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: NETWORK MODELS OF DRIVER BEHAVIOR
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author made thorough revisions on the introduction, especially regarding the following three topics: Difference between measurement model and structural model, difference between general latent variable model and one-factor latent variable model and added a well-described paragraph about ontology and the consequences of a realist interpretation of factor models. With this revision, my former concerns regarding these topics have been met.
Experimental design: My concerns regarding the discussion by the author of between-subjects networks and cross-sectional networks have been met by the author, by clarifying that the data are longitudinal in nature and discuss how the networks are related.
Validity of the findings: The author replied with good arguments on why my suggestion regarding plotting the two networks in the same layout has not been incorporated, therefore I accept the choices made by the author.
Additional comments: The author replied extensively and satisfactorily to my raised concerns.


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: NETWORK MODELS OF DRIVER BEHAVIOR
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I think that the manuscript, which I found very interesting in its first version, has been greatly improved and is now suitable for publication.
I only have a (very) minor comment: L 52-57. How is the possibility that “individual violations and errors may be causally related” a reason for which the latent variable perspective “has gone unchallenged”?


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: CHECKLIST TO THE ELATOSTEMA (URTICACEAE) OF VIETNAM INCLUDING 19 NEW RECORDS, TEN NEW COMBINATIONS, TWO NEW NAMES AND FOUR NEW SYNONYMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Google translation of the attached pdf [done by PeerJ staff]:

"The author of this article has done a checklist of the genus Streptomyces in Vietnam through field surveys, specimen inspections (inspection of specimen museums, and related botanical sites).
There is a large space in the article about the contents of the flora.
Since I (reviewer) is not good at flora, it is not convenient to comment in these aspects, but only comments or suggestions on the plant taxonomy in the article.

1. In this paper, the author proposes 10 new combinations, 1 new name, and 3 new synonyms, but none of the qualified authors and sources, including page numbers and illustrations, that are complete and directly reference their base name or replaced synonyms. And date.
Therefore, the names given by the authors of this article are all unqualified publications.

2. The authors combined the names of the red genus to the stairweed, but barely presented their own experimental data or evidence.
It is recommended that the authors come up with credible data to prove that the merger of the two genus is more reasonable. Otherwise, it is better to keep the two genus.

3. The new synonym proposed by the author does not indicate the basis.
This is unfounded and cannot be convinced.

4. This article lists the voucher specimens under each name, which is beneficial to others' inspection; this article also attaches photos of wild plants to some types, increasing the credibility.
But it would be better if you attach a photograph of a wild plant or a photo of a voucher to each new record.

5. Can you increase the content of taxonomy (including experiments, this article is changed to two articles: one is the taxonomic revision of the genus Streptomyces in Vietnam, and the other is the floristic analysis of the genus Streptomyces in Vietnam.

The above comments or suggestions are for the author's consideration."


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: CHECKLIST TO THE ELATOSTEMA (URTICACEAE) OF VIETNAM INCLUDING 19 NEW RECORDS, TEN NEW COMBINATIONS, TWO NEW NAMES AND FOUR NEW SYNONYMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This is a very well written manuscript, excellent in language and consistency. I marked a couple of minor comments in the attached file that I would like you to consider. Here I would like to mention few major points:
- the format of citation of plant names seems to follow the rules for zoological nomenclature, but not for botanical names. Author names must follow Brummitt/IPNI standard forms, not in brackets, and the year omitted;
- new combinations are only valid if the complete protologue reference for the basionym is cited on the same page;
- the authors complain that full access to the JSTOR Plants website is not available for free. But they do not mention if they ever used other websites that allow free access , sometimes even to the same images, and some in comparable quality (mentioned in my annotations).


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: FOREST FLOOR TEMPERATURE AND GREENNESS LINK SIGNIFICANTLY TO CANOPY ATTRIBUTES IN SOUTH AFRICA’S FRAGMENTED COASTAL FORESTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Lines 136-: what species occurred in these plots? This information is entirely missing.

The raw data are supplied and, while I did not attempt to run the R code, generally look good. You do need to specify the version of R (and all packages) used to run this code, citing R correctly (see “citation()”).

Line 168: “leaf area index” and “plant area index” are not the same thing; clarify.
Line 208-: this is confusing, as the introduction (esp. 102-106) hasn’t prepared us much for this upscaling, and many details are unclear.
L. 272: “are not independent of each other”, i.e. “are correlated”?
L. 275: “Plots whose”?
L. 294-295: right. And this needs to be emphasized more, I think; it doesn’t take anything anyway from this study to note that almost everything here is predictable from such first principles
L. 313-324: interesting and well done
Figure 3: on the one hand, thanks for including this figure, as it’s confusing how sample size varies in this study. On the other, the experiment unit is the plot (yes?) not individual camera sample points. Be careful to use a common unit (i.e. the experimental unit)
Figure 4: how were these models chosen?
Figure 5: need to report p-values of these models in caption
Table 1: nicely done
Experimental design: The “two main questions” in lines 102- are not well defined. First, “interlinked” is vague. Second, “Can we statistically link…” is a poor framing that might open the authors to charges of ‘fishing’ for relationships they want to find a priori. Both of these should be framed as falsifiable hypotheses.

How were the plots (line 122) sites? Randomly? From remote sensing images? Clarify.
Validity of the findings: Line 203: good job on using Bonferroni adjustments

Line 130: this isn’t sufficient, as temperature variations in this 4+ hour window could seriously affect the results. What was the air temperature distribution during your measurements (e.g. from a nearby met station, if available)? How much temperature change occurred in this window, and what percentage of observations fell outside this window? How do you reconcile this with lines 275-278?
Additional comments: This study looks at the relationships between forest floor temperature, canopy greenness, and canopy leaf area across a number of field transects in coastal forests in South Africa. This is a potentially interesting subject, for reasons well laid out in the introduction. The ms is generally well written although there are many minor typographical and grammar errors the cumulatively make for distracted reading.

I’m not used to the PeerJ review format, but in general: there are many points of interest here, but there are many unclear points about the methods; the research questions are poorly (and potentially dangerously) defined; the study is actually quite limited and conclusions extremely tentative. See specific comments above and below.

1.	Line 27 and throughout: “Random Forest” (capitalized) is the name of the algorithm
2.	L. 81-82: but lines 71-72 say vegetation productivity show inconclusive relationships; does this conflict with text here?
3.	L. 98: define NDVI
4.	L. 110: repeated “and April”
5.	L. 124: define MAPIR
6.	L. 354-: “Our data seem to suggest that…”  I agree with this careful language, given the limitations of the study design; this should be emphasized more in the abstract


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: FOREST FLOOR TEMPERATURE AND GREENNESS LINK SIGNIFICANTLY TO CANOPY ATTRIBUTES IN SOUTH AFRICA’S FRAGMENTED COASTAL FORESTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: *Please see separate doc for specific comments*

The language is generally clear and professional - a few confusing sentences but nothing major. References are occasionally missing or incorrect. Figures require more explanation in their legends, and some of the colour choices make them difficult to interpret. Raw data are available (metadata describing the columns and their units would be useful).
Experimental design: *Please see separate doc for specific comments*

Description of the different sites sampled was very confusing, with various different terms used and not all of them appearing in figures. Perhaps thinking about why these sites were chosen to answer the questions of this study would help the authors to settle on clearer terminology.

It's difficult to assess whether the statistical models were appropriate as they are not described in much detail within the ms. The fitted lines in figures seem to be a good overall fit, but were produced using ggplot rather than the models directly.
Validity of the findings: No comment.
Additional comments: This study addresses an important issue in ecology: how we can exploit remotely sensed data to map habitat characteristics that matter to species, at biologically-relevant spatial scales. While there is clearly more work to be done in upscaling these models, the findings presented here will certainly help to guide future research and on this basis I think it is an important piece of work. My main criticisms are in the description of what was done, rather than the study itself.


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: FOREST FLOOR TEMPERATURE AND GREENNESS LINK SIGNIFICANTLY TO CANOPY ATTRIBUTES IN SOUTH AFRICA’S FRAGMENTED COASTAL FORESTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: See below.
Experimental design: See below.
Validity of the findings: See below.
Additional comments: As before, this study looks at the relationships between forest floor temperature, canopy greenness, and canopy leaf area across a number of field transects in coastal forests in South Africa.

The authors have generally responded well to all my previous concerns, clarifying the methods and results in many spots and notably improving the introduction. As a result there is greater reproducibility and transparency, and the text is clearer and stronger throughout. Nice job! I have no further major concerns.

One potential issue: note that the “dat_all_PeerJ.csv” file doesn’t load correctly in many standard CSV readers on my system (Excel, etc). A programming-oriented text editor opened it without problem, but you may want to check the file encoding used.


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: FOREST FLOOR TEMPERATURE AND GREENNESS LINK SIGNIFICANTLY TO CANOPY ATTRIBUTES IN SOUTH AFRICA’S FRAGMENTED COASTAL FORESTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Reads much better now. References have been added and corrected. Clear metadata included with the raw data. Unfortunately I wasn't able to find the revised figure legends; I hope that the authors addressed my concerns there as robustly as they have addressed my other comments. A few minor things below.

Line 18 – ‘above-ground’ shouldn’t have any spaces
Line 29 – repeat of word ‘that’
Line 36 – ‘field-derived’ shouldn’t have any spaces
Line 40 – 'requires that we map' not 'requires to map'
Line 76 – I’d delete the ‘And’ at the beginning of the sentence
Line 104 – great to see the Blonder et al. ref here, I was about to suggest it
Line 325 – should read ‘underestimated’ and ‘overestimated’ (change tense)
Line 327 – ‘whose’ instead of ‘those’
Line 343 – would probably get rid of ‘whilst’
Lines 397-422 – this is a very long para now. Maybe list the four suggestions as a bulleted list?
Experimental design: In the revised version I can see much more clearly what the authors did, and I am satisfied that their experimental design and analyses were robust and appropriate. Hypotheses are much clearer and research questions no longer 'dangerously' worded.

Line 162-163 – following up on comment 14 of reviewer 1, the authors have now specified that "approximate locations of transects were chosen prior to fieldwork based on our knowledge of the landscape and ease of accessibility". I’m curious how they were chosen exactly, within these approximate locations? Randomly?

Line 170 – why does T11 comprise five plots when there are only four habitat types (natural forest – edge - natural grassland – bush habitats)?
Validity of the findings: As before, I think the study meets a need in the literature. The authors are clear in outlining this as a pilot study, so while in some ways it is limited (as noted by reviewer 1), the authors acknowledge this and make several useful suggestions for further work.
Additional comments: Great job on revising this ms - it is a significant improvement. It's now a lot easier to read and follow what was done. The context and implications are also more clearly described in the Intro & Discussion, and the Figures look good too. Well done.

(Please note - the line numbers I quote above are referring to the document with track changes.)


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: MICRORNA-705 REGULATES THE DIFFERENTIATION OF MOUSE MANDIBLE BONE MARROW MESENCHYMAL STEM CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Introduction
  According to Liao et al. 2013, besides miR-705, miR-3077-5p was shown to function as a negative regulatory factor of osteogenic differentiation of BMMSCs derived from long bones of hind limbs. Therefore, the authors should show why they focused on miR-705 not miR-3077-5p in introduction part.

Results
1.	In the paragraph from line 185, there were no explanation about Fig. 3c that shows the adipogenic differentiation of mandibular BMMSCs. They should be added in the text. Also, in Fig. 3b and 3c, the results of statistical analysis should be shown.
2.	Although the sentences in the line 199-200 and 210-212 showed that the authors analyzed the effect of miR-705 mimic, the results of mimic transfection were not shown in Supplementary Fig. 1a and 1b. They should be added.

Representations and grammars
1.	Including the title, there are a lot of phrases “mandible bone marrow” in the text. They should be altered to “mandibular bone marrow”.
2.	Throughout the text, en spaces should be added ahead of the brackets to show abbreviations or references.
3.	The abbreviations such as “BMMSCs”, “miRNA”, “BMD” and “BV/TV” were restored to their full-spelling in the later part. Once the abbreviations were shown, they should be continuingly used. On the other hand, I think the line 58 “BRONJ” need not to be shown because it was not used subsequently.
4.	The phrases “in vitro” and “ex vivo” should be represented in italic letter.
5.	Line 35 and 68 “mark genes” would be “marker genes”.
6.	Line 39 “osteogenic” would be “osteoporotic”.
7.	Line 49 “Despise” would be “Despite”.
8.	Line 60 “~in alveolar bones~” would be “~alveolar bones~”.
9.	Line 79 “regulation” would be “regulatory”.
10.	Line 91 “~anesthesia._After~” would be “~anesthesia. After~”.
11.	Line 107 “0.25% trypsin/1m MEDTA” would be “0.25% trypsin/ 1mM EDTA”.
12.	Please start a new line ahead of line 123 “Real-time RT-PCR of mRNA and microRNA.”.
13.	Please change the font color in the line 129 - 131.
14.	Line 134 “Supplementary Table 1” should not be in italic letter.
15.	Line 160 “adipocyteduring” would be “adipocyte during”.
16.	Line 182 and 201 “---” should be changed to commas.
17.	Line 183 “lipoprteinlipase” would be “lipoprotein lipase”.
18.	Line 198 “Si-miR-705” would be “si-miR-705”.
19.	Line 201 “APL” would be “ALP”.
20.	The beginning of the line 183 “705by ~” would be “705 by ~”.
21.	Line 210 “gain or loss function” would be “gain or loss of function”.
22.	“mir705” and “mir-705” in the line 222, 224, 231, 233 and 234 should be changed to “miR-705”.
23.	Line 289 “MiR-21” should be altered to “miR-21”.
24.	Line 303 and 304 “Transfection of miR-705 into ~” would be “Transfection of miR-705 inhibitor into ~”.
Experimental design: The results shown were interesting and clear. However, in some part, they didn’t match the hypothesis shown in introduction part (line 62-63). The hypothesis was that some kind of significant roles of mandibular BMMSCs cause the different responses to bone loss stimuli between mandibular and long bones. However, the results shown were not enough to demonstrate what made the differences. To clarify the cause of difference, the responses of mandibular BMMSCs to miR-3077-5p should be examined following the study by Liao et al. 2013. The target mRNA of miR-705 in mandibular BMMSCs should also be analyzed. Such kind of analyses enable the authors to compare with the results in long bones and provide profound information to find the causes of difference.
  Considering clinical applications, it is unlikely that miR-705 mimic that might degenerate bone loss would be applied to osteoporosis. However, for comprehensive analysis of the role of miR-705 in bone loss conditions, the effects of miR-705 mimic on the osteogenesis and agipogenesis of mandibular BMMSCs in OVX mouse should be examined.
Validity of the findings: As shown in discussion part (line 276-281), there were different mandibular responses to OVX between in vivo and in vitro. However, I thought that the authors didn't show the consideration of the causes of this difference. Please consider the causes of the difference and add the authors’ interpretation in discussion part.
Additional comments: The authors showed the significant role of miR-705 in lineage commitment of mandibular BMMSCs. I thought that the results shown would bring a lot of important information for the clinical strategies of osteoporosis in the future. However, there were some points to be corrected or added in the manuscript.


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

Input:
Title: MICRORNA-705 REGULATES THE DIFFERENTIATION OF MOUSE MANDIBLE BONE MARROW MESENCHYMAL STEM CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English grammer and proper scientific term (miR/Mir) need to be edited.
The authors owned three out of four manuscript on miR-705 in Pubmed. So,literature and references on miR-705 are very poor.
The premise or significance of this study is not clear and very week
Figures are preliminary, subjective, not mechanistic and very similar kind of experiments.
The quantitative expression of miR-705 is required.How much do they really expressed and how they regulate the differentiation.
Experimental design: Not novel or unique
Validity of the findings: Iam not sure.
Satining and real time PCR are routine analysis.However they are not confirmatory. Western analysis needed.
Additional comments: Fundamentally, the studies mentioned in this manuscript are lacking of miRNA mechanism that regulate the whole process of differentiation.MicroRNAs are post transcriptional repressor through their targets. The authors did not show any of such experiments to prove.Therefore, the studies are very priliminary. More meaningful experiments are needed to show  the direct role of miR-705 in the reugulation mandible mesenchymal cell differentiation.


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: MICRORNA-705 REGULATES THE DIFFERENTIATION OF MOUSE MANDIBLE BONE MARROW MESENCHYMAL STEM CELLS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Representations and grammars
	As I pointed out in the first review, en spaces should be added ahead of the brackets to show abbreviations or references. Also, just after a bracket and a period such as line 79 and 116 respectively, en spaces should be inserted. Thus, lots of mistakes of the use of punctuation are found throughout the text. I highly recommend the authors to get English proofreading.
	Line 21 and 64 “(maxillary)” would be “or maxillary” or “and maxillary”.
	The underlines drawn in the line 264 and 265 should be deleted.
	Line 281 “haemostasis” would be “homeostasis”.
	The phrases “in vivo” in the line 316 should be represented in italic letter.

Results
  I found the authors have added the explanation of Fig. 3c in the revised text. However, the results of statistical analysis are not still shown in revised Fig. 3b and 3c. The expression levels of miR-705 in each time point seem to be significantly different. If they are not significantly different, they should be shown as such in figures and their legends.
Experimental design: No comment.
Validity of the findings: What is the hypothesis of this study? The hypothesis shown in the line 63-65 and those in the line 334-336 and 342-343 are similar, however, they are not the same. According to the hypothesis shown in the line 63-65, the authors think that some kind of significant roles of mandibular BMMSCs cause the different responses to bone loss stimuli between mandibular and long bones. On the other hand, the experiments conducted in this study are for the analysis of the roles of miR-705 in mandibular BMMSCs, not to analyze the roles of mandibular BMMSCs to cause the different responses between mandibular and long bones. The analysis performed in this study rather matches the hypothesis shown in the line 334-336 and 342-343. Therefore, although it is undesirable, the hypothesis shown in introduction part should be arranged to match conducted analyses.
Additional comments: I thank the authors for correcting their manuscript. Especially, in the discussion part, the authors added lots of information from other related studies, and the authors showed their considerations for additional experiment and our suggestions. Also, I understood the authors’ situations concerning the analysis of miR-3077-5p. I look forward to see the findings in the future. However, I found some points to be corrected to improve the authors’ 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: MICRORNA-705 REGULATES THE DIFFERENTIATION OF MOUSE MANDIBLE BONE MARROW MESENCHYMAL STEM CELLS
Review round: 3
Reviewer: 1

Output:
Basic reporting: As I pointed out many times over, there are still lots of adjusting points related to punctuations and use of abbreviations. For instance, in the introduction and discussion parts, absence of en spaces are recognized a lot ahead of the brackets. In the line 93-94, “OVX” is shown as a new abbreviation though it has already represented above in the same sentence.
  I assume that there are similar adjusting points concerning representations more. I recommend the authors to get English proofreading again.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No comment.


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

Input:
Title: MICRORNA-705 REGULATES THE DIFFERENTIATION OF MOUSE MANDIBLE BONE MARROW MESENCHYMAL STEM CELLS
Review round: 4
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment
Additional comments: The adjusting points that we pointed out previously were corrected properly in the revised version. As a result, I think the overall quality of the authors’ work has been remarkably improved. I would commit the final decision to the editor.


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: INFLUENCE OF SUBSTRATE TYPES AND MORPHOLOGICAL TRAITS ON MOVEMENT BEHAVIOR IN A TOAD AND NEWT SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Introduction – Overall the introduction did a good job of presenting the context for the study – changing landscapes may present new challenges for dispersing/migrating wildlife and the magnitude of impact will depend of specific species requirements. However, I feel that some of the main points were not as clearly described as they could be and with some revision the overall flow and clarity could be improved. Finally, because the experiment compared cement to soil, I think the introduction could benefit from a more narrow focus on the impacts of urbanization and roads on wildlife and movement. There are many studies on movement behavior, physiological responses, and geneflow in response to roads. I also have some specific suggests – see below.

Figures – Figure 1 does not match the description in the text.

Raw Data – Is provided and seems adequate

There are many small English grammar specifics that should be edited. I suggest a thorough reread. For example, from the first paragraph:
Line 52: “Since many decades” should be “For decades”
Line 53: “The anthropogenic practices” should be a more general “Anthropogenic practices”
Line 56: “associated to habitat loss” should be “associated with habitat loss”
Line 57: “ensure a sufficient” should be “ensure sufficient”
Line 58: “could be force” should be “could be forced”
Line 61: “associated to dispersal” should be “associated with dispersal”
Line 64: “pattern in the disturbed landscapes” should be a more general “pattern in disturbed landscapes”

Other specific comments on introduction and results
Abstract:
Line 22: Should be, “Traveling through heterogeneous…”
Line 35-36: I would like more detail about these results.

Introduction:
Line 57-60: This is assuming species already have plasticity or the capacity to adapt their behaviors. It may be that species are already functioning at the maximum of their physiological tolerances and/or dispersal ability. More relevant to your study is that fragmentation and land use change could create inhospitable habitat within the current dispersal range/paths of an individual, rather than them traveling farther.
Line 64: While understanding adaption is a key component of evolutionary biology; the biggest implication from this kind of study is management and conservation. This mention of evolution seems out of place within the context of this paragraph and the paper as a whole.
Line 65: I would rephrase this in the context of complex life histories and be specific for pond- and stream-breeding amphibians. Not all amphibians have aquatic larval stages.
Line 67-68: Terrestrial movements are more risky than what? Aquatic movement? But the risks are the same. Also, do you have a reference for the risk of UV-B exposure during dispersal/migration?
Line 73: One of the reference seems wrong – Youngquist and Boone (2014) looked movement behavior not species richness. Youngquist et al. (2016) looked at distribution and gene flow.
Line 99-108: This part feels more like broad introductory information, not a narrowing of focus to your specific experiment. It is also a little redundant with earlier information. I suggest working the information presented here into earlier paragraphs. I would open this paragraph with your specific objectives.
Line 109-110: When I first read this, I thought you were testing multiple substrates of each category. I recommend specifying that you are testing a single surrogate for each category.
Line 112-114: Do you have references for this information?
Line 115: I need more information about how the “specific ecological requirements” differ between these species.

Results:
Line 210: This figure does not show this.
Experimental design: Research Question – The overall research question is well defined. But I would like to see more specific aims/objectives/hypotheses in the introduction.

Methods – The experimental design is appropriate for the question and the methods are well described.

Specific Comments:
Line 124: Reference?
Line 132: Reference?
Line 149-153: Did you collect individual post-breeding? Or did you have gravid females?
Line 166-167: Were substrates dry or wet? B/c amphibians often move during rain events, how would this affect your results?
Line 167-169: How long did you wait between stops before poking? How did this influence your calculation of crossing speed?
Line 196-203: While not strictly wrong, I would recommend model comparison using AICc. I’m interested to know if you end up with different top models when using AIC vs. backward selection.
Validity of the findings: Results: The data seem robust given the experimental design and the results are valid.

Discussion – The discussion is adequate and generally falls within the scope of the study. I would like to see more discussion on the differences between sexes, within the context of this specific study and sex-biased dispersal. I would also like discussion on the limitations of this kind of laboratory study (e.g., mimicking natural environment and conditions during actual dispersal events, translating lab results to actual in-situ behavior and functional connectivity, using adults vs juveniles, etc). Another consideration is that bare dirt could be considered an anthropogenic land use, i.e. bare ground in agricultural plots. Justification for dirt as a natural ground cover is needed or at least acknowledgement that it is not only found in natural land uses. I would also like greater linkage between the introduction and discussion about roads and road ecology b/c this seems to be the focus on your experiment.

Specific Comments:
Line 223: Why does sensitivity to aquatic changes affect terrestrial dispersal?
Line 276: Were your females gravid or post-breeding? Do you have other references showing differences in dispersal ability between sexes? There are a number of papers on sex-biased dispersal, some amphibian specific (e.g., Austin et al. 2003, Molecular Ecology; Lampert et al. 2003, Molecular Ecology; Palo et al. 2004, Molecular Ecology; Smith and Green 2006, Ecography; Liebgold et al. 2011, Molecular Ecology).
Line 295-296: This is a big idea that seems thrown in. I would like a more extended discussion on the limitations of this kind of study. What evidence is there that laboratory responses correspond to actual responses in the field and functional connectivity?
Additional comments: Overall, this was a well executed study and a well written manuscript. I congratulate the authors on their efforts.


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: INFLUENCE OF SUBSTRATE TYPES AND MORPHOLOGICAL TRAITS ON MOVEMENT BEHAVIOR IN A TOAD AND NEWT SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Needs English language editing. Overall concepts can be understood, but choppy to read due to word choice and issues with articles as well as verb-noun agreement. In general, the writing in the Intro and discussion could also be tightened and better organized.

Missing or in appropriate citations in several spots, especially in the introduction. Please see my detailed comments for suggested alternatives or additions.

Article conforms to standard sections and figures are well prepared. The table is visually pleasing, but I would like to see some measure of error (SE), df, and test statistic (F, chi-squared, etc).
Experimental design: How this experiment addresses the knowledge gap could be better defined. Please see my detailed comments for suggestions.
Validity of the findings: I have some concerns about the data analysis.

1. There are lots of problems with this extent of backwards selection. Given the design of your experiment was hypothesis driven, please present the full model. Some might construe this backwards approach as p-hacking.

2.  Why were species not considered in the same model? Would only cost 5 df. Seems like that would be the strongest way of getting at your comparative questions. If your short on df’s could consider doing a PCA analysis to compress BI and leg into a single variable.

3. These animals that didn’t cross seem like they would provide valuable information, especially considering they were a sizable proportion of all animals (~25%). If you have multiple measures from these animals, at a minimum test if individuals were more likely to not cross depending on species x substrate (binomial glmm). An even better approach would be to use a glmm with the full data set, and deal with the 0’s by using a poisson / negative binomial / zero-inflated model. With this approach, crossing speed would not be transformed.

4. Likewise, modeling stops could also be interesting and add another layer to your discussion. Also, might want to consider modelling stops as a response variable to look at if stopping rates differ by any of your covariates.

In regard to the conclusions, a major challenge with this sort of study is how to interpret speed. Are animals moving faster because it is a hostile substrate or are they moving faster because the substrate offers lower resistance? This issue, and specifically the potential for ambiguity needs to be addressed at some point.
Additional comments: 37: It might be stronger to tie this back to your initial questions regarding movement through heterogeneous landscapes and metapopulation dynamics rather than touting the comparative aspect, which while present, isn’t super strong.

50: “for” instead of “on”

52: “For” instead of “since”

53: “with the development of human activities” is awkward. Rephrase.

53: “These anthropogenic”

55: Habitat

56: associated with

57: While increased connectivity is the result of increased movements, I’d argue individuals aren’t moving more to ensure connectivity. Rather, they may need to move more to find resources or mates
in suboptimal, fragmented landscapes, and a by-product of this is increased connectivity. Please rephrase.

68: This citation does not seem particularly relevant to the previous statement. Please replace. The following may be helpful:

Pittman, S. E., Osbourn, M. S., Drake, D. L., & Semlitsch, R. D. (2013). Predation of juvenile ringed salamanders (Ambystoma annulatum) during initial movement out of ponds. Herpetological Conservation and Biology, 8(3), 681-687.

Pittman, S. E., Osbourn, M. S., & Semlitsch, R. D. (2014). Movement ecology of amphibians: a missing component for understanding population declines. Biological Conservation, 169, 44-53.
Rothermel, B. B., & Luhring, T. M. (2005). Burrow availability and desiccation risk of mole salamanders (Ambystoma talpoideum) in harvested versus unharvested forest stands. Journal of Herpetology, 39(4), 619-626.

73: Regarding gene flow in fragmented landscapes,

Cosentino, B. J., R. L. Schooley, and C. A. Phillips. 2011. Spatial connectivity moderates the effect of predatory fish on salamander metapopulation dynamics. Ecosphere 2(8):art95. doi: 10.1890/ES11-00111.1

Cosentino, B. J., Schooley, R. L., & Phillips, C. A. (2011). Connectivity of agroecosystems: dispersal costs can vary among crops. Landscape ecology, 26(3), 371-379.
77-78: Please add in scientific names for these two species.

92: Also, in amphibians. This study also found no evidence of an effect of leg length in a pond breeding salamander on movement distance.

Ousterhout, B. H., & Semlitsch, R. D. (2018). Effects of conditionally expressed phenotypes and environment on amphibian dispersal in nature. Oikos.

105-107: Seems like Rothermel and Semlitsch 2002 should be cited here. Also, perhaps few studies have simultaneously considered multiple species when looking at movement speed across different substrates, but many studies have used the same methods when testing a single species. Please rephrase to make that clear and cite some of that work here

Rothermel, B. B., & Semlitsch, R. D. (2002). An experimental investigation of landscape resistance of forest versus old‐field habitats to emigrating juvenile amphibians. Conservation biology, 16(5), 1324-1332.

Stevens, V. M., Polus, E., Wesselingh, R. A., Schtickzelle, N., & Baguette, M. (2004). Quantifying functional connectivity: experimental evidence for patch-specific resistance in the Natterjack toad (Bufo calamita). Landscape ecology, 19(8), 829-842.

Semlitsch, R. D., Ecrement, S., Fuller, A., Hammer, K., Howard, J., Krager, C., ... & Walker, M. (2012). Natural and anthropogenic substrates affect movement behavior of the Southern Graycheek Salamander (Plethodon metcalfi). Canadian Journal of Zoology, 90(9), 1128-1135.

114: Please explain briefly here why you predict that differences in movement type (hopping vs walking) might affect responses to different substrates.

127: Migrate, not disperse.

149: Given that juveniles, not adults, are thought to be the primary dispersing stage class, you might want to restructure your intro a bit to introduce the concepts of straying (adults that breed in different locations in different years).

Gamble, L. R., McGarigal, K., & Compton, B. W. (2007). Fidelity and dispersal in the pond-breeding amphibian, Ambystoma opacum: implications for spatio-temporal population dynamics and conservation. Biological Conservation, 139(3-4), 247-257.

Trenham, P. C., Koenig, W. D., & Shaffer, H. B. (2001). Spatially autocorrelated demography and interpond dispersal in the salamander Ambystoma californiense. Ecology, 82(12), 3519-3530.

188: There are some issues with using residual from a linear regression, but the fix does not require much more work. See:

Peig and Green. 2010. The paradigm of body condition: a critical reappraisal of current methods based on mass and length. – Funct. Ecol. 24: 1323–1332.

194: It’s odd the animals that stopped more crossed more quickly. Is this a typo? If not, did poking have a strong effect on speed. If so, that would be a pretty big methodological problem.

208: Which of the six tests listed above does these statistics correspond to? How did you compare crossing speed between soil and cement? Add to methods or results.

227: I’d argue that to truly compare the two species, they need to be considered in the same model.

236: Please reiterate your relative crossing speed finding earlier in this paragraph to remind readers.

275-276: Maybe, but this is not something that would have been detected by your study.

295-296: I agree this is important, but not something addressed in your 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: AMELIORATION OF DROUGHT EFFECTS IN WHEAT AND CUCUMBER BY THE COMBINED APPLICATION OF SUPER ABSORBENT POLYMER AND POTENTIAL BIOFERTILIZER
Review round: 1
Reviewer: 1

Output:
Basic reporting: Regarding the manuscript, it is clear and well organized. The language is appropriate and the presentation of the results is nicely summarized in figures and tables.
Experimental design: The main concern for the reviewer is the number of days that was considered for harvesting. It is important to evaluate full-life cycles; however, short-term studies are equally valid with a strong justification. The reviewer considers that such justification is missing in this article.
Validity of the findings: The discussion is clear and cites relevant literature that ratify the results found in the investigation. The narrative is clear and easy to understand. The reviewer suggests incorporating a few references from more recent investigations in the discussion (2018
Additional comments: Specific comments

Language
-	The article is well written and with a structure that is easy to follow. It makes the reading fluid and easy.
-	Throughout the text, there are various cases of missing commas after a list when there is a “and” at the end. Example: Line 47: “solubilization and indole-3-acetic acid (IAA) production” should be written as “solubilization, and indole-3-acetic acid (IAA) production”.
-	Lines 18 and 188, please replace manufacture’s with manufacturer’s
-	Line 239, letter L from L-56 is missing.

Abstract
-	The abstract summarizes accordingly the findings of the investigation.
-	Line 23 reads “both biofertilizers amended with SAP could tremendously promote germination”. The reviewer suggests using a word that can be easily quantified or omit the word tremendously.

Introduction
-	Line 38 reads “Traditional agricultural practices have fatal negative influences on…” Are fatal and negative synonyms?
-	Lines 60-61 “However, some biofertilizers failed to promote plant growth in the field” Could you provide some examples/references for this statement?
-	Line 75, please define Superab A200
-	Lines 76-78 “Most studies only focused on the application effect of SAP alone or mixed with chemical fertilizer, while that of synergistic use of SAP with biofertilizer were rarely reported, particularly under water deficit conditions” Please provide examples of reports with some results. If there are none, write a statement such as “To the authors’ knowledge, there are no reports…”
-	Line 83, please define all elements (or none).
-	Line 91, could you justify why “greatly different crops” were selected? Why specifically wheat and cucumber?

Materials and Methods
The methods are overall clear and well written. Specific details to be modified include:
Bacterial strains and potted plant soil
-	Table 1 describes L56 as obtained from storage in the laboratory. Was this purchased in the past? Who is the manufacturer? Where was it obtained from? Please provide more detailed information.
-	Line 106, please define O.M.
Plant culture and collection
-	In line 124 (and all treatments including SAP), if the weigh of SAP is ignored, how can this experiment be replicated? Could you provide more information on the treatment preparation?
Viability assessment of inoculants in rhizosphere soil
-	Line 141, why 14, 19, 24, 29, 34, 39 and 44 days after sowing were selected?
Sample collection and preparation
-	Why were the samples harvested at day 44? (line 159)
Determination of free proline content and total soluble sugars (TSS) in plant leaves
-	What is the relevance of measuring free proline and TSS. Could you include a short sentence to justify these assays?

Results
The results are clear and nicely summarized in figures and tables.  The reviewer suggests to increase the font size in axis names and legends in figures, as well as showing the standard error bars as ±.

Discussion
The discussion is clear and cites relevant literature that ratifies the results found in the investigation. The narrative is clear and easy to understand.
-	Line 345 “Similar results have been reported (Siddikee et al., 2016).” In which plants?
-	The reviewer suggests to incorporate a few references from more recent investigations in the discussion (2018).

Conclusions
The conclusions are clear and the take-home message is nicely summarized in the paragraph “Our results indicate that SAP addition in biofertilizer is a good strategy for improving the efficiency of biofertilizer, especially in the areas suffered from long-term drought stress.”
-	In line 388, the reviewer suggests to avoid the use of the word tremendously


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: AMELIORATION OF DROUGHT EFFECTS IN WHEAT AND CUCUMBER BY THE COMBINED APPLICATION OF SUPER ABSORBENT POLYMER AND POTENTIAL BIOFERTILIZER
Review round: 1
Reviewer: 2

Output:
Basic reporting: Even though the writing is overall of good quality, the text needs to be improved.  In some cases the use of words is not the appropriate, even though the spelling is correct (e.g. lines 312, 390). Be objective, provide measurable information, facts.  Use articles and connecting words appropriately. The introduction and literature cited are relevant as well as the Figures provided.  However, the quality of the images need to be improved as it is difficult to read the letters/words.
Experimental design: The research is within the scope of the journal and the question is well defined (can SAP help the proliferation of biofertilizers).  The investigation includes high technical standards and the methods are described in detail.
Validity of the findings: Data is robust, statistically sound and controlled.  However, I suggest to double check the error bars.  They are relatively small for having only n=3.  These may be alright, it is a comment as I am used to seeing bigger error bars in plant samples with such a small number of replicates. Speculation was kept to the minimum, the conclusion is linked to the original question and does not bring extra information at last. Also, SAP did improve the viability of the inoculants.  However, is this due to an improvement in moisture content? Why was moisture not recorded? Did I miss it?
Additional comments: Even though the writing is overall of good quality, the text needs to be improved.  Three main concerns: Double check the error bars (details above), Figures’ resolution, and explain why moisture was not recorded, if the whole point was to prove that SAP could help overcome drought issues for the survival of biofertilizers.
Various lines – “i.e.” is not needed in most cases.
Various lines – Check spelling (e.g. lines 27, 239, 453, etc.). Avoid starting sentences with the word “and”.
Various lines – Improve use of articles and other words such as “was, were, are”. E. g. line 77 …or mixed with “a” chemical fertilizer, while “studies on the” synergistic use of SAP with biofertilzer “are” rarely…
Various lines – Check for sentence fragments or statements that don’t belong.  E.g. lines 83-84, 87, etc.
Various lines – Be objective, provide measurable data. E.g. in line 91 justify why wheat and cucumber were selected, if you state they are “greatly” different, point out those differences; line 117 “super clean bench” provide concise conditions; 137 specific details, 161 by how much, 371 what “great” roles, 388 how “tremendously”, etc.
Various lines – Be clear. E.g. in line 95 what do you mean by “generalize”? Improve clarity throughout the text. E.g. lies 265-275, 298-299 do you mean trend? 303-304, etc.
Figures and Tables: Double check statistics, image resolution, remove extra dot in Fig. 4 caption, Table 1 "storage in the lab" may not be appropriate, rearrange Table 5 column 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: EFFICIENT GENERATION OF HUMAN PRIMORDIAL GERM CELL-LIKE CELLS FROM PLURIPOTENT STEM CELLS IN A METHYLCELLULOSE-BASED 3D SYSTEM AT LARGE SCALE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript was well written.
Experimental design: The experiment was well conducted .
Validity of the findings: The manuscript reported a novel method to produce human primordial germ like cells in a large scale
Additional comments: The manuscript titled by “Efficient generation of human primordial germ cell like cells from pluripotent stem cells in a methylcellulose-based 3D system at large scale” reported a novel method to produce human primordial germ like cells in a large scale. The experiment was well conducted and the manuscript was well written. I suggest this paper should be accepted by minor revision.
Minor concern:
1.	In result 1, the authors choose 0.35% MC as the optimized concerntration as refereced (Jiang et al. 2015), because 0.7% MC has a similar yield volume of PGCLC. Still I wonder about the accuracy. To make sure the experiment could be reproduced by other research team; the authors should provide more data to prove it.
2.	There are no scar bars for the immunofluorescence results of Figure 2 and 3.
3.	There are some grammartical problems throughout the MS, the authors should check it carefully.


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: EFFICIENT GENERATION OF HUMAN PRIMORDIAL GERM CELL-LIKE CELLS FROM PLURIPOTENT STEM CELLS IN A METHYLCELLULOSE-BASED 3D SYSTEM AT LARGE SCALE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study focuses on a new cell culture technique to induce human PGCs differentiation, it's an interesting and promising field. The format and figures in this manuscript are pretty good, and the language is also fine.
Experimental design: The project is well designed, and the content in this study is suitable to publish in PEER J.
Validity of the findings: This study provides some fundamental data of human PGCs differentiation using 3D system, which is reliable. But some of these data need further interpreted to get more precise conclusions.
Additional comments: In this manuscript Wang et al. reports a 3-D system to induce human pluripotent cells to differentiate into PGC-like cells in vitro, and compared the differentiation efficiency to traditional system. Currently, the 3-D culture system is a promising method widely used for stem cell maintenance or differentiation in vitro. However, there are still many technique obstacles in this field. The authors chose this new system to test the effect used for induction of human ESCs or iPSCs differentiation, which has significance for assisted reproductive technology. The project is well designed, and the logic is clear. Many observations are interesting, but the some of the data is not well analyzed, especially the difference of hPGCLCs derived from two systems should be discussed.
There are some suggestions for the authors:
Why the authors chose 0.1, 0.35 and 0.7% MC for research, please provide the reasons or references.
In figure 1E and F, the proportion of EpCAM/ITGA6 cells in 0.35MC group keeping on declining during culture, while the control exhibited a different characteristic. And the author chose Day 4 for further investigation, probably because the proportion of EpCAM/ITGA6 cells from two groups is significantly different in cell number. However, based on these data, we can only conclude that 0.35MC condition seams to promote differentiation faster and have a little bit higher efficiency on Day 4, but U96 condition reached the peak value of differentiation slower. The logic need be discussed here.
In figure 4C, why the hPGCLCs exhibited the decreased expression of DAZL and DDX4, while the expression of other germ line markers increased? Please analyze these data or discuss the inconsistence.
In line 329, the conclusion “All these findings demonstrated that iPSCs efficiently differentiated into hPGCLCs using the MC system” is not suitable here. The above observations only suggest that 0.35% MC system has a higher efficiency than the old system, but can not conclude that hPGCLCs were efficiently obtained, since the purity of hPGCLCs is not known.
Moreover, during differentiation in two systems, the expression of stra8 and Sycp3 (at mRNA level at least) should be detected, to make sure which stage these cell achieved.
Finally, the language is good, but more passive voice should be used in the manuscript.


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

Input:
Title: EFFICIENT GENERATION OF HUMAN PRIMORDIAL GERM CELL-LIKE CELLS FROM PLURIPOTENT STEM CELLS IN A METHYLCELLULOSE-BASED 3D SYSTEM AT LARGE SCALE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Well.
Experimental design: Experimental design is good.
Validity of the findings: Data is well.
Additional comments: The authors developed a simple and efficient 3D induction system to generate hPGCLCs from
 human pluripotent stem cells at large scale.The manuscript is well, and I suggest to accept it.


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: EFFICIENT GENERATION OF HUMAN PRIMORDIAL GERM CELL-LIKE CELLS FROM PLURIPOTENT STEM CELLS IN A METHYLCELLULOSE-BASED 3D SYSTEM AT LARGE SCALE
Review round: 1
Reviewer: 4

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript developed a methylcellulose (MC)-based 3D hPGCLC induction system which can efficiently produce hPGCLCs from hESCs/hiPSCs at a large scale compared with traditional differentiation system, U bottom plate. The differentiated hPGCLCs showed similar characteristics of those from the U96 protocol, including gene expression, germ cell markers, and epigenetic properties. This differentiation system will facilitate the study of human germ cell development and stem cell-based reproductive medicine.
Specific comments:
1.	This study compared the efficiencies of hPGCLCs differentiated by iMeLC in their developed system and the traditional system. However, they have three kinds of variables, i.e., different plate shapes (U bottom vs flat bottom), different seeding densities (3.5×10^5 vs 5×10^5) and different media (w/o MC vs w/ MC). It is not convincing to demonstrate that “0.35% MC” is better than “U96” by comparing these two systems with more than one experiment variable. They author should prove the effect of each factor.
2.	In Fig. 1C, the author should provide critical controls including U96 plate+ different concentrations of MC, flat plate+ no MC.
3.	In Fig. 1D, no technical replicates.
4.	In Table 1, check the seeding # in MC group.
5.	On line 251, change “30×10^4~40×10^4” to “3×10^5~4×10^5”.
6.	If the abbreviations were appeared for the first time in the manuscript, it is no need to use the full names from then on. For example, embryoid bodies.


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: EFFICIENT GENERATION OF HUMAN PRIMORDIAL GERM CELL-LIKE CELLS FROM PLURIPOTENT STEM CELLS IN A METHYLCELLULOSE-BASED 3D SYSTEM AT LARGE SCALE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The writing of this revised manuscript is good.
Experimental design: In the updated version of manuscript, several experiments were added to enhance the evidence.
Validity of the findings: Generally, the data is solid and convincible.
Additional comments: All the questions are answered, and the ambiguous points are well explained by the authors, and the updated figures in revised version also help to strengthen the conclusions. Some language errors are also revised. In all, this new version of manuscript should be accepted for publication.


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

Input:
Title: EFFICIENT GENERATION OF HUMAN PRIMORDIAL GERM CELL-LIKE CELLS FROM PLURIPOTENT STEM CELLS IN A METHYLCELLULOSE-BASED 3D SYSTEM AT LARGE SCALE
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript described a method for producing human PGCs from ESC/iPSC in bulk, which will facilitate the study on human germline development and human reproductive biology. The manuscript wrote well, and prepared solid data to prove their 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: SOCIAL NETWORK ANALYSIS FOR THE ASSESSMENT OF PIG, CATTLE AND BUFFALO MOVEMENT IN XAYABOULI, LAO PDR
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall this manuscript needs to be vastly improved in terms of basic reporting for this general biology journal. The use of jargon related to SNA needs to be dramatically reduced, and more literature could do with being included on what previous studies have found out about FMD and what interventions can work in what types of networks to justify why finding out the network is important. Specific comments:

Whilst the abstract is well written scientifically, I am concerned about the extensive use of field specific jargon, which will reduce the ability of those not familiar with social network analysis to actually understand the paper and the meaning of the results. Without knowing the method & the term definitions the abstract is in fact currently completely incomprehensible to a general audience. Given that this is a general journal, I think the abstract should be re-written in a way to make the results understandable to people in other fields; especially as this work is applicable to veterinarians, farmers, policy makers etc.

The introduction contains extremely minimal information, obscured by extensive use of jargon and is not sufficient at all. I would like to see a bit more (something at least) about FMD, what viruses cause it etc, and a mention of how highly contagious and destructive it is, what the clinical signs are and how it is diagnosed etc. to set the scope for the reader. Further, there needs’ to be some mention of what is known about the risk factors for transmission of the disease from other countries. You mention the SNA has been used for FMD in the UK but do not say anything further; how useful was it? What did it reveal that justified your use of it now? Also, please define your terms, I know what a ‘node’ is but a generalist audience won’t, so define it the first time you use it in line 48. Again, the jargon used here (betweenness centrality and the closeness centrality of these nodes) is obscuring actual meaning. What does this mean: “the out-degree centrality of the infected chain of nodes was similar to a fragmented network”, it’s ok to say it, but then you need to explain it.

The lines in the beginning of your discussion (165-168) would be good to know in the Introduction.
Experimental design: Mostly the design of this study seems sound to me, I only have one query which I'd like to see cleared up to provide sufficient detail to replicate it: your sampling method needs explaining better. It’s not exactly clear at the moment what you did to identify 892 nodes from interviewing 189 people, especially if you required each node to reciprocally mention the other…which wouldn't be possible.
Validity of the findings: The data has been reported well and the conclusions appear to be relevant, but could do with explaining further to non-SNA specialists.
Additional comments: I think this is a valuable study, especially for the people of the province. However, further work is required to make this publishable to a standard that allows it to be understood by a general audience. Specific additional comments below:

Line 156 needs rewording, ‘They consisted of both' would make sense, but as it stands it sounds confusing to say ‘They were both'.

Line 174: you should link this statement with data from your study that shows over half of your interviewed farmers did not vaccinate against FMD

Paragraph at 178: this is a good paragraph, you have explained what your results mean to the reader and in practical terms. This is the sort of information/language required in your abstract.

This section needs expanding: what are the nodes with high betweenness centrality? Why do they influence the spread of FMD? “The betweenness centrality was much higher overall. This means that the nodes greatly influenced the spread of FMD. The nodes with a high value of betweenness centrality should be given special consideration.” Your sort of explain it in lines 224-226, but not early enough to justify your conclusion in line 221-223.

Line 233: define GSC before using the acronym.

Figure 2: different colours or shades of the same colour would help interpreting this. Also, the figure legend does not adequately explain the figure to allow a reader to interpret it if they have a general background. Please expand it to contain more detail.

Both above points also apply to Figure 3 A and B.


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: SOCIAL NETWORK ANALYSIS FOR THE ASSESSMENT OF PIG, CATTLE AND BUFFALO MOVEMENT IN XAYABOULI, LAO PDR
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study is a nice application of social network science for understanding infectious and emerging diseases. I would expect that studies like this should or will become more common in the future due to usefulness for understanding disease transmission.  Therefore, there would be a potentially broader audience. Due to the broader audience it would be useful if the authors defined the social network metrics (in-degree centrality etc.) and gave examples. This would allow a broader audience outside of social network scientists to understand the results and use the information.  The intent is to make this work relevant to a broader audience.
Experimental design: The experimental design is appropriate.  More information should be given on how the people (nodes) were chosen to be interviewed and samples sizes for each category or type of nodes.
Validity of the findings: The finding are valid, however, it would be useful to better define or describe what the findings mean.  For example, line 178 can better describe what it means that animals move to and away from each other. Because the metrics do not have units, it is more difficult to describe the meaning of a result and extrapolate outside of the study.  Line 183 is good with giving the exact example of what the results means.

Again, describing what in-degree centrality means in social network science will be helpful to a broader audience.
Additional comments: Line 35 Define more narrowly multi-species. For example, ungulates and not zoonotic.
Line 69 Add to Aims something to briefly describe the data collection and focus group.
Line 76 Questionnaires sent to who and how were they selected?
Line 131 It might be useful to have a table that describes each metric (e.g. in-dgree centrality)  to a general audience.
Line 227 Define what a weak component means.
Line 238 Define cut-point parameters
Line 277 In the Ethical Statement was this research approved by an Institutional Review Board (IRB)? Lao PDR to the institutions may not have an IRB but if they do then this should be included.
Table 1 Can easily add the sample size for group for interviews
Figure 1 Could be decreased in size or zoomed in to just include Lao PDR
Figure 2 and Figure 3A are so similar it difficult to see the difference. One could be omitted to the clarity increased.
Figure 3 requires cleared and larger font and the numbers and units described in the legend.


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

Input:
Title: A PUBLIC DATABASE FOR THE NEW MLST SCHEME FOR TREPONEMA PALLIDUM SUBSP. PALLIDUM: SURVEILLANCE AND EPIDEMIOLOGY OF THE CAUSATIVE AGENT OF SYPHILIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Line 40-43. This statement is unclear. Please rephrase it.
Line 65-68. The in vitro cultivation of laboratory strains of T. pallidum using rabbit epithelial cells for up to 100 generations is a major breakthrough and it appears this method may support cultivation of strains directly from clinical specimens. Unless the authors can justify, “…we are still far away from the in vitro cultivation..”, this statement should be rephrased.
Line 73-75. Are there other examples of “monomorphic bacteria” where fewer MLST targets were better for genotyping versus conventional MLST as alluded to by the authors?
Line 175. The statement “Since TPA is not cultivable in vitro…” is not entirely correct and should be rephrased to reflect clinical strains cannot be routinely cultivated in vitro therefore, MLST or other typing methods have to be applied directly on clinical specimens.
Line 189-190: Does the “genetic variability” possibly reflect the low copy numbers of T. pallidum often seen in blood versus swabs? Could this account for preferential amplification of the tpr genes targets?
Line 194. This statement seems out of place and it does not flow from the previous paragraph.
Figure 2. Does no value mean the clonal complex was not determined?

There are several grammatical and syntax errors in the manuscript that need to be corrected.
Line 111: change “primary” to “primarily”
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors describe a public database for the recently proposed MLST typing system for T. pallidum. As with other MLST schemes, this is an important tool for assigning strain types and will avoid confusion when new strain types are encountered by different groups.


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

Input:
Title: A PUBLIC DATABASE FOR THE NEW MLST SCHEME FOR TREPONEMA PALLIDUM SUBSP. PALLIDUM: SURVEILLANCE AND EPIDEMIOLOGY OF THE CAUSATIVE AGENT OF SYPHILIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript “A public database for the new MLST scheme for Treponema pallidum subsp. pallidum: surveillance and epidemiology of the causative agent of syphilis” by Linda Grillova and colleagues focused on an important topic about the molecular typing schemes for Treponema pallidum subsp. pallidum. In this study, it introduced a harmonized typing tool for consideration of several different molecular typing currently available and described the PubMLST database for treponemal DNA data storage and for assignments of allelic profiles and sequencing types. It would be benefit for molecular typing studies of the syphilis pathogen in the future. But there are some issues which should be addressed:
1) On line 123-124, Ref Grillová et124 al. 2018, it would be not appropriate. It was not the original research on the whole genome sequencing of Treponema pallidum.
2) On line 141, “passed” would be better change for “propagated”; Also on Line 172.
3) On line 155-157, “STs were divided based on the 137 variable sites present in the concatenated sequences of typing loci (2584 bp), where 46 variable positions were found to be parsimony informative for distinction of the two clonal complexes.” The variable site was relative to which reference? Nichols or SS14 ? It would be better described more clearly. And for the 137 variable sites, there would be good to present a summary table to show the detailed information about this position.
4) As the paper described, there was included 31strains propagated by rabbits, but on line 172-173, “Interestingly, five STs were found exclusively among the strains passed through rabbits-ST20-ST24.” It was only 8 strains in ST20-ST24. This section should contain a series of contradictory statements. And the authors mentioned that the phenomenon was interesting, what's suggested imply of this phenomenon?


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

Input:
Title: A PUBLIC DATABASE FOR THE NEW MLST SCHEME FOR TREPONEMA PALLIDUM SUBSP. PALLIDUM: SURVEILLANCE AND EPIDEMIOLOGY OF THE CAUSATIVE AGENT OF SYPHILIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors are to be congratulated for their work to generate a central database for T.pallidum. As the sequencing of this organism grows such approaches may have increasing value.

I have only minor comments

I would have liked to see a more clearly articulated argument about what the database adds to the existing availability of the data/sequences through existing portals. I believe this argument can be made but would like to see if more clearly put forward.

Line 147 - I am not sure it is particularly surprising that a small number of samples were PCR positive/serology negative - this is a well recognised clinical phenomenon.  Suggest re-word this line.
Experimental design: N/A
Validity of the findings: I found the section on associations to be slightly out of keeping with the rest of the manuscript. In particular there is limited/no discussion of the extent to which associations found may be reflective of sampling bias - as the authors themselves are aware there has been limited geographic, epidemiolgical range in sequencing of T.pallidum thus far and the extent to which associations reflect true relationships vs sampling artefacts should be discussed.
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: MLPA IS A PRACTICAL AND COMPLEMENTARY ALTERNATIVE TO CMA FOR DIAGNOSTIC TESTING IN PATIENTS WITH AUTISM SPECTRUM DISORDERS AND IDENTIFYING NEW CANDIDATE CNVS ASSOCIATED WITH AUTISM
Review round: 1
Reviewer: 1

Output:
Basic reporting: The text is somewhat scattered and lacks a clear coherent underlying message. The literature is adequate.  The cohort is relatively small and heterogeneous which contributes to my difficulty in identifying a clear message.
Experimental design: This is primarily an observational study.  The research questions are not well-defined.
Validity of the findings: The data are relatively sparse and given the lack of clear research questions, it was hard for me to understand the authors' conclusions.
Additional comments: I have nothing further to add.


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: MLPA IS A PRACTICAL AND COMPLEMENTARY ALTERNATIVE TO CMA FOR DIAGNOSTIC TESTING IN PATIENTS WITH AUTISM SPECTRUM DISORDERS AND IDENTIFYING NEW CANDIDATE CNVS ASSOCIATED WITH AUTISM
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout:
No. Corrections to the English are needed

Literature references, sufficient field background/context provided:
No. Key references are missing

Professional article structure, figs, tables. Raw data shared.
Yes

Self-contained with relevant results to hypotheses.
Aims and hypotheses are not clearly stated
Experimental design: Original primary research within Aims and Scope of the journal.
Yes

Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.
No. A minimal increase in knowledge is presented in this work.

Rigorous investigation performed to a high technical & ethical standard.
Findings appear to be rigorous in that multiple confirmatory methods are used

Methods described with sufficient detail & information to replicate.
Yes.
Validity of the findings: Data is robust, statistically sound, & controlled.
No.  Evidence for candidacy of DOCK8, CTNNA2 and genes involved in parkinsonism is not shown.  Gender balance of controls may not match a typical ASD cohort where more males than females are expected.

Conclusion are well stated, linked to original research question & limited to supporting results.
Conclusions are not clear.  Emphasis for using MLPA over microarray as a frontline test is not strong.  Discussion states conflicting opinions on this topic
Additional comments: A summary of findings from 92 individuals affected by autism spectrum disorders tested for structural variants by karyotyping, FRAX PVCR test, MLPA of specific targets and chromosomal microarray.  Overall 18 individuals with pathogenic or likely pathogenic variants were detected.  All pathogenic or likely pathogenic variants were detected by CMA except for one FRAX mutation.
The aims as stated in the text "The aim of the study was to assess the efficiency of CMA relative to MLPA in terms of clinical significance and to identify new candidate genes for ASD in the studied cohort of ASD-diagnosed patients" are not clear. There are several interpretations for this: Aim to assess the efficiency of CMA relative to MLPA to detect clinically significant CNV; and/or there are different patient outcomes as a result of using different methods. CMA is established as the frontline test for neurodevelopmental disorders over other methods
Why were CNV less than 10kb excluded?  MLPA at least should be able to accurately detect much smaller CNV than this.
You suggest "CNV encompassing the region 9p24.3 were identified in several earlier studies, but none of these works identified DOCK8 as being potentially associated with ASD." Glessner et al 2017 demonstrate well the potential involvement of DOCK8 with ASD as well as other neurodevelopmental disorders.
How do you conclude that DOCK8, Parkinsonism and CTNNA2 are associated with ASD without providing a calculation of enrichment of any of the loci to be involved significantly with ASD?  Exact tests with appropriate multiple testing corrections to assert novel loci are associated could be performed.
It is asserted that MLPA using 6 probe sets is more cost effective than microarray for excluding loci involved in syndromic autism but no cost benefit analysis is shown to support these claims. I am not convinced that by the time labour is factored in that there is a significant difference.
Why is exclusion of potentially pathogenic loci by MLPA desired over probable detection by CMA (discussion page 9)?
Controls used for DOCK8 are equal numbers of males and females, does this reflect the gender balance in the 92 samples?
What were the selection criteria for inclusion in this study?  What fraction of individuals tested by this department over the 2012-2016 time period does this represent?
Suggest the four CNV that were detected by MLPA but not by CMA should be verified to be real or not using either alternate MLPA probes or quantitative PCR of genomic DNA in those regions.
The individuals with pathogenic CNV have a mixed bag of syndromic neurodevelopmental disorders.  It would be good to have a section in the discussion explaining how they were regarded as primarily ASD or PDD-NOS disorders as opposed to the syndromes described in OMIM.  My interest is, do they in hindsight of molecular diagnosis have the classical syndromic features of those disorders (particularly the male with Becker muscular dystrophy and the female with 22q deletion syndrome) or are the behavioural phenotypes more pronounced than expected in these individuals that made these diagnoses based on phenotype alone difficult?
Was segregation attempted for any of the CNV identified in individuals with affected siblings or family members in the cohort?  This may add evidence towards the likely pathogenicity of those variants.
Suggest remove CTTNA2 from the abstract because no convincing evidence is shown in the manuscript to support it as a genuine ASD candidate gene.  There is another report of an individual with an intragenic CTNNA2 deletion and speech delay that may be similar to the individual you describe (Paganelli et al. 2017)?

Minor Corrections
page 3 line 67: MLPA analysis is fast and easily interpretable, and can be used ... >> MLPA analysis is fast, easily interpretable and can be used ...
Page 4 line 69: ...CNV in a cohort of 92 children treated for ASD. It is not clear what treated for ASD means as to my understanding the disorder can't be treated.  Would 'diagnosed' be an accurate term?
Page 4 line 71: The aim ... >> The aims ...
Page 6-7 lines 133 - 145:  This paragraph beginning with "MLPA and CMA revealed 8 pathogenic CNV in..." seems out of place here.  I suggest this discussion of the overlap between different detection methods would be better placed later in the manuscript.  Only highlighting what was detected by karyotyping and FRAX testing in this first section would make this easier to follow.
Table 1: 'Normal'.  Suggest to use 'negative'.
Table 1: "n = number of the patients with the CNV" is not referring to anything, i.e. the abbreviation (n) does not appear elsewhere in the table.
page 8 line 181: "Of the genes listed above, CTNNA2 and DOCK8 are potentially associated with ASD." References required.
Supplementary table contains multiple spelling errors.
Page 13 Line 3012 counselling >> counseling

References:
1: Glessner JT, Li J, Wang D, March M, Lima L, Desai A, Hadley D, Kao C, Gur RE, Cohen N, Sleiman PMA, Li Q, Hakonarson H; Janssen-CHOP Neuropsychiatric Genomics Working Group. Copy number variation meta-analysis reveals a novel duplication at 9p24 associated with multiple neurodevelopmental disorders. Genome Med. 2017 Nov 30;9(1):106.
2: Paganelli V, Giordano M, Meazza C, Schena L, Bozzola M. An intragenic deletion
within CTNNA2 intron 7 in a boy with short stature and speech delay: A case
report. SAGE Open Med Case Rep. 2017 Feb 15;5:2050313X17693967.


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: MLPA IS A PRACTICAL AND COMPLEMENTARY ALTERNATIVE TO CMA FOR DIAGNOSTIC TESTING IN PATIENTS WITH AUTISM SPECTRUM DISORDERS AND IDENTIFYING NEW CANDIDATE CNVS ASSOCIATED WITH AUTISM
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have compared findings by CMA with those from selected probe mixes to detect clinically relevant variants by MLPA in 92 ASD patients and demonstrated that both methods accurately detect pathogenic variants, but CMA is about 3 times more likely to detect likely pathogenic variants (13% vs 4.35%). This may be significant, since use of MLPA probe mixes is necessarily limited to existing knowledge.  As new knowledge grows the interpretation of pathogenic variants is likely to grow, as it did in this study regarding DOCK8.  It is difficult for me to understand why a less-revealing study would be preferable. The speed with which results are delivered is not a significant advantage when there is a loss of precision. The use of English language is much improved and the literature review is adequate.  Please use the term Asperger syndrome for for Asperger's disorder.
Experimental design: This is an observational study which is adequately explained and interpreted.
Validity of the findings: No comment
Additional comments: No Comment


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

Input:
Title: MLPA IS A PRACTICAL AND COMPLEMENTARY ALTERNATIVE TO CMA FOR DIAGNOSTIC TESTING IN PATIENTS WITH AUTISM SPECTRUM DISORDERS AND IDENTIFYING NEW CANDIDATE CNVS ASSOCIATED WITH AUTISM
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Dear Dr Srovnal & co-authors, you are commended for the improved clarity of the manuscript throughout.  These data are presented well and the summary of your experiences are valuable. The discussion is now quite long (6 pages) and though it is generally interesting it could be trimmed a little to make the manuscript sharper. I would suggest the discussion of the incidental variants occurring in the individual with FRAXA would be a good candidate for shortening.  This is just a suggestion and it can be to the discretion of the editor and you as to the length of the final manuscript.

Minor corrections:
Page 8: Line 127 : The percentages of rare CNV in the males (60.32%) and females (58.62 %) of our cohort were similar.  >> Females 48.62% ?

The use of dystrophinopathia is uncommon, I suggest using muscular dystrophy or dystrophia


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: CLIMATIC NICHE EVOLUTION IN THE VIVIPAROUS SCELOPORUS TORQUATUS GROUP (SQUAMATA: PHRYNOSOMATIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well-written manuscript that addresses climatic niche evolution in lizards.

Tables:

Please round decimal places, it will make the tables more readable. Table 3 could be moved to the SM.

Figures:

Generally, there are too many figures, and some of them will not be readable in the final PDF. Please choose figures wisely and move the rest to the SM. Figure 1 could be combined with Fig. 2b. Captions for Figure 3-4 could explain what these results mean. Figure 5-6 will be very small and difficult to understand.

Supplementary material:

I appreciate the inclusion of the raw data for review and also for the reader eventually. However, in their current state, some of the supplementary material is impossible to decipher. The locality PDF for example, is split up in such a way that cannot be read. I'm not sure if this is PeerJ converting it a PDF, but if PeerJ does not accept excel files as SM, please include in another data repository.
Experimental design: Most of the analyses appear to be sufficient to address their hypotheses and are well explained and rigorous. The paper could be improved by better linking their analyses and results to the cold climate hypothesis and viviparity generally.
Validity of the findings: The discussion section poorly links back to introduction, and seems to restate the results too often, where readers will be interested in how these results support or reject the cold climate hypothesis. In addition, the discussion can better include the background research available for the topic. Lambert & Wiens (2013) is a glaring omission in the discussion, which tests this very same hypothesis in the larger group of Sceloporus lizards. In particular, Lambert & Wiens (2013) directly address this species group, pointing out that they represent an invasion of warmer climates from cooler climates. Therefore, phylogenetic history is a very likely factor that explains the origination and retention of viviparity, while at the same time explaining why there is niche divergence here. I think this is a very interesting and important point missing from the discussion.
Additional comments: Line Item comments:

Line 53-57: This paragraph would be better after the next paragraph (Line ~82).

Line 56-57: Confusing wording; aren't all species viviparous in this group?

Line 82-88: Not clear what the purpose is of each of these specific objectives and they relate to your hypothesis.

Line 95: how did you know the tossed-out records were from misidentifications?

Line 188-196: Why not use BEAST to estimate divergence dates? The dates are quite different from previous studies (e.g. Lambert & Wiens 2013), where the age of the group is half in comparison.

Line 388-389: This makes sense, but the interesting question is how does this phylogenetic history lead to the patterns we see today? Colonization of warmer climates from cooler climates would explain all the niche divergence.

Line 412: What about mean temperature of the warmest quarter (Bio10)? This group of lizards lays their eggs during the warmest months of the year (Jones and Lovich 2009)?

Line 563: Not all climatic variables (i.e. tolerances) would be expected to be constrained by viviparity, only variables relevant to the egg laying season (e.g. Bio10 and Bio5). I think it's not entirely relevant to consider all of the variables, when only a few might be important and conserved.


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: CLIMATIC NICHE EVOLUTION IN THE VIVIPAROUS SCELOPORUS TORQUATUS GROUP (SQUAMATA: PHRYNOSOMATIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors of this study tested for niche conservatism (PNC) on climate occupation in a group of viviparous lizards of the Sceloporus torquatus group. They aim to test if this life history trait (viviparous) might represent an ecological constraint on the occupation of different climate conditions. Although I found their data, sampling and evolutionary questions interesting I think the manuscript needs revision.

I would recommend the authors to re-design some of the analysis they performed using the data they already have, in order to make the design more appropriate to what I understand are the main aims of their study. I found is not clear to me in their manuscript how is that viviparity should represent and evolutionary constraint to occupy cold habitats expecting to find PNC (see comments below for lines 35 - 43). Additionally, the study is sometimes hard to follow because it oscillates between the tempo and mode of diversification and the ecological consequences of viviparity in this group of lizards. These instances should be revised throughout in order to clarify the main objectives of the study and explain what the expected patterns are. Bellow I summarize some comments that I hope can help them to improve their manuscript.
Experimental design: It’s not clear to me why they are expecting that viviparous species should be evolutionary constrained on the climatic niches the species with this trait occupy and thus show PNC. If viviparity is supposed to avoid egg mortality linked to cold temperatures observed in OVIPAROUS species, why is that viviparous should be ecologically constrained? Besides that, these changes on climatic occupation between viviparous and oviparous species might only be reflected in the breeding season

Multivariate estimates of climatic niche occupation might give them more approximate insights about the diversity of ecological conditions (in this case the climate space of the niche) the species occupy and the historical evolution of this occupation. So I would recommend multivariate analyses to integrate different dimensions of the species climatic niches rather than making analyses based on single predictor variables
Validity of the findings: They stated that “there is no evidence that climatic niche differentiation was the main factor in the diversification of the studied group” does this contradict the fact that they did not found evidence for PNC based on their results of lack of PS?

The link of orogenic and glacial periods and the diversification of the group is not well explained. Although this might be discussed as a potential hypothesis I think it should not be included as a conclusion of the study since is not directly linked to their aims and their methods are not the most appropriate to test this hypothesis
Additional comments: I think they should introduce the study explaining the evolution of the viviparous strategy in lizards. Then, discuss the possible evolutionary and ecological consequences of this life history trait and mention what the other studies have found so far. This will introduce the reader to their main question of the study and clarify what this new study will bring to the discussion on the evolution of viviparity on lizards. This would stands as a better introduction rather than discussing ecological niches and PNC as an introduction.

2 do they refer by “actual” to the current distribution?

6 the authors should clarify that their concept of ecological niche is based on abiotic factors, but not that they refer to it through the text as estimates of biotic and abiotic interactions. Clarify

7 which are those ecological components?

9 what kind of speciation? Sympatric, ecological?

2 -15 is a little confusing, I would recommend to introduce their study with the evolution of viviparity and after that explain that PNC is the predicted pattern we should expect to observe when some life history trait represents an evolutionary constraint (in case viviparity really should represent an evolutionary constraint)

40 such as which life history traits?

56 -59 the three aims they mention here are very inter related to one another and to the ones stated at lines 86 – 88 (see comment below). Remove or try to be less redundant

74 mention how many species are present in these groups (grammicus and megalepidurus). The authors also should mention in what direction excluding these groups are expected to bias their results. If these groups habit on very different climatic habitats than the included group, excluding them should bias their results toward PNC making they inferences more robust (i.e. there is lack of PS even when excluding groups ecologically different than the main species group)

86 - 88 the aims 3,4,5, are very related and they might not stand alone as single aims of the study.

97 occurrence points should be removed at the same spatial scale than the environmental layers. A 0.5 km cut does not guarantee that all the points falling in the same pixel were removed because the resolution of the layers they used is ~ 1km x 1km

108 The recommend output for phyloclim analysis is the raw output from MaxEnt niche models, why the cloglog output? Which was the method used for MaxEnt replicates: bootstrap, cross-validation? They should also mention which version of MaxEnt they used or if these analyses were performed in some R package

119 how they choose 11 layers if they previously selected 10 layers according to their MaxEnt results and then remove the most correlated?

128 check previous comment on raw outputs

130 – 138 they might only mention that they performed partial ROC to test their models. Since this is more robust than AUC with pseudo absences (the default performance test implemented by MaxEnt)

234 – 242 the statistical support of the models of evolution could be simplified using likelihood ratio test instead of labeling models as more or less equivalent, distinguishable, etc.

239 242 why all the variables if these are not the most important on MaxEnt analyses

254 a positive and signifficant correlation is the pattern expected if there is PNC (species tend to have more niche overlap as they become more distantly related) and signifficant and negative correlation is the pattern expected if there is not PNC (species tend to have less overlap as they become more distantly related). But not that both signifficant correlations are evidence of PNC as stated in line 254

251 – 263 with which variables they performed these analyses?

266, 269, 271 remove niche tolerance or replace by niche occupation

313 - 314 the authors should also mention what is the general support for the node within these clades to give the reader an idea of how well resolved and supported their phylogeny is throughout

353 remove fails to detect PS since this test is not the most appropriate to do that

358 PNO profiles seem to be not that heterogeneous and they show many overlap among species
386 remove “other statistical bias” or be more specific about which these biases are

481 482 If they want to test if the species occupy different habitats it might be more appropriate to integrate multivariate analysis (for instance testing for phylogenetic signal on PC score values of multivariate PC axes). Since the pattern could be only associated with the breeding season but not in all the variation of climate occupation the species have throughout the year

491 change 20015 for 2015

493 this might not be reflected in the scale of their variables

498 how low niche overlap is evidence of niche conservatism? Isn’t this a contradiction?

524 525 absence of PS is evidence that climatic niche differentiation was not the main factor in the diversification of the group?

527 remove tolerance and change with occupation

529 same here