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: COPROID PREDICTS THE SOURCE OF COPROLITES AND PALEOFECES USING MICROBIOME COMPOSITION AND HOST DNA CONTENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the work, the authors present a bioinformatics method to infer and authenticate the host source of paleofeces from shotgun metagenomic DNA sequencing data.
At first look, this is a very good manuscript. At the same time, after its reading it is clear that the authors where first who implemented the identification system.
The authors present a reproducible method using both host and microbial DNA to infer the host source of fecal material of archaeological contexts. This is extremely interesting in ancestral studies. So also about migrations and interactions between peoples and cultures in different geographical sites.
The introduction is clear and shows the context and general background of the investigation. Literature agrees with the characteristics of the work. References are relevant.
In the document the figures are of high quality and explanatory. (Fig 1 A, B, C) although the size is not visible. I suggest putting a unit of measure in the images.
Experimental design: I think that this work is an original research and very high impact. The numbers of samples is adequate and research aims well defined.
I suggest incorporating a map that indicates the sampling points. Add georeference of each point.
Methods described are very details and high probability replicated.
Line 149 DNA extraction: It would be interesting to know if the DNA of paleofeces was quantified , how its amplification is verified. DNA extracted from feces and soil is characterized by having a large interfering amount.
In Figure 2. Workflow schematic of the coproID pipeline. Is it the same reported by Maxime Borry (2018). CoproID: Coprolite Identification. DOI: 10.5281/zenodo.1458163 ?
Line 263 aDNA ancient DNA?
Validity of the findings: no comments
Additional comments: The article is a very complete and complex work. It is a contribution to the knowledge of the ancestral interactions of man and animals. It presents a high impact system for the analysis of information from difficult to treat materials (feces, sediments). The methodology is detailed and well defined. I did not see obvious speculation in the writing. I think it should be accepted 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: COPROID PREDICTS THE SOURCE OF COPROLITES AND PALEOFECES USING MICROBIOME COMPOSITION AND HOST DNA CONTENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The reporting of article is clear and unambiguous and professional English used throughout. Sufficient background data provided and data is shared appropriately.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The current research provides open-source, documented, tested, scalable, and reproducible method to perform the  identification of archaeological paleofeces and coprolite source. The MS is written according to the guidelines and research questions are well defined, relevant & meaningful. Following are few comments to improve article
Please add citation line 84 "Even well-preserved fecal material degrades over time, changing in size, shape, and color"
Follow format for citation line 104
line 153 "protocol D described" protocol D?????
There are no details of statistical analysis performed. Statistical analysis section should be included at the end of Materials and Methods section and details of analysis performed should be included viz., Kruskal- Wallis H-test and Mann- Whitney U test
line 318 coproID??


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: COPROID PREDICTS THE SOURCE OF COPROLITES AND PALEOFECES USING MICROBIOME COMPOSITION AND HOST DNA CONTENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
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: DEEP CONSERVATION OF PRION-LIKE COMPOSITION IN THE EUKARYOTIC PRION-FORMER PUB1/TIA1 FAMILY AND ITS RELATIVES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article is clearly written, well structured and is consistent in its background presentation, description of aims, methods and results.
Experimental design: This article describes a bioinformatics-based study concluding that the prion-like sequential features of the prion-like Pub1 protein, important in stress granule assembly in S. cerevisiae and its human co-ortholog Tia1, which also features prion-like features and has been shown to play a role in certain human neurodegenerative diseases, are deeply conserved across eukaryotes, as is also the prion-like composition of their close relative Nam8/Ngr1. The methods used are the typical ones in this field, and led to solid results. The investigation is basically descriptive: it only offers the raw evidence of evolutionary persistence, without providing additional insights that might be useful to understand neurodegenerative diseases in humans, or basic mechanisms of granule formation.
Validity of the findings: no comment
Additional comments: Very little is said in the discussion about Nam8/Ngr1, which is a very interesting protein with a prion-like domain and has been found to be involved in or correlate with  a number of human diseases.


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: DEEP CONSERVATION OF PRION-LIKE COMPOSITION IN THE EUKARYOTIC PRION-FORMER PUB1/TIA1 FAMILY AND ITS RELATIVES
Review round: 1
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: DEEP CONSERVATION OF PRION-LIKE COMPOSITION IN THE EUKARYOTIC PRION-FORMER PUB1/TIA1 FAMILY AND ITS RELATIVES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Basic reporting

•	Clear, unambiguous, professional English language used throughout.

The manuscript is clearly written in English.

•	Intro & background to show context. Literature well referenced & relevant.

Introduction and background sound in a good shape. Literatures are well referenced.

•	Structure conforms to PeerJ standards, discipline norm, or improved for clarity.

Yes.

•	Figures are relevant, high quality, well labelled & described.

Figure 1, 2, and sup figure 3: it is necessary to provide tables (e.g. excels files) showing exact scores/numbers from the PLAAC program, the IUPRED2a program and the Pfam database for each protein. Someone may want to know the exact numbers of a particular protein.

Figure 4, figure legend: “The intervals of …. 5 for 1-10, etc”. This sentence is just a copy from the figure legend of Figure 3, and need to be fixed.

•	Raw data supplied (see PeerJ policy).

Yes.
Experimental design: EXPERIMENTAL DESIGN

•	Original primary research within Scope of the journal.

The manuscript fits to the scope of the journal.

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

Not really. The authors should more explain the rationales behind these sequence analyses.

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

I don’t think so. The authors relied only on one program to detect the prion-like domains or IDRs. There are some other programs for these tasks. For annotation of RRM, they used only Pfam. Prosite can do the same task. Using the multiple programs/databases would increase reliability of this investigation.

•	Methods described with sufficient detail & information to replicate.

Yes.
Validity of the findings: VALIDITY OF THE FINDINGS

•	Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.

As describe above, this manuscript will be meaningful for other researchers when the authors provide the tables of all the scores/numbers of each protein.

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

The same as above.

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

Conclusions are well stated. However, since the original research question is unknown, it is not linked to the conclusions.

•	Speculation is welcome, but should be identified as such.

The lase statement in “Conclusion”: The results of the sequence analyses suggest, but not provide evidence, of prion-like aggregation of these protein families.
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: DEEP CONSERVATION OF PRION-LIKE COMPOSITION IN THE EUKARYOTIC PRION-FORMER PUB1/TIA1 FAMILY AND ITS RELATIVES
Review round: 1
Reviewer: 4

Output:
Basic reporting: In this paper, Su et al. develop a pipeline to study prion-like conservation across homologues of Tia1/Pub1 protein family. First, they analyse its evolution inside the Saccharomycetes genus, then they extend it to all eukaryotes. They also check the degree of disorder and number of RNA-binding regions for each homologue. They reach the main conclusion that this protein family tend to evolutionary conserve prion-like behaviour, certain degree of disorder and RRM domains. Despite the technical part of the work is generally correct, it is my opinion that the results provide just incremental knowledge relative to previous studies and therefore that their potential impact in the area do not justify their publication in PeerJ.
Main Comments

1.	The introduction although well-written, fails to provide context to the results section manuscript. It does not mention why authors consider disorder content or why they believe RRM domains conservation is important for function. Moreover, the description of the Nam8/Ngr1 pair is poor and unconnected. The introduction would benefit from a broader description of the Nam8/Ngr1 family or its human homologue TRNAU1AP. In this line, Pab1 protein (for which authors compare sequential identity) is not introduced either.
Phrase in lines 61-63 could is poorly connected with the adjacent context.

2.	Authors do not justify why they check disorder content. They choose IUPred2 as the only methodology behind overall disorder, and the % of amino acids in the whole protein predicted to be disordered as a method to quantify it. PLAAC amino acid scoring system has a first bias towards disordered regions (highly scoring Q,N,Y,G and poorly scoring L,C,F,w,I,V). Although this is not exactly IUPred2's scoring system (for instance, IUPred2 also scores positively charged-residues), their predictions are expected to have a degree of overlap. This could be improved by applying several disorder predictors or a consensus method (like MobiDB, Piovesan et al. 2017). Furthermore, the quantification of disorder percentage for the complete protein and its relevance for the protein behaviour should be better defined and region known to have folded structures excluded from the calculations and comparisons.

3.	It is not clear for unspecialized readers why the authors use the KMDA methodology for the alignments.

4.	Yeast prions are modular. The manuscript will benefit from a study of the relationship between the position of RRMs and that of predicted prion-like domains. In a large majority of proteins, prion-like domains are located at the extremes of the proteins, this allows the assembly of the prion-like domains into fibrillar structures, keeping the globular domains appended to them and hanging from the fibrillar structures. It is difficult to envision how this function can be done by an internal sequence when you have more than two prion-like domains. In the lack of experimental evidence for the functional role of internal predicted prion-like domains, especially in the Tia1/Pub1 family, the relevance of these predictions remains questionable.

5.	Conclusions should be improved to clearly reflect strictly what is demonstrated in the results section.

Other comments:
Where the authors refer to “Uniprot names it should be more correct to state Uniprot Accession number.
-Line 81. Authors should state for which clade they downloaded proteins from OrthoDB and their number. Authors state that “only OrthoDB proteins from the Uniprot reference fungi were considered for further analysis”, but further analysis includes proteins from all Eukarya. The paragraph should be rewritten to reflect how authors acquired and filtered homologues to Pub1/Tia1.
-OrthoDB and UniprotKB and PfamDB releases should be stated.
-"ASTRASCOP" should be changed for "Astral SCOPe". The downloaded release should be stated.
- Clarify if the LLR threshold 15 has any experimental basis or is completely arbitrary?
-Authors should explicitly state how the protein families were compared in BLASTP to conclude the most similar RMM-domain-containing protein family was Pab1. Moreover, a graph showing the top ranked conserved protein families would benefit the manuscript.

Figure 1.
-Authors do not explain what "Support values" represent.
-The support values colour code for black is missing.
-The scores next to I3GXC2 represent LLR? Should be clearly stated either at the top of the histogram axis or specified at the legend.
-Image could be more easy to understand by showing in bold Uniprot Accession numbers for Nam8, Pub1 and Ngr1.

Figure 2.
Same 2 first points as fig 1.

Figure 3
Colour code in legend is difficult to understand.
Probability value represents one or two tailed p-value?
Figure panel should contain same (or similar) size plots. And panel letters should be aligned.

Figure 4
Legend in figure 4 should be rewritten:
 -Legend should state "color coding same as Figure 3", not "2", unless it is clearly stated the regrouping modifications done as it is in figure 3. Moreover, the color scheme in legend is repeated twice.
 -The phrase "The intervals of prion-like scores are labelled with the value of their midpoints, i.e. 5 for 0-10, etc" does not seem to correspond with the Figure or if it does is difficult to understand what does it refer to.
Experimental design: Included in the Basic reporting
Validity of the findings: Included in the Basic reporting
Additional comments: Included in the Basic reporting


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

Input:
Title: DEEP CONSERVATION OF PRION-LIKE COMPOSITION IN THE EUKARYOTIC PRION-FORMER PUB1/TIA1 FAMILY AND ITS RELATIVES
Review round: 2
Reviewer: 1

Output:
Basic reporting: As in first submission.
Experimental design: As in first submission.
Validity of the findings: As in first submission.
Additional comments: The Authors have addressed my suggestion of including background information about Nam8/Ngr1.


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: DEEP CONSERVATION OF PRION-LIKE COMPOSITION IN THE EUKARYOTIC PRION-FORMER PUB1/TIA1 FAMILY AND ITS RELATIVES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The author accordingly responded to my comments and other reviewers' comments. The manuscript is ready for publication.
Experimental design: NA
Validity of the findings: NA
Additional comments: NA


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: DISTINCT GENE EXPRESSION AND SECONDARY METABOLITE PROFILES IN SUPPRESSOR OF PROSYSTEMIN-MEDIATED RESPONSES2 (SPR2) TOMATO MUTANTS HAVING IMPAIRED MYCORRHIZAL COLONIZATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors report about a comparison between wild type and spr2 (JA biosynthesis) mutant tomato plants with respect to colonization by mycorrhizal fungi after about seven weeks of colonization. They analyzed an extremely wide variety of parameters including  a wide range of transcripts but also many metabolites (many with unknown identity) across various biosynthetic pathways. The ms is nicely written and presents its content in a good english but the mere wealth of information and the many ups and downs of transcripts and metabolites  (some significant, some not) makes it very difficult to find out any truly significant and meaningful results. The figures and tables are clear and nicely presented including appropriate statistis in most parts but again it is very difficult to distill relevant information from the huge amount of data presented, much of it having non-significant or contradictory alterations, whose reproducibility is doubtful.
Experimental design: One point that adds to this enormous complexity and complicates conclusions very much is the use of three different colonization levels defined by microscopic analysis of mycorrhizal structures originating from three independent experiments (Fig. 1). The authors themselves admit that they are unaware what caused these variations. Perhaps it is in part due to the different seasons when these experiments/plant cultivations were performed  but the authors do not assign a distinct cultivation season to a distinct colonization level yet this information should perhaps be added.
Validity of the findings: A major problem with this experimental design is that the three colonization levels are not that clearly distinct in all aspects and that non-mycorrhizal controls are shown only in some cases. The „high colonization“ is characterized only by high arbuscule frequency (Fig. 1) whereas frequency of colonization (F%) is not different from „medium colonization“ and only slightly above „low colonization“. Most importantly, many conclusions are made for specific colonization levels only (e.g. even in the title) and this is very problematic when looking at the rather minor differences between colonization levels in some cases (all regarding differences in wild type genotype, spr2 background has no significant differences at all).  I have strong doubts that these results will be reproducible in future experiments. The whole concept of comparing different colonization levels can only work if there are really clear differences between such levels. With the current variability between colonization levels I would limit the analysis to one (strong oder medium) colonization level and compare it to the non-mycorrhizal state (which is actually missing in many but not all  data displays of the current ms, better have it uniform). Some of the molecular marker data (e.g. LePT4 that I also see as a reliable marker for a mycorrhiza with functional arbuscules) are in at least partial contradiction (for medium colonization) with the colonization levels defined by the microscopic analyses. Also, some genes involved in specific biosynthetic pathways appear to be regulated in a strange manner, e.g. repression of CCD7 and CCD1 during high colonization (Tab. 1) and high arbuscule abundance (high LePT4 (Tab. 1) , high arbuscule score by Mycocalc (Fig. 1)) - makes no sense at all. Also, the glycosyl transferase CGT1 (highly uncertain if involved in pathway) is not induced in high colonization roots. Moreover, some of the data obtained from leaves are also very strange, e.g. that LePT4 is induced in leaves – contrary to all previous reports. The only rather clear conclusion seems to be that JA biosynthesis-deficient plants are moderately compromized in mycorrhizal colonization (not a new finding). Moreover, the reduction of tomatine in the strongly colonized state could be interesting but this is then not represented in the title.
Additional comments: The ms adds to rather than solves the current confusion of whether JA is relevant for mycorrhization and if so whether it is acting positively or negatively and in what stages of mycorrhiza development. The ms lacks a clear focus. Already the title is very difficult to understand and misleading and it is not clear why one should have an interest in what is going on during „suboptimal“ mycorrhizal colonization and why a „suboptimal“ state should be important. In many cases results are presented as „tended to be significant“ or similar. Statements should be limited to clearly significant changes with very few exceptions.  These problems are not only due to the use of the different colonization levels, which in my judgements (as discussed above) are not distinct enough, but this concept adds to the problems of the ms. A simple comparison of non-mycorrhizal vs mycorrhizal states for the two genotypes throughout the ms would probably have been the better choice. The discussion is very knowledgeable and phrased nicely but is much too long.

MINOR POINTS
The review of Gutjahr, 2014 does not cover JA. Other references are mostly adequate.


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: DISTINCT GENE EXPRESSION AND SECONDARY METABOLITE PROFILES IN SUPPRESSOR OF PROSYSTEMIN-MEDIATED RESPONSES2 (SPR2) TOMATO MUTANTS HAVING IMPAIRED MYCORRHIZAL COLONIZATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Casarrubias-Castillo and co-workers aims to deepen in the role of JA in the regulation of the AM symbiosis. For that, they analyze the transcriptional and metabolic profiles of the JA-deficient mutant spr2 compared with its corresponding wild-type accession, and at different colonization levels (‘low’, ‘medium’ and ‘high’). The topic is timely and interesting. The authors have done a lot of work, and presented a manuscript with a good and updated Introduction, and well described Materials and Methods. However, probably because they have tried to present too much data, Results and Discussion sections are a bit messy, making the reading and comprehension difficult for readers. It must be more focused. Moreover, the paper is not compensated, as the Discussion is about 50% of the whole manuscript. This section should be reduced and be more focused.
Experimental design: Experiments were well design and, as I mentioned before, Material & Methods are well described. Just one remark, in order to obtain 3 different levels of mycorrhization, instead doing 3 independent experiments, it might be better to performe a time course assay and repeat it twice.
Validity of the findings: As I said, Results and Discussion sections are a bit messy, which difficults the reading and comprehension of the manuscript. In my opinion they should be more focused, specially the Discussion. Moreover, maybe because auhtors wanted to present so many data and information, some conclusions are not well stated. In some cases, marker genes do not behave as expected. I suggest removing the 'medium' mycorrhization level experiment to get rid of the background noise. By doing so, I think data will be more reliable.
Additional comments: The manuscript by Casarrubias-Castillo and co-workers aims to deepen in the role of JA in the regulation of the AM symbiosis. For that, they analyze the transcriptional and metabolic profiles of the JA-deficient mutant spr2 compared with its corresponding wild-type accession, and at different colonization levels (‘low’, ‘medium’ and ‘high’). The topic is timely and interesting. The authors have done a lot of work, and presented a manuscript with a good and updated Introduction, and well described Materials and Methods. However, probably because they have tried to present too much data, Results and Discussion sections are a bit messy, making the reading and comprehension difficult for readers. It must be more focused. Moreover, the paper is not compensated, as the Discussion is about 50% of the whole manuscript. This section should be reduced and be more focused.

As mentioned, my main concern is about the huge amount of data presented, where some of them are not relevant for the main goal. Thus, tables with transcriptomic and metabolic data could be reduced in order to show those that are key for the paper. The whole set of data could be presented as supplemental. Many times ‘less is more’! In the same line, I think that data showing the ‘medium’ colonization levels could be removed, because its mycorrhizal levels are either equal to the ‘low’ or to the ‘high’ samples (see Fig 1). In any case, the two latter are the most contrasting and they would be sufficient for comparison. Moreover, some of the ‘rare’ results come from the analysis of the ‘medium’ samples. Thus, I think that removing them, authors could get clearer conclusions.

The results about the 9-LOX and 13-LOX pathways are very strange. spr2 encodes for a ω3 fatty acid desaturase FAD7, thus blocking not only the 13-LOX pathway producing jasmonates, but also de 9-LOX giving rise to other oxylipins. This fact must be considered, as other JA-deficient mutants are not altered in mycorrhization. On the other hand, even though spr2 is defined as a JA-deficient mutant, I suggest quantifying hormone levels, at least JA, since they are crucial to understand the changes observed along mycorrhization.

Physiological parameters such as plant growth, mycorrhizal dependency, fitness, etc should be included in the manuscript in order to get an idea about the phenological stage of the plants.

Transcriptional results with different marker genes are strange. For instance, DXS2, a well characterized mycorrhizal marker, here is not induced in myc plants. Regarding the metabolic data, the authors could present those that change, independently of the p value (0.05, 0.01 or 0.001). They might also group these compounds by families better than by m/z. This would help to make the results clearer.

Minor points

- Figure legends are poorly described

- Some marker genes used are not properly described in the text. Please explain what the different marker genes are, indicating the corresponding references.

- Line 267, ‘AMF colonization levels coincided with expression of LePT4’. According to Fig.1 and Table 1, this is not completely true. The same happens for the gene CCD7. Please check and correct.

- Instead presenting the results of the a-tomatine as tables, I suggest showing them in a figure with the biosynthetic/catabolic pathway, indicating which compounds are up or down. This would help to globally visualize how this pathway is altered by AM symbiosis.


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: DISTINCT GENE EXPRESSION AND SECONDARY METABOLITE PROFILES IN SUPPRESSOR OF PROSYSTEMIN-MEDIATED RESPONSES2 (SPR2) TOMATO MUTANTS HAVING IMPAIRED MYCORRHIZAL COLONIZATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comments
Experimental design: Please explain why non-mycorrhizal Wt and spr2 roots or shoots haven’t been considered for the gene expression analyses.
Validity of the findings: The authors analyzed gene expression and metabolite profiles of mycorrhizal wild-type and jasmonic acid deficient (spr2) tomato mutants. Colonization levels were determined by a non-vital staining technique (trypan blue staining) and by transcript analyses of one plant (LePT4) and 2 fungal marker genes (18S Gi and EF1-α Gi). The authors used the staining data to classify experiments as yielding “low”, “medium” and “high” colonization and discussed transcript and metabolite data in context of this classification.

Remarkably, the transcript levels of the AM marker genes don’t correlate with the results obtained from the trypan blue staining. How do the authors explain the opposite results obtained with trypan blue staining and qPCR? Could the authors include more marker genes to validate the findings obtained with trypan blue?

Some of the expression data presented in context of the three colonization levels are not in line with previously published data, e.g. CCD1 transcript levels in colonized wild-type plants decrease when colonization levels are higher (line 297). These can be indications that the staining data don’t reflect the colonization status in the part of the root system used for transcript and metabolite analyses (root system was divided lengthwise during harvest). I am highly concerned that current way of presenting the data is misleading. I suggest presenting transcript and metabolite data in context to colonization levels classified based upon AMF-marker genes which reflect the colonization level of the material used for these analyses.
Additional comments: The authors provide an extensive set on transcript and metabolite data from wild-type and spr2 mutant plants that may lead to additional findings in how JA signaling affects AM symbiosis if presented in the correct way.


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: DISTINCT GENE EXPRESSION AND SECONDARY METABOLITE PROFILES IN SUPPRESSOR OF PROSYSTEMIN-MEDIATED RESPONSES2 (SPR2) TOMATO MUTANTS HAVING IMPAIRED MYCORRHIZAL COLONIZATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: In this new version, the authors have addressed all or most of the reviewer’s suggestions and introduced changes accordingly. They have removed some of the previous experiments that were ‘controversial’ and included 2 new mycorrhizal experiments. The MS has been clearly improved. It reads better, and the results, conclusions and final message are much clearer now.
Experimental design: As mentioned, 'strange' data from previous experiments were removed. With the inclusion of 2 new experiments the results are more focused and clear now.
Validity of the findings: Results from 3 independent experiments are represented, so the results are consistent and the conclusions well stated. Only one remark, although the authors made an effort to explain the discrepancy with the results regarding the mycorrhizal marker gene LePT4, they fail to find a 'feasible hypotesis'.
Additional comments: This is a new version of previous MS PeerJ-34877, which has been surely improved. The authors have addressed all or most of the reviewer’s suggestions and introduced changes accordingly. They’ve eliminated some of the previous experiments that were ‘controversial’ and included new mycorrhizal experiments and analyses. The MS reads better, and the results and message are much clearer now.

I only have a few remarks, according to the ‘physiological’ data, spr2 is smaller, and it has less chlorophyll content, etc. The same applies to transcriptional data. The comparison between the two genotypes is not made, and I think is important. On the other hand, it should be nice to analyse the expression of all set of genes in the same samples. Thus, you’ll have a better comparison.

The authors made an effort to explain the discrepancy with the results of the marker LePT4. This gene is also a good mycorrhizal marker in tomato, and there are several papers that prove it.

Minor points:

- In line 158, in addition to the final concentration of P used (7 µM) indicate the ‘final’ percentage to get an idea of the reduction.

- In M&M when describing the qRT-PCR analyses, instead using 3 technical replicates of a pool from 3 biological samples, it should be better to analyse the biological replicates per separate and then calculate the SE.

- I’d include the Trouvelot data from E1, at least as supplemental, not just in the text.

- Since the MS is mainly based on the changes produced in roots, when analysing the metabolic profiles I’d start by the roots instead the leaves.

- In Fig. 3, the letters a, b, c and d are not present.


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: DISTINCT GENE EXPRESSION AND SECONDARY METABOLITE PROFILES IN SUPPRESSOR OF PROSYSTEMIN-MEDIATED RESPONSES2 (SPR2) TOMATO MUTANTS HAVING IMPAIRED MYCORRHIZAL COLONIZATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: Casarrubias-Castillo completely revised their manuscript which aims to explain reduced AMF colonization in spr2 mutants by analyzing transcript and metabolite data. Instead of using three different colonization levels for the interpretation of their data, the authors performed two additional experiments that mirrored the high colonization levels from the first experiment and included these data in the revised version. Additionally, relevant hormone measurements were included (JA and SA). I doubt that the addition of targeted metabolite analyses in roots and leaves (table 2 & 3) really contributes to the aim of the paper. In fact, the discussion of some of these data is very speculative (discussion section lines 734 – 742) and can be removed.
The manuscript is nicely written. However, some parts of the discussion are very speculative and can be shortened. One example was already mentioned, another one is the discrepancy between mycorrhizal colonization and LePT4 expression which will be explained in the “comments to the author’ section.
The figures are relevant. The placing of the lower case letters in figure legends 2, 3 and 5 is confusing. Additionally, figure legend 3 refers to lower case letters (a-d) that are missing in the figure.
Experimental design: As already mentioned, the authors performed two more experiments to validate their data (E2 and E3). For these two studies, an AMF consortium consisting of six AMF species was used. From the Material and Methods part it is not clear if these two additional experiments were run in parallel and started at the same day, or setup at different time points. This information may be useful for the interpretation of some data.
Validity of the findings: Colonization data of these two additional experiments align very well with an experiment performed with only one single AMF species (E1, 50dpi). However, LePT4 expression data for E2 and E3 don’t align with colonization data obtained from fungal staining. The current MS includes expression data only for selected genes where E1 data were added instead of 45dpi expression data. It would be helpful if the authors include all gene expression data from E1 to table 1 to show the reproducibility of the data. This can be easily addressed by adding 50dpi columns for WT and spr2 in Table 1.
Additional comments: LePT4 expression and expression of other genes known to be induced during AMF colonization (e.g. CCD7, LHA1) keeps looking strange and I am not satisfied with the discussion of this discrepancy. As outlined by the authors in their comments to the reviewer, the current way of presenting the expression data reflects the expression relative to the non-mycorrhizal plants (2-ΔΔCt method). I am wondering if useful information, e.g. expression of genes in non-mycorrhizal roots of spr2 plants, is lost when this method is used. I suggest presenting the expression data for each genotype (WT and spr2) and treatment (non-mycorrhizal and mycorrhizal) separately by calculating gene expression relative to the reference gene (2ΔCt). Maybe some genes are differentially regulated in non-mycorrhizal spr2 roots?  This finding may open a more reliable avenue for interpretation. The current way of discussing the discrepancy between fungal staining and LePT4 expression is very speculative.
The authors report testing of other AMF marker genes to support their findings in their comments to the reviewers. Why can’t these data be included in this MS?


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: IDENTIFICATION OF GENES INVOLVED IN THE EVOLUTION OF HUMAN INTELLIGENCE THROUGH COMBINATION OF INTER-SPECIES AND INTRA-SPECIES GENETIC VARIATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language must be improved throughout the article. I provided a few suggestions covering all up to the results section, but ultimately reckoned this to be to daunting a task for me to address. I suspect that no person with English as mother tongue has had any part in writing this manuscript. I strongly advise the authors to submit it to a native English speaker with adequate scientific knowledge for proofreading before resubmission.
The article's context is otherwise relatively clearly stated and the literature references seem satisfactory. I annotated the text in the submission pdf whenever I felt a reference was missing.
The methods and results section intersect each other somewhat. I think the article would benefit from a more clear separation between method description and elucidation of results. The figures are mostly fine. I annotated the submission pdf wherever I found anything amiss with them. Figure 2 could perhaps benefit from higher resolution.
Experimental design: I think the study is original and in line with the aims and scope of the journal. The research question is clear. The aim of this study is quite ambitious. To what extent the highlighted knowledge gap is filled is arguable, but the effort made by the authors is commendable.
The analyses are well described and should be replicable. I think the results should be elucidated more systematically. The authors found 40 genes with intra-species variation and potential evolutionary salience but do not discuss all of them.
Validity of the findings: I think the conclusions of the study are commensurate to its results. The only reservations I have are about the title and the emphasis on the novelty of the method in the abstract. As far as I understood, the authors essentially searched and combined multiple databases. The comparative genomic analysis they performed served only the purpose of pruning existing (fixated) human-specific variation databases (by the way, how much does this pruning actually affect the results? is it necessary?). I think the title should be changed to highlight the combination of inter- and intra-species variation and de-emphasize the use of comparative genomics analysis. Also, the last statement of the abstract should be downplayed or possibly removed entirely.
Additional comments: I was particularly bothered by the ingenuous, Homo-centric language used by the authors throughout abstract and introduction. I feel that phrases such as “the evolution of human intelligence from great apes to humans” or “humans are much more intelligent than chimpanzees” do not belong in a scientific publication. It is common to use our chimpanzee cousins as evolutionary proxy of our common ancestor but it is a misconception to presume a temporal relationship between apes and humans, and one that should not be reiterated in a scientific work, even if only “in a manner of speaking”. Also, intelligence is a very hard concept to define and depends to a great extent on the context in which one attempts to measure it. In fact, it depends just as much on the context as it does on the subjects that are inserted in that context. The journal itself is called “Life & Environment”. One shouldn’t judge a fish by how skilled it is at climbing trees.
To sum up, I think the substance of the manuscript may remain as is, with only those few additions I indicated, but its form should be considerably improved. Make no mistake, I do consider the needed revisions major ones.


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: IDENTIFICATION OF GENES INVOLVED IN THE EVOLUTION OF HUMAN INTELLIGENCE THROUGH COMBINATION OF INTER-SPECIES AND INTRA-SPECIES GENETIC VARIATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors have used clear and professional English in their article.
They have described in detail the various GWAS from which their genes baits are derived. They provide sufficient literature references. However, their work is not merely reporting their findings but they also claim that “the work provides a new method in searching for the key genes in human evolutionary genetics.” In such a case, I would suggest the authors give more background and write about other methods that are currently used in the field and compare to theirs. This will provide a better context of the strength or weakness of their method. I feel the authors have moved rather abruptly to specific information about the genes in line 46-47 and then the authors go back to describing other studies in line 58 onwards. Here, I would suggest writing a bit about the methods and shortcomings of those studies, not just their findings.
In line 72-75 the authors claim the previous reference genomes are not qualified enough but do not expand on why they are not qualified.
Minor point: I was not able to find reference to Figure 2 anywhere in the text. Some typographic errors such as spacing between words in the figure legends 1, 2 & 3.
Experimental design: The investigation seems to be of high standard but again, I had some doubts over the research questions and if they had been answered well. Was the aim to find genes associated with the evolution of intelligence or to devise a new method of finding such genes? Either way, the authors should place their results within the larger context of the field and discuss the strength and weakness of their method.
Validity of the findings: no comment
Additional comments: The authors have utilized a novel method to provide a list of candidate genes associated with evolution of intelligence in humans. While the genetic results are interesting, it would be helpful if the authors could discuss the potential weakness of their method and how they have built upon existing methods. The manuscript could improve with some elucidation and clarification about the method.


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: IDENTIFICATION OF GENES INVOLVED IN THE EVOLUTION OF HUMAN INTELLIGENCE THROUGH COMBINATION OF INTER-SPECIES AND INTRA-SPECIES GENETIC VARIATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have revised all instances of ingenuous or Homo-centric language, which were my major concern in their first draft. The language, however, still needs work. The authors have corrected a few but not all errors and have introduced new ones. I have applied some edits up to the results section in an annotated pdf but do not think the article's form meets the standards for a publication yet. I therefore reiterate my recommendation to submit the manuscript to a native English speaker for proofreading.
A couple of additional issues remain. They are commented on in the annotated pdf.
Experimental design: no comment.
Validity of the findings: no comment.
Additional comments: The descriptions of some of the submitted files appear with typos in the reviewer's dashboard. I'm not sure this is something the authors or the journal editor should fix but it would be best if it was not propagated.


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: IDENTIFICATION OF GENES INVOLVED IN THE EVOLUTION OF HUMAN INTELLIGENCE THROUGH COMBINATION OF INTER-SPECIES AND INTRA-SPECIES GENETIC VARIATIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have improved the language and flow of the manuscript. The authors have provided more background and context after revision.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have given more context and elucidated the points that were asked of them and the language is much better now.


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 PRECLINICAL PHARMACOLOGICAL STUDY ON HX0969W, A NOVEL WATER-SOLUBLE PRO-DRUG OF PROPOFOL, IN RATS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Authors already reported the acitivities of HX0969W,
1) Sichuan Da Xue Xue Bao Yi Xue Ban, 2015 Mar;46(2):214-7.
2) Anesth Analg. 2014 Apr;118(4):745-54.
3) Br J Anaesth. 2013 Nov;111(5):825-32.
4) Bioorg Med Chem Lett. 2013 Mar 15;23(6):1813-6.
5) Eur J Mass Spectrom (Chichester). 2011;17(4):405-13.

It needs to provide sufficient rational reason for this study.
Experimental design: no comment
Validity of the findings: Because the previous report, couldn't found the impressive novelty.
Additional comments: 1. To verifying the preclinical analysis, authors need to detect the metabolic enzymes such as p450 families with mRNA levels.
2. Figure 1 was showed several times in previous publication therefore don't need anymore here.
3. Author need to discuss in detail about differences between previous 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: THE PRECLINICAL PHARMACOLOGICAL STUDY ON HX0969W, A NOVEL WATER-SOLUBLE PRO-DRUG OF PROPOFOL, IN RATS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	As it was described in article, the major objective of this study was to develop a new water-soluble pro-drug of propanol with non-toxic metabolites. (Line 223 & 224). Therefore, I think propofol should be taken as the major reference standard, when they discussed pharmacokinetics and pharmadynamics of the novel substance HX0969W, rather than fospropofol disodium. While surely when it comes to toxic metabolites, HX0969W should be compared with fospropofol disodium.
As we all know (and as the writer pointed in Line 248), propofol has been the most widely used intravenous anesthetic in clinical practices due to its rapid onset and recovery from sedation. Unfortunately, the novel substance HX0969W as a prodrug was deprived the characteristics.
It was described in the article like
“For onset time, propofol (0.4±0.1 min) had a shorter time to generate LoRR than HX0969W (1.8±0.4 min, p=175 0.017) and fospropofol disodium (2.1±0.7 min, p = 0.006). However, the duration of the LoRR 176 for HX0969W (75.8±9.6 min, p = 0.036) and fospropofol disodium (68.5±18.4 min, p= 0.041) 177 were significantly longer than that caused by propofol (27.1±6.0 min).” (Line 173-177)
“In this pharmacokinetic study, the longer onset time and duration of HX0969W and fospropofol disodium compared to propofol were owing to their pharmacokinetic features.” (Line 210-212)
“The time to achieve maximal concentration of propofol by HX0969W and fospropofol disodium was lengthened at 8 to 9 fold than propofol after intravenous administration. “ (Line 234)
In my opinion, the longer onset time and recovery time may not be well received by clinical anesthesiologists. It might not be highlight, but be a disadvantage.

2.	As it was suggested in article, “Therefore, a slow-released propofol from HX0969W and fospropofol disodium reduced the risk of apnea, cardiovascular system depression (bradycardia, arterial blood pressure, systolic blood pressure, cardiac output, cardiac index, stroke volume index, and systemic vascular resistance), and convulsion (Line 244-247)”, which can be a satisfied reason to develop that a prodrug with longer onset and recovery time. Therefore, please provide solid support for the opinion.
It could be widely acceptable literatures (I haven't seen in manuscript), or an additional experiment to compare the effect of HX0969W and propofol on rat circulatory system, like blood pressure, QT interval, etc.

3.	The data list in tables were not matched with figures, making the Cmax is obvious and really confusing. Please do not use mean±SD and mean±SEM alternatively, just use same format in a whole article.
Experimental design: 4.	Quantification of HX0969W, fospropofol disodium and propofol in rat plasma was necessary for pharmacokinetics study, thus the methodology work should be properly displayed.
At least typical chromatogram, linear regression of standard samples and precision and accuracy of quality control samples, etc.
For HX0969W and fospropofol disodium, they were analyzed via LC-MS/MS without stable isotope labeled internal standard, thus a matrix effect test could be taken into consideration.
For propofol, the focal point may be selectivity. Propofol would be or not be interfered by HX0969W and fospropofol in a HPLC-fluorescence assay, which should be clarified.
Methodology could be submitted as supplementary materials.
Validity of the findings: have no comments.
Additional comments: NA


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 PRECLINICAL PHARMACOLOGICAL STUDY ON HX0969W, A NOVEL WATER-SOLUBLE PRO-DRUG OF PROPOFOL, IN RATS
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: The purpose of your experiment was to study the sedative-hypnotic effects of HX0969W, but did you consider the effect of pentobarbital sodium on your results at the end of the experiment.
Validity of the findings: Although HX0969W and fospropofol disodium had a longer time and longer duration to produce LoRR than propofol in rats, according to your pharmacokinetics, they take longer to work, how do you explain this.
Additional comments: You need to add more experiments to prove that HX0969W is superior to propofol in anesthetic effect


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 PRECLINICAL PHARMACOLOGICAL STUDY ON HX0969W, A NOVEL WATER-SOLUBLE PRO-DRUG OF PROPOFOL, IN RATS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors addressed the comments.
Experimental design: Authors addressed the comments.
Validity of the findings: Authors addressed the comments.
Additional comments: Authors addressed the comments.


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

Input:
Title: THE PRECLINICAL PHARMACOLOGICAL STUDY ON HX0969W, A NOVEL WATER-SOLUBLE PRO-DRUG OF PROPOFOL, IN RATS
Review round: 2
Reviewer: 2

Output:
Basic reporting: I have reviewed the resived manuscript and all the supplementary material.
It is still a hypothesis that the pro-drug could show lower incidences of hypotension, but now I accept it is reasonable since the author has given some evidences for supporting their opinion.
I have no further comments.
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: LNCRNA LOC100506178 PROMOTES OSTEOGENIC DIFFERENTIATION VIA REGULATING MIR-214-5P-BMP2 AXIS IN HUMAN BONE MARROW MESENCHYMAL STEM CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the abstract and conclusion, the authors mentioned about in vivo ectopic bone formation. however, the results regarding the in vivo work are not present in the submitted manuscript.

The authors should include that data if available or remove the in vivo claim in the manuscript.

The authors should comment on how LOC100506178 changes during  osteoblast differentiation using ascorbic acid and beta-glycerophosphate, if known.


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: LNCRNA LOC100506178 PROMOTES OSTEOGENIC DIFFERENTIATION VIA REGULATING MIR-214-5P-BMP2 AXIS IN HUMAN BONE MARROW MESENCHYMAL STEM CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study explored the regulatory mechanisms between LINC00968 and miR-3658 during osteogenic differentiation of hBMSCs. The study is interesting.
Experimental design: The experimental design is good, but some experiments need to  improve.
Validity of the findings: Some findings need to  improve.
Additional comments: The study explored the regulatory mechanisms between LINC00968 and miR-3658 during osteogenic differentiation of hBMSCs. The study is interesting, but some concerns need to be addressed.
1.The manuscript mentioned “ectopic bone formation in vivo”, but I do not find the data in “methods and results”, and please add the data.
2. Figure 2B, please provide three times the results of Alizarin Red S Staining and make quantitative analysis.
3. Figure 2C and Figure 3C, please provide the results of ALP activity on day 3 and day 7, because ALP activity is a marker at the early stage of osteogenesis differentiation. In addition, what is the unit of ALP activity in your study.
4. Figure 5 in raw data, the authors need to provide more images of the original western bolts.


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 FOR THE BREAST MUSCLE OF JINGHAI YELLOW CHICKEN AT EARLY GROWTH STAGES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Major issues
- Throughout the manuscript there is an issue with grammar and written English which is very poor in parts - it should be edited by a native English speaker before being re-submitted.
- The discussion section starts nice, with good appointments, but at some point, I think you're lost. And, the final part is a little bit confusing. Re-write the last paragraphs of this section and give better closure.

Minor issues
Some of the suggested corrections are in the .docx file (attached).

Lines 50-52: The background description is poor. Write again, please.
Lines 73-80: Re-write this paragraph with better grammar.
Line 86: change this expression - "biggest factor".
Lines 94-95: Re-write this sentence with better grammar - "Among them, transcriptome...".
Lines 104-104: Split this sentence to better comprehension.
Line 119: explain "lnc" from lncRNAs. All abbreviations should be explained in parenthesis when it appeared for the first time.
Lines 129-134: Let this information for the Materials and Methods section.
Lines 219-221: Re-write this sentence as it doesn't make sense as is.
Lines 236-237: change to "Figure 1 shows the DEG with P-adjust ≤ 0.05".
Line 237: Do not start a sentence with numerals.
Line 240: Do not start a sentence with numerals.
Line 243: Do not start a sentence with numerals.
Line 246: Do not start a sentence with numerals.
Line 250: The Ensemble gene ID is not necessary, add gene description inside the parenthesis.
Line 251: change the word "strains".
Lines 273-275: Re-write this sentence.
Line 286: Change the expression "by many scholars".
Line 403: change the expression "important significance".
Lines 408-409: Re-write this sentence.

Tables
Table 1: change "was very significant" to "was considered as significant".

Table 2: Tables should be self-explanatory, please provide more information in the title (ex. BP = biological process). I found it very vague to present "Some genes related to growth and development". Choose a fixed number of genes to show, or present all enriched genes for that BP.
Experimental design: - Let the goals of your study clearer in the introduction.
Validity of the findings: - The conclusion is weak. You could name some interesting genes that could be investigated in future studies.
Additional comments: I think the work was well done, but some issues have to be clarified. Please review the written English and grammar to improve your paper.


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 FOR THE BREAST MUSCLE OF JINGHAI YELLOW CHICKEN AT EARLY GROWTH STAGES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please review in whole document the way to cite.

Abstract must contain: Background, Methods, Results, Conclusion and key words, this paper hahas not include Colclusion in the Abstract.

Please mention in material and methods that the experiment was approved by ethics Committee.

Please check the lines
152 (poly-N)
338- pH value
Experimental design: That kind of research shed light about genes expression in different kinds of conditions.

They are relevant for future experiment designs and can be guided to propose experimental design on gene function.

The experiment ordering sequential and the results were well interpreted.

Methods were sufficiently described, sequential and corresponding to the aim of the work.
Authors should mention the elapsed time between taking the samples and RNA extraction process.

Software used to stimated gene expression is okey, as well the to use FPKM to normalizated estimation of gene expression.
Validity of the findings: Please mention criteria used to choose the genes that were verified by qPCR, they were nine (9). There is no mention on why they were selected.

It is important to show the different results on pathway between female and male, nothing is mentions on the text.

Discussion lines 253 to 255 should be improved, the paragraphs are non-sequencial, they contain many ideas but cutting the point.

Nothing is mentioned about the gen Novel 00254
Additional comments: Genes regulations  is one most fascinating event in genetics, therefore any effort to understand the nature of gene regulations is invaluable. This paper is important step to know gene expression and regulation in an important economic specie.


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 FOR THE BREAST MUSCLE OF JINGHAI YELLOW CHICKEN AT EARLY GROWTH STAGES
Review round: 2
Reviewer: 1

Output:
Basic reporting: I consider that this paper has been well revised, and the writing is more fluid and pleasant. However, I still have some suggestions to improve the paper.

Suggestions:

Lines 59-60: change the sentence for "The differential gene expression analysis resulted in 364, 219 and 111 DEGs (P-value < 0.05) for the three comparison groups, F8FvsF4F, F8SvsF4S, and M8SvsM4S, respectively."
I believe that this way, the sentence becomes clearer.

Line 63: change the sentence for "such as ANXA1, COL1A1, MYH15, TGFB3 and, ACTC1, were obtained.".

Line 64: change "(58)" for "(n = 58)". Just the number in the parenthesis seems like a reference.

Line 95: change "transcriptome" to "transcriptomics".

Line 96: change "genomics" to "omics" - just a suggestion.

Line 97: change "transcriptome" to "transcriptomic".

Lines 98-101: The sentence is too long and with many repeated words, becoming even a little redundant. I suggest that you summarize this concept a little, making the sentence more direct.

Line 102: change "has become one of the important means" to "has become an important tool".

Line 106: remove "were".

Lines 120-121: change "in muscle tissues, TCONS_00064133 and TCONS_00069348, were identified." to " in muscle tissues, the TCONS_00064133 and the TCONS_00069348.".

Lines 121-123: suggestion, change the last sentence for "Although RNA-seq technology has been applied to study the growth and development of poultry, the specific regulatory mechanisms of skeletal muscle development remain unclear, and the transcriptome sequencing technology still will be increasingly explored in future studies in the field.".

Line 152: Insert a comma after the parenthesis.

Line 191: change "RNA" to "RNA samples".

Line 221-223: suggestion, change the sentence to "Figure 1 shows the DEG with adjusted P-values of ≤0.05. The analysis resulted in 364, 219, and 111 DEGs obtained from the three comparison groups F8FvsF4F (Fig 1A ), F8SvsF4S (Fig 1B), and M8SvsM4S (Fig 1C), respectively."

Line 225-226: suggestion, change the sentence to "And, 27 upregulated genes and 84 downregulated genes in the M8SvsM4S group.".

Line 227: change "of" to "with".

Line 232: insert "groups" after M8SvcM4S.

Line 238-240: change the sentences "The clustering results are shown in Figures 3–5. The figures demonstrate that the three individuals in each group were all well clustered together. The results show that the three individuals selected in each group have good repeatability." to "The clustering results are shown in Figures 3–5, demonstrating that the three individuals in each group were all well clustered together. Also, these results show that the three individuals selected in each group have good repeatability.".

Line 243: remove "the", start with "Significantly enriched GO terms...".

Line 244-246: change the sentence to "For the F8FvsF4F (Fig 6A) and F8SvsF4S (Fig 6B) groups, 12 and 44 GO terms were significantly enriched, within these 4 and 17 were classified as biological processes (BP), respectively. ".

Line 246-247: change the sentence to "In Table 2, we highlighted some important BP terms related to growth and development, such as extracellular matrix organization, extracellular structure organization, cell adhesion, cell division, and fibril organization.".

Line 249: insert a comma before "and".

Line 250: exclude "respectively".

Line 310: insert "mentioned" after "above".

Line 393-394: change "We could find that eight pathways in the top 20 were enriched in both, the F8FvsF4F group and the F8SvsF4S group" to We found that eight pathways in
top 20 were enriched in both F8FvsF4F and F8SvsF4S groups".

Line 395-396: the sentence is a little bit confusing. I suggest re-write it.

Line 410-413: it should be in the conclusion section.

Line 415-418: Conclusion, my suggestion is to change to "This study examined the theoretical basis further to reveal new insights into the growth and development mechanism of chickens. Overall, the common DEGs, including ADAMTS20, ARHGAP19, Novel00254, and significantly enriched pathways, such as ECM–receptor interaction and focal adhesion, showed to be essential to the growth of chickens. Still, their functions need to be further investigated. Therefore, these results could serve as an important guide for future experimental designs of gene function in poultry science."

**I highlighted all the sentences above in the PDF file attached.

Tables and figures:
- Figures description is now ok.
- Tables: I still think that you could write more robust tittles. Like: "Table 3 - The KEGG pathway related to the growth and development of skeletal muscle in
Experimental design: The experimental design is ok and, all my issues have been attended in the revised paper.
Validity of the findings: The authors discussed interesting finds for chicken muscular development and growth. These finds will be crucial for future studies in poultry science.
Additional comments: I consider that the authors have done an excellent job of proofreading. The new version of the paper is much better written and pleasant to read. I believe that all of my previous comments have been answered. However, I still have some suggestions that I think will make the work even better.


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: REG Γ KNOCKDOWN SUPPRESSES PROLIFERATION BY INDUCING APOPTOSIS AND CELL CYCLE ARREST IN OSTEOSARCOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study focused on the effects of  enforced REGgama expression on the proliferation of osteosarcoma. This paper is well organized and well written.
Experimental design: The experiments were properly designed, and the methods were suitable for the aims of this study.
Validity of the findings: This study provides some valuable information about the effects of REGgama overexpression in osteosarcoma. However, the roles of REGgama in cancer cells have been discussed in various kinds of tumors, and the related mechanism has been illustrated in other studies (as described in Instruction). Thus, to improve the novelty of this study, more  study (not limited to cycle-cycle genes expression) are strongly suggested to explore the underlying mechanisms of REGgama.
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: REG Γ KNOCKDOWN SUPPRESSES PROLIFERATION BY INDUCING APOPTOSIS AND CELL CYCLE ARREST IN OSTEOSARCOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors found for the first time that REG γ is overexpressed in osteosarcoma tissues and cell lines and knockdown of REG γ significantly inhibits cell  proliferation and induces apoptosis and cell cycle arrest in osteosarcoma cells. Furthermore, they observed that p21 and caspase-3 are increased while the expression of cycinD1 and bcl-2 are decreased after REG γ depletion in osteosarcoma cells.
Experimental design: This article fails to meet the standards
Validity of the findings: The findings are far from the novelty. All the experimrnts are siRNA results.
Additional comments: The data quality are too low. The authors must use the stable knockdown cells for there study. In addition, xenograph model is reqiued.


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: REG Γ KNOCKDOWN SUPPRESSES PROLIFERATION BY INDUCING APOPTOSIS AND CELL CYCLE ARREST IN OSTEOSARCOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: •	Yin et al. had reported the role of REG γ in osteosarcoma patients and cell lines. The manuscript was written scientifically, but several concerns should be addressed before published.
•	Line 25: Please specify the role of REGγ, like anti-apoptotic, or tumor suppressor protein.
•	Line 31: What do you mean by “cell behavior”? Cell growth/ proliferation?
•	Line 51: Please define those acronyms, like REG, PSME3, PA28g, etc.
•	Line 53: Citation is needed for the statement on REGγ.
•	Please include more findings like Zannini, et al. 2008 (DOI: 10.4161/cc.7.4.5355) on the role of REG γ in chromosomal stability (in introduction or discussion).
•	Line 55: This statement is ambiguous. What cancer-related proteins are involved? Oncogenic proteins or tumor suppressor proteins? Please specify.
•	Line 58: Should be “skin cancer”, not “skin carcinogenesis”.
•	Line 71: The OS and adjacent normal tissues were employed to determine the expression of REG γ. However, I am concerning about the purity and integrity of these tissue samples. How the authors differentiate and confirm those tissues are “OS” or “normal” tissues? Is any histological examination like IHC on the mitotic antigen (Ki-67) or H&E staining performed to confirm the distribution/location of cancer cells? How do authors confirm there is no OS cancer cells had already infiltrated into the adjacent tissues?
•	Line 73: According to the authors, seven OS patients received pre-operative chemotherapy and one is not. The authors may need to mention the main chemotherapeutic agents used as this may be the confounding factor for your apoptosis study. Is it possible that the pre-operative chemotherapy may somehow interfere with the result? Please explain this in the discussion section.
•	Line 73: Besides, clinical features for the OS patients are missing. Information like age, gender, location of OS tumor, histological types of OS, grading and any metastasis should be reported.
•	Line 143: The subheading “flow cytometry” is not suitable as the name of the assay will be a better fit. Please amend this into apoptosis assay and cell cycle analysis (or any similar).
•	Figure 1A: Single IHC staining of REG γ is inconclusive to relate with OS cancer cells as authors did not identify the cancer cells and we are not sure the REGγ is expressed by which cells.
•	Figure 1B: The authors mentioned there were 8 OA patient samples but figure 1B showed 10 results. Please explain this.
•	Figure 1: Please standardize the acronym (AT & T) throughout the figures.
•	Figure 1F: It would be better if the authors focus on osteosarcoma statistics. Describing the expression of REGγ in other sarcomas (leiomyosarcoma, liposarcoma, and fibrosarcoma) is not really contributing to this study. Suggest removing this data or convert this as supplementary data.
•	Figure 2: How the authors calculate the relative REGγ mRNA level? Please include this under “Methodology”
•	Figure 3A: The plot mark for siRNA-1 and -2 are the same.
•	Figure 3A/B: The growth curve of si-NC is identical for both MG-63 and Saos-2 (same OD for 48 & 72h) which sounds weird to me as MG-63 is growing faster with shorter doubling time as compared to Saos-2 cells. Parallelly, the MG-63 cells could produce a larger/greater number of colonies due to its higher growth rate (Figure 3B). Please check again your data on Figure 3A.
•	Figure 3C/D: Is it possible for authors to quantify the EdU levels by using ImageJ software?
•	Figure 4A/B: Please manually set the maximum Y-axis scale (around 1600 in your study) to have a better comparison between groups. The default axis setting is “automatic” and this will make the G0/G1 curve for each group look similar. The G0/G1 peak should be higher for si-REG groups.
•	Figure 4A/B: The asterisk marks are too small.
•	Figure 4C/D: Apoptosis assessment on 48h-REGγ-knockdown cells revealed around 4% increase of apoptosis (for si-REG-2). Is 4% of apoptosis induction clinically relevant? Besides, upon knockdown of REGγ, there is only a 4% increase in apoptosis, is REGγ crucial for OS cell survival? I suggest authors amend the statement in line 40, 218 and 247.
•	Figure 4E/F: The quantitative data for the protein levels is missing as authors claimed that the protein density had been quantified via ImageJ software (line 114-115). Authors may combine these two figures as 1, and repeated labeling is not necessary.
•	Figure 4E/F: How multiple proteins are detected in this study? Stripping and reprobe method? Please describe this in methodology.
•	Figure 4 title: The statement “alters multiple cell apoptosis and cell cycle-related genes in MG-63 and SaoS-2” not suitable as gene expression is not conducted. Suggest “apoptosis and cell cycle-related proteins in MG-63 and SaoS-2”.
•	Line 195: The upregulation of caspase-3 (35kDa) could not directly explain/relate the apoptosis. This is because the 35kDa caspase-3 is an inactive zymogen (pro-caspase-3). The authors should report the cleaved subunit (p12 & p17) which are representing the activated caspase-3. Please amend the western blot and insert the blot with activated caspase-3 at 17 and/or 12 kDa position.
•	Discussion: I am expecting more discussion from the authors. For instance, how REGγ regulates the CyclinD1, p21, and other proteins. Is it by transcriptional or post-transcriptional regulation? Since both MG-63 and Saos-2 cells are p53-mutated cell lines, authors may discuss the p53-independent p21 induction upon knockdown of REGγ. Is REGγ the upstream regulator or the intermediate signaling molecule? What is the transcriptional regulation of REGγ? Can it be upregulated by another oncogene like mutated p53 or kinases? What is the potential role of REGγ in OS carcinogenesis, progression and survival?  What is the limitation of this study?
•	Conclusion: An overall schematic diagram which concludes the findings is much appreciated.
Experimental design: •	The experiment was conducted on OS cell lines and patients with siRNA approach, however, references and details are not adequately provided.
•	Line 80: Any justification on why RPMI-1640 media were used for these OS cells as EMEM is recommended for MG-63, while McCoy’s 5A for Saos-2? Will this affect the REGγ expression and make comparisons across the studies become difficult?
•	Line 88: Why authors need to use the H&E pre-stained tissues for subsequent IHC?
•	Line 89: Please specify the name of the primary antibody used in the first place (combined from line 93-94), together with the catalog number for better reproducibility.
•	Line 90: Is “overnight” in this study 12h?
•	Line 91: Why streptavidin-HRP was used in this study? Is the secondary antibody linked with HRP?
•	Line 99: Forward or reverse transfection? Please specify the amount/conc of siRNA and Lipofectamine reagent used? Is complete RPMI media (with FBS and antibiotics) or serum-free media like optiMEM was used to dilute the siRNA and reagents?
•	Line 100: Is good to have the pictures to visualize the siRNA transfection efficiency as supplementary data. Is 50% transfection efficiency consider a good transfection?
•	Line 109: Please include the PVDF catalog number.
•	Line 113: Please include the chemiluminescent reagent used.
•	Line 115-116: Please include the catalog number for these antibodies.
•	Line 122: Please put in more details for the procedure like temperature and incubation time for each step to ensure good reproducibility.
•	Line 125: What do you mean “Cell transfected with siRNA for 24h were seeded”? The authors trypsinized the cells after transfection for 24h? Why some experiment like apoptosis assay is using 48h-transfected cells?
•	Line 140: Any justification on why 6000 cells/well were used in EdU assay but 2000 cells/wells was used for CCK-8 assay?
•	Line 151: What is the PI concentration?
•	Line 153: Please mention the use of ModFit LT software in the cell cycle analysis.
•	Line 155: How about the normality of data?
•	Line 170: Do authors confirm with the off-target phenomenon? Do authors determine the viability (trypan blue assay) and gene expression of the house-keeping gene after transfection? Cytotoxic effect of siRNA was shown to downregulate gene expression. How authors confirm that the si-REGγ-1 &-2 downregulate the REGγ gene expression is not via the cytotoxic effects of siRNA.
Validity of the findings: •	Most of the findings sound legit and valid, but a second round of checking is required after this correction (refer to the comments in Basic Reporting).
•	Please pay attention on the figure 3A (refer to the comments in Basic reporting).
Additional comments: N.A.


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

Input:
Title: REG Γ KNOCKDOWN SUPPRESSES PROLIFERATION BY INDUCING APOPTOSIS AND CELL CYCLE ARREST IN OSTEOSARCOMA
Review round: 1
Reviewer: 4

Output:
Basic reporting: The manuscript is well-constructed and well-written. The English used is good but the description in the results could be clearer and more concise.
Experimental design: The scope of the investigation is limited but clearly defined. The methods are well-described.
Validity of the findings: Some of the results are purely descriptive, like the western blot and colony formation. Description of the results should be more percise (statistical significance marked and stated in the text).
Additional comments: The authors investigated the role of REGg in the proliferation of osteosarcoma using SiRNA technique. The manuscript is well structured and well written. I only have minor comments as the following:
1. The visual display of western blot results is convincing. Did the author also quantify the band density?
2. Figure 1A: Can the authors point out in the legend and the figure the features to be considered?
3. Why was the relative mRNA expression in Figure 1C and 1E shown in different type of graphs?
4. Were the reduction of REGg expression caused by SiRNA significant? If yes, in Figure 2A and B, why were the significance not marked?
5. Were the colony formation quantified? The authors mentioned "obviously", do they mean "apparently"?
6. The authors should clearly indicate in the text that which results were significantly altered and which were not, maybe by indicating the p-value.
7. Some of the limitations should be clearly stated at the end of the discussion. For example, the mechanism of regulations of REGg in promoting cancer proliferation is not investigated in depth. Please mention also what are the purposed further studies and the current implications of current observation in cancer treatment.


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

Input:
Title: REG Γ KNOCKDOWN SUPPRESSES PROLIFERATION BY INDUCING APOPTOSIS AND CELL CYCLE ARREST IN OSTEOSARCOMA
Review round: 1
Reviewer: 5

Output:
Basic reporting: 1.The author should add the catalog number of antibodies and kits.
2.In fig4 E and F, the results were in same membrane? If yes, could the author describe the details of the methods of western? Because the MW of these proteins are similar, such as cyclinD and caspase3. Stripping buffer can be used, but the methods should be described. If no, the author should add more pic of loading control.
Experimental design: 1.Author should add the protein level of Cl-caspase3 in fig 4 E and F.
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: REG Γ KNOCKDOWN SUPPRESSES PROLIFERATION BY INDUCING APOPTOSIS AND CELL CYCLE ARREST IN OSTEOSARCOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you authors for the 1st revision. I do have some follow-up questions for the previous comments:
-Comment no.9: Authors did not address my question directly. How the authors differentiate and confirm those tissues are “OS” or “normal” tissues based on H&E staining as H&E staining is not specific for cancerous cells. Additionally, Zhiqiang Yin and Hao Jin who performed the experiment, are affiliated from the Department of Orthopedics, not Oncology. Are they the right persons (equipped with proper training and qualifications) to detect and confirm the cancerous OS cells? Kindly advise.

Besides, as mentioned earlier, I am concerning about the purity and integrity of these tissue samples. According to the authors’ statement “Normal tissues were collected as far away from OS tissues as possible”, where in my opinion, it is not scientifically reproducible.

- Comment no.13: Authors claimed that H&E was performed before IHC. Are the same tissue sections were used in both H&E and IHC staining? I didn’t see any overlapping or side-by-side comparison of H&E and IHC in Figure 1.

- Comment no.18 & 19: I didn’t see any correction being done on Figure 3A/B in the amended version.

- Comment no. 20: Thank you authors for the effort to set the Y-axis. This is important to reduce the bias during comparisons between groups in cell cycle analysis. The G2/M peaks in Si- REG γ-2 groups were too low should not be a problem. Please amend the graphs by changing the Y-axis scale.

- Comment no.21: I didn’t see any correction being done on the asterisks of Figure 4A/B.

- Comment No.22: Authors did not address my question directly. I understand that the 4% of apoptosis induction is statistically significant but yet my question is whether this is clinically sound and relevant? Besides, upon knockdown of REG γ, there is only a 4% increase in apoptosis, is REGγ crucial for OS cell survival?

-Comment No.23: I am very appreciated the quantitative data of the protein levels in Figure 4. However, I am not sure how the authors calculated this. Is it by relative to the si-NC group? How many biological replicates that authors perform for Figure 4? Kindly describe in methodology.
Additionally, I suggest authors re-confirm the data. This is because I noticed that the quantitative number of protein bands for MG-63 caspase-3 si-REG-1 (1.90) is smaller than SaoS-2 p21 si-REG-1 (1.92), yet the band for MG-63 is much thicker. The si-NC for both is similar anyway.

- Comment No.24: I cannot find any reprobe description in line 113-114 method. Please correct this.

- Comment no.26: The anti-cleaved caspase-3 antibody (cat no.: 9661) from Cell Signaling Technology is not able to detect the cleaved subunit p12. It could detect p19 and p17 as demonstrated in Figure 4. Kindly change the 17kDa in Figure 4 to 19&17kDa as double bands were detected.
Experimental design: - Comment no.6: From my understanding, streptavidin-HRP is used to detect the biotin-labelled target. I am not sure how streptavidin-HRP peroxidase can be beneficial for quantity and distribution of REG γ in western blot. Kindly explain this.

- Comment no.9: PVDF membrane that used (catalogue no: IPVH00010) has the pore size of 0.45 µm (https://www.merckmillipore.com/MY/en/product/Immobilon-P-PVDF-Membrane,MM_NF-IPVH00010) which is not compatible with protein that smaller than 20kDa. In this case, the result for cleaved caspase-3 (17kDa) is affected where the protein may pass through the PVDF membrane and the blot result is not valid. Authors need to re-run the cleaved caspase-3 expression (17kDa) with 0.2 µm PVDF.

- Comment no.14: Sorry to say I am not sure what authors talking about. “Not so many cells were appreciated to leave cells enough living space”?? Are authors meaning that to use lesser cell conc in longer incubation CCK-8 assay?
Validity of the findings: No additional comment
Additional comments: No additional 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: REG Γ KNOCKDOWN SUPPRESSES PROLIFERATION BY INDUCING APOPTOSIS AND CELL CYCLE ARREST IN OSTEOSARCOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Thank you for addressing my comments. I have no further comments about this manuscript.
I think the authors left out the "*" for statistical significance in Figure 2. This can be amended later.


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: REG Γ KNOCKDOWN SUPPRESSES PROLIFERATION BY INDUCING APOPTOSIS AND CELL CYCLE ARREST IN OSTEOSARCOMA
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have revised comments.
Experimental design: The authors have added the protein level of the cl-caspase3.
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: INVASIVE CRAYFISH DOES NOT INFLUENCE SPAWNING MICROHABITAT SELECTION OF BROWN FROGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript explores the possibility of adaptive egg-laying behavior displayed by two frog species in ponds invaded by the red swamp crayfish. Studies exploring the coexistence between native and invasive species are highly relevant in the context of global changes, especially applied to threatened groups like amphibians. I appreciated this manuscript which is clear and presents well-defined hypotheses. However, while sample sizes are appropriate for the statistical analyses applied, I have concerns with the biological representativity of the results, because of the low number of ponds that were sampled within each factor modality. Additionally, I suggest to reconsider the proxy used for the shelter hypothesis. The main text lacks details on sample sizes and on the global context regarding the invasion pressure. Please see my major comments in the next section.
The text must be edited by a native English speaker.
Experimental design: (1)	15 ponds were considered for this study (8 invaded and 7 uninvaded ponds), for a total of 498 clutches sampled in two frog species. While these sample sizes are sufficient for the statistical models applied, I am worried with their biological representativity, because of the low number of ponds sampled: one species was found in 13 ponds, and the other is found in 8 ponds, with N < 10 clutches found in 7 ponds. In the third analysis, models are conducted separately for each species to test for the effect of crayfish, and clutches belong to a maximum of 7 sites per modality (invaded or uninvaded) and as low as 2 sites for Rana latastei in uninvaded ponds. This limited representativity should be discussed in the manuscript.

(2)	In the introduction, the authors hypothesize that the unavailability of predator-free habitats could force amphibians to breed in invaded ponds, and to adopt adaptive strategies to limit predation on their progeny. This is a sound starting point for the manuscript. However, information on the predation pressure or overall crayfish occurrence in the study area is lacking, while it would considerably enlighten the results of the study. Only half of the 15 sampled ponds were invaded, suggesting that habitats free of crayfish are still largely available.

(3)	The hypotheses of adaptive behavior are well argumented and accompanied of clear predictions. However I have concerns about using distance from pond edge as a proxy for aquatic vegetation cover. Presence of aquatic vegetation notably depends on pond depth, water turbidity and bank slope. The supplementary tables indicate very shallow ponds with a maximum pond depth of 80 cm. Because the authors used visual surveys to detect crayfish, I also assume that water turbidity is low (but see my next comment). Then, there is no particular reason to assume that vegetation is restricted to pond edges, and I suggest the authors to use direct information on vegetation. Alternatively, the authors should better justify the use of this proxy and discuss this point. Additionally, it seems to me that the shelter and deepness hypotheses should not be mutually exclusive, which is somewhat the case in the present version.

(4)	Attaining high probabilities of crayfish detection using visual encounter surveys is highly dependent on water turbidity, habitat complexity, i.e. site characteristics, as well as methodology (e.g. walking pace, counts…), so that more information on the protocol used to detect crayfish is needed. If available, the authors could reinforce the certainty of non-detections if crayfish also went undetected in previous surveys, especially using other techniques such as traps. Additionally, because the effect of crayfish on larval densities has been shown to vary with its density (e.g. Ficetola et al. 2011), the authors should consider using crayfish density instead of presence/absence data. Whether this option is not feasible because of sample sizes, this point should be discussed with point (1).

Litterature cited:
Ficetola, G., Siesa, M. E., Manenti, R., Bottoni, L., De Bernardi, F., & Padoa-Schioppa, E. (2011). Early assessment of the impact of alien species: differential consequences of an invasive crayfish on adult and larval amphibians. Diversity and Distributions, 17(6), 1141–1151.
Validity of the findings: (5)	More information on sample sizes should be directly available to help the reader judge the reliability of the study: number of ponds studied per species, and for each species, the number of invaded and uninvaded sites, directly in the text or in Table 2; mean +/- SD or range of clutches found per site per species. This information can currently only be calculated from the supplementary tables. Means and standard errors of the environmental variables can be added to help the reader visualize the study systems.

(6)	The results on spawning microhabitat differences between species are means of means per site, while Figure 1 looks based on raw data. Please homogenize. Please add standard deviations to all the given means to help the reader judge the significance of the results.

(7)	Line 237 “The invasive crayfish has attained very high density”. Vague sentence, please justify with data or reference.

(8)	 Table 1 could be placed in supplementary material as it stands for a methodological point.

(9)	 Please check values in the supplementary tables: there is a 0.9 maximum pond depth, and pond width is often larger than pond length. Add a legend to these tables that explains variables, units, and acronyms.

(10)	 Please cite the publications associated to the R packages used, which can be found using the citation(“pckg_name”) function in R.

(11)	 Please check the English language and typos: lines 63, 72, 81, 136-137, 154, 197-198, 202, 215-216, 226, 235-236... and column names in supplementary tables.
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: INVASIVE CRAYFISH DOES NOT INFLUENCE SPAWNING MICROHABITAT SELECTION OF BROWN FROGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper by Romagnoli and colleagues reports on a study investigating the microhabitat preferences during egg-deposition in two anuran species, Rana dalmatina and R. latastei, and assessing whether these preferences are influenced by the presence/absence of Procambarus clarkii, an invasive alien species that is a voracious predator of anuran larvae. Because studies on such predator-mediated changes in habitat- and microhabitat use during egg-laying are rare in amphibians, and the spread of P. clarkii is becoming a major problem of conservation biology, the study is novel and interesting. The study design is appropriate, the introduction sets the scene well, and results underpin the interpretations. However, there are a few issues that need to be addressed.

First of all, the English needs to be corrected. I provided suggestions in this respect (please see attachment), but I am not a native English speaker, so that further language editing would certainly enhance the legibility of the paper.

A few important papers that should have been considered thoroughly are only mentioned tangentially, or are not referred to at all. For example, the papers Nunes et al. 2014 Ecology, Nunes et al. 2014 Ecology and Evolution, Nunes et al. 2013  Oecologia, Hettyey et al. 2016 Ecology should be cited and discussed both in the introduction and the discussion.
Experimental design: Lines 75-77: The authors state that according to the shelter hypothesis, because vegetation is usually more dense along the shores of ponds, shelters may be more abundant close to the shore, so that the expectation is that frogs would deposit their eggs close to the shore when P. clarkii is present in order to provide protection to their offspring. It is unclear, however, why the authors used the proxy of distance from the shore instead of directly measuring vegetation density around clutches. That would have been clearly preferable.

Lines 81-82: Regarding the schooling hypothesis: I find it difficult to believe that tadpoles' limited home ranges lead to tadpole aggregations when eggs are laid in close proximity. Tadpoles often can be seen at places where no clutches had been laid before within tens of meters. This statement at least needs some good citations. Or it would need to be changed in the sense that when tadpoles hatch and are present in high density locally during their most vulnerable stage (early post-hatching), their survival probabilities may be enhanced in the ways spelled out in the next sentence.

Lines 111-113: It is mentioned that maximum water depth was measured and clutch density was calculated, but it is unclear, why. Please explain here briefly.
Validity of the findings: Lines 111-113: It is mentioned that maximum water depth was measured and clutch density was calculated, but it is unclear, why. Please explain here briefly.

Line 161: One of the results is that R. latastei laid clutches into deeper water than R. dalmatina. However, Fig. 1b suggests the opposite. Please double-check and clarify.

Line 165: As before, the figure contradicts the result. Please double-check and clarify.

Line 193: The end of the discussion's first paragraph is left in empty space: a conclusion is missing.

Lines 204-208: Female frogs invest a lot of energy into the eggs, and males spend a lot on calling, fighting, maintaining a territory, etc. Also, it is the time factor that may be important here (enough vs. not enough time to reliably assess the presence and abundance of predators), so that the reference to relatively little investment in energy should be deleted.

Lines 211-212: This idea needs to be elaborated a bit more. It may not be imperative to adjust microhabitat preferences during egg-laying because of low predator activity during that cool period, while the devastating effect of the crayfish on theses amphibians may manifest later on during the tadpole stage, when water temperatures rise.

Lines 216-217: Awkward wording. Frogs (or any other organisms) do not evolve something. Evolution is not an active process in this sense. Evolution is not performed, it just happens.

For further minor comments, please see the attached file.
Additional comments: Table 1 presents data of marginal (only technical) importance, so that it should be moved into an electronic appendix.

Table 2: Dependents and independents are messed up, so that this table is difficult to read. Please re-arrange.