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: EFFECTS OF TAPHONOMIC DEFORMATION ON GEOMETRIC MORPHOMETRIC ANALYSIS OF FOSSILS: A STUDY USING THE DICYNODONT DIICTODON FELICEPS (THERAPSIDA, ANOMODONTIA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: See PDF
Experimental design: See PDF
Validity of the findings: See PDF
Additional comments: Review: Kammerer et al., Effects of taphonomic deformation on geometric morphometric analysis of fossils: a case study using the dicynodont Diictodon feliceps (Therapsida, Anomodontia)

The authors aimed to examine the impacts of taphonomy on geometric morphometric analyses of fossils using Diictodon and a truly impressive sample size, not often seen in vertebrate paleontology. Using a combination of empirical and simulated datasets, they assessed the detectability of sexual dimorphism and ontogenetic trends in their Diictodon dataset. Additionally, they examined a dataset of anomodonts in addition to their Diictodon dataset to determine whether variation between higher taxonomic levels is swamped by taphonomic deformation. They found that while biologic trends were detectable in their dataset, it would be difficult to tease factors such as sexual dimorphism, ontogeny, and taphonomic variation apart in practice. Within their larger analysis, they found that phylogeny was not overprinted by taphonomy suggesting that geometric morphometric analyses on higher-level taxonomic groups will likely not be as strongly impacted by taphonomic distortion.

This is a powerful paper and a worthy addition to the literature. I have outlined several points below that would improve this paper, but I strongly feel that it should be published after minor revisions.

-Brandon Hedrick

Comments:

Line 48–51: Check the grammar in this sentence

Line 63: Note that Hedrick et al. (2019) did not use a retrodeformation technique. Rather, our paper attempted to quantify the percent of variation that could be attributed to taphonomy. It in fact has a section discussing the issues with retrodeformation. Hedrick et al. (2019) should be cited elsewhere in the paper, especially because it closely aligns with the goals you present as it also attempts to understand to what ‘degree taphonomic deformation can overwrite the underlying signal of biological shape variation.’

Line 66: Large sample sizes? Large samples sounds like ‘large specimens’

Line 102: ‘to quantify the amount…’

Line 108: Yes, this is a great point. Hedrick and Dodson (2013) discussed this at length, finding different specimens of Psittacosaurus going off in different directions depending on whether they were dorsoventrally compressed, laterally compressed, or a combination.

Line 91–120: Please add some citations here. Angielczyk and Sheets (2007), Hedrick and Dodson (2013), Tschopp et al. (2013), Baert et al. (2014), and Hedrick et al. (2019) discuss many of these ideas at length.

Line 156: add period to the end of the sentence.

Line 256: I wouldn’t say that eigenvalues to the right of the broken stick are random noise, so much as not meaningful to the overall variance structure. They usually represent singleton taxa or single landmark displacements, which are not random noise.

Line 266: How many iterations?

Line 269: DefCat citation?

Line 330: I was having a little trouble following the beginning of this paragraph. Perhaps, it could be reworded in a few places? ‘For each biological signal,…”, How many specimens were in each ‘dataset’ in each group?

Line 354: add ‘(Adams and Otárola-Castillo, 2013)’

Line 423: Interesting that anteroposterior compression is so rare. However, that is what I have qualitatively seen in my work as well. I like your interpretation as to why.

Line 487: It would be better to avoid the use of significant in colloquial terms here. Maybe substantial throughout when referring to results from the broken stick criterion?

Line 595: Was this tested with the K-mult statistic (Adams, 2014) or is this a qualitative observation?

Line 639: ’10.0’

Line 653: ‘largest’ instead of most

Line 670: ‘but not 1 through 4’?

Line 685–687: Grammar, reword

Line 739: I would be wary here. I don’t think that morphometrics should be used to directly assess taxonomy since autapomorphies axiomatically cannot be landmarked since they do not appear on all specimens. It might be worth expanding on that point here.

Line 755–765: I have wondered for a long time at what taxonomic level distortion no longer overprints biological signal and I find this point quite convincing and important.

Line 780: I like that you are up front about the difficulties of discerning biological signals in practice. However, I am not sure that looking at single taphomorphotypes would help to uncover true signals. It might be more likely to generate false signals that have higher (but spurious) correlations (e.g., if you only have laterally compressed samples and are looking for ontogenetic trends, you might get a higher correlation between shape and size than if you used multiple distortion types. However, the trends you would see in the data would be largely related to the lateral distortion). Could you expand on this idea for a few sentences?

Line 801: ‘ontogeny’

Lines 810–817: Yes, I wonder this too and speculated on it in Hedrick et al. (2019). I think future work examining taphonomic variation at different time slices will need to be done in the future to examine this question fully.

Line 834: Add Baert et al. (2014) and Hedrick et al. (2019) here

Figure 8, 10, 12: Unfortunately the colors in A and D are very difficult to discern. I would strongly suggest making these figures larger and the dots larger, especially because space is not at a premium in PeerJ. Perhaps more different colors would also help. As it is now with the resolution of figures I have, I pretty much have to take your word for where the points are. This is somewhat of a problem for figure 14–20 as well, but is helped by the additional figures with Diictodon in blue and the other groups in gray.

Not sure if you did this in R or MorphoJ, but this is a palette that I’ve found works for a large number of groups: palette(brewer.pal(12, "Paired"))


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: EFFECTS OF TAPHONOMIC DEFORMATION ON GEOMETRIC MORPHOMETRIC ANALYSIS OF FOSSILS: A STUDY USING THE DICYNODONT DIICTODON FELICEPS (THERAPSIDA, ANOMODONTIA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This is a very thorough and detailed manuscript and I can happily recommend it for acceptance with some very minor revisions. I would recommend that the authors carry out MANOVAs (for example, with the procD.lm function in geomorph) to test whether in morphology differs among groups in each of the classification schemes (deformation type, size class, sex, and assemblage zone). The interpretations of morphospace occupation are important for the conclusions of the paper and they would benefit from this type of quantitative assessment.
Line 145: Citation needed for “(well-supported sources of morphological variability in actual Diictodon)”
Line 436: why is anterodorsal shear most problematic for morphometrics?


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: EFFECTS OF TAPHONOMIC DEFORMATION ON GEOMETRIC MORPHOMETRIC ANALYSIS OF FOSSILS: A STUDY USING THE DICYNODONT DIICTODON FELICEPS (THERAPSIDA, ANOMODONTIA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: This manuscript was a pleasure to read and I think it is basically ready for publication as-is. Interpreting taphonomic deformation and its effects on morphological disparity within and between taxa is both challenging and underappreciated by many palaeontologists, and this study provides a welcome framework for understanding the effects of taphonomic variation that I think will be very useful to vertebrate palaeontologists. The questions being addressed in this study are clearly presented, and the methods are sound (I particularly liked breaking the specimens into assemblage zones to test for taphonomic signals within the basin, in addition to size classes and sex). I have one minor suggestion that would be a helpful addition to the methods, and that would be a brief discussion about how non-taphonomically distorted specimens were identified from distorted specimens. I don’t doubt any of the classifications, but for the sake of clarity I think a few notes that you looked for things like bilateral asymmetry, elliptical deviations from circular structures like orbits or the foramen magnum, breaks/crushing/displacement, etc., would be very helpful. Thanks for the opportunity to review this extremely interesting 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: EFFECTS OF TAPHONOMIC DEFORMATION ON GEOMETRIC MORPHOMETRIC ANALYSIS OF FOSSILS: A STUDY USING THE DICYNODONT DIICTODON FELICEPS (THERAPSIDA, ANOMODONTIA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I thank the authors for taking the majority of my comments in a previous review. I find that this paper is now ready to be accepted. It is an excellent addition to the literature. I did find a few minor grammar points that should be corrected before the proof stage, noted below.

Line 53: ‘rates’

Line 344: ‘amount’ reads weird. Maybe ‘number’?

Line 407: ‘crania’

Line 560: ‘meaningful’ rather than ‘significant’


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: FACTORS INFLUENCING UNMET NEED FOR CONTRACEPTION AMONGST ADOLESCENT GIRLS AND WOMEN IN CAMBODIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall this is a well written article which has some valuable insights into a key determinant of health – unmet need for contraception. Below are some recommendations to improve readability.

The abstract is clear and concise.

I’m not a fan of the acronym you have used throughout: UC for unmet need. It impacts the readability of the text. In text I would strongly recommend using unmet need - particularly in intro and discussion. Perhaps use UMN if you really need to use an acronym within the results section where it would be most useful (I have seen UMN used in other manuscripts, but it is still not overly common). I realise it is very tempting with word counts being so strict, but UC is very distracting. My mind kept going to universal health care/ universal coverage. Also, in some tables you wrote UNC for unmet need which is inconsistent. I think it is fine to say: unmet need for contraception (hereafter, unmet need). That way you are not writing out the full definition each time, but it will read better.

While I agree with your statements in lines 134-137, it feels slightly clunky here. Here are two suggestions that might work:
1)	Perhaps this argument fits better in the introduction paragraph about unmet need in Cambodia. Specifically, where you mention the CDHS and note young women and girls are bearing the disproportionate burden of SRH issues…but then, as you have noted, are often not included in data collection unless they are married. Hence the suitability of CDHS which has captured this information.
2)	Alternatively, it could work in the methods in the context of explaining why you used the DHS data. A fair amount of space is used describing the methods used in the CDHS which are documented elsewhere (lines 118-126) – perhaps this could be written more succinctly, and the space could be used to explain why you used the CDHS data. The fact that it is nationally representative is not clear – but should be.

The description of the outcome variable (lines 163-176) is long, and really appears to repeat what is said more succinctly in lines 155-161. If you do think this much detail is required – perhaps it is better placed in a clear flow chart or table and moved to the appendix if you have met your max number of figures already.

The description of the statistical analyses is good, clear and concise. Covers the main issues with an mlr model, such as how the variables were introduced to the model, how you dealt with multicollinearity, missing data and goodness of fit.

I really like figure 1, clear and straightforward. I would switch the a, b, c to bullet points because underneath you have references labelled a, b, which could be confusing and lead people to think they are related.

Having the references mid-sentence makes the paper hard to read. Is this a PeerJ requirement? If not, I would consider moving all references to the end of the sentence, unless they are using an in sentence citation, such as “Bradley et al in 2012 proposed….”
e.g. currently:  The birth spacing policy in Cambodia was introduced in 1995 as part of the national family planning program (8) in the wake of the ICPD 1994 (2).
Change to:
The birth spacing policy in Cambodia was introduced in 1995 as part of the national family planning program in the wake of the ICPD 1994 (2, 8).

Appendix - S1 policy implications - refers throughout to 'this chapter' if this is to be published with the paper it should probably be reworded to 'this paper' or this study/ analysis.
Experimental design: The research is novel and is within the scope of the journal. Research question is clear and fills an identified knowledge gap.

Ethical standard is sufficient for a secondary analysis of publicly available data. Methods are clear and sufficient for replication, with changes noted below.

Table 1 – I would like to see a total column for each age,  given that age was a significant factor but urban/rural was not. It also helps with replicability; I know it can be calculated but it’s good to lay out for the reader. I appreciate you included a graph for unmet need by age in supplemental materials- thank you- but it still would be good to see those totals in the table which will be in text.

Thank you for providing the stata output. It would be more useful to readers if it had some further annotation around the variables in each model. This can be easily rectified with a small table at the beginning of the output, so the reader knows which variables relate to which question in the survey.
Validity of the findings: The data used are robust, the statistical methods are sound, and the results are transparent. The findings present a clear benefit to the literature. The conclusions and recommendations need some further refinement, as detailed in general comments below.
Additional comments: Overall this paper is well written and informative. Country level analyses like those presented here have the potential to make a significant contribution to policy direction and research priorities in LMICs. You have presented some interesting and novel findings, interpreted them well, and made some good recommendations. I believe the manuscript could be strengthened with these recommendations:

1.	Is there an opportunity to bring on Cambodian collaborator/s as authors on the paper? University contacts or Non-gov institutions such as WHO, Unicef, UNFPA present good opportunities – if not for direct collaboration, then for recommendations for SRH/ adolescent health experts in Cambodia who you could collaborate with. With in-country collaborators you will be able to strengthen your recommendations/ ensure they are aligned with need and ensure better dissemination.  Engagement in this way will help with enabling of translation to action. Appreciate it is not always easy to find willing and appropriate collaborators, and this does slow down the editorial process, but the opportunity for your study to be impactful is much greater. I suspect if you are doing considerable work with a focus on Cambodia you would have a few key people you would be able to contact. See reference: Beran D, Byass P, Gbakima A, et al. Research capacity building-obligations for global health partners. Lancet Glob Health 2017;5:e567-e568. doi:10.1016/S2214-109X(17)30180-8.

2.	I wasn’t completely convinced by your recommendations. You had some really interesting findings, and your discussion is strong… you are well placed to make some clear and focussed recommendations for policy, intervention, and future research. I wanted to be given some clearer steps for action. Improving SRH literacy is key but how? One point that you touch on briefly is the inclusion of adol & young people in the discussion about SRH needs. This is an important and overarching issue. Especially given what you have shown in the results around autonomy of decision making in general. One way to make some clear recommendations might be to structure them around your theoretical framework. Or link back to how the recommendations can cover the issues raised across the framework. The recommendations are often the hardest part to get right – but very important for communicating your key messages. The strength of the rest of the paper makes it worth the extra work.

Some suggestions/ citations for the in pdf comments:
Employment & education are key - have a look at NEET data and Education completion data for Cambodia and neighbours (e.g. Viet Nam, Lao PDR and Thailand).
These relevant studies/ reports provide some good context and data:
Azzopardi PS, Hearps SJC, Francis KL, et al. Progress in adolescent health and wellbeing: tracking 12 headline indicators for 195 countries and territories, 1990-2016. Lancet 2019;393:1101-1118. doi:10.1016/S0140-6736(18)32427-9. (Has comparison of NEET/ Education/ demand satisfied/ child marriage for Cambodia and relevant neighbours)

Slaymaker E, Scott RH, Palmer MJ, et al. Trends in sexual activity and demand for and use of modern contraceptive methods in 74 countries: a retrospective analysis of nationally representative surveys [published correction appears in Lancet Glob Health. 2020 May;8(5):e648]. Lancet Glob Health. 2020;8(4):e567-e579. doi:10.1016/S2214-109X(20)30060-7 (recent publication which I think confirms aspects of your findings – specifically around the need to focus on those social-structural determinants).

Gender Counts, South East Asia. See:  https://www.unicef.org/eap/sites/unicef.org.eap/files/2019-08/Unicef_East%20Asia_Gender%20Counts%20report_Digital_1.pdf

Discussion could be strengthened by recommendations around Adolescent friendly health services as noted in pdf – see references:
World Health Organization. Making health services adolescent friendly: Developing national quality standards for adolescent friendly health services. 2012. Available at: https://www.who.int/maternal_child_adolescent/documents/adolescent_friendly_services/en/ (this is an older but useful overview of adol friendly health services and why they are important).

Sheehan P, Sweeny K, Rasmussen B, et al. Building the foundations for sustainable development: a case for global investment in the capabilities of adolescents. Lancet 2017;390:1792-1806. doi:10.1016/S0140-6736(17)30872-3. (not specifically related to adolescent friendly health services - this is paper which shows why investment in adolescent health and wellbeing has such a good return on investment for low resource settings – with a specific focus on gender inequity. While this might be a bit further away from the focus on your paper it is a strong argument for the need to invest in SRH in adolescents).


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: FACTORS INFLUENCING UNMET NEED FOR CONTRACEPTION AMONGST ADOLESCENT GIRLS AND WOMEN IN CAMBODIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Let me congratulate the authors for hard working on secondary data analysis where most of such data are not utilize regularly in most of the countries.
I feel, the language needs further improvement to convey message clearly to audiences.
The introduction part contains limited information and missing information on influential factors of unmet need for family planning. I suggest to present these factors in accordance to socioecological model (SEM) and on previous similar studies in the country or your neighborhood and globally.  Simply you can follow Fig 1 for a systemic literature review.
Line 113-115: it is better to have specific objectives e.g. to explore microenvironment factors of unmet need for family planning and to determine macro-environment.
Material and Methods: this section is not structured well. You can briefly describe method applied in DHS and how you select your sample for analysis. Furthermore it is important to
Line 118-139: there is much information that does not necessary to be in the method and material section.
a.	The sentence begin in line 119 and end in line 122
b.	Information provided in line 125-131 seems to be also irrelevant.
c.	Line 132: the name of data file should be cross checked. It is better to use DHS common word that is “individual questionnaire” or in data set it is called individual record.
d.	Line 132-139: information provided in these lines, if necessary, could be shifted to relevant section e.g. important definitions.
Line 155-176: information on definition for unmet need is repeated twice. It is better to present precise definition.
Line 192. The sentence about sample size already been presented in material and methods section. There is no need to repeat information.
Line 193: instead of multiple logistic regressions, it is better to use binary logistic regression.
Line 194: There is no need for reference.
Line 198: If we are presenting weighted data, there is no need to present crude rate.
Line 199-208: there is additional information on statistical tests and procedure. I propose to omit these information and just name them.
Line 155-176: information on definition for unmet need is repeated twice. It is better to present precise definition.
Line 192. The sentence about sample size already been presented in material and methods section. There is no need to repeat information.
Line 193: instead of multiple logistic regressions, it is better to use binary logistic regression.
Line 194: There is no need for reference.
Line 198: If we are presenting weighted data, there is no need to present crude rate.
Line 199-208: there is additional information on statistical tests and procedure. I propose to just name them and no need to provide such information for readers.
Line 211: Socio demographic characteristics of sample are missing. I suggest presenting such characteristics in frequency distribution table along with brief description.
Line 215: it is better to present bivariate analysis by outcome in table along with brief description. It is important to put outcome result in column while row will contain independent variables.
Line 221-224: there is no table to support these finding.
Line 225-228: However, from table 1 it is possible to calculate current use of contraceptive, but such figure is not presented.
Line 237: the AOR and its CI were not matched with table 2 result.
Line 241: No results are presented in binary logistic regression table (table 2).
Experimental design: Data are collected from a cross sectional survey. However the design is not appropriate to infer casual relationship but can predict or estimate the possible association between factors. Therefore the design is seems to be appropriate.
There were small methodological points for improvement that are just presented in the basic report.
Validity of the findings: In order of novelty, many studies conducted outside of Cambodia and as per author statement, this study is first study investigating factors connected to unmet need for family planning.  Below are some points to be considered for validating and presenting finding:
Finding of this study is not presented in systematic way. It is better to start from brief sample description followed by bivariate analysis and then conduct binary logistic regression.
Table 1 is not presented in good way.  I don’t think that column should have age and residence categories.  This table has some typo error for example figure stands for number of urban women aged 15-19 has decimal. However, most of independent variables does not sum up to 4823 probably due to missing information but surprisingly the number of children ever born was exceed from 4823. In addition the last category of the number of children ever born should be ≥3 not > 3.
Table 2 should present only adjusted odd ratio and I think no need to present crude OR. In addition parity variable should have different categories as presented in table 1 e.g. have no children, one child, two child and three and more children. Furthermore, the symbol use for p-value should be P (capital), small p represent proportion in statistics.
Line 254-256: no figure presented as table.
Line 258-260: if there is no difference according between receiving message through media and unmet need for family planning, should not be reported.
Line 262-264: information is not translated into the table form.
Line 267-270: figures in text are not presented in table.
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: LACTOBACILLUS JOHNSONII BS15 IMPROVES INTESTINAL ENVIRONMENT AGAINST FLUORIDE-INDUCED MEMORY IMPAIRMENT IN MICE—A STUDY BASED ON THE GUT–BRAIN AXIS HYPOTHESIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English needs to be improved. For example, neurogenesis should be replaced to neuroplasticity.
Experimental design: No comment.
Validity of the findings: 1. In figure 1 and 5, it is unclear to show the data of PCR and western on same bar.
2. In figure 2, morphological evidences are needed to show the changes of gut permeability, such as HE staining, Staining of gap junction proteins
3. In Figure 4 and Figure 7, more evidences including western or/and immunostaining are needed to confirm the changes of detected factors.
Additional comments: In the manuscript “Lactobacillus johnsonii BS15 improves intestinalenvironment against excessive fluoride intakeinducedmemory impairment in mice - a study basedon the gut-brain axis hypothesis”, the authors found that Lactobacillus johnsonii BS15 intake limited neuroinﬂammation and demyelination in the hippocampus , improved cognitive dysfunction and increased the expressions of neuroplasticity-related factors. It is interesting.  However, I have concerns which I believe should be addressed prior to publication, as outlined below in detail.


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: LACTOBACILLUS JOHNSONII BS15 IMPROVES INTESTINAL ENVIRONMENT AGAINST FLUORIDE-INDUCED MEMORY IMPAIRMENT IN MICE—A STUDY BASED ON THE GUT–BRAIN AXIS HYPOTHESIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: The manuscript has a novel idea regarding the correlation between gut microbiota or probiotic and neurotoxicity induced via flouride. It can be accepted for publication but it needs some major revisions.

First it needs English language editing also there is some spelling mistakes as:
Introduction section:
Line 60 : from not form
Line 96: present not prsent
Line101: learning not leaning
In discussion:
Line 323: resulted in not resulted

So revise the whole manuscript for any spelling mistakes.

Also the abstract must be brief and more precise.

In the discussion section correlation between probiotics and neurogensis must be mentioned in details also references for the effect of probiotics on inflammatory and neurogenic markers must be mentioned.


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: LACTOBACILLUS JOHNSONII BS15 IMPROVES INTESTINAL ENVIRONMENT AGAINST FLUORIDE-INDUCED MEMORY IMPAIRMENT IN MICE—A STUDY BASED ON THE GUT–BRAIN AXIS HYPOTHESIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: ok
Experimental design: ok
Validity of the findings: ok
Additional comments: This manuscript aimed to test whether Lactobacillus johnsonii BS15 improves intestinal environment against ﬂuoride-induced memory impairment in mice. The authors found that BS15 intake could benefit the neuroinflammation and demyelination in the hippocampus by improving the gut environment and ameliorating fluorine-induced memory dysfunction. The manuscript is interesting. Several revisions are suggested to improve the manuscript.
Major concerns:
1. The authors should present the research results following certain logics  such as protection, then the underlying mechanisms.
2. Figure legends didn’t clearly show the meaning of figure. For example, what’s the meaning of a and b in figures?
3. Figure 3C is wrong
4. Figure 5 is not clear. Higher magnification is needed.
5. Data of a molecule of mRNA and protein should put in one figure. And all protein level detection should provide western blot band and corresponding statistical chart.


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: LACTOBACILLUS JOHNSONII BS15 IMPROVES INTESTINAL ENVIRONMENT AGAINST FLUORIDE-INDUCED MEMORY IMPAIRMENT IN MICE—A STUDY BASED ON THE GUT–BRAIN AXIS HYPOTHESIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: It is well written and can be accepted for publication in its current form
Experimental design: Well expressed
Validity of the findings: Valid for publication
Additional comments: The manuscript can be accepted for publication


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

Input:
Title: LACTOBACILLUS JOHNSONII BS15 IMPROVES INTESTINAL ENVIRONMENT AGAINST FLUORIDE-INDUCED MEMORY IMPAIRMENT IN MICE—A STUDY BASED ON THE GUT–BRAIN AXIS HYPOTHESIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript( # 46283) investigated the effects of lactobacillus BS15 treatment on fluoride-induced memory impairment and possible signaling mechanisms. These findings are interesting. However, I remain concerned about the following:
1. The description in Result of Abstract is similar to discussion. Please revise it.
2. Figure-2b,-2c, and -2d are unclear.  Please provide pictures with higher magnification.
3. There is counterstaining in Figure 3. The authors didn’t explain how to decrease the effects of counterstaining on the optical density of CREB staining. In addition, similar to Figure 2, figure 3 is also unclear. Please provide pictures with higher magnification.
4. It is difficult to understand why the authors detected the mRNA and protein levels of certain molecules, but not for other molecules. Please add explanation.
Experimental design: ok
Validity of the findings: ok
Additional comments: The manuscript( # 46283) investigated the effects of lactobacillus BS15 treatment on fluoride-induced memory impairment and possible signaling mechanisms. These findings are interesting. However, I remain concerned about the following:
1. The description in Result of Abstract is similar to discussion. Please revise it.
2. Figure-2b,-2c, and -2d are unclear.  Please provide pictures with higher magnification.
3. There is counterstaining in Figure 3. The authors didn’t explain how to decrease the effects of counterstaining on the optical density of CREB staining. In addition, similar to Figure 2, figure 3 is also unclear. Please provide pictures with higher magnification.
4. It is difficult to understand why the authors detected the mRNA and protein levels of certain molecules, but not for other molecules. Please add explanation.


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

Input:
Title: A DIGITAL MAPPING APPLICATION FOR QUANTIFYING AND DISPLAYING AIR TEMPERATURES AT HIGH SPATIOTEMPORAL RESOLUTIONS IN NEAR REAL-TIME ACROSS AUSTRALIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: The criteria for not rejecting an AWS (lines 122-125) suggest that each iteration of the evaluation process the number fo stations actually participating in the TPS procedure would differ. Tamperatures at stations in regions with sparse coverage, could end up being estimated from data coming from quite distant areas, and thus presenting large estimation errors. Could the authors discuss the potential significance of this effect?
Narrowing down the validation period and AWSs used, to those "overall commonly available" might not prove practical. However, an estimate might be obtained by employing an additional completeness criterion based on the percentage of "active" nearby stations during each estimation step.
This could also be relevant to the discussion in lines 203-204 and perhaps lead to an additional comment after line 345 on the possible effect of a reduced number of stations available during the TPS interpolation procedure .
Additional comments: A very interesting article with great potential for application. Proper data handling and analysis, very well writen and presented. Apart from the comment in the "Validity of the findings" section, just a few more, minor, comments/suggestions:
1. Air temperature is measured with an accuracy of one decimal so using two decimals to report the respective MAE might be considered an exaggeration. In that respect, an estimation of the combined uncertainty -instead of simply the MAE- of the estimations made by the method, might also be of interest.
2. There are but a few grammatical errors and missing/misused words (i.e. lines 38, 54, 123, 124, 171, 299)
3. There is a narration gap in lines 42-43, from generaly speaking about the dependence of (environmental) variables on geographical factors to the specifics of air temperature. Perhaps a bridging sentence could be added.
5. Please indicate how many AWS stations were discarded (line 125)
6. Concordance coefficient is indexed as Pc, pc (capital and small p, in the text) and CC in the raw data. Please choose one.
7. The setup of the mapping systems seemed clearly described (lines 196-208), however it was only after lines 288-291 that ts operation was really understood. This is a very important feature of the system. Perhaps the description could be improved and the rationalle behind the overall design, better explained.
8. Line 210: perhaps the authors meant "visualize"
9. Line 243: less than 10% is hardly a large proportion
10. Line 328: "minimum" should be removed from here
11. Raw data provided were easy to identify and use, except for the Excel version of figure 8 (my, older, version of Excel could not display that) and the MAE tiffs (evaluation\data\spatial\mae) that seem to be blank (instead of showing the plates of Figure 5).


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

Input:
Title: A DIGITAL MAPPING APPLICATION FOR QUANTIFYING AND DISPLAYING AIR TEMPERATURES AT HIGH SPATIOTEMPORAL RESOLUTIONS IN NEAR REAL-TIME ACROSS AUSTRALIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Comments on “A digital mapping application for quantifying and displaying air temperatures at high spatiotemporal resolutions in near real-time across Australia”

The authors applied the thin plate spline method to interpolation T2m that were observed at a sparse in situ meteorological monitoring network across Australia toward the creation of high resolution T2m maps. The paper is generally well written and the analysis sounds meaningful. However, the technique is a bit outdated given the available of more contemporary yet advanced data fusion and assimilation methods.
1.Line 43-44: The variations in temperature in space is also highly regulated by factors such as aerosols via direct radiative effect (Mitchell et al., 1995, doi:10.1175/1520-0442(1995)008<2364:OSTGGA>2.0.CO;2; Li et al. 2017, doi:10.1093/nsr/nwx117 ), clouds (Xue et al., 2019, doi:10.1007/s00382-018-4505-8), synoptic-scale circulation (Redmond and Koch 1991. doi: 10.1029/91WR00690; Domonkos et al. 2003, doi:10.1002/joc.929; Liu et al., 2018, doi: 10.1175/JCLI-D-17-0608.1), which is suggested to be mentioned at least.
2.Line 67: thin plate smoothing spline performed poorly with limited observations, why didn’t have a try on the widely applied Kriging method?
3.Line 128: as per your description, air temperature should be the commonly applied 2m temperature which should be denoted as T2m to be in line with the common usage in meteorology. Please change the usage throughout the paper.
4.Methodology: currently, especially at the time where both satellite observations and meteorological reanalysis are widely applied, creating a gridded field of geophysical parameters simply using a 2D interpolation method seems to be out of date and less accurate, though it could be a timely way. A popular method is to fuse/assimilate the in situ observations with a given background field either from satellite observations or model output since they may provide more accurate estimates over remote regions with limited or even no in situ observations.
5.Line 248-249: As stated by the authors, larger interpolation errors would be introduced in regions with limited neighboring AWS sites. How to deal with this drawback, or just providing estimations with large uncertainty?
6.Line 263–264: why large errors were observed in the coastal regions during afternoons in summer and spring? Is this due to instrumental bias or region-specific anomaly? Please try to give some explanation.


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: COVID-19: MOLECULAR AND SEROLOGICAL DETECTION METHODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Is the review of broad and cross-disciplinary interest and within the scope of the journal?
The review proposes a description of the SARS-CoV-2 virus genome, a summary of transmission mechanisms, and an overview of direct and indirect methods of viral infection detection.
The topic is of great interest, broad and interdisciplinary, and within the scope of the journal.

Has the field been reviewed recently? If so, is there a good reason for this review (different point of view, accessible to a different audience, etc.)?
Reviews are of great interest in order to help readers to make sense of the large information content present in the scientific literature. Since the number of publications related to COVID-19 is extremely high and growing fast, reviews published within few months have a large amount of novel information to discuss. That said, an example of a review that discusses diagnostics approaches for COVID-19 is https://doi.org/10.3389/fcell.2020.00468. The value of this published review is that it clearly lists advantages and disadvantages of every approach discussed and clearly differentiates test used in the clinics from experimental assays that are under development.
Does the Introduction adequately introduce the subject and make it clear who the audience is/what the motivation is?
The audience is clearly identified. Both the abstract and the introduction do not set a clear expectation for the following sections: “SARS-CoV-2 structure” and “Transmission and symptoms of COVID-19”. If the sections are to be retained, they should be announced in the introduction and in the abstract.
Experimental design: Is the Survey Methodology consistent with a comprehensive, unbiased coverage of the subject? If not, what is missing?
Survey Methodology clearly describes the databases used for literature research and the keywords used in the search. It would be useful to state the date the searches were conducted. A nice addition to the diagnostic methods might have been a discussion on neutralization methods.
Are sources adequately cited? Quoted or paraphrased as appropriate?
Sources are adequately cited and paraphrased. The bibliography includes 104 references.
Is the review organized logically into coherent paragraphs/subsections?
The review is logically organized and easy to follow. More emphasis of advantages and disadvantages of every testing approach and a clear delineation of what tests are currently used in the clinics and what assays are experimental would have been very helpful.
The paragraphs on the structure of the virus and on transmission and symptoms need to be announced in the Introduction and Abstract. On transmission, current estimation of the number of viruses needed to infect a human host might be relevant and interesting to the readership. A stratification of symptoms according to the WHO might be helpful.
Validity of the findings: Is there a well developed and supported argument that meets the goals set out in the Introduction?
The introduction states that the goal of diagnostic efforts is to achieve a rapid and accurate diagnostic method. Nowhere in the review it is stated that serological methods are not fulfilling the scope of early detection because seroconversion happens weeks after infection. Antibody measurement indicates exposure to the virus.
It is also very important to clarify the concept of “serological method” that in the present review appears to be detection of proteins in serum. Antibody measurement indicates exposure, viral antigen testing indicates presence of the virus and likely active infection. This very important concept needs to be clarified throughout the manuscript and in the abstract. If a discussion of serology diagnostic methods is retained in the manuscript, the goal in the abstract and introduction of rapid testing needs to be rephrased and expanded.
Does the Conclusion identify unresolved questions / gaps / future directions?
The conclusion is that further research is needed to overcome present limitations of diagnostic approaches but it is not very specific nor lays out future directions. Interesting points might be: including need for standardization, need of coordination with regulatory agencies, accessibility, reagent quality are not discussed. Additionally, the use of testing in a scenario where a vaccine is available would be interesting.
Additional comments: General comments.
The review covers recent information about the SARS-CoV-2 structure, COVID-19 symptoms and transmission, and diagnostic tests including 1) PCR and other nucleic acid amplification technologies, 2) serology, and 3) direct antigen test. The intended audience is clearly defined. The authors compiled a good amount of information on the testing approaches currently available.

Major issues with the manuscript (issues are listed in order of importance):
-	Serology tests do not include direct antigen tests in serum. The concept of serological tests should be clarified.
-	The need for COVID-19 testing should be clarified in the abstract and introduction. If the goal is to achieve early diagnosis, antibody testing is not the preferred test.
-	Tests should be critically discussed and advantages and disadvantages including costs, turnaround time, sensitivity, specificity should be presented. The fact that PCR is the gold standard for virus detection should be stated.
-	Introduction should mention that a description of the virus and its symptoms is present.
-	A distinction between clinically used tests and research assays should be clearly defined.
-      Language is at times imprecise.


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: COVID-19: MOLECULAR AND SEROLOGICAL DETECTION METHODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: this paper represents good academic work on summarizing existing techniques to detect SARS-CoV-2 in real life samples collected form patients or prospective patients. This field has been extensively reviewed recently, for example in examples below. Many of the published review go into much further details on technologies reviewed, and provide a very detailed landscape of futuristic trends, which current review lacks. Also, this review is too general, and contain parts on biology of SARS-COV-2 coronavirus, which are commonplace, and should be removed

1. PMID: 32729549
2. PMID: 32729494
3. PMID: 32641875
4. PMID: 32609256
5. PMID: 32607246
Experimental design: Also, this review is too general, and contain parts on biology of SARS-COV-2 coronavirus, which are commonplace, and should be removed. Sources are cited adequately, but some parts are present in incorrect sections, i.e. LAMP PCR is reviewed in two different sections, depending on subtype of the method
Validity of the findings: I think this review is too general, and, because of that, short of expectations.
Additional comments: nice effort, but not specific (focused) enough for PeerJ


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

Input:
Title: THE HIGHLY REARRANGED MITOCHONDRIAL GENOMES OF THREE ECONOMICALLY IMPORTANT SCALE INSECTS AND THE MITOCHONDRIAL PHYLOGENY OF COCCOIDEA (HEMIPTERA: STERNORRHYNCHA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Line 42. Please, rephrase “Coccoidea exhibit complete metamorphosis” as so it can mislead readers in relation to the Coccoidae belonging to Hemi- or Holometabola.
Line 159-160. Please, rephrase “The CREx analysis predicted the alternative scenarios how the three mitogenomes rearranged from that of D. yakuba”, otherwise it can be understood that D. yakuba is an ancestral organism for Coocoidae as the authors only speak about the ancestral type of mitochondrial genome.
Line 192-194. Genes COI as well as cytB have been used as phylogenetic markers for insects for a long time and their efficiency is already confirmed and widely recognized. I would recommend removing this phrase or, please, specify the underlying ideas. This remark remains at the discretion of the authors.
Line 200-201. Please, add “C. rubens” into the sentence: “Length and A+T content of the tRNA genes were subequal between U. yanonensis and P. citri, whereas the lengths of tRNA genes were generally shorter than U. yanonensis and P. citri.” In this sentence it is not obvious what species’ tRNA genes are shorter than U. yanonensis and P. citri.
Line 247-250.  “The length, nucleotide composition, number and types of structural elements in CRs of the three mitogenomes 249 were found highly variable, which implied that the scale insect mitogenomes were regulated in different ways during the mitogenomic replication and transcription processes”. I am not sure that the difference in nucleotide organization of CR allows making conclusions about the difference in the replication/transcription regulation even though this output is logical. This remark remains at the discretion of the authors.


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

Input:
Title: THE HIGHLY REARRANGED MITOCHONDRIAL GENOMES OF THREE ECONOMICALLY IMPORTANT SCALE INSECTS AND THE MITOCHONDRIAL PHYLOGENY OF COCCOIDEA (HEMIPTERA: STERNORRHYNCHA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Please find herewith a few observations;
The scale insects are hard to deal with because of its waxy surface coating; especially for molecular work, as it can act as a PCR inhibitor. Overall, this paper is very informative except for the phylogenetic analyses.
To me, it is highly immature to state the monophyly of Aphidoidea by merely analyzing a few aphid species mitogenome (that too Aphididae alone and has nothing to do with the paper!!!). The same applies to the other superfamilies as well, as all of them were underrepresented with very few species. Hard to understand why the authors employed the whole of Sternorrhyncha, for their phylogeny? Is it merely to increase the sample size? (The whole paper is on Scale insect, except this analysis!)
It is better to do the phylogenetic analyses using the coccids from this work and publicly available along with the aphids (a couple of species) as outgroups rather than the present one.
Otherwise, the paper looks solid with mitogenome analyses, figures (except phylogeny), and tables.
Few minor corrections have been added in the PDF version (please find the attachment).
Best regards,


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

Input:
Title: THE HIGHLY REARRANGED MITOCHONDRIAL GENOMES OF THREE ECONOMICALLY IMPORTANT SCALE INSECTS AND THE MITOCHONDRIAL PHYLOGENY OF COCCOIDEA (HEMIPTERA: STERNORRHYNCHA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript can be accepted as it 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: MASTER REGULATOR GENES AND THEIR IMPACT ON MAJOR DISEASES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript "Master regulator genes and their impact on major diseases" presents a revision of the literature on master regulator genes and its relationship to human diseases. The authors declared that the definition of MRG remains "ambiguous". Actually this is not the case.

Though it may seem strange, given the authors broad coverage of the literature, there are some key references missing. They refer to well known works that define MRGs in quantitative and measurable terms and applied them in biomedical research. These methods are based on the results of omic technologies, for instance whole genome transcriptomics, ChIPSeq and ATAC-Seq and well established bioinformatic analyses such as GSEA and its variants. We strongly encourage the authors to look up at the following references for analytical and computational methods and applications:

Lefebvre, C., Rajbhandari, P., Alvarez, M. J., Bandaru, P., Lim, W. K., Sato, M., ... & Basso, K. (2010). A human B‐cell interactome identifies MYB and FOXM1 as master regulators of proliferation in germinal centers. Molecular systems biology, 6(1), 377.

Lim, W. K., Lyashenko, E., & Califano, A. (2009). Master regulators used as breast cancer metastasis classifier. In Biocomputing 2009 (pp. 504-515).

Boboila, S., Lopez, G., Yu, J., Banerjee, D., Kadenhe-Chiweshe, A., Connolly, E. P., ... & Yamashiro, D. J. (2018). Transcription factor activating protein 4 is synthetically lethal and a master regulator of MYCN-amplified neuroblastoma. Oncogene, 37(40), 5451-5465.

Tomljanovic, Z., Patel, M., Shin, W., Califano, A., & Teich, A. F. (2018). ZCCHC17 is a master regulator of synaptic gene expression in Alzheimer’s disease. Bioinformatics, 34(3), 367-371.

Dinkelspiel, H., Iyer, A., Lefebvre, C., Wright, J., Lewin, S., Herzog, T., ... & Califano, A. (2013). Identification of master regulators of cisplatin resistance in ovarian cancer. Gynecologic Oncology, 130(1), e4-e5.

Lefebvre, C., Rajbhandari, P., Alvarez, M. J., Bandaru, P., Lim, W. K., Sato, M., ... & Basso, K. (2010). A human B‐cell interactome identifies MYB and FOXM1 as master regulators of proliferation in germinal centers. Molecular systems biology, 6(1), 377.

Tovar, H., García-Herrera, R., Espinal-Enríquez, J., & Hernández-Lemus, E. (2015). Transcriptional master regulator analysis in breast cancer genetic networks. Computational biology and chemistry, 59, 67-77.

Tapia-Carrillo, D., Tovar, H., Velázquez-Caldelas, T., & Hernandez-Lemus, E. (2019). Master regulators of signaling pathways: An application to the analysis of gene regulation in breast cancer. Frontiers in genetics, 10, 1180.

Hernández-Lemus, E., Baca-López, K., & Tovar, H. (2015). What makes a transcriptional master regulator? A systems biology approach. In Physical Biology of Proteins and Peptides (pp. 161-174). Springer, Cham.

Alvarez, M. J., Giorgi, F., & Califano, A. (2014). Using viper, a package for Virtual Inference of Protein-activity by Enriched Regulon analysis. Bioconductor, 1-14.

Baca-López, K., Mayorga, M., Hidalgo-Miranda, A., Gutiérrez-Nájera, N., & Hernández-Lemus, E. (2012). The role of master regulators in the metabolic/transcriptional coupling in breast carcinomas. PLoS One, 7(8), e42678.

Aside from considering these additional information sources, I would like to suggest the authors to include one or perhaps two figures as aids to explain in a semi-mechanistic way the functional roles of MRGs.

Aside from these conceptual and presentation issues, the paper may become a relevant source of information on the role of MRGs in biomedical research.
Experimental design: This is a "traditional" review paper (i.e. not a systematic revision nor a meta-analysis) so the "design" is quite straightforward. I only suggest the authors to revise some additional references and try to add some illustrative figures to broaden the scope and readability of their manuscript.
Validity of the findings: This is a review paper
Additional comments: Aside from a couple of comments already made, this looks like a timely contribution to a (slowly) nascent field.


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: MASTER REGULATOR GENES AND THEIR IMPACT ON MAJOR DISEASES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The review talks about the Master Regulator Genes (MRG) in view of different disease conditions. The review is within the scope of Peer J. It describes many aspects of MRGS and I commend the authors for doing a large sum of literature review, although the English writing is not up to the mark of a reputed journal. Further, it fails to put forward any new insights or describe details in a critical manner. The content is mere description or paraphrasing of existing articles. The introduction section is small with very less background.
Experimental design: The authors did a good job in incorporating a large volume of references. It represents unbiased coverage of the subject. The writing is non-coherent in many instances. I mentioned few of those errors in general comment. I would encourage the authors to consult a science writer to improve the English. I would also encourage to use pictorial representations whenever possible to make it more interesting to the reader.
Validity of the findings: The review has a very short conclusion/outlook section that fails to identify any unresolved questions or gaps. The conclusion is an important part and the authors need to give more importance in writing this section. The writing in this section need major restructuring. For e.g.,line 219 “ ….gene regulation in all aspects of individual development is becoming apparent .“ – what does ‘individual development’ mean here ? Does the author mean development of an organism?
Also, the last sentence “ For the application of MRGs in clinical medical treatment, it is necessary to strengthen the in-depth understanding of disease-related MRGs to develop target-specific and more effective treatment programs.” This sentence does not convey the message well. I would not recommend starting the sentence with ‘For’. There are many such examples throughout the article.
Additional comments: There are some grammatical mistakes and other issues as described below:
Line 45 “ MRGs has been reported…….” : It should be MRGs have been.
Line 46 “ in major clinical diseases..” : Please remove one fullstop.
In section 3 Overview of MRGs: Inclusion of a figure showing the different MRG families in pictorial representation will make this section more interesting.
Line 138-153: The paragraph is filled with gene names and references. This make the section hard to read, I would suggest to use tables for such descriptions.
In Section 4, regulation of major diseases by MRGS is discussed but it is not clarified why the author choose to focus on only tumor and cardiovascular diseases while the MRGS can regulate many other diseases.
Line 171-172 “ RUNX family, which was recently discovered to be a new mutant gene in human…….” : new mutant gene is not a proper terminology, I would suggest writing - A  new mutation in RUNX gene was discovered. Use of proper terms and structure of sentence is very important and the author should review this criteria throughout the article.
Line 179-180 “ MRGs have been reported as associated……”: MRGs have been reported to be associated.
Line 209-210 “ There are some reports on the……” : Please add references.
Line 209-214 : This paragraph closes the section ‘Regulation of Major Diseases by the MRGs” but the writing seems abrupt without continuation to the previous writing. Need rewriting.

In table 2: what does ‘whole signal pathway’ mean? I would suggest some alternative terminology.


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: MASTER REGULATOR GENES AND THEIR IMPACT ON MAJOR DISEASES
Review round: 1
Reviewer: 3

Output:
Basic reporting: There is a growing interest in the concept of master regulator genes (MRGs). This review appears to be relevant in the current situation and for future considerations during efficacious treatment development for various clinical diseases. Introduction highlights the importance of MRGs, the lacuna in the area and the portion of coverage provided by this review to fulfil the deficit.
The review is well within the scope and it will be of interest to the readers of the journal. Although, there are many reviews covering a specific MRGs in diverse area, the current review is one of its kind that spans different aspects and provides classification of MRGs. However, the most recent literature available in diverse areas are missing. The specific details are mentioned later in the comments. It will be in the interest of authors to cite most recent literature to enhance the value of manuscript.

The review is well structured, and English is clear, unambiguous, and professional. There are some minor punctuation issues that needs to be corrected. The details of which are provided along with other comments.
Experimental design: The literature survey is unbiased and well organized into sections and sub-sections. However, to be comprehensive and up to date, recent developments in the area should be included. Authors have covered MRGs involvement in tumors and cardiovascular diseases. Recently, the MRGs in various diseases have been identified, which can be adequately covered.
Validity of the findings: The authors have systematically categorized MRGs and elaborated their involvement in various processes. MRGs involvement in tumors and cardiovascular diseases is well covered and the requirement of in-depth understanding for their application in clinical medical treatment is highlighted. To increase the impact of the manuscript, the MRGs involvement in other diseases can be incorporated. For instance, the information for MRGs in metabolic and neurodegenerative diseases can be covered.

Below are some recent advances in the area:
NRF2: Cores Á, Piquero M, Villacampa M, León R, Menéndez JC. NRF2 Regulation Processes as a Source of Potential Drug Targets against Neurodegenerative Diseases. Biomolecules. 2020 Jun 14;10(6):E904. doi: 10.3390/biom10060904. PMID: 32545924.
Nrf2-ARE pathway: Buendia I, Michalska P, Navarro E, Gameiro I, Egea J, León R. Nrf2-ARE pathway: An emerging target against oxidative stress and neuroinflammation in neurodegenerative diseases. Pharmacol Ther. 2016 Jan;157:84-104. doi: 10.1016/j.pharmthera.2015.11.003. Epub 2015 Nov 23. PMID: 26617217.
The Keap1-Nrf2 pathway: Deshmukh P, Unni S, Krishnappa G, Padmanabhan B. The Keap1-Nrf2 pathway: promising therapeutic target to counteract ROS-mediated damage in cancers and neurodegenerative diseases. Biophys Rev. 2017 Feb;9(1):41-56. doi: 10.1007/s12551-016-0244-4. Epub 2016 Dec 6. PMID: 28510041; PMCID: PMC5425799.
The ubiquitin proteasome system and schizophrenia: Luza S, Opazo CM, Bousman CA, Pantelis C, Bush AI, Everall IP. The ubiquitin proteasome system and schizophrenia. Lancet Psychiatry. 2020 Jun;7(6):528-537. doi: 10.1016/S2215-0366(19)30520-6. Epub 2020 Feb 12. PMID: 32061320.
Additional comments: General Comment: Overall, the study is interesting and a useful contribution to the area. The strength of the manuscript is the diverse coverage and systemic classification of the master regulator genes. A general comment on the manuscript is the need of citation of recent references. These changes will present the current scenario to the reader. However, there are some minor concerns, which are detailed below. A revision is recommended for improving the manuscript.

Page 3, Line 78-84: The authors have provided the definition that partly overlaps with already existing definition in an article (Chan SS, Kyba M. What is a Master Regulator? J Stem Cell Res Ther. 2013 May 4;3:114. doi: 10.4172/2157-7633.1000e114. PMID: 23885309; PMCID: PMC3718559.). The definition presented in the published article states that, “We propose that the best interpretation of the term master regulator is a gene that is expressed at the inception of a developmental lineage or cell type, participates in the specification of that lineage by regulating multiple downstream genes either directly or through a cascade of gene expression changes, and critically, when misexpressed, has the ability to respecify the fate of cells destined to form other lineages”.
Although in the next sentence, the authors have cited the above reference but only for a portion of its definition. It will be appropriate to provide due credits to what already exists and defined. Therefore, rephrasing is recommended. The addition to this existing definition by authors can be highlighted.

Page 3, Line 71: It will be appropriate to add recent reference for this pathway as a master regulator for cancer. "Schaefer T, et al. SOX2 and p53 Expression Control Converges in PI3K/AKT Signaling with Versatile Implications for Stemness and Cancer. Int J Mol Sci. 2020 Jul 11;21(14):E4902. doi: 10.3390/ijms21144902. PMID: 32664542."

Page 4, Line 99-105: Incorporation of the recent articles on HOX family and regulation mediated by them will be more relevant. For instance, the following articles are already available.
Brotto DB, et al. Contributions of HOX genes to cancer hallmarks: Enrichment pathway analysis and review. Tumour Biol. 2020;42(5):1010428320918050. doi:10.1177/1010428320918050
Collins EM, Thompson A. HOX genes in normal, engineered and malignant hematopoiesis. Int J Dev Biol. 2018;62(11-12):847-856. doi: 10.1387/ijdb.180206at. PMID: 30604854.
Li M, et al. Epigenetic upregulation of HOXC10 in non-small lung cancer cells. Aging (Albany NY). 2020 Jul 19;12. doi: 10.18632/aging.103597. Epub ahead of print. PMID: 32687064.
Qu X, et al. HOX transcript antisense RNA (HOTAIR) in cancer. Cancer Lett. 2019;454:90-97. doi:10.1016/j.canlet.2019.04.016

Page 4, Line 106-109: Similarly, in other sections please include recent articles for GATA family members. For, GATA1 and GATA 2:
Gutiérrez L, et al. Regulation of GATA1 levels in erythropoiesis. IUBMB Life. 2020;72(1):89-105. doi:10.1002/iub.2192
Leonards K, et al. Nuclear interacting SET domain protein 1 inactivation impairs GATA1-regulated erythroid differentiation and causes erythroleukemia. Nat Commun. 2020 Jun 12;11(1):2807. doi: 10.1038/s41467-020-16179-8. PMID: 32533074; PMCID: PMC7293310.
Siegwart LC, et al. The transcription factor NFE2 enhances expression of the hematopoietic master regulators SCL/TAL1 and GATA2. Exp Hematol. 2020 Jun 25:S0301-472X(20)30245-9. doi: 10.1016/j.exphem.2020.06.004. Epub ahead of print. PMID: 32593672.
Castaño J, et al. GATA2 Promotes Hematopoietic Development and Represses Cardiac Differentiation of Human Mesoderm. Stem Cell Reports. 2019 Sep 10;13(3):515-529. doi: 10.1016/j.stemcr.2019.07.009. Epub 2019 Aug 8. PMID: 31402335; PMCID: PMC6742600.
Bresnick EH, Johnson KD. Blood disease-causing and -suppressing transcriptional enhancers: general principles and GATA2 mechanisms. Blood Adv. 2019 Jul 9;3(13):2045-2056. doi: 10.1182/bloodadvances.2019000378. PMID: 31289032; PMCID: PMC6616255.
Katsumura KR, et al. Human leukemia mutations corrupt but do not abrogate GATA-2 function. Proc Natl Acad Sci U S A. 2018 Oct 23;115(43):E10109-E10118. doi: 10.1073/pnas.1813015115. Epub 2018 Oct 9. PMID: 30301799; PMCID: PMC6205465.

For GATA 3:
Nomura S, et al. Pyrrothiogatain acts as an inhibitor of GATA family proteins and inhibits Th2 cell differentiation in vitro. Sci Rep. 2019 Nov 22;9(1):17335. doi: 10.1038/s41598-019-53856-1. PMID: 31758034; PMCID: PMC6874683.
El-Arabey AA, et al. GATA3 as a master regulator for interactions of tumor-associated macrophages with high-grade serous ovarian carcinoma. Cell Signal. 2020 Apr;68:109539. doi: 10.1016/j.cellsig.2020.109539. Epub 2020 Jan 11. PMID: 31935430.
Mirlekar B. Co-expression of master transcription factors determines CD4+ T cell plasticity and functions in auto-inflammatory diseases. Immunol Lett. 2020 Jun;222:58-66. doi: 10.1016/j.imlet.2020.03.007. Epub 2020 Mar 24. PMID: 32220615.

Page 5, Line 126-128: The novel concern for mTOR is also because it is a master regulator of the inflammatory response in immune and non-immune cells and implicated in a number of chronic inflammatory diseases, especially rheumatic diseases, such as systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, sjogren syndrome and seronegative spondyloarthropathy.
Suto T, Karonitsch T. The immunobiology of mTOR in autoimmunity. J Autoimmun. 2020 Jun;110:102373. doi: 10.1016/j.jaut.2019.102373. Epub 2019 Dec 9. PMID: 31831256.

Page 9, Line 220: It will be appropriate to write, “(2) signaling pathway MRGs,..”

Page 2, Line 55 and Page 13, Line 369: The referencing format needs to be consistent with other references.  https://pubmed.ncbi.nlm.nih.gov/109750/
Suggestion: In text cite (Ohno, 1978) and in reference Ohno, S. (1978). Major sex-determining genes. Monogr Endocrinol 11, 1-140.

Table 1: A suggestion to incorporate the master regulator Group 3 innate lymphoid cells. Group 3 innate lymphoid cells (ILC3s) have emerged as master regulators of intestinal health and tissue homeostasis in mammals. (Zhou W, Sonnenberg GF. Activation and Suppression of Group 3 Innate Lymphoid Cells in the Gut. Trends Immunol. 2020 Jul 6:S1471-4906(20)30133-2. doi: 10.1016/j.it.2020.06.009. Epub ahead of print. PMID: 32646594.)

Table 2: It will be more relevant if the recent articles are referenced. For instance, NF-κB signaling pathway: Schnappauf O, Aksentijevich I. Mendelian diseases of dysregulated canonical NF-κB signaling: From immunodeficiency to inflammation. J Leukoc Biol. 2020 Jul 17. doi: 10.1002/JLB.2MR0520-166R. Epub ahead of print. PMID: 32678922.
For Yap and Hypoxia signaling pathway: Zhao C, et al. Yes-associated protein (YAP) and transcriptional coactivator with a PDZ-binding motif (TAZ): a nexus between hypoxia and cancer. Acta Pharm Sin B. 2020 Jun;10(6):947-960. doi: 10.1016/j.apsb.2019.12.010. Epub 2019 Dec 19. PMID: 32642404; PMCID: PMC7332664.

Supplementary Table 1: It will be more relevant if the recent articles are referenced. For instance, regarding MicroRNAs the following article is available. Liang Y, et al. MicroRNAs Modulate Drug Resistance-Related Mechanisms in Hepatocellular Carcinoma. Front Oncol. 2020 Jun 30;10:920. doi: 10.3389/fonc.2020.00920. PMID: 32695666; PMCID: PMC7338562.
Similarly, for NRF2 many recent articles are available. For example, Cores Á, et al. NRF2 Regulation Processes as a Source of Potential Drug Targets against Neurodegenerative Diseases. Biomolecules. 2020 Jun 14;10(6):E904. doi: 10.3390/biom10060904. PMID: 32545924.

Other minor formatting issues:

Page 2, Line 46: “…roles in major clinical diseases..” Authors can remove extra full stop.
Page 2, Line 52: “ 2. WHAT IS THE MASTER REGULATOR GENE ?”. Here, authors can bold “2” to make it consistent with their manuscript format and it will be appropriate to write “ 2. WHAT IS A MASTER REGULATOR GENE?”.
Page 3, Line 68: “… within the cells (Witczak et al., 2008)”, Please use a full stop before starting the next sentence.
Page 6, Line 155: “ 4. REGULATION OF MAJOR DISEASES BY THE MRGs”. Here, authors can bold “4” to make it consistent with their manuscript format.


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: PHYLOGEOGRAPHIC ANALYSIS AND SPECIES DISTRIBUTION MODELLING OF THE WOOD FROG BATRACHYLA LEPTOPUS (BATRACHYLIDAE) REVEAL INTERGLACIAL DIVERSIFICATION IN SOUTH WESTERN PATAGONIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this study, Nuñez et al. explore the main phylogeographical patterns in the wood frog Batrachyla leptopus using a combination of the  analysis of a multilocus dataset (3 mitochondrial genes + 2 nuclear loci) using both tradicional phytogeographical analyses as well as more modern ABC methods to test for specific hypotheses regarding patterns of population size and connectivity. These results were complemented by past and present species distribution modelling. The study is very well-written and the tested hypotheses are provided explicitly.
Experimental design: In general, both genetic and SDM methods agree with the best practices in their fields. The only exception perhaps is the mismatch distribution, given that the other used methods (EBSPs and ABC) have been shown to be vastly superior.
Validity of the findings: The conclusions are well-supported and provide an important contribution to the understanding of the evolution of species from southwestern Patagonia, as well as to the conservation of the wood frog.
Additional comments: A few minor comments:

L66: there is an extra comma before “have”
L92: patchy?
L202: Please provide information on the number of generations, their intervals, and  parameter ESSs, as you indicated for the BEAST analysis on line 229-231.


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: PHYLOGEOGRAPHIC ANALYSIS AND SPECIES DISTRIBUTION MODELLING OF THE WOOD FROG BATRACHYLA LEPTOPUS (BATRACHYLIDAE) REVEAL INTERGLACIAL DIVERSIFICATION IN SOUTH WESTERN PATAGONIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an interesting and meaningful study focused at testing biogeographic hypotheses associated with Patagonian paleo-refuges, using as model the wood frog Batrachyla leptopus, with in-depth analyses covering phylogenetics, phylogeography, population genetics, divergence dating and ecological modelling. The tests are well-thought and the steps explained in a clear manner. Nevertheless, the conclusions are strongly based on an important bottleneck, the divergence dating analysis, which results are extrapolated for downstream ABC and niche modelling; because the authors use only a rate prior, extrapolated from a previous study, without checking for a a likely larger mitochondrial evolutionary rate, and without incorporating any fossils/biogeography as node date priors, their conclusions about the importance of past refuges should be taken with a grain of salt. Without further testing the effect of a faster evolutionary rate, no conclusions can be drawn temporaly, although spatial conclusions seem reasonable (population genetics, phylogenetic pattern).
Experimental design: Phylogenetics, phylogeography, population genetics, and ecological modelling analyses are all interesting and in line with what the authors aim to test.

However, the dating analysis may bear important biases. Because rate extrapolation is used without imposing fossil/biogeographic constraints, rate biasesshould have been considered, as the posterior times could be strongly affected by them (due to an inverse relationship between times and rates); and because times are an important issue in this ms, I strongly suggest an upwards correction (Nabholz et al., 2009) in BEAST, to check if correlation to paleo-refuges still holds. The Irisarri 2012 study apparently did not employ such a correction either.

Therefore, the divergence dating analysis assumes too much in its current configuration, and because downstream analyses assume the posterior time ranges reported as correct (when there's the possibility that they are not due to rate underestimation), it's very important that the authors re-run Beast with an alternative rate prior embracing (likely) larger rates for mitochondrial genes (as Nabholz et al. 2009 indicate that these are larger than commonly assumed).

Further questions/suggestions:

- The authors tested for saturation, but do not report if they found it for any marker in the results. In case there's saturation, older times could be underestimated, possibly inflating the tendency to infer recent population growth even if there is none (or if there is but it's an older one). If only a subset of the markers suggest a linear relationship with the best fit model, exclude the markers with more extreme signs of saturation, or else exclude positions with largest entropy (e.g., using Tiger or the likes), and re-run.

- Have the authors tested for clusterization by sampling tissue (i.e., swab vs. liver) in the phylogeny (or by genetic distances)? Given that there may be nuclear copies of mtDNA, or even multiple D-loop copies in a mtDNA (depending on the vertebrate group; and I couldn't find a complete Batrachyla nor Batrachylidae mtDNA genome at NCBI to testify for a single-copy D-loop in the species), it'd be important to show that there isn't such a clustered pattern of inferring an inexistent clade due to paralogy. Testing if clades are associated with tissue type shall give a hint at this.

- Regarding population structure, please also provide Fst measures by mtDNA concatenated, and by each nuclear locus (could be a SM table). Discuss if the 3 measures disagree and, if so, how such disagreement is in line or not with current conclusions.

- Do mtDNA concatenated vc nuc1 vs. nuc2 trees agree? Showing a SM figure with the 3 topologies would be enough. If they disagree, how does it impact conclusions?

- Does PhyML support partitioning? If not, how did the authors estimate the tree, assuming at least some partitioning had better fit than none (as often is the case)? If not, please run a partitioned ML using either RaxML, RaxML-ng, or IQTree.

- Were the 2 different nucDNAs concatenated in a single partition, or not? Please clarify. Also, tests of partitioning do not mind if nuc and mt partitions are mixed, but in fact they shouldn't be. If so, there's the chance that *BEAST run may be incorrect. There should be at least 2 partitions (mt plus 1-2 nuc partitions), and eventually more if the best partitioned model suggests so, e.g. by codon position within nuclear genes (regarded mt+nuc partitions are avoided). One nuc partition is acceptable if there's low nuclear variation.

- Still regarding the different types of loci in Beast: was the correct ploidy number assumed for mtDNA, and for nucDNAs?
Validity of the findings: Because the authors base their conclusions on the importance of paleo-refuges on dates that can be biased, it is hard to judge the validity of their findings in the current ms version.
Additional comments: A meaningful and important ms, using relevant techniques to test paleo-refuges using an anuran as model, but which needs further sensitivity analysis of divergence dating to become more credible. I recommend major revisions, mostly due to dating analyses.


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: PHYLOGEOGRAPHIC ANALYSIS AND SPECIES DISTRIBUTION MODELLING OF THE WOOD FROG BATRACHYLA LEPTOPUS (BATRACHYLIDAE) REVEAL INTERGLACIAL DIVERSIFICATION IN SOUTH WESTERN PATAGONIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article Phylogeographic analysis and species distribution modeling of the wood frog Batrachyla leptopus (Batrachylidae) reveal interglacial diversification in south western Patagonia; It deals with a topic of particular importance to scientists interested in the evolutionary history of herpetofauna in southern South America. The particular case of the species of the Batrachyla genus, endemic to the Nothofagus forests, in southern Chile and Argentina has been of great interest by many scientists, who have revealed aspects of their natural history, characteristics, chromosomes, behaviors, larvae, etc. In this context, this article contributes significantly to a better understanding of aspects of the evolutionary history of one of its members, Batrachyla leptopus. This is a work very well documented in the extensive bibliography that exists on the matter. Furthermore, it is very well structured and therefore easy to read, even for someone who does not know molecular technologies in depth, but who, by understanding the context well, can realize that the objectives are well achieved and fully explained. For all of the above, this work deserves to be published without major corrections than those that could be derived from the very action of writing a scientific article.
Experimental design: The experimental design is appropriate for a job of this nature. The authors use three mitochondrial regions (D-loop, cyt b, and coI) and two nuclear loci (pomc and crybA1) to test their hypothesis. Indeed, taking into account that one of the questionable aspects of molecular methods is their robust statistics, this paper uses those most reliable methods, and incorporates a coalescence analysis, highly recommended nowadays as there is much confusion, due to the variability of results when a single molecular marker is used. On the other hand, the sample design incorporates specimens from the entire distribution, which makes the work very representative of the reality of B. leptopus. One aspect that could perhaps be lacking in this regard is some additional populations in the Andes Mountains, at 38 ° latitude (Curacautín) (Tohuaca National Park) and from north of Los Queules, but it does not diminish the importance of the work.
Validity of the findings: The results are valid since they are in line with other investigations carried out on the herpetofauna of southern South America, and have been obtained by applying an appropriate sampling design. Within the southern batrachofauna, the Batrachyla species, including Batrachyla leptopus, are part of a group of amphibians of tropical origin, and therefore their diversification may have occurred as a consequence of orographic processes subsequent to the current constitution of the continents. This is absolutely consistent with those expressed by the authors of this article. Furthermore, the historical antecedents that allow the evolution of South American amphibians to be contextualized do not provide many other additional hypotheses. On the other hand, the authors do not suggest in the text, if these four different lineages that appear in Batrachyla leptopus, present enough differentiation to be considered as possible different species. Perhaps it would be appropriate for the authors to discuss their results considering this hypothesis.
In this same line, It would be interesting to know, if the authors noticed any correspondence between lineages and some morphological distinction that suggests the presence of different taxonomic entities in Batrachyla leptopus. This question arises from the fact that during our extensive campaigns in the forests of southern Chile, we noticed some different morphs in B. leptopus, for example, some smaller specimens, the texture of their skin smoother.
Additional comments: I would like to congratulate the authors of this work for their important contribution to the knowledge of the evolutionary history of an important species in the herpetofauna of southern South America. One aspect that I would like to see at work is what consequences these results could have on the number of species in the genus. Could it be suggested that these four lineages represent four different species? If the molecular analyzes have a robust support, and the results obtained are consistent with other historical antecedents, can it be suggested that within species B. leptopus there is more than one species?
I would like the authors to discuss this point in some depth.
However, the authors mention that Batrachyla leptopus presents a very wide distribution (discontinuously ~ 1000 km) , it is important for the reader that the figures where maps are represented have a reference bar in km.
Do the authors have a photograph of a specimen from each of the lineages?


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: PHYLOGEOGRAPHIC ANALYSIS AND SPECIES DISTRIBUTION MODELLING OF THE WOOD FROG BATRACHYLA LEPTOPUS (BATRACHYLIDAE) REVEAL INTERGLACIAL DIVERSIFICATION IN SOUTH WESTERN PATAGONIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: My previous assessment of this study was already positive on the first round of reviews.
Experimental design: The experimental design follows the best practices in the field.
Validity of the findings: The conclusions are well supported by the data.
Additional comments: I believe that the authors addressed all of the issues I raised in my review.


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: PHYLOGEOGRAPHIC ANALYSIS AND SPECIES DISTRIBUTION MODELLING OF THE WOOD FROG BATRACHYLA LEPTOPUS (BATRACHYLIDAE) REVEAL INTERGLACIAL DIVERSIFICATION IN SOUTH WESTERN PATAGONIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article used population, phylogeography, phylogenetics, divergence dating, and distributional modelling to study evolution of four populations of B. leptopus in the Patagonian region, arriving at relevant clues about association of their evolution to paleo-refuges.

*Conclusions section*

I think that the section "Conclusions", as it is now, is repetitive regarding what has already been said in Discussion. So a suggestion is to either remove it, or to use it expanding the discussion by comparing to the distribution of other organisms in the Patagonian region, and giving some perspectives of conservation and on what future studies could reveal further in terms of open questions.

*Text suggestions*:

Line 73: route that may have originated.

Line 86/87: re-structure phrase: "... that at least four Pleistocene glaciations occurred in the southwestern..."

Line 103: that tend to facilitate.

Line 122/123: Quaternary glaciations may have contributed to a phylogeographic.

Line 148: SDM approaches have.

Line 149: to assess species ranges, and to evaluate special.

Line 248: Due to the fact that. it is not possible.

Line 249: as there are no fossils available nor conspicuous dated biogeographic events.

Line 251: million years for the other mitochondrial.

Line 257: estimate divergence times.

Line 266: both approaches because small sample.

Line 296: corresponded to those.

Line 374: the latter with moderate support.

Line 376: Please add legend to Fig. S1, to which marker each tree corresponds to?

Line 441: probably due to lower effective.

Line 450: glacial advances, in which.

Line 452: could facilitate population divergence.
Experimental design: A few methodological doubts remain:

- one of the genes apparently was not included for dating, if so state it clearly, and why so.

- how comparison of models was done? Simply using marginal likelihoods is not acceptable, AICM is not as bad, stepping-stone is much better. If some other method, please clarify.

- does SH-test show that tree differences are related to low-support nodes? If not, best to say is that "visually" differed among non-supported nodes, or "probably" differed...
Validity of the findings: The steps taken are sensible, and the choice of tools as well. The results and discussion provide reasonable evidence for past paleo-refuges influencing population divergences within B. leptopus.
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: PHYLOGEOGRAPHIC ANALYSIS AND SPECIES DISTRIBUTION MODELLING OF THE WOOD FROG BATRACHYLA LEPTOPUS (BATRACHYLIDAE) REVEAL INTERGLACIAL DIVERSIFICATION IN SOUTH WESTERN PATAGONIA
Review round: 2
Reviewer: 3

Output:
Basic reporting: As I mentioned in my first review, the work of Núñez et al. Is an important contribution to the knowledge of the batrachofauna of southern South America. Batrachyla leptopus is a species with a very wide distribution throughout the temperate forest and also outside it. The samples analyzed in this work are representative of this wide distribution. Therefore, it is a very important contribution to the knowledge of this species, and could be understood in part, as an explanation of the morphological variability that is known about it. I think it is a great and valuable effort.
Experimental design: No comments on this section. I think authors explain totally this point to other referee.
Validity of the findings: I read the entire manuscript, and checked the tables and figures again. As well as the comments and responses to the other referees, and it seems to me that the methods used by the authors of the article are appropriate to the scientific question that they are trying to answer, therefore, in my opinion the results are valid and the findings absolutely relevant in context.
Additional comments: Again I want to congratulate the authors for their work, and I strongly urge them to try to go a little further in solving this type of problem (in another future article) about the specific problem. The boundaries between species are often confusing, and in my scientific opinion, it is necessary to establish clear positions regarding this fact. Mainly because this background is of the utmost importance when dealing with amphibian conservation issues.


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

Input:
Title: THE TALE OF SPRINGS AND STREAMS: HOW DIFFERENT AQUATIC ECOSYSTEMS IMPACTED THE MTDNA POPULATION STRUCTURE OF TWO RIFFLE BEETLES IN THE WESTERN CARPATHIANS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I have carefully read read the paper entiled „The tale of springs and streams. How different refugial ecosystems impacted the present molecular population structure of two riffle beetles in the Western Carpathians“.
The study is interesting in the aspect of phylogeography of aquatic animals in Europe. Authors used the largest sequence data on the riffle beetles in Europe, and the main hypotheses of the study are testable by performed analyses (excepting question b-Line 102). The study will be a valuable addition to knowledge how different animal taxa survived Pleistocene glaciations, and authors also stressed out the conservation implications of their findings. Writting style is clear and proffesional. Materials and methods are well described. Sampling strategy was well chosen to investigate differences in distribution patterns and genetic structure of two elmid species. Authors also made a comprehensive comparison with other research studing influence of Pleistocene glaciations on aquatic invertebrate taxa.

Here I comment on specific points.
Title
I propose to change „refugial ecosystem“ to something more specific e.g. Pleistocene refugial biotopes/environments/habitats … Springs and streams cannot be considered as separate ecosystems because the flow of matter and energy between them is high. In any case, there is no single definition of what constitutes an ecosystem so it is better to use more specific terms such as habitat or biotope.

Lines 60-63. The sentence is not easy read and understand. Please rewrite this sentence. I suggest „In this context, studies upon the diversity and evolutionary history of aquatic biota studies should be more focused on springs, that are now heavily understudied compared to other aquatic biotopes.“

Lines 88-90. Please add „aquatic beetle family Elmidae“ in this sentence. Maybe to change „of two riffle beetles“ to „two members/species of aquatic beetle family Elmidae“ or „two aquatic beetles of family Elmidae“

Lines 102-103. It is questionable that connectivity between subpopulations can be tested only using COI mtDNA marker. This marker cannot be good for estimation of demographic history over thousands of year but recent connectivity must be analysed with more variable markers such as microsatellites. Better to remove this question. Moreover, I did not found any analyses of gene flow to test connectivity between subpopulations

Line 131. Add „Trenčín (Slovakia)” after “Environmental Care”

Line 251. Please add GenBank Acc. No. of this problematic sample

Figures
>>> Figure 1. It is not clear from presented maps what is the exact position and size of examined area. Also, Switzerland have the same color as seas.

>>> Enlarge fonts in every figure in case that text is hard to read.

>>> Please add scale in kilometres in all figure with geographic maps.

>>> Legends in figures are not well designed. Rectangles in various colors which represent legend for haplotypes have to be separated from squares which represent geomorphological units… It can be explained as text what these rectangles are showing.

>>> Identical colours used in subfigures in Figure 2 and 3 are representing different things: Haplotypes and Geographic areas. Every occurrence of color must be explained by text not only by graphical legend.

>>> Figure 4. Fonts in figure are small. Consider to enlarge them to make them readable, especialy for E. aenea. If enlarged text overlap, it possible to organise text differently, e.g. move laterally every second word or use arrows for indication.

>>> Figure 6. change the time scale from Mya to show value in Kya
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments