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: CHARACTERIZATION OF AMPHOTERIC BENTONITE-LOADED MAGNETIC BIOCHAR AND ITS ADSORPTION PROPERTIES FOR CU2+ AND TETRACYCLINE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have solved most of the problems raised by the reviewers, however there are still many mistakes should be revised. Although the authors said they have proofread and redescribed the unclear description with the help of a native speaker, but the English still needs to be further improved. Major revision should be considered.
Experimental design: 1. Line 110-114,“The pH, CEC, and Brunaer–Emmett–Telle specific surface area (SBET) of BC… were analyzed by scanning electron microscopy (SEM), thermogravimetry (TG), Fourier transform infrared (FT-IR) spectroscopy, and vibration sample magnetometry (VSM), respectively.” Wrong statement, these parameters cannot be tested by the instruments listed.
2. Line 125, the temperature range 25-1500 °C was not consist with the result of Fig. 4 TG curves of test materials.
3. Line 133-134, “The experiments were carried out at 20 and 40 ℃ respectively for calculating the thermodynamic parameters of Cu2+ and TC adsorptions” The common method to analyze thermodynamics is to require adsorption isotherm data at more than 3 temperatures, because the slope needs to be calculated during the analysis. However, the author only carried out adsorption experiments at 20 and 40 ℃, it is difficult to calculate the thermodynamic parameters accurately.
Validity of the findings: no comment
Additional comments: 1. Line 13-16, such a long sentence.
2. Line 21, two spaces between surface and area.
3. Line 28, “lagest”, spelling mistake. The author should not make such elementary mistakes again in the revised edition.
4. Line 46, chargeed, wrong spelling.
5. Line 51, “The adsorption capacities of BC for phenol and tannic acid are multilayer and monolayer sorption, respectively”, grammatical error.
6. MBC or MBCs？
7. “Change in the TG curve showed three stages at the temperatures of 0–250, 250–600, and 600–900”, missing unit.
8. Line 227, Remove Spaces before punctuation marks.
9. Line 238, “indicats”, spelling mistake.


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: CHARACTERIZATION OF AMPHOTERIC BENTONITE-LOADED MAGNETIC BIOCHAR AND ITS ADSORPTION PROPERTIES FOR CU2+ AND TETRACYCLINE
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
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: VIOLENCE, RUNAWAY, AND SUICIDE ATTEMPTS AMONG PEOPLE LIVING WITH SCHIZOPHRENIA IN CHINA: PREVALENCE AND CORRELATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Grammar editing may be done. The title can be ' ,,,people living with schizophrenia' , instead of  '...people living schizophrenia' . 'With' might have been left out from the title inadvertently.  Grammar edit will improve the clarity of the observations presented in the paper.  .
Experimental design: ' no comment'
Validity of the findings: no comment'
Additional comments: This is a relevant area and the methodology is good. Editing for the English language and grammar will improve the quality of 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: VIOLENCE, RUNAWAY, AND SUICIDE ATTEMPTS AMONG PEOPLE LIVING WITH SCHIZOPHRENIA IN CHINA: PREVALENCE AND CORRELATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Line 52-56: were these studies conducted in China or across the world? Please clarify and point it out.

Line 60 - 62: It would be good to give a definition of and contrast schizophrenia vs. mental illness, in order for the audience not in the immediate field to understand the differences. A general description of schizophrenia would be very helpful.

Line 187: I suggest either reformatting or adding grids for the tables to improve readability.

Line 197: I suggest authors make an effort to use data visualization to tell the stories instead of just showing these tables. For example, consider using pie chart/bar chart or combinations.
Experimental design: Line 219: For completeness of the results, all factors and their OR/CI/P value should be reported. It can be included in the supplementary. Also, consider using forest plot to show these plots.

Line 270: It would be good to point to what is the suicide rate for the general population for comparison.
Validity of the findings: Supplementary file 1 raw data: The full names for each of the abbreviations column names should be provided (perhaps in a separate file)
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: VIOLENCE, RUNAWAY, AND SUICIDE ATTEMPTS AMONG PEOPLE LIVING WITH SCHIZOPHRENIA IN CHINA: PREVALENCE AND CORRELATES
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1)	It is suggested that the statistical values of the full text be in a unified format and in italics.
2)	The line 175 and line 197 should indent.
3)	The layout of the table 1 & 2 is not clear enough.
4)	Table 3 shows a multivariate logistic regression for correlates of disruptive behaviors (Line 219), but the title of Table 3 is “Multivariate logistic regression for correlates of violence”. Please check it again.
Experimental design: 1.	Regarding the inclusion and exclusion criteria:
1)	There are two exclusion criteria (4), please check again. Besides, why exclude the people who absent for five medicine refills?
2)	In inclusion criteria (4), it mentioned that living with a family member. If the patient lives with more than one family members, could he be able to attend the study?
3)	In inclusion criteria (5), it mentioned that speaking Chinese. But it would cause bias because many patients aren’t able to attend the study who speak dialect only.
4)	The exclusion criteria are effective supplements to the inclusion criteria, not repetition of the opposite of the inclusion criteria. Please check the criteria as follows.
①	The inclusion criteria (3) vs the exclusion criteria (4)
②	The inclusion criteria (4) vs the exclusion criteria (1)
③	The inclusion criteria (6) vs the exclusion criteria (3)
2.	Regarding the Instruments:
1)	You defined physical violence as any form of aggressive behavior toward other people such as scolding, cursing, scratching, punching, and hitting peoples. However, scolding and cursing should be categorized to language violence than physical violence.
2)	The GAD-7 scale score ranges from 0 to 21, not from 0 to 27. Please check it again. (Spitzer RL, Kroenke K, Williams JB, Löwe B. A brief measure for assessing generalized anxiety disorder: the GAD-7[J]. Arch Intern Med, 2006, 166(10): 1092-1097.)
3.	This cross-sectional study was conducted in Changsha, but the title of this paper describe it is in China. Please add the relevant description in the part of limitations.
4.	In the discussion, it’ s better to add the innovation of the study.
5.	There are a lot of types in the schizophrenia, such as “catatonic schizophrenia” which the manifestation is catatonic schizophrenia. I doubt if these types are comparable?
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: SEPALLATA-LIKE GENES OF ISATIS INDIGOTICA CAN AFFECT THE ARCHITECTURE OF THE INFLORESCENCES AND THE DEVELOPMENT OF THE FLORAL ORGANS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I have no significant objections to the present article using these 4 objective criteria.
Experimental design: In this work, the authors sequence the likely orthologs of two SEPALLATA genes from the crucifer species Isatis indigotica (woad).  They perform qRT-PCR studies to quantify the expression of one of the two cloned genes (IiSEP2)  in floral and other organs of woad, and they also perform over-expression studies of this gene in transgenic Arabidopsis thaliana.  The DNA sequences and RT-PCR results presented in this work are valid scientific data which add incrementally to the huge mass of data currently available on MADS-box genes in flowering plants.  The studies performed in the present work are, however, extremely limited, notably as there are four SEP genes in Arabidopsis- so are there also SEP1 and SEP3 orthologs in woad, or have these genes been lost in the woad lineage?  This might be an interesting question, but the present authors fail to address it.   Also, the qRT-PCR results presented here reveal only that the woad ortholog of SEP2 is expressed in all floral organs, as is its Arabidopsis ortholog.  This is hardly surprising, and provides no insight into the phenotypes (petal color, seed number, fruit morphology etc) cited as important differences between Arabidopsis and woad. In particular, the overexpression experiments perfomed here do not provide any information to significantly elucidate the role of SEP2 in woad.  To clarify what I mean by this: if Hypothesis 1 was that SEP2 in woad performed exactly the same functions as SEP2 in Arabidopsis, and if Hypothesis 2 was that SEP2 in woad performed at least partially different functions to those of SEP2 in Arabidopsis, in my view, the present manuscript provides no means of choosing between Hypotheses 1 and 2 (so what was the point in even reporting these uninformative experiments?).
Validity of the findings: As I said above, the sequence and (very preliminary) expression data communicated in this paper are valid additions to what is known of plant MADS-box genes in general. We learn from these data that at least two SEP genes are present woad and that at least one of them (SEP2) is expressed in all floral organs, as is its Arabidopsis ortholog. But that is really all we learn  (i.e. not very much).
Additional comments: I am very unenthusiastic about this paper.  The results within it could be submitted directly to a DNA database with a brief note on the expression results- they are hardly worthy of a specific publication on their own.


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: SEPALLATA-LIKE GENES OF ISATIS INDIGOTICA CAN AFFECT THE ARCHITECTURE OF THE INFLORESCENCES AND THE DEVELOPMENT OF THE FLORAL ORGANS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: The authors clarified that liSEP2 and liSEP4 are key regulators for flower development in the woad, which is a novel study. Moreover, liSEP2 and LiSEP4 could be genes related with the flower development in the woad in terms of the results of transgenic Arabidopsis. However, I could not understand what main roles of liSEP2 and LiSEP4 in the flower development from this study.

The authors described “As a cruciferous plant species, the flowers of woad are similar in structure to Arabidopsis. However, yellow petals are produced in woad flowers, and the fruits of woad are indehiscent. In addition, the fruits of woad contain only one seed and are known as silicles. Considering these different phenotypes, it makes plenty of sense to clarify the roles of woad MIKC-type MADS transcriptional factors, including SEPALLATA proteins, in controlling the flowering time and the reproductive growth.” in Abstract. These sentences were not described in the main text and not correspondent with the results of this study. The author should rewrite the Abstract to correspond with the results in this study. Additionally, the described results are not well described by the Figures. I have listed specific comments which may guide the authors through revisions.

L307-312：These sentences are unclear.

L307: Please explain why the angle of leaves and stem were changed by overexpression of liSEP2 gene in the Arabidopsis in the Discussion section.

Fig.4: I do not understand what Figure 4A-C meant in this Figure. Please explain the meanings of Figure 4A-C. It appeared that the angle of leaf in Fig. 4F and 4G looks different, both pictures are the liSEP2 overexpressed plants? What is the difference between Figure D and E? It does not make sense to me. Please compare the wild type with transgenic plant in a same picture, or same stage or same organs, or same scale of picture, if the authors highlight the differences in the flowering development in woad.

L326：The authors have described “two congenitally fused carpels that form the central gynoecium in the fourth whorl (Figs.6A-6C)”, but it’s hard to understand from Fig.6A-6C. It should be better to indicate only gynoecium like Fig. 7F.

L328: “A number of flowers carrying the wilted floral organs were male sterile (Fig. 5D)” does not make sense to me. Please rewrite and make it clearer for readers.

L329-330: Authors mentioned “At the same time, the inflorescence meristems were transformed into defective terminal flowers without sepals and petals, and the number of stamens was reduced (Figs. 5E-5F).”, but the number of stamens was not shown in Fig. 5E-F. Please show the number of stamens in Fig. 5E-F and replace the picture of flower buds with a new picture of flower stamen.

L333-334：Authors described “In addition, some apical flowers with secondary flower lacked the sepals and the petals, and the number of the stamens was decreased,”, and the authors should show the data about apical flowers with secondary flower.

L332-337: The abnormal phenotype of 35S::liSEP2 transgenic plants are shown in Fig. 6, and these phenotype are mentioned in L332-237. However, the line numbers and Figures are not corresponding. Fig. 6G is 5, 6, 7 or 8?
L332: line 7 and 8 (Fig. 6E-6G)
L334: line 3 and 4 (Fig. 6D,6F)
L337: line 5 and 6 (Fig. 6G-6H)

Fig. 6: Authors should describe “The sepals have been removed from of Arabidopsis flower.” in the legend of Fig. 6C.  The explanation of Line 333-337 is not corresponding to Figure 6. The authors should add information about line number of transgenic plants in each Figure.

L363：Authors described “In some transgenic flowers, sepals are fused with petals, and stamens are fused with petals (Figs. 8C-8H)”, but it does not make sense to me from Fig. 8. It should be better to show each floral organ like Fig. 9 F, G, H, J,K and L.

L365: Please explain why the authors performed the exp. of VIGS targeting only liSEP4and ap1 cal double mutant for overexpression of liSEP4. Both liSEP2 and liSEP4 should be analyzed to understand the function of both genes on flower development in woad. The authors should mention why liSEP2 was not carried out in this experiment.

Fig S5：Why AP3 and P1 were decreased by liSEP2OX?
Additional comments: Fig. 1A, B, and C is enough as a supplementary material. Furthermore, Figure 1C is fuzzy and low quality, and unclear. Please repaint it and make it clear for understanding for readers.

Fig.9: Please add a scale bar. The picture of F, G, H, J, K and L should be better upside down.

Minor point
L131: Thalictrum thalictroides (Ranunculaeceae) -> Ranunculaeceae (Thalictrum thalictroides)
Fig S5: The asterisk where is a significant difference is missing. Please add the asterisk in the Fig S5 for making it clear.

There are some unclear points in this article, and it is necessary to show more evidence to fully support the function of liSEP genes. In my opinion, the article is interesting and presents valuable information about flower development in woad. Overall, I strongly suggest revising the text for English, and the clarity of exposition. It is also recommended to thoroughly review the manuscript and to consult with a professional translator or with someone with the relevant expertise to give guidance on English writing. Please check the flow and structures by a native English speaker to better understanding the paper. Unfortunately, English and clarity of the manuscript needed to be improved so much, and the text sounds quite unprofessional.


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: SEPALLATA-LIKE GENES OF ISATIS INDIGOTICA CAN AFFECT THE ARCHITECTURE OF THE INFLORESCENCES AND THE DEVELOPMENT OF THE FLORAL ORGANS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The submitted manuscript entitled that "SEPALLATA-like genes of Isatis indigotica can affect the architecture of the inflorescences and the development of the floral organs" describes phenotypic changes of Arabidopsis and I. indigotica plants when IiSEP2 or IiSEP4 is ectopically overexpressed or silenced. Overall the manuscript is very descriptive but the phenotypic changes are well documented. English is readable enough. Because I am not a native speaker of English, I don't evaluate the language quality any more. Background information is explained adequately but some parts of Discussion should be incorporated into Introduction. Figures seem to be properly prepared except for Fig.9 which lacks scale bars.
Experimental design: Overall experimental design looks appropriate. However, the experiment about IiSEP4 in Arabidopsis may have some unclear points like no description about the number of plants used in the experiment and the number of plants which showed actual phenotype. Likewise, VIGS experiment in I. indigotica also lacks such basic information.
Validity of the findings: I thought the conclusion which the authors want to mention is properly supported by the presented data but some experimental design should be clarified as mentioned above.
Additional comments: 1.	L350: The authors should describe what they did and its motivation before stating the observed results.
2.	L358: I couldn’t understand why the authors are motivated to transform 35S::IiSEP4-GFP onto ap1 cal double mutant. Explain the background briefly first.
3.	L366: Again, the authors should mention what they were going to do and its motivation before stating the results.
4.	L366: Explain what is “TRV”.
5.	L374: No actual quantitative data is presented about the bolting time of the IiSEP4 silenced lines.
6.	Discussion looks too long! I thought L408-419, 470-490 are not necessary.
7.	I thought the statistical tests in Fig.2 and 3 need multiple testing correction.
8.	No description about what kind of statistical test was employed for Fig.3.
9.	Fig.9 lacks scale bars.


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: SEPALLATA-LIKE GENES OF ISATIS INDIGOTICA CAN AFFECT THE ARCHITECTURE OF THE INFLORESCENCES AND THE DEVELOPMENT OF THE FLORAL ORGANS
Review round: 2
Reviewer: 1

Output:
Basic reporting: After reading the new version of your MS, I am happy with the authors taking into account most of my previous comments, and the presentation of results cleared up significantly.

However, I am still having troubles with the scale bar of Figure 3.

I think the scale bar in all Figure 3 is misdescribed. The author indicates 1 cm for the scale bars in Figure 3A to 3C, however I don't think both image is the same size. It can be misdescribed in both 3A and 3C, or 3A or 3C. Furthermore,  scale bars of 3D to 3F represent as 1mm, but they do not appear as 1 mm. If so, the plant in Arabidopsis can be too small. Please check them again, carefully and modify them for the new version of the manuscript.
Moreover, the author should replace the sentence “The sepal have been” with “The sepal and petal have been” in legend of Figure 5.
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: SEPALLATA-LIKE GENES OF ISATIS INDIGOTICA CAN AFFECT THE ARCHITECTURE OF THE INFLORESCENCES AND THE DEVELOPMENT OF THE FLORAL ORGANS
Review round: 4
Reviewer: 1

Output:
Basic reporting: minor point
Columbia (italic) should be changed to Columbia (non-italic).
Ler (non-italic) should be italicized to only "er" (italic), not "Ler".
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: SARGASSO SEA BACTERIOPLANKTON COMMUNITY STRUCTURE AND DRIVERS OF VARIANCE AS REVEALED BY DNA METABARCODING ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: peerJ review
Review of paper: “Biogeographic analysis of bacteria in the Sargasso Sea using
high-throughput sequencing”. The paper presents some interesting findings from the Sargasso Sea regarding the drivers of bacterial community assembly. The authors applied a range of data analysis methods to investigate the relative role of environmental selection over stochasticity in shaping community assembly. The overall aim of the study is interesting, however I have some concerns with the amount and structure of the data available as well as the suitability of data analysis methods to support the questions set and the findings of the study. Due to insufficient amount of information in the methods, I’m not able to assess at this point whether the manuscript could be publishable. I would expect the concerns raised below to be addressed before further consideration.

Lines 60-62 & Lines 77-79. There are plenty of more recent studies on the topic applying novel methodologies from both bacteria and protists. Reading here seems a bit limited.
Lines 94-103: Need to explain why this area is distinct by contrasting it with adjacent seas. Are there barriers that make dispersal limited…eg ocean currents, or some geomorphological features?
122-128: The hypothesis is unclear as the authors are not comparing the dispersal limitation processes within the euphotic zone with a different condition, eg in the disphotic zone. Also the information presented in lines 94-103 seem rather disconnected from the hypothesis whereas the hypothesis should rather build on the info previously provided. In short, it is unclear what the authors are hypothesising and why.
127-128: In my mind, a community is either assembled based on a dominant force or by stochastic processes, however, this last phrase seems to imply that it can be both, which I find confusing. The reasoning behind this last paragraph needs to be much more thought out and connected to background presented higher up.
132-134: The “sampling” section should provide adequate details on the areas sampled, stations within areas. The map should provide also a map of the broader geographical area of the easter states inside which this smaller frame should be indicated. …”Sargasso Sea and adjacent areas”..you need to explain what are the similarities differences between these Seas and the limits of these areas on the map and why they were selected -connect your original research questions and hypotheses with your experimental design. The total number of samples n=40 needs to be broken down to the detailed experimental design that it was derived from so the readers can understand what samples were collected from where and why (eg 2 areas x 10 sites x 2 depths? = 40). Also provide more detail on the legend of figure 1: eg what does the gray gradient color stand for? I guess depth, but you need to be precise.
146-148: You need to be upfront about the bias of using different methodologies can pose on your findings (if any).

151-153: you need to state according to which protocol of analysis and add appropriate reference.

155-167: Ok now I understand why the areas were not defined in the first paragraph of the methods. You used the env info collected to categorise the stations within different areas a posteriori. In which case I am wondering what was the reasoning behind this transect design in the first place? You will need to better connect your hypothesis with the reasoning behind the way you did your sampling.
I will leave the details of the molecular analysis to someone more expert on this topic.
223: You need to state which a-diversity metrics you used and what they do/show, justify why you selected the specific ones and add appropriate references.
224-225: It is unclear why the authors chose to do a correlation analysis instead of fitting a statistical model with the diversity metric as the response variable and the env variables as explanatory also adding depth and area as factor variables. The model selection procedure would help them test the effect of all these geographical and env variables on the diversity simultaneously. This cannot be done with correlation analysis as such I don’t think this analysis is appropriate here.
226-229: you will need to explain what the analysis does and how it helps you address your research question. Eg what do you mean compared across categories, what are the categories in your case, need to put this into the context of your own study.
231-233: Again it is unclear how this analysis helps address the research questions. In the Mantel test, geographical distances are included as covariates not accounting for the fact that two stations that are very close can actually belong to two areas with widely different characteristics. This analysis does not thus take into account dispersal limitations between the sites.
236-237: You use Venn to determine the overlap between which things and why? You need to be more specific in your description of methods and how these connect to your objectives.
237-242: This analysis seems much more suitable to understand the role of environmental variables in driving community similarity as the role of geographical distance and its overlap with env variables is accounted for. Thus to me the mantel tests seems rather obsolete, unless I’m missing something here.
246: Again it is unclear what the question is behind this method and how it relates/complements the rest of the analysis carried out here. As it stands it seems rather disconnected from the rest of the text. You should clearly explain how this approach was employed to address a specific question as well as what the results would look like and how you will use them to address the question.
General remark: the authors need to explain how the unequal number of samples across areas (eg see table 1) is affecting their analysis. In my opinion this unequal sampling design and the fact that in some areas some variables were not measured, would have an important bias in the validity of the results.
268-273: Unsure how chi squared analysis was used to test for significant differences between depths here…Again a GLM with consequent post hoc tests seems more suitable.

329: Are you able to say which regions are significantly different from which?
Tables 1 and 2 need to say what numbers represent, eg mean, median and add some measure of variation eg range
343-344: This sentence is very confusing. Not sure how temperature differences can be correlated with something.
349-352: Why doesn’t this model include area? Is it because it can only include numerical variables instead of factors? The structure of this model should have been described in the methods.

357: What is meant here by spatial effects? Even though spatial effect had a smaller contribution than env drivers, it would be useful to visualise in which axes did most of the effect occur so as to be able to unravel the mechanism. Eg northeast or from south to north?
Experimental design: See above
Validity of the findings: Discussion
First paragraph of the discussion: Please first state your main findings and how these address your research questions and hypotheses.

391-392: these conclusions are unsupported by the results as a GLM analysis is needed instead of correlation.

410-413: this aim should be stated also clearly in the aims and objectives paragraph of the intro…

It would be useful to see some discussion whereby the relative role of spatial structures and the environment is contrasted between bacteria and protists. There have been several interesting studies on the later that can provide further insights here.
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: SARGASSO SEA BACTERIOPLANKTON COMMUNITY STRUCTURE AND DRIVERS OF VARIANCE AS REVEALED BY DNA METABARCODING ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript agrees with PeerJ’s requirements for basic reporting. The study presents a biogeographic analysis of microbial communities in the Sargasso Sea and adjacent regions and aims to identify the environmental, spatial and stochastic factors that influence the observed distribution. The raw sequencing data and associated metadata were deposited into the Sequence Read Archive database however these will be released on 30/9/2022. Overall, the study has merit although there is space for improvement on structure, clarity and conciseness.
Experimental design: The research lies well within the aims and scope of the journal. The research question is well-defined but the authors could state more clearly how this research fills an identified knowledge gap given the fact that the microbial communities of the Sargasso Sea are among the best-studied worldwide. Concerning the bioinformatic analysis, I would expect a more up-to-date sequence clustering method resulting in ASVs rather OTUs however, I will not insist on that. What is more important is to make the manuscript more reader-friendly and I am referring mainly to the statistical analysis. Reading the article, the impression I got was that every single statistical test available has been used without really having thought of the question that it will answer. Several times, the same or similar question, was addressed by a number of statistical methods resulting in a confusing mix. Also, insignificant results are presented when there is no need for that. The authors should think carefully which methods or tests they are using and why. Lastly, there is one sample that is completely different from the rest, the coastal Bermuda sample. It would be best to remove this sample altogether, it cannot be used in any stats and including it resulted in unnecessarily complex tables of statistical results with or without this sample.
Validity of the findings: The findings are valid but some restructuring and reduction in length is needed. I would recommend presenting the “big picture” first, i.e., the beta-diversity analysis, then go into the detail of the factors and taxa that may be important in the observed differences. The core finding is that microbial communities differ only slightly and, on this basis, the authors can then explore, the variation partitioning between environmental and spatial or stochastic factors that cause these subtle differences.
Additional comments: Line 94-96: Please expand on why the Sargasso Sea, which is a distinctly bounded open water mass, is an appropriate ecosystem to study biogeographic processes. I presume due to the subtropical convergence zone?
Line 113: The last paragraph of the introduction is a good place to address the novelty aspects of this study compared to previous ones (see comment in #2 Experimental design). What new does this research bring? Also, the word “vertically” is somewhat misleading, one expects a vertical profile of the whole water column but it’s just two depths that are considered here.
Lines 117-121: This part spoils the flow of the text here. It would fit better in the Materials and Methods.
Line 124-128: It is better to rephrase this part and refer to all the environmental parameters tested (including stochastic effects) to confirm your hypothesis, rather than an expectation that one of them is significant among the tested factors. This is something to be tested in the analysis later.
Figure 1: It would help to show the wider area in a smaller map on the side of the main one.
Line 149: typo in colorimetric
Line 173: Please mention the region of the 16S rRNA gene that your primers are targeting.
Line 209-210: please reconsider this sentence to improve clarity
Line 214: Do you mean prior to statistical analysis? Wasn’t the rarified dataset used for all downstream statistics?
Line 216: Did you use Unifrac or weighed Unifrac?
Table 1. Please remove the line ALL, it is not informative provide an average of T or chlorophyll of all regions.
Lines 267-294: Please use more concise language. Here for example, the take-home message is that there was a statistically significant difference between surface and DCM attributed to the SSS samples. This could be conveyed in one sentence. Also please use the same number of decimals throughout the manuscript for the same metric, e.g., 3 decimal places for all p values.
Line 285: Is it not Bacteroidetes?
Line 286-288: Please remove any mention to Archaea if your primers were not targeting them. This was part of the filtering process and should not be in the Results.
Line 283: This is where it starts becoming confusing. According to SIMPER we expect these communities to be quite dissimilar but then ANOSIM and PCoA give a different impression.
Line 329: Again, please be more concise. The whole paragraph should be shortened to convey a single outcome: only surface communities differ between regions. You could then perform pairwise testing to see where the significant difference stems from. You can do that with Permanova and pairwise permanova. I am not sure what “overlapping but separable” means from an ecological point of view.
Line 338: About Mantel tests: since you have high correlation between two variables and you need to use partial Mantel test controlling for that, then only this result should be presented. It becomes more confusing further on with db-RDA (I presume on the same distance matrix as Mantel) and other factors become significant. Also, it is not clear to me how you ended up with the best fit model. What kind of selection process was used and were there any interaction terms? It is mentioned that 3 terms are in the best fit model but only two are significant. Is that because of an interaction term? If this is the case then only the significant interaction is mentioned and not the individual variables that make up this interaction.
Line 359: Why is the residual variance so high when 58% of the variation is constrained?
Lines 367-378: In the first part of the paragraph, it is mentioned that diversity peaks at a certain point and then declines which agrees with your data. I think the statement below about deep water stable conditions etc. cannot be supported by your data since you don’t have any deep water samples. The paper you are referring to (Ghiglione et al. 2012) is comparing surface to deep ocean (>200 m depth) phylogenetic diversity and speciation patterns. In this study, all samples belong to the surface ocean. I think you should focus on the special characteristics of DCM rather than depth-related changes in diversity.
Line 431: if there is no reasonable oceanographic reason that these two are similar, I would avoid these statements. Other samples are close temperature-wise but appear to be dissimilar.
Line 444-446: this sentence is unclear, please explain better. Also consider if Sargassum density is really significant or a statistical artifact with no ecological meaning.
Line 453-455: This should be the core of the discussion i.e., the acknowledgement that this is a well-bounded water mass and differences in microbial communities are subtle. On this basis, you can then explore, the variation partitioning between environmental and spatial or stochastic factors.
Line 459: Is there an agreed dissimilarity level over which the contribution of legacy factors are considered important? How about the partial Mantel test that shows no effect of geographic distance when controlled for temperature? This is what I meant before about becoming lost in the statistics, finding significant differences and factors causing dissimilarity, to then reach the conclusion that is mentioned in the previous comment.
Figure 4: I am not familiar with distance-decay models but it seems that there are points having a similarity of 1 and 0 distance. I presume it refers to one community being compared to itself, if that makes any sense. Shouldn’t these be excluded from the regression?


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: EXTENSIVE SHEEP GRAZING IS ASSOCIATED WITH TRENDS IN STEPPE BIRDS IN SPAIN: RECOMMENDATIONS FOR THE COMMON AGRICULTURAL POLICY
Review round: 1
Reviewer: 1

Output:
Basic reporting: I generally like the manuscript, probably because I love both - sheep and birds and try to find interactions between both group of organisms
Experimental design: I have only one general comment - the manuscript based in fact mainly on correlation
Validity of the findings: no comment - I add directly on the MS
Additional comments: I add directly on the MS


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: EXTENSIVE SHEEP GRAZING IS ASSOCIATED WITH TRENDS IN STEPPE BIRDS IN SPAIN: RECOMMENDATIONS FOR THE COMMON AGRICULTURAL POLICY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript would benefit from some language improvement. Also, the structure/scope of the introduction could be better organized,as you jump between topics in a not very organized manner. For example, the details on the overall impacts of grazing on biodiversity might not be needed here… But it depends on the major issue of clarifying what is the scope of the paper.
Experimental design: There is a major issue with this paper: Following the abstract and introduction, it is supposed to address issues related to birds and sheep in Iberian natural steppes, but the title and data analysis focus on country-level data on livestock and bird trends. This makes the paper confusing and not adequate for publishing as it is.
Validity of the findings: The main issue is that authors use a dataset of steppe bird abundance and sheep numbers that relates to national-level data. In that perspective, both total sheep numbers and overall bird abundance (many species are not exclusive of shrub steppes) may not represent what happens in natural steppes. This relates to the key issue; what is the geographic scope of these natural steppes? Both sheep and bird data should focus on these areas.
Additional comments: If analysis are to be carried out at national level, and if sheep numbers are an indicator of the amount of available habitat, why authors don’t focus on the latter, rather than the former ? Surely, the solution to the bird decline is not to increase the number of sheep, but the area of pastures/fallow. If sheep were replaced by cattle, would that be a problem ?


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: EXTENSIVE SHEEP GRAZING IS ASSOCIATED WITH TRENDS IN STEPPE BIRDS IN SPAIN: RECOMMENDATIONS FOR THE COMMON AGRICULTURAL POLICY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The research question is clear, the data and the methods are suitable for addressing the question, the results are not particularly open to interpretation, and the discussion and conclusions are well supported by the results. Well done!

I have checked the data, and I can only say that it seems to be available and correspond to the paper. Did the authors use the ‘FBI_Index’ or the ‘FBI_Index_Annual’ for their analyses? Please, clarify. Likewise for the variables ‘SBI_Index’ and ‘SBI_Index_Annual’ and for the ‘sheep-dupontvF’ sheet – which variables were used in the analyses? Please, clarify to ensure reproducibility.

Figures: Presumably, the baseline year (corresponding to annual change = 0) is the first year of each period mentioned in the figure captions, right? For example, in Fig. 2 the baseline year is 1998, right? It might be worth clarifying it in the figure captions and perhaps mentioning that the value of zero represents the baseline against which the change is estimated.

This is a bit of a cliché, but I think the writing needs a bit more work. I am not a native English speaker, but I found quite a few typos and some sentences that don’t seem to make much sense. I have included some suggestions and comments.
Experimental design: Some clarifications and further explanations on the statistical methods used are needed. In particular, what is Johansen’s method? (lines 176-177) How am I supposed to interpret the results of the Johansen test presented in lines 195-198? There are statistically significant p-values there; what do those values mean? That the variables are cointegrated? It is confusing because, in many applications, a significant p-value could imply the opposite. Please clarify since this was the method used to test for cointegration, which is essential for the paper. Likewise, what is the Durbin-Watson statistic (line 170)? Talking about statistics, please provide the point estimates, standard error, and 95% confidence intervals of all the parameters of your linear regressions. Uncertainty measures and point estimates are fundamental, and at the moment, the authors only report on the F-statistic. The authors could consider a table detailing these values.

The authors mention that the positive effects of sheep grazing on steppe birds are higher at intermediate sheep densities. Have you considered fitting models to accommodate this effect? It is relatively straightforward to by including a square sheep change rate. It would be something along the lines of SBI ~ sheep abundance + (sheep abundance)^2. This could also be done using the raw sheep abundance presented by the authors in their dataset. I reckon the authors should consider it as it could make their results and conclusions stronger.
Validity of the findings: Although the authors have done a nice job explaining the potential mechanisms underpinning their results, I think that the direct conclusions and implications are less clear. Therefore, I consider that it would be good to provide some explicit and straightforward assessment of the role of sheep in determining steppe bird declines. Do the authors think that increasing the number of sheep will result in a parallel increase in steppe birds? Are the authors recommending investing in maintaining sheep in Spain as a conservation measure? These will imply a direct role of sheep on the management and decline of steppe birds. Or can the ecosystem services provided by sheep be substituted by other conservation interventions? For example, how about regular burning? Or perhaps fostering the recovery of native herbivores such as the rabbit – would that replicate the effect of sheep on the ecosystem and help recover steppe birds?
Additional comments: I am nit-picking, but how come “natural” steppes are so reliant on human activity? I found it a bit contradictory, to be honest. Would some of those “natural” steppes revert to forest or scrubland without human use? In that case, would those be actual “natural” steppes?

Title: I think it should be “in Spain in the PAC context” The authors could add EU PAC to indicate that they are referring to the EU programme. Moreover, I suggest not using acronyms in the title since it can be confusing for the readers not familiar with the meaning of PAC.
Abstract, lines 23-24, and throughout the manuscript: I am pretty sure it should be “sheep numbers” (plural) instead of “sheep number”.
Lines 56-57: I am not sure what the authors mean by “both directly through its depositions and indirectly”. Is it that sheep dung affects it directly, and there are other indirect effects? By the way, what are those indirect effects?
Line 60: Coprophagous species? Beetles?
Line 68: the main populations within Iberia or a different geographical area? Please, clarify.
Line 86: Change “supposed” to “caused” or “produced”
Line 90: I suggest changing “reduced” to “limited”
Line 96: I suggest changing “with” to “on” – “sheep numbers on the abundance”
Line 100: I suggest changing “compiled” to “collected”
Line 118: I don’t think “husbandry” is the right word here.
Line 119: Change “reduced” to “less”, although I think this sentence could be re-written to improve clarity.
Line 124: Change “cero” to “zero”
Line 128: Perhaps change “partnership” to either “partner” or “representative”
Line 130: Change to “a set of”
Line 181: I suggest reordering the words so it reads “response variable”
Line 192: I suggest changing to “In 2020, there were 37.3% fewer sheep than in 1992”
Line 226: I don’t understand this sentence very well. What are casuistic correlations?
Line 231: I think it should be “to cite a few”
Line 246: I suggest changing “plant consume” to “plants consumed”
Line 262: I don’t understand what the authors mean by “which are usually hosted”. Please, clarify.
Line 265: I don’t understand what the authors are discussing here. Please, re-write the sentence to make it clear.
Line 275: I think the authors mean “this study”


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

Input:
Title: EXTENSIVE SHEEP GRAZING IS ASSOCIATED WITH TRENDS IN STEPPE BIRDS IN SPAIN: RECOMMENDATIONS FOR THE COMMON AGRICULTURAL POLICY
Review round: 1
Reviewer: 4

Output:
Basic reporting: The paper is structurally sound and of good ecological, conservation and management importance. It uses a national dataset which gives a general overview of the tendencies in the country, strengthened the conclusions.
I definitely thing is a publishable paper although I have some concerns about the lack of support and detailed information of some decisions and thing some conclusions are somehow pushed beyond what the paper results tell us.
I also thing that, including 79 citations in a paper addressing a simple concept is too much. 40-45 citation would be reasonable and will give sufficient background and context.
Lastly, please check again the common and Scientific names of the species in Tables S1 and S2 since some names do not match (e.g., S2: Crested Lark, designed as Sylvia conspicillata), and check the trend in Spain so is the same in the two tables. Additionally, the Common Linnet as changed genus and is now Linaria cannabina, however, this change is not necessary if you add a note to the table referring you are using the older/traditional classification.
Experimental design: The research questions are well presented and explained.
The method section is clearly explained. Both sheep and bird data were collected at a country level, with the best available information considering the spatial and temporal scale of the work.
The use of an independent dataset for the Dupont’s Lark shows that the authors considered the under representation of the SACRE Dupont’s lark data and worked around it to get a sound dataset for this species.
Validity of the findings: This paper puts in highlight what has been discussed in the scientific community for a few years now, which is the decrease of both the traditional sheep extensive grazing and steppe and farmland birds.
Respecting to conclusions, the authors focus mainly on the sheep-steppe birds relationship, although they have also evaluated the relationship for farmland birds (which are not all steppe birds). More attention should be given to farmland birds in the discussion and conclusion.
In addition to that, the strong direct association of cause-effect made between both sheep and bird declines need to be revised. As the authors also mention, there are other factors that can contribute to the bird declines and that were not measured in this work (lines 224-226). Therefore, although the cause-effect is unquestionable, it should be presented as one of many other factors already known that contribute for steppe and farmland birds declines.
Additionally, I have a particular concern with the fact that the authors do not give enough attention to the shift from sheep-based to cattle-based systems occurring in the Iberian Peninsula, which is already known to negatively affect farmland bird densities and distribution (Reino et al. 2010) and is associated with the CAP subsides that the author briefly mention.
Additional comments: The lines numbers are made considering the PDF version provided

1.	Title: I'm assuming PAC mean the same as CAP (Common Agricultural Policy) in which case I suggested changing to the English abbreviation.
Additionally, the authors recognise, in the discussion, that more research is needed to better understand the association of bird declines and sheep declines. They also mention the existence of other factors that lead to the observed bird decline (Lines 224-226). In this sense, I think the title should be rethought in order to not be misleading of the paper main conclusions.
On a second note, if the authors want to focus on CAP (which I agree they should), some more context of it is needed, particularly in the introduction; and further discussion between CAP and the paper main results, should be added.

2.	Keywords: not sure why the authors included the “paramo” as keyword. Is not used as a term in any part of the paper.

3.	Lines 76-82: is not clear to me how agricultural intensification leads to shrubs encroachment. That may be true for agricultural and grazing abandonment (as stated in lines 80-82), which tend to happen in the marginal regions mentioned, due to the low income but not do to agricultural intensification.

4.	Lines 87-90: There are some recent works addressing the relation between grazing and farmland and steppe birds in Mediterranean landscapes missing throughout the manuscript. Please see papers from Faria et al., Reino et al., Beja et al. and Moreira et al.).

5.	Lines 94-95: The term “common natural steppe bird species” can be misleading, since some of the species in the index are not considered steppe birds, although they can occur and are typical of Iberian natural steppes. Please use the term used in the methods “common species typical of Iberian natural steppes”.

6.	Lines 150-152: The SBI has an index to measure the relation between sheep numbers and birds, that occur in natural steppe areas, is correct. However, using this index to “further explore the relationship of steppe-specialist birds with the variation in sheep number” in not correct and should not be done. First, there are some steppe-specialist birds missing from index (eg., Great bustard, Otis tarda). Second, at least half of the species used in the index are not classified has steppe birds and most are common in a diverse range of habitats (Little Owl, European Stonechat, Common Linnet). That said, using this species in the index will not allow to draw conclusion about the steppe-specialist birds. I don’t think you need to change the index, just rephrase the sentence and change the conclusions to account for this.

7.	Line 256-258: If the relationship including both sheep and goat was evaluated, why not present those results? If the number of goats is “much reduced” and was not evaluated because of that (lines 117-120), why discussing those results at all?

8.	Lines 277-285: The discussion and conclusion of the subject addressed in this line, although it may be true, goes beyond this study scope and findings. The authors did not measure in what extent the different types of grazing affected birds’ trends neither they worked with grazing intensity.


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: EXTENSIVE SHEEP GRAZING IS ASSOCIATED WITH TRENDS IN STEPPE BIRDS IN SPAIN: RECOMMENDATIONS FOR THE COMMON AGRICULTURAL POLICY
Review round: 2
Reviewer: 1

Output:
Basic reporting: I already commented it it the first turn. However now I see really improving version, although from the beginning I was quite optimistic.
Experimental design: OK, it looks like correlate study, but anyway, quite interesting. The authors underlined potential limits.
Validity of the findings: In local, as well as European scale, we need similar results to better protection of farmland birds.
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: EXTENSIVE SHEEP GRAZING IS ASSOCIATED WITH TRENDS IN STEPPE BIRDS IN SPAIN: RECOMMENDATIONS FOR THE COMMON AGRICULTURAL POLICY
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: Please explain in the main text what is the Durbin-Watson statistic and how to interpret it. As it is written currently (lines 207), it is impossible to know what this means or what does it imply. The authors have provided additional details in their responses letter, but they should add further explanations in the main text for the sake of interpretability and repeatability.
Validity of the findings: I have some minor comments that the authors might want to consider. These are suggestions for clarifying the interpretation and meaning of some of the statistics presented in the paper.

First, I don’t think the analysis can entirely rule out spurious correlations. Cointegration is helpful but not perfect, and as the paper is currently written, it gives the impression sometimes that the results are causal. I think the authors should be more careful and use expressions such as “to mitigate the impacts of potentially spurious relationships” or “to test for the potential effects of spurious relationships” (e.g., abstract) and “indicates that our results are unlikely to be due to spurious relationships” (line 235 in results).

I might be nitpicking, but I really dislike the concept of “marginally significant” (e.g., abstract and line 246) to refer to a p-value = 0.06. In my opinion, p-values are either significant or not, even more so when the authors themselves indicate that they are using the p < 0.05 cut off (line 228). Therefore, I suggest deleting instances of “marginally significant” throughout the manuscript, perhaps changing to “trend negatively although it was not statistically significant” or something similar.
Additional comments: Line 235: “posterior regression results” - posterior usually applies or refers to the results of a Bayesian analysis. Did the authors do a Bayesian analysis? If not, please delete the word “posterior” as it is confusing.

I have reviewed this revised version of the manuscript, and I think the authors have done a good job addressing my comments. In general, the paper is interesting, and the results are properly contextualised. I am looking forward to seeing it published in PeerJ.


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

Input:
Title: EXTENSIVE SHEEP GRAZING IS ASSOCIATED WITH TRENDS IN STEPPE BIRDS IN SPAIN: RECOMMENDATIONS FOR THE COMMON AGRICULTURAL POLICY
Review round: 2
Reviewer: 3

Output:
Basic reporting: In Table S2, the Spectabled Warbler, has a typo, it should be Spectacled Warbler.

Although there was a big language improvement, some sentences are still hard to read and too long. The paper will benefit from a second revision focusing on the sentence length and structure.
Experimental design: Nothing to add
Validity of the findings: Nothing to add
Additional comments: 1.	The CAP section of the manuscript, particularly in the discussion, was greatly improved and know gives a good idea about that problem while making suggestions about how to improve it in the future.

2.	L. 185-190 : Since the authors selected species for the SBI index taking into consideration those species that can be found in “uplands” in UK, this need to be justified in the methods. There is a lot of research on this topic in the UK, which makes it appealing to compare to, however, the Iberian Peninsula has a particular type of natural and semi-natural open habitats which are not found in the UK (steppes). Considering that and the fact that important steppe bird specialists are missing from the index (like the Great Bustard) I wonder if the authors should re-think their position about what species to include or better justify the species included in the index.
In addition, the authors maintained a misleading sentence from the previous version: “to further explore the relationship of steppe-specialist birds with the variation in sheep numbers.” As they said in the response to my previous comment the 20 birds selected are “typical of shrub-steppes” and are not all steppe-specialist birds”, so this term should be changed.

3.	L. 300-302: regarding the goat results. Since the authors mention in the methods (L. 155-157) that “did not include goat livestock, since goat numbers are low in Spain” the reader will not “ask themselves if the same pattern would have been obtained if including goats in the analyses”. Moreover, including these trends in the discussion is questionable, if the numbers of goats are low, then the relationship found is because of the sheep and not the goats. Secondly, since goats are “especially linked to specific regions" should be consider since the paper looks at trends at the country level.
Considering all the above, I think the lines 298-302 should be removed from the discussion.


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: ANALYSIS AND COMPARISON OF PROTEIN SECONDARY STRUCTURES IN THE RACHIS OF AVIAN FLIGHT FEATHERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The aim of this study was to determine protein secondary structures (PSS) of flight feathers of seven bird species. Although potentially interesting, there are several points that preclude recommendation of acceptance.

The use of the English language needs to be improved. There are also typos. For example, in the abstract: he.
Experimental design: The research question is not well defined.

The data analysis is not convincing. Which standards were used for PSS?

The curve fitting in Figure 3 appears very questionable. A detailed description and validation of the approach is necessary. Statistical analysis is required.

Legend of Figure 5: Was hair investigated?
Validity of the findings: The validity of the findings is not demonstrated by the authors.
The data analysis is not clear.
Statstical anaylsis is missing.
Conclusions are not well stated.
Additional comments: No additional comments


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

Input:
Title: ANALYSIS AND COMPARISON OF PROTEIN SECONDARY STRUCTURES IN THE RACHIS OF AVIAN FLIGHT FEATHERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Analysis and comparison of protein secondary structures in the rachis of avian flight feathers” is a timely publication at the height of crystallography. Furthermore, the comparison of the species is well thought out and provides a encompassing view of flight feather’s secondary structure. However, I have several general comments and one major revision that should be addressed before publication.
Major revision:
1)	The authors attribute the identified secondary structures to only alpha-keratins, beta-keratins and collagen. However, we now know that proteins comprising the EDC genetic locus are widely expressed in feathers (Strasser et al. 2014 and more recent papers). The authors need to justify their attribution of these structures to just three types of proteins. Secondary structures of proteins are easily calculated using online bioinformatics tools. What are the secondary structures of these other EDC proteins? Could any of the beta-sheets or alpha-helixes be attributed to those proteins? Beta-sheets and alpha-helixes are very common among proteins performing a myriad of functions. Therefore, I think the authors should calculated the secondary structures of the EDC genes identified in the chicken (Strasser et al., 2014) in Results and justify their conclusion of those structures only being attributed to three protein types in the Discussion.
Minor revisions:
1)	The manuscript should be carefully read over by a native English speaker. There are several grammar issues and/or awkward sentences throughout the ms. For example these sentences should be revised (these are only some examples): Lines 31-32, 56-58, 89-90.
2)	The introduction should include specific information on the flight modes of these birds. Also and related, the information in the first three paragraphs of the Discussion and first paragraph of the “Collagen in the rachis” of the Discussion should have been in the Introduction.
3)	The information in the paragraph found on lines 201-210 should have references as this is not novel information.
4)	The first figures cited in the text (lines 193-195) are Figure 1 and Figure 3. Therefore, Figure 3 should be renamed Figure 2.
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: ANALYSIS AND COMPARISON OF PROTEIN SECONDARY STRUCTURES IN THE RACHIS OF AVIAN FLIGHT FEATHERS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The language: the English language should be improved to ensure that an international audience can clearly understand your text. Some examples where the language could be improved include for example lines 31-34, 40-42, 53-56, 76-78, 80-81, 89-90 (the adaption of the rachis medulla for flighting), and many other throughout the ms, where the current phrasing makes comprehension difficult, some sentences are ungrammatical or simply constitute sentence fragments. I would like to suggest you ask a colleague who is proficient in English and familiar with the subject matter review your manuscript, or contact a professional editing service.
The Introduction is well written and adequately introduces the research questions.
The selection of literature is good and relevant. See section 4 “Additional comments” for minor remarks on the subject.
In my opinion, the structure of the manuscript meets PeerJ standards and disciplinary norms.
I thank you for supplying the raw data, which is accordance with the PeerJ standards. However, your supplemental Excel sheets require a detailed description of the variables contained therein, e.g. in the form of a legend, to be useful to future readers and users.
Experimental design: The aim of this study was to analyze the molecular structure (protein secondary structure, or PSS) of flight feathers in seven bird species, using up-to-date spectroscopy methods (ATR-FTIR and SR_FTIRM). Then the Authors sought to establish whether there was a relationship between the flight mode used by the species studied and the secondary protein structure composition of their feathers. Previous studies showed that mechanical properties of avian flight feathers (e.g. stiffness) depend mainly on the rachis structure. The Authors rightly suggested that the keratin and corneous protein composition is also important here. The research questions are well defined, relevant and meaningful.
To the best of my knowledge, the methods you used to molecularly analyze the feather composition are appropriate and up-to-date. These methods are described with sufficient detail and information to replicate.
My reservations about the methods concern the selection of feathers for analysis. You wrote that you analyzed “flight feathers”. But which feather? Rectrices, remiges, primaries or secondaries? Please specify.
Another problem is the choice of bird species – you sampled feathers from aviary birds (Mallards and chicken) and from wild birds (ibises). You did not account for the possibility that unfavorable conditions during molt in captivity may cause birds to deposit low quality keratin in growing feathers (e.g. Murphy et al. Can J Zool 66:1403-1413, 1998; Dawson et al. Proc R Soc Lond B: 267: 2093-2098, 2000). This could affect the results of your analysis. I warmly suggest you to address this issue in the paper.
You have classified the Mallard as a weak flier. This is not a good classification. Mallards are strong fliers, performing daily migrations between foraging and resting sites, migrations to molt sites, and even long-distance seasonal migrations. They have short, but high aspect ratio and high wing loading wings. Such wings produce high induced power and are adapted for fast and sustained flight over long distances (e.g. Rayner, Form and function of avian flight, Current Ornithology, 1988). Wing flight feathers in Mallards are certainly structurally adapted to withstand high aerodynamic forces.
And last but not least. You aimed at looking for a relationship between feather structure and flight mode in study birds. But you did not perform such an analysis. I would suggest you to use statistics to compare flight feather morphologies between strong and weak flying species in your sample.
Validity of the findings: The results obtained in this study are undoubtedly worth publishing. The rationale and benefits of peer-reviewed research to the scientific discipline are clearly stated and justified.
Feather molecular structure analysis data are well presented. They are robust. However, I see no quantitative or statistical justification for the claims (lines 362-369 and 378-380) that differences in feather protein composition were found between good and poor flyers.
Conclusions are well stated in terms of the molecular structure of the feathers. However, I see no statistical justification for the statement that good and poor flying birds differ in the protein composition of their flight feathers. I strongly encourage you to provide such a justification.
Additional comments: Citations:
Lines 60-61: in this case, the following papers should be cited: Feduccia & Tordoff Science 203:1021–1022, 1979; Norberg, Vertebrate flight, Springer, 1990.
 Reference list:
The names of the journals are given usually in abbreviated form, but sometimes in full (e.g. lines 414-415). Please standardize.
Bonser, R. and P. Purslow (1995). "The Young's modulus of feather keratin." J Exp Biol 198(Pt420 4): 1029-1033 – this citation contains an error – what is “PT410” that is shown in parentheses?
Please check all citations and make them uniform.


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: ANALYSIS AND COMPARISON OF PROTEIN SECONDARY STRUCTURES IN THE RACHIS OF AVIAN FLIGHT FEATHERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The quality of the text has been improved.
Experimental design: no comment
Validity of the findings: The reporting of the results has been improved.
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: ANALYSIS AND COMPARISON OF PROTEIN SECONDARY STRUCTURES IN THE RACHIS OF AVIAN FLIGHT FEATHERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: I think the authors adequately addressed all of my initial concerns. Therefore, I think the ms is ready for publication.
Experimental design: I think the authors adequately addressed all of my initial concerns. Therefore, I think the ms is ready for publication.
Validity of the findings: I think the authors adequately addressed all of my initial concerns. Therefore, I think the ms is ready for publication.
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: COMPARATIVE PROTEOMIC ANALYSIS FOR REVEALING THE ADVANTAGE MECHANISMS OF SALT-TOLERANT TOMATO (SOLANUM LYCOPERSCIUM)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article includes sufficient introduction and background information. It fits into the scope of Peer J. All standard sections are included according to the format required by the journal. It is self-contained and raw data are provided.
Experimental design: Several method sections do not have sufficient details. For example, the reviewer could not evaluate the results of the enzymatic assays because there is no description about sample preparation.
Validity of the findings: Thank authors for providing the raw data for Figures 1, 8 and 9. Please provide more description about the statistical analysis, eg. the number of replicates and how the p values are calculated.
Additional comments: Salinity is a major stressful condition that causes a significant decrease in crop production worldwide. In response, plants have evolved complex salt-responsive signaling which involves significant proteome remodeling processes at the cellular level. Wang et al. analyze how global proteome abundance vary across the salt-tolerant and salt-resistant tomato cultivars and relate the protein expression profile changes to cellular metabolism. They report the following main findings: 1) significant increase in expression of proteins involved in Brassinolides and GABA biosynthesis; 2) higher expression level of antioxidant-related proteins; 3) overexpression of proteins functioned in chloroplast; 4) identification of three key proteins as important salt-resistant resources for breeding salt-tolerant cultivars. In general, the introduction section is well written with recent literature and the proteomic workflow is well established with qRT-PCR verification of gene expression. However, significant modifications of several sections are needed, especially that several method sections do not have sufficient detail and information to replicate.

Major comments
1.Root is the main organ for carrying water and mineral nutrients to the rest of the plant and the primary site of perception and injury for salt stress. Accordingly, root proteome variations in tomato have been a target for study of the molecular mechanism underlying plant salt stress tolerance and adaptation. Examples are from Manaa et al (J Exp Bot. 2011 May;62(8):2797-813) and Gong et al (Biochem Biophys Res Commun. 2014 Mar 28;446(1):417-22). It would be beneficial if the authors would expand the introduction section by explaining the choice of four true-leaf as target of research. Specifically, the authors may compare the effects of salt stress on tomato seedlings (OMICS. 2011 Nov;15(11):801-9), root, leaf, as well as on fruit (OMICS. 2013 Jun;17(6):338-52) to provide a better picture of the proteomic alterations of tomato upon change in salinity.
2.Quantitative LC-MS/MS based proteomic workflow in this study represents a significant technical advantage over previous reports. However, experimental details are needed for the Sample preparation section and the Protein Identification section. Given that the authors are focusing on identifying key proteins as important salt-resistant resources for breeding salt-tolerant cultivars, I believe special attention should be paid to the TMT labeling procedure to minimize potential artifacts. No description of the TMT labeling procedure and corresponding data analysis in the current version of the manuscript.
3.Related to Point #2, in section Sample preparation for TMT proteomic quantification. It has long been recognized that protein carbamylation occurs in urea-containing buffer above 37 degree celsius. Even on ice, sonication can increase local temperature in protein sample, thus significantly enhancing the undesired carbamylation on N-termini of proteins/peptides and at the side chain amino groups of lysine and arginine residues, which may blocks protease digestion and affects protein identification and quantification in mass spectrometry analysis by blocking peptide amino groups from reacting with the amine-reactive NHS ester group on TMT label. It would be beneficial if the authors would provide Venn diagrams of identified proteins among technical replicates for each sample treatment.
4.Some arguments in the Results need clarification since they do not match the data in figures. For example, Lines 192-194 read “Remarkably, the content of H2O2 displayed a lower basal level in Salt-tolerant tomato compared with the Salt-sensitive tomato (Fig.1B)”, however no difference is observed between bar 1 and 3 on Figure 1B. And the following sentence “One hour after imposing, the content of H2O2 in Salt-sensitive tomato was still significantly higher than that in Salt-tolerant cultivars (Fig.1B), which may cause damage to these cultivars.” It is not clear from the Figure 1B what data groups the P values are referring to.

Other comments
5.It is impressive that 7663 proteins are identified using TMT proteomics and, of which, 6501 proteins are quantifiable. It is not clear whether all of the 6501 proteins are quantifiable in all 18 mass spec replicates. Thus it is less obvious that for the section starting from Line 222 (Proteins located at chloroplast exhibited significant difference between SS and ST), are the authors trying to draw a baseline of the proteome differences between SS and ST at time 0 (before treatment)? If so, please make it clear in the text.
6.Lines 118-120: please explain what are the concentrations of salt stress applied and the reason for choosing 200 mmol/L NaCl as final experimental condition.
7.Lines 126-130: please provide the experimental procedures briefly instead of just referring to the assay kits.
8.Lines 131-134: please provide the experimental procedures briefly instead of just referring to the assay kits.
9.Line 139: what is the final conc. of urea after adding trypsin? Trypsin should not work in 8M urea.
10.Line 139: please provide details for peptide extraction and lyophilization.
11.Line 144: Is it a C18 column?
12.STRING database is essentially a literature data-mining resource, which not only contains physical protein-protein interaction data, but also contains functional data. Thus is is crucial to provide the settings when retrieving data from STRING.
13.Line 173: there is no description in the main text whether the three key proteins identified are autophagy-related.
14.Line 253 and Line 270: citations should follow same scheme. Eg. MSANNE et al should be Msanne et al.
15.Line 271: duplicate word “important”.
16.Line 293: “Superoxide (Q7XAV2)” should be “Superoxide dismutase (Q7XAV2)”
17.Line 298: Response should be Responses
18.In Discussion, I feel the tone should be softer in several cases, such as the words on Line 382 “proved”, on Line 395 “guarantee”.
19.Line 502: Brassinolides were also … (missing sentences)
20.Lines 505-506: This sentence is not a good summary since salt-tolerant cultivars, by definition, have more advantage in resistance to salt stress. It would be more appropriate if the authors would change this sentence to similar words like “our results provide mechanistic insights into the advantages of salt-tolerant cultivars vs. salt-sensitive cultivars via comparative proteomic analysis”.
21.Figure 1: Please spell out “ST and SS cultivars” as “salt-tolerant and salt-sensitive cultivars” in the title.
22.Figure 1: Please provide number of replicates used in the figure.
23.Figure 1: “SS0” and “SS1” et al. is confusing without reading the manuscript in detail. Thus I suggest to change them to “SS 0h” and “SS 1h”.
24.Figure 8C-E: please also provide the name of the protein of interest.
25.Figure 8B: Please considering removal of the names for the nodes other than the three proteins of interest for clarity.


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: COMPARATIVE PROTEOMIC ANALYSIS FOR REVEALING THE ADVANTAGE MECHANISMS OF SALT-TOLERANT TOMATO (SOLANUM LYCOPERSCIUM)
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.1 In this manuscript, scattered throughout are words used incorrectly that detract from the reading. Furthermore, I found several subject/verb agreement erros.

For example:

tomato growing ´was` limited by various factors relevant to salt stress` (L 65-66)

mechanisms of tomato resistance to salt stress ´was` still unclear (L 31-32)

Meanwhile, the repair and reconstruction of cell membrane system of seeds ´was` also impaired under saline-alkali stress (L 80-81)

The membrane ion selective absorption capacity ´was` decreased under salt stress (L 82)

Well studied is that salt stress (L 86)

The accumulation of Na+ in soils will result in osmotic stress in plant cells ´will` result in the destruction` (L 68-69)

salt stress ´will` significantly influence the structure of plasma membrane` (L 85)

an in-depth proteome ´were` performed (L 201-202)

only ´protein` in which the log1.5(fold change) > 1 or < -1 and the P 224 value < 0.05 ´were` identified as differentially expressed proteins (L 223-224)

These are mostly things and subject/verb agreement erros that detract from the reading of the manuscript. Thus, I suggest the authors read the manuscript carefully, sentence by sentence, to eliminate grammatical and stylistic errors. I also recommend the authors identify and fix all the subject/verb agreement erros.

1.2 In the introduction, I suggest the authors also cite recent articles (e.g., from 2015 on). Regarding the sentence below, recent studies should also be cited:
´Currently, various mechanisms of plant tolerance to salt stress were elucidated, including reprogramming the photosynthetic pathway (Allakhverdiev et al. 2002; Mittal et al. 2012),enhancing the antioxidant protection ability (Gupta et al. 1993) and synthesis and accumulation of osmotic regulating substances (Yang et al. 2007)` (L 89-92).

1.3 Regarding the sentence ´The physiological indexes of tomato plants were measured using four true-leaf at 0, 1, and 12 h after treatment` (L 122-123), I did not find the results concerning 12 h after NaCl treatment in the topic ´Observable physiological changes between Salt tolerant strain (ST) and Salt sensitive (SS) strain`.
Experimental design: 2.1 There are other articles available on proteomics of tomato plants under salt stress. For example:

´Identification of Proteins for Salt Tolerance Using a Comparative Proteomics Analysis of Tomato Accessions with Contrasting Salt Tolerance` ( https://doi.org/10.21273/JASHS.138.5.382)

´Proteomic analysis of salt-stress responsive proteins in roots of tomato (Lycopersicon esculentum L.) plants towards silicon efficiency` ( https://doi.org/10.1007/s10725-015-0045-y )

So I recommend explaining the novelty of the manuscript in the introduction part, citing other articles on this topic.

2.2 Justification for application of 200 mM NaCl and why the physiological indexes of tomato plants were measured at 0, 1, and 12 h after treatment is not clear to me. I recommend explaining (and citing references) on why this NaCl concentration and these times of exposure are suitable for examining tomato genotypes for tolerance/sensitivity to salt stress.

The authors observed: ´one hour after imposing, the content of carbohydrate, H2O2 and total protein in both salt-sensitive and salt-tolerant cultivars were determined. The results showed that no significant change was observed in the content of carbohydrate among both cultivars (Fig.1A). ` (L 189-192). Perhaps the authors would see differences in carbohydrate content if a later exposure time was used instead of 1 h after treatment
Validity of the findings: 3. A proteomic analysis of tomato plants is reported in this manuscript. However, I am not sure if salt tolerance mechanisms in tomato were unraveled or at least adequately investigated.

3.1	The authors should provide more details about the tomato cultivars (in the introduction and methods), rather than just mentioning 'tolerant' or 'sensitive'. How did the authors conclude that one tomato cultivar is salt stress-sensitive and another one is tolerant? Have previous studies used these cultivars? Was plant growth of these cultivars under salt-stress conditions previously studied? Once again: I believe that these details should be presented in the introduction or in the methods in order to clarify the contrasting salinity tolerance between cultivars.

3.1.1 Quantifying the effects of salt stress on plant growth is crucial to asserting about the contrasting tolerance. On this topic, I recommend reading the article ´Evaluating physiological responses of plants to salinity stress` ( https://doi.org/10.1093/aob /mcw191). Thus, the authors should present phenotypic and plant growth differences between the tomato genotypes before asserting on contrasting salt tolerance. Please see the following article as another reference on this topic: ´Proteomic Analysis of Seedling Roots of Two Maize Inbred Lines That Differ Significantly in the Salt Stress Response` ( https://doi.org/10.1371/journal.pone.0116697 ).

3.2 Regarding the results on proteomics and antioxidants (figure 03), I recommend the authors present results from control tomato plants (NaCl exposure-free condition) in order to compare salt stress responses that are induced within each genotype, better understand the contrasting salt tolerance, and provide enough information on salt stress-induced responses.
Additional comments: 4. Has the proteomic dataset been deposited in any publicly available repository?


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: COMPARATIVE PROTEOMIC ANALYSIS FOR REVEALING THE ADVANTAGE MECHANISMS OF SALT-TOLERANT TOMATO (SOLANUM LYCOPERSCIUM)
Review round: 1
Reviewer: 3

Output:
Basic reporting: Dear Editor,

The review of the manuscript "Comparative proteomic analysis for revealing the advantage mechanisms of salt-tolerant tomato (Solanum lycoperscium)” was completed.

The manuscript possesses major flaws that avoid its publication at the current stage. For instance:

1- ALL TEXT
Authors should carefully check the text of files that were subjected to PeerJ in order to correct typographical mistakes [which could be noticed even in the title (“lycoperscium”), in the first lines of Introduction (“worlf” – line 58), as well as in Supplemental files (“Descrption” – Supplementary Table 2)]. There are glued words too.

INTRODUCTION
2- L68
Please, insert the citation of following statement “The major harmful ions in saline and alkaline soils is Na+”.
What is the source of the Na ions in these soils?

3- Only works from 1985-2011 were cited in the “Introduction” section. Thus, authors should include citations from recent publications in the first part of the manuscript.

MATERIALS AND METHODS
4- L113
4.1- What were the tomato cultivars? Please, insert the names of these cultivar in the M&M.
4.2- What of them is tolerant cultivar to salinity? And the sensitive one?
4.3.- Based on which (previous) work or assays, authors identified these cultivar as tolerant/sensitive to salinity?

5- L114
There are information missing in “peat, perlite and vermiculite”, since authors cited 3 substrates and described the  proportion of only two of them “(1:1)”. In addition, what is the unity of such proportion? volume(v), mass (g)? Authors should correct these mistakes too.

6- L122
Based on which works, did authors selected the dose (200 mmol·L-1 NaCl) and time of exposure to stress plants?

7- What was the experimental design?

8- Did authors performed analysis of variance (ANOVA) for evaluation of differences among treatments?
8.1- If so, were data in accordance to ANOVA's assumptions (normal distribution, error independence, and variance homogeneity)?

8.2- Were data subjected to transformation and/or exclusion before ANOVA?

8.3- According to the manuscript, a two-way ANOVA is preferred over the one-way ANOVA, since two factors (i) “tomato cultivars”, and (ii) “salinity levels” were tested.

8.4- What was the p-value used as cut-off in ANOVA?

8.5- What was the value of significance used for differences among treatments in LSD test?

8.6- What test comparison of treatments’ means was used? LSD, Tukey etc? What was the value for significance?

9- The number of biological replications is very low, especially for the biochemical data (SOD, POD etc), so that the freedom degree is unsuitable.

10- Please, there is missing information about PCA in M&M. Please, cite that and other missing information about the statistical procedures in M&M.

RESULTS

11- The “Results” section is very long. For instance, the text of the first subsection can be easily rewritten in 5 lines in one or two setences.

12- L189
It is not usual to use strain in this cases; therefore, replace “strain” by “cultivar”.

In all Figures and Tables
13- Include the meaning of each abbreviation that were included in the Figures and Tables (for instance, “PPO”, “SS”, “ST” etc).

14- Include the number of replications (n) that were used in Figures and Tables?

DISCUSSION
15- This section is very long. In addition to reduce it, authors should separate “Discussion” into subsections according to the subject.

CONCLUSIONS
16- Please, exclude statements like “Our results suggested that salt-tolerant cultivars have more advantages in resistance to salt stress than that of salt-sensitive cultivars.”
Experimental design: See comments above.
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: COMPARATIVE PROTEOMIC ANALYSIS FOR REVEALING THE ADVANTAGE MECHANISMS OF SALT-TOLERANT TOMATO (SOLANUM LYCOPERSCIUM)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has been improved by authors. Thank authors for addressing my questions. There are still some typos. For example:
1) in Abstract: “were investigated to deciphered” should be “…to decipher”;
2) L224. Proteome cannot be "performed". Please change to "Mass spectroscopy based proteomics was performed.
3) L510 limit
4) Table 1, what is the meaning of "b" in "94.99±3.15b" ?
Experimental design: With the revision, methods are now described with sufficient detail & information to replicate.
Validity of the findings: All sample collection and preparation procedures were performed in triplicate, and each independent biological experiment has 3 technological replicates, Please show the type of the error bar (S.D. vs S.E.M) used in Figures 1, 3, 8, and 9.
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: COMPARATIVE PROTEOMIC ANALYSIS FOR REVEALING THE ADVANTAGE MECHANISMS OF SALT-TOLERANT TOMATO (SOLANUM LYCOPERSCIUM)
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors have considered all my comments. I can see the substantial improvements that have been done in the manuscript. I have no more comments and recommend acceptance.


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: RESPONSE OF BACTERIAL COMMUNITY STRUCTURE TO DIFFERENT ECOLOGICAL NICHES AND THEIR FUNCTIONS IN KOREAN PINE FORESTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors studied how the bacterial community change in soil and roots of Korean pine in pure forests at different ages. They wanted to related community composition/structure and putative functionality to climate and soil chemistry. The introduction focus on different topics all important to understand why the authors choose to study this particular topic and which aspects are known and which are instead the open questions. I appreciate that the authors clearly state their objectives at the end of the introduction. In the material and method, authors should describe better how many samples were processed at the end as they state multiple cores were sampled but then pooled together.
Finally, the abstract need major revision, compared to the main text, it is difficult to read and do not provide the right information, too many sentence for results and no take home message.
Experimental design: In results: table are presented with average and SD but the authors should also include the number of samples used for this (n value= XXX). Table 1 authors state correctly that two sampling have the higher richness and diversity however this phrase is not supported by any statistic analysis please include. In section 3.1.2 it is not clear if the figure 2 include only data from CS1 or all the sampling points, while in the following part of the section authors analysed the data dividing them in CS1 alone and all the samples together, please clarify. Addtionally for table 1b CS1 does included only August samples or the total? And if the latter how the authors have taken in account the difference in number of samples. In general for alpha diversity data authors do not support their statement with statistical analysis. This trend is also continued in section 3.2.1 e.g. line 315/316 significantly higher but not stats are provided.
Figure 4, 5 should be redone. Relative abundance data are better presented as stacked histogram, the present figures are hard to ready and understand.
Validity of the findings: Discussion authors state that they did not found clear evidence of climate influence on the bacterial community however the difference in temperature is quite limited as all the sampling points are relative close, if winter sampling was performed a more marked difference in temperature would be found and maybe the results would have been different. Authors should state the difference in climatic variables are quite small and this could be the cause of the lack of effect. Line 450-451 how the authors can justify this phrase? Please explain better. Discussion is quite short and does not provide any additional information compared to the results, some interesting points are just briefly discussed and should be better explored. At the moment there is a clear lack of development of this section.
Additional comments: Sometime the text is repetitive or over wordy and can be simplified for an easier reading. E.g. line 102 Recently,…. In recent years.
Line 154 sapling -> sampling?


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: RESPONSE OF BACTERIAL COMMUNITY STRUCTURE TO DIFFERENT ECOLOGICAL NICHES AND THEIR FUNCTIONS IN KOREAN PINE FORESTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Understanding the composition, diversity and functions of bacterial community is a long-standing topic in ecology, and the authors provide a detailed study on these respects of bacterial community in Korean pine forests. Experimental design provides an intuitive perspective from which the complete dynamics of bacterial community can be obtained. Results are reasonable.
Experimental design: 1.	Why the authors do not show the soil bacterial and physicochemical properties of the blank plots (i.e. plots without planting trees)?
2.	Not only the RDA, but also other models can be used for evaluating the relationship between bacterial community structures and environmental factors. Please to explain why the RDA method is used here for your purpose.
3.	Line 160-165: There are reference for collecting rhizosphere soil samples of forests. Please to supplement relevant references here.
4.	Line 167: Does “ice” mean the dry ice here? If not, it will affect the subsequent extraction of soil or plant bacteria, and the authors should ensure the accuracy of cryopreservation methods.
Validity of the findings: Overall, the research results are reliable, and the research conclusions are somewhat innovative.
Additional comments: INTRODUCTION
1.	The objectives of this study seem too much and scattered, and it is suggested to further refine the research objectives.
RESULTS
2.	Line 277-278: It can be seen from Table 1 (b) that CS1-G has the lowest Shannon index (2.73 ± 0.01). Please consider correcting the statement here.
3.	Line 314-320: Authors should consider selecting the dominant bacterial taxa as the biomarkers of the different forest ages according to their feature importance.
4.	Line 330-331: The five differently aged forests should be five different forest sites.
5.	Line 378-395: Please to consider showing a table or figure to illustrate the relationship between climatic factors and soil physical and chemical properties separately, because of that the readers can see the result more visually.
6.	In Figure 9 (a): RDA1 and RDA2 account for a small proportion, why?
DISCUSSION
7.	The authors should further strengthen the analysis of the relationship between bacterial community functions and dominant bacterial taxa in different forest ages, and analyzed the difference of dominant bacterial groups in soils of different ages.