you mean or do you mean side-side or up and down.
Validity of the findings: The only word of caution I would give on your otherwise excellent results is that you have measured occurrences and not the duration of behaviours, which means a behaviour of a long duration and a short duration are given equal weight. This could impact your results, particularly when considering individual variability, and should be acknowledged in your discussion section.

The conclusion and the abstract could do with a little bit of work to clearly link back to the aims and hypotheses, and to summarise the results. For example, in the conclusion, it would be great if you could add a sentence or two about your findings related to the diameter of branches and size of the food item.
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: TRANSCRIPTOME ANALYSIS PROVIDES A BLUEPRINT OF CORAL EGG AND SPERM FUNCTIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a timely and important study on gene expression in gametes of a reef building coral.  The methodology is sound as are the analyses.  The findings are interesting on their own, but have value in supporting much-needed research on molecular responses of different life-history stages of corals to environmental stressors.
Experimental design: The protocols are all appropriate to addressing the questions posed.  The collection and handling of gametes were properly performed.  The analytical techniques are sound as are the data analyzes.
Validity of the findings: At a time when coral reef throughout the world are in steep decline, understanding the mechanisms underlying documented problems, including in gamete quality, fertilization success and larval recruitment, is very important.  I like the use of the KEGG and GO databases in the analyses, as these provide important insight.  This is a well-designed and executed study.
Additional comments: My editorial suggestions are included in the manuscript for their consideration.


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: TRANSCRIPTOME ANALYSIS PROVIDES A BLUEPRINT OF CORAL EGG AND SPERM FUNCTIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: These are interesting data sets and should certainly be published in some form. However, I feel the authors have not completely found their story yet, and some analytical choices seem peculiar given the stated hypotheses. I looked at the authors’ rebuttal letter to the previous round of reviews and was not surprised to find that the scope and analyses had changed quite a bit from the original intent. That comes across in the manuscript in a few ways. My greatest concern is that some of the experimental design choices undermine the validity of the few conclusions that are made.

Major Concerns

1. There are so many sources of gene expression variation that weren’t accounted for. The sperm, adult, and egg samples were collected across different years (2015, 2016, and 2018, respectively), collected from different sets of genetically distinct colonies and then pooled from different numbers of colonies (I could be wrong here—the methods are quite vague regarding collection), processed very differently (e.g. some with Qiagen kits, others with Zymo kits), and run on different high-throughput sequencers with different sensitivities (MiSeq produced 30 million reads for sperm while HiSeq produced 125 million reads for eggs). I realize that pooling and genetic variation might not be a big deal in gamete samples, which are by their nature composed of millions of genetically distinct cells, and that normalizing to TPM should account for read depth differences. But when you think about how many sources of variation there are, it’s all the more surprising that eggs and sperm were found to express pretty much all the same gene pathways. I worry that this might be a bias stemming from 1) high variation within and among samples washing out the signal and 2) the somewhat arbitrary 100 TPM cutoff for detecting “expressed” genes.  Is it possible that only a subset of the most highly expressed house-keeping genes were retained after the filtering step, whereas many other important, variable, and somewhat more lowly-expressed genes were missed? I don’t know the answer—perhaps some sort of rarefaction analysis could be performed. I know its not that useful to say I have vague concerns about the validity of the results, but as this is the main conclusion of the study, I’m giving it more scrutiny and ultimately I think it needs more support.

2. The first line of the discussion reads “On the most general level, the egg and sperm of M. capitata each differ considerably in their gene expression when compared with RNA-seq data from adult cells but differ much less when compared with each other.” To me, that implies that three DEG analyses were performed: egg vs. adult, sperm vs. adult, and most importantly egg vs. sperm. But as far as I can tell this was not the case; there was no direct egg vs sperm comparison, and it really begs the question as to why. Instead the authors looked at overlap in DEGs from the sperm vs. adult contrast and the egg vs. adult contrast, but this set does not necessarily capture all of the genes that could have been differentially expressed between sperm vs. egg directly. For example, Gene A could be 2-fold upregulated in sperm relative to adults and 2,000-fold upregulated in eggs vs adults. Since they are both differentially upregulated relative to adults, they would both fall in the “shared” portion of the Venn diagram of Figure 2A. But if they were compared directly it would be obvious that their expression patterns were quite distinct, and lumping them as sharing an expression pattern would be an oversimplification. Again, eggs and sperm sharing patterns is pretty much the major conclusion of the paper, but it wasn’t tested directly. The authors either need to run that test or provide a very clear explanation as to why it doesn’t need to be done.

3. A full half of the discussion is devoted to speculation about the role of two genes in fertilization. It’s too much—I’d reduce this section down to two or three sentences, and perhaps move some of the material up to the introduction. Overall I think this is a minor finding and doesn’t have very much solid evidence to support it. If you make this change, there’s really very little discussion left. It’s an unbalanced paper in that respect, which is one of the reasons I don’t think the story has been fleshed out very well.
Validity of the findings: Please see "Experimental Design" section, since I covered "Validity" aspects in most comments.
Additional comments: Minor Concerns

4. I really don’t understand the value of Figure 2. It is meant to show similarities and differences in sperm/egg expression relative to adults, but it is restricted to only the top 10 most highly upregulated or downregulated genes, which may not be all that representative. I would rather see counts of the total number of upregulated or downregulated genes that are distinct or shared across sperm/eggs relative to adults. That would give a much more representative summary. I think one of the goals was to show that many of the top genes of interest weren’t annotated, but you don’t need a figure to get that point across.

5. Figure 1 looks very nice but I find it hard to interpret. The text suggests order matters but the caption doesn't provide details. Originally I thought a heatmap would be better but the text indicates there are no major differences. So what exactly is being highlighted? If it's just a list of shared expressed pathways, wouldn't a pathway map be a better visual representation?

6. In the methods, can you provide any details about the sperm/egg concentrations in the final samples? If they were maintained for a half hour in 1.5 mL tubes at very high concentrations, I worry about anoxia kicking in, causing stress, and potentially harmonizing expression among sperm and eggs. Although if that happened, I imagine we’d see more enrichment of stress-related genes. I’d also appreciate a clearer explanation for how sample pooling happened, and whether the three replicates were per sample, or only per treatment.

7. L295: Are the “egg up-regulated genes” inclusive or exclusive of the sperm up-regulated genes that overlap? Same with the down-regulated genes.

8. L296: I’d keep LF2C capitalized throughout the manuscript to be consistent.

9. L324-325: I’m having a hard time wrapping my head around whether the sperm/egg percentages of the exome are comparable to the adult percentage. Which TPM cutoff was used for the adult data set in this case, 100 or 60?

10. L332-334: Need to make clear that the DEG lists in Figure 2 are similar in identity but not necessarily similar in expression level (all you can infer is a similar direction). This is why I think it’s a bit disingenuous to say that sperm and egg have similar expression without testing it directly.


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: TRANSCRIPTOME ANALYSIS PROVIDES A BLUEPRINT OF CORAL EGG AND SPERM FUNCTIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors did an excellent job addressing my concerns. I was very glad to see that the new DEG results were consistent with the initial conclusions. With that extra level of support, I found the revised manuscript's story to be more cohesive and convincing than the original. Great work.


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

Input:
Title: GENDER-SPECIFIC ASSOCIATIONS BETWEEN APOLIPOPROTEIN A1 AND ARTERIAL STIFFNESS IN PATIENTS WITH NONALCOHOLIC FATTY LIVER DISEASE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is overall well-written and organized. Only the style of reference is not always homogeneous
Experimental design: I’ve read with attention the paper of that is potentially of interest. The background and aim of the study have been clearly defined. The methodology applied is adequately described and overall correct
Validity of the findings: The reviewer is not sure about the results obtained and discussed. Infact, it seems not biologically plausible that ApoAI is related to cardiovascular disease in general population, but related to a worse PWV (directly related to CVD risk!) in NAFLD patients. Otherwise, the authors limit their discussion to the fact that it is contrary to what known on ApoAI.
Additional comments: The authors should consider what raised on the validity of the findings


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: GENDER-SPECIFIC ASSOCIATIONS BETWEEN APOLIPOPROTEIN A1 AND ARTERIAL STIFFNESS IN PATIENTS WITH NONALCOHOLIC FATTY LIVER DISEASE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.

Literature references, sufficient field background/context provided.

Professional article structure, figures, tables. Raw data shared.

Self-contained with relevant results to hypotheses.

All the points mentioned above were taken into consideration by the authors.
Experimental design: Original primary research within Aims and Scope of the journal.

Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.

Rigorous investigation performed to a high technical & ethical standard.

Methods described with sufficient detail & information to replicate.

All the points mentioned above were taken into consideration by the authors.
Validity of the findings: Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.

All underlying data have been provided; they are robust, statistically sound, & controlled.

Conclusions are well stated, linked to original research question & limited to supporting results.

Speculation is welcome, but should be identified as such.

All the points mentioned above were taken into consideration by the authors.
Additional comments: 1. Original Submission

1.1. Recommendation

Minor Revision

Manuscript Number: 48102

Title: Gender-specific associations between apolipoprotein A1 and arterial stiffness in patients with nonalcoholic fatty liver disease

2. Comments to Authors:

2.1. Overview and general recommendation:

The aim of this manuscript was to elucidate potential biomarkers for the prevalence of CVD, its related complications and the role of the arterial stiffness in NAFLD patients. Their results showed that serum ApoA1 concentration was associated with arterial stiffness only in male NAFLD patients, unlike in Female NAFLD patients after controlling for multiple risk factors.
The manuscript is well written and original. The scientists have used relevant tools to assess NAFLD and arterial stiffness. Besides, they took into consideration lots of excluding criteria which make the findings more accurate and relevant. The methods section is pretty well described along with the statistical analysis. This work presents interesting and novel results pertaining to the importance of this subject.
This paper has a potential to be accepted, but some important points have to be take into consideration.

2.2. Minor Comments:

1)	It is unclear which stage of NAFLD/NASH authors are referring to, is it steatosis along with inflammation or fibrosis is there too. A good assessment would be serum AST/ALT ratio which is usually <1, but can increase as fibrosis advances. Patients can be divided into 2 groups AST/ALT ratio lower than 1 and a group with an AST/ALT ratio higher than 1. As such, they can correlate serum ApoA1 concentration with the specific NAFLD/NASH stage and it can become an important diagnostic tool linking fatty liver to arterial stiffness.

2)	The altered (Apo B/Apo A-I) has been suggested to be a good predictor of cardiovascular risk, even better than HDL/LDL ratio. Studies have shown that there is an increase (Apo B/Apo A-I) ratio among patients with NAFLD. Have the authors assessed these parameters to link them with arterial stiffness?

Thus, the paper can be accepted after addressing the above-mentioned 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: GENDER-SPECIFIC ASSOCIATIONS BETWEEN APOLIPOPROTEIN A1 AND ARTERIAL STIFFNESS IN PATIENTS WITH NONALCOHOLIC FATTY LIVER DISEASE
Review round: 1
Reviewer: 3

Output:
Basic reporting: PeerJ paper review:

The is a professionally written tight story that reveals interesting male-female discripances in the association between ApoA1 and arterial stiffness in NAFLD patients in China.

Thanks for referring to the studies that contradict with the present study within the discussion.

The authors use “sex” and “gender” terms within the manuscript. Example: the authors mention in the results section that data were corrected for sex, whereas the main finding related to gender differences. Please note that sex and gender should not be used interchangeably. NIH has distinct definitions for each of these terms. The authors should stick to the term that was tested in the current study.

The current study reports a male-female differenced in baPWV. What does that mean?

The authors bring up an interesting point to the discussion, which the potential male-female difference in ApoA1 distribution. Please expand the discussion to cover how that may provide an explanation for the present findings

In lines 70 and 71: ApoA1 is the major protein component of HDL. Please expand on this central concept for rationalization of the study.

Clarify whether data are presented as mean +/- standard error OR mean +/- standard deviation.

In the results section. Lines 146-199: values are mentioned without inclusion of their standard deviation or error. Please include +/- values within text as within the tables.

In the Discussion. Lines 193-195: how was this statistical comparison performed between the current finding and the previous one? The comparison should not rely only on comparing the mean values without consideration for the variability within the studies.

The English language should be revised to ensure comprehension of the reader (especially for the abtract). For example: there are some missing letters in the abstract page “stiffness, effects”

Abbreviations should be spelled out the first time they are mentioned within the manuscript (Examples include: NAFLD, baPWV, CV, CVD, DBP, SBP, HDL, LDL..)

Many words are written with a capitalized first letter, although this is not needed and they are witten in the middle of a sentence (this is very frequent across the manuscript. examples: Males, Females, Education, …
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: GENDER-SPECIFIC ASSOCIATIONS BETWEEN APOLIPOPROTEIN A1 AND ARTERIAL STIFFNESS IN PATIENTS WITH NONALCOHOLIC FATTY LIVER DISEASE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper is overall well-written and organized.
Experimental design: I’ve read with attention the paper of that is potentially of interest. The background and aim of the study have been clearly defined. The methodology applied is adequately described and overall correct
Validity of the findings: The authors have clarified the validity of their findings.
Additional comments: The authors have considered the reviewers’ suggestion and improved the paper accordingly. I’ve no further comments on it.


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: GENDER-SPECIFIC ASSOCIATIONS BETWEEN APOLIPOPROTEIN A1 AND ARTERIAL STIFFNESS IN PATIENTS WITH NONALCOHOLIC FATTY LIVER DISEASE
Review round: 2
Reviewer: 2

Output:
Basic reporting: all the criteria are met
Experimental design: all the criteria are met
Validity of the findings: all the criteria are met
Additional comments: 1. Original Submission: Recommendation: Accepted

Manuscript Number: 48102
Title: Gender-specific associations between apolipoprotein A1 and arterial stiffness in patients with nonalcoholic fatty liver disease

2. Comments to Authors:
The aim of this manuscript was to elucidate potential biomarkers for the prevalence of CVD, its related complications and the role of the arterial stiffness in NAFLD patients. Their results showed that serum ApoA1 concentration was associated with arterial stiffness only in male NAFLD patients, unlike in Female NAFLD patients after controlling for multiple risk factors.
This work presents interesting and novel results pertaining to the importance of this subject.

	The authors did provide a satisfactory answer regarding the AST/ALT ratio comment. Unfortunately, they do not have ApoB values available in their database currently but still the paper can be accepted without that parameter. In addition, they did handle well the crucial comments given by the other 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: SIGNIFICANCE OF HUB GENES AND IMMUNE CELL INFILTRATION IDENTIFIED BY BIOINFORMATICS ANALYSIS IN PELVIC ORGAN PROLAPSE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Zhao et al analyzed a published transcriptomic data set from GEO to select the most important genes (hub genes) having a possible essential role in Pelvic organ prolapse (POP), besides the authors discovered the immune cells infiltration and significant changes have been found during the disease compared to the healthy state. Not only Weighted Gene Co-expression Network Analysis (WGCNA) has been used to find the hub genes in the network but also RT-qPCR helped to validate the results.
The authors collected the most important articles in the topic, they published their work mostly in an understandable way. However, the language was not accurate and scientific enough in every case (and should be rewrite at some parts), this did not disrupt their work.
The figures and tables are numbered good, however the sequence of the figures is not satisfied. The quality of them is adequate, but I missed a general figure about the workflow which could help the interpretation of the work. Another general problem, that at some points the description is not detailed enough, it is difficult to understand the method, after rereading the section neither.
Experimental design: The research question has been well defined, looking for hub genes / potential biomarkers in POP is an essential step to identify and treat the early stage of the disease successfully. The authors collected the most relevant publications in this research area. The main gap has been identified and the written method tried to answer it. The bioinformatic analysis used various areas of biology (hierarchical clustering, WGCNA, GO and pathway analysis, CIBERSORT etc.), however not every step was understandable enough due to the lack of documentation (e.g. parameter description), therefore I suggest to expand the text with the provided information in the pdf.
Validity of the findings: The authors published a well-built pipeline to collect potential biomarkers from the published data set. Nonetheless, I have concerns about the unbalance regading the number of samples in different conditions (at the beginning it was 18 vs 16 but it could not be followed after removing the outlier samples), while by the CIBERSORT analysis these numbers became 4 vs 8.
In spite of that the workflow may contain mistakes, by the discussion part the authors explained that the found 8 genes could be highly related to the POP supported by the literature.
Additional comments: I would like to describe my comments in the PDF document.


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: SIGNIFICANCE OF HUB GENES AND IMMUNE CELL INFILTRATION IDENTIFIED BY BIOINFORMATICS ANALYSIS IN PELVIC ORGAN PROLAPSE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The writing Language need to be improved by professionals.
background, reference, and study design are meet the academic level.
Experimental design: the experimental design is insufficient.
Only one dataset involved in this study, which contained only 34 samples. The dataset is too small, more dataset is need to show the reliable of results.
And the authors need provide the search strategy, which means how this dataset was chose (including database, inclusion criteria, exclusion criteria, key words).
Which and how these analysis software used, the authors need described briefly.
Validity of the findings: The quality of sample in GSE12852 needs to be analyzed and described.
How the normal sample of GSE12852 used in data analysis, which need to be provided this study?
The authors described some RNA were extracted from POP samples? Where are the POP tissues from? Human？ Animal? There is no information about how to collect, where to collect the how the sample process. This part is very confused.
Additional comments: Some contents in the article is very confused, and cannot be understand. For example, line 45 “the incidence of the POP will be as high as 46% in 2050(Wu et al. 2009)”. Or line 59 ” complex network theory of high-throughput data emerges as the Times require”
Some details need to be provided when described for the 1st time, such as in the results part of abstract” WGCNA revealed that blue module”. The authors need to describe the blue module


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: SIGNIFICANCE OF HUB GENES AND IMMUNE CELL INFILTRATION IDENTIFIED BY BIOINFORMATICS ANALYSIS IN PELVIC ORGAN PROLAPSE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Zhao et al analyzed a published transcriptomic data and created a Weighted Gene Co-expression Network Analysis (WGCA) to identify hub genes which can have an effect on Pelvic organ prolapse. The updated version of the manuscript describes very well not only the disease itself with current statistics but the method part became much more understandable. The language is improved, I have found only a few typos/mistakes. The new workflow figure is appreciated, it helps to follow the analysis. The network figure (Figure 5) should be improved because the message of the network can not be seen (neither the name of the nodes). I would suggest to avoid the usage of the simple circle layout, rather grouping the proteins with high degree and arrange their connections around them. Figure 2 has a small mistake in the description: menopause state is assigned with 1 not with 0!
Experimental design: The research question has been well defined, the methodology is well-described. However, the definition of the final beta value ( I assume it is 7 based on the figures but it would be great to describe) is missing from the text and the hub gene module identification paragraph is a bit confusing (why ME and not MS has been used for the selection of hub module). In the functional enrichment, the background dataset is still not good. Finally, the limitation of CIBERSORT algorithm using the described samples was not mentioned in the text.
Validity of the findings: The outcome of the whole pipeline was a list of 8 hub genes which were validated by RT-qPCR. With the extended description of methods/parameters, I do not have any concern regarding the results.
Additional comments: PDF is attached containing the 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: CMTM6 SIGNIFICANTLY RELATES TO PD-L1 AND PREDICTS THE PROGNOSIS OF GASTRIC CANCER PATIENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: 1.The authors mentioned IHC score in the Method section. Was this score used for the assessment of CMTM6 expression?

2.The authors classified low and high expression of CMTM6. How did you classify the CMTM6 expression? The authors should showed the cut-off value of CMTM6 expression in the method section.

3. The authors mentioned that CMTM6 is co-localizes with PD-L1 at the plasma membrane in the Discussion section. However, the authors did not show this phenomenon. The author should showed the merge of immunofluorescence image of CMTM6 and PD-L1.

4. In the Discussion section, the authors mentioned that  Recent research shows that CMTM6 displays132 specificity for PD-L1 and maintains its cell surface expression. CMTM6 depletion decreases PD-L1 without 133 compromising the cell surface expression of MHC class I and inhibits T cell activation and anti-tumor 134 responses in both in vitro and in vivo [15]. These sentences were already mentioned in the Introduction section. The authors should delete these sentences from the Discussion section.

5. In the Discussion section, the authors mentioned that the low expression of CMTM6 also increased the prognostic value of PD-L1 expression. This sentence give misunderstanding that low CMTM6 expression increased PD-L1 expression. The author should revise this sentence without misunderstanding.


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: CMTM6 SIGNIFICANTLY RELATES TO PD-L1 AND PREDICTS THE PROGNOSIS OF GASTRIC CANCER PATIENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: 1. Standardization of the new immunohistochemical protocol is very critical. It is better to use widely used antibodies for immunohistochemical study. Previous studies using Novus Biologicals (NBP1-31183, United States) and Biolaboratories (ARG57681, Taiwan, China) antibodies should be cited and positive controls also added.
Validity of the findings: 1. Gastric cancer is mainly classified according to Lauren classification. The author should add Lauren classification and perform comparative analysis with CMTM6 expression.
2. In table2, the TNM stage does not affect prognosis. However, in general, the TNM stage is the most important prognostic factor, and it is difficult to understand that TNM tages I and III do not differ in prognosis. Therefore, the authors need to describe what selection bias they had when collecting samples.
3. The author should describe when the patient was operated and when the sample was taken.
4. In lane 115, the author said that the cut-off level for CMTM6 expression was determined using X-tile software. It is necessary to describe in detail how the cutoff is determined by X-tile software and accurately describe the cutoff value. A detailed explanation in Figure 2A is also needed.
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: CMTM6 SIGNIFICANTLY RELATES TO PD-L1 AND PREDICTS THE PROGNOSIS OF GASTRIC CANCER PATIENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: The manuscript was carefully revised according to my suggestion.
Validity of the findings: The manuscript was carefully revised according to my suggestion.
Additional comments: no comment


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

Input:
Title: MICROBIOME DYNAMICS IN THE TISSUE AND MUCUS OF ACROPORID CORALS DIFFER IN RELATION TO HOST AND ENVIRONMENTAL PARAMETERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper could use revision for grammar and better use of the active voice over the passive voice.
Not all sections are adequately referenced.
Further details are provided below in the general comments.
Experimental design: Some of the research questions were well defined, while others were intentionally vague, especially when drawing inferences of cause and effect which were not correct for this experimental design. Not all methods are described with sufficient detail. Further details are provided below in the general comments.
Validity of the findings: Many of the findings are presented as direct cause and effect relationships which is too far of an over-reach with the given data. Further details are provided below in the general comments.
Additional comments: This paper provides some useful but incremental information to the growing field of coral microbiome interactions. The largest weakness of the paper is that the authors swing back and forth from discussing possible correlations between microbial diversity patterns to possible external and internal variables and how external variables act as “controls,” “drivers,” “responses,” or “affects” to the microbial diversity. While testing for possible correlations between microbiome diversity and other variables are germane here, the authors must remove all language that implies any sort of cause and effect relationship, as they have no evidence for this, nor did they specifically have a proper experimental design to make such determinations.

Many of their correlations are weak and the variables chosen are so limited that the authors cannot make many solid conclusions as to how many of these variables influence the patterns in microbial diversity. In summary, the authors can certainly address their first hypothesis (microbial composition will vary based on compartment). While the relationships between the mucus layer and the overlying water column are germane (but perhaps not fully supported), the discussions concerning the tissue microbial community and “changes in the physiology of the host” are exceptionally weak and not justified without better data and a much more sophisticated controlled study.

Other issues:
The concepts of alpha and beta diversity are not mentioned in the introduction clearly, so the context of why they are germane for this paper is weak.

There is confusion in how some terms are used which makes it difficult to understand what the authors are referring to. For example, chlorophyll a was sampled from the algal symbionts in the corals but was also sampled from the water column above the corals. The authors use several terms (and abbreviations) and it was not always clear which pigment data they were referring to in the paper.

The entire paper could use some tightening and better use of the active voice over the passive voice. I have provided a few examples below.

Abstract
-Line 37: Rather than calling these “environmental” factors, could you choose a better word? Perhaps “water column parameters”?
-Line 38: can you really say these were “drivers”?
-Line 39–41: This is far too weak and requires some specificity so the reader can understand the significance (or lack thereof) of this aspect of the study.
-The last sentence of the abstract seems redundant.

Introduction:
-Line 47: Remove “eukaryotic” as this term is redundant since all dinoflagellates are eukaryotic.
-Line 51: change, “can act in defense” to “defends”
-Line 52: There are several examples in the text where the active voice should replace the passive voice. This will also help to lower the word count and to increase readability. For example, the ending sentence on this line should be simplified to: “The coral microbiome is influenced by…” Please pay attention to extensive overuse of the verb to be throughout the paper.
-Line 67: Remove “animal” as we know that the coral is an animal.
-Line 73: Can you really say this information is crucial toward understanding the symbioses or is it more general than this since not all consortia of the microbiome are necessarily “symbiotic”?
-Line 83: I believe the correct grammatical term here would be “Symbiodiniacid” since the family name is in the plural.
-Line 95: simply saying “dinoflagellate” or “algal” here sounds much better than using the family name. Again, you are using a plural followed by another plural word and this is not grammatically correct. A comma should go after “cells”.

Methods:
-More details are needed on the water and sediment samples and how they were collected. At the very least, some citations should be provided here. Simply stating that you took these samples “according to standard procedures of AIMS” with no citations is not sufficient for another person to replicate your study.
-Line 165: This section is missing an appropriate reference for the chlorophyll a extraction and quantification protocol.
-Line 178: Please specify that these are “Soluble” proteins.
Please remove the last sentence in this section, as this is redundant and assumed for the protocol.
-Line 198: Please state the number of independent hemocytometer loadings as the technical replicates here for your counts. Providing the number of squares is not as informative.
-Line: 206: I think you mean a Venn diagram was “constructed” 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: MICROBIOME DYNAMICS IN THE TISSUE AND MUCUS OF ACROPORID CORALS DIFFER IN RELATION TO HOST AND ENVIRONMENTAL PARAMETERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I very much enjoyed reading this work by Marchiroro/Glasl et al. It was easy to read, had interesting visuals and the results were compelling and interesting. Overall it is a nice piece of microbiome science that adds to our knowledge about how the portions and key taxa respond to individual variables such as nutrient concentrations and/or particulate carbon. This aspect of the work makes it novel and insightful.

There are a few issues with the text and visualizations that need to be conducted to make the work acceptable for publication.

1. For the visualizations I found it strange that despite having ‘season’ being a primary variable in the experimental design, none of the visualizations have samples split by season. If the authors can prove that season was not a main effect I might understand this, but based just on the ANOVA richness data (p=0.053….think about what this really means in the context of your design) I suspect season has an interaction that limits your interpretation of some of the other factors driving changes in your community composition and individual groups of taxa changes.  So please revised your visualizations (Figs. 1, 2, 3) to include from which season the samples came.

2. Further, while I always like bubble charts use of mean relative abundance in figure 1 does not show the large amount of variation of the taxa across the different sample categories. For full transparency you need to revise this to show how certain taxa are present in a wide range of abundances despite being in similar variable categories (e.g., host, season, compartment). For example, in your descriptions the Endos exhibit a very large range of variation but the reader can’t tell what the variation is being driven by (see Lines 271-273). If you use a box plot or even just show every sample in clusters on the bubble chart we’d be able to see this more completely to really access what is going on here.

3. Also Figure 2 is both underwhelming and somewhat inaccurate. You can make Venn diagrams scale so that the overlap of each category represents the relative number of similarities. Thus in your diagram there would be a very large overlap in mucus and seawater and little overlap in tissue and seawater….which I believe is the point of this figure. Right?

4. For Figure 4, I think this would be easy to follow if you removed the legend and put headers above and on the left side to indicate if the figure was tissue or mucus or from A tenuis or A millepora. Lastly my favorite part of this paper is your last figure. Again showing the variation might be nice.  You can use a heat map style for this which would add more columns arranged by sample type (essentially you’d show all three of each sample category instead of the mean). To simplify it you could also only show the significantly different taxa.
Experimental design: The major flaw of this paper is its experimental design, although I don’t think that this warrants a rejection. The authors, despite the low replication for each compartment/species/time (n=3) still find important positive results that are interesting, novel, and worthy of dissemination. However, I think the authors should be wary of making strong statements concerning the negative results they find. For example, the differences between significant tests for the A. millepora and A. tenuis maybe due to lower sample sizes (lines 244-245) in one host. The low power of the experiment may also be driving the absence of differences in seasonality which is a unexpected and somewhat surprising finding that raises some red flags. To this point, the authors state in the richness section that ‘season’ was not a significant factor, but then they list a p value of 0.053.  Now that’s pretty darn close, begging the question of whether this ‘negative’ result was due to the small number of samples. As you will see in the section on validity of the findings, I think the use of a GLM would help in this regard. You may see more clear main and secondary effects of difference variables, with season, I imagine, being a significant interactor. But currently it’s difficult to tell because the data are not visualized this way.
Validity of the findings: I have some questions about the microbiome analysis.
1.	Why did you choose to use primers that are no longer standard?  The V4-V5 (515-806) has become the most widely used and thus databases are likely to contain more taxonomic information using this region instead of the one you chose 27F-515.  I think a description for the rational for this is important.
2.	Line 148.  Does this mean that you removed any sequences with one or more N’s or that you limited to those with more than a defined number of N’s? If the later please state the number of N’s that was permissible.
3.	Did you ensure that when you generated the zOTUs/ASVs table that you trimmed to the identical sequence length to ensure that additional bases were not considered additional zOTUs? If not…you must do this. If yes please state this.
4.	Why did you use an ANOVA for your diversity metrics? Most microbiome data is not normal and unless a transformation was done (none was mentioned) it is unlikely that your data will meet the assumptions of the tests. Further, in our experience GLMs perform better (aka better residuals) than an ANOVA for coral microbiome data (see Maher et al., Scientific Reports 2019 as an example). I recommend that you consider this approach in place of the ANOVAs when comparing your richness data.
5.	Line 215. I did not know that these normalizations were “common.” In fact I suspect that they are not really standard. Please further defend the use of these as they are essential to your data analysis and interpretation. Also please cite additional literature other than your own paper regarding these normalizations.
6.	Did you use a multiple comparisons correction for your composition data analysis?
Additional comments: I really enjoyed reviewing this paper and think that with some edits it could be a really nice contribution to our knowledge about what drives variation within and between coral microbiomes. However, I feel like the emphasis on the finding that the tissue compartment is less sensitive to the environment is not very compelling. Several papers have done this and with much better datasets to confirm it, including our own that used over 650 samples. In fact when I first read the title I kind of groaned cause I felt like this was a settled topic, and I didn’t want to review another paper on this topic. But when I read the paper I found that it was much more compelling than the title and abstract let on. Basically I think you undersell your work here. Importantly the novelty of your work is in the linkages between shifts in community members with different environmental factors that you measured. These findings have important implications in management and restoration implications and thus should be highlighted more completely.

Minor comments:

Line 91. Prokaryote? Are we still using this word? I think given the recent revisions to the tree of life, this term needs to be retired completely. Please use bacteria and archaea if that is what you mean.

Line 91. In our Pollock et al., paper we actually found that when you look across phylogenetically diverse coral hosts, the mucus is actually less diverse than tissue across the board!

Line 97-99. I would revise this hypothesis as it has been addressed several times before and is not the most compelling hypothesis you could come up with based on your introduction. This is not a requirement, it’s a stylist thing, but one I think would elevate your work.

Line 116. What size were the nubbins?

Line 121. I’m confused. Where the nubbins and sterivex snap frozen or just the sterivex?

Line 141. Again you need to address why you used these primers and not the Prada/Apprill et al., ones.

Line 206. zOTUS?

Line 248. Did you look at richness at any other level of taxonomic classification (e.g., genus?). Also did you look at phylogenetic diversity?  Seems appropriate.

Line 256-257. I am a tad confused by this statement. Do you mean that species x time and water x time had no effect on the community?  Or do you mean that when you look at all samples there was no effect? Seems unlikely that the water did not change across time. Again maybe using a GLM would clarify these main and interacting effects.

Lines 289-321. I really like this section and think you could expand on it to increase the impact of the work. Maybe some of the supplements could be brought to the main text and that Venn Diagram get moved to the supp.

Line 338-342. These are very cool findings! You don’t talk about it at all in the discussion! You maybe want to elaborate.


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: MICROBIOME DYNAMICS IN THE TISSUE AND MUCUS OF ACROPORID CORALS DIFFER IN RELATION TO HOST AND ENVIRONMENTAL PARAMETERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Overall, this paper has been improved. I still feel as though the link between the microbial community and the coral "physiology" is tenuous, and the authors could do a better job framing this. For example, why / how does the Chl a. level of the symbiotic algae relate to the bacteria? There is probably some interesting hypotheses out there if one digs into the phytoplankton literature and how other microalgae (perhaps dinoflagellates that are notoriously mixotrophic) live with some forms of bacteria. I have tried to offer other suggestions below as well. I am also not happy with some of the language that still reaches a bit too far and is assigning causation with you really need to adhere to the correlations and let them speak for themselves, and I have suggested alternative phrases.
Experimental design: no comment, please see section 4.
Validity of the findings: no comment, please see section 4 and my comments above in section 1.
Additional comments: Last sentence of the abstract is a bit vague. It would be nice if you could quickly encapsulate a few of the "novel insights" "implications" for restoration or management that you allude to here. Critically, you really do not ever tell the reader how exactly your results fit this point later in the paper either.

Line 322: remove "significantly"

Line 357: I would add "in the Caribbean" after "coral species"

Line 395: remove "have previously been shown to"

Line 397: for "were associated with Symbiodiniaceae in the genus Durusdinium," remove "Symbiodiniaceae in the genus"

Line 398: change "was associate to the genus" to "harbored" and add .sp following Cladocopium.

Line 399: Remove the sentence that begins, "These corresponded…"

Line 405: change "structured" to "correlated to"

Line 413: change "were concomitant" to "corresponded"

Line 425: add "the" between "study," and "abundances" and drop the s in abundances.

Line 449 and 450, I am still not in favor of using words like "responded" here since you did not test for a response per se. I would stick to "corresponded" or "correlated"

Line 455–460: It may be useful here to be more explicit about this relationship and perhaps the different symbiotic algae are contributing different kinds and levels of organic C and N that may also affect your microbiome here.

Line 474: This seems like a good place for a paragraph break.

Line 477: The paper by Da-Anoy and colleagues is a spurious argument, as multiple studies are now documenting physiological variability among different genotypes of the same algal species. Dig deeper and you will find examples across different strains of Breviolum minutum, Durusdinium trenchii and others. Please do not assume that having the same ITS2 sequence means you are working with only one genotype of alga, as this would be incorrect. This also weakens your point here.

Line 485: What if what you are calling total suspended solids also include particulate organic matter (POM). There is an point to be made that considerably more work is needed in comparing coral trophic dymanics to their microbial consortia here.

Line 500: change "responses" to "patterns".

Line 505: change "we begin to disentangle" to "this study has identified several intrinsic and extrinsic factors that contribute…"


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: DELIMITING THE BOUNDARIES OF SESAMOID IDENTITIES UNDER THE NETWORK THEORY FRAMEWORK
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors present an interesting comparison of the many sesamoid bones in an anuran skeleton. They considered the topological place of sesamoids within the context of the entire skeleton, finding that these skeletal or cartilaginous elements show some connectivity features that set them apart from other skeletal elements.

The introduction and discussion provide adequate context for the study. If anything, I would suggest citing the work of Étienne Geoffroy Saint-Hilaire, who proposed the principle of connections as a rule to identify same structures in different organisms (i.e. homology in modern language) instead of Gutman & Esteve-Altava (2014) in Line 349.

I would suggest revising the writing style to make some ideas clearer. At times, there are unnecessarily complicated sentences. For example, [line 317] “Joints impose spatial neighborhood relationships between the skeletal pieces”, where I guess the meaning is ‘Joints connect nearby skeletal pieces’. Or [line 319] “Sesamoids as skeletal pieces that are, in general terms, characterized by not being articulated to other pieces of the skeleton,” which either is missing some verb (?) or would mean ‘Sesamoids are skeletal pieces that do not articulate to other skeletal pieces’.
Experimental design: The driving hypothesis is interesting and justifies using a network-based analysis. However, I have some concerns about the specific questions: 1b [line 87] “Is it possible to identify anatomical modules in the network?” and the cross-testing of 2a [89] and 2b [91].

Regarding question 1b. The community detection algorithm used will find modules even when a modular partition makes no sense or is not ‘truly’ present, such as in a random network or in a regular network. For example, if one uses this algorithm on a regular ring network of 10 nodes, cluster_optimal(make_ring(10)), it produces an output with 3 modules and a fairly good modularity quality value (Q=0.36). Because of that, we know a priori that the answer to this question is yes.

There are other community detection methods that will provide a better answer to this question, for example, OSLOM (http://www.oslom.org/index.html). This method has the potential to unveil also non-modular networks, as well as other features that would be interesting evaluating here, for example, if sesamoids contribute to the overall modularity of the skeletal network or are singletons without contribution. References to this algorithm are available it the software webpage. An example of its use and interpretation in anatomical networks is available in https://doi.org/10.1002/jmor.20690.

If authors decide to use algorithms  based on maximizing modularity Q value (like cluster_optimal), then it would be necessary to estimate the error of Q to know if the parition is of enough quality. This can be done using a jackknife procedure where each connection is treated as an independent observation. Details about estimating Q error are available in https://doi.org/10.1002/jmor.20690 or https://doi.org/10.1103/PhysRevE.69.026113

Regarding questions 2a and 2b. The authors test whether non-sesamoid and sesamoid bones (on the one hand) and non-sesamoid vs embedded sesamoids vs glided sesamoid (on the other hand) show different values for network parameters. I have different concerns here with the statistical testing. The first one is that the testing of eigen-centrality is omitted (Supplemental Table S2), but I suppose that they also tested it. Why are eigen-centrality tests not shown? The second one is about the type of tests selected (Kruskal-Wallis tests and Wilcoxon). The authors do not justify the reason to mix both types of tests. Why not a partially nested ANOVA? Moreover, because multiple tests are performed (on various overlapping groups and for 3-4 variables each), I believe a correction for multiple testing  is necessary (e.g. Bonferroni or similar). I suggest that this part of the study is evaluated by a professional statistician.

Regarding the building of the anatomical network. It is not clear why the authors decided to [line 98] “built an adjacency matrix considering the whole skeleton of the right half of the body” only. Because they are including many skeletal elements aligned in the midline of the body plan (e.g. crania, vertebra), making only a network of half the body has a big impact on the output of the analysis. For example, midline elements will be half important (half central) if we only analyze half the body. This can also impact modularity results: as the left-right axis is omitted, modularity will emphasize the A-P axis. What is the reason to make a network of half the skeleton and not of the whole skeleton?

Finally, I found a problem in the R script for the analysis that may have had a substantial impact in the measurement of some network variables. The authors load their raw edge list data from an .csv containing all the connections between elements. Some of these pairs of elements have more than 1 connection of a different type (e.g. V3 and V4 have an articulation contact and a muscular contact). Then, they used the function graph.edgelist to create the main network for analysis. However, this network is not simple and contains multiple links (this can be checked with function is_simple). Multiple links have an impact on the quantification of parameters density, degree, betweenness centrality, and eigen centrality. For example, edge_density function (to calculate density) compares actual number of connections with the maximum theoretically possible; however, if multiple links are allowed, then the maximum becomes infinite. Consequently, density is overestimated. Some of the functions used to calculate other parameters may also have similar problems.

The details in the Methods section suggest that the authors are not aware of this issue. For example, in line 110, they state that “A weighted version of the network serves as a simpler graphical display in figure 1”. I guess this network weights come from the multiple links (2 links = weight value of 2).

To solve this problem, I suggest either working with the simplified network without multi-links or using the weighted version of the network explicitly. Both actions will change the measurements for some of the parameters. There is a third way. Authors could acknowledge that they are using a multigraph and analyze it accordingly with specific algorithms for this type of networks.
Validity of the findings: Because of the methodological problems mentioned, I am not certain whether the findings of this study will hold.
Additional comments: This study addresses very interesting questions: (1) what is the topological nature of sesamoids? And (2) what is their impact on the organization of the skeleton? An anatomical network analysis is the right tool to answer these questions. However, to answer these questions properly, the authors need to address important methodological issues regarding the building of the network and the algorithms used to analyzed it.


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: DELIMITING THE BOUNDARIES OF SESAMOID IDENTITIES UNDER THE NETWORK THEORY FRAMEWORK
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	More context is needed in the intro. Why are anurans a good system to study sesamoids (73-81)? “A driving hypothesis of our work was that sesamoids' nature relies profoundly on their connections” (lines 83-84) – what evidence supports this hypothesis?
2.	In discussion, compare with results of other network analyses that included sesamoids, e.g. Diogo et al. 2015
3.	371-372: “Beyond their intrinsic morphological properties, connectivity patterns incorporate key aspects of sesamoid identity.” This statement (and others elsewhere in the ms) implies that connectivity could be useful in identifying and/or defining sesamoids, but this idea is never explicitly stated.
4.	357-358: Are the authors arguing that glide sesamoids are not sesamoids? Please clarify.
5.	The article is clearly structured and the figures and underlying data make sense. However, the language requires editing throughout to correct grammatical mistakes and improve clarity. In particular, parts of the introduction and discussion are very difficult to follow (e.g., 317-325).
Experimental design: 1.	The investigation seems to be technically valid and well done, and the methods are described sufficiently. However, I have a serious concern about the choice of methodology, which treats muscles as links (lines 106-107). As far as I’m aware, the existing literature treats muscles as nodes, not links (e.g. Diogo et al. 2018, Molnar et al. 2017 – cited in the current ms) or does not consider them at all (e.g., Esteve-Altava et al. 2013). This may be a perfectly valid choice, but there is no explanation or justification offered. Furthermore, this difference must be taken into account when comparing the results with previous studies that used different methods (e.g., lines 241, 245).
2.	86-87: Research questions 1a and 1b do not really add anything to the study
Validity of the findings: 1.	The conclusions are well supported by the data. I do have several suggestions about how the conclusions are stated: first, I think there are too many conclusions and that it would be better to number them than use bullet points. I would remove points 1 and 3 and combine points 5 and 6. Also make sure they are clearly linked to the original research question.
2.	Line 368: “antero-posterior differentiation along the vertebral axis” is a confusing phrase. Do you mean differences in network structure between the pectoral and pelvic limbs?
Additional comments: The manuscript describes an anatomical network model of the (postcranial) musculoskeletal system of the frog Leptodactylus latinasus and its implications for the evolution of sesamoids. L. latinasus has an unusually large number of sesamoids in the hindlimb compared to other tetrapods. The study found that sesamoids, particularly gliding sesamoids, have low connectivity, possibly making them more evolutionarily labile. Exploring sesamoids from a network perspective is an interesting idea, but I have concerns about the methodology and I feel that more context and clarity are needed in the introduction and discussion.


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: DELIMITING THE BOUNDARIES OF SESAMOID IDENTITIES UNDER THE NETWORK THEORY FRAMEWORK
Review round: 1
Reviewer: 3

Output:
Basic reporting: •	Clear and unambiguous, professional English used throughout.

Although I am not completely competent to criticize the use of English, from my experience it seems to me to be fine, without apparent deep grammatical errors that make reading unintelligible.

•	Literature references, sufficient field background/context provided.

Yes, very good literatue with a contest and suffiicent background.

•	Professional article structure, figures, tables.

It seems to me a well written paper. The parts it is made of, are clearly structured, easy to follow. In general, then, it seems to me a pleasant and very interesting article, with great possibilities of being imrpoved, both in its results, and in both its discussion and conclusions (see below my observations on this matter). Figures and tables are clear.
Experimental design: •	Original primary research within Aims and Scope of the journal.

Yes.
•	Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.

Yes. The questions they pose are relevants. In fact, it is interesting that they pose many questions in an explicit way.

These are the questions they posed:

Can embedded and glide sesamoids, be defined under the same criteria?:
I did not see the answer to this question in the text

1.a) How is the network structured?:

 Answered in the lines 160 y 166.

1.b) Is it possible to identify anatomical modules in the network?:

Answered in the lines 169 y 178.

1.c) Is the antero-posterior differentiation of the anuran skeleton reflected in their element connectivity pattern?:
 Answered in the Modularity and sub.networks comparisons items

and the last ones relating to sesamoid interactions within the network: Answered in the sesamoid patterns item

2.a) Do sesamoids share common network properties that make them distinguishable from other skeletal pieces?: Answered in the sesamoid patterns item

2.b) Do embedded and glide sesamoids differ in network parameters? Answered in the sesamoid patterns item, and in the last conclusion.

•	Rigorous investigation performed to a high technical & ethical standard.

Yes

•	Methods described with sufficient detail & information to replicate.

Although there is not, strictly speaking, an experimental design, there is a methodology of how they did to develop the modules and anatomical networks. However, I still have the question of whether they only used the R package to build both the modules and the anatomical networks, or they also used, for example, a main components analysis (PCA), in addition to other statistical packages that were not explicit in the methodology. In summary, I think the methods for these anatomical network and module constructions should be more detailed (see, for example: Evolutionary parallelisms of pectoral and pelvic network-anatomy from fins to limbs. Science Advances  08 May 2019: Vol. 5, no. 5, eaau7459 DOI: 10.1126/sciadv.aau7459).
Validity of the findings: •	Impact and novelty not assessed.

I think there is not enough evaluation of the impacts and news generated by this study. For example, in developmental biology and systematics. Although they include something, they could be expanded This article shows that there are cases when a clear independence of the characters cannot be shown. The study shows that there is interdependence between sesamoid elements in the construction of modules and anatomical networks. We see it when they say "sesamoids nature relies deeply on their connections". Where is the proof of this hypothesis? Where's your answer? What would be its systematic application, even if it is from a functional point of view?
Although it is based on a species, could these modules and networks be extrapolated to other species of anurans, or can it only be per species?
It would be possible to make comparisons with studies in mammals and reptiles, for example, and even humans?

•	All underlying data have been provided
Yes.  Maybe a short description of the sesamoids is missing, taking into account that they affirm the importance of their morphology for the construction of networks and modules.
Although in Ponssa et al.(2010) there is a description of sesamoids in this species, I think it would be important to highlight here, in a synthetic way, the fundamental elements of this description that are the basis for the construction of modules and networks. I say this taking into account that, in line 34, they stated that “The loose connectivity pattern of sesamoids could be related to their evolvability, which in turn seems to be reflected in their morphological diversity and their facultative expression”. How does morphological diversity explicitly influence these constructions?

•	All underlying data are robust, statistically sound, & controlled.

Apparently yes, but they need to deep in the methodology about both the modules and antomical networks construction.
•	Conclusions are well stated, linked to original research question & limited to supporting results.

Yes, they are well stated and linked to the research. However,
I cannot see where this conclusion comes: “These results support the role of sesamoids as morphological innovations generators”.

I think that they need to respond in the conclusions to the questions and hypotheses they did in the introduction. Although the answers are included in t he discussion, I think they should be made explicit in the conclusions.
Additional comments: The study of the sesamoid elements is still very important, both from a morphological and functional point of view. However, little has been explored compared to other parts of the skeleton of tetrapod vertebrates, such as the skull, spine, or limbs. As they are structures that are mostly considered floating, sometimes difficult to observe, and apparently distributed without patterns, they give the impression that they are of little use in solving biological questions. I think that studies like this, in which it puts a different context for its application in biology, should be used much more to try to solve problems of kinship relationships, of the evolution of frogs, of their biomechanics, of the biology of development, etc. I hope the authors maintain this line of research.


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: DELIMITING THE BOUNDARIES OF SESAMOID IDENTITIES UNDER THE NETWORK THEORY FRAMEWORK
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made a thoughtful revision of the analyses in first manuscript following all my suggestions. I am fully satisfied with the results and I have no further comments.
Experimental design: In my opinion the new, revised analyses are sound and make the overall results stronger. I do not have further comments.
Validity of the findings: The now revised results are more compelling. The fact that they do not change substantially after improving the methods mean that the authors were already into something with a real biological meaning. Now, better supported in the revised version. I have no further comments.
Additional comments: I congratulate the authors on a very thoughtful and through revision of the methods and analyses used. I rarely see authors acknowledging and amending these problems when they are made aware of them. Also, the manuscript now is much easy to read and figures look nicer (specially Figure 1).