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: COMPREHENSIVE ANALYSIS AND IDENTIFICATION OF KEY GENES AND SIGNALING PATHWAYS IN THE OCCURRENCE AND METASTASIS OF CUTANEOUS MELANOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Melanoma is a malignant tumor of melanocytes, and the incidence has increased faster than any other cancer over the past half century. Most primary cutaneous melanoma can be cured by local excision, but metastatic cutaneous melanoma has a poor prognosis. Therefore, the research on the mechanism of cutaneous melanoma metastasis will be beneficial to diagnose the metastatic melanoma early, improve the treatment, and prolong the survival of patients. In this study, Dai Hangying and colleagues identified the key genes and molecular pathways involved in the occurrence and metastasis of cutaneous melanoma. The study provides the conclusion, which showed that some DEGs were found from normal skin, primary melanoma and metastatic melanoma samples. These hub genes may play an important role in the generation, invasion, recurrence or death of CM, providing new ideas and targets for the diagnosis and treatment of metastatic cutaneous melanoma. The paper is improved and most concerned raised by the reviewer have been addressed. But there are still questions to be answered.

Major concerns:
1. Too many pictures need to be deleted, too many network pictures, lack of presentation meaning.
2. Make sure the text is clearly visible in the figures.

Minor concerns:
1. Figure 11. Please convert the data logarithmically.


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: COMPREHENSIVE ANALYSIS AND IDENTIFICATION OF KEY GENES AND SIGNALING PATHWAYS IN THE OCCURRENCE AND METASTASIS OF CUTANEOUS MELANOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: My previous comments have been addressed.


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: WING MORPHOLOGICAL RESPONSES TO LATITUDE AND COLONISATION IN A RANGE EXPANDING BUTTERFLY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English is mostly clear and unambiguous. For suggestions for improvements please see annotated MS.
The background needs to be clarified, by paying more attention to plastic versus genetic variation. Hypotheses need to be clarified also. For some data, the relevance for the questions addressed is unclear.
Figures and Tables can be optimized, for details please see annotated MS.
Experimental design: Methods are sufficiently described. However, not all research questions are well defined. It was also not clear to me which gap of knowledge is targeted, as there are earlier studies on P. aegeria but also various other species on similar matters.
Validity of the findings: Replication is fine. I have several problems with the interpretation of statistical results and the conclusions drawn. Please see annotated MS for details.
Additional comments: The authors collected an impressive set of data, which has been analysed with sophisticated methods. Nevertheless, I have several concerns the authors may wish to consider. A major weakness of the paper is that it is impossible, using field-collected individuals, to distinguish among genetic and plastic effects. This is only briefly mentioned in the discussion. The study is also quite narrow in focus, and several previous study have addressed the same questions even using the same model. For details, please see annotated manuscript. My most important concerns are:

The Introduction needs to be rewritten. Please make a clear distinctions between plastic and genetic sources of variation. State which source of variation you are targeting, and which law / rule / prediction relates to plastic or genetic effects. Some concepts are not correctly or sufficiently explained in the introduction. It needs to be clear for the reader which type of variation you are targeting and what your predictions are. Currently, the hypotheses are partly very weak and do not follow from what is given in the introduction.

The authors should pay more attention to the implications of morphology for dispersal. Although often assumed, what is actually the evidence for a strong link between the two? Other factors (intrinsic ones, weather etc.) may be much more important. Supporting evidence should be given in the Introduction. Much more information is needed on the relevance of wing size and especially shape and colour.

I had repeatedly problems with the presentation of results, and especially the interpretation of statistical results. Several times, significant findings are claimed while this is not the case, or statistical trends are considered significant results. The whole result section needs a thorough revision. Please restrict yourself to statistically significant results. Based on these problems, I think that some interpretations and conclusions are not supported by your data.

The discussion should focus on the own results and put them into context. Currently, large parts give general information on the traits considered.


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: WING MORPHOLOGICAL RESPONSES TO LATITUDE AND COLONISATION IN A RANGE EXPANDING BUTTERFLY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors present a detailed and coherent study on the morphological responses of Pararge aegeria to range expansions, which is comprehensively written, well-structured and provides a sufficient background of the study question. Yet I, not having English as mother tongue, often struggled to get the meaning of the sentences. This is a pity, because it seems as if the authors present some remarkably interesting ideas, which are concealed by unprecise wordings. Mostly, my struggle results from an over-complication of sentence structure with the addition of highbrow words, which are not always necessary and/or could be replaced by simpler and more frequently used words. Thus, I think the whole text would benefit from being written in a simpler, more straight-forward manner, with shorter sentences and consistent use of terms. Please refer to the minor points in “general comments” for examples where I did not (directly) understood the authors statements.
In best scientific spirit, the authors not only provide their data, but also their scripts and macros. Unfortunately, the zenodo-link to the photographs which would have been needed to run the analysis did resulted in an internal server error.
Experimental design: The research question is well defined. However, the authors could point out the reasoning for investigation more clearly. The notion, that “climate-driven range expansions often covary with latitude (and temperature), making it hard to disentangle the effects of these processes” (i.e. demographic and environmental impacts on morphological traits), line 145-146, is very interesting and disentangling the effects seems to be the most important contribution of this study. However, this notion gets lost in the shuffle of the introduction. It deserves a more prominent position and surely, a greater resumption in the discussion.
Concerning the assessments of the used morphometrics and environmental variables, I applaud to the technical effort and detailed description thereof.
Four variables are used as independent variables: latitude, years colonized, mean developmental temperature and 10 year mean annual temperature. From the nature of those variables, I would expect issues with multicollinearity, and I don’t see whether such problems were looked at or addressed. Thus, the results might be highly biased. If, however, the authors considered multicollinearity and did check for it (which seems to be the case according to their R-script) , I strongly recommend adding a pairs plot of the chosen variables or something similar in the Appendix.
Validity of the findings: The authors discuss their results very carefully, and I really like this refreshingly non-over-selling. Maybe this had the unfortunate side effect, that the conclusions were not as concise as they could be; as stated in the “Experimental design section”, it would benefit from a more clear and basal discussion of the relative contribution of demographic and environmental effects on morphometrics.
There are some methodological / statistical issues which need to be addressed or discussed, specifically: multicollinearity, peaks close to the end or beginning of a month (see general comment Line 211-216), variation of developmental time along latitude (see general comments Line 216-220), and size standardization (see general comments Line 247-250).
Moreover, the authors should consider that they could not include the colour lightness of the body – probably the most important part in thermoregulation (which might explain the contrasting results to Zeuss et al. 2014)
Additional comments: Discussion in general: You make some good points. However, you often present them block wise, e.g. from line 451 to 467, you compare your found slope to that of similar studies. Then, from line 169-484 you outline the ecological effect of a certain shape in general and in line 186 – 496 you are more specific about the behaviour of P. aegeria. However, the latter two blocks are not directly connected to your results anymore. It would be better to link them more closely and be more straight forward. i.e. result: rounded forewing in younger populations -> the contrast of what one would suspect when looking at migratory species -> maybe because of the behaviour in P. aegeria

Considering that the divergence in cream-coloured spot and brown background colour of the wing takes considerable space, the reasoning behind this divergence is superficially introduced.

Line 60: Please replace “thermal tolerance” with “colour lightness”, given that Zeuss et al. (2014)  did test the phylogenetic component of colour lightness but not thermal tolerance (in terms of temperatures which the species can cope with) per se .

Line 62-63: I got lost in this sentence. The relative importance of which response mechanisms? Please restructure the sentence to be less complicated for people like me who don’t have English as mother tongue.

Line 69: Maybe “is a product of” instead of “is predicted by”?

Line 74-75:  I do not get this sentence.

Line 75-77: When you talk about dispersal traits before, and then mention Bergmann’s rule, you must point why a pattern in body size is connected to dispersal (i.e. body size is regarded as a dispersal trait)

Line 85-87: On first view, you give contradicting statements here. In parentheses, you give a clear direction (smaller individuals at higher temperatures) but then you say: “less clear is how the […] slope of this reaction norm is likely to be affected by temperature selection”. If “reaction norm” refers to the relationship between developmental temperature and body size, then you already have the slope, don’t you? Or is temperature selection something different than developmental temperatures? If the latter, can you be clearer what you mean with “temperature selection”?

Line 91-93: “thermal reaction norms may evolve in different directions” – can you call it a norm, if it is not consistent? I really struggle with this term.

Line 111-114: I would suggest moving this sentence behind the following, so that the sentences dealing with the TMH (general description and thermal properties of wings) are not interrupted by other mechanism descriptions.

Line 134: which range are you referring to? Dispersal?

Line 146-148: I did not get the point of this sentence

Line 211-216: This is an interesting and smart approach. However, I am wondering how much variation in Julien day of peak there is within 10 years. As you used month as a an unit to get the developmental temperatures, there might be problems if the sampling dates are close to the end or beginning of a month: For example, if you have the peak on the edge between June and July, it will make a big difference which month to choose, as the former then includes a quite cold month (April), the latter doesn’t. Maybe using the temperature of 90 Julian days would be a more suitable approach? It would be nice to see a graph in the Attachment depicting the phenology of the butterflies based on your data.

Line 216-220: Assuming a steady development time even under different developmental temperatures is seems problematic: as you mentioned yourself, southerly populations have more generations, and should have, at least under the temperature-size-rule, shorter development times. Hence, including also three months prior to emergence peak in southern generations will result in an underestimation of the developmental temperatures that were actually experienced.

Line 235-237: That’s a good point. Here, it would be good to say more about how well the recorder effort and the geographic coverage of the records was. For the latter, it would be of utmost importance to have a good, consistent geographic coverage when dealing with range-expansion. For example, the gap between the English and Scottish populations could be a sampling effect (i.e. not enough people which participated in the surveys living there) – unlikely, but it would be better to really see some sampling-coverage-map in the Appendix to be assured that it is no sampling effect.

Line 247/ Line 250: If the method used standardises specimens through controlling size, how can centroid size be used as a measure of wing size?

Line 309: Why did you exclude years since colonization? According to Mattila (2015) also dispersal might be influenced by melanism. Thus, I do not understand why you handle body size and melanism differently.

Line 345: The question is, whether 1.5% and 2.5% of change in wing size are biologically meaningful

Line 352-353: What do you mean with “Individuals […] fall along the same regression line”?

Line 392-394: if the cream has a steeper increase in colour lightness compared to the brown, than the slopes can’t be parallel

Line 397-398: Put the reference to Dennis and Shreeve (1989) to the discussion

Line 436: what do you mean with “they”?


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: WING MORPHOLOGICAL RESPONSES TO LATITUDE AND COLONISATION IN A RANGE EXPANDING BUTTERFLY
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment, everything is fine
Experimental design: great experimental design
Validity of the findings: everything fine
Additional comments: The authors have submitted a thoroughly revised manuscript and apparently, have put large effort into addressing the raised concerns. It reads now very well, especially the introduction. I now could follow every sentence even before my first morning coffee! (Excuse my non-professionality here, but that really means they did a good writing). In circumstances were issues or suggestion raised by the reviewers were not implemented, the authors expressed fair reasons why this was not possible or desirable from their point of view. Overall, this manuscript was good to start with based on the detailed methods, but now even got better due to a profound writing style and argumentation. There are only minor things with which I struggle, but I am confident that the authors will continue their superb efforts in addressing them without any problems.
1)	The cream spot hypothesis.
There is still a part in the introduction missing, why the authors (or Dennis & Shreeve, 1989) are expecting cream spots to become larger with latitude.

Dennis & Shreeve have some interesting ideas about this hypothesis, which might also add substance to the reasoning for the divergence in cream-coloured spot and brown background colour. I have extracted some passages which I think could be especially useful:
-	“expectations are that as summer conditions become cooler and less predictable to the north and west, selection has resulted in modifications in adult phenotype in response to thermoregulatory requirements and the needs of mate advertisement and predator escape.”
-	“Cooler, cloudier conditions result in the lower activity of adults (rate, duration), which in turn influences their ability to locate mates, to court conspecifics successfully, to release their egg load, to nectar efficiently, to escape predators and to colonize new habitats. Depression of any of these biological functions adversely affects reproductive success. Some modifications in phenotype are known to be advantageous in cool climates. These changes depend on species-specific basking modes.”
-	“The upper wing surfaces in dorsal baskers are often darker at the base and outer margin, but have more extensive brighter […] areas in the outer portion of the wings, which also allows the larger apical ocelli to be highlighted […]”

2)	The discussion might benefit from a paragraph or sentences which discuss which effect demographic impacts on wing morphology might have under the expected change in environmental conditions. Let’s take the striking results for body size (being both shaped by range expansion process and latitude, but not subjected to temperature-size rule) as an example. While the authors carefully evaluate why there might been a mismatch between the latter two effects (latitude vs developmental temperature), a discussion of the range expansion effect in combination with latitude and developmental temperature could raise also interesting questions. E.g., does the Scottish South-East expansion (and thus selection for larger-bodied animals in south east) outdo the latitudinal effect? One or two sentences per trait would do to underline the important statement drawn in the conclusion: “Interpretation of the patterns observed here [and elsewhere!] must take account of the dynamic nature of this recent and ongoing range expansion”.


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: OXYTOCIN WEAKENS SELF-OTHER DISTINCTION IN MALES DURING EMPATHIC RESPONSES TO SADNESS: AN EVENT-RELATED POTENTIALS STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript “Oxytocin weakens self-other distinction in males during empathic responses to sadness: An event related potentials study” focuses on a very interesting research question. However, as detailed below, I have several main concerns regarding this manuscript:
- Introduction. The conceptual framework in the introduction is very limited. The authors should include relevant literature on empathy and self-other distinction at implicit and explicit level as well as on oxytocin and self-other distinction. In addition, the authors would need to justify the rationale for selecting sad facial expressions based on previous studies on oxytocin and empathy. The authors are invited to revise the manuscript to remove all the misleading information (e.g., introduction: “extreme masculine cognitive style”. The authors did not measure it; the references to autism is misleading. No measures of autism traits were included).
- The Methods section needs more details. “We believed that the peptide would increase its concentrations in the cerebrospinal fluid during this time.” Please include relevant references with respect to the time elapsing between drug administration and testing.

- I commend the authors for controlling baseline group differences with respect to IRI,; however, they do not consider possible important group differences with respect to the affective state/mood and to the awareness of one’s own emotions (self-awareness/alexithymia). It is difficult to make strong inferences without these controlling measures.
The manuscript should be revised throughout for clarity. A few examples: abstract: “..influence the normally high level of self-other distinction”, what does “normally high levels” mean?; introduction “problem”, perhaps “issue” or “question”? Methods:” the other–task, we told the subjects to press the F button if the emotion expressed by the face was sad and the F button if neutral” “F” and “F” ? Discussion: “Although the predecessors claimed that OXT could reduce the difference wave in the self-task by decreasing brain reactivity to negative emotion and increasing neural activity involved in emotion regulation (Ma et al., 2016), it's hard to explain” Predecessors?

The authors claim that oxytocin reduces self-other distinction. The discussion section would be strengthened by situating the results of the current study within the context of studies highlighting that oxytocin affects self-other distinction at explicit level but not at implicit level (e.g., Pfundmair, M., Rimpel, A., Duffy, K., & Zwarg, C. (2018). Oxytocin blurs the self-other distinction implicitly but not explicitly. Hormones and behavior, 98, 115-120. Colonnello, V., Chen, F. S., Panksepp, J., & Heinrichs, M. (2013). Oxytocin sharpens self-other perceptual boundary. Psychoneuroendocrinology, 38(12), 2996-3002). In addition, I suggest expanding the limitation section: the sample size is very small and no measures of self-awareness and mood were included.
Experimental design: The research question is not well defined. I suggest the authors include relevant literature on empathy and recognition of sad expressions.
Overall, the methods are well described. However additional references are needed (e.g., drug administration procedure)
Validity of the findings: The conclusions should be revised. The results should be considered in the context of the current literature on oxytocin and self-other distinction.
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: OXYTOCIN WEAKENS SELF-OTHER DISTINCTION IN MALES DURING EMPATHIC RESPONSES TO SADNESS: AN EVENT-RELATED POTENTIALS STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is well-written and places the present study and results in the appropriate context of prior studies.
Experimental design: The experimental design is sound, although the samples sizes could be larger.
Validity of the findings: Despite the small sample sizes, there were several significant findings.  This work builds on previous studies on this topic and addresses important questions.  The conclusions are strongly supported by the present results without excessive speculation about their relevance and impact.
Additional comments: 1.  There is a need for more introductory material on the merits of ERP and general background on the technique for those not experts in this area.  It should be made clear when discussing fMRI vs. ERP findings. and the relationship between these findings should be explained.

2.  The discussion would be strengthend by a brief summary of the findings in the first paragraph, beyond just noting differences which are discussed in later paragraphs.

3.  Were there any analyses evaluating interactions between IRI values and the treatment effects?  This might provide additional useful findings.

4.  It would seem that these findings could be relevant beyond autism.  What other populations, conditions, or disorders might these findings be relevant for?  It would seem important to connect these findings to maternal behavior topics given the common use of OXT in the peripartum setting.  While the present study only includes males, they all had mothers.


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: OXYTOCIN WEAKENS SELF-OTHER DISTINCTION IN MALES DURING EMPATHIC RESPONSES TO SADNESS: AN EVENT-RELATED POTENTIALS STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The quality of the manuscript has significantly improved. However, I have a few more concerns and comments:
Abstract: “to investigate whether OXT can weaken men’s excessive self-other distinction”. Is it really “excessive”? Based on what? Please clarify or delete here and in the text of the manuscript.
Introduction: “men display a lesser degree of concern about others than women because of their higher intrinsic psychological significance of self relevance information”. Please clarify this sentence and its relation to the self-task, if any.
Conclusions: “indicating OXT’s role in integrating the self with others instead of separating them.” This statement is very strong. Please revise it or elaborate a bit more on it.
It looks like that an additional round of English editing is needed (e.g., “Of these 40, one subject withdrew at the drug application stage because of rhinitis, leaving 39 subjects remaining.” …leaving and remaining?)
Please upload a more recent certificate of English editing.
Experimental design: The experimental design and the methods are described with sufficient information.
Validity of the findings: No additional changes are required
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: OXYTOCIN WEAKENS SELF-OTHER DISTINCTION IN MALES DURING EMPATHIC RESPONSES TO SADNESS: AN EVENT-RELATED POTENTIALS STUDY
Review round: 3
Reviewer: 1

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


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

Input:
Title: HIGH CYTOPLASMIC YAP1 EXPRESSION PREDICTS A POOR PROGNOSIS IN PATIENTS WITH COLORECTAL CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript broadly meets all the criteria and expected standards. However, I recommend authors to provided a English-translated version of the written consent form (currently in Chinese), as this has been presented as a Supplemental evidence.
Experimental design: No Comments
Validity of the findings: I have a few comments on the results presented and conclusions drawn by the authors.

1. There is an instance of minor image duplication in Figure 1E. In the upper panel comprising of Normal (Upper) Cytoplasmic (Left) and Nuclear YAP1 (middle), authors use slightly shifted field-of-views of the same image to extract the inset crop. If possible, provide an image of another field for the Nuclear YAP1 data. If you intend to use the same image, please provide the exact same field of view for both data set and mention the same in the figure legend.

2. Authors report overall similar trends in their results when scoring for either ‘High cytoplasmic YAP1’ or ‘Low YAP1 NCR’. While these two are intuitively linked, the ratio could be biased by differences in Nuclear Levels of YAP1. Authors have provided independent quantification of the same in Fig 1 B-D. I feel this result could be bolstered by adding a correlation scatter plot of Cytoplasmic v/s Nuclear YAP1 H-score, to rule there are any non-trivial relations between them, which might bias their results.
Additional comments: The differential effects of chemotherapy on high cytosolic YAP1 cells constitute the key finding of this manuscript. Please highlight this in the abstract more clearly.


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: HIGH CYTOPLASMIC YAP1 EXPRESSION PREDICTS A POOR PROGNOSIS IN PATIENTS WITH COLORECTAL CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	Clear legend should be provided for Figure 1E. It is not clear what the colors represents, and it is not clear what are the differences between the images presented and what is the message each image is delivering to the readers.
2.	Comparing the H-scores in Figure 1E with that in Figure 1B-D, it seems that images presented in Figure 1E are examples of extreme H-scores. Those images are not representative staining.
3.	In Line 62 of the Introduction, the authors mentioned the need of new biomarkers for CRC prognosis. I think the authors should introduce if there are other biomarkers for CRC prognosis and discuss about how YAP1 is different from or better than existing biomarkers.
4.	In Lines 66-74. The authors first introduced the paradoxical reports of YAP1 either as an oncogene or a tumor suppressor gene, then proposed the need of establishing a clear relationship between YAP1 expression and its clinical relevance. I think the authors should discuss why they think the relationship they have established is clearer than those in previous reports instead of simply one more example being added to the existing paradoxical reports.
5.	Raw data. A lot of information, including TNM stage, differentiation grade, manual grading of H-score before averaging, etc., are not provided in the raw data used for Figures 2-3 and Tables 1-2. Raw data for Figure 1B-E are not provided.
6.	The font size in Figure 2 is too small.
Experimental design: 1.	In Lines 88-95 bioinformatics analysis, the authors used publicly available gene datasets. The authors should describe how and why they select those specific datasets for their analysis. Are those all the related datasets available from the database, or are they randomly selected from the database? In Line 90, the authors should provide more details on how the data are normalized.
Validity of the findings: 1.	In Lines 193-194, the authors concluded that high cytoplasmic YAP1 expression may contribute to CRC aggressiveness. This conclusion is not justified as the data only suggest for a correlation but not for a causation. It is equally likely that YAP1 expression is modified as CRC progresses to a more advanced stage, i.e. CRC progression contributes to cytoplasmic YAP1 expression.
2.	The conclusion (Lines 215-216, Lines 228-229, and Lines 264-265) generated from Figure 3 is not justified. Firstly, the sample size for the groups without chemotherapy is too small, it is premature to draw any conclusions based on the current data (i.e. Fig. 3C-D, Fig. S3C-D). Secondly and most importantly, to evaluate whether chemotherapy has an effect on cytoplasmic YAP1-low group or cytoplasmic YAP1-high group, and whether the effect of chemotherapy is different due to the expression level of cytoplasmic YAP1 level, the authors should compare the group with chemotherapy (either high cytoplasmic YAP1 or low cytoplasmic YAP1) to the corresponding group without chemotherapy first, and then check, for example, if chemotherapy is effective for the cytoplasmic YAP1-low patients but not effective for the cytoplasmic YAP1-high patients. The current comparison in Figure 3 only answers the question whether chemotherapy influences the prognostic role of cytoplasmic YAP1 level, but not the question whether cytoplasmic YAP1 level influences the chemotherapy effect.
Additional comments: In this manuscript, Dong and colleagues investigated the relationship between YAP1 expression and colorectal cancer (CRC) prognosis. They found a correlation between CRC progression with cytoplasmic but not nuclear YAP1 expression, with high cytoplasmic YAP1 corresponding to poor prognosis. This is an interesting finding as YAP is a transcriptional co-activator which regulates gene expression when transported to the nucleus. However, some of the conclusions are not justified. Please see specific comments for details.


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: HIGH CYTOPLASMIC YAP1 EXPRESSION PREDICTS A POOR PROGNOSIS IN PATIENTS WITH COLORECTAL CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors have investigated the subcellular loacalization of YAP-1 protein in CRC samples and examined its potential correlation to CRC prognosis and Disease specific/free survival. They calculated YAP-1 staining intensity h scores and studies TMA.

The language is simple, clear and non ambiguous. Authors have provided proper references. A little descriptive background about why YAP-1 plays a role in CRC, its dual role in CRC as an oncogene and a tumor suppressor,  and its association to hippo pathway might be insightful.
Experimental design: The overall observational analysis suggesting that higher nuclear and cytoplasmic YAP-1 expression is associated with aggravated CRC.
1. The authors suggest that YAP1 mRNA expression was elevated in 3 GEO and 1 TCGA dataset upon analysis following which they mention that other 5 GEO datasets also exhibit similar results. Is there a significance to segregating the first 3 and other 5 GEO datasets while analysis and result interpretation?
2. Suppression of  YAP-1 phosphorylation leads to its higher nuclear accumulation and has been previously reported to be correlated to CRC prognosis. However, the authors have shown that CRC samples had both high nuclear and cytoplasmic YAP-1 expression. What is the significance of higher cytoplasmic YAP-1? YAP-1 regulates and drives multiple pathways and It would add to the study if authors look into the pathways that drive its high cytoplasmic expression in CRC.
3. Authors observe that subjects that underwent adjuvant chemotherapy and had high cytoplasmic YAP-1 had a shorter DFS and DSS. Authors can extrapolate this observation to in vitro studies to understand the actual mechanism of how cytoplasmic YAP-1 expression can determine the efficacy of adjuvant chemotherapy. If authors are able to verify this observation through mechanistic evaluation, it is the most important and interesting finding of this study.
4. Did authors see any age dependent or gender dependent cytoplasmic/ nuclear YAP-1 variability?
5. In the discussion section, authors have reported previous literature on how the upregulation of EGFR by YAP1 confers chemoresistance to esophageal cancer cells . It will be intersting to see if their observation on effect of high cytoplasmic YAP-1 on adjuvant chemotherapy is also due to EGF-R upregulation or other factors.
6. If I understand correctly, the cytoplasmic YAP-1 expression was high in all malignant CRC irrespective of stage. What is the prognostic value of YAP-1 in differentiating the stage based on its expression.
Validity of the findings: The study is interesting in many facets owing to its clinical significance however it is mostly observational and bioinformatic. To assess and confirm these observations,  elucidating the underlying cause of YAP-1 cytoplasmic up-regulation in CRC will be highly useful.
The english needs minor revision and language editing. Overall the paper is easy to understand and direct. Fine tuning the discussion can help interpret the finding of the study more significantly. Authors can add the most recent work on correlation between YAP-1 and CRC.
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: HIGH CYTOPLASMIC YAP1 EXPRESSION PREDICTS A POOR PROGNOSIS IN PATIENTS WITH COLORECTAL CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: Authors have taken a good-faith effort in addressing comments from the initial submission. I have now reviewed the changes they have made in the revised version and quite happy with the overall manuscript.


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

Input:
Title: HIGH CYTOPLASMIC YAP1 EXPRESSION PREDICTS A POOR PROGNOSIS IN PATIENTS WITH COLORECTAL CANCER
Review round: 2
Reviewer: 2

Output:
Basic reporting: Raw data. The authors declined to provide raw data such as TNM stages, differentiation grade for each patient due to the concern for patient confidential information, although in principle information such as TNM stage should not be any different from the already provided DFS, DSS, Chemotherapy raw information. The authors provided the manual grading of H-scores before averaging. However, the nuclear H-score appear weird. All nuclear H-scores are in the increment of 5, which should be the case if it is calculated in the same way as the cytoplasmic H-score.
Experimental design: No comment
Validity of the findings: 1.	The authors said they had compared the group with chemotherapy (either high cytoplasmic YAP1 or low cytoplasmic YAP1) to the corresponding group without chemotherapy as suggested, and revised their conclusion based on this comparison to “Stage III CRC patients with high cytoplasmic YAP1 expression did not receive significant survival benefit from adjuvant chemotherapy”. However, they did not make any update to Figure 3 to reflect this comparison.
2.	The authors rewrote their final conclusion of Figure 3 to “therefore, as compared to the patients in stage III with lower cytoplasmic YAP1 expression, those with high cytoplasmic YAP1 expression did not receive significant survival benefit from adjuvant chemotherapy”. This conclusion is exciting, but it is not supported by their data at all and is misleading. It sounds like for patient with lower cytoplasmic YAP1 expression, they did receive significant survival benefit from adjuvant chemotherapy; and for patient with high cytoplasmic expression, they did not. Related to the point above, the author did not show this comparison in their Figure 3. And based on the data presented in Figure 3, chemotherapy did not seem to benefit patient with lower cytoplasmic YAP1. Instead, chemotherapy even appeared to have an adverse effect on the survival of patients with low cytoplasmic YAP1, although this may largely be due to the small sample size of the group with low cytoplasmic YAP1 and without chemotherapy. Either way, the data does not support the conclusion drawn by the authors.
Additional comments: In this revised manuscript, Dong and colleagues addressed some of my questions. However, they did not address my concern on the adjuvant chemotherapy result, which is a particularly important part of this study. Their conclusion is not supported by the data presented. Please see specific comments for details.


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: HIGH CYTOPLASMIC YAP1 EXPRESSION PREDICTS A POOR PROGNOSIS IN PATIENTS WITH COLORECTAL CANCER
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have incorporated my suggestions and answered all my queries.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: No additional comments


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

Input:
Title: FOUR SESQUITERPENE GLYCOSIDES FROM LOQUAT (ERIOBOTRYA JAPONICA) LEAF AMELIORATES PALMITIC ACID-INDUCED INSULIN RESISTANCE AND LIPID ACCUMULATION IN HEPG2 CELLS VIA AMPK SIGNALING PATHWAY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Results are of interest to those working in this area of research. A clear report was presented.
Experimental design: Sound.
Validity of the findings: OK.
Additional comments: The reported influence of SG's on glucose uptake and lipid accumulation in insulin-resistant HepG2 cells is well-presented. As Eriobotrya japonica is often used in the traditional medicine of China, it is recommended to refer to a review on the use of it against diabetes [J. Ethnopharm., 92, 1-21 (2004)].
Some linguistic corrections need to be performed:
- line 81: delete 'natural'
- line 186: 'In the H1-...'
- line 187-188: 'all corresponded to ..'In addition, use superscripts for R)substituents in the formulae.


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: FOUR SESQUITERPENE GLYCOSIDES FROM LOQUAT (ERIOBOTRYA JAPONICA) LEAF AMELIORATES PALMITIC ACID-INDUCED INSULIN RESISTANCE AND LIPID ACCUMULATION IN HEPG2 CELLS VIA AMPK SIGNALING PATHWAY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This MS by Dr. Li and colleagues reports on the effects of sesquiterpene glycosides (SGs) on palmitic acid induced insulin resistance in HepG2 cells via AMPK signaling pathway.   The experimental model involves treatment of HepG2 cells with palmitate + ethanol + BSA  or with BSA alone.   There does not seem to be challenge with insulin at any stage.
It has been reported previously that ethanol inhibits AMPK (You M, matsumoto M. et al Gastroenterology 2002; 127:1798-1808) but palmitate activates AMPK (Kawaguchi T. et al., JBC 2002, 277; 3829-3836).  The authors therefore need to consider whether the decrease in AMPK by palmitate/ethanol (Fig. 4B) is due to the ethanol rather than the palmitate and whether the activation of AMPK by SG1-4 is independent of reversal of insulin resistance but is due to direct activation of AMPK.  The model proposes that the activation of AMPK by SGs is upstream of IRS and Akt signalling.  This can be tested using an allosteric AMPK activator such as A-769662.  This would clarify whether the increase in glucose uptake (presumably glucose phosphorylation) is secondary to activation of AMPK or other mechanisms.

Basic Reporting:

Clear unambiguous except for the lack of clarity whether the model can be described as insulin resistant if:  (i)  insulin was not tested;  (ii) whether the trigger was palmitate or palmiate plus ethanol, with the palmitate responsible for the TG and cholesterol storage and the ethanol for inhibition of AMPK as shown by Crabb DW et al 2004 Gastroenterology; 2008, Am J Physio).
Experimental design: Experimental design:  If AMPK is considered to be upstream of IRS1, AKT (See Fig. 5) then an activator of AMPK should be tested in parallel in both the Palmitate/ethanol and control.

Authors should consider revising title to SGs ameliorate palmitate induced lipid accumulation and IRS signalling.

Fig. 3C and 3D are both labelled TC presumably, Fig-3D should be TG
Validity of the findings: The SGs appear to cause activation of AMPK.   They have been tested in conjunction with palmitate and ethanol. The latter has been shown to inhibit AMPK.  The SGs may be effective in conditions of lipid overload and attenuated AMPK.  It would be of interest to test them with palmitate alone (without ethanol).  A palmitate solution can be prepared without ethanol by warming 20mM sodium palmitate in water and then diluting it in cold BSA.
Additional comments: This MS by Dr. Li and colleagues reports on the effects of sesquiterpene glycosides (SGs) on palmitic acid induced insulin resistance in HepG2 cells via AMPK signaling pathway.   The experimental model involves treatment of HepG2 cells with palmitate + ethanol + BSA  or with BSA alone.   It demonstrates that SGs activate AMPK and IRS>AKt signalling.  Whether phosphorylation of IRS1 is downstream of  activation of AMPK could be tested with an AMPK activator.

It has been reported previously that ethanol inhibits AMPK (You M, matsumoto M. et al Gastroenterology 2002; 127:1798-1808) whereas palmitate activates AMPK (Kawaguchi T. et al., JBC 2002, 277; 3829-3836).  The authors therefore need to consider whether the decrease in AMPK by palmitate/ethanol (Fig. 4B) is due to the ethanol rather than the palmitate and whether the activation of AMPK by SG1-4 is independent of reversal of insulin resistance.  The model proposes that the activation of AMPK by SGs is upstream of IRS and Akt signalling.  This is an assumption but can be tested using an allosteric AMPK activator such as A-769662.  This would clarify whether the increase in glucose uptake (presumably glucose phosphorylation) is secondary to activation of AMPK or other mechanisms.

The SGs may be effective in conditions of lipid overload and attenuated AMPK.  It would be of interest to test them with palmitate alone (without ethanol).  A palmitate solution can be prepared without ethanol by warming 20mM sodium palmitate in water and then diluting it in cold BSA.

Fig. 3C and 3D are both labelled TC presumably, Fig-3D should be TG


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: FOUR SESQUITERPENE GLYCOSIDES FROM LOQUAT (ERIOBOTRYA JAPONICA) LEAF AMELIORATES PALMITIC ACID-INDUCED INSULIN RESISTANCE AND LIPID ACCUMULATION IN HEPG2 CELLS VIA AMPK SIGNALING PATHWAY
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Their Editor,
The article concerns the isolation and structural elucidation of four sesquiterpene glycosides from loquat (Eriobotryajaponica) leaf. It evaluates the beneficial effects of these four single SGs isolated from loquat leaf, including SG1, SG2, SG3  and  one  novel  compound  SG4  against  palmitic  acid-induced  insulin  resistance  in  HepG2 cells. SG1, SG3 and SG4 could significantly enhance glucose uptake of insulin-resistant HepG2 cells at non-cytotoxic concentration.
The article is interesting and very well written but several imperfections should be corrected.

1)	“D” and “L” used in IUPAC name should be lower size
2)	δH and δC should be used instead of δ and should be italicized.
3)	The carbon should be rewritting using one decimal instead of two.
4)	Described acid hydrolysis and sugar analysis.
5)	Why was the extraction and isolation done for two months.
6)	In table one, number shoulb be put under the abreviations of sugars.
7)	For abreviation of glucose, Glc should be use instead of Glu.
8)	Rha I and Rha 2 should be used as abreviation for Rhamnose in bot the table and the text.
9)	“ . ” should be used instead of “ , ”  In table one in the Coupling constant of carbon two .
10)	The coupling constant should be rewritting in the one decimal place.
11)	In line 200-201, the correlation is in the HMQC spectrum.

Yours Sincerely


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: FOUR SESQUITERPENE GLYCOSIDES FROM LOQUAT (ERIOBOTRYA JAPONICA) LEAF AMELIORATES PALMITIC ACID-INDUCED INSULIN RESISTANCE AND LIPID ACCUMULATION IN HEPG2 CELLS VIA AMPK SIGNALING PATHWAY
Review round: 1
Reviewer: 4

Output:
Basic reporting: Figure 3 C D has some typos. I supposed that Figure 3C should be "TG" content and Figure 3D should be "TC" content. However, two figures had the same labels.
Experimental design: In general, the experimental design could be improved in following aspect.
1. When insulin resistance is claimed, there should be evidence showing that insulin signaling is impaired by palmitate treatment. Therefore, in addition to basal condition, insulin stimulated condition should be also considered. This part of the data was not shown in this manuscript.
2. Basal glucose uptake was reduced by PA treatment. Is this due to "insulin resistance" in the absence of insulin stimulation? Besides, the rationale to put glucose uptake data with TG and TC content is not clear. Glucose uptake should be related to glycogen deposition and separated from PA-induced lipid accumulation issue.
3. How do SGs sensitize PA induced insulin resistance via AMPK activation remained to be clarified. Inhibitor study should be employed.
4. Due to the observation that SGs compounds can activate AMPK and reduce TG and TC content in PA treated cells, AMPK down stream substrate should be also analyzed.  Acetyl-CoA carboxylase (ACC) is one of the key substrate for AMPK to regulate beta-oxidation. Therefore, effects of SGs on ACC phosphorylation should be also measured. Such effect should be blocked by AMPK inhibitors to demonstrate that SGs action is really mediated by AMPK activation.
5. SREBP1 downstream genes related to lipid metabolism should be also measured in the current study to claim that SGs inhibits lipid deposition via SREBP1 .
Validity of the findings: no comments
Additional comments: In general, findings are too preliminary and experiments need to be expended.  Insulin stimulation condition should be included in "Insulin resistance" study. Inhibitor for AMPK should be used to strengthen the validity of finding in the investigation.


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: FOUR SESQUITERPENE GLYCOSIDES FROM LOQUAT (ERIOBOTRYA JAPONICA) LEAF AMELIORATES PALMITIC ACID-INDUCED INSULIN RESISTANCE AND LIPID ACCUMULATION IN HEPG2 CELLS VIA AMPK SIGNALING PATHWAY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript was sufficiently corrected and adapted.
Experimental design: -
Validity of the findings: -
Additional comments: The manuscript was sufficiently corrected and adapted.


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: SEED MORPHOLOGY AND SCULPTURE OF INVASIVE IMPATIENS CAPENSIS MEERB. FROM DIFFERENT HABITATS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Rewicz and colleagues performed seed morphology and sculpture of Impatiens capensis Meerb. from different habitats.
The authors organized the manuscript quite well, with a good introduction, that reports information about family and the genus which Impatiens taxa belonging to, and important historical citations. Moreover, tables and figures are clear and readable.
Experimental design: Material and methods are briefly enough. Research question and aim of study well defined.
Validity of the findings: Results and discussion are adequately structured to clearly describe their findings. Conlusions are well stated.
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: SEED MORPHOLOGY AND SCULPTURE OF INVASIVE IMPATIENS CAPENSIS MEERB. FROM DIFFERENT HABITATS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Comments to author
	In the present research work, the authors did work on the species Impatiens capensis seed morphology (SEM), their ultrastructure, and the relationship and variability of various environmental factors have been discussed. The paper is according to the scope of the journal, but I have some suggestions and comments on the present manuscript.
The introduction is to the point but need one paragraphs to explain the morphological and molecular difficulties of the genus Impatiens and specifically focus on the specie I. capensis.
Experimental design: In methodology some paragraphs are more looking background then the methodology. The subtitle “The Species” should be the part of introduction. Methodology is up to the point and well explained the study. This section is well explained the methodology section of the article despite with the results.
Validity of the findings: The results should be written in the past tense, change the present tense to the past tense. The results are written concise, and well explained with relation with methodology. Discussion is well explained and provide some basic information for the study of the species. I would like to suggest comparing your study with the recently published articles of other groups of plants i.e. 1) 10.1002/jemt.23393  2) 10.1002/jemt.23167
Some articles missing volume number and pages number, provide page and volume number in the revised manuscript.
Additional comments: The manuscript is well written, but i would like to suggest some major comments on the present manuscript. After that i will be agree to accept the manuscript.


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

Input:
Title: SEED MORPHOLOGY AND SCULPTURE OF INVASIVE IMPATIENS CAPENSIS MEERB. FROM DIFFERENT HABITATS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript is clear and easy to read. The background is also enough to follow and understand the story.
Experimental design: Good experimental design.
The methods are clear.
Validity of the findings: For me, the manuscript is a bit too simple for PeerJ. It is not very interesting to me. Many publications report on seed morphology of Impatiens.
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: SEED MORPHOLOGY AND SCULPTURE OF INVASIVE IMPATIENS CAPENSIS MEERB. FROM DIFFERENT HABITATS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I read the revised article and I am agree to accept the manuscript in current form. This manuscript is clear to understand and easy for the readers. The manuscript is have enough data to understand the scientific method of the article.
Experimental design: The experimental design is enough to understand the article.
Validity of the findings: The findings are well written and understandable.
Additional comments: I would like to accept the manuscript in current form.


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: SEED MORPHOLOGY AND SCULPTURE OF INVASIVE IMPATIENS CAPENSIS MEERB. FROM DIFFERENT HABITATS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript aims to investigate the seed morphology and sculpture of invasive species Impatiens capensis collecting from different locations. It also aims to show the effect of environmental variables on the seed features of the species. The text is well written but lacks clarity and sharpness in the discussion conclusion.
Experimental design: The methodology and statistical analyses are care, and interesting data are provided for the single species studied.
Validity of the findings: I don’t know the editor’s and journal policy, but in my opinion, this is very simple with a very narrow scope for the readers of PeerJ. As per the literature cited in the manuscript, the seed morphology of Impatiens capensis and related species is already published, and in my knowledge environmental factors including soil, characteristics have little but obvious effects on the seed morphological traits. They found no variation in ultrastructure including shape and structure of rib. The variation in seed size and mass is superficial might depend on the collection time, maturity, condition of the mother plants, and some other factors. The comparative study including some other related species would have been interesting though.
Additional comments: Specific comments are presented below, to guide the authors in the revision.
Abstract:
The abstract lacks key results and conclusions of the study.
Line 26: Our research shows also two types of cells on the ultrastructure of seeds, between the ribs and on the ribs, not yet described in the literature. I think this is a normal feature in the seed sculpture.
Line 27: The paper gives new data about the seed morphology of I. capensis growing in different habitat conditions as well as the information about the area – the circuit that has not been reported before. For me, this is a very general statement.
Same for line 29: The relationship between seed variability and various environmental factors was shown.
Introduction:
The introduction is very long. It covers the unnecessary history of the genus and species should be specific on the problems and objectives of the study. There are lots of unnecessary citations as well.
Materials and methods:
Line 149: anthropogenic? How about cultivated or human used land?
Line 152-153: the difference between almost the entire day and the entire day is confusing? What are the criteria for the condition?
Line: 167: how do you measure the thickness of the coating layer? Is it instrument-specific? I don’t think it is necessary to mention here.

Results: Tables 3 and 4 can be removed as the statistical data are presented in the text.

Discussion:
Discussion lacks clarity and sharpness. In several parts, the writing is also confusing.
Line 304-306: the seed measurement is very much comparable. The difference margin is small so it is very difficult to say the larger seed than previous reports.
Line 320-327: Writing is very confusing and vague. The discussion and comparison with I. nolitangere are superficial. The author needs to specify how the surface sculptures of both species differ.
Line 355-342: Not related to the discussion of this study. Please delete or you can adjust in the introduction part.
Line 346-367: The author discussed about the phenotypic character like height which is I think is beyond the scope of this study. Authors need to discuss how the seed morphology of I. capensis varies in different populations and what is the most influential environmental factor?
Line 368-374: I think this part is not related to the result of this study or not linked properly with the result of this study.
Conclusion: The whole part is just a general statement. I think this is not the conclusion of this study.


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: SEED MORPHOLOGY AND SCULPTURE OF INVASIVE IMPATIENS CAPENSIS MEERB. FROM DIFFERENT HABITATS
Review round: 2
Reviewer: 3

Output:
Basic reporting: I appreciate the general structure of the paper. The subject is properly introduced and the background knowledge about the studied species is rich. However, I have several points to report.
In the abstract is explained the kind of data revealed by the study but I would expect that the authors explicitly detail what are the main results obtained in one or two sentences (regarding relation to environmental variables and seed morphology variations between populations). Moreover, I am surprised that the emphasis is put on the methodology used (SEM) but less on the questions addressed, and would suggest to better balance the abstract towards hypotheses and results.
In the manuscript, I am surprised to find very limited details about molecular differentiations between Impatiens species, although the taxonomy of this group of species is discussed.
I understand the motivations of the authors to focus on Impatiens capensis, due to its invasive nature, however I am frustrated that the study only report data about seed morphology restricted to this species without including measurement and comparison with closely related species at least, or populations from the native area to make more relevant the study.
Experimental design: The statistical analyses performed are appropriated, well conducted and explained. Figures are appropriate and well detailed (maybe check Figure 4, Ordination diagram, it seems that the asterisks are missing on the statistically significant variables, and maybe verify the caption of this figure as I do not understand what the “Anthrop” refers to, no such variable in the diagram).
My main concern is about the very limited geographical range within which the seeds have been collected. The prospected area cover only approximately 20 x 60 km, and I am concerned about the representativeness of the data (trait mean and variations) at a larger scale (country, and European range) for the species considered. To address the requirements of the journal, I would suggest including to the study seeds from more populations, either from different part of the country (to make more relevant the effect of environmental factors on seed traits) or from different ranges (to confirm your observations apply to native population in the same extend).
Indeed, as evolution of life history traits (reproductive characters) is being more and more supported as an evolutionary explanation for plant invasiveness, this aspect is important to consider, at least to discuss. Although the authors acknowledge that plant is naturalized for about 30 years in Poland, rapid evolution as been reported for several plants and can occur very quickly under the effect of climatic factors, and reproductive characters being central to invasiveness, I would expect this to be verified (Beheemen et al., 2019, doi: 10.1111/nph.15564; Molina-Montenegro et al., 2018, doi: 10.3389/fpls).
I am surprised to see that moisture content has been hand-felt assessed and wonder how precise this method is. This variable being a fundamental resource for plant development its variations may be important to consider.
Validity of the findings: The results regarding inter populations variations of seed traits with environmental factors is interesting and should be emphasized. Morphological characterization seems less relevant because no comparison with closely related species is directly made in the study. For someone intending to address taxonomical differentiation issues between Impatiens sp. it would require to perform proper comparisons (e.g. through morphometric measurements on the different species concerned).
Additional comments: In my opinion, this study brings significant and relevant results although it may miss an hindsight with the inclusion of populations from different locations.


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: SEED MORPHOLOGY AND SCULPTURE OF INVASIVE IMPATIENS CAPENSIS MEERB. FROM DIFFERENT HABITATS
Review round: 2
Reviewer: 4

Output:
Basic reporting: Authors of the reviewed manuscript examined Impatiens capensis, an annual plant native to eastern North America which spreads across Europe and in Poland it is considered a locally invasive species. The species is an interesting and worth studying taxon, especially according to seeds which most probably play an important role in a rapid spread of that invasive species into secondary localities. Although the important differentiation in seed size and weight between examined populations was proved, the studies concerning seed biology and seedling recruitment in different environmental conditions were not undertaken.  With such additional studies the work would be much more complete and would allow to evaluate the importance of seed size differentiation in spreading strategy of that invasive species. Anyway, the above mentioned problem might be an interesting aim of the future studies on I. capensis.
The actual aims of the reviewed work were as follows: the first aim was to examine seed morphology based on the standard biometric measurements; the next aim was the examination of the seed coat ultrastructure with the use of SEM; the third aim was the evaluation of the potential variability of seed morphology between individuals growing in different populations and in different habitat conditions.

Abstract:
It is not fully in agreement with the obtained results. The Authors stated in lines: 24-25 that “The current work presents a detailed description of the morphology of the seeds of I. capensis using SEM for the first time”. In my opinion such really complete description is not given in the manuscript– for the details concerning several lacks please see some of my comments given to the paragraphs Results and Discussion. Additionally, to make the Abstract more concise and clear I would suggest to unit all new and all the most important findings and underline together their taxonomic significance and/or the novelty to science.
Introduction
The Introduction is to the point, however it is sometimes too detailed in my opinion, like e.g. the information concerning the botanical description of the stem and leaves which are not the subject of the study.
Please make the text more concise and do not describe the obvious facts: e.g. lines:74-75; also devoid the repetitions: e.g. lines: 51-52 and 53; 130-132 and lines: 120-123 on the previous page; I also suggest to use proper descriptions for the 3-dimensional objects which the seeds are (instead of those suitable for the 2-dimensional ones): e.g. line 120: “seed are oval to lanceolate with …. “– instead I suggest the following description: seeds were narrowly ovate and tapering to a point at the apex; you may also use the description:  seeds were laterally compressed, prolate spheroids, with four strong ribs.
There are also some inconsistences in the text e.g. in line: 93 it is written that …” One of the morphologically undescribed species is Impatiens capensis” while later in line: 125-126 the text is as follows …” Numerous studies (over several hundred – see Adamowski 2016 onward and the literature cited therein) have been devoted to the morphology, ecology, biology and genetics of this species…” – maybe the Authors meant the whole genus Impatiens, not the examined species? But it is not clean and should be corrected.
The aim of the study described in the last paragraph of the Introduction is awkward.
The English language should be improved in my opinion; it should be checked by the native speaker.
Results and Discussion:
I have listed below just few examples/observations (not all of them …) to be corrected:
-	lines: 221-222: the sentences: “The seeds from the G (Police) population is characterized by the biggest seeds” and “This population characterized by the highest average values of the following traits:…” are not clear;.
-	Lines: 219-220: “In the B (Lubin) population was observed the shortest and narrowest seeds (respectively:…)” again the English language correction is necessary
-	 The paragraph : The structure of seed surface must be rewritten according to the given below suggestions; in the present form it is not clear and the interesting results are not fully and properly described;
-	The finding concerning the clear difference between seed morphology of the examined species and the closely related I. noli-tangere is important, but it would be also interesting to compare the observed type of the seed sculpture with the findings concerning other Impatiens species described by different authors (many of them were cited by the Authors, but I also recommend to see the additional following articles:

1. Three New Species of Impatiens L. from China and Vietnam: Preparation of
Flowers and Morphology of Pollen and Seeds
Author(s) :Yu-Min Shui, Steven Janssens, Su-Hua Huang, Wen-Hong Chen, and Zhi-Guo Yang Source: Systematic Botany, 36(2):428-439. 2011 and

2. 45 (5): 708–712(2007) doi:10.1360/aps06037 Acta Phytotaxonomica Sinica
Impatiens macrovexilla var. yaoshanensis S. X. Yu, Y. L.
Chen & H. N. Qin, a new variety of Balsaminaceae
from Guangxi, China.
1,2YU Sheng-Xiang 1CHEN Yi-Lin 1QIN Hai-Ning*

But to do such comparison it will be necessary to improve the present description of the seed coat micromorphology with several missing elements. Based on the observations of the structure and the microornamentation pattern of the epidermal cells of the testa, the following characters of the primary/secondary sculpture should be described or corrected: cellular/microornametation pattern should be defined; the cell outline was not clearly described; specific characters of the anticlinal walls should be described (degree of their elevation and thy type of their boundary) - by the way, from which literature comes the description anticyclic walls?; specific characters of the outer periclinal walls should be described (flat, convex, concave etc.); the secondary sculpture (if present) should also be described (e.g. cuticle striations, perhaps the  presence of some protrusions etc ….).

The present descriptions of SEM images (figure captions) are not clear enough, they should be more informative, and the arrows pointing particular details should be described.
Experimental design: It is an original primary research which fits to the scope of the journal.

However the methodology needs some additional information:
-	How big were the examined populations (How many individuals were growing in them approx.? Were there only few, several or more individuals, e.g. not less than 50?)
-	Did you do any preparations of the seeds before examining them in SEM; were they anyhow dried?  Was the seed dehydration necessary? If yes, how was it done?
-	From how many individuals in each population were the seeds collected?
-	line 171: instead “pictures of seed obtained using SEM’ you may write: SEM images of seeds or  SEM micrographs etc. …
Validity of the findings: Concerning the variety of the examined plant material the performed studies are not too extensive, but the additional aspects concerning the relationships between the examined seed morphological characters and different environmental conditions gives the reviewed work an additional value.

The results are important and interesting. I suggest to complete and discuss the results of seed SEM analysis according to the given comments and to prepare the revised version of the manuscript. While preparing the description of the seed micromorphology and testa sculpture I would advice to follow the terminology of Barthlott W. (1981; 1984).
Additional comments: I would like to suggest Authors some minor revision of the manuscript, and encourage them to take under consideration my comments.


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

Input:
Title: SEED MORPHOLOGY AND SCULPTURE OF INVASIVE IMPATIENS CAPENSIS MEERB. FROM DIFFERENT HABITATS
Review round: 3
Reviewer: 1

Output:
Basic reporting: No more comments on this part.
Experimental design: No more comments on this part.
Validity of the findings: No more comments on this part.
Additional comments: I appreciate that the comments made on previous version of the manuscript have been taken into account.  I understand that the work presented here is partial and will be included in a much larger study, coming in series of more articles. I have no more comments, and would be satisfied that the work is published as is.


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: CHANGES AND SEX- AND AGE-RELATED DIFFERENCES IN THE EXPRESSION OF DRUG METABOLIZING ENZYMES IN A KRAS-MUTANT MOUSE MODEL OF LUNG CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: Age, sex and disease-state could have an impact on the expression of drug metabolizing enzymes, which could then contribute to the differences in drug efficacy and toxicity. In this study, Li et al. attempt to profile the effect of sex, age and disease-state on the expression of DMEs in a lung cancer model. While it is important to profile the age-and sex- related differences in DME expression in a lung tumor model to predict adverse drug reactions or lack of response of chemotherapeutic agents, the study presents numerous limitations. Incoherent language and grammatical errors throughout the paper make it very difficult to read. I advise the authors to kindly revise the language of the manuscript thoroughly to make it clearer. The references provided were inadequate and had several errors. Few references were cited multiple times and most of the papers cited were review articles and not original papers. It was unclear whether independent experiments were performed to validate the study. While they show differences in DME levels with respect to age, sex and disease state, they fail to discuss any possible hypothesis for these observations.

Please see my specific comments below:

Line 51-52 “Therefore, it is necessary to investigate the expression of DMEs for drug safety and availability.” This sentence is abrupt and misleading. Please amend the sentence to reflect that you are specifically investigating the age and sex-related differences in DME expression levels in a lung cancer model.

Line 53-60. Please revise the language and sentence structure in this paragraph.  Provide justification to choosing KRAS model to study differences in DME expression in a lung cancer context in a concise manner.

Line72. Please change the sentence to “Sex and age are important factors influencing the expression level of DMEs”. Include references.

Please include references for the following lines: Line 65, 67, 72

Line 76.  Please be more specific about which enzyme levels/ oral drugs you are referring to. Also, please find an appropriate reference for this. Waxman and Holloway 2009 is a review paper and it does not conclude that there is an increase in clearance rate of oral drugs by 17-26% in females. Cite original and not reviews articles whenever possible.

Line 91. Change the sentence to “.. can increase the sensitivity and throughput..”
Experimental design: Methods:

Line 112. Correct typo in C57 BL/6

Line 168-168. Change the sentence to “… were diluted to a recommended dilution of 1:1000 with 5% milk..”

Line 130. What microscope was used for the histopathological analysis?

Please include details the following details in your methods/results section:
You report average of technical replicates in most of the figures.  How many independent experiments were performed? This is very important to determine whether the data was reproducible.

Please specify the number of animals used per group in EACH study.

Results:
Fig 3 and Supplemental Fig 4- Please mention the sex of the mice on the graph.

Supplemental Fig 3. Why is there an increase in many of the enzyme expression levels in female KRAS mice at week 15 in the liver? (Not seen in male mice at the same time point). If this data is reliable, please discuss the possible reasons for this.

Fig 4. What are the possible reasons for the enzyme activity of Cyp3a11 not correlating with the protein levels in the liver? Your attempt to discuss this in Line 301 is not satisfactory.

Fig. 5 – It is not acceptable to have so much variation in your beta –actin bands (loading control) across the samples. Please label your figures appropriately.  Which lanes correspond to WT and KRAS mice? What about the receptor expression in female WT and KRAS mice?

Fig 6- Why did you choose 10 weeks as your time point for this study?
Line 248 “ the differences in DME content based on sex are similar in KRAS mice and WT mice” This statement is not clear. Are you implying that sex-related changes in DME enzymes have a similar trend both in WT and KRAS mice? While this is true at 10 weeks, you see different trends in male and female KRAS mice after week 15 (Fig 3 and supplemental Fig 4) and this needs to be discussed.  According to the literature, Cyp3A4 (human ortholog of Cyp3a11) is expressed at higher level in females than males.  Can you discuss why you do not see a similar trend (at least in the WT mice at week 10) in your study?
Validity of the findings: Discussion:

Line 272. You statement does not reflect that the downregulation of Cyp2c29 and Cyp3a11 was observed only in male and not female KRAS mice at 26 weeks.

Line 277, 310 and 50 -you cite Kusunoki 2015. Is it Kusunoki 2015 or 2014? In Line 281 you refer to the same paper as Kusunoki 2014.  Please double check ALL your references.

Line 285-286. Again, upregulation in Sult1a1 and Sult1d1 enzyme levels was seen in only male but not female KRAS mice after week 20.

What is your hypothesis for altered DME expression is KRAS mice?

How do you explain the sex-related differences in expression of hepatic enzymes in older KRAS mice? Do hormones have a role to play in the expression of DME?

I do not agree with the conclusive remarks of this study. This study attempts to profile sex-and age-related differences in DME expression in KRAS mice. However, female KRAS mice do not show the same age-related changes in DME expression as the male KRAS mice. This needs to be highlighted and discussed in detail. All the conclusions of altered DME expression in KRAS mice were drawn from the data with the male mice (with the data from female mice merely provided as supplemental data).
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: CHANGES AND SEX- AND AGE-RELATED DIFFERENCES IN THE EXPRESSION OF DRUG METABOLIZING ENZYMES IN A KRAS-MUTANT MOUSE MODEL OF LUNG CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is largely well written and structured. However, it could use a proper revision for grammar and sentence structure in a few places, for example lines 42, 43, 53, 73, 74, 83, 91 etc.

The results and conclusions are within the stated hypotheses and do no make any exaggerated claims.

Points to address:
- please state what is S9 fraction
- some nomenclature is inconsistent. For example, pay attention to case in gene and protein names. in vivo/ in vitro should be consistently italicized
- Fig 2b, what is the x-axis? The figure is not clear
-Fig 5a, what are the different lanes in the western blot? Actin expression is not consistent. Is the higher expression normalized with other genes in that lane?
Experimental design: The question is well defined and the authors present relevant data to address the aims of the study.
Validity of the findings: The conclusions are clear and discussion of the data has been made in a sufficient way with proper links to existing literature.
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: CHANGES AND SEX- AND AGE-RELATED DIFFERENCES IN THE EXPRESSION OF DRUG METABOLIZING ENZYMES IN A KRAS-MUTANT MOUSE MODEL OF LUNG CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: In this work by Li et al. the authors investigate the sex- and age-dependent differences in the expression of 21 drug-metabolizing enzymes in WT and KRAS mice. The findings are of interest; nonetheless, I do have few questions and suggestions regarding the study to clarify these points.
1.	There are various repetitions in the text-lines 47, 66, 73, 76 and lines 51, 78
2.	Please provide the references for the statements in lines 54, 56, 57, 61, 65, 72, 87
3.	The language in the present manuscript requires major improvement – it is full of errors and typos. There are many grammatical, linguistic and spelling mistakes throughout the manuscript.
Some examples where language can be improved include lines 115, 116, 119, 121, 122, 129, 132  etc
4.	Please add scale bars to the images - Fig. 1 and Supplemental Fig. 2
5.	It would help if the histopathology described (line 189-190) is marked on the images (fig. 1b and supplemental fig. 2) with arrows.
6.	Did the authors see any difference in the size and the number of lung nodules across different age groups in KRAS mice?
7.	Please provide a more detailed description of the results part.
For example, the description of the PLS-DA analysis should be improved (line 191-193)
-line 223-226 please provide a detailed explanation
8.	The authors refer to the expression of Sult1a1 (20 weeks) and Sult1a1/Sult1d1 (26 weeks) in the liver as significant (p< 0.05); however, the asterisk is missing from the figure (Fig. 2b).
9.	Symbols for genes should be italicized, for example Cyp3a11
10.	 Please explain, in the results part, based on what criteria were only Cyp2e1, Cyp3a11, Ugt1a9 and Sult1a1 selected for the enzyme activity - Fig. 3?
11.	 Fig. 5, are the first 5 wells for WT mice? Please indicate that on the figure
- It is not clear, does Fig. 5b represent a normalization to actin?
- Please explain the choice of these particular receptors.
- Actin is unequal between samples, even in the same group, I would suggest
  to use another loading control or to re-do the blot.
- The authors did not specify which age group was used for WB analysis.
12.	It would be interesting to check if there is any difference in the protein
       abundance of these receptors in  the female mice.
13.	 In the discussion, authors use couple of sentences to discuss the major finding-sex- age- and tissue-related differences in the expression of DME. Please elaborate it more and include a paragraph illustrating a relevance of the main finding.


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: CHANGES AND SEX- AND AGE-RELATED DIFFERENCES IN THE EXPRESSION OF DRUG METABOLIZING ENZYMES IN A KRAS-MUTANT MOUSE MODEL OF LUNG CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed the questions satisfactorily. The language has been improved considerably, with more details added to the methods section. I was satisfied with the changes made to the discussion as well.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: No additional comments


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

Input:
Title: CHANGES AND SEX- AND AGE-RELATED DIFFERENCES IN THE EXPRESSION OF DRUG METABOLIZING ENZYMES IN A KRAS-MUTANT MOUSE MODEL OF LUNG CANCER
Review round: 2
Reviewer: 2

Output:
Basic reporting: The study presented here is much clearer and polished compared to the initial submission. The authors have made the changes to convert it into a more professional manuscript. The references are improved and figures have been made clearer. My comments on the initial submission have been addressed satisfactorily.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I wish you the best for your future endeavors


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: CHANGES AND SEX- AND AGE-RELATED DIFFERENCES IN THE EXPRESSION OF DRUG METABOLIZING ENZYMES IN A KRAS-MUTANT MOUSE MODEL OF LUNG CANCER
Review round: 2
Reviewer: 3

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: The authors addressed all concerns raised by the reviewers and the manuscript has been significantly improved. However, there are some points which still need further clarification;
1.	Please correct
-	line 59: change  ‘’detoxifivcation’’ to detoxification
-	line 167: change the sentence to ‘’before the experiment, animals were fasted overnight but allowed free access to water’’.
-	Line 168: change the sentence to ‘’all procedures were performed…’’
-	Line 180: change ‘’fixated’’ to fixed
-	Line 224: change the sentence to ‘’Then, the solution was vortexed and thereafter centrifuged for…’’
-	Line 226: change the sentence to ‘’ the enzyme activity was measured from 2 independent experiments’’
-	Line 249: add ‘’was performed from 2 independent experiments’’

2.	Line 169: it is not clear what do the authors mean by: ‘’at the conclusion of the experiment, the animals bodies were harmlessly treated by professionals’’
3.	Line 199, the authors state that 10 isoforms of UGTs were analysed, however they show only 9 isoforms in Fig. 2 (F, G, H), UGT1A2 is missing
4.	Line 269, the authors mention that cell membranes were broken in H&E images, however this is not clear from the image, please clarify or mark it on the image.
5.	Please mention in the results part, for Fig. 2, what do the arrows correspond for
6.	Authors state that the cancer stage of 20-week old KRAS mice corresponds to the advanced stage of human lung cancer. Could you please explain why did you then choose 26-week old mice mice for your experiments (western blot, histology), taking into consideration that these mice live 28 weeks and as Fig. 1 in the rebuttal letter shows, it is already difficult to distinguish lung morphology in mice at 26-weeks of age. And how does this age group (26-weeks) correlate to cancer stage in humans?
7.	Figure 4, did the authors correlate the enzyme activities of 26-week old mice? Please mention the age in the figure legend
8.	I would suggest to add to the main manuscript the explanation from the rebuttal letter, (Reviewer 3- question 10) of the criteria for selection Cyp2e1, Cyp3a11, Ugt1a9 and Sult1a1 for the enzyme activity - Fig. 3
‘’In our present study, we found the CYP2C29/CYP3A11/SULT1A1/SULT1D1 displayed significant changes in protein expression in the liver of male WT and KRAS mice. Therefore, we are intended to research their activities in the liver tissue. We failed to find an authoritative and specific probe to study the activity of Cyp2c29. Chlorzoxazone and Propofol are usually used as specific substrates to study the activities of Cyp2e1 and Ugt1a9. Their good correlation between protein expression and activities indicated the protein quantification results are credible. Therefore, we select them for the enzyme activity test, intending to show a good correlation between protein expression and activity’’.


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: CHANGES AND SEX- AND AGE-RELATED DIFFERENCES IN THE EXPRESSION OF DRUG METABOLIZING ENZYMES IN A KRAS-MUTANT MOUSE MODEL OF LUNG CANCER
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors have addressed all raised points.
I would only ask the authors to rewrite/rephrase the sentence (line 70-71) using correct English.
Experimental design: -
Validity of the findings: -
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: ADAPTIVE DIVERGENCE, NEUTRAL PANMIXIA, AND ALGAL SYMBIONT POPULATION STRUCTURE IN THE TEMPERATE CORAL ASTRANGIA POCULATA ALONG THE MID-ATLANTIC UNITED STATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript (#47962), entitled “Adaptive divergence, neutral panmixia, and algal symbiont population structure in the temperate coral Astrangia poculata along the Mid-Atlantic United States”, Aichelman and Barshis aim to address a central question in marine molecular ecology: what are the forces shaping the pattern of genetic structure within species?
Focusing of a temperate and facultative symbiotic coral, the northern star coral, Astrangia poculata (= A. danae), they “use high-throughput mRNA sequencing to consider patterns of neutral and adaptive differentiation” in the host and the symbiont, Breviolum psygmophilum (l.101-103). More particularly, they “characterized population structure of the A. poculata host and its symbiont (B. psygmophilum) both by population (VA vs. RI) and by symbiotic state (brown vs. white) using SNPs derived from mRNA-Seq data” (l.121-123). Interestingly, this work complements a recently published paper (Aichelman et al. 2019) suggesting the occurrence of adaption to local thermal conditions in this species.
The Authors focused on symbiotic and asymbiotic individuals from two populations: “10 brown and 10 white Astrangia poculata colonies were collected each from Virginia (VA) and Rhode Island (RI), USA” (l. 130-131). These individuals were submitted to a common garden experiment (l.165-204) “designed to consider differential gene expression in A. poculata across temperatures and populations” (l.152-153). Here, the Authors present “the sequencing data from this experiment and the resulting single nucleotide polymorphisms (SNPs) to consider population structure” (l.153-155).
The Authors sequenced 96 libraries (84 for gene expression analyses + 12 for population genetics analyses) from 10 symbiotic and 10 asymbiotic individuals from two localities. They conducted the de novo transcriptome assembly and SNP calling and filtering. They analyzed the population genetic structure contrasting the pattern between “neutral” and “outlier” loci in the host and the symbiont using an AMOVA, F-statistics and PCA (l.272-315). In addition to gene ontology analyses was conducted on outlier SNPs to “determine if genes containing outlier SNPs were enriched for any particular GO categories” (l.318).
Based on these analyses, the Authors discuss: 1) the occurrence of contrasting patterns of genetic structure among the host and the symbiont; 2) the potential impact of physical barrier on connectivity among the two populations; 3) the influence of local temperature regime on the pattern of genetic differentiation; 4) identify putative genes that may be involved in adaptation to local thermal conditions.

Basic reporting:

The paper is well written (except some typos, eg. populationS on l. 332, 333).

Ideas are clear, reasoning is well exposed.

The Introduction section gives an interesting perspective starting with general considerations on marine connectivity, issues linked to the study of holobionts and the potential role of temperature on the pattern of intra-specific diversity. The model and the objective of the study are well presented and described. One potential flaw may be the lack of reference to works already published in temperate corals (except Costantini et al. 2007). The Authors may consider the following papers focused: i) on the neutral genetic structure in temperate corals: Ledoux et al. 2010 Mol. Ecol., Ledoux et al. 2018 JBiogeo, Aurelle et al. 2011 Genetica, Arizmendi-Meija et al. 2015 Plos One, Masmoudi et al. 2016 EcolEvol; ii) on the potential for local adaptation to thermal conditions in temperate corals: Ledoux et al. 2015 Ecol Evol, Arizmendi-Meija et al. 2015 Coral Reefs, Haguenauer et al. 2013 JEMBE, Crisci et al. 2017 Scientific Rep. The Introduction and the Discussion sections may be strengthened by adding some of these references.

In addition, one of the main originalities of the paper is to be focused on a facultative symbiotic coral species. Whether these models have been previously studied in the context of population genetics or local adaptation remain unclear based on the Introduction. This may be improved.

Details on the previous work conducted by the Authors (Aichelman et al. 2019) may be mentioned in the Introduction instead of being presented in the Material and Methods.

The structure of the paper is conformed to PeerJ Standards. Figures and tables are relevant. I would suggest to complete the Figure 1 with the localities discussed from l. 428-468 (e.g. Cap cod, Virginia province).

I probably missed them but the raw data do not seem to be available (no depository number).
Experimental design: Experimental design:
The issue addressed by the Authors perfectly fits the scope of PeerJ.

The research questions are relevant. However, it would be nice to explain why do the Authors focused on the potential impact of temperature in this particular species (e.g. potential impact of climate change?).

Investigation is rigorous in line with high ethical and technical standard.

The methods are sufficiently detailed in most of the manuscript. However, I have some minor concerns regarding the design of the common garden experiment as well as the filtering of molecular markers.
In my understanding, the idea of common garden would be used to name the whole experimental design (e.g. Villemereuil et al. 2015 Heredity; Kawecki 2004 Ecology Letters) and not only the holding tank as did by the Authors. This may be confusing and should be rephrased.
The Authors did not replicate the experimental conditions (i.e. only one warm and one cold tank). Logistic may be a limiting factor for replication, however this lack of “experimental tank” replication and potential logistical limitations should be mentioned.

Did the Authors genetically confirm the individuals used in the experiment were not clone?

Regarding the SNP markers, the Authors conducted the SNP filtering at two levels: considering two and four populations, respectively. It is not clear from the manuscript why the Authors choose this approach resulting in two different sets of SNPs, instead of considering the same set of SNPs for two and four populations and to what extend the SNPs that are not shared among the two levels influence the results of the analyses. This should be considered by the Authors.

The Authors stated “It should be noted that the dataset presented here had relatively low read counts, resulting from an error in the library preparation that led to sequencing of rRNA in addition to mRNA. To combat this, we have used detailed screening methods at every step of the data analysis pipeline to address these issues” (l.277-280). While they mentioned “detailed screening methods”, I was not able to identify these particular screening methods in the present version of the MS. Moreover, the potential implications of the errors in library preparation should be explained.

The number of filtered SNPs is relatively low (1,506 and 1,562 considering 2 and 4 host populations, respectively and 68 for the symbiont). From these pools of SNPs and following the Baypass results, the analyses were conducted on 226 and 9 neutral and selected SNPs and 213 and 12 neutral and selected SNPs for the four and two populations analyses, respectively.
The difference in the number of high-quality SNPs and the SNPs used for analyses should be explained both for the host and the symbiont.

It may have been interesting to consider the whole pool of SNPs first (neutral + selected) and then to conduct the analyses on neutral vs. potentially selected markers, separately.

The Authors mentioned “high-quality SNPs”. What do the “high-quality” refer to?

Moreover, considering the high dispersal capacity of Astrangia poculata, the expected level of genetic structure should be very low. Accordingly, the lack of significant genetic structure reported from neutral SNPs may potentially be linked to the low number of SNPs analyzed. Did the Authors consider this hypothesis?
Validity of the findings: Validity of the finding:
Overall, this is an interesting study focused on an original model (temperate facultative symbiotic coral). Conclusions are globally well supported by the analyses and fully linked to the research question.

I would suggest the Authors to rephrase parts of the manuscript addressing the contrasted patterns of genetic differentiation among the host and the symbiont to better fit with the analyses and results. Indeed, the pattern of “selected” genetic structure was not characterized in the Symbiont.

The Authors may also consider to precise the type of genetic markers used when discussing the results of previous population genetics studies conducted in the same region (paragraph 4.2).

It would be interesting to develop the idea that selection may drive the pattern of differentiation at the neutral markers in the Symbiont (l.476).

The Authors stated “Interestingly, the stronger signal of divergence in the algal symbiont is in contrast with our previous results demonstrating the strongest physiological population divergence in coral host respiration” on l. 541-543. In the present form, this sentence may appear a bit vague. The Authors should consider to develop a bit more this issue and its implication in term of local adaptation both at the symbiont and host levels.
Additional comments: General comments:
As mentioned, the study presented by Aichelman and Barshis is interesting and well written. In my opinion, it fits the standard of publication in PeerJ. However, I encourage the Authors to account for the comments previously made in order to clarify some parts of the paper.