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: SUBCELLULAR COMPARTMENTALIZATION OF THE PLANT ANTIOXIDANT SYSTEM: AN INTEGRATED OVERVIEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written, professional English was used throughout the manuscript.

Basically the paper put together a revision of the plant antioxidant system in an integrative way. The main concepts are correctly explained according to up to date understanding of antioxidant enzyme families and antioxidant molecules. The major point is to look and integrate knowledge about this system at different cell locations, like the cytosol, mitochondria, chloroplast and peroxisomes.

In line 310, the name of the fungi is misspelled, is it Botrytis cinerea Pers.?

In line 323, the word arabidopsis should be Arabidopsis

In line 368, it says in chloroplasts associated, I suggest: .. in chloroplasts are associated ...

In line 441, do you mean ascorbate and glutathione are present or presented in vacuoles?
Experimental design: The method to select experimental work published through the years is clearly described after the Introduction.
Validity of the findings: Novel analysis of available information about the antioxidant system is presented. As part of this analysis the discussion is reasonable and authors synthesized the information in an organized way. Although the analysis was kind of historical, the length is appropiate to get a sense of how this field has advanced. It also, authors add insight to what could be done ahead to make more integrative studies.

I consider important the way they uncover current opportunities to set up enzymatic assays that are still lacking.
Additional comments: My suggestion is about information at Table 1, I am not sure about the relevance of the pathway link column, perhaps if this part is explained in the text it will help to understand how it can be helpful.


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: SUBCELLULAR COMPARTMENTALIZATION OF THE PLANT ANTIOXIDANT SYSTEM: AN INTEGRATED OVERVIEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is well written and the english language is good
Experimental design: no comment
Validity of the findings: Authors described their methodology and made a really good work of synthesis in the AOS components part. This paper also shed the light on modeling approaches to study antioxidant system and this is a very interesting part.
Additional comments: I only have a minor suggestion/modification: I was wondering why thioredoxins and glutaredoxins did not appear in the introduction part, in abbreviations and in table 1. First I was thinking that authors have forgotten these components since they talk about them in the main features of the antioxidant system components. Maybe authors could consider thioredoxins and glutaredoxins as the other components, because their choice of presnetation it is a little disturbing, as if thioredoxins and glutaredoxins were of less importance.


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: SUBCELLULAR COMPARTMENTALIZATION OF THE PLANT ANTIOXIDANT SYSTEM: AN INTEGRATED OVERVIEW
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a very interesting review which summarizes much of the scientific knowledge about antioxidants systems in plants, putting together aspects of biochemistry, plants biology and subcellular integration.
 The review is correctly organized in sections including different fields of the Antioxidant System (components, regulation, compartmentalization, modelling).). It is appreciable that a large bibliographic research was done, including both up-to-date and also some outdated bibliography
English grammar requires extensive editing. Authors should pay careful attention to some phrases or sentences which are confusing.
Experimental design: The survey methodology is very simple and concise and it is well described.
Bibliography is very abundant, covering all the aspects of the topics reviewed.
Some minor mistakes were found such as sources missing in the bibliography section and some citations incorrectly written.
Validity of the findings: no comment
Additional comments: Some suggestions were included in the attached pdf which, in my opinion, are important for the understanding of this review.


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

Input:
Title: THE CHEMOTACTIC SWIMMING BEHAVIOR OF BIRD SCHISTOSOME MIRACIDIA IN THE PRESENCE OF COMPATIBLE AND INCOMPATIBLE SNAIL HOSTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English is generally fine, but some passages need rephrasing or polishing. For example, Table 1, “Variants of filling the side arms” is extremely awkward writing. And so is “The place of closing the arms” in the caption of Figure 1.

The Introduction is narrowly focused on digeneans, specifically schistosomes. There is an extensive literature on transmission interference by non-compatible hosts acting as decoys, sinks, etc (see Thieltges et al. 2008 Parasitology). It would be nice to have a broader context presented in the introduction.
Experimental design: The snail Potamopyrgus antipodarum is exploited by a rich fauna of digeneans in its native New Zealand. It would be important for the authors to tell us about the parasites of this snail. Are any of those schistosomes? Could it be that this snail is already compatible to other schistosomes?
Validity of the findings: The results of all statistical tests should be presented by including the test statistic, and not just the P-value.

Also, I don’t think Kruskal-Wallis tests are appropriate. Because the output data are categorical with three possible outcomes (miracidium chooses one arm, the other arm, or neither), the authors should use a multinomial GLM, using each replicate trial as an observation, with the three series as random factor.

The possibility that Potamopyrgus antipodarum interferes with miracidial host-finding and reduces the risk of swimmer’s itch depends entirely on its abundance and distribution. The authors provide no data on this. Is this non-native snail occurring in lakes with swimmer’s itch at sufficiently high densities to cause interference? If not, then the conclusions are a little exaggerated.
Additional comments: This is a simple but well-designed experiment testing the attraction of miracidia of two species of schistosomes to the odours of their snail host versus the odours of non-compatible snails. I feel it could be accepted, but first it requires some revision.


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

Input:
Title: THE CHEMOTACTIC SWIMMING BEHAVIOR OF BIRD SCHISTOSOME MIRACIDIA IN THE PRESENCE OF COMPATIBLE AND INCOMPATIBLE SNAIL HOSTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	There are some minor typographical and grammar errors including:
a.	Line 191 – the space is missing between “regenti” and “larvae”.
b.	The caption for Figure 1 is missing a closing parenthesis.
c.	In Lines 20, and 80-81, the authors have written “incompatible snails species”, the plural of snails is superfluous.
d.	Line 27 – A sentence should not start with an abbreviation
e.	Line 96 – Suggest using the appropriate term for an autopsy performed on an animal.
f.	Lines 143/149 – Suggest removing the first-person phrasing.
g.	Line 78 – Suggest replacing “check” with a more appropriate word.
Experimental design: 1.	There is some additional information required for another researcher to be able to successfully replicate this study.

a)	i) Did the larva have to be fully in the choice chamber to be counted as such? What about larvae that remained in the “C” area or the arms leading to the “B” areas of the choice chambers? What distance from the choice chambers was chosen for the larva to be considered to have preferred that water treatment?
ii) In the supplemental tables the authors provided, all of the series values for each variant add         up to 10, does this mean that in every single replicate the larva ended up entirely in one of the arms?
b)	Were the choice chambers cleaned thoroughly between each replication? If not, it is possible that a higher concentration of snail excretory products was present than intended.
c)	The authors state that one larva was used for each repetition, does this mean that each larva was used only once and replaced, or that one larva was consistently used in all 10 repetitions in a series?

2.	The sample size for this study is adequate only if a new larva was used in each repetition (n=60), otherwise with pseudoreplication, n=6 (3 series for each parasite species) which is not sufficient.

3.	In the statistical analysis section of the methods, the authors state that they used the sum of the larvae in the arms from each of the variants to get a value for every arm in each of the six series. But, in Figure 2, they report the average number of miracidia in the arms per series. It is misleading, because when the authors report the results, they give the p-value from the statistical analyses and cite Figure 2, but those should be two separate entities. These need to match.

4.	In the results section, the authors report the p-values for the variants, after which they have a statement regarding the percentage of larvae found in the specific choice chamber for that variant. If the percentages are mentioned, they should be included as a numerical value. It is not necessary to mention the percentages when the results are insignificant (i.e. variants V, VI, VII).
Validity of the findings: 1.	A possible confounding factor in this study is that the Potamopygyrus antipodarum snails may have had pre-patent infections. Is it possible that a snail with a pre-patent infection excretes a different chemical cue than a snail that is free of infection?
2.	Why were young individuals of the lymnaied snails and adults of P. antipodarum used in this study? Since the lymnaeid snails were taken from lab colonies, why not keep it consistent across all snail species and use adult lymnaeids?
Additional comments: This paper discusses using an incompatible host species of snail to prevent miracidia from finding their compatible host species as a method for controlling swimmer’s itch. The research gap identified in the study was the use of miracidial behaviors and preferences as a means of reducing the risk of swimmer’s itch instead of killing hosts or releasing a chemical into the environment. This was evident in the Introduction and Conclusions but was lacking in the Discussion section. The research outlined in this paper is within the scope of this journal and the authors have well-defined research questions. The proper controls were in place, the required ethical information was present, and the experiments were carried out ethically. There is some information missing from the Materials and Methods section that would be required to replicate this experiment, which is addressed below. The Conclusions section linked back to the original research questions and acknowledged that there were still further studies that were required to answer those questions.

The authors found that miracidia of both Trichobilharzia szidati and T. regenti preferred the lymnaeid snail-conditioned water over the dechlorinated tap water, whether it was from the compatible or incompatible host. And, when given the choice between the compatible or incompatible lymnaeid snail-conditioned water, there was no significant difference between the two chambers for both species. As for the tests with the non-native snail-conditioned water, T. regenti appeared to avoid it, while T. szidati chose the snail-conditioned water more than the dechlorinated tap water. When given the choice between the non-native snail-conditioned water and lymnaeid host-conditioned water, compatible or incompatible, there were no significant differences between the choice chambers for the miracidia of both species.

In the introduction, the authors clearly explained their reasoning as to why the reader should be concerned about swimmer’s itch, and why it is important to find a way to combat it without negatively impacting the local fauna. The authors clearly articulate why the incompatible snail species may be able to act as a shield for compatible snail hosts. However, there are a few ways to improve the introduction, including explaining what the miracidia and cercariae larval stages are with respect to the schistosome life cycle. This will help the reader to better understand the authors’ points in the first paragraph. Also, it would help to affirm the authors’ message if it was explained that snails can release thousands of cercariae during a trematode infection. Therefore, decreasing snail infections through the use of methods explained in this paper could greatly help in reducing the occurrence of swimmer’s itch. The authors touch on this point very briefly in Lines 77&78, but it should be expanded upon.

Aesthetically, the figures are clear and easy to understand. As for the content, there is some confusion on the results reported in Figure 2, which is addressed below. Table 1 does a really good job of explaining all of the variants used in the study and was the most concise way to describe them, as writing them out in the results section likely would have been cumbersome and confusing.

For the most part, the manuscript was well written and straightforward. There are some places where the phrasing could be improved, such as in Lines 27-29, 59, 62-63, 68-70, 71-74, and 125-126 to aid in reader comprehension.


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

Input:
Title: THE CHEMOTACTIC SWIMMING BEHAVIOR OF BIRD SCHISTOSOME MIRACIDIA IN THE PRESENCE OF COMPATIBLE AND INCOMPATIBLE SNAIL HOSTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Please find my detailed remarks summarized in the general comments section below.

The English language is generally understandable but could benefit from some proof-reading and editing, e.g. in lines 62-63.

Raw data is provided.
Experimental design: The experimental setup is well-designed and appropriate to test the authors’ research question. Materials and methods are described in adequate detail to allow replicate studies (see some minor comments below though).

L110-113: It would be helpful to highlight which of the snail species represent compatible/incompatible hosts to the respective trematode miracidia.
Validity of the findings: See below.
Additional comments: The authors present a study on the chemotactic swimming behaviour of bird schistosome miracidia in the presence of compatible and incompatible snail host species. Since bird schistosomes are important infectious disease agents in recreational water bodies in Europe, this study is of relevance and should be of interest to the scientific community.

However, the manuscript has some shortcomings and weaknesses, in particular in the introduction and discussion sections, that would need to be addressed before considering the work for publication. Unfortunately, I have to recommend major revisions of the manuscript in its current from. I hope these comments can be useful for a successful re-submission of this undoubtedly relevant and interesting work.

*********************************

In detail, I have the following main concerns and objections: Overall, the introduction and discussion sections seem a bit rushed and could be expended.

1.	I believe it would be very helpful to include a brief overview of the general bird schistosome life cycle, as not all readers will be familiar with it. Furthermore, I suggest explaining the typical host-specificity of these parasites to their first intermediate snail host, since this is a central element of the study’s research question.

2.	One of the key aims of this study was to test whether “miracidia of bird schistosomes exhibit chemotactic swimming behaviour” (L78-79). However, the authors already answer this question in the introduction (L58-59). Furthermore, this has also been investigated for bird schistosomes specifically and some of the relevant literature is already cited by the authors. The following reference could be added in this context: Hertel, J., Holweg, A., Haberl, B., Kalbe, M., and Haas, W. (2006). Snail odour-clouds: spreading and contribution to the transmission success of Trichobilharzia ocellata (Trematoda, Digenea) miracidia. Oecologia 147, 173–80.

3.	I am critical of the interpretation that “as a result of the closer micro-habitat of suitable snail hosts (Radix spp.) and non-host snails (P. antipodarum), T. regenti probably developed a tool to avoid penetration of a non-host snail” (197-199).
Potamopyrgus is a rather recent invader in European and North American waterbodies, and likewise, lymnaeid snails were only recently introduced into New Zealand freshwaters, the native habitat of P. antipodarum. Accordingly, the host-parasite co-evolution of bird schistosomes and their snail hosts (or potential dead-end diluters) has occurred for the longest time without contact to P. antipodarum. In the case of Potamopyrgus/Trichobilharzia such an adaptation would therefore have occurred quite recently and very quickly, which would require strong selective pressure. I am absolutely in favour of speculation but I would suggest a more cautious interpretation of these results, if possible backed by and discussed in the context of findings from comparable studies on such evolutionary processes.

*********************************

Furthermore, the following minor comments should also be considered by the authors:

•	L1: The title seems a bit wordy to me. Suggestion for a slightly shorter version: “The chemotactic swimming behavior of bird schistosome miracidia in the presence of compatible and incompatible snail hosts”
•	L13, and throughout the text: Please change “invasive” to “infective” when speaking of cercariae/miracidia. Potamopyrgus antipodarum is an invasive species.
•	L16: Please change “application” to “presence”
•	L57: Please change “between” to “in sympatry with”
•	L72: “Marszewska et al., 2018b” is cited before “Marszewska et al., 2018a” (L208)
•	L90-95: Move to separate ethics section.
•	L106: Please add the number of snails used.
•	L173: change “Digenea” to “digenean”
•	L178: Suggestion “Our study supports these findings and shows…”
•	L181 and following: Please remove “statistically significant” in the discussion.
•	L191: add space between “regenti” and “larvae”
•	L195: pleases change to “covering the lake bottom in the shallow littoral zone, similar to…”
•	L196: Is “mimic” the correct term here? I would suggest “are adapted to”
•	L215 following: Please check the references. Species names should be in italics; journal names should use capital letters.
•	Figure 1: I would suggest labelling the side arms “B1/B2” to show that they contain different treatments.

*********************************

Despite my critical points, I commend the authors on their work and I hope my suggestions can be useful for a successful re-submission of 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: THE CHEMOTACTIC SWIMMING BEHAVIOR OF BIRD SCHISTOSOME MIRACIDIA IN THE PRESENCE OF COMPATIBLE AND INCOMPATIBLE SNAIL HOSTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English writing remains generally poor in many places (grammar and spelling), and ok at best elsewhere. It needs improving either by a fluent English speaker familiar with science writing, or by a professional editing service.
Experimental design: My earlier concerns have been satisfactorily addressed.
Validity of the findings: My earlier concerns have been satisfactorily addressed.
Additional comments: My earlier concerns have been satisfactorily addressed, but the writing (in many places) still falls short of what I would consider acceptable.


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

Input:
Title: THE CHEMOTACTIC SWIMMING BEHAVIOR OF BIRD SCHISTOSOME MIRACIDIA IN THE PRESENCE OF COMPATIBLE AND INCOMPATIBLE SNAIL HOSTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: I acknowledge the authors’ efforts to include all suggestions and feedback from the three reviewers in the revised version of their manuscript. I don’t have any further major comments and can suggest the manuscript for publication in PeerJ.

Congratulations to the nice study.

The authors might want to consider the following minor comments though:
-	L52: Some more information on the disease could be added here, e.g., “bird schistosome cercariae can accidentally penetrate the skin of people swimming or wading in the water, where they can cause an allergic reaction and an itchy rash.”
-	L60: please add a comma after “applicable”
-	L74: Maybe “non-host species” is more suitable than “potential targets,” since the following examples include predators, filter feeders etc.
-	L76-77: I feel the beginning of this sentence is a bit vague. Maybe something like “Studies have shown several ways how these non-host organisms can disrupt parasite transmission, e.g., by acting as physical barriers....” would work?
-	L81: I would suggest adding “as diluters” after “importance”
-	L94: Please change to “schistosome miracidia”
-	L95-103: I feel this paragraph might be better placed in the discussion section.
-	L148: “were kept in 100 ml of dechlorinated water for 2 h.”
-	L203: “It is also well known that many digeneans use chemo-orientation to find suitable hosts in the water column.”
-	L231: Full species name at the beginning of sentence should be used.


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

Input:
Title: REVIEW OF THE GENUS LOIMIA MALMGREN, 1866 (ANNELIDA, TEREBELLIDAE) FROM CHINA SEAS WITH RECOGNITION OF TWO NEW SPECIES BASED ON INTEGRATIVE TAXONOMY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language is overall clear and unambiguous but some spelling and structural issues that are highlighted in the General Comments section. I would further advise to replace "huge" and "enormous" with more toned synonyms.

The introduction should mention how Loimia is identified compared to other Terebellidae (uncini, branchiae, lobes), so that it is clear how material was assigned to this group. There are further some nomenclature inconsistencies because some of the species in Chinese seas were not originally placed in Loimia and the author name and publication year should therefore be enclosed in parentheses to follow nomenclatural rules. This applies to 3 species (L.76-77), Loimia ingens (Grube, 1878), Loimia medusa (Savigny, 1818), Loimia montagui (Grube, 1878).
Experimental design: A main issue is that not all available sequence data is being used. While sequences for two other terebellids are included as outgroups from published data, available sequences for the species under investigation are not considered. Genbank has COI, 16S and one 18S for species of Loimia (Loimia ramzega, Loimia medusa, Loimia arborea, Loimia ingens) that were not used here. If there is evidence that these sequences should not be used (e.g. suspected misidentifications of the species) that would be valid but it should be justified why publicly available data is not being used here to gain the most insight into Loimia species.

The phylogenetic analyses are carefully performed but I would encourage to add a maximum likelihood analysis of the data. The analysis should involve selecting the best fit model for each data partition (COI, 16S, 18S) rather than estimating a substitution model across all three partitions because these genes are from different genomic compartments (mitochondrial, nuclear genome) and have different rates and patterns of substitution. The partitioned dataset can then be used in a maximum likelihood framework with bootstrap supports. RAxML or IQTREE can be used for these purposes and both approaches have free-to-use servers that can be used with small datasets like this one. IQTREE even performs model selection with Modelfinder as part of the analysis.

I discourage the use of "basal" when describing phylogenetic relationships among species (albeit this term being very commonly used in the literature). Krell & Cranston 2004 Systematic Entomology describe the problem clearly: "Every branching in a (phylogenetic) tree is rotatable (see Fig. 1). Of course, the tree has a base, and there is a most basal branching and a next most basal branching, but there is no such thing as the most basal clade. Because branchings are rotatable, there are always two most basal clades (if the most basal branching is completely resolved, Fig. 1) or even more most basal clades (if the most basal branching is not completely resolved, e.g. in Polyneoptera in Fig. 1). Both branches originating from a node (i.e. the two sister groups) are of equal age and have undergone equivalent evolutionary change. Whether a group has branched off early (basal) or later in the phylogeny contains no information about this particular group, but information about both this group and its sister group, because both branched off at the same time." I encourage the authors to describe the phylogenetic relationships among Loimia species using descriptions about sister groups. This applies to the Abstract and the paragraph L.388-394, L.456-462.
Validity of the findings: no comment
Additional comments: Thank you for a carefully conducted and easy to follow study.

Minor comments on language and structure:
Abstract
- Add Annelida, Terebellidae after first mention of Loimia
- "collected from the coasts of Shandong..."
- Remove point after "respectively: Loimia."
- 2 spaces missing: "COI, 16S rRNA and 18S rRNA"
- small case on "phylogenetic tree"
- upper case on "identifying Loimia species"
- "China seas" could be better as Chinese seas

Abstract, L.264, L.266, L.363, L.460, L.468: I believe it is uncommon to start sentences with an abbreviation (of Loimia to L.)

L.79: add space after Malmgren, 1865
L.75: capitalize China
L.102: maybe better "from coastal areas of
L.114: "using the primer pairs:
L.142-143: repeated reference to Gblocks compared to L.136
L.180: maybe "convoluted" or "complicated taxonomic history" rather than "confused"
L.181: remove dot before reference
L.181: "occur in tropcial areas" or "are distributed in tropical areas"
L.183: Add "The most useful character"
L.196: Add space after August 21,
L.216: remove ")"
L.217: Add space after October 22,
L.219: lower case specimens
L.252: replace "and" with "," in Figure reference
L.277: Add "." at end of sentence.
L.281: remove ")"
L.286: remove ")", add space after April 21,
L.291: add space after August 21,
L.304: remove ")"
L.376-394: all occcurrences of L. arborea and L. bandera misspelled
L.396: maybe better "reidentified" than rechecked
L.397: remove "out" after finding, remove "very"
L.398: "new to science"
L.400: add space segment 3
L.409: misformatted reference
L.411: "does not dwell in Chinese seas"
L.411-413: sentence is difficult to understand and the last references seem misformatted.
L.414 : remove . after Loimia
L.416: we referred to the description"
L.438: "where notopodia end"
L.446: "species that have been deposited"
L.460: lower case phylogenetic
L.467: suggest to remove "independent" because COI and 16S are not independent because they get inherited together on the mitochndrial genome. Could be stronger to say " from three mitochondrial and nuclear molecular markers"
L.473: why "preliminary", remove?
L.477: add "pairs" after third to be consistent with L.479-480
L.496: remove "extremely"

Table 4: add space L. arborea and before Japan
Table 5: misspelled arborea and bandera, inconsistent spacing sp.nov.


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: REVIEW OF THE GENUS LOIMIA MALMGREN, 1866 (ANNELIDA, TEREBELLIDAE) FROM CHINA SEAS WITH RECOGNITION OF TWO NEW SPECIES BASED ON INTEGRATIVE TAXONOMY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is poorly written- Intro very confusing
latest references are not quoted

the format for new species is not up to current standards for new species
Experimental design: not applicable
Validity of the findings: ok but fail to understand that molecular data from material outside of type locality is not useful
Additional comments: a major revision is needed have added sticky notes throughout the manuscript


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

Input:
Title: REVIEW OF THE GENUS LOIMIA MALMGREN, 1866 (ANNELIDA, TEREBELLIDAE) FROM CHINA SEAS WITH RECOGNITION OF TWO NEW SPECIES BASED ON INTEGRATIVE TAXONOMY
Review round: 2
Reviewer: 1

Output:
Basic reporting: see attached file still some typos and some changes still needed
Experimental design: ok
Validity of the findings: ok
Additional comments: see my attached file


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

Input:
Title: LIGHT DEPENDENT COURTSHIP BEHAVIOR IN DROSOPHILA SIMULANS AND D. MELANOGASTER
Review round: 1
Reviewer: 1

Output:
Basic reporting: I’m not native English spiker but I think that the language is good enough, it is clear and fluent.
The introduction is generally well written. Specific comments are provided in the pdf file.
Figures match the criteria and raw data was provided.
Experimental design: The paper covers the material well known to the scientific community. However, the authors believe that the previous researches do not cover a large enough sample to represent a whole species. In my opinion, the sample size in this research is small too. If the authors aimed to increase the sample size, in addition to increasing the number of strains, the number of pairs needed to be increased too. The 1320 pairs are still not enough to represent the whole species. It is necessary to either change the approach, not emphasize the sample size, or increase the number of pairs. Authors could switch the focus to the correlation between light-dependent copulation success and collection variables (longitude and latitude).
Why singing and copulation attempts have not been analyzed and discussed? What is the connection with this work?
Specific comments are provided in the pdf file.
Validity of the findings: Underlying data have been provided. Statistical analysis is appropriate. The conclusions are clearly defined.
Additional comments: Generally, the paper is interesting well and writhen. However, it is necessary not to underline the sample sizes. There are more interesting findings. One of them is the correlation between light-dependent copulation success and demographic history of species.


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

Input:
Title: LIGHT DEPENDENT COURTSHIP BEHAVIOR IN DROSOPHILA SIMULANS AND D. MELANOGASTER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The ms is clearly written except the second part of the discussion which is vague. The literature is mostly relevant but several critical references are missing. The illustrations are OK except for a color code detail. The raw data are supplied.
Experimental design: This ms presents relatively original data given that a couple of similar papers appeared in the late 1970’s (Joe Grossfield) also testing the inter- and intraspecific effect of light on Drosophila mating. The research question is well defined. The investigation was performed with great rigor. The methods are described with enough details to allow replication.
Validity of the findings: The novelty is not completely true (see my comment above). The data are statistically sound but there is some misinterpretation related to the longitude parameter. The speculation is questionnable. Some of the conclusions are well stated, but not completely supported by the data shown. Some supplementary data should be added to provide a clear answer and to support more firmly the current conclusion.
Additional comments: Paper by Shahandeh and coll

This paper deals with the effect of light on a set of populations of two sibling Drosophila species: D.melanogaster and D.simulans. These populations, obtained from stock centers, were initiated by flies collected at different years. The authors have tried to compare pairs of populations (in the two species) collected nearby. The principal experiment consisted to indirectly measure mating frequency (based on fecundity) between pairs of flies either tested under white light or under darkness, during 2 hours and during 6 hours. Based on the ratio between “mating” frequency under the two light regimes (only for 2-hour long tests) populations were classified based on their ratio (between mating frequencies under light/darkness) during two hours. Authors have tried to establish correlations between this ratio and various geographical parameters (longitude, latitude). They also measured the “real” courtship response in the two extreme “ratio” lines in each species under each light regime and they performed a reciprocal cross experiment between two extreme ratio D.simulans lines.

I found generally the experimental design of the paper solid, but the most intersting findings were not developed enough to convey the authors to a strong conclusion. Moreover, a large part of the Discussion is too vague and should be completely rewritten.

Major points:
A better description of the Drosophila populations tested should be provided into a complete table showing not only the coordinates but also the altitude at which the founder flies were collected, together with the type of vegetation and climate.

LL 225-236 : It would be worth to complete this paper with cross experiments with the two extreme D.melanogaster lines (similarly to what was done with D.simulans).

The Discussion is loose and too vageu, specially in its second half.

Minor points:
L 43: the reference “Savarit et al., PNAS 1999” needs to be added

L 95-96: Keeping males together until the mating test can strongly affect their wiliingness to mate depending on male genotype (Svetec et al., Genetical Research Camb, 2005). Please explain why you have kept together before the test.

L102: The presence of food in the test can interact with the presence/absence of light as shown with two D.simulans populations and two D.melanogaster populations (Cobb & Ferveur, Behavioural Processes; Table 1). Please discuss.

L158/L177: “...more successful...” than what?

LL 206-207: The longitude scale is not a natural parameter per se given that it is only based on the convention of the 0° at Greenwitch meridian. If, for D.simulans (Figure 4B), one shifts the world map representation starting from left to right in Oceania (100-150-with AUS004 + NG009 lines) then continuing with Pacific ocean and America and ending with Africa/Europa, I would be curious to see the level of the correlation?

I generally find that the longitude is not a real biological parameters, and I think that oceans and deserts are more likely to cause separation between populations. To me, the latitude would represent a more potent factor to investigate, despite the fact that the number of populations investigated here is too limited to establish a real strong correlation. Moreover, the pairs between sibling species should be examined more carefully than just belonging to same country (another artificial non-biological factor) but should rather be related to ecological and geographical parameters such as altitude relatively to the sea level, type of vegetation (forest vs desert), seasonal variation of temperature etc...

L235 : delete « interestingly ».

LL272-273 : I found completely non-realistic to test the effect of collection date given that it can be confounded with so many other parameters examined, or not examined here (longitude, latitude, light x food interaction, group effect...). Otherwise, the authors could always carry a study similar to that performed on a smaller geographical range— Houot et al. J.Exp. Biol. 2010—to test the effect of acclimation on mating and pheromones after several years spent in the laboratory).

L276-280 : I do not agree with this conclusion made and with the explicative scenario provided (LL281-286).

L314 « absences » or « absence » ?

L317 : One cannot conclude this since complementary tests are missing (see above and below).

LL333-334 : I found that the cross population mating experiment probably provides the most exciting data ofthe paper. To validate this result, the authors should test another cross (reciprocal crosses between MAL261 x GEO288 or MAL261 x GRE181).

L337 : « ...possible...are... »

L351-352 : 7-T is not very different between D.simulans sexes, but only males produce cis-vaccenyl acetate (Jallon, Behav Genetics 1984).

L357 : « ...suggest that male latency to initiate courtship... »

L367 : « « ...what other signals cause... » please rewrite.

LL367-377 : the style is loose : the authors should rewrite (not « we’ve » !)

L373 : « we’ve » what does this mean ? It seems that « other authors have identified... »

L369 : « smell-blind males in the dark ? Tarsi-less males ? ». The authors should refer to published papers: Robertson Experiencia 1983; Grillet et al., R.Soc. Open Sci. 2018.

LL377-378 : I feel that the authors have not exploited the most interesting point of their study: instead of inter-specific difference, they should have explored more deeply inter-populations (intraspecific) variation. This is specially worth when looking at the data shown on the Figures 5B & C (see also my point above).


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

Input:
Title: LIGHT DEPENDENT COURTSHIP BEHAVIOR IN DROSOPHILA SIMULANS AND D. MELANOGASTER
Review round: 1
Reviewer: 3

Output:
Basic reporting: This paper addresses the use of light (and by inference, vision) in courtship behavior in Drosophila melanogaster and D. simulans. Although mating in the dark has been explored extensively in these species (much more so in D. melanogaster than D. simulans), what is unique in this paper is the use of multiple strains from different longitudes. This is probably the best assessment of species variation in this trait that I have seen (if not the only one).  The raw data are solid, and most is included (the data for longitude, latitude, or age of strain are missing), but the analyses (problems detailed below) seems to have many problems.

In general, the discussion of visual assays, except within a neurobiology context, is very dated. Many people have done more recent assessment of visual dependency using other approaches and assays (such as painting over eyes, e.g. Giglio & Dyer 2013, Ecology and Evolution 3: 365).
Experimental design: For the most part the experimental design is good, but the sample sizes are too small for the light/dark observations (N=10 for D. melanogaster and N=3-7 for D. simulans without the same numbers done for comparisons). I didn’t find reports of the sample sizes in the text—they are in Table S2. I do not think the sample sizes are sufficient for strong conclusions to be drawn from the data. Moreover, with careful observation in the dark, many of the questions about the visual cues necessary for males to start courtship can be addressed. The authors missed the opportunity to calculate courtship latency in these assays (they probably have the data), which might provide clues about how males court without vision. A recent paper (Roy and Gleason, 2019, Behavioural Processes 158:89) measured courtship parameters in the dark in other melanogaster group species providing evidence that males of some species start courtship in the dark more slowly than in the light (but some are equally fast).  Such analyses might provide insights into the differences between D. melanogaster  and D. simulans.

There are a few areas where the materials and methods are not clear. These are below with the line comments.
Validity of the findings: I have many questions about the data analysis, particularly the calculation of Pdark/Plight and the way it is graphed. Looking at the data in Table S1, some of the these values are negative, which is not possible with this data.  I recalculated the values and mine match what is in the table for the 2 hour D. simulans, but after that the numbers are incorrect.  The values are graphed in Figure 2C.  First, I don’t understand why the y-axis has a scale of 0-110 and not 0-1.1 (the correct scalce for a percentage divided by another percentage scale). Secondly, if there a negative values, these should appear on the figure. Thirdly, the numbers are clearly not correct in the figure, both based on the numbers in Table S1 and the values in Figure 2A and Figure 2B.  For example, after 6 hours, NUE006 mated more in the dark than the light, thus Pdark/Plight should be greater than 1. This strain appears in Figure 2C nearly at the bottom of the figure, where as it should be closer to the top. Thus all the analyses are questionable.

This is also a problem in Figure S1A.  Only three of the points are in right place on the Y axis (if assuming the scale has been multiplied by 100).  Thus, I don’t think the analyses done with longitude, latitude, or age of strain are correct (and the data for these analyses are not included, but they should be).  I think it is possible that the authors transformed the data, but this is not explained (and also does not explain why three points are correct and the rest are not).

At this point I stopped evaluating the paper for the results based on the 2-hour and 6-hour data because I think all the analyses have to be repeated.

I was also looking for a discussion of the biological relevance of doing 2 and 6 hour assays. I was expecting a discussion of how short these observations are with respect to many that Grossfield and others have done.  In addition, I was expecting an explanation for changing to an only 30 minute assay for the observation tests.
Additional comments: Line 50: use of the word “surprising.” Although it is true that many species have some light dependency, there are also a large number that are light independent. Basing D. melanogaster behavior on that of other species does not seem to be a great argument.

Line 55: Please explain what a “postural display” is.  I am unfamiliar with this.

Line 61: Please avoid the use of contractions. Also, the males in this experiment were not physically blinded, but were mutant for a particular gene. The point that when males cannot see they do not do as well as wt males is valid, but make it clear that this is a genetic mutation. To further support this, mutant white males, which are blind, also do not court as well as wt males.

Line 65: “assays of variable lengths and designs.”  It would be helpful to the reader to understand what are the different past approaches to these kinds of experiments. In addition, the citations here, from 1959 and 1950, predate even Grossfield’s (1971) approach.  Moreover, many people have studied light dependency more recently, thus variability in studies should be updated.

Line 93: How close to eclosion were virgins collected?

Line 101: How long after lights on was the experiment performed?

Line 103: Given that this is a paper about vision, a description of the light used would be helpful (For example, wavelengths, light source, Lumosity, distance from vials—all these things may not be necessary but provide at least a description of the light bulb).

Line 111 (and throughout the paper): The two measures are not different timepoints, but rather different exposure times.  When I read the abstract I assumed that different timepoints were used such as 2 hours post lights on and 12 hours post lights on.

Line 125: use of collection date: not all strains in Table 1 have collection dates.

Lines 130-142: Were the dark and light experiments performed on the same day?

Line 134: How long after lights on was the experiment performed?

Line 136-139: I found this description confusing.  I think what happened is that for each minute, the presence/absence of the three courtship behaviors was scored.  Rephrase to make that clear.  In addition, why collect all this data when what is reported is only whether or not the males courted.  How was it decided that males did or did not court?  There is a lot that can be done with the data here, such as calculating courtship and copulation latency (do they differ in the light and dark).

Line 141: Dark room with red light: make it clear that the sole illumination in the room was red light.

Line 143: What timepoint was used to determine “most” light independent and light dependent?

Line 147: Please identify the strain

Line 150 : “their own females” – slightly confusing given that one strain did not have the same females.

Results: throughout, please specify the test used when reporting P values.

Line 159: The value I obtained here was 0.45 (Pdark/Plight)

Line 164: The value I obtained here was 0.78 (Pdark/Plight)

Line 267: Replace “space” with “range.”

Lines 361-370: a list of questions are hard to read.  You have a single point: vision needs to be combined with other sensory modalities for analysis to determine how the senses interact.

Line 373: avoid contractions. Also, the use of “we” is misleading here.

Author contributions: the contribution of one author is not mentioned.

Table 1: Organizing this table by collection location would help the reader considerably.

Figure 1: the experimental design is simple enough that I don’t think the figure is necessary.

Figure 5:  Because D. simulans results are always presented first in the rest of the paper, present them first here.

Figure S1: An indication of units on the X-axis would be helpful.  The label on the Y-axis is confusing.

Table S1: the Fisher’s corrected P-values are missing for all the D. melanogaster comparisons. The last cell that is occupied as an incomplete formula and not any values.


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: LIGHT DEPENDENT COURTSHIP BEHAVIOR IN DROSOPHILA SIMULANS AND D. MELANOGASTER
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors did their best to respond to all requests of the reviewer. The paper is now acceptable for publication in PeerJ.
Experimental design: The authors justified the experimental design.
Validity of the findings: Underlying data have been provided. Statistical analysis is appropriate. The conclusions are clearly defined.
Additional comments: The paper is interesting, well writhen and acceptable for publishing.


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: LIGHT DEPENDENT COURTSHIP BEHAVIOR IN DROSOPHILA SIMULANS AND D. MELANOGASTER
Review round: 2
Reviewer: 2

Output:
Basic reporting: Clear language.
Experimental design: Questionnable specially after reading their reply to my requests.
Primary research not original and not so rigorous.
Validity of the findings: The findings miss some key experiments to allow any interpretation.
Conclusion remain vague.
Additional comments: I am sorry to say that I am not satisfied with the way they dealt with my request. While they took into account most of my minor remarks (concerning the typo and grammatical problems), they did not answer, or they negatively answered to my requests. I just give soma exemple below.

To my question: Keeping males together until the mating test can strongly affect their willingness to mate depending on male genotype (Svetec et al., Genetical Research Camb, 2005), the authors replied: The study the reviewer provides compares white-eyed flies harboring a known mutation that affects male sex discrimination behavior during courtship to wild-type strains. It is also known that the white mutation significantly impairs vision which we know will also impact courtship. It would be surprising if the authors didn’t find an effect of genotype here. In fact, it is is not true since wild-type red eyes males were also tested in this paper (Cs, Di2, Cs/B42 and Di2/B42; Table 2) and showed a moderate to strong effect either in homo- or heterosexual courtship (or in both) between isolated vs grouped condition.

To my question: "The longitude scale is not a natural parameter per se given that it is only based on the convention of the 0° at Greenwitch meridian", the authors replied: "The convention of 0 at the Greenwich meridian does however place 0 very close to the source populations (sub-Saharan Africa and Madagascar) for both of these species, so it is not entirely without meaning in this case". So does this reply bring any fuel to my question?


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: LIGHT DEPENDENT COURTSHIP BEHAVIOR IN DROSOPHILA SIMULANS AND D. MELANOGASTER
Review round: 2
Reviewer: 3

Output:
Basic reporting: In the text, the change in mating from light to dark is discussed as a percentage, but in Figure 1C and 2C it is graphed as a ratio. I prefer the ratio so that the point being discussed is not confused with the data points at each time and condition. Regardless, the text and figure should be consistent with each other to help the reader.

Although I like the addition of courtship latency and courtship effort, I would like some more details about how these were calculated. First, did any of the males copulate in the observation period. If so, this should be mentioned, particularly because it affects the calculation of courtship effort (which the time spent in active courtship divided by the time from the initiation of courtship to copulation, not to the end of the observation period). The calculation of courtship effort (often called “courtship index”) is not clear.

In addition, the sample sizes in Table 2, I believe, are for the total number of pairs observed.  If this is for the number of pairs in which the males copulated, then the sample sizes for Figure 4 are unknown.  I think these sample sizes are actually the right sample sizes for Figure 4 (which should have sample sizes included in the legend or the figure) and the sample sizes here are even smaller if only the courting males are included (Line 170).  This is then problematic because only ~15% of SA169xSA169 males courted in the dark.  With a sample size of 7 (Table 2), this is equivalent to 1 male courting and the values in Table 2 have a standard deviation, which is not possible.  I’m not sure where things are disconnected, but I’d like to see some clarity on sample sizes.

P-values were corrected for multiple tests but never is it stated for how many tests each P-value was corrected. I’d like to see that stated explicitly. The uncorrected P-values should also be reported in Table S1
Experimental design: The experimental design seems mostly solid (with the exception of some of the analysis described above). The geographic and date analysis is not explained in the materials and methods.
Validity of the findings: I do not find any problems with the findings. Quite a bit of it is speculation, but I do not feel it goes beyond the data available anymore.
Additional comments: Overall, this is a nice revision of the paper.  The authors handled the criticisms well and I’m glad to see that the changes necessary in calculating the data did not change the conclusions. I think this paper will be noted by people interested in variation in courtship behavior.

Some specific comments:

Paragraph starting Line 47: This paragraph is difficult to read because it shifts ideas so many times. Breaking it up and concentrating on the message would help.

Line 53: Use of the word “surprising” not warranted. D. melanogaster uses many sensory modalities in courtship and has never been demonstrated to depend on one solely. Most tests of success in light and dark are measures of copulation success and not measures of courtship ability. This paper, as well as others recently, shift the emphasis on short term effects of vision from the long-term effects that have been tested before.  Thus, “surprising” isn’t the right word when the previous analyses have been so different.

Line 221: “counterparts”: I found this confusing.  Did you mean that you compared a D. simulans strain with the D. melanogaster from the same geographic location?  I think this could be stated more clearly.

All figures: the font is so small that they are very hard to read.

Figures 1 and 2: yielding is not spelled correctly (y-axis)

Figure 1C and 2C: the contrast is hard to see (light gray and dark gray)

Table 2: what are the units for courtship latency?

Table 2 figure legend: “Mean courtship latency is prevented with standard deviation in parentheses.  I think “presented” is meant instead of “prevented.”


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: LIGHT DEPENDENT COURTSHIP BEHAVIOR IN DROSOPHILA SIMULANS AND D. MELANOGASTER
Review round: 3
Reviewer: 1

Output:
Basic reporting: The figures are well presented and the raw data is provided. The writing is mostly clear.
Experimental design: The experiments are straight forward and the revision makes the procedures clear and easy to understand.
Validity of the findings: There are problems with the conclusions because many of the conclusions are unsubstantiated.

The conclusion that there is variation within species for copulation success in the dark is robust, and should be of interest to people studying these species. The result that copulation success in the dark varies with longitude in melanogaster is surprising, but seems genuine.

However, the sample sizes for the observations are so small that no conclusions can be drawn.  This is an unfortunate aspect of Drosophila behavior that the flies do not always behave when they should.  The only aspect of this with nearly a reasonable sample size is the comparison between courting/non courting in the light/dark. With so few males courting, nothing can be concluded for courtship latency and courtship effort. The data could be reported in a supplementary but should be left as inconclusive. The point is that males might court less in the dark but it may be dependent on the courtship target, implying that failure to mate in darkness lies with the females. Nonetheless, is there power in the sample size to detect a difference?  Whether males or females are driving reduction in mating remains an open question. The interpretation of these data needs to be reduced and adjusted to fit what the study can test, which with such a small sample size, is very limited. Some specific areas that need adjusting are below, but overall, the discussion of these results should be considerably shortened.
Additional comments: Line 127-128: Instead of “approximately 30 pairings,” give the range so that the reader knows that the sample size is sufficient (I had to go to the supplementary table to check this—make it obvious).

In Figure 4, the melanogaster results are presented before the simulans results, where in all the rest of the paper simulans comes first.  The reader would be helped if these were reversed (Table 2 is set up with simulans first and it is the same data).

Line 263 (and elsewhere in the paragraph): Please do not claim that there was an increase if it is not statistically significant.

Line 376: “…SA169 is less willing or able to court in the dark than MAL26…” This is clearly not true if the courtship target is changed (Figure 4C).  Please change the statement.

Line 385: “While we observed a reduction in MAL261 courtship overall when paired with SA169 females…” Where are the statistics that support this claim?  The experiments were not performed at the same time (intra- and inter- strain courtship) so it is not possible to determine if the effect is real (if significant) or an artifact of performing the experiments at different times.

Line 418. “It is also clear that strains vary in the importance of visual perception to initiate male courtship success.”  This is not clear.  Only two lines were tested and male courtship initiation seemed to vary with courtship target more than with visual signals and cues.


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

Input:
Title: A COMPARISON OF LODGEPOLE AND SPRUCE NEEDLE CHEMISTRY IMPACTS ON TERRESTRIAL BIOGEOCHEMICAL PROCESSES DURING ISOLATED DECOMPOSITION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript text is clear, references adequate, for some comments how to possibly improve tables and figures see General comments for the author, please.
Experimental design: The manuscript brings original primary research with well defined questions. For some shortcomings in Methods and data interpretation see General comments for the author, please.
Validity of the findings: The manuscript is novel and data are robust. For more details see General comments for the author, please.
Additional comments: General comments:
The manuscript (MS) summarizes results of two-year field experiment with decomposition of spruce and lodgepole needles on chemical and microbial composition of soil. The needles were removed from forest plots and incubated under identical natural conditions on soil surface of subalpine meadow (Colorado, USA) to avoid influence of differing tree roots and rhizosphere effects. Authors evaluate effects of needle type (lodgepole vs. spruce and healthy vs. bark beetle attacked spruce) and chemical composition on CO2 (and CH4) fluxes, leaching of C and N, and soil microbial communities. The MS represents a complex study, useful for other scientists interested in biogeochemistry of forest ecosystems and modelling. However, some important information, especially details on measurements of soil moisture and temperature, and CH4 fluxes are missing. Some contrasting results (e.g., higher CO2 production and DOC leaching from lodgepole vs. spruce needles despite lower total C loss during decomposition) deserve explanations. More details, including some minor changes and specific comments how to improve the MS quality are given chronologically below:

Specific comments:
Line (L) 32 and throughout the MS: “a lower subalpine meadow”. Lower than what? Either use “a subalpine meadow” or better explain, please.
L 34 and throughout the MS: Several terms are used for needle incubations (weathering, decomposition, natural decay). Is there any reason for using synonyms? If not, the most frequently occurring “decomposition” should be used consistently.
L 61: Change “pine” to more general “conifer”.
L 69: “Fraterrigo et al., 2018.” or “Fraterrigo, Ream, and Knoepp, 2018”?
L 75: Year “2019” is missing (according to the reference; L 563).
L 81: Porewater composition?
L 83: “at a subalpine meadow environment”
L 86: Change “has since intensified since” to “has intensified since”.
L 97: “(Dendroctonus ponderosae)” repeats (see L 84).
L 105: “(Dendroctonus rufipennis)” repeats (see L 86).
L 126: Explain abbreviation “SNOTEL”, please.
Ls 128–129: There is: “Mean annual values over the study years of 2016-2018 recorded averaged temperatures of 3.9 ºC, maximum air temperature 9.8ºC, minimum air temperature -0.6ºC”. Are these maximum and minimum values means of all daily maxima and minima, respectively, observed through 2016–2018?
Ls 131–132: Unclear how was soil temperature measured. There is: “Soil temperatures recorded on-site averaged 6.7 ºC during the study period, reaching a maximum of 28 ºC and a minimum of -0.3 ºC.” Is it a temperature of soil surface measured by a thermometer without a radiation shield? Why are average and maximum soil temperatures so much higher than air temperature? Are they really expressed in °C (and not in °F)? The annual mean soil temperature of 6.7 ºC at an elevation of ~3,000 m seems to be high. What does the maximum temperature represent – an average of all daily maxima through 2016–2018 or a single value observed only once? Does mean soil temperature refer to annual or summer data? Are soil mean, maximum, and minimum temperatures computed from a dataset similar to air temperatures (i.e., based on the same number of readings per day in identical times)?
L 134: There is: “The collars were 17.78 cm in height”. The number could be safely rounded to ~18 cm.
Ls 200–201 and 222–223: “samples were … and frozen for transport where they were stored at 4 ºC until analysis”. Were they frozen until analysis or melted earlier (if yes, how long)?
L 226: “Parada et al., 2016” or “Parada, Needham, and Fuhrman, 2016”?
L 239: “by Honeyman et al. 2018” or “Honeyman, Day, and Spear, 2018”?
L 280 and further in the text: The decrease is given in percent (… by 6% and 15%), which is somewhat misleading because also original concentrations are given in %. It would be better (and easier for a reader) to provide all concentrations in mg per g, as it is in Table 1.
L 314: There is: “…with lodgepole needle decomposition releasing the most CO2.”  However, this highest CO2 production from lodgepole litter contrasts with the lowest decrease in C concentrations compared to both types of spruce needles (Table S1). Why? Without explanation this disproportion could question the data coherence.
L 328–329: Similar disproportion as above. There is: “… green spruce needles released more CO2 than red spruce in 2017; however, this trend reverses in 2018”.  Fig. 3A shows relatively small differences in the 2018 CO2 release from green vs. red needles. However, Table S1 shows a pronounced difference in the C loss during 2017–2018 (70 vs 32 mg/g in the green vs. red needles, respectively).
L 331: A potential problem with soil temperature again. There is: “Soil data revealed 10% lower moisture content and 12 ºC higher temperatures in the later 2018 months”. It is difficult to believe to such a high between-year (or between-season) difference in soil temperature. More details in Methods are necessary on how it was actually measured.
L 335: Not clear. How can be CH4 flux negative? Did you measure CH4 production or uptake? Define “removal rate” in Methods and add more details on how it was measured, please.
L 346: There is: “There were no clear differences in total nitrogen”, but TN was lower below the red than green spruce needles in all 3 available sets (Table 2).
Ls 351–354: The observed highest concentrations of soil extractable DOC below lodgepole needles are in contrast to the total C loss from the needles that was only 18 mg/g for lodgepole but 32–70 mg/g for spruce needles (Table S1). Why?
Ls 382–384: The sentence: “In addition … the controls” is not clear.
L 415: “(Millard, 2010)” or “(Millard and Grelet, 2010)”?
L 452: “Šantrůčková et al. in 2006” or “Šantrůčková, Krištůfková, and Vaněk in 2006”?
L 471: “Fraterrigo et al., 2018” or “Fraterrigo, Ream, and Knoepp, 2018”?
L 527: I do not understand the term “root deposition”.
L 536–537: I do not understand the terms “tree hydrology” and “rhizosphere deposition”.
L 589: The reference “D. Eldhuset T, Kjønaas OJ, Lange H. 2017” is in different format.
Ls 647–649: Is this a valid reference?
Fig. 3: (1) Legend: “Soil data was available” or “Soil data was not available”? (2) It is rather difficult to see differences in lines and points. Try to use larger points and more intensive colors (e.g., red for red spruce, green for green spruce, blue for pine, black for control). (3) Soil temperature is surprisingly high for elevation of >3,000 m. Is it actual temperature of soil surface during CO2 measurement? What were weather conditions and time (full sunshine at noon)? Were thermometers in radiation shields, or exposed to the direct full sunshine?
Table 1: Are results based on a dry weight (105 °C) or air-dried basis? Provide all data in mg/g and add to Methods that all results on litter composition are expressed on a DW or an AD basis. What does unit “g dry litter/mg*M” mean? Explain “*M”, please.
Table 2: Explain unit “L/mg-M”, please. Data on C concentrations could be safely rounded to one decimal place.
Table 3: Are results based on a dry weight (105 °C) or air-dried basis?

Supporting information: Legends are missing for all Figs.
END OF REVIEW


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

Input:
Title: A COMPARISON OF LODGEPOLE AND SPRUCE NEEDLE CHEMISTRY IMPACTS ON TERRESTRIAL BIOGEOCHEMICAL PROCESSES DURING ISOLATED DECOMPOSITION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, this paper meets all of PeerJ's basic reporting standards. The English is clear, references are recent and appropriate, the paper is appropriately structured, raw data is completed and shared, and the study contains the results necessary to test the hypotheses.

I do have a few minor suggestions regarding the layout of the figures.

For Figure 1, I would suggest adding either a legend to B and C (the FTIR spectra), or at least mentioning which colors are which in the caption. It is implied that they'd be the same as the boxplots above, but it would be more thorough to add it for B and C.

For Figures 3 and S2, consider separating the Soil Moisture and Soil Temperature labels and making the right side of the figure its own axis. Even though these are on the same scale in terms of the number value, it is more conventional to have separate axes for separate variables. Consider also eliminating gridlines from the background of these figures.

For Figure 4, to clarify, are panels A and C from August 2017, and panels B and D from July 2018? It might be worth being more explicit about this.

For Figure 5, could you specify the units of abundance?
Experimental design: This paper does meet the aims and scopes of PeerJ and represents complete and original scientific research. The research questions are sound, and this paper does address them, and the methods are detailed.

Perhaps the most concerning gap for me in this experimental design is the lack of information on the morphology and mineralogy of the soils in this study. It makes sense to move the needles to another environment to isolate the biogeochemical role of these needles. In this case, the organic matter content and composition of the soil would clearly be different (this might be worth mentioning for transparency – if OM is low in both alpine meadows and forests in this region, perhaps it is not too important), but to what extent would the mineralogy of the soil be different? If the mineralogy is similar to what we’d expect in a lodgepole pine or spruce forest, and the only difference is lack of trees, then the interpretation that these results represent the isolated influence of litter is stronger. An easy way to address this would be to include a more detailed soils description in your Materials and Methods section, and to indicate that this soil type is similar, at least in bedrock and mineralogy, to where these needles were harvested. Interactions with the existing soil microbial community in the meadow and with the different assortment of organic compounds in the meadow is one factor also limiting the extrapolation to forests, but I believe that what you have done is reasonable, probably the best you could do to isolate litter from the forest while also keeping "field" conditions.

I would also recommend adding a bit more information regarding your gas flux measurements in the methods, in particular, mentioning perhaps when the sample was taken (time of day) and for how long, roughly, as this probably also influences the gas flux in soil.

I would also recommend considering clarifying how you ensured randomization in your study, if you can succinctly do so. How did you ensure that there would be no bias towards the edges of the collar?
Validity of the findings: This paper reports different chemical changes over time in lodgepole pine and spruce needles, and reports a greater change between species than within species that have been affected by beetle infestation, suggesting that changes in soil biogeochemistry as a result of beetle disturbance might be relatively small. This paper also demonstrates differences in soil biogeochemistry in general as a result of needle litter addition to soil (e.g., higher DOC in soils, higher proportion of organic N). Soil microbial communities also seemed more strongly defined by litter species, and there were no differences among green and red spruce needles, suggesting limited effects of beetle infestation on soil community composition as a result of chemical changes in needle litter. This paper also documents the effect of moisture on the clarity of biogeochemical signals, which is reasonable, given that water is often a necessary component of soil biogeochemical weathering reactions. These findings are supported by the data presented in this paper.

I recognize that one of the main challenges with this study is the relatively low sample sizes (n = 3). I commend you for pointing this out and not reporting significance values anyway, as that would be misleading. I think you still can and should, however, report measures of uncertainty in your results. You display this well in your figures by reporting standard deviation. I would recommend adding standard deviations or confidence intervals to your findings reported in the written text as well, to give readers a better sense to the amount of variability you had in your study.

One minor comment to aid readers in your interpretation, I would recommend clarifying in the Methods or Results section the connections you are making between the individual, chemical components that FTIR spectra detect and the molecules you believe that these represent (e.g., lignin, cellulose). You mention changes in terms of labile and recalcitrant compounds, but for an audience familiar with litter decomposition, it may be effective to tie into your explanation how your FTIR results link to these familiar carbon compounds early on and before describing these implications without this explicit context in the Discussion.

I have a few specific questions and recommendations for portions of your interpretations from this study:

Lines 431-438: Why might this be, that lodgepole pine and spruce needles behaved in different ways, chemically? Can you expand on the relevance of the He et al. 2019 work to your work? Or provide evidence of other factors that might cause this difference?

Lines 478-490: This is an interesting thought, but feels a bit speculative and requires quite a few assumptions that, as written, are not either clearly stated as assumptions or backed up by literature. It may also be important to acknowledge that you only collected senesced needles under healthy lodgepole pine trees, and the assumption that there’d similarly be little difference between litter from healthy and infected pine trees (as there was little difference between litter from health and infected spruce trees) would probably need to be spelled out here.

In general, that is another limitation of the study (testing of needles from only health lodgepole pine trees, as opposed to both healthy and infected) that might be worth addresing as a caveat in your discussion. It does not make your study less publishable, but helps identify the limits to which your conclusions of the effects of tree species ID vs. influence of beetle infestation are generalizable.

Throughout, exemplified in 506: I would be very clear about the fact that you are, for spruce, testing the impact of beetle infestation on the tree’s litter, specifically, and can only comment on effects of nutrient cycling as they relate to litter only.
Additional comments: Great job on this study; overall, I think it is fairly thorough and well researched.

Beyond what I have already expressed above, I have a few additional comments.

First, I had not come across a reference to "litter weathering" before reading this paper. I see what you mean, and see now that it is a term that is sometimes (rarely?) used, but I think you are capturing biogeochemical effects of litter decomposition, and so it is appropriate and potentially less misleading to be consistent about using the term "decomposition" instead of "weathering".

Second, this is very minor, but "red spruce" is also the common name of another spruce species, and so the use of "red spruce" to refer to needles that come from beetle-infested trees was a bit confusing at first. I wonder if "healthy" and "infested" might be better? Or is there is another set of terms that would be better? I understand the reasons for 'green' and 'red' given how these trees are typically referred to when they are infested... this again is a minor comment.

Third, for your Results, I notice that you often repeat your methods in a sentence or two before stating your results (e.g., lines 267 to 272 feel like methods). I do not think it is necessary to do this. Instead, I would try to emphasize your results as soon as you can, and maybe try to weave these methodological details into that sentence, or omit them altogether (since this all should be clear in your methods).

Line 473: I recommend clarifying what you mean by a "significant shift" . Significant shift in biogeochemistry?

Minor Line by Line Comments that do not require a response:

Line 129 and beyond: Add a space between the number and the unit (e.g., 73 cm instead of 73cm)

Line 130: Omit the space between / and October (September/October)

Line 227: add "by" after "as done"


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

Input:
Title: A COMPARISON OF LODGEPOLE AND SPRUCE NEEDLE CHEMISTRY IMPACTS ON TERRESTRIAL BIOGEOCHEMICAL PROCESSES DURING ISOLATED DECOMPOSITION
Review round: 2
Reviewer: 1

Output:
Basic reporting: Overall, this paper meets all of PeerJ's basic reporting standards. Changes to references that I overlooked were caught by the other reviewer and addressed by the authors, and my earlier suggestions regarding the layout of the figures were accepted.
Experimental design: This paper does meet the aims and scope of PeerJ and represents complete and original scientific research. Modifications made by the authors to address comments that I and the other reviewer made help further clarify the experimental design and research; the manuscript has improved.
Validity of the findings: The modifications that the authors made in order to address my concerns and the other reviewer's concerns regarding interpretation of the findings were addressed. Findings are valid and reasonable, and interpretations are sound.
Additional comments: My only comment might be, in Line 506, to consider referring to the variability of climate and native soil microorganisms as "reduced" rather than "removed" in this study, as soil communities can be quite heterogeneous, even within very small areas.

Otherwise, the revision in response to the two reviews was very thorough and the manuscript improved. Great work!


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

Input:
Title: ENDOPHYTIC BURKHOLDERIA SP. SSG AS A POTENTIAL BIOFERTILIZER PROMOTING BOXWOOD GROWTH
Review round: 1
Reviewer: 1

Output:
Basic reporting: The reporting of methods and the description of how this bacterium is positioned to potentially be very useful as a treatment for boxwood in production and landscape is very clear. Information is presented in step-by-step fashion and could be easily repeated by another scientist. Background is provided for each of the aspects of pathogen biology that relate to plant growth promotion, and even for the question of whether this microorganism could be used safely by humans. The data is presented professionally and the raw data is available for backup. Although references are made to some other recent by the same authors, this paper covers a distinct set of questions and provides clear answers. The isolate that they are working with has obvious growth-promotion abilities, and the tests carried out and reported here give specifics for some of the mechanisms that may be involved in the observed improvement in inoculated boxwood growth, for three cultivars with different rates of growth. Suggestions were listed for comma changes, typo correction, and slight rephrasing to make the communication more smooth, but the communication in the manuscript is very good. Detailed suggestions for improvements in wording follow:

Summary at beginning
Giving this endophyte great potential as a treatment for plant protection
Growth promoting bacterium (not -a)
The degree of growth promotion was significantly

Abstract
Line 21. Great potential as a treatment for plant protection
Line 24. Growth promoting bacterium,
Line 25. Comma after solubilization
Line 35. The growth rate of treated plants
Line 36. Notably more pronounced with the slow and intermediate
Line 57. Strains, such as those
Line 58. Low human health risk
Line 64. Biocontrol product or other potential biocontrol agents evaluated
Line 78. Add (PGP) after spelling it out because used in line 79
Line 88. Add comma after (PDA)
Line 90. From the stored culture
Line 94. What age was the colony used to inoculate?  You might also say Inoculated ‘from’ rather than ‘with’ if a portion of the colony was used
Line 116. Seven days, then
Line 118. Broth, to which 0.3 ml of an overnight. [also, perhaps define ‘overnight’ as ‘overnight (16-18 hrs)’ or whatever you want here at first mention, since it is used throughout]
Line 123. Solution, then
Line 139. (fast), were
Line 142. Paper toweling.  [or ‘a paper towel’]
Line 144. Soil,
Line 155. Measured
Line 160. A homogeneity test
Line 193. Winter Gem
Line 194. Winter Gem
Line 195. Promoting the growth of the slow
Line 210-11. [perhaps delete ‘and one another’. Not sure I get your meaning.]
Line 213. Cultivar, which
Line 220. Producing IAA, as other
Line 222. Ml, compared to
Line 229. Genes, although
Line 236. Nitrogen, following
Line 250.  Spp.
Line 257. [meaning is not clear; making the 3 items more parallel would help the reader. “ …for iron nutrition, soil heavy metal stress alleviation, and plant pathogen suppression.] [Alternatively, ‘for iron nutrition and to alleviate the stresses imposed on plants by high levels of heavy metals in soil and for suppression of plant pathogens”]
Line 262.  ‘these bacteria’ - not clear which ones you are referring to.   . . . limited research on siderophores in Bcc. (?)
Line 270. Were confirmed
Line 275. Highly beneficial biofertilizer [the traits mentioned go beyond fertilization, so they are not making it a better biofertilizer, they are making it a biofertilizer with additional advantages].
Line 281.  Delete ‘the’ before Saunders Bros.
Line 282. Suggest you add an initial or a first name to Ms. Richardson.
Line 289. not Burkholderia cepacia?
Line 334. not Burkholderia?
Line 385. Extra space
Line 389. P availability rather than p
Line 405.  Late blight is Phytophthora capsici???  Also, journal name should be capitalized.
Line 408. Words run together after Latin name
Experimental design: The work is original and contributes some key knowledge of the functions of the bacterium being tested for its plant growth promotion abilities. It is clear how this fits with other studies of disease reduction with this same organism, and with studies of the genes present in the bacterium. The studies all are replicated and repeated, usually three times, to dispel fears that results are not robust. Methods are described quite clearly.
Validity of the findings: The scientific standards of the researchers appear to be excellent. Work is repeated over time, and data is subjected to statistical analysis. Appropriate controls are provided for each study. Treatments were designed to substantiate or reject the hypotheses that arose after  seeing possible growth promotion in boxwood. The biochemical tests were appropriate given other work that explored the genetic makeup of he organism. The tone of the paper is straightforward rather than speculative: evidence is presented that substantiates the earlier observations that suggested growth promotion potential with the isolate.
Additional comments: This paper builds additional evidence that the Burkholderia isolate being studied has potential for use as a biocontrol agent. It is certainly interesting work. One wonders how widespread this organism might be in nature. It should be possible to learn more about biocontrol in general while investigating the utility of this organism for use in nurseries or landscapes.


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: ENDOPHYTIC BURKHOLDERIA SP. SSG AS A POTENTIAL BIOFERTILIZER PROMOTING BOXWOOD GROWTH
Review round: 1
Reviewer: 2

Output:
Basic reporting: Although very concise, the manuscript is substantiated in the literature and the experimental design is accurate, with the appropriate statistical treatment of the data. The hypothesis that Burkholderia sp. SSG is a plant growth-promoting bacterium is clearly demonstrated using boxwood as a model. The authors also performed a mechanistic investigation of the process, revealing that Burkholderia sp. SSG has the ability to grow in N-deprived medium, produces indole-3-acetic acid (IAA), the main auxin in higher plants, and siderophores. Figures, tables, and raw data are neat.
Experimental design: The experimental design is generally appropriate. To measure the plant growth-promotion ability, a very standard approach was used and thoroughly described. Although the assays performed to detect IAA, siderophores, and the ability to fix atmospheric nitrogen are very basic bacteriological approaches, they provide some evidence to support the main finding, which is the plant growth-promoting ability of Burkholderia sp. SSG.
Validity of the findings: No comments.
Additional comments: Some minor revisions are needed:
1) Lane 32: “atmospheric oxygen” should be replaced with “atmospheric nitrogen”
Lanes 193 – 195:
2) “Justin Brouwers (P = 0.0014) and Winer Gem (P = 0.0190), respectively. Justin
Brouwers, Buddy and Winer Gem Buddy are slow, intermediate and fast-growing cultivars. SSG appeared more effective in promoting the slow and intermediate rather than fast growing plants.”
This statement is unsubstantiated and could be withdrawn, without any prejudice to the article.
3) Lanes 271 - 272
"These traits along with other features such as potent antagonism against pathogens and low human health risk demonstrate its potential as a biofertilizer."
I strongly suggest future studies on Burkholderia sp. SSG should include the genomic prospection and assessment of protein secretion systems, especially the Type VI Secretion Systems. They are widespread in Burkholderia spp. and can act as powerful antagonists to other bacterial or eukaryotic cells, including plant pathogens.


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: BIOFILM RELEASED CELLS CAN EASILY BE OBTAINED IN A FED-BATCH SYSTEM USING ICA+ BUT NOT WITH ICA- ISOLATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Some errors found in file in red font so re-send the revised file to the journal


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: BIOFILM RELEASED CELLS CAN EASILY BE OBTAINED IN A FED-BATCH SYSTEM USING ICA+ BUT NOT WITH ICA- ISOLATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Comments

1.Is there any approach to conduct the experiment under static condition in addition to shaking condition culture? If so what was difference?
2.Is it 24h or 28h?

Overall the authors made nice attempt to characterize Brc from ica+ and ica- isolates with distinct abilities to form biofilms.


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: BIOFILM RELEASED CELLS CAN EASILY BE OBTAINED IN A FED-BATCH SYSTEM USING ICA+ BUT NOT WITH ICA- ISOLATES
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this study, Gaio and Cerca address an open question, which arose from their previous study on the feasibility of a fed-batch system to obtain S. epidermidis cells release from biofilm from strains with lower abilities to form biofilms. I have a positive feeling about the publication as the study is well written, introduction is clear with the relevant literature being referenced, data are well presented and conclusions are appropriately stated
Experimental design: I have few suggestions/questions, which might help improving the manuscript:

Major points:
1) The fed-batch method is the focus of the manuscript; it includes various steps and different measurement at different times (described in lines 116-135). I think including a general scheme, which summarize this method (with the measurements, the naming of the samplings….), would be helpful for the reader as it is hard to keep track of the steps. For example:
- does the Planktonic culture in line 133 refer to the “supernatant” from line 129?
- To what do planktonic cells from line 138 refer?

The authors already have Figure 1, which summarized part of the method but not the whole.

2) Table 2. I am not sure I understand the ration Brc/Bio. This ratio is very high in the ica- isolates because these strains form no biofilm and the few cells just live has planktonic cells. Therefore, I would not call these cells “biofilm released cells” as there was no biofilm in the first place. These are cells, which detached from the almost not formed biofilm (as the authors also observed in the following experiment).  Maybe the authors can just rephrase, and make it clearer?

Minor points:

Line 31: “To form biofilms ON the surface” and not ““To form biofilms IN the surface”
Line 78: “for all THESE reasons” and not “for all THE reasons”
Table2: Why did the authors not measure the Ration Brc/bio also on 72 hours?
Table 1: line 5, rpoB has to be written in italics
Figure 2 legend. “with α representing statistically significant differences” and not “with a representing statistically significant differences”
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: BIOFILM RELEASED CELLS CAN EASILY BE OBTAINED IN A FED-BATCH SYSTEM USING ICA+ BUT NOT WITH ICA- ISOLATES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
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: BIOFILM RELEASED CELLS CAN EASILY BE OBTAINED IN A FED-BATCH SYSTEM USING ICA+ BUT NOT WITH ICA- ISOLATES
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors nicely answer all the questions/comments arose from the reviewers


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: CHANGES OF DIAZOTROPHIC COMMUNITIES IN RESPONSE TO CROPPING SYSTEMS IN A MOLLISOL OF NORTHEAST CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Introduction and background prepared to show context that the experience were developed.
Literature well referenced and relevant. It has some ancient references, but concerning the subject of study.
The figures are relevant, high quality, well labeled and described.
Experimental design: Methods described with sufficient detail and information to replicate.
Validity of the findings: All underlying data have been provided; they are robust, statistically sound, and controlled.
Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: If it is possible, eliminate ancient bibliography, or part of it.


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

Input:
Title: CHANGES OF DIAZOTROPHIC COMMUNITIES IN RESPONSE TO CROPPING SYSTEMS IN A MOLLISOL OF NORTHEAST CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
Literature references, sufficient field background/context provided
Professional article structure, figures, tables. Raw data shared.
Experimental design: Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.
Rigorous investigation performed to a high technical & ethical standard.
Validity of the findings: All underlying data have been provided;
Additional comments: 1 a)]. Please, re-word and discuss accordingly.
20)	Lines 283 – 285: Please, relate Shannon index and AP back to the cropping systems.
21)	Line 292: Please, replace “breeding” with “yields”.
22)	Line 294: In fact, Bradyrhizobium japonicum was, by far, the dominant species in all treatments (Table 4). Please, correct and discuss accordingly.
23)	Lines 296 – 297: Please delete commas after systems and soybean.
24)	Line 308:  “… dominant diazotroph under 35 years of monoculture of corn in a black soil…” this is good information. It would be interesting to state why was Skermanella dominant under the mentioned conditions.
25)	Line 328: “…previous report has shown that soil phosphatase activity was significantly increased in crop rotation…” If this is true, why SS (continuous cropping) and CSC (intercropping) were not significantly different in terms of available phosphorous (Table 1)? Please, correct and discuss accordingly.
26)	Line 335: Please, relate your conclusions to the three objectives stated in the introduction.

Reference

 1.	Fehr, W.R. and Caviness, C.E., Stages of soybean development. 1977: Iowa State University of Science and Technology Ames, Iowa.


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: CHANGES OF DIAZOTROPHIC COMMUNITIES IN RESPONSE TO CROPPING SYSTEMS IN A MOLLISOL OF NORTHEAST CHINA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Manuscript # 44497
Title: Changes of diazotrophic communities in response to cropping systems in a Mollisol of Northeast China
Authors: Jiaxun Zou, Qin Yao, Junjie Liu, Yansheng Li, Fuqiang Song, Xiaobing Liu, Guanghua Wang

In this study, the authors examined diazotrophic microbial communities in different cropping systems. The study, overall, is well designed and has strong potential in expanding our understanding of how these specific microbial communities compare in different cropping systems. The manuscript is very well written - clear, professional and articulate English has been used throughout. Authors have used good literature citations throughout. Sufficient background, rationale and discussions for the work has been included in the manuscript. The figures and tables look good and support the study and the collected dataset well.

I have the following comments about the manuscript, please see below.

Lines 68-69: needs a few references after this line.

Line 73: I suggest you write “rarely specifically related to the diazotrophic community”

Section 2.1: Study site descriptions and soil sampling - Were samples taken only once? Authors need to include this information.

Section 2.3:
•	Line 119-127- qPCR conditions – Authors need to include more detail on how the qPCR standards were generated and used: e.g., What was exactly used as a standard? What exactly is “known number”? What concentrations and range of standards were used? Was there an internal control used in the study? What other controls were used for your qPCR reactions?

•	Line 123: Instead of volume of primers, please provide the final primer concentrations in your final mastermix volume. Volume of primer really does not inform much if someone would like to refer to your manuscript in future.

Line 265: Please elaborate on the levels of C and N here (in your reference article) and how exactly that compare to this particular study.

Line 267-268: Please elaborate in a sentence the exact findings from this reference and how specifically this compares with your data.

Figure 3: From what I gather, this tree has been constructed with sequences across all samples. If possible, I suggest you make this separate: make one neighbor joining tree each for each treatment. That comparison will be highly informative for future references. If space doesn’t permit, you can also include the three separate trees as supplemental information.

Table 3: I suggest you make/add a plot of this instead of the table. On sequence analysis platforms such as QIIME, you should be able to generate stacked plots of each of the diazotrophic bacterial groups based on each of the cropping systems. That certainly will be more informative and serves as a better way to display this particular result.
Experimental design: See all my comments above
Validity of the findings: See all my comments above
Additional comments: No additional comments