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 STRUCTURE IN THREE NORTH AMERICAN TENT CATERPILLAR SPECIES (LEPIDOPTERA: LASIOCAMPIDAE): MALACOSOMA AMERICANA, M. CALIFORNICA, AND M. DISSTRIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Tables and figures too confusing and not well explained
Experimental design: Research questions diffuse and not particularly well articulated

Purpose of particular methods not clear
Validity of the findings: Claims not well supported and mostly based on assumptions rather than robust tests of alternative hypotheses
Additional comments: Review of Lait and Hebert

Overall critique
I had difficulty making sense of the figures and the purpose of the paper. It strikes me that the authors set out to test whether particular historical events influenced the abundance and distribution of genetic variation. For instance, in the discussion beginning with line 295, the authors discuss whether there is evidence of glaciation effects on the distribution of variation, and they introduce some new data and describe an analysis that was not included in the paper (isolation by distance). I have a hard time seeing the pattern they describe in their figures. The statistical parsimony networks are, in my opinion, uninterpretable and the colored circles maps were also difficult to interpret. Additionally, the “genetic breaks” are difficult to see because they are wholly or partially obscured by the circles. Moreover, contrary to their claim, for M. dissitria, there seems to be LESS variation in the south than in the north.  My recommendation is to make a specific testable prediction: all three species have lower genetic variation in the north because of the effect of glaciation. It should be relatively straightforward to make a graph with latitude on the x axis and genetic diversity on the y-axis, indicate the southern boundary line of the glacial maxima, and simply assess whether the expected association exists. If it does, that’s interesting and adds more species to the list of species that were greatly impacted by the last glacial maximum. If there is no association, that’s interesting and suggests a number of different scenarios (species not dispersal limited). Finally, isolation-by-distance does not, by itself, “indicate that recolonization happened in stepping-stone fashion.” Overall, I would argue that the claims are not supported (at least by what is presented) and the inferences are weak and overstated.

Further, the authors reported that “The fact that M. americana lacked fixed differences among populations, while M. disstria had only a few between the western and eastern populations, suggests a single refugial origin for both species, likely in the south-eastern United States.” I am unaware that this evidence enables a claim about the number of refugia. It strikes me that the authors would need to establish alternative models that different in the number of refugia and to use some objective criteria for evaluating the fit of the data to each model. Only then would I be convinced that there was a single refuge. (See papers by Steele and Storfer 2006; Waltari et al. 2007; Bao et al. 2015, etc).
The authors did a phylogenetic analysis. It would have been nice to see a full phylogram tree of all haplotypes rather than the un-informative triangles (Figure 2). Also, it is curious why the authors did not include all seven clades in their analysis since each clade appears to be variable. This would have provided more power for estimating the dependence of diversity on latitude.
I was also unclear why the PC plot is so complicated and what, if anything, we are supposed to come away with after trying to figure out what all the colors and symbols mean. Overall, the authors did a poor job of constructing visualizations of data that effectively and efficiently communicate relevant information (in my opinion).
Table 2 is also nightmarish. I would recommend using the data to construct a simple visualization.

I would favor a much more focused story, perhaps one that uses the data to address 1) whether there is a general signal of north-south gradient of genetic variation, and 2) the number and possible location of refugia. These results can then be used to compare with other taxa to assess whether the inferences gain for these species were evident for other species with similar or different biological characteristics.
Overall, I can not recommend acceptance of this manuscript until the authors make the connect between the evidence and claims more robust, or alternatively, emphasize the great amount of uncertainty associated with the claims.
Introduction
The authors begin with the statement… “While phylogeographic structure has been examined in many North American vertebrate species, insects have received much less attention. The present study begins to address this gap by examining population structure in members of the moth genus Malacosoma, an important group of forestry pests.” I am not convinced that the fact that insects “have received much less attention” is sufficient justification for pursuing a study of an insect. Another weak justification for the research: “study represents the first step in a broad investigation of phylogeographic patterns in North American Lepidoptera.”  I think it would be much more compelling to justify the work because the biology of the insect can add to our general understanding of phylogeographic structure of temperate organisms.
Methods
I think it would be useful to briefly explain WHY a particular statistical approach is adopted and the parameter that is being estimated.


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 STRUCTURE IN THREE NORTH AMERICAN TENT CATERPILLAR SPECIES (LEPIDOPTERA: LASIOCAMPIDAE): MALACOSOMA AMERICANA, M. CALIFORNICA, AND M. DISSTRIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The second version of this manuscript is much better and clearer than the previous one. All the main critics and suggestions made by the reviewers were considered and included by the authors.
Experimental design: Methodology is better described and objectives are well defined.
Validity of the findings: Results are much more clearer than the previous version, with data well described and discussed. In the Conclusion section, the main achievements are highlighted properly and appropriate suggestions for further studies are made.
Additional comments: In my opinion, both authors carefully considered the critics and suggestions made by the reviewers and the editor. The quality of the manuscript was greatly improved and it is, in my opinion, now suitable 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: PHYLOGEOGRAPHIC STRUCTURE IN THREE NORTH AMERICAN TENT CATERPILLAR SPECIES (LEPIDOPTERA: LASIOCAMPIDAE): MALACOSOMA AMERICANA, M. CALIFORNICA, AND M. DISSTRIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: First, I'll begin by congratulating the authors on their revision: they did a good job of addressing all of the criticism . Importantly, they clarified the purpose of the paper. They wrote the work was motivated "...to determine whether limited dispersal abilities and contemporary barriers are preventing movement in these species, what role the Pleistocene glaciations may have played in their current structure and distribution, and if these three Malacosoma species show concordant phylogeographic patterns or if there are differences in the patterns observed that may be explained by their life history characteristics."

I have no problem with the authors publishing this paper. There is good information that needs to be published.

But do I want to be very clear that most of the revision failed to help me understand what the data show, why they do the analyses they do, and whether any hypotheses were tested. For example, one of their goals (stated above) is testing whether their is an effect of life history on patterns of genetic variation. With three species, n = 3. I am certain that there is insufficient power to estimate the effect of life history traits on the distribution of mtDNA sequence variation.

Similarly, the data are DNA sequences from a short section of one gene. I am not sure why they used a multi-locus method (BAPS) for inferring "clusters". I would have preferred a more conventional approach that includes making haplotype trees for each of the three species (33, 64, and 42 "taxa") and perhaps color code taxon labels by latitude or distance from the edge of the ice during the last glacial maximum. I have a hard time see anything in the data visualizations that tell me anything about the role that "Pleistocene glaciations may have played...". In response to my criticism of the largely (and still) confusing statistical parsimony trees, the authors noted that "The full phylogram is not appropriate to include for the purposes I was using it for (to test monophyly). The full figure contains almost 500 samples - this would make it entirely unreadable. and lose the point of the figure." I wasn't asking for ALL sequences to be included, only the unique haplotypes for each species and there are fewer enough the tree will likely be easier to interpret, with appropriate taxon labels, than multi-colored tiles in figures 3-5. Being color blind makes these figures a challenge for me.

The authors remain intent on making very specific claims without evidence of support for alternative hypotheses. For example, "M. americana...shows limited structure with a north-south trend and greater diversity in the south. This is consistent with its expansion from a single southern refugium following the last glaciation with limited ongoing gene flow between distance regions." What other historical events, processes, might generate the same pattern? What does it mean for data to be consistent? Finally, we should lose site of the fact the data are a single gene and so there are no degrees of freedom associated with historical inference. Typically phylogeographic inference necessarily requires MULTIPLE independent loci.

Finally, there were really no changes in the data visualizations to help me better see patterns in the data. The PC graphs have a lot of colors and symbols and I am not sure WHAT the purpose of the graph is relative to the goals of the paper. It does not relate to life history, dispersal ability, or the Pleistocene as far as I can tell.

Overall, while I have no problem with the paper being published, I do think that the inferences based on limited data go far beyond the evidence. What I see is nicely summarized in table 1: three species with different amounts of genetic variation for a bar-code locus.
Experimental design: No comment
Validity of the findings: See above
Additional comments: See above


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

Input:
Title: BIOSYNTHESIS OF SILVER NANOPARTICLES USING CAESALPINIA FERREA (TUL.) MARTIUS EXTRACT: PHYSICOCHEMICAL CHARACTERIZATION, ANTIFUNGAL ACTIVITY AND CYTOTOXICITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: -The article is written clear and unambiguous.
-Literature references are sufficient, however the discussion part need more relevant work to be addressed and compared.
- The structure of the article is acceptable with respect to the journal standard format.
-All results are presented well and they are relevant to the article hypothesis.
Experimental design: All experimental work is well designed.
Validity of the findings: All findings/results are novel.
Statistical results are presented well.
Additional comments: The author has to improve the discussion part, adding more relevant/new work with respect to the anti-fungal and cytotoxicity findings.


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

Input:
Title: BIOSYNTHESIS OF SILVER NANOPARTICLES USING CAESALPINIA FERREA (TUL.) MARTIUS EXTRACT: PHYSICOCHEMICAL CHARACTERIZATION, ANTIFUNGAL ACTIVITY AND CYTOTOXICITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript is well written, at least, with professional English
Experimental design: The concept of silver nanoparticles is not new; however, the authors made use of C. ferrea seed extract for preparing such nanoparticles and they showed its biological application.
Validity of the findings: Nanotechnology is gaining importance and the present finding is timely in the context of nanotechnology.
Additional comments: The manuscript by Serna et al. is interesting and well written. However, much information are lacking: (i) the authors state that the C. ferrea seed extract is a reducing compound, evidence confirming its reducing property is lacking or elsewhere has it been shown? (ii) what is the composition of this C. ferrea seed extract? (iii) lines 311-318, authors do state that the secondary metabolites could be responsible for the fungicidal activity; however, what is the (at least speculated) mechanism of action during antifungal activity in the present experimental context?

Other comments:
1.	Abstract: ……demonstrated that C. ferrea may be used for…., correct is at ……demonstrated that C. ferrea seed extract may be used for….
2.	Line 216, …….in an oven at 37 ± 2ºC in an……., correct it as in an incubator at 37 ± 2ºC in an……..
3.	In Figure 1 the authors do show the UV spectra for AgNO3:NaOH solution, but was it after incubating AgNO3:NaOH solution for 12-24 h?


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: BIOSYNTHESIS OF SILVER NANOPARTICLES USING CAESALPINIA FERREA (TUL.) MARTIUS EXTRACT: PHYSICOCHEMICAL CHARACTERIZATION, ANTIFUNGAL ACTIVITY AND CYTOTOXICITY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The work is interesting and suitable for publication after minor revision and modifications.
Experimental design: Please explain in detail how the UV spectra were obtained. Was a colloidal suspension used? How was contribution from turbidity accounted for?
Validity of the findings: Baseline correction of spectra in figures 1 (UV-Vis)  and Figure 2 (SERS) can help to better show the peak /location absorbance of the final product.

Why is there a loss in viability at higher concentrations? does the compound/product affect cell function? (Figure 5) . The plotting of the control and AgNP in the same figure could help to better show the effect of AgNP. What is the effect of uncoated AgNP?
Additional comments: Please check for the term oxygen-reactive species (Line344). Us it reactive oxygen species?


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: HOMOLOGY MODELING AND DOCKING STUDIES OF A Δ9-FATTY ACID DESATURASE FROM A COLD-TOLERANT PSEUDOMONAS SP. AMS8
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript, Garba et al report a structural model for a bacterial fatty acid desaturate, as well as docking studies to its palmitic acid substrate. Specifically, they identify two mammalian desaturases of known structures, which they use as template to model the bacterial homologue. They then refine their model my energy minimization, and verify its geometry. Finally, they use this model for docking studies with Palmitic acid.
Predicting the structure of membrane-embedded enzymes is clearly an important and challenging problem. In particular, bacterial lipid modifiers such as desaturates are potential antibiotic targets, and therefore understanding the molecular details of their mechanism is highly relevant, However, here the authors employ a very simplistic methodology based on automated servers. Furthermore, the complete lack of biological insights from this bioinformatics analysis renders this study rather moot. Finally, the figures are very poorly rendered, and largely consist of screenshots from the various programs and/or servers used.
Experimental design: - The abstract and introduction mainly discuss the difficulty of protein structure determination and the advantages of homology modeling. This is widely known, and has been reviewed in many articles. The authors should rather focus on their target enzyme: what is known about desaturases mechanism, how the bacterial enzyme differs from other desaturases, and how a structural model could explain its difference in mechanism.

- While YASARA is a commonly used program for homology modeling, it has not been developed specifically for membrane proteins, which represent a specific challenge. Using programs specifically designed for membrane proteins, such as Rosetta, Medeller and/or MEMOIR would be better suited for this study. Similarly, it is not clear if Zn ions and lipids are present in the docking experiments reported here. These would be essential to make sure that the results are accurate.

- In the sequence alignment shown in figure 2, it seems that the target enzyme possesses a ~120 amino-acid extension at the C-terminus. What is the predicted structure of this region, and does it have sequence homology to any other proteins? Was this domain included in the structural model?
Validity of the findings: - The discussion barely shows any biological insights. The authors mention the presence of iron in some membrane-bound desaturates, but the two structures used as template for the modeling clearly have zinc, and therefore the results of this study cannot be used to argue for a difference in ion coordination.

- For such study, the structural model and ligand pdb files should be included in the supplementary material, so that it can be used by readers.
Additional comments: - Figures 1 and 4 seem to be simple screen shots of a server results page, and should be re-done, including labels specific to this work. Figure 7 is unreadable and needs to be re-done. Figures 3 and 6 should be improved, with better color coding, labeling important residues/domains etc. For example, the side-chains in Figure 3 are superfluous, and so are the hydrogen atoms on figure 6.


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: HOMOLOGY MODELING AND DOCKING STUDIES OF A Δ9-FATTY ACID DESATURASE FROM A COLD-TOLERANT PSEUDOMONAS SP. AMS8
Review round: 1
Reviewer: 2

Output:
Basic reporting: The approach is good but outcome is not supported by the strong in-silco methods  as well as experimental validation therefore real result might be different from what is presented here.
Experimental design: Experimental design is poor. Docking result need to be validated with molecular mechanics methods and molecular dynamics simulation.
Validity of the findings: In my view, finding can not be validated from result provided.
Additional comments: The approach have novelty but lack design and experimental validation therefore in my view, the present work is good start but author have to perform high throughput computational methods to reach at conclusion.


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: HOMOLOGY MODELING AND DOCKING STUDIES OF A Δ9-FATTY ACID DESATURASE FROM A COLD-TOLERANT PSEUDOMONAS SP. AMS8
Review round: 1
Reviewer: 3

Output:
Basic reporting: The introduction needs more detail. I suggest that you improve the description abot the enzymatic mechanism of Δ9-fatty acid desaturases.

Please, provide newer references about molecular docking calculations (line 85).

I commend you to provide a description for the initials GCMS (analysis) on line 138.

Figure 1 does not present clear relevance, thus, I suggest you to remove it and solely keep the mention on the text.

Figure 2 presents high relevance on your work and I commend you to improve it, since the given format is the automatically provided by the Clustal software.

The English language is well written, but I recommend the revision of caption 3 (figure 3).

Figure 6 highlights important findings of you work, please, provide a more ilustrative structure, using different colors for secondary structures and for Zn ions.

Figure 7 should also be improved for a better understand of your data. Sticks over transparent cartoon (or only sticks) in a white background, instead of lonely lines in a black background, are more clear representations.
Experimental design: I commend the authors to provide a better description of structure prediction methodology.

I encourage you to provide the specific parameters applied for docking calculation in order assure the results reproducibility.

Since most results are based on docking calculations, I strongly commend the authors to perform redocking calculations (using the mentioned crystal structures) to validate the docking system, in view of the high flexibility of the analysed ligand.
Validity of the findings: The reported findings need to be well discussed to highlight its relevance. Discussion related to the findings are too brief, and the modeled structure is not properly compared to the available crystal structures, especially about ligand binding. Please, provide a extended discussion comparing the molecular basis for ligand binding among the modeled and the crystal structures.
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: HOMOLOGY MODELING AND DOCKING STUDIES OF A Δ9-FATTY ACID DESATURASE FROM A COLD-TOLERANT PSEUDOMONAS SP. AMS8
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revied version of the manuscript is significantly improved, with clearer biological insights and much better figures.

There are two labels in figure 2A that seem to show the protein termini, but are clearly out of place. This should be fixed. In addition, the few lies at the top of figure 3 are not required and should be cropped out.
Experimental design: - The modeling procedure is now well supported, except for the fact that two models were generated (AMX8-em1 and AMX8-em2) but it's not clear what is the difference between them. Were they generated using the two different templates?

- For docking experiments, a few additional controls are required: With and without iron for the model, as well as a control docking in one of the experimentally-determined stearoyl-coA desaturase structure.
Validity of the findings: The overall conclusions are supported, provided the above controls are performed.

A few concept could be explored further:
- The MD simulations show significant changes over time, which part(s) of the protein is(are) affected?
- The authors mention that a three-TM desaturase is unusual. Is it unique to this specific enzyme, or more broadly applicable to a certain sub-family?
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: FATIGUE AND PERCEPTUAL RESPONSES OF HEAVIER- AND LIGHTER-LOAD ISOLATED LUMBAR EXTENSION RESISTANCE EXERCISE IN MALES AND FEMALES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have provided a succinct, clear report of their study with well described methodology and rationale. Thank you for providing the raw data. The group codes in the supplementary file currently use numerical values (1 vs 2), that could be unmasked to what I assume is male and female based on the values.
Experimental design: No comment, adequately designed and described methodology.
Validity of the findings: Line 343-345: Participant flow is adequately documented in the study, and that the target sample size was not attained. Consequently, the between-sex comparisons may be under powered. This could be substantiated by providing a post hoc power calculation and discussion as to what degree of difference one should consider meaningful between the sexes. Is there a clinically important difference for fatigue values, or a difference that at least exceeds the measurement error such that we would need to consider the power of the comparison?
Additional comments: -Lines 355-358: The authors conclude that based on the differential fatigue response between the sexes, that training program modifications may be required based on sex. It is not necessarily clear what outcomes would be expected to be different between males and females in this instance. Many studies have demonstrated comparable hypertrophic and improved (but differential) strength responses with varying training loads taken to the point of failure, would there be other relevant adaptations that should be considered in the context of this data or muscle group? Discussion on this may be somewhat speculative in nature and beyond the scope of the article but would enhance the existing statement.

-Line 27/28: Inconsistent spacing between numerical symbols and numbers, this is also evident in figure legends and tables (PDF rendering issue?)

-Line 186: Unnecessary footnote, could be included in the paragraph

-Line 369/ Line 45: Fatigue occurred following both high and low load exercise, but only in the low-load condition is fatigue referred to as "debilitating". What differentiates the use of the term "fatigue" and "debilitating fatigue". Stating "increased fatigue" may be more appropriate.

-Line 367-70: Is there a common misconception in rehabilitation settings that low-training is required to strengthen the musculature? This may have less to do with a relationship to desired training adaptations and relate more to clinician perceptions of loading tolerance/pain. The data on fatigue and discomfort is useful clinically, however, is there any connection between RPE-D and pain (i.e. NPRS)? Is the perception of clinicians that the use of low-loads reduces the risk of increasing pain relative to high loads, irrespective of discomfort?

-Line 371: "....in other rehabilitation environments". It is not clear what rehabilitation environment this was completed in. Were they asymptomatic, healthy controls? If so, should read "....rehabilitation environments"


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: FATIGUE AND PERCEPTUAL RESPONSES OF HEAVIER- AND LIGHTER-LOAD ISOLATED LUMBAR EXTENSION RESISTANCE EXERCISE IN MALES AND FEMALES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Reporting is overall very well done.
Abstract: final sentence - lighter load is in fact used in rehabilitative settings to increase 'endurance' of the lumbar extensors as an extension from Biering Sorensen's original finding of decreased endurance and its association with back pain. However, as load is required for strength the statement is flawed. Lighter load may increase endurance, but not strength. Please revise the sentence.

Some disparity in the wording of the aims in the final sentence of the introduction and the first sentence of the discussion. Restating may be easier for the reader.
Table 1 - please change Kg to kg (SI units)
Line 156 - Comma after incurred
Line 158 - Comma after effort
Line 275 - A reference is needed in here.
Line 442 - Capitalize Journal
Experimental design: Line 130 - How was lumbar ROM assessed? Seeing there are numerous ways of assessing this, some explanation for reproduction may be warranted.
Validity of the findings: Not a criticism as the authors have reported on both central and peripheral influences to fatigue, some of Professor Samuel Marcora's newer work on the central influence of fatigue (if its published yet) could be added. This could be an nice addition to the discussion around Line 321.
Additional comments: Congratulations on a well written paper. Some minor modifications are suggested.


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 USE OF NUTRITIONAL SUPPLEMENTS TO INDUCE KETOSIS AND REDUCE SYMPTOMS ASSOCIATED WITH KETO-INDUCTION: A NARRATIVE REVIEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors aimed to review the current literature available on nutritional ketosis, the use of ketone supplements and how they can be used to mitigate negative symptoms associated with keto-induction. Considering the recent expansion in commercially available products aimed at facilitating the adherence to KD, it is crucial to better understand the actual scientific evidence supporting the claims currently made.

The manuscript is well structured and well-written. However, a few edits would improve the quality of this report:

1.	References:
1.1. No reference has been listed to substantiate any of the sentences written in the ‘Short chain fatty acids’ section (Lines 92-98)
1.2. Lines 111-114 also need reference(s)
1.3. St. Pierre et al. mentioned in Line 122 is not listed in the References section

2.	The exogenous ketones are simply described as either salts or esters of BOHB. Since describing the use of ketone supplements to facilitate nutritional ketosis is a key objective of this review, it would be helpful to have a more detailed description of these supplements (types of salts, types of esters, biodegradation and bioavailability, adverse effects, proposed applications etc.)

3.	Additional and relevant data comparing ketone salts and MCT (described in Kesl et al., Nutrition & Metabolism, 2016; 13:9) is available and can be included to further improve this narrative review on supplements used to induce ketosis

4.	It is relevant to address that there’s lack of consensus on whether achieving greater ketosis necessarily means better physiological outcomes. Some of the questions in the field that are of importance and could be expanded upon in the text:
4.1. Would achieving higher levels of ketosis (by supplementing with exogenous esters, salts or MCTs) shorten the keto-induction period and/or enhance the physiological benefits observed with nutritional ketosis?
4.2. Is there any available scientific evidence that ketone supplements do actually mitigate the ‘keto-flu’ symptoms associated with the keto-induction period?
4.3. Does chronic supplementation with exogenous ketones impair endogenous ketogenesis (as observed in Balasse et al., 1975)?

5.	When discussing MCTs, the authors describe their ketogenic effects, suggesting that acute ingestion of MCTs induces nutritional ketosis. However, it is important to differentiate between hepatic production of ketone bodies due to acute ingestion of MCTs and the biochemical shift in fuel utilization that occurs during prolonged nutritional ketosis (the physiological process described as keto-induction or keto-adaptation). I suggest addressing this difference and possibly emphasizing the acute/transient effects elicited by single or acute consumption of exogenous ketones, resulting in peripheral ketosis but not necessarily keto-adaptation. On that note (and according to lines 159-161), would the MCT ketogenic diet likely shorten the keto-induction period when compared to the classic KD?

6.	The language is appropriate but a few typos were found:
6.1. Abstract: Line 20: dose-dependant instead of dose-dependent
6.2. Introduction:
6.2.1. Line 46: references 22 and 23 are listed not separated by a comma
6.2.2. Line 50: Fibre instead of fiber
Experimental design: A summary table with relevant articles on ketogenic diets (classic, modified MCT diet, MCT ketogenic diet and supplements) listing preclinical and clinical studies and main observations (levels of ketosis, time to achieve ketosis, physiological outcomes, adverse effects etc.) would provide greater support and structure for this narrative review.
Validity of the findings: 7. The topic of ketogenic diets and ketone supplements has risen in popularity due to well-known benefits in weight management, diabetes, athletic performance and metabolic health. However, it is important to emphasize that, although this approach is promising as potential therapeutic intervention for many other conditions, clinical data is still lacking. For instance, the positive effects in anxiety described by Kashiwaya et al., (2013) were observed in transgenic models of amyloid and tau deposition. An additional report of ketone supplementation reducing anxiety-related behavior in rodents was published last year (Ari et al., Front Mol Neurosci, 2016; 9:137) using two different rat strains. Clinical studies on ketogenic diets and supplementation in populations of mental disorders patients are still lacking. Studies based on animal models of mental disorders have limited generalizability when translated into the same conditions in humans. Caution should be exercised and clinical studies remain necessary.

8. The same caution can be extrapolated to other areas such as cancer, autism spectrum disorders, Alzheimer’s disease etc., where mostly case reports are currently published and the vast majority of data is originated from animal studies and anecdotal reports. This does not invalidate these approaches, as sound medical hypothesis have been published (as in the cases of metabolic dysfunction associated with cancer and Alzheimer’s disease) but it does highlight the need for well-designed randomized, clinical trials to support the available hypothesis and preclinical data.
Additional comments: Given the abundance of anecdotal reports on the health applications of ketogenic approaches, this review is of particular interest and value to the scientific community. I thank the authors for addressing such an important topic, compiling evidence for such a promising metabolic therapy implicated in diverse health conditions.


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 USE OF NUTRITIONAL SUPPLEMENTS TO INDUCE KETOSIS AND REDUCE SYMPTOMS ASSOCIATED WITH KETO-INDUCTION: A NARRATIVE REVIEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: I believe there is insufficient background and context to the subject provided;  the diet used for mainstream and athletic use is generally a very different diet from the one used for neurological disorders such as epilepsy.   In order to write this paper - these 2 separate diets need to be explained; the rest of the article then should follow suit: which diet along the course of this paper are the authors referring to?
IF it is the medically managed KD then I disagree with many statements that have been made: For example: there is a wealth of literature that reports the length of time to "nutritional ketosis" and the side effects that relate to this - none of which have been listed by the authors.  This is important as it is the question of the study.  What Is meant by an "improvement in time to ketosis?" line 65?  Is a short time an improvement?  Without a parameter for time to ketosis I fail to understand how you could study improvements in time to ketosis!

If it is a low carbohydrate diet then the parameters for carbohydrate and fat need to be provided.
The mechanism for ketone production by the Classic KD is not explained; it is the combination of high fat AND low carbohydrate - causing a shift from burning glucose as first from of fuel to fat; this shift in metabolism is what leads to all of the short and long term side effects.   Many statements written in the paper appear to have negated this basic premise.
Many words are not considered scientifically "sound" in keto world - words such as "keto-flu" "nutritional ketosis" and Very low CHO KD are not considered scientifically credible.
Experimental design: Research question is not well defined; see basic reporting above; more background needs to be provided in order to understand the study aims better.
Would suggest to be considered scientifically more credible EMBASE should have been searched.
I struggle to comment on the manuscript regarding leucine; it may be a Ketogenic AA but needs to be given in the context of a ketogenic environment to work?  There is no need for it to act as such unless stores are depleted and it is being used as an alternative source of fuel.  Vinegar - interesting!   BUT the only reason it is prescribed as a "free food" in KD is it is carbohydrate free; from a practical point of view; so to are dried herbs and spices, salt and pepper and lettuce!
MCTs are useful in the space of KD - the reason they are more Keto yielding has been stated incorrectly - it is merely because for some metabolisms the capacity of B oxidation has been exhausted and so another form of fat; metabolised via a different pathway can effectively produce ketones.  MCT used for CPT II and II is used as the oxidation of LCT is not possible and those patients require an alterative source of fuel to avoid metabolic decompensation.
Exogenous ketones - their role has been established; this is not clearly defined.   D'Agostino is the expert and should be considered extensively here; unfortunately only mice studies have used those EK used in research - these appear to work but are unavailable to the public and taste awful!
Validity of the findings: Believe this study could be interesting but basic parameters need to be addressed in [particular what diet is being referred to and is supplementation on top of a KD or on top of a "normal diet".
Authors need to examine literature more thoroughly to establish the length to ketosis ) it is defined in the medically managed diet; as to are the side effects of this stage.
Additional comments: Agree the authors have chosen a topical subject: many people are seeking a "quick fix" to feel better; lose weight, feel fuller etc.  a supplement that could do that would be great.  This paper needs more work to establish the basics and the context before discussing the potential for nutritional supplements.


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 USE OF NUTRITIONAL SUPPLEMENTS TO INDUCE KETOSIS AND REDUCE SYMPTOMS ASSOCIATED WITH KETO-INDUCTION: A NARRATIVE REVIEW
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I thank you the authors for addressing the comments made by the reviewers. The manuscript has been significantly improved by the changes in the revised version submitted.


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 USE OF NUTRITIONAL SUPPLEMENTS TO INDUCE KETOSIS AND REDUCE SYMPTOMS ASSOCIATED WITH KETO-INDUCTION: A NARRATIVE REVIEW
Review round: 2
Reviewer: 2

Output:
Basic reporting: The language used is ambiguous and is not the vocabularly used in this field.  The terms "nutritional ketosis" "keto-flu" "Very Low carbohdyrate ketogenic diet (incorrect!) " "natriuresis, kaliuresis" are rarely if ever used amongst health care professionals looking after patients on the medically managed Classical ketogenic diet.  This is the diet that I am believe the authors refer to as stated: line 48 - "consists of a 3:1 to 4:1 ratio".  The article starts with VLCKDs are becoming increasingly popular for mainstream athletic use.....Line 31/32 but never refers to these group again.   The literature cited has been taken largely from studies based on the KD for epilepsy and has stated that "there is a paucity of research available that identifies specific time points to NK" - line 73.   In the epilepsy community functional "ketosis" ie: that which has the potential for a beneficial effect on seizures and be considered a point at which the body has truly started to use fat as the first form of fuel is determined as > 2.0mmol/L not 0.5mmol/L; as stated by the authors - line 75.  Again, amongst this community - it is well known that a 3:1 to 4:1 ratio diet followed strictly for 2-4 days will achieve levels 2-4mmol/L - which makes me question what diet you are reviewing?
I do not believe the article is structured well and does not report on key studies regarding Ketosis and time taken to achieve.   The evidence for ketosis can be witnessed in many studies on dietary efficacy in the epilepsy community.
Experimental design: To review the literature the article uses various search engines.  It is unclear how the words that were put into the search engines were linked to the "ketogenic supplements"..  A ketogenic diet search alone will retrieve 100s of articles.  No discussion is made of this data and reasons for excluding a huge body of literature.
Much of the  literature that has been reported is either inconsequential or irrelevant to this discussion.   This is evidenced by line 74 - Bergqvists paper - it states that participants fasting achieved mean levels of >0.5mmol/L BOHB the day after......the conclusions you have drawn suggest a lack of understanding in the clinical delivery of the diet.   In addition, this paper is again children with epilepsy.   The aim in these chidlren is > 2.0mmol/L.   A level of 0.5mmol/L can be achieved in most of us by fasting overnight and having a delayed breakfast!  Line 80 - Wirrell and colleagues, what ratio diet were they using?  It would be more common to report in the literaure on those who do not achieve ketosis on a 3:1 - 4;:1 ratio quickly than those who do!
Validity of the findings: These findings may be true but I fail to see relevance in the article;   For who is it to be used for?  Children and adults with epilepsy who we know we can achieve ketone levels > 2mmol/L over a number of days, or athletes and the general public?

In addition, time to the ketosis level in children with epilepsy has little impact on tolerability of the diet.   The difficulty in managing a diet which is providing 90% fat is one of the main reasons for dietary disbandoment.  This is well documented also.
Additional comments: The article sets out to review scientifc knowledge on effects of methods listed on "time to NK" and on symptoms during keto - induction phase.
It may have achieved the goal of identifying MCT as ketogenic yielding (nothing new in the literature here) and excluded others but I fail to see the value of this research to anyone.


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 USE OF NUTRITIONAL SUPPLEMENTS TO INDUCE KETOSIS AND REDUCE SYMPTOMS ASSOCIATED WITH KETO-INDUCTION: A NARRATIVE REVIEW
Review round: 3
Reviewer: 1

Output:
Basic reporting: Several improvements to this article have been made but key issues remain

1) Language often used contraindicated to topic and not used in scholarly articles; Very Low Carbohdyrate Ketogenic Diet is ambiguous; "keto-flu" - why discussed; why not symptoms as per aim of the study?
2) Existing literature in the area is missing and that employed often irrelevant or not the most appropriate (ie see lines 81-95 - these studies are taking groups of patients that are not comparable, hence no conclusion understandably drawn)

3) Improved structure required - very hard to follow - suggest: What is the aim of the study; why are you studying it? ie what is your experience with length of time to ketosis and what is considered an improvement?   Currently without a defined length of time to ketosis how can you say what is an improvement? In addition, How debilitating are the side effects of keto induction (this needs defining as gives meaning to the paper)  and would decreasing length of time really help this (practical experiecnce at an interantional level in the area of KD in epilepsy has shown not). Why were leucine etc chosen as supplements? ie number of articles from literature search please.   For each supplement chosen address the hypotheses: link to length of time to keto induction and side effects.  This will give your paper meaning.
Conclude with either use, consider using and why....

4) Results may be relevant to hypotheses but there no statements linking the two.   An extensive literature search was conducted - what were the results of this?   How many articles and in what journals highlighted leucine, SCFAs etc as relevant to time to NK? Symptoms during Keto induction phase?  In addition; at conclusion of these sections - ie line 126-127; discusses leucine reducing seizure activity and not independently increasing blood levels of BOHB; neither statements linked to hypotheses and do so wonder why leucine was chosen?
Experimental design: Research question not well defined; Remain unsure as to the target group this work is trying to assist, in addition not clear that reserach fills any knowledge gaps.

Rigourous investigation not performed; studies have been reviewed without meaningful consistent; comparisons made.  understanding of practical implication of diet appears to lack

Methods not replicable - as per results - see point 4) basic reporting
Validity of the findings: Replication of results would not be possible as per point 4) basic reporting
Unable to conclude any point to article: are any supplements worth or not worth considering as part of dietary management for the athlete? The child with epilepsy?
Could not say with conviction to use or not to use in either of these target groups after readig paper.

Conclusions from results sections do not link to oringial hypotheses; which itself is not well established.

Line 103 states "paper reviews the available scientific literature relevant to improvements in time to ketosis......" but nowhere is it defined what the time to ketosis is (as stated by the aurthor) and to what degree this is an issue.

Many changes have been made to protocols for ketogenic diet intiation in the patient with epilepsy over the past 10 years; studies report extensively on the success of switching from fasting to non-fasting initiation and the move from inpatient to outpatient initiation.   Both of these changes were made to reduce symptoms of ketogenic diet initiation, occasionally at the expense of increasing time to ketosis - these protocols are tailored to the individual based on their preferences BUT generally most patients (with epilepsy) are able to achieve high ketosis (>2mmol/l) chronically on a KD protocol which has been initiated over a 2-5 day period; very few with any side effects at all.   This work is all published.   I would very much like to know how your work links in with this vast amount of literature, some at the highest level of reporting.
Additional comments: See glimmers of success with the publishing of this article but structure and improved linking statements are required to ensure its' success.


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: PCR: AN R PACKAGE FOR QUALITY ASSESSMENT, ANALYSIS AND TESTING OF QPCR DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: - The manuscript clearly summarizes the functionality of the presented R-package and outlines basic use cases. The background section may be shortened as these methods have been extensively reviewed (as also cited) elsewhere (please see http://relative.gene-quantification.info/, http://gene-quantification.com/hkg.html#reviews)

- Line 49 - please improve grammar
Experimental design: - The relevance of the package is not clearly presented. Many R-packages with much greater functionality (also including the functions presented in the manuscript) are available and in my view this package does not fill any “knowledge gap”. Already published R-packages include: https://cran.r-project.org/web/packages/qpcR/index.html, https://bioconductor.org/packages/release/bioc/html/EasyqpcR.html, https://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-13-296, …

- The authors argue that a new unified interface is provided, but this interface still requires a certain input format.

- The authors may include more data preprocessing methods to directly import data from different qPCR vendors.

- The package is clearly outlined, also the vignette presents many details
Validity of the findings: - The provided sample data allows users to explore the included package functionality

- Authors should respect the MIQE guidelines (https://www.ncbi.nlm.nih.gov/pubmed/19246619), including their proposed naming convention
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: PCR: AN R PACKAGE FOR QUALITY ASSESSMENT, ANALYSIS AND TESTING OF QPCR DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Article is written in clear, structured and professional manuscript schema with appropriate background, rationale, methods, examples, and importantly limitations. Authors have throughout adhered to most commonly used best-practices for developing and documenting R package.
Experimental design: Authors presented a R package to include two of commonly used statistical approaches in interpreting PCR data with an easy-to-understand concepts behind each of statistical framework for new users. Authors have explained in detail core concept, biological context and limitation of each of these two methods before describing implementation of those in their R package.
Validity of the findings: Authors have made source code available on CRAN and github and did an impressive job of adhering to best-practices for developing and documenting R package. Authors provide both visual and tabulated form of data for interpreting PCR data for both irrespective of one's expertise in statistics. I have a few minor comments or rather suggestions in interpreting results:

1. For comparative Ct method, besides assuming similar amplification efficiency for reference and gene of interest, a resulting Ct value typically above 40-45 (depending upon calibration of machine) may not be reliable and should be flagged accordingly, i.e., Ct value for gene of interest beyond 40-45 cycle is non-deterministic as that gene may not be expressed at all or would have very minimal expression.

2. For t-test, unless pcr_test function inherits t-test arguments, I suggest to add an option for user to provide either "one-tailed" or "two-tailed" t-test depending of an experimental design.
Additional comments: Authors did an impressive job at structuring and writing this article in easy-to-understood language but yet providing details on interpreting PCR data with both biological and statistical context. I second and upvote their suggestions in limitations to improve this package and also suggests to add R-Shiny interface to analyze PCR data in interactive manner.

Thanks!


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: PCR: AN R PACKAGE FOR QUALITY ASSESSMENT, ANALYSIS AND TESTING OF QPCR DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: - Background section: The authors reply that they want to present a complete and stand-alone document for readers. Unfortunately, the section is not complete and might not stand on its own. I still would suggest to shorten it.

- I would very much recommend adding a section that clarifies where your method is applicable and where it is not. Since it only covers a very small part of the analysis workflow, potential users need to be informed what scenarios and data they can analyze with the package.
Experimental design: - Concerning data format: Can the authors reason, why they did not use the AnnotatedDataFrame? (https://www.rdocumentation.org/packages/Biobase/versions/2.32.0/topics/AnnotatedDataFrame) There is even a package for reading: ReadqPCR (https://www.rdocumentation.org/packages/ReadqPCR/versions/1.18.0/topics/read.qPCR)

- The authors reply that “input data are always the raw ct values”. Can they please elaborate (also in the manuscript) why raw Ct values are taken for statistical testing when a reference gene is used?

- How will the authors include new preprocessing methods to directly import data from different qPCR vendors in their package?
Validity of the findings: - I think by adding a short statement that Cq will be used instead of Ct no reader will be confused. (MIQE has been cited over 6000 times.

- Novel diagram: Why does the diagram start with statistical testing (on the left side). What is the input? What is the output?

- Table: How does “pcr” perform error propagation? How does it comply with the MIQE guidelines?

- Spelling mistakes: line 236
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: PCR: AN R PACKAGE FOR QUALITY ASSESSMENT, ANALYSIS AND TESTING OF QPCR DATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comments
Experimental design: Thank you for consideration of comment. In my knowledge, there are no set rules for cycle threshold from method perspective. In theory, depending upon sensitivity of PCR machine and type of experimental design with proper negative controls, it can detect even the minute levels of transcript in the lysate, i.e. at much higher cycle threshold than 40-45. However, in practical scenario, e.g., at Ct value of 45, for tissue-specific expression of a given gene transcripts, it is questionable if that transcript is expressed at a minute but functional level or tissue does not express that isoform at all. I like the idea of providing warning message with base R function but leave the decision on authors' end if they do not find compelling reference to support such argument.

Related references about cycle threshold but not regarding formal cut-off:
1. https://www.qiagen.com/mx/resources/faq?id=783d4566-9ad9-4fab-9936-182beda65617&lang=en
2. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 2008;3:1101–8. PMID: 18546601
Validity of the findings: no comments
Additional comments: No additional comments


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

Input:
Title: EVOLUTIONARY PERSISTENCE IN GUNNERA AND THE CONTRIBUTION OF SOUTHERN PLANT GROUPS TO THE TROPICAL ANDES BIODIVERSITY HOTSPOT
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the manuscripts is well executed.
Experimental design: Some of the methods need more details and clearer justification.
Validity of the findings: The findings are interesting and robust (although there are some concerns).
Additional comments: The paper addresses a significant question on plant diversity in the Neotropics, that of the history of diversification of plants in the mega-diverse Andean hotspot. The manuscript is well written, with a good structure and flow of ideas. The objectives are quite clear and the methods are appropriate for the questions being asked. The methods seem reasonable and overall robust, although I have some concerns in this area (see comments below). The discussion is adequate, but in need of careful revision. In addition, some of the interpretations of the results are, in my opinion, a bit to far-fetched; specially the section of stasis vs. rapid speciation.
Finally, I would like to see a more integrated discussion on the history of Gunnera, as is I found the sections somehow loosely connected.
Nevertheless, I think the present manuscript would represent a significant contribution to the understanding of Andean (an other montane regions) mega-diversity and I would recommend its publication pending revisions.

My main concerns are related to some of the methods used and some of the interpretation in the discussion. The methodological parts do not appear to be critical, but I am a bit skeptical on several fronts. So some clarifications are needed and extra details on specific analyses would be welcomed. Several comments below.

Dating analyses:
I’m not very familiar with the STACEY algorithm, but it appears that its main strength comes when using many loci, for which the coalescent is the best way to go. My question goes to the fact that you are effectively using 3 loci (cpDNA + two nuclear genes), so what are the real advantages of using STACEY over other dating algorithms?

According to Wanntrop & Wanntrop, Tricolpites fossils show a great resemblance to specific extant groups of Gunnera, but the authors did not perform a proper phylogenetic analyses to place these fossils within the phylogeny. So I am a bit skeptical whether the fossil might belong to some stem member of Gunneraceae. From the original description of the fossil grains, these probably belong to crown Gunnera due to their resemblance to different pollen types within the family. Although this assignment might actually hold, since the dating is so crucial here I think more justification is needed on the placement of the fossil in crown group Gunneraceae.

I'm a bit concerned about your interpretation of the age of Gunnera because of the very wide interval you obtained. The mean age of 90 Mya for CG Gunneraceae is reasonable, but as said before I have doubts regarding this particular fossil calibration. What happens if the fossil goes to the stem group instead? Remember that the first unambiguous evidence of Core Eudictos is ca 100 Mya, so having CG Gunneraceae older than this is quite surprising. More so, Gunneraceae going back into the Early Cretaceous-Late Jurassic is at odds with everything we know about the fossil record of angiosperms: 165 will push Core Eudicots to a time prior to the first fossil angiosperms.

BAMM analyses:
More detailed is needed for the BAMM analyses and the corresponding results.
Moore et al. (2016) showed that the posterior distribution of range shift estimated in BAMM is highly influenced by the prior. How did your analyses behave in this respect? and more importantly, what prior on the number of shifts was used?
A major drawback of BAMM (as with other methods that estimate diversification rates) is that rates of speciation and extinction are hard to tease apart and thus interpretations based on these are limited. Authors should at least acknowledge this problem when discussing speciation dynamics.

Climatic niche evolution:
I’m a bit concerned on the way authors selected climatic variables. Did authors estimate the correlation matrix among variables for all species pooled together? If so, I’m not sure this works because the correlation matrix would be heterogeneous in different areas and for different species. I never been particularly concerned by correlated variables in MaxEnt (but I may be seriously wrong), so I would go with the 19 climatic variables (maybe eliminating the monthly variables only).
The cumulative output of Maxent seems reasonable, but a bit of explanation would be useful.
If I understood correctly, the climatic history of Gunnera was reconstructed under a BM model, but the tests of PNC indicate that this is best modeled by a OU model. Please clarify.
What would be the possible effect of missing species in the reconstruction (14 out of 41 for the Panke group)? I'm not concerned on BAMM estimates, but trait reconstruction is highly conditioned on the observed traits (sampled species). Are the missing species climatically similar to others?
Figure S2 is hard to follow. Maybe incorporating climatic niche information into figure 2 might help, or viceversa. Authors might have other ideas. By the way, why are only figures for temperature and precipitation shown in figure S2?

Results and discussion:
Lines 336-337. How do you reconcile the fact (from your own figure) that Gunnera fossils are found in the Andean region during the Cretaceous. Why did the genus not start to diversify earlier? Are you assuming these fossils do not belong to Panke?

Lines 346-348: So, Gunnera is effectively a northern migrant, rather than a southern group that colonized the Andes. This seems interesting. Please clarify, this goes strait to the main objectives and definitions of the manuscript.

Lines 359-361. No, this argument is very very tricky. By stating this you are implying a very high confidence on your selection of some fossils over others (besides doubts that can be raised regarding the assignment of those fossils to particular nodes in the phylogeny). Your dated phylogeny relies in assuming certain fossils are part of specific groups. So, if these fossils turn out to be part of Panke, your dated tree would be wrong. Hopefully, the point I'm trying to make here is clear.

Lines 362-363. But you do not know where this lineage goes in the phylogeny, which is crucial to support or reject your dating hypothesis. So, you have to be more careful here.

Lines 372-274. Low rates of diversification do not mean stasis or low speciation. Extinction is crucial here.

Lines 382-383. I do not see the connection.
First, the fossils have not been properly assigned to a particular branch in the Gunnera tree, it is just mere resemblance we are taking about here. Second, these fossils are evidence of extinct members of Gunnera, which might turn out to be part of the G. herteri clade. I think you are pushing your results to much. To me, the diversification results of BAMM are interesting enough, without going into speciation or extinction (which by the way are not shown anywhere). The two basal branches show very contrasting histories of diversification. The question here would be why the Panke lineage is diversifying more rapidly than the G. herteri lineage?Perhaps the climatic history can identify something here.

Lines 384-385. To me, the 165 Mya is a highly unreasonable age (it is misleading in figure 2). And if you are using the confidence interval to make a point, you can certainly also make the opposite point with the 40 Mya lower bound, which would make your results a lot less contrasting in terms of age. I think there is not much to say (confidently) about the age of G. herteri given the wide confidence interval, or just stick to the mean.

Lines 394-396. You have to keep in mind that you have older fossils in northern latitudes. I think this goes to the heart of another of my main concerns. I feel that the fossil record is somehow disconnected from the rest of the discussion. It would certainly be nice to include a more explicit account of the distribution of fossils throughout the discussion.

Lines 398-399. To me, the key element here is that Gunnera in the Andes comes from the north, not the south. So perhaps, the ability to colonize the Andes came from the first northward shift in the distribution of the genus.

Lines 424-427. To me, the pattern observed in Ceroxylon is quite different from yours, with a south to north (stepping-stone like) diversification. Whereas the pattern you uncovered in Gunnera is more complex, a south to north expansion, followed by a north to south diversification. Again, I think that explicitly integrating the data on the distribution of fossils would be highly valuable.


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: EVOLUTIONARY PERSISTENCE IN GUNNERA AND THE CONTRIBUTION OF SOUTHERN PLANT GROUPS TO THE TROPICAL ANDES BIODIVERSITY HOTSPOT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors present a case study (the genus Gunnera) to investigate the role of southern immigrants in the assembly of the Andean biodiversity hotspot flora. This is an interesting case study since it has been hypothesized to represent a Gondwanan origin with vicariance by continental drift to explain the present-day distribution of most Gunnera species. The authors present a sound analytical and interdisciplinary approach. The article is very enjoyable to read and is mostly well written. I have a few comments aimed at improving the clarity of the manuscript, providing more detail on the methodology that should allow someone to replicate the study, and some comments on the interpretation of the results and sentences that sound too speculative to me. If correctly addressed, in my opinion, the manuscript is suitable for publication in PeerJ.

The Abstract gives the impression that one of the main results of the paper is that Gunnera colonized the Andes or northern Andes from the south. Please re-rephrase the last sentence to be consistent with what you say in the discussion lines 409-410, colonization from the north (Central America). In the last sentence of the abstract, the authors say “composite origin”, does this refer to a northern and southern colonization of the Andes? I would argue that this is not an example of an Andean colonization from the south.
This is my main interpretation comment. From your figure 2, I see Andean colonization from the north (Central America) not the south (node IV and inner nodes). I provide a further explanation below (Discussion, Lines 327-328).

Introduction:

Line 71. “…below and above the treeline”. Do you mean north and south of the treeline?

Lines 73-75. Sentence needs revision since it is unclear. “Unlike the numerous examples of páramo species from the southern regions of the Andes colonized the northern Andes, fewer montane forest dwellers, such as Gunnera, have successfully dispersed to the region”. From which region to which region?

Lines 100-101. “We also discuss why so few southern hemisphere montane forest dwellers have entered the northern Andes….” The sentence sounds vague. Can you provide an idea of how many is few? What is the proportion of those southern hemisphere montane forest dwellers that have colonized the northern Andes? Just to get an idea of how rare this event is.

It is unclear whether there is a previous molecular phylogeny of Gunnera and how much DNA data or information was taken from a previous study.
Experimental design: Methods:

The authors should give a percentage of taxon sampling of Gunnera in the text. Total percent taxon sampling of the genus?

Are all DNA sequences newly generated for this study? How much was obtained from Genbank? I did not find a table with this information: Taxa names, voucher specimens, Genbank numbers, collecting place, etc.

Lines 136-138. “The substitution model was GTR with a gamma prior distribution (0.05, 10) on each substitution type…” Is this information the same as the information given after the comma, which describes another gamma distribution? For all three linkage units? Not loci.

Line 148. Mean age of 90 Ma. Is this all the information needed to replicate the dating analysis? Any other settings used? Type of prior distribution?

Line 173. Was the DEC +J model selected according to the AIC? BioGeoBEARS implements several other range evolution models. An explanation of why the DEC+J model was chosen is needed.

Lines 173-178. Time slices and rates/probabilities of dispersal between biogeographic areas are not clear or enough in the description provided. Readers shall be able to replicate your analysis; in its current state, this is not possible. I suggest to add a dispersal cost matrix table.

Line 196. “climatic variables were” , same for Line 199, replace with “were”

Line 200. Please revise this sentence, English is awkward. Replace with “We chose”, “because of their biological”. It is unclear how climatic variables were eliminated after the PCA. Did the authors take axes 1, 2 and 3 and selected the environmental variable with the highest load on each? What was the cutoff value of the load? How many axes did the authors choose?
Isn’t the purpose of a PCA to use the first 2 or 3 axes as the new environmental variables instead of the original Worldclim variables?

Line 203-205. This sentence needs revision. It is unclear why this test was conducted. Perhaps start the sentence with something like this “To infer the climatic history of Gunnera….”.
It seems that the mean values of each of the 8 bioclimatic variables for each Gunnera species was obtained after the Maxent models were obtained? A better description is needed and a better integration with the next sentence (Lines 206-208) since it seems that it is part of the same analysis.

Line 209. Unclear which variables were used for the PNC analysis. I am guessing the 8 bioclimatic variables.

Line 223 “combined with the comparison of BM and OU models…” but not with the white noise model?

Results:

Line 247-248. Which area(s) are represented by the white sections? Does it mean all areas or combination of areas with LESS than 5% probability?

Lines 273-275. Consider deleting “between temperature variables” and “between precipitation variables” in this sentence since it reads awkward. Also, the bioclimatic variable acronyms are not working because these are too long (e.g. MTCQ, PWETQ, PWARMQ). I would rather see them spelled out completely like the authors did on the last sentence of that paragraph “Minimum temperature of coldest month (Bio 6) and mean annual precipitation (Bio 12)”
Validity of the findings: Discussion:

Line 323. Another long distance event is from area E (Central America) to C (southern Andes) at node VI.

Lines 327-328. “By the Mid-Miocene the stem node of the Panke clade (15 Ma; Node IV) was  distributed in the Andes…..”
Based on your Figure 2, node 4 (and subsequent inner nodes) had a higher probability for area E (Central America). We can thus infer that Central America was the ancestral area for the Andean Gunnera species. Can the authors provide an explanation on why they chose to interpret that this node IV represented an Andean-distributed ancestor?

Lines 361-366. I like the interpretation here. It shows how important morphological studies are to correctly place a fossil in a phylogenetic tree.

Lines 382-283. “This pollen evidence suggests a lack of extinction bias and hints to PNC as an important mechanism behind evolutionary stasis.” I don’t follow the logic or conclusion of this sentence. The fact that PNC may have existed for the exine (empirically untested) does not exclude extinction bias. How can PNC of one character exclude extinction bias? And how can PNC of the exine explain the evolutionary stasis? (given that there could be several other phenotypic characters not under PNC).

Lines 419-420. I would delete this part of the sentence “as our results show more generally for Gunnera”. This last part contradicts what the authors wrote in the previous paragraph (Lines 409-410). I agree with the interpretation of the biogeographic analysis that colonization of the Andean Gunnera species came from the north (Central America).

Lines 424-427. The last sentence in the Discussion sounds weak. “there are several taxa that are similar to Gunnera” Can the author name some? Isn’t the goal of this last paragraph to provide examples of lineages that colonized the Andes or northern Andes from the South? Ceroxylon colonized the northern Andes from the south. This is a different pattern than the one shown here for Gunnera.

Conclusions

Line 435. Delete “morphological” since no test of PNC was conducted on morphology.

Line 437-438. This sentence sounds too speculative. “best explained by the massive increase in the area of suitable habitats and opportunities for allopatric speciation…” Since there is no test presented to address allopatric speciation, I would replace with “could be explained” and delete “ and opportunities for allopatric speciation”.
Additional comments: Figure 2: What does the white part of the pie charts mean? What is that number 165 Ma in node I? An explanation should appear in the figure caption to allow the figure to stand-alone.

Figure 3: Add genus name (Gunnera) to each taxa in the phylogeny and italicize names throughout.

Supplementary figure S2 and Table S1 could go in the main text, not as supplementary. Since this is an open access journal, I don’t think there is a space limitation, is there? This figure and table disclose important results of the paper.

A few typos:
Line 67 “into”
In reference 15: “evolution”, “fire”. 	In reference 75: “Trachycarpeae”


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: EVOLUTIONARY PERSISTENCE IN GUNNERA AND THE CONTRIBUTION OF SOUTHERN PLANT GROUPS TO THE TROPICAL ANDES BIODIVERSITY HOTSPOT
Review round: 1
Reviewer: 3

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

Line 31: Note that the use of “hottest” is subjective. Please consider revising and use “…is the richest and most diverse--”. Please consider “ancestral area reconstruction (lines 89–90). In general, the text is clear, unambiguous, and professional English language is used throughout.

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

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

Figures are relevant, high quality, well labelled & described. Perhaps a better photograph of a Gunnera species is desirable; the one included is low quality and blurred.

Raw data supplied (see PeerJ policy). 26]. All new sequences generated in this study have been deposited in GenBank (Appendix 1). Though occurrence records should be publicly available, perhaps at Dryad. The records are illustrated in Fig. 1, but the resolution at the scale used is not very helpful.
Experimental design: Original primary research within Scope of the journal.
Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap.
Rigorous investigation performed to a high technical & ethical standard.
Methods described with sufficient detail & information to replicate.

Lines 81–84. According to the authors, Gunnera had a wide geographic distribution during the Cretaceous, as demonstrated by numerous fossil pollen records from the Antarctic Peninsula, Australia, the Kerguelen Plateau, as well as in both North and South America according to Jarzen’s (1980) data of Gunnera pollen occurrence in the fossil record (lines 81–84). The oldest of these dates to the Turonian (ca. 90 Ma) of Peru [22] and ten million years later Gunnera became widespread across all landmasses that previously formed Gondwana (Jarzen 1980). However, they only used one date for tree calibration. Please explain why the other more recent dates of pollen fossil records shown in Fig. 1 are not useful for calibration.

Lines 144–151. Also, the use of pollen records may be problematic for divergence time estimation (lines 144–151). The shortcomings of fossil pollen use in divergence-dating analysis must be acknowledged. Fossil pollen often has very high representation, especially for wind-pollinated species, and therefore, pollen produced by members of a given clade can appear in the fossil record relatively soon after the origin of that clade. However, fossil pollen is typically identified with relatively low taxonomic resolution, a problem exacerbated by the fact that the identification of fossil pollen types is rarely supported by synapomorphies and is often based solely on gross similarity. As a result, extinct plesiomorphic types can be confused with extant groups. This problem can be alleviated when fossil pollen data are accompanied with macrofossils or mesofossils (i.e., leaves, flowers and fruits). Please indicate whether or not these concerns apply to your study.

Lines 144–151. An alternative for tree calibration is to increase taxon sampling as to include secondary calibrations from tree-calibrated phylogenies that used many fossil calibrations. For example, Magallón et al. (2015) reports a split between Gunneraceae and Myrothamnaceae dated at 104.6 MA and the split of Gunnerales at 129 Ma.

Lines 108–110. It is unclear whether you have used outgroups for estimation of phylogenetic relationships and divergence timing. Please clarify this and indicate the taxa that were used as outgroups of Gunnera.

Lines 166–178. Please consider comparing your results of the dispersal–extinction-cladogenesis model (J-DEC) for a likelihood framework with those using a Bayesian Binary MCMC analysis (BBM). The BBM analysis takes into account the inherent uncertainty of the phylogenetic inference by optimizing ancestral areas over multiple trees (Nylander et al., 2008).

Lines 181–190. From my reading, it is not clear whether you have estimated both speciation and extinction rates for phylogenies simulated under constant-rate birth-death process in BAMM. BAMM (Bayesian Analysis of Macro-evolutionary Mixtures) has become widely used for estimating diversification rates and rate shifts. At the same time, several papers have concluded that estimates of net diversification rates from the method-of-moments (MS) estimators are inaccurate. Meyer & Wiens (2017) have recently compared the ability of these two methods to accurately estimate clade diversification rates. They uses simulations to compare their performance, and found that BAMM yielded relatively weak relationships between true and estimated diversification rates. This occurred because BAMM underestimated the number of rates shifts across each tree, and assigned high rates to small clades with low rates. Errors in both speciation and extinction rates contributed to these errors, showing that using BAMM to estimate only speciation rates is also problematic. In contrast, the MS estimators (particularly using stem group ages), yielded stronger relationships between true and estimated diversification rates, by roughly twofold. Furthermore, the MS approach remained relatively accurate when diversification rates were heterogeneous within clades, despite the widespread assumption that it requires constant rates within clades. Overall, they caution that BAMM may be problematic for estimating diversification rates and rate shifts. Please discuss some of the caveats discussed by Meyer and Wiens (2017) Evolution DOI: 10.1111/evo.13378.
Validity of the findings: Their conclusions are well stated, linked to original research question and limited to supporting results. However, there are some important methodological issues indicated in the Experimental design section that must be corrected or clarified before this manuscript is recommended for publication in PeerJ.
Additional comments: I really enjoyed the reading of this manuscript. I have no major concerns regarding the quality of the study.


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

Input:
Title: EVOLUTIONARY PERSISTENCE IN GUNNERA AND THE CONTRIBUTION OF SOUTHERN PLANT GROUPS TO THE TROPICAL ANDES BIODIVERSITY HOTSPOT
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This is my second time reviewing this manuscript and again found a very well executed analysis.
As for my original review, my concerns were dealt with appropriately in the new version of the manuscript and thus I have no further comments. The methods are clearer and better justified and I found a much more cohesive argument in the results and discussion.
The present manuscript is very interesting and addresses a relevant question for the understanding of biodiversity in the Neotropics. Thus I am happy to recommend its 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: EVOLUTIONARY PERSISTENCE IN GUNNERA AND THE CONTRIBUTION OF SOUTHERN PLANT GROUPS TO THE TROPICAL ANDES BIODIVERSITY HOTSPOT
Review round: 2
Reviewer: 2

Output:
Basic reporting: I am satisfied with the way the authors have addressed my comments.
I have no further suggestions and think the manuscript is ready for publication.
Experimental design: All good
Validity of the findings: All good
Additional comments: All good.


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 RIGHT THALAMUS MAY PLAY AN IMPORTANT ROLE IN ANESTHESIA-AWAKENING REGULATION IN FROGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The Right Thalamus may Play an Important Role in Anesthesia-Awakening Regulation in Frogs

The present manuscript reports a well-designed simple and clear experiment. Authors aimed to investigate the neural correlates underlying the state of General Anesthesia in an amphibian species and to test whether its regulation is lateralized in the thalamus. Authors devised a single experiment in which the complexity of brain activity (estimated using approximate entropy) was recorded in fourteen frogs (Xenopus laevis), implanted with six EEG recording electrodes (three per each hemisphere positioned in: telencephalon, diencephalon and mesencephalon) during four different stages (before, during and after administration of anesthesia). Authors report main effects of stage and sex for the level of approximate entropy. Indeed higher level of complexity were observed before and after recovery from anesthesia and those levels were significantly higher than during the recovery stage. Moreover, they showed that overall females exhibited significantly higher levels of estimated approximate entropy than males. The level of complexity measured before anesthesia positively correlated with the time anesthesia required being effective only in the right hemisphere. The level of complexity measured in the right hemisphere during the administration stage negatively correlated with the recovery time. Eventually, a negative correlation was found between the complexity measured in the right diencephalon only and the recovery time. From these results, authors conclude that thalamus (part of diencephalon) seems to have a distinctive asymmetrical role in anesthesia-awakening regulation in frogs, suggesting an old phylogenetic origin in the regulation of arousal and consciousness.
MAJOR COMMENTS:
1.	In the Introduction, authors state that non-linear behaviors exhibited from neurons can only be adequately described using non-linear parameters (starting from line 74), “Among these methods” (line 76-77) they chose approximate entropy to estimate complexity of the EEG signals. It would be extremely useful to provide the reader with more information about other nonlinear methods used for estimating the complexity of EEG signal and particularly why among all the others approximate entropy was chosen. What are the characteristics of this method that made authors chose it instead of choosing others? What are the advantages of using approximate entropy in this work instead of using other nonlinear parameters?
2.	In line with previous point, in section Materials and Methods, authors should stress the advantages of using approximate entropy instead of other nonlinear methods. I would strongly recommend providing a second nonlinear statistical method and applying it to raw data in parallel to approximate entropy. I believe this could increase enormously the validity of the findings. Comparisons between results obtained with the two different methods would enhance authors’ conclusions.
3.	Results section is difficult to follow, too concise and lacks in coherence with the statistical analyses’ part. Results should be reported in order of relevance from the main effects to the minor ones. Results should be compared between significant and non-significant one. Moreover, the text in the results section should only describe what was observed and not explain it that pertains to the Discussion section. I highly recommend re-writing it completely.
4.	Lines 284-292 in the Discussion section look odd and it is difficult to follow, the reason why habenular nuclei were discussed is put at the end of the paragraph and the reader risks to lose the point. I strongly suggest rephrasing it.
5.	In the Discussion section within the “Sex differences in GA” paragraph (starting from line 348), authors discuss the differences found between sex from a comparative perspective with humans. Indeed, sexual differences exist in the effectiveness of anesthesia between males and females in humans and the same differences are found in frogs. Authors argue that sex hormones may play a role in the sex differences found in frogs as well as they play it in humans. “The results of the present study indicate these phenomena include amphibians as well.” (lines 367-368). I would highly recommend the authors to stress that this a speculative conclusion. Moreover, the raw data provide a more parsimonious explanation of these sex differences in the susceptibility to anesthesia (higher doses needed for female and higher recovery time for males), by looking at the weight of subjects it is clear that females on average weight twice a male. This information suggest that difference in the dosage and in the recovery time may be dependent on subjects’ weight rather than on their gender.
6.	I would avoid adding a section Conclusion if it is so short, I would suggest instead adding it as a summary part of the discussion.

MINOR COMMENTS:

a.	Lines 43-44 “Previous studies have indicated that specific brain regions are more sensitive to anesthesia..” please provide some examples of the brain regions you refer to.
b.	i.e. line 52 use of the verb “poise” please re-consider the word choice.
c.	i.e. line 58 paper cited “Salva et al., 2012” the citation is wrong, correct citation is “Rosa-Salva et al., 2012” please change it throughout the text.
d.	Line 62 I think a type error occurred and a comma was placed instead of a full stop.
e.	Line 84 I believe a reference is missing to support the homologies between the general pattern of connectivity between amniotes and amphibian diencephalon.
f.	Line 88 after the full stop I believe a new paragraph should start addressing the choice of anesthesia.
g.	Section Material and Methods “Statistical analyses” lines 220-222 the selected levels of significance are stated “A significance level of p < 0.05 was used for all comparisons; p values > 0.05 and < 0.1 were considered as marginally significant (Altman et al., 1983; Utts & Heckard, 2005)”. Whereas in the Results section and in the tables different levels of significance are used, I strongly recommend being consistent throughout text.
h.	In the Results section at the beginning, I would suggest authors to point out that from line 225 to line 231 is a descriptive level of analysis.
i.	In the section Results values of significant post-hoc tests will be easier to follow if inserted in the main text body where they are presented and not only in the tables, which delays reader understanding.
j.	Figure 3 is not easy to read and I believe is not providing new information to the reader. I strongly suggest removing it, or to change it completely.
k.	In Figure 3 for instance, different recording sites are labelled as LT, RT, LD…using the letters of the corresponding brain subdivision. Whereas, in the Materials and Methods the same sites are labeled R1, R2, R3.. To enhance the readability I suggest keeping abbreviations consistent throughout the text.
l.	I strongly recommend in Figure 4 to add to the picture and not only to the caption to which brain subdivision the plots refer (i.e. Right Telencephalon). Moreover, I suggest to put axis labels for each plot and to write to which stage it refers. I.e., first plot on the upper left side: title of x-axis could be “time stage II” and y-axis “ApEn Stage I”. Figure caption should be complete. Eventually, authors should really re-consider whether the picture is really informative to the reader and helps the understanding or not.
m.	In the Discussion section lines 311-313 the reference is referring to humans whereas the sentence is speaking about frogs and amphibians, I believe “as it was described for humans” is missing.
n.	Line 360 type error “for instence”.
Experimental design: no comment look above
Validity of the findings: no comment look above
Additional comments: no comment look above


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

Input:
Title: THE RIGHT THALAMUS MAY PLAY AN IMPORTANT ROLE IN ANESTHESIA-AWAKENING REGULATION IN FROGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: is very simple
Validity of the findings: Brain region specific findings are not convenient, the differences between stages and a potential role of sex might be fine, but the analysis needs to be worked out and summarised better.
Additional comments: Review of the manuscript

 “The right thalamus may play an important role in anesthesia-awakening regulation in frogs (#22293)”

submitted for a publication in PeerJ.

The presented study explores the brain activities in a common laboratory frog species (Xenopus laevis) during different stages of anaesthesia and awakening measured based on EEG recordings from implanted electrodes in different brain areas. The EEG data was analyzed by calculating approximate entropy (ApEn) values, which reflect the complexity and regularity of brain activity. The main finding of the study is, that brain activity regularity and complexity is reduced during a general anaesthesia and is increasing during awaking stages. Moreover, some trends were indicated which reflect brain region specific and sex dependent variations. E.g. some correlations are reported to be significant in the right thalamus between the duration and ApEn for some stages as well as brain activities in females are higher than in males. The importance of thalamus for regulation of consciousness are discussed by considering also the functional lateralisation of the brain and the effect of sex differences.

After a brief reading, as such these results, would be suitable for a publication in PeerJ (given the philosophy of the Journal as far as I remember). However, in my opinion the authors should significantly revise and overwork the manuscript. Although the experiment was very simple, the manuscript in its present form is very difficult to follow, in particular the results section is not very clear, contains unnecessary complexity and at the same time is missing important information’s, which overall makes it very difficult to understand the findings. The authors should also carefully revise the introduction/discussion and check the validity of their statements and corresponding references (E.g. line 315 the authors cite a study on caimans (Pritz 2016) by considering these crocodile species as amphibians). Also, the authors should be more careful with over claims by making interpretations of the overall very weak results. As far as I can see apart from the activity reduction of the brain activity during anaesthesia, the potential effect of factors such as region specificity, sex and lateralisation is very week in the given data. The manuscript should be more focused e.g if the study is intended to validate the ApEn approach, the manuscript should be written as a methodological paper. If it is about lateralisation or the specific functions of the thalamus during anaesthesia, more data need to be collected. However, it is of course possible to report the findings as a it is and to speculate on the potential region-specific functions in the discussion, but the results need to be presented very clear, the main findings should be visible and it must be made clear when it is a speculation about additional potential findings.

Very Important: 5% error probability is a widely-accepted convention and already a very “soft” measurement of significance. I would strongly recommend the authors not to consider any values with p>0.05 as significant!  However, it would be acceptable if the values closely  p>0.05<0.1 would be discussed as marginally non-significant trends which need to be confirmed in future studies but these trends should not be the core of the main results for a scientific publication and should not be put in the same categories as other significant results.

Other more specific comments are here below:

-	Line 39: To many shortenings reduce readability. I would recommend not to shorten ‘general anaesthesia’ into ‘GA’ and instead to write the whole wording in the whole manuscript.
-	Line 71: EEG is not the most direct approach, please rephrase e.g. instead of ‘is the most direct approach’ write ‘allows’
-	Line 117: instead of ‘…water bath using MS-222 solution (3.5g/L, 500ml)…’ would be better ‘‘…water bath using 500ml of MS-222 solution (3.5g/L)…’
-	Line 133, specify how deep the electrodes were implanted
-	Line 221, see comment above on the significance of results.
-	Line 232, Table 1 should be a figure and contain the results for all four stages (negative results should be reported)
-	Line 234-239 and Table 2: Interaction of brain area*stage*sex need to be reported.  if there was no such significant interaction, it is at least statistically not allowed to split the data into stages, areas and sex for any post hoc analysis. In such a case the authors should try a simpler approach by removing one of the factors e.g. stage*sex might be potentially promising.
-	Line 240-253: these are a lot of correlations, of which as far as I understand only a part of all possible comparisons is reported and even here with only few significant values. If it is true, that the correlations (negative or positive) are more often occurring in the right hemisphere, than the authors should think about how to summarise these results into a simpler and more convenient analysis. In any case the authors should be very careful about the interpretation and discussion of these correlations by making conclusion about the role of specific brain regions.
-	Line 244:  ‘ApEn values in Stage I were positively correlated with the duration of Stage II ‘. The authors should explain better the potential meaning of such correlations of activities between different stages in the discussion.
-	Line 274: As it is stated here ‘…ApEn values in the right thalamus and telencephalon were positively correlated with the duration of Stage II…’, however the discussion is focusing on the specific role of the thalamus, which in my opinion is not so clear based on the data. The authors should be more careful with over claims and identify this part of the discussion as rather speculative.
-	Line 291-293: ‘…habenular nuclei could not be responsible….’ is a very strong claim, the explanation in the paragraph line 294-293 does not provide enough evidences for this conclusion.  The authors should be more careful with such short cuts in the discussion.
-	Line 315: caimans are not amphibians. See also the comment above.
-	Figure 3: The y axis should contain the sex e.g. in A: ApEn (males) and B: ApEn (females). Also, please remove any ‘a-d’ form the graphs, here it is very difficult to understand what was compared with what. Instead please make a line above the compared bars with one or two asterisks for significant results. Do not specify non-significant trends here. Overall, it seems that all results among different brain regions are very similar and I am wondering if it is meaningful to divide into brain region specific plots here.
-	Figure 4: As already mentioned, I am not sure if it is useful to split the analysis into different brain regions, since also the correlations in the measured different brain areas are very similar.
-	Table 2: please report also the interactions and if significant also the detailed post hoc results