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: HOW MANY ONTOGENETIC POINTS ARE NEEDED TO ACCURATELY DESCRIBE THE ONTOGENY OF A CEPHALOPOD CONCH? A CASE STUDY OF THE MODERN NAUTILID NAUTILUS POMPILIUS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript adheres to all PeerJ policies as far as I can judge. The ms is well-written and well-organized. I would not judge the English as I am not a native English speaker. Figures are appropriate and helpful. I think few references could be added in the introduction about measuring the ontogeny of cephalopods and the relationship with absolute age such as Dommergues (1988, Lethaia, 21).
Two minor comments:
-	“10 mmm” l. 108 should be “10 mm”
-	 “dimeter” l. 158 should be “diameter”.
Experimental design: The question asked by the authors is in the title: “how many points are needed to describe the ontogeny of a cephalopod conch”. The authors measured one conch of a modern nautilid with several resolutions (by varying the angle between measurements), described the events of the ontogenetic trajectories and for which resolutions they could be observed or not. The final advice is to use 30° or 45°, being the good balance between resolution and data collection time accompanied with a quick discussion on other taxa and problematics.
In my opinion, this work has two major weaknesses. The first one concerns the way the accuracy of the representation of the ontogenetic trajectory is evaluated. As the authors used only one specimen, I would expect the errors brought by the different resolutions to be measured while, throughout the manuscript, only qualitative descriptions of errors are provided. I know that most authors do not take into account ontogeny as precisely as it should be and only describe it qualitatively. However, a paper dedicated to measurement errors of the ontogeny should treat it as precisely as possible. For example, there is, for this specimen, an exact location in ontogeny of each growth change. How far are we from this exact location by using a given angle? Does the error change depending on the phase (location of the first angle)? Should we model ontogenetic stages to get theoretical locations of these growth changes? Is the acceptable degree of error related to the structures, events or stages under study? I think that the quantification of the errors is unavoidable.
Secondly, the procedure undertaken by the authors is not well-suited to answer the problematic that is way wider. As it is, the paper answers that a given resolution is enough to capture some growth changes of the specimen PIMUZ 7825. However, we only have a limited insight of how general this conclusion can be? The authors are clearly aware of this limitation as developed in the sections “Dependence on the research question” and “Measurements in other taxa” but these two sections need to be extended as they are, in my opinion, the main core of the argumentation to treat the problematic. In which way some other specimens, some other species with other growth changes may be measured with a given resolution? Indeed, it is linked to the variability of what you measure and for what goal. For example, consider the angle at which the trajectory pass from a given growth stage to the following one in the ontogeny. I guess that the resolution to be used will be very different if you just want to detect that this change exists in a given specimen (this paper) or if you want to use the variability of this location in a phylogenetic analysis or to recognize dimorphs or anything else. I really expected to find in the paper a attempt of a procedure suitable to a wider variety of situations with quantification of variability. Something like: 1) identify the hypothesis to test, 2) evaluate the variability of the event(s) or stage(s) used in the hypothesis testing, 3) based on the first two points, deduce a reasonable resolution to use and ensure the test of the hypothesis will be robust enough.
Validity of the findings: Several conclusions are not based on the data and analyses from the manuscript as, for example, the fact that fine details may be lost by poor resolutions. It is likely but not tested here. On the opposite, much more can be extracted from the data herein collected, especially some statistics on errors.
Additional comments: I think that the problematic is extremely interesting and deserve some papers. For now, the ms does not support its claims enough. The analyses are not suited to answer the problematic. I hope my comments will not be too discouraging as I really think a proper treatment of this question may be of great interest for the community. Quantifying the errors should not be that time-consuming and I think it would give greater consistency to the paper.


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

Input:
Title: A TAXONOMIC REVISION OF THE SOUTH-EASTERN DRAGON LIZARDS OF THE SMAUG WARRENI (BOULENGER) SPECIES COMPLEX IN SOUTHERN AFRICA, WITH THE DESCRIPTION OF A NEW SPECIES (SQUAMATA: CORDYLIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Excellent with very few mistakes.
References complete.
Well illustrated with clear maps and drawings.
Experimental design: The authors use several museum "acronyms" but do not indicate a bibliographic reference to explain which acronym list they follow - this is important since many museums have several distinct acronyms.
Authors have to precise if colour is a character that can be used on preserved collection specimens and if that character is still obvious in older preserved specimens - this is important since many of their identification characters are based on colour and colouration pattern. Authors have to distinguish between colour pattern observed on live and preserevd specimens or to explain that the colour pattern of live specimens is preserved in collection.
Experimental design of that paper is correct.
Validity of the findings: Findings are robust.
Conclusions are correct.
Additional comments: Muscles have to be written in italic.
You indicate ratio of all characters in percentage but not for TL/SVL.
Check since authors cited as reference in the paper are not all in italic.
Can you not provide some illustrations for two or three morphological characters used in the identification key?
The first part of the discussion is just a repetition of what was previously said and looks more like a summary than a discussion. Discussion could include some references about other animal or vegetal groups that follow the same distributional and endemicity pattern than the S. warreni group.
Bibliographic references are not homogeneous e.g. the last author of a paper (if several) is not indicated in the same way throuhout the references.
It should be indicated that colouration of the holotype is taken on a more than 10 years preserved specimen not on a live specimen.


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 TAXONOMIC REVISION OF THE SOUTH-EASTERN DRAGON LIZARDS OF THE SMAUG WARRENI (BOULENGER) SPECIES COMPLEX IN SOUTHERN AFRICA, WITH THE DESCRIPTION OF A NEW SPECIES (SQUAMATA: CORDYLIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper looks at a lineage of the Smaug warren complex that was found to be distinct in a previous molecular phylogenetic study. Using external morphology and ct scans to describe osteology, the authors provide a nice taxonomic work describing a new species for eswatini. The paper is well written, but I have some concerns that should be addresses before this paper is published. First, more details are needed on the molecular phylogenetic evidence that is used to support the hypothesis presented here, so the reader does not have to go to the original paper. Hopefully several independent nuclear markers are used in a multi-species coalescent context, to deal with potential incomplete lineage sorting. Also, the morphological data could be analyses in a more rigorous way, using proper multivariate statistical analysis (e.g. linear models) and possibly species delimitation analyses like iBPP. Below I outline some more minor issues.

1. I find odd putting taxonomic author names but not year of description following species, like in S. warreni and S. barbetonensis in abstract.

2. The type of molecular data used in the referenced phylogeny in the abstract should be mentioned. Was it multi-locus, just mitochondrial, phylogenomic, etc…

3. I think a general broader introduction to girdled lizards is warranted to put the reader in context, rather to jumping straight to the Smaug warren complex. Maybe start with cordylids briefly, then introduce Smaug and then this species complex.

4. Line 55: I am not a native English speaker, but have never heard the term “rupicolous”, and if it means the same as saxicolous, I suggest using that term instead since I think it is more familiar to a broader audience.

5. I think the convoluted taxonomic history of the complex can be well summarised in a table, as a supplement to the text.

6. Line 76: If want to add an alternative spelling to Eswatini, do it upon the first mention of the country

7. Lines 81-92: Definitely more details are needed for this molecular study, in particular what kind of data were used, since it seems to be central to the conclusions of this paper. I would be careful if this is based just on mitochondrial loci, since it has been increasingly shown how susceptible these data is to introgression and incomplete lineage sorting.

8. Line 118: how is TM an abbreviation of a museum with no T word in it?

9. Line 123: missing “the” before “American”

10. Line 137: I think Ethics approval can just go in acknowledgements.

11. Indicate if all measurements were performed by the same person or not.

12. Line 188: “Statistical and multivariate analyses”. Multivariate analyses are also statistical, and first line of the paragraph says “univariate”. Maybe just call it statistical analyses.

13. Line 189: It is weird to say what program you used before describing the analyses.

14. Line 190: first time I read about “mensural characters” (again I admit, I am not a native English speaker, but I do read a lot of taxonomy). Maybe use continuous character.
15. Line 191: state based on what are specimens over 70 mm considered adults. Its just an arbitrary call?

16. The section on statistical analyses needs more detail on what actual analyses where performed. PCA is just an ordination/visualization tool. I think proper multivariate statistics (e.g. MANOVA) are needed to assess differences in morphology between species.

17. It is better for readership is sections of the results match sections of the methods.

18. Line 269-274: I think finding something “slightly more spinose” sounds extremely ambiguous. Can you measure this?

19. PCA shows extensive overlap between the new species and S. warren. These results do not convince me the species have different morphology. I am not saying this means they are not different species, but this indicates stronger support from molecular data is needed.

20. I think a more detailed molecular assessment is needed, the phylogenetic pattern found might be a product of past mitochondrial introgression.

21. Lines 915-917 more detailed analyses would be good. I also sugest trying proper species delimitation methods, like BPP and iBPP (which can handle morphology as well as molecular data).
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: A TAXONOMIC REVISION OF THE SOUTH-EASTERN DRAGON LIZARDS OF THE SMAUG WARRENI (BOULENGER) SPECIES COMPLEX IN SOUTHERN AFRICA, WITH THE DESCRIPTION OF A NEW SPECIES (SQUAMATA: CORDYLIDAE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a well written manuscript describing a new Smaug from southern Africa. The authors do a good job of providing sufficient taxonomic history information and comparing morphologically similar species using both traditional techniques and high resolution CTscans. However, I have a few comments and suggestions that pottential will benefit the paper.

Material and Methods section:
When referring to the coordinates system the authors should provide the formart that they are using through the text as well as the datum.

Systematics section:
I recommend that the figures that are cited right before the chersonymy should be cited in the subsequent text, where appropriate.

In the diagnosis section the authors simply provide a comparison of the new taxon with other species of the genus. While this is needed and important its not sufficient as a diagnosis. This should start with a combination of unique characters that generally describe the species, only after that the comparison with other species should appear. For the case of Smaug warreni the diagnosis 1) does not need the comparative part because the comparison between it and the new species was already been done in the new species diagnosis, 2) and the species is already been described.

Format in the text:
The structure of the article is conform to an accepteable format of "standard sections" with exception of:

line 94 : delete page number from FitzSimons 1943 is irrelevant

line 128: substitute co-ordinates by coordinates

line 146: FitzSimons 1943 should be in italics

line 681: Jacobsen 1989 should be in italics

line 692: Bates et al. 2014 should be in italics

line 971: Boycott, 2019 should be in italics

Please check the Tables and Figures titles they must be in bold.

Missing title on figures 10 & 13, also they must be identified with A and B, and not with left and right term (see PeerJ Standards).

References:
Please check the literature section, some references are not in full agreement with the PeerJ Standards.

e.g.

line 1007
ADOLPHS, K. 1996. Bibliographie der Gürtekechsen und Schildechsen (Reptilia: Sauria: Cordylidae & Gerrhosauridae). Squamata Verlag, Sankt Augustin, 255 pp.

PeerJ
ADOLPHS, K. 1996. Bibliographie der Gürtekechsen und Schildechsen (Reptilia: Sauria: Cordylidae & Gerrhosauridae). Squamata Verlag, Sankt Augustin.

line 1022
BOULENGER, G.A. 1908. On a collection of fresh-water fishes, batrachians, and reptiles from Natal and Zululand, with description of new species. Annals of the Natal Government Museum (Pietermaritzburg) 1:219˗235.

PeerJ
BOULENGER, GA. 1908. On a collection of fresh-water fishes, batrachians, and reptiles from Natal and Zululand, with description of new species. Annals of the Natal Government Museum (Pietermaritzburg) 1:219˗235.
Experimental design: The experimental design is standard and adequate to such type of study.
Validity of the findings: All the data supports the recognition of the new species.
Additional comments: This is a well written manuscript describing a new Smaug from southern Africa. The authors do a good job of providing sufficient taxonomic history information and comparing morphologically similar species using both traditional techniques and high resolution CTscans. However, I have a few comments and suggestions that pottential will benefit the paper.

Material and Methods section:
When referring to the coordinates system the authors should provide the formart that they are using through the text as well as the datum.

Systematics section:
I recommend that the figures that are cited right before the chersonymy should be cited in the subsequent text, where appropriate.

In the diagnosis section the authors simply provide a comparison of the new taxon with other species of the genus. While this is needed and important its not sufficient as a diagnosis. This should start with a combination of unique characters that generally describe the species, only after that the comparison with other species should appear. For the case of Smaug warreni the diagnosis 1) does not need the comparative part because the comparison between it and the new species was already been done in the new species diagnosis, 2) and the species is already been described.

Format in the text:
The structure of the article is conform to an accepteable format of "standard sections" with exception of:

line 94 : delete page number from FitzSimons 1943 is irrelevant

line 128: substitute co-ordinates by coordinates

line 146: FitzSimons 1943 should be in italics

line 681: Jacobsen 1989 should be in italics

line 692: Bates et al. 2014 should be in italics

line 971: Boycott, 2019 should be in italics

Please check the Tables and Figures titles they must be in bold.

Missing title on figures 10 & 13, also they must be identified with A and B, and not with left and right term (see PeerJ Standards).

References:
Please check the literature section, some references are not in full agreement with the PeerJ Standards.

e.g.

line 1007
ADOLPHS, K. 1996. Bibliographie der Gürtekechsen und Schildechsen (Reptilia: Sauria: Cordylidae & Gerrhosauridae). Squamata Verlag, Sankt Augustin, 255 pp.

PeerJ
ADOLPHS, K. 1996. Bibliographie der Gürtekechsen und Schildechsen (Reptilia: Sauria: Cordylidae & Gerrhosauridae). Squamata Verlag, Sankt Augustin.

line 1022
BOULENGER, G.A. 1908. On a collection of fresh-water fishes, batrachians, and reptiles from Natal and Zululand, with description of new species. Annals of the Natal Government Museum (Pietermaritzburg) 1:219˗235.

PeerJ
BOULENGER, GA. 1908. On a collection of fresh-water fishes, batrachians, and reptiles from Natal and Zululand, with description of new species. Annals of the Natal Government Museum (Pietermaritzburg) 1:219˗235.


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 TAXONOMIC REVISION OF THE SOUTH-EASTERN DRAGON LIZARDS OF THE SMAUG WARRENI (BOULENGER) SPECIES COMPLEX IN SOUTHERN AFRICA, WITH THE DESCRIPTION OF A NEW SPECIES (SQUAMATA: CORDYLIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Attached the pdf file with few minor suggested corrections.


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 TAXONOMIC REVISION OF THE SOUTH-EASTERN DRAGON LIZARDS OF THE SMAUG WARRENI (BOULENGER) SPECIES COMPLEX IN SOUTHERN AFRICA, WITH THE DESCRIPTION OF A NEW SPECIES (SQUAMATA: CORDYLIDAE)
Review round: 2
Reviewer: 2

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: I am pleased to see a much-improved version of the manuscript, where most of the reviewers comments were properly addressed. However, I still believe, given that the authors have plenty of molecular and morphological data, that a species delimitation method should be implemented. Also, a genetic Structure analysis, to see how much admixture there is between these potential taxa, will reveal how genetically isolated these lineages are. If they strongly believe this is not suitable, then they should argue it in the manuscript.  We are now moving into an era of systematics where lots of species we have considered real based on phylogenetic trees and morphological differences are actually not evolutionarily independent entities, therefore I think it is better to include a more robust suite of analyses to support the taxonomy.


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: ASSOCIATIONS OF MODERATE-TO-VIGOROUS PHYSICAL ACTIVITY WITH PSYCHOLOGICAL PROBLEMS AND SUICIDALITY IN CHINESE HIGH SCHOOL STUDENTS: A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Even not being a native English speaker, I found several inconsistencies in the grammar. Therefore, I strongly recommend another grammar revision.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Major

Overall: even not being a native English speaker, I found several inconsistencies in the grammar. Therefore, I strongly recommend another grammar revision.

The authors should avoid the term “psychopathologies” , as they did not evaluated this in the present study.

Even with an extensive literature review in the introduction, the literature gaps that the present article will focus are not clear, please revise this.

The authors should better describe the sampling process, it is not clear in the current version.

It is not clear why the authors adopted the cutoff point of 3 days/week with 60 min of MVPA, it is lower than the recommendations (7d/wk) and even the widely used 300 min/wk.

The authors should avoid the use of dichotomic p-values, it is not necessary to insert p for trend of OR values (it is possible to infer potential differences analyzing 95%CI). It is also possible to infer potential associations using 95%CI from OR values.

The authors did not analyze the sex-differences that they presented as an important objective. For this, it would be necessary to insert interaction terms of sex in the analysis, the authors should revise this.

I am not sure if it is necessary to repeat the results of Table 3 using a figure.

Minor

The title “Gender, physical activity, psychopathologies, and suicidality in Chinese students” is not in line with the manuscript. I suggest something in the line of: Sex-differences in the association between physical activity…

- The authors should better describe the methods on abstract. How MVPA was collected? Which indicators of psychological problems did the authors adopted? Statistics?

- The authors should insert informative numbers in the results of abstract, p-values are not informative and its dichotomic interpretation should be avoided.

- The authors should re-write the conclusion of abstract (it is not clear).

Line 69: There is a more recent paper showing this (GBD 2018)


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: ASSOCIATIONS OF MODERATE-TO-VIGOROUS PHYSICAL ACTIVITY WITH PSYCHOLOGICAL PROBLEMS AND SUICIDALITY IN CHINESE HIGH SCHOOL STUDENTS: A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an inetresting study that examined the association of physical activity and psychological problems in high school students in 10 Chinese cities. The strength include relatively large sample size, valid measurements, analyses stratified by gender, including psychological problems other than depression and anxiety (for instance, ADHD). However, there are several major limitations.
1. Language is a problem and proof reading is needed.
2. The representativeness of the sample is doubtful. Sampling method should be described in detail. All participants are from 10 cities, therefore the results are not generalizable in rural students or other cities in China.
3. There are important potential confounders that have not been addressed in this study. For example, the pressure on academic performances is a well-known stress in high school students. The pressure may lead to less physical activity and anxiety at the same time.
4. Line 214, 'The participants' gender was not significantly different'. I cannot understand this sentence.
5. As shown in table 2, it seems that the prevalence of depression in male were higher than those in female, across all three MVPA groups. Please explain this.
6. The authors used 'prevalence rate' in line 233 and 278. However, prevalence is not a rate. Please use 'prevalence'.
7. Using self-report data is a limitation, particularly when the authors did not report test-retest reliability. Please mention this in the limitation.
8. It would be helpful to examine the association between MVPA and psychological problems combining both genders.
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: ASSOCIATIONS OF MODERATE-TO-VIGOROUS PHYSICAL ACTIVITY WITH PSYCHOLOGICAL PROBLEMS AND SUICIDALITY IN CHINESE HIGH SCHOOL STUDENTS: A CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: None.
Experimental design: None.
Validity of the findings: None.
Additional comments: None.


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

Input:
Title: ASSOCIATIONS OF MODERATE-TO-VIGOROUS PHYSICAL ACTIVITY WITH PSYCHOLOGICAL PROBLEMS AND SUICIDALITY IN CHINESE HIGH SCHOOL STUDENTS: A CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: My concerns have been successfully addressed by the authors. I have no further 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: MITOCHONDRIA-TARGETED TRIPHENYLPHOSPHONIUM-BASED COMPOUNDS DO NOT AFFECT ESTROGEN RECEPTOR Α
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is well written.
Experimental design: I suggested to check if the compounds bind to ERα.
I suggested to add PPT as selective estrogen receptor ERα agonist in addition to E2.
Validity of the findings: It is hard to get the conclusion.
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: MITOCHONDRIA-TARGETED TRIPHENYLPHOSPHONIUM-BASED COMPOUNDS DO NOT AFFECT ESTROGEN RECEPTOR Α
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Figures should be relevant to the content of the article, of sufficient resolution, and appropriately described and labeled.
2. Several original data (Lines 171, 249 and 262) should be given as supporting information even though they are not critical for this research.
Experimental design: The exposure concentrations used in this research should be more reasonable.
Validity of the findings: The conclusions should be appropriately stated, should be connected to the original question investigated, and should be limited to those supported by the results.
Additional comments: Zinovkina and coworkers report TPP-based SkQ1 and C12TPP, as well as the rhodamine-based SkQR1 do not directly interact with ERα and do not induce estrogen-like changes in MCF-7 cells. The present study confirms the general fact that TPP-based compounds are safe to use. I like the idea and methodological concept of the study. However, some major concerns that in my opinion markedly reduce the impact of the study. These can be found below.
Major concern:
(1) In the present study, the MCF-7 cells (estrogen receptor alpha positive) and MDA-MB-231 cells (triple-negative, also known as estrogen receptor alpha negative) were selected as in vitro models to investigate the influence of triphenylphosphonium (TPP)-based compounds and the underling mechanisms as well. By assessing the cell proliferation, cell cycle progression and gene transcription, the authors showed that mitochondria-targeted TPP-based compounds have no direct action on the estrogen receptor α. Herein, no significant differences were found between MCF-7 and MDA-MB-231 cells based on the resazurin assay, which should be deeply explained. As you known, other than the estrogen receptor alpha, both cells are expressed other membrane estrogen receptors (mERS) such as estrogen receptor alpha 36 (ERα36) and G protein coupled receptor (GPR30). Those mERs may also participate in TPP-based compounds-mediated proliferative effects, which should be further investigated.
(2) In the present study, all experiments were conducted 5-6 replicates independently, so the statistical significance should be evaluated and be marked accurately in the figures.
(3) The authors showed TPP-based compounds at higher concentrations significantly inhibited cell proliferation, but these original data and underlying mechanisms were not presented.
(4) The authors showed that TPP-based compounds had no significant effects on the cell cycle distribution unlike that of E2. What I'm focusing on is that dose these TPP-based compounds also have no effects on the gene expression of several cell cycle-regulated protein. In fact, different cell cycle stages (SubG0, G0/G1, S and G2/M phases) should be investigated separately.

Minor issues:
(1) Different concentrations of C12TPP were used in MCF-7 and MDA-MB-231 cells (Line 175-185), is it correct? If correct, the authors should give some basis.
(2) How many duplicate samples were used in the real-time cell analyzer assay? From Figure 4, I cannot find this formation.
(3) English should be improved on some places - for instance, the sentences in Reagents are somehow weird and I recommend rewriting it for better clarity. This also applies to other portions of the text, such as Lines 203-204 and Lines 215-217.


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: MITOCHONDRIA-TARGETED TRIPHENYLPHOSPHONIUM-BASED COMPOUNDS DO NOT AFFECT ESTROGEN RECEPTOR Α
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a descriptive study where the authors demonstrated that triphenylphosphonium-based compounds do not act as estrogen receptor α modulators. Manuscript is in general well delineated, very objective and has appropriate references. Results are in agreement with the methodology and the hypothesis proposed. Although results are negative, it is important to the field to have this information.
Experimental design: Authors added as control a transform cell line; however, results might be different in primary naive cells.
Validity of the findings: No comment
Additional comments: No 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: MORPHOLOGY AND MOLECULAR PHYLOGENY OF THREE NEW DEEP-SEA SPECIES OF CHRYSOGORGIA (CNIDARIA, OCTOCORALLIA) FROM SEAMOUNTS IN THE TROPICAL WESTERN PACIFIC OCEAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Morphology and molecular phylogeny of three new deep-sea species of Chrysogorgia (Cnidaria, Octocorallia) from seamounts in the tropical Western Pacific Ocean
Yu Xu1,2,3,4, Zifeng Zhan1,2,3, Kuidong Xu1,2,3,4

Reviewer Comment
1.	This is an excellent paper that contributes significantly to our taxonomic knowledge of the Octocorallia, and offers descriptions of three new species of the genus Chrysogorgia.

2.	They have created some terms, but unfortunately have not defined them (i.e. amoeba-shaped sclerites, skateboard shaped scales, tree-shaped colony).

I think that when creating a new term, it is necessary to examine the terms of the previous authors and then define the term properly.

3.	For octocoral taxonomy, sclerites need to write down the shapes, sizes, amounts, and arrangements for each of the following portions.

Polyp body, tentacle, pinnule, possibly the basal portion of the polyp, surface layer of the axis or branches (e.g. Pinnules sclerites can be species characters, but are not included in this manuscript.).

It is necessary to write them properly, or if there is a series of common bones, it is necessary to accurately describe the state.

   Sclerites pictures must also be shown for each portion.
 (There is concern about “Sclerites of the polyp neck and tentacles” in Fig. 4A, 6A.
In this case, it is necessary to distinguish between “polyp neck” and “tentacle, and information on the “poly body” is required).

4.	Chrysogorgia is a genus of “sympodial branching”.

In contrast, Pseudochrysogorgia Pante & France, 2010, has been established for species that have the characteristics of Chrusogorgia and are monopodial branching.

While they write that C. dendritica is monopodial, and they cited Pante & France, 2010, moreover they introduced Pseudochrysogorgia in Fig. 9, but there is no textual explanation.

   Need the explanation that C. dendritica is Chrisogorgia, not Pseudochrysogorgia.

   I also suggest that the picture which can confirm that C. dendritica is monopodial is needed.

5.	The References section needs some modification.

6.	I suggest that they refer to the comments I wrote in the manuscript, and that they will be modified.

I hereby recommend this paper for publication in the Peer J.


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: MORPHOLOGY AND MOLECULAR PHYLOGENY OF THREE NEW DEEP-SEA SPECIES OF CHRYSOGORGIA (CNIDARIA, OCTOCORALLIA) FROM SEAMOUNTS IN THE TROPICAL WESTERN PACIFIC OCEAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comments
Validity of the findings: no comment
Additional comments: The description of three new species from deep waters is an important contribution to the biodiversity research and make clear the relevance of these deep water environments that should be preserved.  Every new exploration gives new species to science. The authors follow the taxonomic standards for the identification and description of the new species with an integrative approach. I just have two comment about the figures:
1-Personally, I like to see the size of the scales on the plates, not in the caption of the figure.
2-About the SEMs of polyps and branches, images like the ones in Fig. 3, do not show much detail, the structures do not look clean and they are collapsed. I suggest to fix the fragments for SEM in formalin for  some hours, wash, and follow a dehydration protocol for critical point drying. I think that you will obtain very nice pictures in the future.


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: QSEBC IS INVOLVED IN THE BIOFILM FORMATION AND ANTIBIOTIC RESISTANCE IN ESCHERICHIA COLI ISOLATED FROM BOVINE MASTITIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: English usage in the manuscript:
Clearly, the manuscript needs English editing to avoid confusion and/or misunderstandings. This is especially obvious in the Materials and Methods section but also makes it difficult to understand the meaning of many sentences throughout other parts of the manuscript. For instance lines 79/80: “The status of antibiotics is questioned in the emergence of multidrug resistance.”

Background information:
It is not clear why in the first place a qseBC deletion mutant was created and then tested in the way the authors describe in the manuscript. Also, information on the signal that is sensed by QseC (although this can be found in the literature) is not provided. Furthermore, a paper published in 2014 in AEM by Sharma and Casey (DOI: 10.1128/AEM.03198-13) is not mentioned. This for me at least is a major issue since these authors created a similar mutant in EHECD O157:H7 and examined the effects on colonization, motility and expression of motility and virulence genes. Although from a different E.coli strain, the data published are highly relevant for this manuscript and should have been included and discussed.
Especially since there are conflicting results: csgA and fliC genes are reported here to be downregulated in the mutant when compared to the isogenic wt strain whereas in the manuscript mentioned above fliC transcript levels were unchanged (Fig. 4) and csgA was upregulated in the mutant (Fig. 6).
Experimental design: 1. Although the original test for antibiotic susceptibility was performed using WT and isogenic XL4 strains (without additional plasmid) all other experiments were done using the stains containing pSTV28 or pCqseBC using Chloramphenicol selection. To be able to draw conclusions from the results shown in Figs. 1-4 I would consider it to be an absolute requirement to show data for WT and XL4 without additional plasmids and without antibiotic selection.
2. A standard for evaluation of the effect(s) of an introduced mutation should be a simple growth curve (for all strains) to demonstrate that all strains grow equally well (growth rate) and to a similar final OD.
Validity of the findings: Statistics: It is not clear how many biological replicates (not just repeated measurements = technical replicates) were tested to generate mean values. E.g.: Fig. 3A. Was the biofilm assay only performed once? This is also relevant for all other data that are presented.

Figures: Fig.1: What is “Microbal concebtration”. What is the definition of “survival rate” (also used in the text lines 161, 202, 204).

Conclusions: From the presented data it cannot be concluded that efflux pump genes or biofilm associated genes are regulated directly by the QseBC two-component system. More experimentation is needed to show a direct involvement of QseB in transcriptional regulation of target genes, for instance promoter fusions and/or DNA interaction studies.
Additional comments: The manuscript by Li et al. describes the genetic manipulation of an E.coli isolate isolated from a cow with mastitis. A qseBC deletion mutant was generated, copmlemented, and tested for effects on antibiotic susceptibility and biofilm formation. The data are complemented by qRT-PCR of some genes involved in efflux pump activity or biofilm formation.
The data are rather preliminary and need more experimentation to allow meaningful conclusions about the role of the QseBC system in antibiotic resistance and biofilm formation of E.coli.


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: QSEBC IS INVOLVED IN THE BIOFILM FORMATION AND ANTIBIOTIC RESISTANCE IN ESCHERICHIA COLI ISOLATED FROM BOVINE MASTITIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: General Comments: The authors address the possible role of the two-component system QseBC in antibiotic susceptibility and biofilm formation, and determine the transcription level of a series of genes known to be related to both phenotypes in the presence and absence of this system. Their results show that the qseBC mutant shows decreased resistance to some antibiotics, and is partially deficient in biofilm formation. qPCR results indicate lower transcription levels of the studied genes in the mutant strain. The work is well presented, the addressed subject is interesting, and the experiments are well designed and executed in general. However, there are some issues which need to be addressed.

Main objection: in qPCR, all of the tested genes are downregulated in the mutant. How can we discard that the mutant is affecting transcription in general? A control gene should be included which does not show differences between the wild type and the mutant, so that we can conclude that there is a specific effect of the lack of the two-component system on the transcription of the target genes. Without this control, I think the final conclusion cannot be drawn.

Please revise
-	Introduction needs to include previous works on QseBC roles in different E coli strains. A very quick search in PubMed rendered several articles which are relevant for the present work. In particular, in lane 94 the authors do not cite the work by Gou et al (doi: 10.1139/cjm-2019-0100) addressing precisely this point. Moreover, the authors find no effect of qseB mutant in biofilm formation. This very relevant information should be included and the authors should discuss this discrepancy with their own data.
-	Also, the works relating efflux pumps and antibiotic susceptibility, and the relationship of the studied genes with biofilm formation, should be included in the introduction rather than in the Discussion (256-263; 271-276), since they provide the reasoning for targeting the transcription levels of these genes.
-	Different effects of complementation in recovery levels of Ab susceptibility for each Ab (Fig1), and qPCR results (Fig 2). Please discuss.
-	How many times were the antibiotic susceptibility tests performed? It is not mentioned in either MM or Table 3. If only once, they should be repeated.

Minor comments
line 90: other in place of others
91-93: indicate the microorganisms for which these effects have been shown in each case
122: into instead of ioto
131: remove frame (That´s what the F of ORF stands for)
156 last two words don´t match
161 exposed, not exposd
209: please cite the appropriate references reporting the association of these genes with antibiotic resistance.
223: transcription levels were restored in the complemented strain only in certain cases. Please enumerate and discuss.
247: add: … except for wcaF (Fig 4). Please write wcaF properly in Fig 4 (it reads wacF).
Throughout the MS: milliliter is ml, not mL. Same with microliter.
Table 3: why is gentamycin abbreviated as CN? It is confusing.
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: QSEBC IS INVOLVED IN THE BIOFILM FORMATION AND ANTIBIOTIC RESISTANCE IN ESCHERICHIA COLI ISOLATED FROM BOVINE MASTITIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Language issues
1) In the revised version of the manuscript English language issues have been addressed in most cases and resolved. However, the meaning of some sentences is not clear. E.g.: 77: "The efficacy of antibiotics is suspected in the emergence of multidrug resistance." Maybe the authors mean "The efficacy of antibiotics in the treatment of bacterial infectious diseases is heavily affected by the emergence of multidrug resistance."?
80: "In bacteria, the two-component system is one of the strategies that helps them to respond and adapt to various environmental stimuli quickly, particularly through changes in target gene transcription (Beier & Gross 2006)." An understandable alternative could be: "Two-component systems are essential for bacteria to quickly respond and adapt to various environmental stimuli, particularly through changes in target gene transcription (Beier & Gross 2006)."
256 and elsewhere: authors use "complementary" to indicate that the effect of a mutation has been complemented
283 and elsewhere: qseBC is not a "gene"
To avoid confusion and misunderstandings I suggest professional language editing prior to publication.

Background information:
In the revised version several references were incorporated to provide a better overview on the relevant literature.
Experimental design: All issues have been addressed and properly resolved. Additional results have been added to improve the manuscript.
Validity of the findings: 1) An additional EMSA experiment included in the revised version clearly demonstrates binding of QseB to a target sequence in the marA promoter region. From that, however, it cannot be concluded that "QseB activated the expression of marA ..." - because this is speculative and should be identified as such (Discussion: lines 341-342; Conclusions: lines 357-359; and lines 40-41).
I agree with the notion that QseB binds to the marA promoter. This suggests a possible role of QseB in activation of marA expression, encoding a known activator of acrAB efflux pump genes. In addition, this proposed function of QseB (as a direct activator of marA) is supported by the effects of the qseBC mutant on the transcription of efflux-pump-associated genes.

2) Table 3 shows a reduced resistance of the XL4 qseBC mutant strain toward Ciprofloxacin. None of the other tested antibiotics showed a change in the MIC when comparing WT/XL4 strains. This should be commented in the results section (lines 249-260) and discussed. Why is only CIP resistance affected? Obviously, QseBC is not involved in general AMR but rather has a very specific effect on a rather limited spectrum of antimicrobials.
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: QSEBC IS INVOLVED IN THE BIOFILM FORMATION AND ANTIBIOTIC RESISTANCE IN ESCHERICHIA COLI ISOLATED FROM BOVINE MASTITIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have successfully addressed all my previous concerns on their work
Experimental design: The authors have successfully addressed all my previous concerns on their work
Validity of the findings: The authors have successfully addressed all my previous concerns on their work
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: TEMPORAL CHANGES IN THE MOST EFFECTIVE POLLINATOR OF A BROMELIAD POLLINATED BY BEES AND HUMMINGBIRDS
Review round: 1
Reviewer: 1

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

Line 31: Note that the use of “efficacy” “efficacious” (e.g., line 288 and elsewhere) might be confusing for the general reader. Please consider explaining somewhere in the MS the difference between most efficient pollinator, most effective pollinator and most efficacious pollinator. Also, the use of “intermediary” (line 52) might be misleading, try “intermediate”. In general, the text is clear, unambiguous, and professional English language is used throughout.

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

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

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

Raw data supplied (see PeerJ policy). 26]. All data generated in this study have been presented. Though morphological data should be publicly available, perhaps at Dryad or as supplementary in PeerJ? Some of the videos of floral visitors would be neat to see at PeerJ.
Experimental design: EXPERIMENTAL DESIGN
Original primary research within Scope of the journal.
Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap.
Rigorous investigation performed to a high technical & ethical standard.
Methods described with sufficient detail & information to replicate.

Line 147 and elsewhere. Please explain if flowers last one day.

Table 2. Based on the list of floral visitors, it would be interesting to see whether all hummingbird species are similarly effective pollinators and abundant over time.
Validity of the findings: VALIDITY OF THE FINDINGS
Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
Data is robust, statistically sound, & controlled.
Conclusions are well stated, linked to original research question & limited to supporting results.

Lines 282–297. The occurrence of PL was recorded in E. lindenii only in the year in which there was a strong decrease in the frequency of visits by hummingbirds, when large bees were the most frequent visitors. Thus, the effectiveness of large bees alone was not enough to achieve the potential seed set fully. This is correlative. An experimental approach might be needed to tease apart the causal factors incl. an experiment in which flowers are pollinated by both functional groups.

Lines 290–292. Lastly, combined measurements of PL and pollinator assemblages along time and space is an interesting approach to evaluate the effects of variable pollination environments (see Gómez & Zamora, 2006 for other suggestions in this regard). Indeed. Hard work is needed to get both temporal and spatial variation of pollinator environments in plants with generalized-pollination systems and parthenocarpic plants as the one studied. Please explain the implications of these in the Discussion.

Line 307 and elsewhere. It might be useful to discuss further the meaning of the positive response to nectar removal in the studied species in terms of its pollination dynamics and what has been found in other species of bromeliads (e.g., Ordano & Ornelas 2004, Oecologia).

Lines 318–324. Wessinger and Kelly (2018) found a relationship between self-compatibility and attributes related to the attraction of hummingbirds, including red flowers and loss of floral aroma and UV-absorbing pigments. Most hummingbird-pollinated species are self-compatible (Wessinger and Kelly 2018; see also Raduski et al. 2012 Evolution, Wolowski et al. 2013 The Science of Nature). If the studied plant species is more specialized on hummingbird pollination, bird-flower traits might be selected with increased hummingbird visitation over evolutionary time, resulting in specialization to (e.g., replenishment of removed nectar, large amounts of nectar) and the evolution of floral traits present in hummingbird-pollinated species (Wessinger and Kelly 2018, Salas-Arcos et al. 2019 The Science of Nature). It seems that your data points toward that direction.

Lines 329–331. Do you have data on pollen transfer? Are hummingbirds most effective in pollen transfer among plants?

Speculation is welcome, but should be identified as such.
Additional comments: Review of PeerJ-2019-35011

Roberta L B Leal, Marina M Moreira, Alessandra R Pinto, Julia O Ferreira, Miguel Rodriguez-Girones & Leandro Freitas

“Temporal changes in the most effective pollinator on a bimodal system involving bees and hummingbirds”

The authors evaluated the pollination effectiveness of the floral visitors of Edmundoa lindenii (Bromeliaceae) across four reproductive events. They analyzed the frequency of floral visitors (large bees, small bees, and hummingbirds) through focal observations and their single-visit efficacy (seed set). Pollen limitation (PL) index was estimated comparing seed set after hand cross and natural pollination. Edmundoa lindenii is self-incompatible and parthenocarpic, requiring the action of pollinators for reproduction. Hummingbirds have greater efficacy than large bees, and small bees act as pollen robbers. The frequency of floral visitors varied among years, and overall hummingbirds were more effective than large bees. The PL index also varied over time, with limitation only occurring in the reproductive event of 2017, when hummingbirds were scarce. The authors conclude that a generalist species can suffer or not PL in different reproductive events, in response to variations in the pollinator assemblage. Although the evolution of a generalized pollination system is expected when different pollinators play the same role as selective agents, their results support the hypothesis that generalization may also be favored when less effective pollinators provide reproductive assurance, lightening fluctuations of the most effective pollinators, such as could be the case for large bees and E. lindenii. Their conclusions are well stated, linked to original research question and limited to supporting results. However, there are few important issues (mainly in the Discussion section) that must be considered before this manuscript is recommended for publication in PeerJ. I offer the following broad areas of criticism and suggestions for improvement:


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: TEMPORAL CHANGES IN THE MOST EFFECTIVE POLLINATOR OF A BROMELIAD POLLINATED BY BEES AND HUMMINGBIRDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Writing in general is good. However, as a non-native writer, I suggest a deeper proof reading by a native writer. However, I see the major problem in the conceptual grounds. Albeit the study have not deep complex matters, the approach is in some sense a list of good measurements on the reproductive biology of an interesting system. Thus, the link between the pollination modes and the factors explaining the variation in phenotypic traits involved in the interaction and in the plant reproductive success, would be articulated in a deeper way,
In one way, the study rely on the reproductive biology of an interesting system, and less in the drivers of pollination limitation and their relationship with pollinator functional groups.
I am some uncomfortable, because the idea sounds good, but in each section the manuscript have presentation problems. This appears as a study case of reproductive biology, more than the title and abstract promet to readers.

In other aspects, I agree with data sharing. However, lack codes of sheets and variables in the datasheet. Also, revise English, there are minor finger mistakes.
Following with this transparency guidelines, it would be fine see our studies meeting the three basic rules of modern reporting, repeatability, reproducibility, and verifiability. However, this study fails respect to these points. Some points of methods must be revised and reported. Data analysis is foggy, and together data, you would be show R scripts. I found here some point for reviewing or for deeper explanations about why and how you did that you did.

Figures may be improved. Moreover if you are an R user.
Experimental design: The sampling protocol in the field is in general good. And by my experience, is generally difficult get a lot of data from this type of systems. However, several points in the methods section require a clearer presentation.
Validity of the findings: I search several times what is the novelty of this study. I find the system very interesting, also with "appeal". However, the conceptual aspects are entangled in the introduction and in the discussion. Moreover, some results referred as conclusions, have not their corresponding statistical table (for example, models). Because hypothesis and predictions are lost in the sea of a good list of traits and matters of the system, then the conclusions appear poorly linked to those conceptual grounds.
Additional comments: This is a pretty system. I encourage you to do a deeper work for improve the presentation and clarity of the idea that you wish to communicate.


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: TEMPORAL CHANGES IN THE MOST EFFECTIVE POLLINATOR OF A BROMELIAD POLLINATED BY BEES AND HUMMINGBIRDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The new version of the manuscript is better than before.
Experimental design: The data analysis is confusing.

The analysis of variance is a case of linear model. Then, you should re-write the sentences where you refer to ANOVA and linear modelling.

If you apply glm to the frequency, then you might to do the same to other response variables. Thus, you may preclude the ANOVA and apply the same approach (glm or lmer) for all cases, with differences in the error structure (Normal, Poisson, others).

On the other hand, modelling is also confusing. For example, year is a random factor when you analyze the success, but year is a fixed factor when you analyze the frequency.

You must be congruent to analyze the response variables. Then, you should take care for writing in a conceptually correct way.
Validity of the findings: In the new version of the manuscript, discussion and conclusions are better stated than before.
I found a bit of noise in the fact that the findings are not completely articulated. For instance, at the conclusions the authors wrote: "The factors that influenced the temporal variation in pollinator frequencies in our system of study are not well understood and cannot yet be predicted." The year-to-year variation is treated as a "black-box", with unmeasured factors possibly driving this variation. Perhaps the main point of the study is represented by the differences between bees and humingbirds, and the functional roles of the different groups.
Additional comments: The presentation of the study is clearly better than before. However, I found some noises yet. You should improve the modelling and/or their corresponding explanations in the Data Analysis section.
On the other hand, in conceptual terms, the relationship between the differences among different types of pollinators and the temporal variation need a deeper work.


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

Input:
Title: EFFECTS OF AFFORESTATION WITH PINUS SYLVESTRIS VAR. MONGOLICA PLANTATIONS COMBINED WITH ENCLOSURE MANAGEMENT ON SOIL MICROBIAL COMMUNITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is very clear and interesting descriptive study on soil micro biota under different treatments.
There is a clear background, but more details may be needed (see below)
Experimental design: the experimental design seems to be appropriate. See below for comments on some clarifications that may be needed.
Validity of the findings: Results are robust and clear, but some extra analyses may be needed .
Additional comments: This is very clear and interesting descriptive study on soil (micro) biota under different treatments in China. This provides very useful information of how different scenarios may drastically change soil biota. I consider that this text is very clear, but some aspects may need some clarification.

There are some typos in the text (not many, but some), so please take a look at it in detail again. For example, in the “Determination of vegetation type and soil samples collection” section, the first sentence in not clear. I suggest you change it to “We selected four 1-ha plots of Pinus sylvestris var. mongolica plantations…”. In line 180, change the comma for a dot. It should read “existed. In order”.  Change the word castration after litter. I am not exactly sure what you mean there, but castration is generally use in animal ecology for something very different, so it is confusing. These are just examples; please take a closer look at the text.

Explain better why Arachis hypogaea is an ideal control. This is not clear why you used it. Is it because it is may vegetation when pines are not there? I think that in “Site information” you need to explain more about the local ecosystems (e.g. pines are native there or they are just use for forestry?).

Since you worked with pine afforestation, I think is important if you talk about mycorrhizal species. Specially ectomycorrhizal species. You mention Wilcoxina in the text, that is a very common mycorrhizal species. You do a very detailed analysis of the diferent groups of fungi and bacteria, but it would be super interesting to know the role that each have. For mycorrhizal species, this may not be too hard to do and I think can add a lot (do mycorrhizal species decrease or increase under grazing?)

All in all, I think that this is a very solid contribution, but some details may be needed to make it even more interesting and relevant to the international scientific community.


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

Input:
Title: EFFECTS OF AFFORESTATION WITH PINUS SYLVESTRIS VAR. MONGOLICA PLANTATIONS COMBINED WITH ENCLOSURE MANAGEMENT ON SOIL MICROBIAL COMMUNITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: I was looking forward to reading this manuscript. It turned out to be  a rather disappointing because of the lack of clear objectives, hypotheses and by the experimental system (peanut - a cultivated species of South American origin vs. Pinus).
The manuscript is wholly descriptive, and the findings are readily predictable (verge on the commonplace). The langue requires considerable polishing.
Experimental design: I found the experimental set-up rather strange. Why did the authors choose to compare enclosed and unenclosed Pinus plantation with a peanut field, instead of native (degraded?) vegetation?
Research questions are not well defined, or relevant / meaningful. Nor is stated how the research reported fills identified knowledge gaps.
The Methods are somewhat difficult to read to replicate field design / sampling.
Validity of the findings: As there are no well-defined original research questions the discussion and the conclusions are difficult to judge.
Additional comments: The authors have failed to establish:

(1) why the submitted work is important and timely (what lacunae in knowledge it addresses),
(2) what hypotheses or questions the reported work addresses ,
(3) how the work fills the identified gaps in knowledge and how the work overall advances our understanding.


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

Input:
Title: EFFECTS OF AFFORESTATION WITH PINUS SYLVESTRIS VAR. MONGOLICA PLANTATIONS COMBINED WITH ENCLOSURE MANAGEMENT ON SOIL MICROBIAL COMMUNITY
Review round: 1
Reviewer: 3

Output:
Basic reporting: This article is well writing. The research topics are clear and the article structure conforms to the structure of scientific articles. However, the topic of this research is not very innovative; it is a common issue with several similar reports. However, relatively few related references have been cited. I suggest that this study can compare previous studies and put forward more direct hypotheses and address the differences and novelty of this study from other studies.
Experimental design: The experimental design for the manipulation is not a big problem. Nevertheless, I have some questions in statistical analysis. Since RDA is performed, I would like to see the proportion of explanation and significance of each constraint factor (the significance of each factor can be tested by type-II ANOVA). Besides, since constrained factors may be collinear between each other because various soil factors may interfere or affect each other. I recommend that you exclude collinear factors before performing RDA to avoid unnecessary weighting caused by multicollinearity.

In addition, I think it is necessary to explain why RDA and CCA are done.
Validity of the findings: no comment
Additional comments: Some other comments are listed below:
Introductions
1.	What is enclosure management? How is enclosure management performed in this forest?
2.	Addition to "better understand the afforestation effect combined with enclosure management", are there any clearer research questions and hypotheses of this study?

Materials and Methods:
1. RStudio is a program to help conduct R. You should use functions in packages of R to perform the analyses with the assistance of RStudio instead of using packages in RStudio for the analysis.
2. R is sensitive to the upper and lower cases, so the packages used in R should be written carefully in upper/lower case. Packages vegan and car, for example, should be written in lower case (vegan and car), instead of Vegan and Car.
3. How many samples used in this study?
4. L233-234: Write the F statistic behind each soil factors rather than put all of the F values together.

Results and Discussion
1.	In Table 2, the P value should not be <0.00.
2.	Detailed data (including statistical data) should be displayed in the tables, not in the main text. In the main text, you should put the summarized comparisons and statistics instead of details.
3.	In the discussion, the soil microbes mediate the carbon sequestration is interpreted. The discussion about causality should be more cautious, because correlation cannot be directly equivalent to causality, it is just correlation.
4.	Many paragraphs are too long, especially the discussion. I would suggest segmentation.
5.	The authors believe that afforestation can increase soil carbon and nitrogen concentrations. Although the authors have data to support higher soil carbon and nitrogen concentrations in afforested land, this composition may also be that the original carbon and nitrogen concentrations of these grounds are high. Also, vegetation restoration and enclosure management were suggested to be able to increase the richness of microbial communities in soils. Are there more direct evidences that afforestation will directly increase soil carbon and nitrogen concentrations? Furthermore, is there any evidence that the increase of microbial richness is not just due to the high richness of microorganisms in these plots originally? I have read that the authors have cited several papers to support these inferences. However, if just citing various references to support these inferences rather than providing more manipulated or statistic evidences, what is the value of this research data? Therefore, I would expect to see more proper manipulated or statistic evidences, rather than just providing more references.
6.	Species compatibility, cooperation, and competition among microorganisms are quite high. When the external environment changes, the change of microorganisms will not be in a single taxon, but a set or sets of functional groups. These comparisons of microbial community diversity before and after the afforestation or enclosure management should take into account interactions between species, at least some discussion should be provided.
7.	I think that the beta-diversity should also be compared directly, not just comparing the difference between alpha-diversities of different communities.


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

Input:
Title: EFFECTS OF AFFORESTATION WITH PINUS SYLVESTRIS VAR. MONGOLICA PLANTATIONS COMBINED WITH ENCLOSURE MANAGEMENT ON SOIL MICROBIAL COMMUNITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: the authors have responded to all my concerns and now the manuscript is stronger and clearer. Thanks for your work on this!
Experimental design: the authors have responded to all my concerns and now the manuscript is stronger and clearer. Thanks for your work on this!
Validity of the findings: the authors have responded to all my concerns and now the manuscript is stronger and clearer. Thanks for your work on this!
Additional comments: the authors have responded to all my concerns and now the manuscript is stronger and clearer. Thanks for your work on this!


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: ITERCLUSTER: A BARCODE CLUSTERING ALGORITHM FOR LONG FRAGMENT READ ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1) The manuscript needs serious editing and proofreading to be more readable.
The main idea of this paper was already presented in the following manuscript:  Minerva: An Alignment and Reference Free Approach to Deconvolve Linked-Reads for Metagenomics (https://www.biorxiv.org/content/early/2018/07/21/217869)

The similarities and differences of this method with Minerva should be addressed and discussed:

2) In addition to comparison with Minerva, there should be a comparison with alignment-based method since a human reference genome or an assembly is available.
Experimental design: 1) It’s not clear how the -g value is determined?

2) What is exactly a divide-and-conquer assembly?

3) There should be a test on full human genomes.
Validity of the findings: 1) stLFR data characteristics are not discussed. All the way through the results section, it seems to me that it’s similar to the 10x Genomics data, but then the authors could not run supernova on this data set? Why? what are the unique features of sfLFR?
Additional comments: The stLFR data is a new linked-read technology but there is not really a discussion about it. This should be discussed in details.


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

Input:
Title: ITERCLUSTER: A BARCODE CLUSTERING ALGORITHM FOR LONG FRAGMENT READ ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: - In this paper, authors presented a method for clustering the barcoded long fragment reads generated from linked-read sequencing technologies. The proposed method is based on alignment-free paradigm and utilizes the k-mer content of reads to cluster them into target regions in the genomes. To evaluate the performance of their method, authors used two datasets from BGI stLFR and 10X genomics chromium read data. Partitioning reads with a single barcode into clusters that correspond to a single long fragment of DNA is one particular issue common to all applications of the linked-read technology. The problem and idea of clustering long fragment range data is very interesting and will be widely encountered in the area. However, the manuscript has a number of shortcomings, ranging from Introduction and Method details to the presentation of results and conclusions. There are several major concerns to need a substantial amount of time to be resolved as follows. Here are several examples falling into each category.

- There are so many typos/mistakes in English of the manuscript. So, I suggest asking a professional proofreader to improve the English of the paper.

- Authors should modify Introduction/literature to include recent studies on clustering linked read/ read cloud data. For example, there is a new tool Minerva recently published for barcode deconvolution of linked-read/read-cloud data: "Minerva: An Alignment and Reference Free Approach to Deconvolve Linked-Reads for Metagenomics"
Experimental design: - Have authors considered and tested other clustering approaches instead of MCL?

- Runtime and memory efficiency are two important features on clustering large-scale linked-read/read-cloud datasets. I don’t see any runtime and memory usage performance analysis of the proposed method. Author should provide comprehensive analysis on these features.

- There is no justification on choosing the k-mer length from the statistics of k-mer coverage profile. How do the authors choose the optimal value of k for the comparison? I would also like to see the runtime/memory performance for different values of k.

- Based on the hash map data structure presented in Fig 2, the required memory for large-scale long-range data could be very large and may not fit on a single compute node. Therefore, the scalability of the proposed algorithm to large-scale datasets could be an important challenge.

- There is no specification for the datasets used in the experiments. Author should provide a table for dataset specification. Further, the URL links to the datasets used in the experiments are not working.

- Author should provide a help manual for running IterCluster in Supplementary Data and on the GitHub page. They also should explain all details and options for running the tool.
Validity of the findings: - Results section is too preliminary unfortunately to convince that the proposed method is a significant tool to take advantage of long fragment range data to improve downstream results such as de novo assembly. Using the proposed method on just chromosome 19 data for evaluating the assembly performance improvement is not robust enough for validation of the proposed tool. So I recommend to use a larger dataset for this section.

- Runtime and memory usage should also be reported in the Results section as a table.

- It would be interesting to see the performance comparison of IterCluster and Minerva in clustering the barcoded long fragment reads.
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: ITERCLUSTER: A BARCODE CLUSTERING ALGORITHM FOR LONG FRAGMENT READ ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Overall Evaluation
The authors seem to have produced a fine tool with a meaningful use case and an interesting approach. With the revision, this paper is likely to be publishable. However, the manuscript as is still fails to clearly describe how the algorithm works and how it was evaluated.

In particular, the authors do not include an overall description of their algorithm in the methods section. This is by far the biggest issue with the current manuscript and is the root of most other issues.

In their response, the authors protest that making modifications requested by the reviewers would take excessive compute time. While this may be justified for Minerva which is designed for microbial samples (see notes below) it is not justified for alternate clustering algorithms or for comparisons to the human reference genome. The current paper does not broadly enough establish a comparison between the presented technique and other reasonable methods.

Major Points
The authors do not clearly explain their algorithm. The section describing the algorithm includes many section explaining subcomponents of the algorithm but it is not obvious how these components are used together. The authors should add a description of the algorithm overall.

As requested before, the authors should compare their method to direct alignment with the human reference genome. We understand that this method does not require a reference genome but the comparison is still necessary to establish a performance baseline.

The authors confude programmatic parameters (i.e. -g) with the mathematical concepts they represent. The main text should not include references to programmatic parameters and should clearly explain what user-defined values can be supplied to the algorithm and how these are chosen.

The description of Markov Clustering (lines 195-206) explains the issues associated with clustering in this context but it does not explain why Markov clustering is useful in this context. This description is critical as Markov clustering is a family of related algorithms. The authors must both define why Markov clustering is useful in this context and explain how Markov clustering works in their method.

The authors seem to present their issues with 10x reads as failures of the data rather than their algorithm. The authors focus too much on the differences between 10x and BGI stLFR in their algorithms performance. This paper is absolutely not an evaluation of the differences between these two data types and should not be presented as such.

The Description of Data Analysis and Data Sources should be more clearly separated from the description of the algorithm. Though both of these are, arguably, methods sections the Data Analysis and Data Sources methods would be more naturally included with the results section since that is where they are relevant.

The Data Analysis section is far too abbreviated. The authors should much more clearly explain their methodology and evaluations. In particular, the authors should:
•	Separate data filtering and quality control and justify why these parameters are different for the two datasets.
•	Move all commands and parameter lists to the supplements. These choices should be explained in the text but merely presenting the parameters does not justify the choice of filter. Few readers will be intimately familiar with the exact interface of the tools being used.
•	Because the overall algorithm is not clearly described it is nearly impossible to follow the exact steps used in the evaluation. After adding an overall description of the algorithm the authors should directly reference which steps match which parts of the algorithm
•	More clearly define what indicates a true-positive and false-positive in this context.
•	Show the cluster size.
•	Include comparisons to other reasonable techniques (like using the reference genome or other clustering algorithms.)

Minor Points
The authors have misspelled ‘clustering’ as ‘clusting’.

Not always clear what the authors intend you to look for in figures. It would benefit readers if figure references included ‘hints’. I.e. the reference to figure 1C in line 109 is a reference to a cartoon showing how false positives can occur but from context I expected a figure showing the false positive rate

The caption on figure 3 is not informative. It’s not clear what k, f, and g are from the figure.

The choice to filter reads which span fewer than 500bp (line 232) feels aggressive. How many reads does this choice remove?

The use of precision/recall to measure cluster accuracy could be supplemented by other simpler metrics.

Comments on Minerva
-	Minerva is designed for metagenomic samples which are typically much smaller than human genomes. The authors should mention this.
-	The manuscript would benefit from a more clear distinction between barcode deconvolution and barcode clustering as this seems to be confusing
-	When running on a single chromosome (19) it is likely that most of the barcodes are already deconvolved which is likely why Minerva produces little output
Experimental design: no comment (see 1. Basic reporting)
Validity of the findings: no comment (see 1. Basic reporting)
Additional comments: no comment (see 1. Basic reporting)


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

Input:
Title: ITERCLUSTER: A BARCODE CLUSTERING ALGORITHM FOR LONG FRAGMENT READ ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed some of my comments in the previous report but the manuscript still needs more improvement.
Experimental design: I tried several times to build the binaries and perform some tests and comparison of the proposed method but due to some hardcoded environmental variables in the scripts I was not able to do so. Authors should resolve all errors and bugs in building the binaries.

$ sh make.sh
make.sh: line 3: /ldfssz1/MGI_ALGORITHM/assembly/huangweihua/bin/cmake-3.9.1/bin/cmake: No such file or directory
Errors executing CMake. Fix errors and run again.

$ sh build.sh
CMake Error: The current CMakeCache.txt directory /var/tmp/IterCluster/IterCluster/CMakeCache.txt is different than the directory /ldfssz1/MGI_ALGORITHM/assembly/wengjiancong/My_software/IterCluster-MCL where CMakeCache.txt was created. This may result in binaries being created in the wrong place. If you are not sure, reedit the CMakeCache.txt
make: /ldfssz1/MGI_ALGORITHM/assembly/huangweihua/bin/cmake-3.9.1/bin/cmake: Command not found
make: *** [cmake_check_build_system] Error 127

Modify all make scripts such as makefile.cmake, build.make, make.sh, Makefile, CMakeCache.txt, … and change the hardcoded vars like:

CXX=/share/app/gcc-5.2.0/bin/g++
DBOOST_ROOT=/ldfssz1/MGI_ALGORITHM/assembly/huangweihua/bin/boost_1_46_1
Validity of the findings: The experimental results are not complete.
- What would be the runtime and memory usage of the proposed method for larger k values? I would also like to see the runtime and memory usage of IterCluster for k values like k=20, 30, and 40.
- How scalable is the proposed method on 50X human whole genome dataset with different number of threads?
Additional comments: There are some typos in the text:
Line 179: frequency --> frequencies
Lines 262, 263, 277, 283: clusting --> clustering
Line 273: fragment --> fragments

Further: change all “kmer” to “k-mer” to be consistent.

There is no link to the GitHub page of IterCluster in the main text. Please add https://github.com/WengJianCong/IterCluster to 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: ITERCLUSTER: A BARCODE CLUSTERING ALGORITHM FOR LONG FRAGMENT READ ANALYSIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: See the attached PDF
Experimental design: See the attached PDF
Validity of the findings: See the attached PDF
Additional comments: See the attached PDF


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: ITERCLUSTER: A BARCODE CLUSTERING ALGORITHM FOR LONG FRAGMENT READ ANALYSIS
Review round: 3
Reviewer: 2

Output:
Basic reporting: My comments for the previous version have been addressed.
Experimental design: I have no further comments.
Validity of the findings: I have no further comments.
Additional comments: No additional comments


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

Input:
Title: OOCTONUS VULGATUS (HYMENOPTERA, MYMARIDAE), A POTENTIAL BIOCONTROL AGENT TO REDUCE POPULATIONS OF PHILAENUS SPUMARIUS (HEMIPTERA, APHROPHORIDAE) THE MAIN VECTOR OF XYLELLA FASTIDIOSA IN EUROPE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article uses clear and unambiguous, professional English. The submission includes introduction and background enough to put the work fits into its broader field of knowledge. Relevant literature is appropriately referenced. The text has a proper structure, figures, tables and raw are offered for sharing, and are consistent and technically sounded to the content of the article.
The submission is self-contained with relevant results to hypotheses.
Experimental design: The proposal is primary research within the Aims and Scope of the journal, and the research question is well defined, relevant & meaningful.
The investigation must have been conducted to a high technical standard. The research must have been conducted in conformity with the prevailing ethical standards in the field.
Methods described with sufficient detail & information to replicate.
Validity of the findings: Impact and novelty are assessed.

not all the data on which the conclusions are based are made available in an acceptable discipline-specific repository. Some flaws in the data are discussed.
Conclusions are well stated, linked to original research question & limited to supporting results.
Speculation is identified as such.
Additional comments: Please consider the annotated .pdf or the Word file


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: OOCTONUS VULGATUS (HYMENOPTERA, MYMARIDAE), A POTENTIAL BIOCONTROL AGENT TO REDUCE POPULATIONS OF PHILAENUS SPUMARIUS (HEMIPTERA, APHROPHORIDAE) THE MAIN VECTOR OF XYLELLA FASTIDIOSA IN EUROPE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Well written and organized, interesting results. Please see attached pdf file with comments and suggested changes to improve the manuscript.
Experimental design: ok
Validity of the findings: ok
Additional comments: see attachment


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: OOCTONUS VULGATUS (HYMENOPTERA, MYMARIDAE), A POTENTIAL BIOCONTROL AGENT TO REDUCE POPULATIONS OF PHILAENUS SPUMARIUS (HEMIPTERA, APHROPHORIDAE) THE MAIN VECTOR OF XYLELLA FASTIDIOSA IN EUROPE
Review round: 2
Reviewer: 1

Output:
Basic reporting: English is clear, unambiguous, and technically correct. Courtesy and expression is respected. References are sufficient for the context.

Introduction and background demonstrate the work fits into the broader field of knowledge, prior literature is appropriately referenced. There is enough structure, figures, and tables.

The article is structured in standard sections.

Figures are not abundant but enough relevant to the content of the article.

Any raw picture is available, the study is self-contained with appropriate results to hypotheses.

The coherent bodies of work is appropriately subdivided.
Experimental design: This is one original primary research within the Aims and Scope of the journal.
The research question is well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.

The submission clearly defines the relevant and meaningful research question. The knowledge gaps are identified and investigated, the study contributes to filling that gap.
Rigorous investigation performed to a high technical & ethical standard.

The investigation has a rigorous and high technical standard, given in conformity with the prevailing ethical standards in the field. The methods are described with sufficient detail & information to replicate to be reproducible by another investigator.
Validity of the findings: The submission defines a field of interest and encourages to explore the topic by a rational approach.

The data on which the conclusions are based are available in an acceptable discipline-specific repository. The data should be robust, statistically sound, and controlled.
Conclusions are well stated, linked to original research question & limited to supporting results.

The conclusions are appropriately stated, connected to the original question investigated, and limited to those supported by the results.

Speculations are identified as such.
Additional comments: please consider the comments added into the U/L .pdf that comprehends the submission, the rebuttal letter, and the AppendixS1


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: OOCTONUS VULGATUS (HYMENOPTERA, MYMARIDAE), A POTENTIAL BIOCONTROL AGENT TO REDUCE POPULATIONS OF PHILAENUS SPUMARIUS (HEMIPTERA, APHROPHORIDAE) THE MAIN VECTOR OF XYLELLA FASTIDIOSA IN EUROPE
Review round: 2
Reviewer: 2

Output:
Basic reporting: ok
Experimental design: ok
Validity of the findings: ok
Additional comments: Please find below my comments on the manuscript entitled “Ooctonus vulgatus (Hymenoptera, Mymaridae), a potential biocontrol agent to reduce populations of Philaenus spumarius (Hemiptera, Aphrophoridae) the main vector of Xylella fastidiosa in Europe”. The manuscript is well written and organized. I found no flaws that make me doubt of the author’s interpretation of results. Please find below some minor editorial changes to improve the manuscript.

L30: Replace “spectrum” by “range”
L33: Change to “This report serves as a …”
L42-43: Change to “wine and grape industries.”
L61-63: Check journal guidelines for citations with more than two authors. Should these be cited as “Halkka et al. 1975” and so on?
L64: Replace “recently” by “previously”
L65: Add “Severin 1950” here and in the references cited. Severin, H.H.P. 1950. Spittle-insect vectors of Pierce’s disease virus. Hilgardia 19: 357-382.
L79: Delete “Carabidae” as no other families were reported.
L82: Provide authorship and Order for Zelus renardii
L92: Change to “…because it renders the host…”
L103: Replace “efficiency” by “efficacy”
L108-109: Add parenthesis to “i.e. geographical coordinates…O. vulgatus”
L118: Change “Cistus” to “C.”
L138: Delete “50 fps”
L219: Replace “came out” by “emerged”
L230: Replace “discriminated” by “distinguished”
L264: Place countries in parenthesis
L270: What is 197? Please explain or delete.
L286: Replace “what are the drivers” by “what is the cause(s)”.
L292, 298, and 300: Provide Order and Family for Gonipterus scutellatus, Edwardsiana froggatti, and Homalodisca vitripennis, respectively.
L299: I suggest changing “Gonatocerus” to “Cosmocomoidea”. See Zootaxa 3967 (1): 001–184 for the “World reclassification of the Gonatocerus group of genera (Hymenoptera: Mymaridae)” by John Huber”.
L304: Replace “addressed” by “determined”
L554: Replace “larvae” by “nymphs”
L555: The pie charts have only P. spumarius. It does not show the dark and light grey portions for adults and nymphs.


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: GENOMIC ANALYSIS OF VARIABILITY IN DELTA-TOXIN LEVELS BETWEEN STAPHYLOCOCCUS AUREUS STRAINS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment the manuscript fits the required standards
Experimental design: need improvement (see general comments)
Validity of the findings: Additional work needed to support the conclusion (see general comments)
Additional comments: This study attempts to identify genetic loci potentially associated with variation in δ-toxin production across S. aureus strains. Four GWAS approaches were applied on a set of 124 S.aureus isolates of the NARSA collection. Despite the fact that none of the four GWAS approaches found any significant genetic loci in common, the authors decided to screen the δ-toxin production phenotype of transposon mutants of 9 of the 42 genes identified by GWAS, the rational for this choice being not obvious. The most striking phenotype being that observed with the carA tn mutant, the author focused on this gene. The experiments based on Tn mutant and complemented isogenic strains convincingly demonstrate the carA has a major impact on delta toxin production (I would have looked at RNAIII expression to see wether the effect of carA on agr is trancriptionnal or translational). However, the fact that GWAS identified this gene among other as potential determinant of delta toxin expression is far less convincing because only synonymous mutation in the carA gene were identified. The proposed explanation that such single nt change may have affected RNA stability or RNA binding to a putative unknown target is highly speculative. Only site directed mutagenesis of the carA gene to recreate the single nt mutation observed would be convincing.
Beside, the rest of the paper is interesting, notably the attempt to apply a machine learning approach to predict δ-toxin from genome sequences is promising. However a number of concerns needs to be addressed to validate the model: (i) The splitting of data into training and validation sets with 70/30 proportions is acceptable but leads to high bias in the validation performance estimator. Please consider using 10-fold cross-validation, which should be feasible given the reasonable sample size. (ii) Use of 'accuracy' to report model performance is deceptive here because the binary outcome is not balanced and a 85% validation accuracy can occur by chance. Please provide detailed model performance estimators including ROC-AUC, precision, recall and Cohen's kappa and its associated statistical support. Please only discuss model performance in terms of ROC-AUC and its confidence interval.

Minor comments
line 97. I suggest to reformulate these sentences to better highlight the fact that RNA is a 514 nt regulatory RNA which is the main effector of the agr system. RNA III also encodes  the 26 aa delta toxin (which is not the case in every staphylococcus specie), and it is generally admitted that the level of delta-toxin production is a marker of RNAIII expression.

line 112. A specific table showing some detail on the 124 NARSA strains would be appropriate: type of infection, clonal assignment, antibiotic resistance, city or country of origin…

line 150. Please state wether the culture used here was the 15hours agitated TSB described above.

Line 164 : threshold of 20000 ? arbitrary according to Fig S1 ? At least write once in the paper that you have 109 Low versus 15 High -producing strains

line 167. Hemolysis assay. Why not using the synergistic hemolysis assay (cross-streaking with a strong beta-hemolysis producer such as RN4220) which is slightly more specific for delta toxin than the crude hemolysis diameter?

line 182. Concerning SEER GWAS method: « Dimensions were chosen… » How many dimensions ?  and is it possible to show the plot in Supplementary data ?

Line 219 : 14 predictors = CCs + MRSA + Agr + 11 predictors from Figure 4 ?

Line 226 & 283. Two different Pagel lambda values for the phylogenetic signal of d-toxin production are given (lambda = 0.50 line 226, = 0.99 line 282). Please clarify the differences between these models.

Line 238. Statistics. Did you perform multiple test corrections for the pairwise Mann-Whitney U tests?

line 281 and Fig.2. Some red barrs that represent delta-toxin assessment on fig2 are apparently missing for some CC (ie CC 8). Is it because the production was undetectable ?

Line 283 regarding Blomberg K and Pagel lambda, "for both measures, a value of 1...": this sentence is unclear given that K << 1 and lambda ~ 1, please detail the interpretation (the current sentence reads as if Blomberg model contradicted the Lambda model)

line 291. I suggest to illustrate "high δ-toxin producing clades » by adding: "such as CC45, CC890 or CC72.."

line 317. "87 strains in the “low” toxin and 19 in the high toxin category ». This totalizes 106 strains; where are the remaining 18 strains ?

line 376. " δ-toxin accumulation by the carA mutant could be rescued by a cloned version of the gene on an expression plasmid (but not the empty vector) » : it would be nice to include the rescued strain in Fig 4.

line 385. « complementation with a different carA allele » What is this allele ? how was it chosen ?….. the rational for these experiment is not clear.

Of note I could not find the figure legend of Fig.S1 and S2


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: GENOMIC ANALYSIS OF VARIABILITY IN DELTA-TOXIN LEVELS BETWEEN STAPHYLOCOCCUS AUREUS STRAINS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I believe the article meets your standards
Experimental design: •	Why did you choose to look at the delta toxin?
•	How did you choose the 124 strains that you examined, which infectious syndromes were they from (was this a single infectious syndrome or many different ones?)
•	Was there a reason to believe that many of the strains would be delta toxin positive – did you perform some statistical analysis before ordering the strains to determine how many needed to be delta toxin positive and delta toxin negative? What was the statistical plan for determining the sample size?
•	Does 124 strains provide enough strains given that you were analysing the whole genome?
•	I would suggest that a proper case control study needs to be performed where you examine strains causing only atopic dermatitis compared with stains carried in the anterior nares to understand any association between delta toxin and atopic dermatitis, the numbers for each group needs to be determined using power calculations.
Validity of the findings: •	It’s interesting that none of the four GWAS approaches found any significant genetic loci in common, why do you think this is?

Toxin identification using HPLC:
-	why did you use an arbitrary cutoff of 20,000? Is there supporting evidence for this cutoff?

GWAS:
-	Why did you chose a MAF of 0.2 because of the low sample size, please explain further
-	What was the reasoning behind using strain N315 rather than other strains?

Phylogenetic Regression:
-	Explain further why you assumed a Brownian Model for the evolution of  toxin given the estimate did not entirely fit under the BM assumption
Additional comments: Additional comments are listed below:

Discussion:
-	I’m interested to understand why the variants that passed the family-wise correction cutoff were thought to be likely false positives
-	Why did the DBGWAS variants exclude CC45

Figure 1 – there are only a few strains with >30,000 delta toxin on the x axis – these are interesting, which strains are these and what are the units?