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 OF OVARY CULTURE-INDUCED EMBRYOGENESIS IN CUCUMBER (CUCUMIS SATIVUS L.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript entitled “Analysis of transcriptomic regulation mechanisms underlying cucumber (Cucumis sativus L.) embryogenesis during ovary culture” by Deng and co-authors, reports on the types of genes differentially expressed in key steps of plant regeneration from ovary culture in Cucumber.
The study is a transcriptomic analysis by RNAseq. The text is clear, and mostly in professional language. However, some sentences would benefit to be rephrased, such as in lines 71-74 and lines 99-100.
Conventional writing is sometimes missing as the names of proteins should be in upper cases. For example, this is the case in lines 88-89.
Background information is sufficiently included. Literature is mostly provided, maybe a bit old sometimes. In some cases, references are missing. For example, the relationship between microtubules and zygote development (line 429) could benefit from referring to:
Kimata Y, Higaki T, Kawashima T, et al. Cytoskeleton dynamics control the first asymmetric cell
division in Arabidopsis zygote. Proc Natl Acad Sci U S A. 2016;113(49):14157–14162. doi:10.1073/pnas.1613979113

The involved of WUS in shoot formation has been published in connection to cytokinin signalling (line 475). Some references (not exclusive):
Dai, X, Liu, Z, Qiao, M, Li, J, Li, S, Xiang, F ( 2017) ARR12 promotes de novo shoot regeneration in Arabidopsis thaliana via activation of WUSCHEL expression. J Integr Plant Biol 59: 747– 758
or
Meng, W. et al., 2017. Type-B ARABIDOPSIS RESPONSE REGULATORs Is Critical to the Specification of Shoot Stem Cell Niche by Dual Regulation of WUSCHEL. The Plant cell, 29(6), pp.1357–1372. Available at: http://www.plantcell.org/content/29/6/1357.long

It would be good if the authors may look into this connection in their study.

The article is properly structured with figures and tables of good quality. I could find the raw data of the RNAseq (I was looking for them to look at genes in auxin production). And I would miss a figure (something like a matrix graph as in Figure 5) compiling the results on transcription factors. Those are discussed in the discussion part but not in the results.
Conclusion of the article is overwise supported by the results.
Experimental design: This manuscript fits the Aims and Scope of the journal as original primary research in Biological Sciences.
The research question is well defined: what groups of genes are differentially expressed during the different steps of plant regeneration from ovary culture in cucumber. It aims at helping to improve the efficiency of the process by understanding the molecular mechanisms during each step. The gap in knowledge is here clearly established. However, the explanation in lines 73-74 why microspore-derived embryogenesis may not be a good method: is it true for cucumber?
However, the abstract would appreciate rephrasing. For example, you selected six key time points in three stages. A clarification would be required for the reader what these time points are. Also, the data are presented as a differential expression between successive time points. Then a very little emphasis is made in the abstract about the cytological analysis.
May I ask what is pre-embryo (abstract and introduction)?
This could be connected with my previous commented. Here no methods are provided for these results: what staining used, what microscope, live or fixed material, etc.
In the methods part, there are also missing details about
-	amount of starting material for RNA isolation
-	Method of RNA isolation (Trizol and then what?)
-	RNAseq library preparation,
-	Number of replicates for each sample
-	RNA QC check,
-	qPCR primer efficiency
-	RNA for qPCR: from freshly collected samples or from the same RNA?
-	qPCR: How many biological replicates? Technical replicates? Was it performed any statistical analysis?
The ovary culture method is presented as unpublished (line 123). If the policy of the journal is that a ready should be able to reproduce the presented data, the in vitro ovary culture method would need to be provided in the method part with sufficient details: renewal of the culture medium (timeframe?), any change in composition during each step (same medium from the ovary at T0 and germinating embryo with a shoot, in term of hormone composition for example?)
Validity of the findings: The validity of the results appears correct. However, I could not access the raw data to look for a specific pathway of interest, as I was missing them from the figures (see below). The results are interesting and will be to improve the culture of the ovary and plant regeneration.
RT-qPCR results are missing a solid statistical analysis, especially that they are compared to the results of RNAseq. The authors claim the RNAseq and qPCR results are consistent (line 239). Figure 2 (as it stands) lacks to demonstrate it for all the presented genes. Could it be an idea to use a double y-axis (one for DE in RNAseq and on for fold-change in qPCR)?
Concerning the results, please try to be accurate in referring to the genes in the text. For example,
-	line 264: “cytokinin transfer protein” is, in fact, Histidine phosphotransferase protein;
-	line 265: “cytokinin response proteins” are response regulators (RR)
-	line 267: “ethylene-binding proteins” are EIN3 binding (F-box) proteins (EBF)
-	line 268: “ethylene-responsive protein transcription factors” are ERF
And you mention nothing about auxin production (YUCCA, TAA1, others), auxin signalling (TIR1, ARF, Aux/IAA) or cytokinin production (LOG, CKX), despite the presence of a KEGG Zeatin biosynthesis enrichment.
In addition, transcription factors are a big part of the discussion, not they are not analyzed in the results. An analysis with a matrix graph would be welcome. It may also help to understand the process of plant regeneration.
Finally, in the discussion (around lines 400), I do not agree with the conclusion that “two genes related to hormone regulation that were highly expressed only at the stage of early embryo development (Csa6M147590.1 and Csa3M389850.1)”. From Figure 4, I could find that genes Csa1M600830.1, Csa1M006300.1, Csa3M822100.1, Csa1M0516890.1 and maybe others after analysis of the raw data, rather than the picture in the figure.
The authors do not over-interpret their data. The conclusions are solid and supported.
Additional comments: No additional comments


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

Input:
Title: TRANSCRIPTOME ANALYSIS OF OVARY CULTURE-INDUCED EMBRYOGENESIS IN CUCUMBER (CUCUMIS SATIVUS L.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English is generally good, though uneven. Ironically, the least clear part of the manuscript is the abstract. In the pdf file I have uploaded, I have made numerous comments about how to make the abstract more clear, and some grammatical corrections.

The raw data is not shared, as far as I can tell. This is totally essential for publication. There is a link in the cover letter that goes nowhere.

The background and literature references are fine.

There isn't a hypothesis. It's a manuscript reporting a transcriptome analysis, with validation of 10 genes by qPCR. The microscopic analysis of induction of embryos from ovule culture is interesting.
Experimental design: The aim of the article is to study transcriptome changes associated with induction of embryo calli from ovules of Cucumber. The authors point out the importance of the technique for double haploid production from ovule culture in cucumber breeding. The point of the article is to determine the transcriptome changes associated with ovule culture that accompany this important technique (double haploid induction). Thus, the research question is meaningful, and the data are worth publishing.

The methods are well described, except for several glaring omissions, which absolutely need to be addressed before publication:

1) the raw data are not available (link in the cover letter does not work)
2) The authors do not state how many biological replicates were used for their RNAseq analysis. From Table S2 I think it is probably three biological reps. If this is so, this needs to be stated clearly in the text.
3) Regarding qPCR replicates: Looking at the excel file with the source data for the qRT-PCR, it looks like they have three technical replicates for each sample, and possibly biological replicates. In the legend for Figure 2, they say they did three 'independent experimental replicates'. What does this mean? They need to refer to technical and biological replicates, so it is clear what they did.
4) On line 155, the refer to a 'highly efficient in vitro ovary culture technology system of cucumber (unpublished)'. This is not acceptable. They need to put a description of this culture technology here, or publish it somewhere else and cite the publication.
5) On line 185, the authors use the abbreviation 'TDZ'. I supposed this is some kind of cytokinin (zeatin). Whatever it is, it must be the key factor for induction of embryogenesis from ovaries, and must be spelled out. Also the supplier of TDZ must be mentioned.
6) The authors should provide more detail in the methods on exactly which part of the tissues were used for the different RNAseq and qPCR samples. I.e. how much of the ovule? Was carpel tissue included also? Were the maturing embryos dissected from the undifferentiated callus tissue, etc.
Validity of the findings: Underlying data have not been provided. Once they are provided, the data will be of value to the field of embryogenesis, in particular for studies of inducing embryogenesis, and apomixis.

Regarding conclusions, many analysis of GO categories and KEGG analyses are provided. But this kind of analysis isn't that informative, even though it's the only kind of analysis that is possible with the data in its current state. I think that it would be good to make the article more concise.

Things that must be changed to be acceptable for publication. These are also listed as comments in the pdf file that I have uploaded.

1) Remove 'mechanism' from the title. There are no mechanisms here. An appropriate title would be something like 'Transcriptome analysis of ovary culture-induced embryogenesis in cucumber (Cucumis sativus L.)'
2) Line 46: state what the timpoints where
3) Line 61: delete 'shoot formation might be regulated by' too speculative
4) Line 180: define 'papilloma'
Additional comments: My only other suggestion would be that it would be interesting to put the authors' findings in the context of other papers focusing on asexual induction of embryos (tissue culture and apomixis). And especially to discuss the paper from the Sundaresan lab on induction of rice embryos using Baby Boom: 	Khanday I, Skinner D, Yang B, Mercier R, Sundaresan V (2019) A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds. Nature 565(7737):91–95.


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 OF OVARY CULTURE-INDUCED EMBRYOGENESIS IN CUCUMBER (CUCUMIS SATIVUS L.)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors addressed all comments from the initial review. I only have uploaded an annotated pdf version of the text for the spelling or text structure.
Experimental design: All comments were addressed. The method part is now fully described.
Validity of the findings: no comment
Additional comments: The authors addressed all concerns raised by the review of the original manuscript. However, there are a series of grammar issues that I have annotated in an enclosed pdf. I do not need to see the text further for scientific issues if English is proofed properly.


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 OF OVARY CULTURE-INDUCED EMBRYOGENESIS IN CUCUMBER (CUCUMIS SATIVUS L.)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The English grammar, style, punctuation and formatting need to be improved. In fact, they seem worse than the previous version.

Literature references are ok.

Article structure ok. Raw data still not shared, as far as I can tell- I could not find the reference for the raw data accession in the manuscript. If it is there, it is difficult to find.
Since this is the second time they have not shared the raw data (I made this comment on the last revision), I would recommend rejecting the manuscript on this basis.

Article is self contained, the addressed my previous comment about an unpublished protocol, since the protocol is now published.
Experimental design: This is all ok, as I mentioned in my last review.
Validity of the findings: No underlying data for RNAseq provided, for the second time. As I mentioned above, I would recommend rejecting the article on this basis.
Additional comments: The response letter to the reviewers' criticisms lacks almost all pertinent detail. There was no point-by-point response to my comments. The only comment they addressed was the need to cite their new method (which they did, it is now a published paper), and a few sentences in the materials and methods. As far as I can tell, the other reviewer's comments were not addressed at all.

I could not find the accession number for the RNAseq data. This is the second submission of this manuscript without a link to the raw data. The most useful aspect of this article would be the availability of the raw data. On this basis, I recommend rejecting the article, because the authors are clearly trying to avoid releasing the raw data.


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 OF OVARY CULTURE-INDUCED EMBRYOGENESIS IN CUCUMBER (CUCUMIS SATIVUS L.)
Review round: 3
Reviewer: 1

Output:
Basic reporting: All comments are now adressed.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: As mentioned in the previous review, I have no scientific comments in this review. All have been previously addressed. I appreciate the effort made for the English style.


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 OF OVARY CULTURE-INDUCED EMBRYOGENESIS IN CUCUMBER (CUCUMIS SATIVUS L.)
Review round: 3
Reviewer: 2

Output:
Basic reporting: • The style, punctuation and formatting still need to be improved. Indeed, I detected several minor errors, in lines: 164, 177, 213, 238.

• Sentences 97-101 would benefit to be rephrased

• Discussion is quite repetitive, and since it is based only on discussion of GO analysis and KEGG analyses I think it could be more concise. I suggest shortening without removing the articles suggested by previous reviewers which have instead helped reviewers helped in improving the discussion section.

• Conclusions section is too repetitive and they should not contain references to figures.
Experimental design: •This manuscript fits the aims and scope of PeerJ.
•The research question is well defined: obtaining a transcriptome landscape of early embryo development in cucumber ovary culture is of high interest for plant developmental biologist and for agricultural research.
•Some concerns about experimental procedures raised by previous reviewers still need to be addressed:

RNA quality control check results are not reported, please provide RNA integrity number (RIN) data if available or at least provide the values obtained from nanodrop analysis (260/280 and 260/230 ratios).

qPCR primer efficiency is still not reported.

It is still not clear in qPCR experiments the distinction between technical and biological replicates. Were the three technical replicates of qPCR related to only one biological replicate of the RNA-seq? Were the sample of RNA-seq and qPCR the same?

In qPCR experiments would be useful to show a gene whose expression level remains more or less the same along the different stages. This might be use in the future by other researchers as a possible housekeeping gene in the same developmental context.

Along results section, please repeat the value of fold change that has been considered for the selection of DEGs.
Validity of the findings: •Now that raw data has been provided, they will be of great value for other studies in the field of embryogenesis.
•The results obtained would increase their value if it were easier for the reader to extrapolate the results of the most interesting DEGs from the manuscript and from the figures. Here my suggestions:

Figure 3 is not very informative; I would prefer instead a table with a list of the principal DEGs between stage T0 and T1 which is for sure the more interesting time points of this study.

Results from 325 to 348 do not refer to any figure. I suppose that this part refers to Figure 9. This part should refer to a new figure or table that must be inserted in order between figure 5 and 6 and not at the end of the manuscript. This new figure or table should be, as already suggested by previous reviewer, a sort of matrix graph similar to that of figure 5 compiling the results on transcription factors.

In Fig. 5 change CTK with CKs (typical abbreviation for cytokinins)
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 OF OVARY CULTURE-INDUCED EMBRYOGENESIS IN CUCUMBER (CUCUMIS SATIVUS L.)
Review round: 3
Reviewer: 3

Output:
Basic reporting: In general I think the manuscript meets the basic reporting criteria. Grammar and spelling have been improved in successive revisions. There are however 3 points which I think must be addressed for the manuscript to be accepted: 1. the word papiloma in the methods section sounds incorrect as a previous reviewer pointed out. Do the authors refer to the papilla/papillae which form on the stigma of plant species-this needs to be checked. 2. I tested the link to access the data on the NIH website and it worked, however as a previous reviewer also commented the link is not included in the text of the manuscript or at least I could not find it. 3. the discussion section could be much more concise. The authors cite many other related studies in relation to their own data, I think such detail is unnecessary.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: TRANSCRIPTOME ANALYSIS OF OVARY CULTURE-INDUCED EMBRYOGENESIS IN CUCUMBER (CUCUMIS SATIVUS L.)
Review round: 3
Reviewer: 4

Output:
Basic reporting: The manuscript needs major English language revision.
Experimental design: The material analysed contains a number of different tissues/cells and non-embryogenic samples have not been included, making it difficult to assign gene expression profiles specifically to embryogenic egg cells/ovules
Validity of the findings: There are some small points to be addressed with respect to the experimental set-up, data analysis, presentation of the data and conclusions/discussion.
Additional comments: The manuscript by Deng et al presents a transcriptome analysis of different steps in haploid embryo induction (gynogenesis/parthenogenesis) from cucumber ovules. Although it is a descriptive/archival study i.e. it does not offer any insight into the mechanism behind haploid induction and development, it seems to have been fairly well performed and is still a good resource for researchers in the field. My main issues with the manuscript are the quality of the writing and the lengthy discussion.

After reading the Abstract I was quite intrigued and eager to read more as there is little known about the molecular regulation of gynogenesis systems. The abstract was well written, which seemed promising for the rest of the manuscript. Unfortunately, I was much less enthusiastic after reading the entire manuscript. Although the Abstract is well written, the other sections in the manuscript are not well written at all. The major problems are the phrasing and lack of attention to detail. The phrasing is often so awkward or the conclusions out of context that I even had difficulty understanding the background literature that they supplied for my own field of expertise (androgenesis). The manuscript needs extensive editing by a native speaker with a scientific background.
Examples:
“In Brassica napus, the expression of embryogenesis-related genes such as BBM, LEC1 (LEAFY COTYLEDON1 ) and LEC2 (LEAFY COTYLEDON2 ) were detected after culture
under heat stress at 32 °C for three days; after incubation at 24 °C, embryo-specific expressed genes, such as LEC1 and
LEC2 were detected (Malik et al., 2007).”
why is the distinction 32/24 relevant – do you mean during induction (32) and later, during outgrowth (24) ?
“ The BBM gene was the first key gene to be isolated in the process of spore cell division; and
was first expressed in zygotic embryogenesis and microspore embryogenesis (Pechan et al.,
1991; Boutilier et al., 2002). Khanday et al (2019) showed that BABYBOOM 1 (BBM 1) is a
member of the AP2 family of transcription factors”
“spore division” = microspore embryogenesis/haploid embryogenesis
“first expressed in zygotic embryogenesis and microspore embryogenesis” = doesn’t make sense
“showed that…is AP2 family” They did not show that it is an AP2 domain gene, it was already known. They showed that the gene is paternally-expressed (as did a group working in maize) and that egg cell expression induces parthenogenesis.

Some of the mistakes are the result of a non- critical eye and should be easy to fix. For example (there are many more):
Li et al.(2014), FAA( formalin
biological microscope ( = stereo microscope?),
heart chamber (= heat?),
magnetic beads with oligosaccharide (DT) ( = oligo dT?)
and embryo-like structures with integuments (do embryos have integuments?)
embryo maturation stage a nd shoot (= and)

Below are a number of specific questions/concerns

1. Material that was sequenced

Since the authors did not isolate egg cells for the transcriptome analysis, it is difficult to assign any of the changes that they observe specifically to egg cell parthenogenesis rather than effects in other sporophytic tissues. Likewise, what is the frequency of haploid embryo induction in this line? If a certain proportion of the ovules do not make embryos, then this will also confound interpretation of the transcriptome data.

I think these points should be made clearer throughout the manuscript, including the Discussion. For example, was the T0/T1 material used for sequencing from isolated ovules or from whole ovary slices? How does this affect interpretation of the expression patterns/gene functions?

2. Figure 1

The resolution in the images is very low. I am not sure if the resolution is low in the original figure or due to the pdf conversion process.

Showing the various treatments on the timeline e.g. heat stress would give a better overview of the culture procedure.

It would be helpful to provide more labels
a-f provide more labels for orientation purposes ovary, ovule, embryo. In f there is quite a bit of callus, a shoot and an embryo.
g-k, provide more labels in general. Specifically, in sections outline embryo sac, indicate ovule integuments/ovary, female gametophyte structures e.g. egg cell.
panel g- label ‘g’ extends beyond boundary of the image

What evidence is there that the egg cell enlarges and not one of the other female gametophyte cells?

What do non-responsive (non-embryogenic) egg cells/embryo sacs/ovules look like. A negative control would help convince me that the changes you observe are specific to the cells that undergo embryogenesis.

3. qRT-PCR

The authors indicate that they used the 2ddCt method ((Ct sample-Ct actin reference gene) –(Ct calibrator sample -Ct actin reference gene)). If the 2ddCt method was used, then the expression value for the sample used as a calibrator should be set to 1. I do not see this, so I suspect they used a different calculation.

The sentence “The results showed that the relative expression levels revealed by RNA-seq and qRT-PCR were closely correlated (Pearson's r = 0.53, 0.77, 0.57, 0.74, 0.79, 0.69, 0.81, 0.81, 0.86, 0.62, 0.81). The avlues are in the figure and do not need to be repeated here, where it is not even clear which value belongs to which gene. I also wouldn’t consider r=0.53 “closely correlated”.

Why were technical replicates used instead of biological replicates?

4. Figure 3
legend should be ‘upregulated’ and ‘down regulated’ not “upregulate” and “down regulate”

5. Figure 5
To improve the readability: move the sample legend to the top of the figure and show average expression values, not replicates.

6. Figures 4, 7, 8

Kegg pathway enrichment figures are not very informative– combine into one figure with 3 panels.

There are some pathways that are no applicable to plants e.g. ‘renin-angiotensin’, ‘lipolysis in adipocytes’. Were the pathways mapped to the correct organism?

7. Figure 9

What is meant by ‘specific-expressed’ gene?

8. Discussion:

The Discussion can be reduced by about 2/3. Given the experimental set-up it is quite speculative to discuss all the genes/pathways in detail. Just highlight similarities with other studies and the novel findings. Please also discuss the limitation of this study.

9. I would not use the term maturation. This is reserved for storage product accumulation phase. the term morphogenesis is more in line with the literature.


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

Input:
Title: TRANSCRIPTOME ANALYSIS OF OVARY CULTURE-INDUCED EMBRYOGENESIS IN CUCUMBER (CUCUMIS SATIVUS L.)
Review round: 4
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: All my comments have been addressed. I have really appreciated the effort to improve the English style and the general structure of the manuscript.


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

Input:
Title: FOREFLIPPER AND HINDFLIPPER MUSCLE RECONSTRUCTIONS OF CRYPTOCLIDUS EURYMERUS IN COMPARISON TO FUNCTIONAL ANALOGUES: INTRODUCTION OF A MYOLOGICAL MECHANISM FOR FLIPPER TWISTING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The clarity of the manuscript could be improved by changes to the structure, additional figures, language editing.

1. Most importantly, the manuscript's structure is confusing. The divisions between introduction, results, discussion, and conclusions seem arbitrary, and the order of the sections is hard to follow. For example, the detailed results of previous plesiosaur muscle reconstructions (90-122) and the summary of functional anatomy of extant analogues (124-309) from the introduction both seem more appropriate for the discussion. Also, the paragraph about sea turtle humeri (346-358) does not belong in Methods. Also, the Myology section at the end of the discussion (2573-2631) really belongs in methods. Finally, the subject matter seems to jump around a lot, making it difficult to find relevant information or follow a thought to its conclusion. Having a clear, strong structure is especially important in a long manuscript like this one.

2. The figures provided are good, but they are insufficient and poorly referenced. There are only two figures showing the reconstructed origins and insertions of muscles on the foreflipper and hind flipper, and only one reference is made to figures within the text (p58). At the very least, there should be a figure showing the actual muscle reconstruction used to deduce muscle function – this would require reconstructing the course/line of action of the muscles in addition to their attachments. Figure references should be added as appropriate; for example in lines 70-86. Additional figures that would strengthen the manuscript include: phylogenetic tree showing EPB of plesiosaurs, appendicular muscles in extant analogues, joint surfaces in plesiosaurs. (Also, what does “?” indicate in figures 1-2? And please indicate anterior-posterior in the figures.) If additional figures and tables were added, the text could be greatly reduced and the results would be easier to understand.

3. It is not clear what raw data were used in the study. Limb skeletons and histological sections referred to in Methods were not described or figured (392-394).

4. The English is generally good, but in certain passages grammatical mistakes and word choices make the meaning unclear. Examples include:
-	Abstract: “possible to obtain” – do you mean identify, or reconstruct?
-	63: I think you mean compressed, not depressed
-	66: hemispherical, not round
-	2290-2292: Confusing sentence, please re-word
-	2305: “which could be corroborated” – does this mean your results agree?
-	2360: “could be supported” – again, does this mean your results agree?
-	2365 and throughout: these figures are not muscle reconstructions, they are muscle maps.
-	2610: I think you mean relatives, not predecessors
-	2623: “in the course of reduction” – what does this mean?
-	2646: delete “partially”
-	Please spell out muscle abbreviations the first time they are used in a section.

5. -	The introduction is generally complete and well referenced. A reference and/or explanation is needed in line 67 for “joints are relatively stiffened.”
Experimental design: The research question is well-defined and fills a knowledge gap, and the choice of methods is appropriate. However, the methods are not well enough described to allow replication. For example: were osteological correlates in fossils used, and if so which ones? Do the “limb skeletons” and histological sections examined refer to extant or extinct taxa, and which particular taxa (391-394)? How were decisions made about equivocal EPB results (e.g., 376-380)? How exactly were muscle functions deduced, particularly rotation (e.g., 419-423)? Precisely what criteria were used to establish homology (line 315) and what does “deducible” mean in this context (lines 363-364)? Which homologies were established by topology, and which were from this study as opposed to previous ones? What is meant by “if EPB was little informative, Sphenodon was considered as well” (lines 324-325)?
Validity of the findings: The results add to the literature by providing a more comprehensive, phylogenetically based muscle reconstruction in plesiosaurs. Provision of underlying data and clearer explanation of interpretation of results would demonstrate their validity.

1. Most importantly, the data used to deduce muscle function are not described or provided. Secondarily, EPB uses osteological correlates in fossils, but no fossils other than the taxon in the reconstruction or osteological correlates are listed. In addition, a table showing which muscles are present in which bracket taxa according to which source would be extremely helpful.

2. It is sometimes unclear what is speculation. For example, the section “Limb cycle and myological mechanism for flipper twisting” does not clearly state whether it is based on extant taxa, literature, modelling, or something else. The underlying data and logic need to be more clearly presented. In general, more justification needs to be provided in the discussion section: not just “we agree” or “we disagree” with previous interpretations, but presenting arguments for each and the logic for preferring one or the other (e.g., 2174-2179, 2206, 2209-2210, 2326).

3. The conclusions section is generally well written, concise, and well supported. However, it does not fully address the original research question: what muscles contribute to flipper twisting? I suggest shortening this section and focusing on the research question and its broader implications.
Additional comments: The research question is, what muscles could have produced flipper twisting in plesiosaurs? To address this question, they authors produced the most comprehensive muscle maps for a plesiosaur to date using extant phylogenetic bracketing and deduced muscle function based on musculoskeletal geometry. The results fill a knowledge gap and could help explain plesiosaur functional morphology and locomotion. There are substantial problems in the methods section and the overall structure of the manuscript that need to be addressed, plus some additional issues with the way data and conclusions are presented and with the manuscript’s language. Currently, the results are difficult to understand and impossible to replicate. In addition, readability and thus impact could be improved by shortening the text and presenting some of the information as figures, tables, or supplementary information.


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: FOREFLIPPER AND HINDFLIPPER MUSCLE RECONSTRUCTIONS OF CRYPTOCLIDUS EURYMERUS IN COMPARISON TO FUNCTIONAL ANALOGUES: INTRODUCTION OF A MYOLOGICAL MECHANISM FOR FLIPPER TWISTING
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript needs extensive proofreading throughout the whole text. Please pay attention to present tense, past tense, and passive voice. This manuscript was written by more than one author, so please refer to “we” instead of “I”. Examples for the above-mentioned problems can be found in my comments in the pdf. The text varies between paragraphs consisting of sometimes extremely short and normal to long sentences. Please unify your writing style.
The literature was properly cited and the topic was well covered by the cited literature.
Restructuring the manuscript would really help to make some things clearer. The texts about extant animal groups in the introduction should be shortened, moved to a new chapter and illustrated by a figure. The abbreviations of all muscle names should be placed where they can be more easily found. Regrouping them in hind- and forelimb would make the lists shorter and easier to overview.
The existing figures need extensive work. Please redraw all lines and close gaps in the lines. Add directions like anterior or posterior to clarify what exactly the reader looks at. Add bone names (in full length or abbreviations) and the thyroid fenestra. This would greatly improve the overview over your figures. Select colors which can be distinguished more easily (for example p, bb, and ecu and pte and ecr might be quite hard to tell apart). Add the missing muscles (b, sl, fdls). Explain the dotted lines and the question marks. Add Roman numerals at the end of each finger/toe to make the finger count clearer. Is there a special order in which you sorted the muscles in your list? If yes, please explain. If no, please sort in a comprehensible order. Resize the whole figure.
Your figures are not mentioned anywhere in the text. Please add mentions to the texts of all described muscles (for example “see Fig 2 a”)
Please add figures of the muscle locations (like your Figures 1 and 2) in the main groups you used as EPB. It would be way clearer if the states in the different groups could be seen in a comparative illustration.
Please add figures for the comparison of all plesiosaur muscle reconstructions. This would allow a direct comparison without looking up everything in several different publications.
The text about EPB misses basic information about EPBs, why you used these groups, and any explanations about the level of inference. The latter should also be mentioned for each individual muscle to describe how similar the insertion and origin is in the different groups used for the EPB.
There is nothing written about the used specimen, except its inventory number and the museum where it is located. Where was it found? What’s its age and find location? Please mention the first describer Phillips 1871 directly behind the name of the species. How complete is the specimen? Please add a photograph or illustration of the specimen you worked with. Your specimen is IGBP R 324. What does IGBP stand for? Please mention this in your list of abbreviations. Add all this information to a new chapter.
It looks like this work was written for a thesis and copied for publication. The manuscript would greatly benefit from shortening and adding illustrations of the muscle settings in the other groups used in the EPB, extant taxa, and reconstructions done by other authors. Overall, the work would profit from a much clearer separation of the EPB, extant, and extinct taxa.
Experimental design: The authors did extensive literature work for their study. The topic is very interesting, and the quite different reconstructions made in the past show that there are still many open questions. Plesiosaurs are the only group of Sauropterygia which survived the past the Triassic and roamed the oceans until the Late Cretaceous. Therefore, they are a very interesting research topic and I greatly support any research performed in this topic.
It is not clear to me how much of this study is based on own experimental work and how much on the comparison of existing literature. The use of histology sampled was mentioned in the introduction (line 303) but there was no further mention anywhere is the text. Was this discarded? And if so, why? The literature comparison can be easily checked by other colleagues and therefore this part of the work is very transparent. Unfortunately, IGBP R 324 itself remains quite unclear. See chapter before. Pictures of muscle scars and rugosities would have helped to make the drawn muscle positions clearer understandable. The Goldfuss Institute in Bonn owns a MicroCT scanner, have you thought about scanning the bones and doing a 3d reconstruction? Fine muscle scars and fine rugosities can be nicely made visible by these techniques… either by studying the slices themselves or by applying hard side light to untextured 3d scans. This would greatly help to support your positionings of each muscle.
In case of some reconstructed muscles, the muscle origin probably lies on the sacrum or caudal vertebrae (for example cfl and cfb). This is not shown in the figures. Why? Showing would be relevant for understanding and checking the geometrical derivation of the muscle functions you made.
Validity of the findings: There is some novelty in this study because the previous reconstructions differ so much from each other. A comparative work is therefore helpful and interesting for all researchers working on Sauropterygia. Unfortunately, no information about the true comparability between the previously studied plesiosaurs and C. eurymerus can be found. The authors state that the muscle setup (head counts etc) in crocodiles or turtles may vary within the group. Can this also be the case in plesiosaurs? There is no information about that.
A large amount of speculation can be found in the results, comparison, and conclusions. The authors generally made this clear by using terms like maybe, potentially etc. Personally, I find it difficult to reconstruct muscles with bad support by the EPB (for example addV, comments about the level of inference by the authors would have helped here) and add them to the final reconstruction without marking them as speculative in the figure.
Additional comments: Although the topic is very interesting, I recommend rejection because the manuscript is flawed and unlikely to be salvageable through revision. Think about rewriting and restructuring the whole manuscript and add illustrations and figures. Maybe think about splitting the monography into two manuscripts, maybe published as part I and II: Part one about the EPB and probably the comparison with extant taxa, part two about the comparison of your results with previous reconstructions.


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: FOREFLIPPER AND HINDFLIPPER MUSCLE RECONSTRUCTIONS OF CRYPTOCLIDUS EURYMERUS IN COMPARISON TO FUNCTIONAL ANALOGUES: INTRODUCTION OF A MYOLOGICAL MECHANISM FOR FLIPPER TWISTING
Review round: 1
Reviewer: 3

Output:
Basic reporting: Given the review order, I assume this section is the first thing you read, so I'd like to first say that this is a really great study and much needed for the field of marine reptile functional anatomy/morphology and I am eager to see it published!

With the Results section, I recognize that this is a comparative anatomy paper and it is very well done, but some sections read as a description of the analog’s anatomy moreso than Cryptoclidus' with its anatomy not mentioned until the last few sentences of each muscle heading. Instead of the way it is currently written where for example for the Musculus subcoracoscapularis, where you first delve into the anatomy of turtles, crocs and lizards and why, based on that, you have inferred its reconstruction on the posterodorsal proximal plesiosaur humerus, maybe start out with “we reconstruct the Musculus subcoracoscapularis on the posterodorsal proximal plesiosaur humerus…. based on….” and then go into the analogous/homologous comparisons.

In general, and I understand the reasoning, much of the paper reads more as a summary of the anatomy of analogs more than a study of the plesiosaur anatomy. I would do a decent amount of reorganization and some shortening of the extant anatomical description sections focusing very explicitly on what is relevant to the plesiosaur reconstructions and why. This will help shorten the paper.

The current figures are good, though I would not have abbreviations for the figures. However, I do think the results section could be greatly augmented with additional or improved figures highlighting the functions of the reconstructed muscles. Perhaps two large fore- and hindflipper figures with A, B, C, D sections highlighting static views of some key functional actions of the muscles. Though, I do really like the tables for this same purpose. Instead of, however, having “function after x study” in the columns maybe just “function” with the studies in parentheses or "function inferred from (study)." It would also be good to include a column of the assigned abbreviation of the muscle groups so the table can connect better to the main text. In Table 2 the third row says “function, this study” which differs from the language used in Table 1.

Additionally, and perhaps this is a personal preference, but due to the large usage of abbreviations and very numerous muscle groups in this study, the manuscript may flow better with abbreviations akin to what Cieri et al. 2020 utilized, for example abbreviating Coracobrachialis brevis as CoracoBrev. I found myself having to stop and go back to the abbreviations list quite a lot, as not all were intuitive. Additionally, definitely place the abbreviations section much earlier in the text as some abbreviations where used prior to when they were indicated (line 99).

Line 127 and 128, modern day paraxial swimmers may not have four similarly sized and shaped flippers, but this sentence makes it sound like in the extant realm it is either caudal flukes in axially swimming groups like cetaceans OR webbed paddle feet. Paddles are drag-based and there are of course flippered animals today which have more than webbing, rely on lift, and can barely/don't use them on land, including penguins and sea turtles which of course you know as this manuscript delves into them as analogs. Given that I find this sentence’s wording in not highlighting these animals as just a little odd given the content of the rest of the paper. Very easy fix though.

The Muscle Function section of the Discussion and the previous, lightly touched on muscle functions section of the Results create redundancy. Additionally, there are elements of the Discussion that might be included in the Results section. Overall there is a lot of length which could be shaved off by having these sections better complement one another and much would be gained with integrating them in a way that the discussion section doesn’t repeat inferred functions, origins, and insertions indicated in the Results section. The Discussion section might be improved by highlighting how this study differs or aligns with studies past and especially, given the title and presented problem, delving into how the reconstructed myology relates to flipper twisting and why that is important for plesiosaurs' locomotion (which you absolutely discussed throughout the manuscript, it's just not explicitly organized in its own section and highlighted). Again, this may entail taking elements of the Results and putting them into the Discussion and vice versa. All of these things are definitely already highlighted in the manuscript, it just needs organizing in a way that will make the findings more straightforward and less repetitive.

The plesiosaur joint morphology, osteology, and myology section (Line 2462) feels out of place in this current Discussion section. As the manuscript is right now, I believe that portions of this section would be better included with the previous section dedicated to the highlighted functional analogs prior to the Results as I find it distracts from the general functional and Cryptoclidus-focused ending of the manuscript.

There are quite a few grammar, flow, and textual issues throughout, with some examples highlighted by line below:

Line 46, strange wording with the rise of plesiosaurs and their extinction.
Line 55, I would merge these two sentences by eliminating the word today. It will also make the paper timeless. “Today” as the 2020’s won’t be very accurate when this paper is read in a decade or two. Something like “…have been debated until Krahl 2020….”
Line 65, extent not extend
Line 99, abbreviations are used prior to them being defined in-text or referenced, so I would move abbreviations to after the introduction and be sure not to abbreviate in the intro.
Line 124, Should this be “Functional analogues <of the> [plesiosaur] locomotory and musculoskeletal system”?
Line 291 and 292, again extent not extend, and repetitive use of “at least”
Line 293, sentence starting with “However” seems out of place. Perhaps meant to be at the end of two paragraphs previous?
Paragraph starting at 295, seems out of place for this section
Line 303, proof, not prove
Line 319, odd wording, I am not sure what this sentence means.
Line 323, replace sometimes with “may” or equivalent
Line 330, instead put “...who sampled and compared…” and remove “and also compared them.”
Line 411, make clearer what “the former” is in this case.
Line 415, I don't think "Therefore" is needed to start this sentence.
Line 563, don’t start sentence with “Decided.”
Line 1426, starts out as “pit was chosen” when other section had the full muscle name written out to start.
Line 1744, add a space after this paragraph.
Line 2181, cumbersome grammar

Citation of “Wittmer,” 1995 should be Witmer with one "t"
Experimental design: I believe this research to be within the aims and scope of PeerJ.

This is a wonderfully comprehensive study with a sound experimental design based on well-done comparative anatomy that fills a major gap in marine reptile functional morphology. Plesiosaurs as having no true analogs have stumped paleontologists for decades with regards to their locomotion and this study is a great push forward and logical continuation of/sequel to studies like Araújo and Correia, 2015 in understanding the myology which relates to how plesiosaur limbs would have moved in the context of potential swimming locomotory styles. The problem is well-defined and some moving around of elements in the manuscript's Results and Discussion section and new/modified sub-sections (recommended under the Basic Reporting and Validity of the Findings comments) would help to make clearer how the problem is addressed.

Some light critique is in some of the analogs. Though similar in function, otariids and cetaceans as mammals have critical differences in this pectoral girdle osteology and myology, lacking entire bones, the large cartilaginous extensions of reptiles, and entire muscles. For that reason, I wouldn’t put as much weight into the osteological and myological comparisons between plesiosaurs and these groups, but the functional strokes of the flippers, especially in otariids is still great comparison for plesiosaurs.

Line 474, “therefore it is presumed to have become superfluous.” In crocs the medial scapulosternal ligament is the originator of some forelimb muscles relevant to locomotion (Jasinoski et al. 2006). I understand the limitations of the reconstructions of this study, and not reconstructing the scapulosternal ligament for the purposes of this project is definitely fine, but I would just further highlight precisely why you believe this ligament to be functionally superfluous given its inferred course, especially given that you did use comparisons with crocs in other areas of the results section.

Line 2634, what are “the three” extant taxa? This paper highlights lepidosaurs, crocs, cetaceans, otariids, sea turtles, and penguins for comparisons.

The literature background used to inform the reconstructions is very solid as based on current phylogenetic bracketing. It may not be entirely harmful, however, to go slightly deeper into avian literature aside from just penguins given that flying birds are of course generating lift and lots of good functional anatomy work has been done on birds in the context of flying that may assist in informing some more obscure and ambiguous muscles and ligaments. Just a light suggestion or worth a passing mention in text.
Validity of the findings: I do find there to be some slight deviation and tangents from the stated purpose of the study at points within the manuscript. Again, there is some dwelling on the taxa used for comparisons. I interpret this paper as being dedicated to making robust muscle reconstructions of the fore- and hindflippers in plesiosaurs with the goal of determining what the underlying myological mechanisms for flipper twisting and general movement in plesiosaurs are. As taken from the abstract:
“The question whether plesiosaurs employed their four flippers in underwater flight, rowing flight, or rowing has not been settled yet. Plesiosaur locomotory muscles have been reconstructed in the past, but neither the pelvic muscles nor the distal fore- and hindflipper musculature have been reconstructed entirely.”
The muscle reconstructions and the inferred functions are excellent and I think the manuscript clearly addresses the second sentence. I feel as though neither the discussion nor conclusion take these functions and infer precisely what type of swimming these functions would have enabled and address the first sentence directly. I think this could be welcome speculation given the robust results. The word "flight" is used only twice in the discussion section, once used in the context of previous studies, the second used in the context of sea turtles. Similar for use of the word "rowing" used only in the context of analogs. The fourth paragraph of the Conclusion comes close to this, but does not explicitly define this as a lift or drag-based stroke. Lift is greatly inferred, but should be made explicitly clear.

It may be worth adding a sentence or two on encouragement of using the myological reconstructions within this study to further assess plesiosaur swimming function with hydrodynamic and biomechanical methods. To fit the journal's needs.

I would restructure the conclusion to particularly how the myological reconstructions of this study inform the flipper twisting and beat cycle. The conclusion should also restate the problem at hand. Why did you do this study? Currently it only summarizes the results, it does not really *conclude*. Why do this? How did you do this? What were your results? How did they differ from/complement previous studies? How would you encourage future work in this realm?
Additional comments: I really commend the authors for this undertaking that will no doubt expand our knowledge and fill a large hole in the field of Mesozoic marine reptile functional morphology. The reconstructions, functional and anatomical comparisons, as well as inferred beat cycle are sound and quality, and I am excited to have reviewed this and for it to be published. The manuscript, however, does have a fair bit of pacing, flow, length, repetitiveness, and grammar issues which hopefully I provided decent constructive critique on.

Line 2377, I believe this section is really the crux of the paper, what the results lead to, and it should be brought out more. Currently it just feels like a generic section especially given the sort of out of place section which follows it. The Muscle Function section in general should be the "main course" of the Discussion section, but currently feels a bit buried overall. It may also be good to slightly pull back on the definitiveness of this section (Line 2377), though I do not disagree with the assertions. Instead of "At the beginning of the downstroke the flipper leading edge is rotated..." perhaps something along the lines of, "Our reconstructions suggest that at the beginning of the downstroke the flipper leading edge is rotated by..." It would additionally be helpful to restate why it is important that plesiosaurs were able to twist their flippers in the context of their currently estimated locomotion style, and then tie in the results to that problem in estimating a plesiosaur fore- and hindflipper stroke. Additionally, definitely restate the ~19 degrees rotation based on Witzel, Krahl & Sander, 2015 and Witzel, 2020 for the foreflipper in this section. It was mentioned very early on in the manuscript, then for the hindflipper only in this section, then restated in the conclusion section as for both.


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: FOREFLIPPER AND HINDFLIPPER MUSCLE RECONSTRUCTIONS OF CRYPTOCLIDUS EURYMERUS IN COMPARISON TO FUNCTIONAL ANALOGUES: INTRODUCTION OF A MYOLOGICAL MECHANISM FOR FLIPPER TWISTING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The clarity of the manuscript is much improved; it is focused and organized around a central hypothesis. The structure is strong and the figures are very good, although some information is still missing from the figures and text. A few additional references are needed. The text is still very long and some portions could be reduced.

1. The organization of the manuscript is logical, but some text could be removed or reduced to make it more concise. For example:
- In the introduction, the significance of the paragraphs on girdle homology is not clear to me (lines 160-188)
- In the results section, each muscle description begins with a list of synonyms and a justification for which term is used in the current ms. This could be replaced by a single statement that the current work uses the term most commonly used for that muscle in the recent sauropsid literature and only the exceptions discussed.
- in the Discussion section there are several paragraphs that comprehensively list previous studies (2027-2046, 2142-2148). I think these are unnecessary because this is not a review paper.
- the Conclusions section reiterates the Discussion section

2. References for muscle homology are insufficient; at the very least Walker (1973) should be listed in this section (199-205) and not just as an afterthought (249). Also see, e.g., Hattori, Soki, and Takanobu Tsuihiji. “Homology and Osteological Correlates of Pedal Muscles among Extant Sauropsids.” Journal of Anatomy 238, no. 2 (2021): 365–99 and Rui Diogo’s work on muscle homology in turtles and lepidosaurs.

3. The figures are very good. However, some important information is not figured, namely the muscle scars referred to in the text (e.g., 566-567, 608, 761-762). This is important because these scars influenced the muscle reconstructions, so they should be figured or a reference provided. In addition, some of the figure captions are extremely long and should be reduced (Figs. 4, 5). Fig. 1c and d should include Sphenodon because it is also used for phylogenetic inference. Finally, in Fig. 4b the humeral head of triceps brachii appears to be missing.

4. Some key background information is lacking from the introduction:
-	Lines 45-46: What are the relationships between Plesiosauria, Sauropterygia, and Diapsida? (could be added to Fig. 1)
-	Lines 151-156: Why was this species of plesiosaur chosen as a basis for the reconstruction, and what are its relevant anatomical features and phylogenetic relationships within the group?
Experimental design: The research question is well-defined and fills a knowledge gap. The methods are described in detail, but some methods were not rigorously followed and/or explained (establishment of muscle homologies and EPB).

1. Most importantly, the method the authors refer to as “EPB” appears to be simply inferring the condition found in the majority of the 3 extant taxa studied. EPB as defined by Witmer (1995) and Bryant and Russell (1992) is a method of phylogenetic inference that follows a series of steps, taking into account the precise relationship of taxa and the presence of osteological correlates throughout the fossil record of the group. So, for example, the presence of a trait in both crocodylia and testudines is weaker evidence for its presence in sauropterygia than its presence in crocodylia and lepidosauria because lepidosauria is the sister group of either sauropterygia + archosauria (Fig. 1b) or of sauropterygia (fig. 1c). Please clarify the methods used here in the context of the literature and justify differences in methods. Throughout the results section the relationship between extant taxa should be considered more carefully and the most parsimonious inference (not necessarily the most common character state) should be given. (I doubt that this will affect the results noticeably, but it should still be considered.)

2. Secondarily, the methods described for determining muscle homology are vague and insufficient. It is implied that most of the muscle homologies are proposed here for the first time (lines 204-205), but Remes 2007 is cited heavily as well. Furthermore, it is stated that homology was established by topology, whereas innervation and development are generally considered to be the most important criteria for muscle homology (e.g., Russell & Bauer 2008, Walker 1973). This section should be reworked with a comprehensive discussion of a) what parts are original to this study, b) the basis for the homologies where original to this study, including innervation patterns, and c) any controversies and their potential effects on the reconstruction.

3. I don’t understand the logic that chameleons are functionally comparable to plesiosaurs (lines 631-632). Their musculature and locomotion are highly specialized for grasping and perching.
Validity of the findings: The results add to the literature by providing a more comprehensive, phylogenetically based muscle reconstruction in plesiosaurs. However, there seems to be some bias introduced into the methods that undermines the conclusions. Also, it is not always clear when something is an inference or speculation and what it is based on, and the logic behind reconstruction decisions is not always explained.

1. Most importantly, there appears to be some circular reasoning leading to the conclusion that plesiosaurs possessed muscles that could enable flipper twisting and thus underwater flight. For example:
- Lines 315-320: “we tried to find muscles that would enable underwater flight ... some muscle attachment areas can be supported by two of the extant sauropsid groups by the EPB, but favorable for flipper twisting is a muscle attachment area which is only supported by one” (these cases are noted in the results but still used in support of the overall conclusion)
- Lines 412-416: “Due to the flipper shape … plesiosaurs were most likely relying only on lift-based locomotion (underwater flight) … therefore, muscles employed in individual digital movement are not reconstructed”
- Line 1042: “a lepidosaur-like insertion to metacarpal I is functionally favorable as it contributes to flipper twisting”
I strongly suggest not making any reconstruction decisions based on functional hypotheses (especially the one you're trying to test), but at least make it absolutely clear how these decisions affected the result and consider the alternatives.

2. Be careful with language when discussing function in extinct animals; it is always uncertain. E.g., lines 342-343 “The whole flipper is rotated by approximately 19 degrees by flipper rotators and twisted by flipper twisting muscles (refs).” This is a hypothesis based on simulations. Line 1965: Before describing the hypothesized muscle functions it would be good to summarize what the hypotheses are based on.

3. The logic behind some decisions is not clear: Lines 910-911: “The state of edc as represented by turtles poses the plesiomorphic condition” – why? Lines 1937-1940: why is m. adductor digiti quinti reconstructed in plesiosaurs when it is only present in lepidosaurs?
Additional comments: In this revision, the authors have reorganized the manuscript and greatly improved its clarity. Several excellent figures have been added. I still have some questions about the methods as they have taken an unconventional approach to muscle homology and EPB, and I'm concerned that some decisions about the methodology may have been influenced by the result the authors were hoping to find. While these are serious issues, their effect on the results is most likely minor. Therefore, I see this version as being much closer to publication.


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

Input:
Title: FOREFLIPPER AND HINDFLIPPER MUSCLE RECONSTRUCTIONS OF CRYPTOCLIDUS EURYMERUS IN COMPARISON TO FUNCTIONAL ANALOGUES: INTRODUCTION OF A MYOLOGICAL MECHANISM FOR FLIPPER TWISTING
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript was greatly improved by the authors. Grammar and spelling were also greatly improved and, except from a few typos, I see absolutely no problems in publishing the manuscript like this. The restructuring done by the authors helped a lot in improving the overall readability and clarity of the manuscript. The newly added figures provide valuable information about the lines of action of the reconstructed muscles and are therefore a very good addition. The results are well presented. I’d recommend moving the description of the complete flipper beat cycle to a new paragraph inside the conclusions to highlight this relevant information a bit more.
Experimental design: The authors addressed my concerns really well. Thank you for that! Everything is fine now.
Validity of the findings: Thank you for the explanations. The manuscript profited from the explanations added by the authors and the previously existing uncertainties were addressed and removed.
Additional comments: You cite Krahl (2021) in several places in the manuscript. This citation doesn’t appear in the reference list. In some cases, there is also a publication named Krahl (2021, in review). Is it the same? If so, please correct or add the publication the reference list.
In my opinion, the manuscript is totally publishable now. The few typos in the text and the one in Fig 2 b can be easily corrected. This also applies for the very few mistakes in the reference list and one wrong reference to a Figure (Fig 3 instead of Fig 4). See comments in the pdf.
Thank you for investing so much work again. The manuscript was greatly enhanced.


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: FOREFLIPPER AND HINDFLIPPER MUSCLE RECONSTRUCTIONS OF CRYPTOCLIDUS EURYMERUS IN COMPARISON TO FUNCTIONAL ANALOGUES: INTRODUCTION OF A MYOLOGICAL MECHANISM FOR FLIPPER TWISTING
Review round: 3
Reviewer: 1

Output:
Basic reporting: I'm happy with the current version, and I defer to the authors on style issues. The figure revisions look good, and I appreciate the paragraph on Cryptoclidus. 3 minor questions/comments:
Line 45. What is meant by "most derived"?
220: Where are homology lists with citations -  does this refer to table 3?
Results section: I meant to suggest that the phrase "The term ____ was chosen because it is the most commonly used one in recent works on Sauropsida" only needs to be used once in this section, e.g., "we use the muscle terminology most common in recent works on Sauropsida" in the beginning of results, and then don't mention muscle terminology again unless you're choosing something different.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have fully addressed my concerns. I appreciate their careful explanations and revisions, and I now am confident that this study has been rigorously carried out and well reported.


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: ANXA9 AS A NOVEL PROGNOSTIC BIOMARKER ASSOCIATED WITH IMMUNE INFILTRATES IN GASTRIC CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: I think it is a very interesting finding that in gastric cancer, the prognosis is worse in the ANXA9 low-expression group, contrary to that in colon cancer. However, I feel that the impact of the other analysis results is weak, so I think the paper will be more accurate if the authors conduct experiments using knockdown and forced expression of ANXA9 in gastric cancer cell lines, and then add a discussion of this confliction between in gastric cancer and colon cancer.
Experimental design: Authors noted that the clinical specimens were analyzed using UALCAN website and that the classification by some clinical information did not show significant differences in ANXA9 expression. I propose to test whether significant differences in the expression of ANXA9 occur when using the gene expression-based classifications (GC, MSI, CIN, and EBV) or the Lauren classification (diffuse or intestinal) described in the TCGA report (Nature 513, 202–209 (2014). https://doi.org/10.1038/nature13480).
Validity of the findings: The authors analyzed most of the data sets except for Figure 1 and 2 using the TCGA data set; does the data set registered in GEO contain any survival data or clinical information? I think that adding the validation results of other datasets will make the results more reliable.
Additional comments: The five boxplots in Figure 1 are all different in size. It would be better to unify them.
 The captions in Figure 1 and Figure 4 are difficult to read because the text is small and the font is not properly selected.
 In Fig. 8, the width of the image is wider to fit the Legend, but I think this unfriendly due to the small size of the heatmap.
 In Figure 7b, the red text is not only difficult to decipher because it is covered by dots, but also some of the numbers are obscured. I suggest that the correlation coefficient and p-value be listed elsewhere.
 In Figure 9B, the individual gene names cannot be deciphered because the letters overlap. How about  adding the gene names to the volcano plot in Figure 9A and omit Figure 9B?
 There are some typos of gene names.


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: ANXA9 AS A NOVEL PROGNOSTIC BIOMARKER ASSOCIATED WITH IMMUNE INFILTRATES IN GASTRIC CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1-The English language should be improved.
2-In some parts authors mentioned AHNAK2 while the study was related to the ANXA9 gene, for example in abstract.
3-The figure quality could be improved.
Experimental design: no comment
Validity of the findings: Did Author use the same tumour samples compared to their matched normal tissues for all bioinformatic analysis? If no, the use of the same samples could strengthen their results and data validity.
Additional comments: 1- In abstract with following sentence :"Although the mRNA expression level of ANXA9 is associated with the poor prognosis of colorectal cancer, the function of ANXA9 in GC remains largely unknown", it was better to indicate that lower mRNA expression of ANXA9 is associated with the poor prognosis of colorectal cancer.
2- Line 302-303: "Kaplan-Meier survival analysis showed that the OS time were prolonged in gastric cancer patients with a high expression of BUB", it seems that BUB should be BUB1.
3- Lines 330-334 could be summarised and merged.
4- Did author observe correlation between ANXA9 expression and OS in other cancers with the TCGA data analysis. It could be performed using UALCAN.


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: ANXA9 AS A NOVEL PROGNOSTIC BIOMARKER ASSOCIATED WITH IMMUNE INFILTRATES IN GASTRIC CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: I think it is good that this manuscript is more detailed than the first draft.
Experimental design: no comment
Validity of the findings: In the added Fig. 3G, the data showed that the expression level of ANXA9 was lower in diffuse gastric cancer specimens than in intestinal specimens. Can you confirm the possibility that the expression of ANXA9 is elevated in the intestinal specimen but is less elevated in the diffuse specimen when normal and tumor tissues are paired?
Additional comments: The size of the dots in Figure 3 is different in several figures, and I request that they be unified.
Although improved, Figures 8 and 9 are still difficult to read. I suggest that the methylation levels of the ANXA9 promoter region and expression levels of that shown in Figure 8 be shown in a separate figure as a scatter plot.
In Fig. 9a and b, the gene names are illegible due to the small size of the diagrams. We suggest that a part of the network diagram be enlarged to create a diagram that clearly shows the gene names.


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: ANXA9 AS A NOVEL PROGNOSTIC BIOMARKER ASSOCIATED WITH IMMUNE INFILTRATES IN GASTRIC CANCER
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Authors made notable changes, however, in line 222, it is better to mention that also lower expression of ANXA9 in UCEC is correlated with good prognosis and better OS.


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: ANXA9 AS A NOVEL PROGNOSTIC BIOMARKER ASSOCIATED WITH IMMUNE INFILTRATES IN GASTRIC CANCER
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: 1. While there have been improvements compared to previous drafts,
 there are still a few areas where the text is small and difficult to read.
 I suggest to enlarge the size of texts in several figures.

2. In Supplementary Table 1, there is a gene with an unfamiliar notation. This is probably due to an automatic correction by the spreadsheet software when it should be SEPTIN11. Please rewrite it to the correct gene name.


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: ANXA9 AS A NOVEL PROGNOSTIC BIOMARKER ASSOCIATED WITH IMMUNE INFILTRATES IN GASTRIC CANCER
Review round: 3
Reviewer: 2

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


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

Input:
Title: THE CNIDARIAN PARASITE CERATONOVA SHASTA UTILIZES INHERITED AND RECRUITED VENOM-LIKE COMPOUNDS DURING INFECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this study, the authors enumerate in the present study, that Cnidarians are the most ancient venomous organisms. They store a cocktail of venom proteins inside unique stinging organelles called nematocysts, and are present in all Cnidaria, including the morphologically simple Myxozoa, which are a speciose group of microscopic, spore-forming, obligate parasites of fish and invertebrates. The authors identified 9 VLCs, all of which have closely-related sequences in other myxozoan datasets, suggesting a conserved venom profile across Myxozoa, and an overall reduction in venom diversity relative to freeliving cnidarians.

The overall composition of the manuscript is good, is written in a clear and understanding manner with a very nice introduction to the topic. The quality of the figures and tables is also good. All used procedures are correct. The paper is scientifically and methodologically accurate, and the conclusions drawn are convincing. The linguistic style is sufficient for publication and the reference section is adequate. This manuscript will find the interest of many readers. The presented study can be proposed for publication when the authors check the following minor concerns:
Minor comments:
I suggest that the figure 1 should be described in the main text. The figure is not described in the main text.

Supplemental files need more descriptive metadata identifiers to future readers, and the figure should be improved using panels A, B, C. Besides, the authors should be providing figure legends for each figure described in the supplementary files.


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

Input:
Title: THE CNIDARIAN PARASITE CERATONOVA SHASTA UTILIZES INHERITED AND RECRUITED VENOM-LIKE COMPOUNDS DURING INFECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors studied the venom-like proteins and transcripts of the myxozoan parasite, Ceratonova shasta. Using the already published C. shasta nematocyst (=polar capsule) proteome data, and the time-series transcriptome dataset obtained in the present study, the authors identified 9 venom-like compounds (VLCs) and detected their intrapiscine expression profiles from 1 to 21 days post exposure. Furthermore based on the outcome of phylogenetic analyses, the authors intended to predict if these VLCs are of cnidarian origin.

The MS is well written in general, although the wording is “lab-slang”-like sometimes (e.g. most heavily expressed). The figures are of adequate quality.

The main criticism is concerning the presentation and the discussion of findings. I suggest re-organizing completely the Results and Discussion parts. The Discussion contains results (and some sentences even refer to methods) which are not mentioned before, and the authors’ speculations regarding the putative functions of the detected VLCs are often not supported by the presented results. Therefore, the Results should be completed with all obtained data in detail, including the outcome of the structural modeling (mentioned in M&M L117-118). Please clarify if you obtained the full-length transcripts/proteins for predicting the structure. On the domain detection graphs (Figure 5 and Suppl. 3), some of the VLCs look partial (lactahedrin-like, metallopeptidase-like transcripts, Kunitz-type protein like ones). Please explain their status.

In M&M, the authors state that the outgroup of phylogenetic analyses is the poriferan Amphimedon queenslandica, but it is not the case for the hyaluronidase-like transcript. Please explain. In several cases, the low bootstrap values do not support the position of C. shasta transcripts (e.g. Figure 5 A, Suppl. 3 C-type lectin-like, lactadhedrin-like, Kunitz-type protein-like transcripts). I suggest discussing the issue, and please „soften” your conclusions regarding the inheritance of the examined proteins.

In the text, I would suggest discussing the “inherited” and “recruited” VLCs separately. Thus L248-253 would come after L300.

Some in-text citations are missing from the reference list (e.g. Hartigan et al. 2001, Eszterbauer et al. 2001). Please check thoroughly all references (both in text and in ref. list).
Experimental design: see above
Validity of the findings: see above
Additional comments: L96 Please check the sentence. Did you mean “…mapped to the C. shasta TRANSCRIPTOME…”?

Table 1 Please re-organize the table. In the first column, I would suggest adding an individual abbreviation for the examined VLCs that reflects both to the name of the organism (C. shasta) and the putative function of the analyzed transcript/protein (and then refer to this name throughout the MS). As the functions are predicted using in-silico, sequence-based analyses only, and the detected aa (??) sequence identities are rather low in most cases, I suggest mentioning in the annotation column that the identification is more a likeliness than a confirmed fact (i.e. C. shasta lactadherin-LIKE protein). Are the contigs obtained from Trinity RNA-seq assembler are available individually in Genbank? If not, please submit, and include here their Genbank Acc. No. Please add also the length of the contigs in Table 1.

Figure 4 What do the three squares per timepoint mean? Three parallels? Please explain in figure legend.

L219-221 Please be more precise with wording, name the referred proteins to help prospective readers to follow your thoughts (certainly, the same applies to the other parts of the MS).

L237 myxosporean

L246-248 multiple-times repetitions, please avoid

L251 serine protease INHIBITORs (serpins)

L252-253 Please add example (with reference) why you came to this conclusion.

L264-265 “We selected…” The sentence belongs to M&M. Use specific identifier, not a Trinity assembler code (the same applies to all transcripts, see the comment for Table 1).

L279 Do you mean over-expressed?

L295 at later timepoints (please add when exactly)

L326-327 It is result. Remove or re-word.

L341-341 “…not similar in sequence identity….”?? Please re-word. The correct reference here is Eszterbauer et al 2001, btw. How do you mean that the outcome of the two studies are comparable, if Kunitz-type proteins were not studied by Eszterbauer et al. 2021?

L347-348 Please explain why you think that Kunitz-type serpins are involved in evading host immunity. Could you add reference that support this theory?

L363-364 This is a crucial statement of the MS, therefore please explain in the previous parts how this 90% reduction was calculated. Is it possible that transcriptome sequencing was not complete, or during the filtering of raw reads, some potential VLCs were removed? Or your 4-step selection procedure was maybe too strict? Please discuss all these possibilities.

L368-374 repetition!


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

Input:
Title: THE CNIDARIAN PARASITE CERATONOVA SHASTA UTILIZES INHERITED AND RECRUITED VENOM-LIKE COMPOUNDS DURING INFECTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: Professional article structure, figures, tables.
Experimental design: Rigorous investigation performed to a high technical & ethical standard.
Validity of the findings: Conclusions are well stated, linked to original research question.
Additional comments: The authors modified the observations adequately and the manuscript is substantially improved and can be accepted.


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: MORPHO-PHYSIOLOGICAL EFFECTS OF ENVIRONMENTAL STRESS ON YIELD AND QUALITY OF SWEET CORN VARIETIES (ZEA MAYS L.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: English is generally good, but does need editorial work.  I made numerous suggestions, but it will need more work.

Literature and structure are appropriate. Raw data is shared.

results are relevant to hypothesis, although the hypothesis is not clearly stated.  This can be handled in the rewrite stage.
Experimental design: It appears to be original research.  I leave it to the editors to decide whether the research was within the Aims and Scope of the journal.  I really hope that they had decided that before they ask me to spend my time reviewing the paper.

Research questions are well defined and the need for the research is made clear.

My only significant concerns are within this box.

1.  Are two years (environments) adequate for this type of work.  I leave that question to the editors.  I do believe that the results reflect what would be expected in those two seasons.

2. The authors must supply more information regarding the germplasm used in this experiment and also more detail regarding the statistical analysis.  Without the knowledge about the germplasm it is impossible to determine th generalizability of the data and repeat the experiment.
Validity of the findings: I believe the authors have done well in addressing this area,
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: MORPHO-PHYSIOLOGICAL EFFECTS OF ENVIRONMENTAL STRESS ON YIELD AND QUALITY OF SWEET CORN VARIETIES (ZEA MAYS L.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language of reporting is not clear and lacks scientific integrity.
Experimental design: Satisfactory
Validity of the findings: Methods section needs more details, so that the reproducibility of results could be ensured.
Additional comments: There should not be repetition of title words in abstract or any other section. Abstract is poorly written, please use standard way to write abstract section. No objectives and methods mentioned in abstract.

The introduction section is too lengthy and so much general information. Please be specific and highlight the study problem, how present study will fill the gap and answer which questions. What are the specific and general objectives.

Why such a high dose of N (400 kg) was applied to plots? What was the exact does of NPK?

L203 when the 10 cobs were sampled?

L219 write the exact date of sampling.

Give more details about the statistical procedures.

If the authors had written Results and discussion as separate sections, then no need to justify results in results section, please remove the cited references.

In the correlation section, please just mention the strong relations and the ones which are of interest, no need to mention the values as already mentioned the tables/figures.

The discussion section is too brief and general. Authors are advised to please elaborate the underlying mechanisms and reasoning while justifying their results.

L394, please clearly mention the range of high temperature which is lethal to crop plants.

Figure 1 gives no or poor information about site.


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: MORPHO-PHYSIOLOGICAL EFFECTS OF ENVIRONMENTAL STRESS ON YIELD AND QUALITY OF SWEET CORN VARIETIES (ZEA MAYS L.)
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this manuscript, the authors studied the effects two environmental stresses, temperature and humidity, on the morpho-agronomic traits of several sweet corn varieties doing the experiments in two successive years, 2019 1nd 2020. The work is interesting and data are promising. However, there are some major issues, especially in writing, as I pointed out below:
1.  Although the authors have provided the key results of this study in the abstract, analysis and overall discussion are not sufficient to understand what are the stress conditions considered and how they affected the results. Since the title of this paper is “effects of stress on …..”, the content of the abstract should be improved accordingly to get a better reflection of the title in the results.
2. L#83-84: “High water (72.7%) and total solids (27.3%) contents of sweet corn kernels significantly contribute to the nutritional value”. Do the authors indicate both moisture and solid as “high”? How can both be high simultaneously in the same kernels?
3. The unit “nmol/g-1 DW” should be either “nmol/g DW” or nmol g-1 DW”
4. Please make proper sub-sectioning in “materials and method” sections. Provide the information on the varieties used in this study and more details on the design of experimentation (plot size, plant numbers in each row, replications etc.). How the data were compared statistically among the varieties and between the years?
5. Results section: analysis and presentation of the results should be stress-oriented rather than emphasizing individual varieties or years.
Experimental design: The authors mostly considered temperature and humidity as the environmental stresses in this study. However, they did not control these conditions artificially as it was a field study and fully depended on the natural fluctuations in two seasons. More importantly, as per the data presented in Table 1, such fluctuations seem to be not so significant. Based on the overall context and aim of this work, my major concern is, why did the authors selected the same time period (June-November) of two cropping years if they really wanted to investigate the effects environmental stress on the morpho-agronomy of the plants? Wouldn’t it be better to consider different seasons for studying such effects?
Validity of the findings: Good
Additional comments: As above


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

Input:
Title: MORPHO-PHYSIOLOGICAL EFFECTS OF ENVIRONMENTAL STRESS ON YIELD AND QUALITY OF SWEET CORN VARIETIES (ZEA MAYS L.)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Good.
Experimental design: Good
Validity of the findings: Good
Additional comments: Now the authors have revised their manuscript with all the suggested changes, it can be considered for publication.


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

Input:
Title: MORPHO-PHYSIOLOGICAL EFFECTS OF ENVIRONMENTAL STRESS ON YIELD AND QUALITY OF SWEET CORN VARIETIES (ZEA MAYS L.)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have made substantial efforts in addressing the reviewer's comments and revising the manuscript. Therefore, I recommend acceptance of the revised manuscript.
Experimental design: Good
Validity of the findings: Okay
Additional comments: None


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

Input:
Title: GENOME-WIDE IDENTIFICATION, EVOLUTION, AND EXPRESSION ANALYSIS OF THE NPR1-LIKE GENE FAMILY IN PEARS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Interesting and considerable work, but needs significant improvement. It is noted that the manuscript needs careful editing by someone with expertise in technical English editing paying particular attention to English grammar, spelling, and sentence structure so that the goals and results of the study are clear to the reader.
Experimental design: Check the below comments.
Validity of the findings: no comment
Additional comments: Manuscript entitled "Genome-wide identification, evolution, and expression analysis of the NPR1-like gene family in pears“, it has been identified nine NPR1-like genes in Chinese pear genome using bioinformatics and molecular biology approaches.This study provided evidence to assist the preliminary PbrNPR1-like gene family information and functional annotation of the nine discovered PbrNPR1-like genes from pear.Below I have highlighted some vital points.
-This potentially interesting study has been marred by an inability to communicate the finding correctly in English and should like to suggest that the authors seek the advice of someone with a good knowledge of English, preferable native speaker.
-The abstract needs to be reorganized. The abstract is wordy and needs to be simplified.
- The last paragraph of the Introduction needs to be rewritten. The results of this research can not be here.
-The results of  “Pbrgene33340” were subsequently lost in Figure 4,Figure 5,Figure 6,and Figure 7.


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

Input:
Title: GENOME-WIDE IDENTIFICATION, EVOLUTION, AND EXPRESSION ANALYSIS OF THE NPR1-LIKE GENE FAMILY IN PEARS
Review round: 1
Reviewer: 2

Output:
Basic reporting: NPR1-like gene plays an important role in stress response. Wei et al conducted the conserved domain and phylogenetic analyses, and identified nine PbrNPR1-like genes. They analyzed the conserved domain and motif, promoter and expression profiling of the NPR1-like genes. In general, the sequence analysis of the gene family is convincing. However, I have some concerns need the author to address. The protein interactions may not be reliable and need to be conducted again. Additionally, the manuscript has many grammatical and phrasing errors, and needs to be reworded by a native speaker or professional editing service.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Abstract
The methods should be removed in the abstract. For example: ''The expression profiles of nine PbrNPR1-like genes in the different tissues were determined through qRT-PCR.''

''The PbrNPR1-like genes structure, conserved motifs, phylogenetics, chromosomal distribution, duplication events, and expression patterns were completely examined.'' These results should be described briefly in the Abstract rather than show what you did.

''The Pbrgene12425 interacted with Pbrgene6286 and Pbrgene8895'' is repeated.

There are many grammatical and phrasing errors, and the English language should be improved by a native speaker or professional editing service. For example, Line 47-51.

Line 227-231, I don’t know how the author named these PbrNPR1-like genes, I suggest the author named these genes with consecutive numbers like previous gene family research or used the gene ID directly.

Line 228-230, ''The length of the PbrNPR1-like proteins ranged from 498 amino acids (Pbrgene40077/34018) to 591 amino acids (Pbrgene12425). The molecular weight of these proteins ranged from 5.45kDa (Pbrgene40077/34018) to 6.54kDa (Pbrgene12425)'' The author had better confirm the molecular weight of these proteins again.

Line 254, ''Fig. 2D'' should be ''Fig. 2C''.

Line 254-257, ''The Clade Ⅰ (Pbrgene12425) with the Clade Ⅱ (Pbrgene6286/8895/8896/2529/8341/33340) have NPR1-like-C terminal region which was proved to be an important part in NPR1-like gene family.'' The author must cite a reference.

Line 264, ''Fig. 2C should be Fig. 2D''.

The legend of Fig. 4, Fig. 5, Fig. 6 and Fig. 7 should be more detailed.

Line 320-321, ''The expression level of Pbrgene6286 gene was the highest in this family'' How did the author know? Each of these genes was just compared to themselves.

The protein interactions make me confused, and the experiment is not reliable.
In the manuscript, the author used Pbrgene12425/6286/8895 gene names, whereas NPR1, NPR3 and NPR4 were used in the Figure 8, these must be consistent.
Line 376-377, ''Green fluorescence was observed in the cytoplasm and nucleus in these combination'' The author used the YFP fusion vector, why they observed it with Green fluorescence.
What is the marker the author used? Could the chloroplast autoﬂuorescence (RFP) be used as the marker of cytoplasm and nucleus?
The negative control is inappropriate. I suggest the author used the YINPR1-YFPn + YFPc or YFPn + YINPR1-YFPc combination.


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

Input:
Title: GENOME-WIDE IDENTIFICATION, EVOLUTION, AND EXPRESSION ANALYSIS OF THE NPR1-LIKE GENE FAMILY IN PEARS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I suggest to accept this manuscript.


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

Input:
Title: GENOME-WIDE IDENTIFICATION, EVOLUTION, AND EXPRESSION ANALYSIS OF THE NPR1-LIKE GENE FAMILY IN PEARS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The new version of the manuscript was greatly improved after the authors' modification. It can be accepted for publication after minor revision.
The legend of Figure 7, "the same'' was repeated.
The author should pay attention to the spaces between data and units, and spaces after punctuation throughout the manuscript, for example, line 311 21:00 pm; line 122 0 h; line 123 0.2 mM.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment