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: URBAN–RURAL DIFFERENCES IN PERCEPTION OF TREES DESCRIBED BY PARENTS BRINGING UP CHILDREN IN WARSAW AND JEDLIŃSK, POLAND
Review round: 2
Reviewer: 2

Output:
Basic reporting: •	Line 77: „complaints“ rather than „complains“?
•	Your introduction is very well written, comprehensive, detailed, and unusually long. So, the description of the aim of your study is placed late in the introduction. I suggest starting your long introduction with the purpose of being so detailed about reporting previous findings and contradictory results. In which way are all these previous findings important to understand your study? An alternative may be to move your long literature review from the introduction to the results part (and label it as literature review or literature summary). Subsections 1.2, 1.3, 1.4, 1.6 may suit well in the results part, as you use them to answer your research question, defined in “1.5 Aim of the Study”.
•	There seems to be a mistake in subsection numbering in the introduction: “1.5 Aim of the Study” is placed after “1.6. Differences between contemporary and previous generations”.
•	The sentence in line 208 (“City children do not have ideal rates.”): Would “City children do not have ideal rates of physical activity” be more precise?
•	Line 240: “all of them” rather than “all them”?
•	Lines 341 and 342 (“relationship”): Isn’t it differences (between urban and rural parents) that you investigate rather than “relationships”?
•	Line 348: I would prefer “the R Commander (Rcmdr) package (Version 2.6.0; Fox, 2005)” instead of “the R Commander add-on”. The full reference is: Fox, J. (2005) The R Commander: A Basic Statistics Graphical User Interface to R. Journal of Statistical Software,14(9): 1–42.
•	Line 479: “on the verge of a significant difference” rather than “on the verge of finding a correlation”? Another alternative would be: “…which means that the result represents a nearly significant difference”.
•	Lines 480-481: “were found” instead of “we found”.
•	Lines 490-491: I suggest “This indicates a statistically significant difference between urban and rural parents” instead of “This indicates a statistical relationship between the results and the place of residence.” It may be confusing for the reader when you switch between “relationship” and “difference” while meaning the same thing. I would recommend consistently talking about “differences” here.
•	Line 489: “stronger disagreed” instead of “stronger agreed” in this context.
•	Line 510-511: I would prefer “In three out of five categories of tree benefits and disservices only one statement was evaluated differently by urban and rural parents” instead of “In three out of five categories of tree benefits and disservices there was only one statement with statistically significant difference in relation to respondents’ place of living.”
•	Line 523: I would not use “their peers” here, because you are talking about children of a previous generation. I would suggest: “…than did children who were born a generation earlier”.
•	Line 526: Isn’t it “attitude towards trees” rather than “approach towards trees”?
•	Line 527: “agreed to” rather than “agreed with”?
•	Line 533: “attitudes” instead of “attitude”?
•	Line 533: “higher appreciation” rather than “more appreciation”?
•	Line 545: I suggest “…the benefits that nature provides…” instead of “…the benefits that contact with nature contributes to…”.
•	Line 569: What do you mean by “gender criteria”? Isn’t it just “gender”? You could also formulate the sentence as follows: “Future research may select a sample which is representative with regard to gender.”
•	What do you want to say with the sentence in lines 574-576 “whether the valid theories about the advantages of trees in the environment will be reflected in numerous scientific articles more widely”? This part of the sentence is unclear to me. There are many scientific articles on this topic (and they strengthen theories). What do you mean by “more widely”?
•	Line 576: Is there a better word for “space organization”?
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: FUNCTIONAL HISTOLOGY OF THE SKIN IN THE SUBTERRANEAN AFRICAN GIANT MOLE-RAT: THERMAL WINDOWS ARE DETERMINED SOLELY BY PELAGE CHARACTERISTICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comments
Experimental design: no comments
Validity of the findings: no comments
Additional comments: Manuscript entitled “Functional histology of the skin in the subterranean African giant mole-rat: Thermal windows are determined solely by pelage characteristics” for PeerJ.
 The authors have studied the comparison of ventral body part (where higher heat dissipation is expected) with the dorsal body side (where heat dissipation should be limited due to the isolating effect of denser fur) of the giant mole-rat Fukomys mechowii. For that, they analyzed the thickness of epidermis and dermis and presence, extent and connectivity of fat tissue in the dermis which were examined using routine histological methods, while vascular density was evaluated using fluorescent dye and confocal microscopy.
The paper is clear and well detailed. The purpose of the study is correctly defined. The details of most methods are comprehensible. The results are clearly presented. The discussion is relevant and complete. The conclusions are appropriately stated and connected to the original question investigated. The manuscript contains sufficient and proper references. The figures are good and help to analyze and comprehend significantly the results. Therefore I would suggest accepting the manuscript for publication in PeerJ after minor revision.
a)	Materials and Methods.
It is not clear the use of “freshly thawed cadavers stored under -20 °C”. How do the authors freeze the specimens? How were they thawed? Do the authors use any cryoprotectants?
Freezing damages cell membranes, and reduces the histological readability of biological specimens. Slow freezing can cause distortion of tissue due to ice crystal formation that replaces the normal morphology.

It should be described the methodology used in detail.

b)	The format of the references in the text should be checked and corrected. The use of and/& should be checked. Check the names of the journals, they must be changed to italic letter.


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: FUNCTIONAL HISTOLOGY OF THE SKIN IN THE SUBTERRANEAN AFRICAN GIANT MOLE-RAT: THERMAL WINDOWS ARE DETERMINED SOLELY BY PELAGE CHARACTERISTICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Subterranean rodents that excavate their burrows deal with the problem of heat dissipation in underground tunnels with high humidity. In a previous study the authors recognized that the giant mole-rat Fukomys mechowii uses mainly the sparsely haired ventral part of their body to dissipate heat. In the current study, the authors followed the question whether the animals show differences in their skin characteristics in the dorsal and ventral parts.
The manuscript is well written, but the text needs some language editing (especially the use of articles should be revised). I give some examples below, but it should be noted that I am not an English native speaker. The introduction provides sufficient background information to follow the authors’ rationale.
Experimental design: The research is original and falls within the aims and scope of PeerJ. The data presented is new. The research question is well defined and the methods have been described in detail, so that replication would be possible.
My major issues are the following:
1. The sample size is very low (n=5 for general description of skin histology; n=3 for studying the vascularization density) and information is partly confusing. Although the authors claim in the material & methods section that they have used five individuals for skin histology (L. 140-141), they present details of only three individuals (Figure 4, Table 1, Table S1) which I find highly confusing. Therefore, the authors should give the sample size for each mean value they present in the result section.
(further criticism see below)
Validity of the findings: 1 (continued): Furthermore, the authors claim that a statistical analysis is not possible due to low sample size. However, without a proper statistical analysis their statements are not valid. The authors come for instance to the conclusion that the difference in vessel density between the ventral and the dorsal part are not so prominent (L. 264-265). But taking a look at Figure 6, invokes the impression that the difference is significant. Thus, the research question has not been solved. I suggest that the authors increase the sample size (at least for histology) in order to run statistical tests (no sacrifice is needed if you use frozen carcasses).
2. The labelling of Figure 2 is not complete and partly wrong. The measured thickness of the dermis seems to include the measured thickness of the epidermis, which is wrong! Did the authors measure the dermis as indicated? The authors should indicate the glands (also which type of glands?) and hairs. What is the extent of the subcutis? From the labelling one gets the impression that the muscle layer is directly beneath the dermis. Is that correct? Which histological textbook did the authors follow?
Additional comments: Here are some minor suggestions:
L30: “to test” sounds better than “to find”
L45: “If they perform energy consuming activity...” specify “they”
L49: I would use “ineffective” or “limited” instead of “restricted”
L59: Plural of “plexus” is “plexus” (not “plexuses”)
L64: “the bats” delete “the”
L76: “We may estimate” sounds a bit weird
L82: “facilitate the heat radiation” delete “the”
L85: “adpressing” never heard of this word before – is that correct?
L90: “of the African mole-rats” delete “the”
L92 and throughout the text (also in the figure labelling): “ventral body part” instead of “ventral body side” (the same for “dorsal body side”)
L99: “on the belly”
L100: “in the latter species” (not “later”)
L101: “on the ventral body part”
L103: “The importance”
L106: “probably highly relevant”
L110: Name the authors (and year) instead of “Later authors compared…”
L112: “the situation”
L115: “the thickness”
L118: “fat in the hypodermis” instead of “fat containing hypodermis” (the latter can be very misleading)
L125: “There is a question” is a weird expression
L168-169: “A square grid with a grid length of 50 µm…”
L174-178: This part of the method section is not very clear. Please rephrase.
L205: “the low number”
L209: “The dermis”
L211: “The subcutaneous layer”
L213: “within the dermis”
L217: What does “the most represented fat layer thickness category” mean?
L223: “is given in Table S2”
L227: “on the dorsal”
L230: “which is a typical activity”
L232: “in the skin”
L239: Which fossorial rodent species do you refer to?
L245: “mice and humans”
L253: “at an ambient temperature of”
L262: “play a major role”; “body parts that facilitate heat dissipation”
L272: “Absence of subcutaneous fat”
L277: “fat in this layer” specify which layer
L292: “across the body”
L298: “from the present 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: SYNTHETIC DIETARY INULIN, FUJI FF, DELAYS DEVELOPMENT OF DIET-INDUCED OBESITY BY IMPROVING GUT MICROBIOTA PROFILES AND INCREASING SHORT-CHAIN FATTY ACID PRODUCTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The objective of this study was to evaluate the potential beneficial effects of the administration of the synthetic inulin, Fuji FF on energy metabolism and in the modulation of the gut microbiota by employing an in vivo rodent model of diet induced obesity. The manuscript follows the structure of the journal although English should be revised because some parts though the text are not clear. In addition, there are some aspects that should be improved and revised to improve the quality of the article.
Abstract: Line 32-43 are not clearly explained. Previous data indicate that synthetic inulin has beneficial effects in glucose and lipid metabolism and modifies gut microbiota but I cannot understand why the authors include in the sentence the comparison with maltodextrin.
The literature employed in introduction and discussion should be improved and extended because the study of complex carbohydrates non digested by the host but fermented by the gut microbiota it has been extensively studied in the last years. The term prebiotic should be also described through the text, including the most recent definition for this term (G.R. Gibson et al., Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics, Nat. Rev. Gastroenterol. Hepatol., vol. 14, no. 8, pp. 491–502, 2017).
In addition, the importance of the employ of dietary fiber such as inulin in the prevention and treatment of obesity should be critically documented. There are recent review articles on this topic that they should be cited (As example: Delzenne et al., Nutritional interest of dietary fiber and prebiotics in obesity: Lessons from the MyNewGut consortium, Clin Nutr. 2019 Mar 9. pii: S0261-5614(19)30115-3 .2019). The authors only select some articles but I would like to know if there are no more recent articles studying synthetic inulin and if it is not the case they should clearly indicate it. The importance of SCFA in energy metabolism and potential mechanism of action should be better explained because it is currently an active research area.
The figures are appropriate and generally well described but some mistakes should be corrected and some improvements made:
Figure 4A: Please write correctly abundande
 Figure 4 D,E, F: The families should we written in another colour, for example black because the grey colour is not easy to read
Experimental design: The article covers the scope of the journal and the research questions are clearly stated. The tests performed answered only some of the objectives of the article.
Methods are not well described and detailed in some cases:
Although the objective of the study is to evaluate differences of the administration of cellulose and Fuji FF inulin in a murine model of diet-induced obesity I would have included another third group that received only the high fat/ high sucrose diet to compare. Please also indicate why they have supplemented the high fat/ high sucrose diet with 10% of cellulose or Fuji FF. This dose has been previously tested?
Please indicate the method employed for SCFA measurement in blood.  The authors have only described the method employed for SCFA quantification in feces.
Since the amount of food ingested by the two groups was not different it would be helpful to know the daily energy intakes of the different groups.
I understand to check differences in the weight of the adipose tissue from the different areas but please better explained why they have also taken liver, caecum, kidney and testis.
Concerning the gut microbiota analysis, Have the authors not seen statically significant changes in the Phylum Actinobacteria or at lower taxonomic levels in the same phylum comparing both bacterial groups? If you check Figure 4A the relative abundance of Actinobacteria seems to change and be higher in Fuji FF group. Moreover, in the heatmap Bifidobacteriaceae is enriched in Fuji FF group and this group is normally modified by certain prebiotic supplements such as fructooligosaccharides, including in this group inulin.
SCFA profile: The authors observed differences in the concentration of the main SCFAs in faeces and blood but please indicate if the fecal SCFA results are for example concordant or not with the gut microbiota profiles obtained in Fuji FF group.
The statistical analysis should be better explained. The authors indicate that they have employed parametric tests but it is not clear if they have checked normality of the samples.
Validity of the findings: The experiments performed do not cover some of the data indicated in the results and conclusion sections.
Moreover, it is not clear why the authors have decided to test the Fuji FF synthetic inulin and why they have chosen the cellulose substrate to compare. It may be necessary a brief more accurate justification of the importance of the study: What is new?. I would like to know if Fuji FF inulin has been previously tested in another in vitro or in vivo models to evaluate the novelty of the article.
To understand the objective of the article it is important to clearly explain what are prebiotics in the introduction section and the importance of the study of prebiotics to treat obesity.
Some results are not clear and more experimentation should be performed. For example, in line 227 the authors indicate that there is no observable effect on insulin resistance but they have not measured insulin or performed HOMA-IR index or additional  tests such as insulin tolerance test to affirm that. They have only measured fasted glucose levels in both groups. This sentence is too speculative and I would remove it.

Please indicate that the employ of the ratio Firmicutes/ Bacteroidetes in obesity is controversial. Different data from animal and human studies do not corroborate the results obtained from Ley et al. 2005.

The authors conclude that the employ of Fuji FF inulin is useful to prevent diet-induced obesity by modifying the composition and the metabolic activity of gut microbiota but for the experimentation performed it is too speculative to indicate that the responsible factors are SCFAs. I would modify the sentence “that SCFAs are likely the responsible factor for the benefits of Fuji FF…” and indicate that the potential role of SCFAs in the anti-obesity action of Fuji FF inulin should be further explored by evaluating the effects of SCFA in GPR41 and GPR43 receptors.
Additional comments: Other comments and some typos and grammatical errors:
Please standarize the text, sometimes sentences are written in present, another time in past to facilitate the reading.
Line 32 please change insulin producing enzyme for inulin producing enzyme….
Line 89-92:  Please rewrite the sentence, it is not clear.  Please indicate the specific results obtained  in plasma after the administration of synthetic inulin.
Line 87: Please change insulin for inulin…
Line 98: Please change ani-obesity for anti-obesity.
Line 99: I would change elements of the gut environment for gut microbiota. The authors have not measured markers of inflammation, gut permeability or examine histologically the gut, They have focused in the gut microbiota profile.
Line 256-258: Please rephrase the sentence. It is not clear.
Line 278: I would say that dietary Fuji FF significantly modifiers or reshapes  the gut microbiota instead of Fuji FF significantly improves…
Line 286: I would change improved for modulated…


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: SYNTHETIC DIETARY INULIN, FUJI FF, DELAYS DEVELOPMENT OF DIET-INDUCED OBESITY BY IMPROVING GUT MICROBIOTA PROFILES AND INCREASING SHORT-CHAIN FATTY ACID PRODUCTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript was written in clear and unambiguous english, and supplied with professional article structure, figures and tables. But some improvements are needed.
The introduction needs more details. I suggested supply more detailed information about Fuji FF, such as the average DP and so on, and summarized reported researches about Fuji FF.
Experimental design: The detailed procedure of plasma SCFAs measurement was not stated.
Validity of the findings: 1. Raw data about Figure 4 should be supplied.
2. PCA of taxonomic groups at all levels should be analyzed, not only at family level.
Additional comments: 1. Levels of acetic acid, propionic acid and butyric acid were elevated in the feces of the mice fed a Fuji FF diet, but only propionic acid was increased in the plasma. please explained why.
2. Fuji FF diet reduced the body weight and white adipose tissue, how Fuji FF affected the plasma lipids?
3. At the genus level, if there are some different fecal microbiota were identified?
4. Fuji FF diet increased the cecum weight, please explained why.
5. The authors should stated what's novelty the study added.


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: SYNTHETIC DIETARY INULIN, FUJI FF, DELAYS DEVELOPMENT OF DIET-INDUCED OBESITY BY IMPROVING GUT MICROBIOTA PROFILES AND INCREASING SHORT-CHAIN FATTY ACID PRODUCTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Igarashi and colleagues evaluate the effect of the synthetic inulin Fuji FF in delaying the metabolic and physiological effects associated with a high-sucrose/high fat western diet. Authors monitor weight gain and energy intake over 12 weeks and energy expenditure at week 9 of treatment. They also studied the gut microbiota profiles associated with the different dietary interventions, as well as some bacterial-derived metabolites. The major finding of this study is that animals supplemented with synthetic inulin (Fuji FF) get less weight over the time, reduce the amount of white adipose tissue and improve metabolic parameters compared with cellulose supplementation. These changes seem to be associated with specific gut microbiota compositional profiles that produce more SCFAs.
Authors provide a sufficient background of the current understanding and an extensive description of the methodology used; the later being highly appreciated by this reviewer. The manuscript is well written and the data clearly presented. However, there are a number of issues that should be clarified by the authors before recommending this paper for publication.
Experimental design: My major criticism to this work is that strictly speaking, authors do not show that Fuji FF inhibited the development of obesity (line 44), reduce white adipose tissue and improve energy expenditure (line 44-45). This is because authors did not induce metabolic syndrome in these animals (e.g. by feeding all the animals with only high-sucrose/high fat diet) first and after the establishment of metabolic syndrome, they did include the supplementations in the diet. This approach would be more similar to a real-world scenario in which a dietary intervention is set up to treat a patient with metabolic syndrome. Given this limitation, at the best, authors can conclude that the supplementation with Fuji FF delays the development of obesity. This limitation should be discused and the claims made reviewed accordingly.
Animals in this study exhibited a similar weight gain until week 6 of treatment. From week 7 onwards, animals supplemented with Fuji FF significantly gained less weight. This seems not to be associated with the caloric intake. Energy consumption in the Fuji FF animals trended to be reduced (although not statistically significant) compared to the cellulose arm. However, authors provided the overall energy intake. I would like to see if Fuji FF suplemmented animals ate less from week 6 onwards. Therefore authors should present energy intake over the course of the experiment. Accordingly with the methods section (lines 120-121) authors measured food intake weekly and each cage contained 2 animals. Thus, authors should have enough data to longitudinally provide a mean, SD and appropriate statistical comparison, of energy intake over the course of the experiment.

Additional comments:
1- Why did authors use really young (3-weeks old) animals?
2- Why unclassified OTUs were eliminated from the final OTU table? Was the percentage of these unclassified OTUs similar between interventions?
3- Which method did the authors use to normalize the OTU table?
4- In figure 4 A, include the bar chart for all of the samples, instead of using an average composition plot.
5- Figure 2B. Which distance matrix was used for the calculation of the PCA? please include in the figure legend. Authors should also perform a PERMANOVA analysis to complement figure 2B.
6- Figure 2C does not provide any useful information. I recommend the authors performing a LEfSe analysis to identify discriminant feature between interventions. Authors can then plot the abundance of these discriminant OTUs between treatments. This will provide the reader a more clear picture about which OTUs are associated with each dietary supplementation.
Validity of the findings: No comment
Additional comments: I have found some typos that should be amended:
Line 98: ani-obese should be anti-obese.


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: SYNTHETIC DIETARY INULIN, FUJI FF, DELAYS DEVELOPMENT OF DIET-INDUCED OBESITY BY IMPROVING GUT MICROBIOTA PROFILES AND INCREASING SHORT-CHAIN FATTY ACID PRODUCTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author  answered the question reasonably.
Experimental design: The author  answered the question reasonably.
Validity of the findings: The author  answered the question reasonably.
Additional comments: Generally, the author  answered the question reasonably. It would be better to discuss the possible mechanism that how Fuji FF affect the plasma lipids as well as why only propionic acid was increased in the plasma of the mice fed a Fuji FF diet.


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: SYNTHETIC DIETARY INULIN, FUJI FF, DELAYS DEVELOPMENT OF DIET-INDUCED OBESITY BY IMPROVING GUT MICROBIOTA PROFILES AND INCREASING SHORT-CHAIN FATTY ACID PRODUCTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript has been improved and in my eyes meet the standards of the journal.
Experimental design: Methods are appropriate and clearly explained. They have substantially been improved after resubmission.
Validity of the findings: All the claims made by the authors are supported by the data and figures presented.
Additional comments: The authors have addressed all my comments and I am happy to recommend this paper for publication.


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

Input:
Title: ESTABLISHMENT OF BROWN ANOLES (ANOLIS SAGREI) ACROSS A SOUTHERN CALIFORNIA COUNTY AND POTENTIAL INTERACTIONS WITH A NATIVE LIZARD SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: fine
Validity of the findings: fine, with interpretational issue discussed in review
Additional comments: This paper importantly demonstrates that the brown anole has the prospect of becoming an invasive species in California. To me, this is somewhat surprising, as I would have predicted that the area was too dry for this species (although the fact that three of the sites are nurseries raises the possibility that the anoles are colonizing areas with atypically high moisture levels).

My only concern with the paper is that it doesn’t consider the alternative possibility that brown anoles are inhabiting areas not suitable for fence lizards. I am reminded of the situation in Florida, where brown anoles are very common in urban areas and the native green anole is much less common. Although the two species do interact, a major factor is that brown anoles are better adapted to parking lots and similar urban habitats—the disparity in population size has resulted because humans have changed the environment to make it suitable for brown anoles and not so much for green anoles.

Obviously, the best approach would be to do an experiment to confirm that the absence of fence lizards is the result of presence of brown anoles, but that’s a next step—the point of this paper is to suggest the hypothesis for further testing. Still, this manuscript should discuss the possibility and provide whatever data or observations are available that indicate that the habitat is suitable for fence lizards such that their absence is a result of the presence of the anoles.

Some minor points (numbers corresponding to line numbers):

35-43: sagrei is native to other islands in the Caribbean beside Cuba and the Bahamas, and it has invaded other places besides the US and Taiwan.

71: it probably should be noted that occidentalis is substantially larger than sagrei, which one would expect would tip the competitive (or predatory) balance toward the native 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: ESTABLISHMENT OF BROWN ANOLES (ANOLIS SAGREI) ACROSS A SOUTHERN CALIFORNIA COUNTY AND POTENTIAL INTERACTIONS WITH A NATIVE LIZARD SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: see comments below
Experimental design: see comments below
Validity of the findings: see comments below
Additional comments: Writing:
Overall, the manuscript is well-written. However, there, are, too, many, commas. The language could be simplified in some places as well. Also, report and discuss your results in the order you initially discuss them in methods/intro.

Methods:
Was it just one surveyor observing in each survey?
How do you define spatial overlap? You should specify what spatial overlap is in your methods, especially since the distance between species is sometimes rather low.

General Comments
The native range of brown anoles includes more than just the Caribbean. Technically, the Bahamas are not part of the Caribbean. You might be able to use Greater Caribbean Region instead. In general, this is fine. The Authors report some basic information on the distribution of a global invader, the brown anole in southern California. These observations are important and worth reporting. That said, I think the authors should eliminate the interaction portion from the title. There are no data that there were, or were not, interactions among species. This makes the title a bit misleading. Given that you don’t provide much data I think you should have something like: Establishment of brown anoles (Anolis sagrei) in Orange County California, USA. And again, I think you should consider reporting perch height data (or whatever other data you have) for both species. Further, are there any endangered species in Orange County that might also be impacted by brown anoles in California?

Minor Comments:
Line 36: The range includes more than Cuba and Bahamas. Make sure that you are specific.

Line 40: Be careful not to give the impression that brown anoles could establish in all states.

Line 68: Will likely have trouble in dry habitats. Annual precipitation is less than 10% of the annual rainfall in much of their native and invaded range. It’s possible that their spread will be limited by precipitation in southern California. Saying that brown anoles are ‘well suited’ seems like something you’d have to demonstrate with numbers.

Maybe simplify the language of your secondary objective? For example, is expropriation the best word choice here?

Line 133: Is this the mean?

Line 180: You’d probably want to know what western fence lizards eat as well, right?

Line 184: Brown anoles are probably not spider specialists. If specialized on anything, it’s probably ants (see Giery et al 2014, and Schoener 1968).

Line 106: 10 meters seems pretty close. Is this not spatial overlap?

Line 107: You should report these data as means, median, etc and put the raw data on dryad if possible. These habitat data are important to show and make available for others.

Line: 165: Is this the correct wording?


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: ESTABLISHMENT OF BROWN ANOLES (ANOLIS SAGREI) ACROSS A SOUTHERN CALIFORNIA COUNTY AND POTENTIAL INTERACTIONS WITH A NATIVE LIZARD SPECIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: In the abstract and in the text the authors tend to refer to the studies in Taiwan to illustrate spread and other issues pertaining to the brown anole. Since this study was done in the USA and the results is of conservation significance in the USA the authors should also make more reference to studies in the USA. Below are some suggested references by Cambell and Goldberg and his collaborators.

Keywords: Since Anolis sagrei already appears in the title, it would maybe be better to use the other synonym Norops sagrei since this nomenclature is used by some researchers. Also, it would it a good idea to list the family name of this species

Line 37: Note, an invasive population of this species has also managed to establish in Singapore (Tan et al. 2012)  and this should be listed.
Lines 52 – 54: Why do the authors not also list the impacts of this species in Anolis carolinensis? That would be of more relevance.
Line 58 and elsewhere in the manuscript: the authors refer to the authors (researchers) of studies and does not indicate the year. This is confusing since it looks like an incomplete citation. It should be rephrased so that the correct citation format can be inserted.
Experimental design: Lines 87 to 92: This information would be easier to read and compare in a table format.
Validity of the findings: Line 145: The phrase “going up” seems very colloquial. Replace it with an appropriate synonym.
Lines 150 – 160: Interesting, but I wonder whether this should be included here. The impact is not known and highly speculative. Diet studies have found that brown anoles will prey on whatever prey is available and conspicuous. Since they will prey on tiny prey such as collembola it is also possible that they will prey on the ticks. For example, Norval et al (2010) recorded mite as prey.
Lines 184 – 185: Brown anoles are not spider specialists! Spiders are common prey but they prey on ants to a greater extent.
Additional comments: Here are some references that can be of use:
Campbell, T.S. 1996. Northern range expansion of the brown anole (Anolis sagrei) in Florida and Georgia.  Herpetological Review 27: 155-157.
Campbell, T.S. 1999. Consequences of the Cuban brown anole invasion in Florida: it’s not easy being green. Anolis Newsletter 05: 12-21.
Campbell, T.S. 2003. The introduced brown anole (Anolis sagrei) occurs in every county in Peninsular Florida.  Herpetological Review 34: 173-174.
Goldberg, S.R., and Bursey, C.R.  2000. Transport of helminths to Hawaii via the brown anole, Anolis sagrei (Polychrotidae). Journal of Parasitology 86: 750-755.
Goldberg, S.R., Bursey, C.R., and Kraus, F. 2002a. Seasonal variation in the helminth community of the brown anole, Anolis sagrei (Sauria: Polychrotidae), from Oahu, Hawaii. American Midland Naturalist 148: 409-415.
Goldberg, S.R., Kraus, F., and Bursey, C.R. 2002b. Reproduction in an introduced population of the brown anole, Anolis sagrei, from O’ahu, Hawaii. Pacific Science 56: 163-168.
Greene, B.T., Yorks, D.T, Parmer-Lee, J.S. Jr., Powell, R., and Henderson, R.W. 2002. Discovery of Anolis sagrei in Grenada with comments on its potential impact on native anoles.  Caribbean Journal of Science 38: 270-272.
Norval, G., and Mao, J.J. 2007. Can Anolis sagrei be unintentionally transported to new localities in bamboo stem bundles? Sauria 29: 51-54.
Norval, G., Hsiao, W.F., Lin, C.C., and Huang, S.C. 2011d. Ambushing the supply line: a report on Anolis sagrei predation on ants in Chiayi County, Taiwan. Russian Journal of Herpetology 18: 39-46.
Norval, G., Huang, S.C., Mao, J.J., Goldberg, S.R., and Slater, K. 2012d. Additional notes on the diet of Japalura swinhonis (Agamidae), from southwestern Taiwan, with comments about its dietary overlap with that of sympatric Anolis sagrei (Polychrotidae). Basic and Applied Herpetology 26: 87-97.
Norval, G., Tso, I.M., and Huang, S.C. 2007. A study of predation by the brown anole (Norops sagrei) on spiders in Chiayi County, Taiwan. Russian Journal of Herpetology 14(3): 191–198.
Tan, H.H. and K.K.P. Lim. 2012. Recent introduction of the brown anole Norops sagrei (Reptilia: Squamata: Dactyloidae) to Singapore. Nature in Singapore 5: 359–362.


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: DECONVOLUTE INDIVIDUAL GENOMES FROM METAGENOME SEQUENCES THROUGH SHORT READ CLUSTERING
Review round: 1
Reviewer: 1

Output:
Basic reporting: the paper has a clear structure and is sufficiently self-contained. the language is mostly clear and unambiguous (there are just few typos: see below). figures and tables are clear and of sufficient quality.

unfortunately the authors seem unaware that the idea of using coverage in multiple samples to improve metagenomic binning appeared already in 2014, and is by now well understood: see e.g. [1] and the papers citing it, e.g. [2]. that line of research actually performs assembly (rather than binning) on the union of all samples, so it might even be more accurate in practice, although possibly slower. the approach in the submitted paper might thus still be competitive in terms of speed, space efficiency, or amount of parallelism allowed, but no experiment comparing it to [1,2] or similar approaches is described.

[1] Alneberg, Johannes, et al. "Binning metagenomic contigs by coverage and composition." Nature methods 11.11 (2014): 1144.

[2] Lu, Yang Young, et al. "COCACOLA: binning metagenomic contigs using sequence COmposition, read CoverAge, CO-alignment and paired-end read LinkAge." Bioinformatics 33.6 (2017): 791-798.

TYPOS
PAGE|LINE|OLD|NEW|COMMENT

1|10|”deferentially affect”|”differentially affect”|
2|2|”de bruijn”|”de Bruijn”|
2|8|”then assemble each cluster”| ”then assembles each cluster”|
2|17|”under-clustering”| |shouldn’t it be “over-clustering”?
2|11|”We refer the new”|”We refer to the new”|
2|-7|”neither space or time”|”neither space nor time”|
2|-7|”To improve the computing efficiency”| ”To improve computing efficiency”|
2|-3|”3.3-fold of speed-up”| ”3.3-fold speed-up”|
3|5|”under-clustering”| |shouldn’t it be “over-clustering”?
4|2|”we expect their vectors”| ”we expect that their vectors”|
4|6|”the sparsity nature”|”the sparse nature”|
4|11|”the same way in the local clustering”| ”the same way as in the local clustering”|
5|-1|”the number of sample increases”| ”the number of samples increases”|
7|3|”The detail of these 7 recovered genome can be found in the Supplemental Table 1”| ”The details of these 7 recovered genomes can be found in Supplemental Table 1”|
7|6|”number of representative k-mers are used”| ”number of representative k-mers used”|
7|13|”In each of the parameter set, only one parameter varies while the other three were held constant”| ”In each of the parameter sets, only one parameter varies while the other three are held constant”|
8|9|”improve both the cluster size”| ”improve both cluster size”|
8|21|”improve clustering purity strain”| ”improve clustering purity”|
Experimental design: the paper is concerned with the relevant problem of unsupervised read clustering in metagenomes: given a set of metagenomic samples with similar content, partition their reads into bins that are in one-to-one correspondence with the bacterial species in the samples. the paper merges all metagenomes into a single read set, it clusters such read set by iteratively merging two reads if they share enough k-mers, and finally it merges the resulting, pure, clusters, based on their frequency in different samples. specifically, for each cluster C and each sample S, the paper builds a k-mer histogram, and uses the second peak in the histogram as a proxy for the abundance of C in S. the abundance vectors of all clusters are then represented as a weighted graph, where the weight of an edge is the (thresholded) cosine similarity between the adjacent clusters. the graph is eventually partitioned using label propagation.

the research topic is within the scope of the journal. methods are described with sufficient detail.
Validity of the findings: the experiments are described thoroughly. conclusions are clearly stated and limited to the data. data and code are provided, so it seems to me that the experiments can be replicated.
Additional comments: No additional comments


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

Input:
Title: DECONVOLUTE INDIVIDUAL GENOMES FROM METAGENOME SEQUENCES THROUGH SHORT READ CLUSTERING
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall comments:
The paper is nicely written and has a clear structure. The proposed new step to the clustering method seems to make a great improvement. This naturally implies a trade-off between completeness and purity.

Results are assessed by considering purity and completeness at the genome level. However, as noted by the authors, strains from the same species may differ only slightly and are extremely difficult to distinguish. I’d love to see results where purity and completeness are determined at the species level rather than at the genome level. This would also make more sense: when clustering reads you’d often wish to cluster the reads that belong to the same species, rather than the same strain. Furthermore adding species-level results would show the validity of two statements in the paper (“Because almost identical … species-level clustering performance.” and “This is likely caused … very similar strains.”).

The discussion is very short. In my opinion it would largely benefit from a discussion of the method’s limitations, rather than only mentioning its benefits. For example, the authors do mention that the global clustering procedure improves clustering completeness, but do not mention that this comes at a cost of lower purity. This is an interesting trade-off (inherent to the clustering problem) that in my opinion should be discussed here.

The paper does contain quite a number of typos and issues with sentence structure, please revise or consider revising by a native speaker.

The figures and tables are very clear and easy to interpret.

Specific comments:
- On page 2 it is written that "clustering metagenome reads based on k-mer statistics across samples is very challenging". Has anybody shown this? Please substantiate with references. The same holds for "The application of … for unassembled reads" in the discussion.
- I do not understand the first sentence of the section “Estimating cluster coverage”. Please revise.
- Description of the MBARC-26 dataset: “real-world”. How can the data be real-world if the dataset is synthetic? Did you mean simulated?
- I cannot find results on the MBARC-26 dataset. Is the dataset actually used?
- “Short read clustering performance is greatly improved with multiple samples from the same microbial community.” This header does not match the contents of the paragraph. Also the first sentence of the paragraph does not match the results presented here.
- Page 5: “without a big drop”: a drop of 10% is big, so please do not say it is not. However this big drop is not a problem: there is simply a trade-off to be made between completeness and purity.
- Page 5: “In contrast, higher … for read clustering.” I do not understand how higher sequencing coverage translating to higher completeness suggests this. Please explain.
- Page 7: “The unclustered reads … presumably … low sequencing coverage.” Can you verify this?
- Page 8: “This problem may … for Bayesian optimization.” Why would Bayesian optimization be a good idea here, and not something else? Please motivate.

Minor comments:
- abstract line 2: "Clustering short reads" please add "by species" or “by genome” (dependent on what your focus is)
- abstract: deferentially -> differentially
- page 2: “data not shown”. Why not? Please consider including it in the supplementary material.
- Figure 2B: add units to the vertical axis.
- Figure 2 caption: “x-axis is cluster size” measured as number of reads, number of bps?
Experimental design: The experimental design is well carried out and results are presented in a nice way. However I do feel that the experiments are rather limited: only one dataset is used (from the results section I deduce that the MBARC-26 dataset was not used). I'd like to see more experiments on different datasets. Besides, it is unclear how this approach performs compared to other read clustering methods. In my opinion the paper would largely benefit from a comparison of methods.
Validity of the findings: The results are clearly presented. The CAMI mouse gut data is publicly available. There is a bit too much speculation and the results that are not so supportive of this method (a reduction in purity) are ignored in some parts of the paper (see comments in block 1).
Additional comments: No additional comments


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

Input:
Title: DECONVOLUTE INDIVIDUAL GENOMES FROM METAGENOME SEQUENCES THROUGH SHORT READ CLUSTERING
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Li et al. here extend a method that uses k-mer abundance correlations
across metagenome samples to cluster out "read clouds" based on
co-abundance. These read clouds should represent bacterial/archaeal
genomes.

This tool is presented against the dominant "MAG" background of
approaches to genome binning, where metagenomes are first assembled
and then the resulting contigs are binned into "metagenome assembled
genomes" based on abundance, tetra-nt frequency, etc.  Given the
widespread utility of the MAG approach in recent years, we think the
authors should spend more time comparing and contrasting the utility
of their approach vs the dominant MAG approach. This would put the
paper in a fuller context.

Thoughts/comments:

The authors present their tool's performance on synthetic data sets,
but not on any real data sets. This limits the power of the
presentation.

There are good reasons to go with read clustering over assembly, and
they could be presented more thoroughly. Assembly by e.g. MEGAHIT is
not that memory challenging in practice, so the authors' interest in
claiming decreased memory requirements is not convincing on its own.
Additional points could be mentioned, such as being able to assemble
the reads with multiple assemblers, rapidly trial different assembly
parameters for each cluster, and avoid strain confusion (where strain
variation interferes with assembly.

Conversely, there are reasons to maybe go with assembly first, such
as the (potential) ability to recover parts of genomes that have low
or high abundance (strain variation, repetitive elements).  Some of these
are mentioned in the paper already.

The authors should do a comparison against megahit for the reads that
are left out. What is this method capturing that megahit misses? What
does megahit capture that this method misses? What do they both miss,
on data sets where there is ground truth?

Why do some genomes with very low coverage (e.g. 4) have very high
completeness while others do not? What is biologically/computationally
different about them (e.g. divergent k=4 abundance?)?

Detailed questions/requests --

Please archive the version of software used in this paper and provide
a DOI; bitbucket is not archival.

What is the license under which this software is released? Please
specify in repository, and also in paper.

What is the definition of a reliable k-mer? (page 2, paragraph 3)
High abundance?

"they are likely to have a uni-modal k-mer spectrum, where the main
peak after the minimum inflection point represents the sequencing
coverage of the genome region where these reads are derived." This will
eliminate some strain variants, correct?  Is this considered a positive,
a negative, or a user choice?

The tool is hard to work with and hard to install, it seems. It would
be ideal to provide some kind of docker container or cloud tutorial.

"Furthermore, this approach can potentially recover almost the full
set of reads of microbial genomes with sufficient representation in
the data, an ability influenced by several parameters including the
number of available samples.": This statement is very vague; could it
be sharpened, please?

Typos --

'deferentially' should probably be 'differentially'

The word 'unseen' is used (2nd paragraph), maybe this should be 'are
not known'? Or have not been cultured?

It should be MEGAHIT, not MegaHIT.

50, 10, 20 an 50 samples => 'and'


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: DECONVOLUTE INDIVIDUAL GENOMES FROM METAGENOME SEQUENCES THROUGH SHORT READ CLUSTERING
Review round: 2
Reviewer: 1

Output:
Basic reporting: CONTENT
PAGE|LINE|OLD|NEW|COMMENT

7|250|"To test this hypothesis"||i don't think you are testing the hypothesis that the assembler can "optimize the assembly process of each individual genome": this sounds like the assembler automatically tuning its parameters or algorithms to each cluster.
8|293|"r4.16xlarge"||what is this?

TYPOS
PAGE|LINE|OLD|NEW|COMMENT

4|116|"those have"|"that have"|
4|120|"maped"|"mapped"|
4|120|"contains dominant part"|"contains the dominant part"|
8|274|"On average clusters"|"Clusters"|
8|278|"we applied the two approaches on"|"we applied the two approaches to"|
8|308|"there are a few existing solutions might be leveraged"|"there are a few existing solutions that might be leveraged"|
13|Panel B|"median ompleteness"|"median completeness"|
14|1|"alliterative"||?!
Experimental design: ok
Validity of the findings: ok
Additional comments: No additional comments


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

Input:
Title: DECONVOLUTE INDIVIDUAL GENOMES FROM METAGENOME SEQUENCES THROUGH SHORT READ CLUSTERING
Review round: 2
Reviewer: 2

Output:
Basic reporting: See report.
Experimental design: See report.
Validity of the findings: See report.
Additional comments: No additional comments


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

Input:
Title: ECUADOR—EASY CURATION OF ANGIOSPERM DUPLICATED ORGANELLAR REGIONS, A TOOL FOR CLEANING AND CURATING PLASTOMES ASSEMBLED FROM NEXT GENERATION SEQUENCING PIPELINES
Review round: 1
Reviewer: 1

Output:
Basic reporting: I found that the paper is well written and reports a bioinformatics tool that could be useful in large phylogenomics studies, especially using available data on GenBank. A do have a few corrections to suggest both to the text and the code.
While seems counter-intuitive the usual order of plastome is reported as LSC-IRb-SSC-IRa. Please check the paper for OGDRAW (https://doi.org/10.1093/nar/gkz238) and references 21, 22 within for the background of this. I believe following this would only take switching IRa and IRb labels in your code.
I think you should mention Fast-Plast in your introduction, as after short read assembly it outputs the plastome in the canonical LSC-IRb-SSC-IRa order, but cannot handle already assembled GenBank data. RepeatFinder in Geneious could also be mentioned as this will identify the inverted repeats but any necessary rearrangements would still need to be done by hand.
It might be useful, if possible, to give the option to output the assembly as LSC-IR-SSC, i.e. without the second IR copy. This could allow easy data formatting for phylogenetic analysis to avoid overestimating the signal from the IR.
Experimental design
Sequence data submitted to GenBank are often included in two source databases: INSDC (GenBank) and RefSeq. RefSeq accession numbers normally start with NC_. This means that your Dataset3 contains duplicated sequences e.g. NC_030299 and KU351660 are the same Ziziphus jujuba assemblies. I recommend that you rerun the Dataset3 analysis with only the INSDC (GenBank) records to get a more accurate success rate.
Experimental design: Sequence data submitted to GenBank are often included in two source databases: INSDC (GenBank) and RefSeq. RefSeq accession numbers normally start with NC_. This means that your Dataset3 contains duplicated sequences e.g. NC_030299 and KU351660 are the same Ziziphus jujuba assemblies. I recommend that you rerun the Dataset3 analysis with only the INSDC (GenBank) records to get a more accurate success rate.
Validity of the findings: While I believe your work will be useful I think it contains some errors in reporting the length and starting positions in the inverted repeats. As far as I can see the length is calculated by subtracting the start position from the stop. This results in 1 bp shorter IRs than it should be as subtraction is inclusive only on one end. I suggest either subtracting the stop position of LSC or SSC from the stop positions of the IRs or just simply add one to the existing calculations.
If assemblies are correct the end of the sequence, i.e the 3’ end of IRb in your paper, should match the 5’ end of IRa. I randomly checked this in ten sequences, one of which I believe is a poor assembly. The start position of IRa in the other nine is 1 bp off from what is suggested by the end of the whole assembly. I think it would be useful to run another analysis to compare the recovered IRs between your script and RepeatFinder or any other programmes that you can think of.
Moreover, length of the IRb calculated as the difference of stop and start position does not match the reported length in nine out of the ten sequences.
Additional comments: Line 71: It is ambiguous what to date is. I would recommend including the date sequences were downloaded, just like in Dataset3
Line 72: I think7,000 is incorrect due to RefSeq duplicated sequences
Line 80: I think inverted repeats can only be palindromic if there are no intervening bases between the two parts, which is not the case for the plastome. I think it would be simpler to just use inverted repeats and IRs for short.
Line 81-83: I think you are describing the isomeric copies of the plastid genome, which should have the original citation (10.1146/annurev.ge.19.120185.001545) and probably some of the newer ones where isomeric copies were mistakenly thought to be inversions (doi:10.3732/ajb.1500299).
Line 84: I am not entirely sure what each of these refer to
Line 93-94: This is really only one of the reasons, though probably the most common one. With small inversion and changes in gene order the non-coding intergenic spacers require laborious checks for homology.
Line 100: I think this should be non-organelle origins and the NUPTs and NUMTs abbreviations should be resolved in the first mention.
Line 167-168: This suggest that by default the shorter repeat will be taken as correct, while this is not necessarily correct.
Line 245: I think it would be useful to make the in-house script available on github
Line 268: There is an extra bracket here
Line 278 & 287: I found these difficult to follow. It might be simpler that accuracy was above 90% with 22 or fewer alterations.
Line 324: This should be plastome data
Line 387: It might be better to use large genomic datasets


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: ECUADOR—EASY CURATION OF ANGIOSPERM DUPLICATED ORGANELLAR REGIONS, A TOOL FOR CLEANING AND CURATING PLASTOMES ASSEMBLED FROM NEXT GENERATION SEQUENCING PIPELINES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Reporting seems well done, please see general comments.
Experimental design: Experimental design seems well done, please see general comments.
Validity of the findings: Validity seems well done, please see general comments.
Additional comments: This paper describes a new program for finding orthology among regions in the plastome. This includes not only coding sequences, but can also identify non-coding sequences. Essentially it feels like the now defunct program DOGMA (Wyman et al. 2004), with phylogenetic data assembly step. From what I can tell this is a valuable niche to be filled, and although some programs do similar procedures, it’s always good to have multiple programs that do something and this is the only one I am aware of specifically capable of combining new data and genbank data.

Wyman, Stacia K., Robert K. Jansen, and Jeffrey L. Boore. "Automatic annotation of organellar genomes with DOGMA." Bioinformatics 20, no. 17 (2004): 3252-3255.

Throughout the paper it would be good to have another run through for grammar and spelling, I did not notice any major issues but I believe it could benefit from another examination.

One thought and this is strictly up to the authors, I don’t by any means is necessary for publication, is that it would be good for the program to also be able to combine with large transcriptome datasets and add in the coding sequences from those. Transcriptomes seem to be an abundant source of this chloroplast material also and although it does not provide the non-coding DNA being able to complement the coding from chloroplasts and the non-coding may be valuable.

Just a quick note it seems as though for the phylogenetic analysis I would imagine it would be bad to include both IRa and IRb as this represents redundant data. This could be something to ensure is mentioned in the paper or on the authors Github to avoid this mistake being made. One of the issues with mass processing is the ability for simple errors such as that to emerge.

At line 340 the discordance between this and the work of Ruhfel et al. makes sense based on studies that are finding conflict among genes and dataset assembly such as Goncalves et al. 2019 and Walker et al. 2019.

Gonçalves, Deise JP, Beryl B. Simpson, Edgardo M. Ortiz, Gustavo H. Shimizu, and Robert K. Jansen. "Incongruence between gene trees and species trees and phylogenetic signal variation in plastid genes." Molecular phylogenetics and evolution (2019).

Walker, Joseph F., Nathanael Walker-Hale, Oscar M. Vargas, Drew A. Larson, and Gregory W. Stull. "Characterizing gene tree conflict in plastome-inferred phylogenies." PeerJ 7 (2019): e7747.

I believe the authors have done a good job finding an area where there is clearly a need for a program and putting together a good program to fulfill that need.


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: ECUADOR—EASY CURATION OF ANGIOSPERM DUPLICATED ORGANELLAR REGIONS, A TOOL FOR CLEANING AND CURATING PLASTOMES ASSEMBLED FROM NEXT GENERATION SEQUENCING PIPELINES
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this study, the authors present software for the detection and organisation of chloroplast regions across multiple assembled sequences, streamlining analyses that are normally time consuming and potentially prone to error. As far as I am aware, I believe this software is novel in that in removes the necessity for manual correction and will likely be of value to future studies in chloroplast NGS studies.

I think that the manuscript is well written and structured clearly, and software testing was comprehensive and informative for potential users. With some very minor changes I feel this should be accepted for publication and I look forward to trialing it on my own data.

I only have minor comments on the manuscripts as outlined below.

Lines 71-73 – when and how was the number of plastomes on GenBank accessed?

Lines 93-95 – add references to “most of the plastomic studies” and “above mentioned”

There were a couple of acronyms that I didn’t understand. It might be worth spelling these out in full. Line 100 – NUPTs and NUMTs. Line 120 – BGT.

Line 114 – I thought the line beginning “Despite that it can usually be done” sounded odd but this might just be me. Perhaps change to “While reconstruction can be easily done for…”

Line 179 – “The amount of low quality in the original data…”. Perhaps specify “low quality sequences” or “low quality base calls” for example.

I thought it made sense to refer to the sliding window parameter “-w” on line 170, at the start of the paragraph, rather than on line 180.

Line 245 – Am I correct in thinking random substitutions introduced as way of creating noisy/low quality datasets? If so, I thought it would be good to state this clearly for the reader.

In figure 3, refer to how the tree was generated in the figure heading, and detail all the usual information such the values on the branches and branch length scale. Also, specify why some families are highlighted in red. I realise this is discussed in text but it would be good to clarify in the figure heading too. The size of the text is quite small, so I expect the quality of the figure will need to be good for publication.


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

Input:
Title: ECUADOR—EASY CURATION OF ANGIOSPERM DUPLICATED ORGANELLAR REGIONS, A TOOL FOR CLEANING AND CURATING PLASTOMES ASSEMBLED FROM NEXT GENERATION SEQUENCING PIPELINES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article has been improved greatly. I have very few comments below mostly suggestions to improve the text. I would be happy to see the paper published after these are incorporated.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Line 47, 53, 171: Irb should be changed to IRb
Line 83-84: I think ref 10 should be cited for this sentence.
Line 94: It should be quick assessment of sequences
Line 99: delete of
Line 106: I think “Accurate and complete molecular sequence data are essential in phylogenomic reconstruction” could be easier to read
Line 110: There is an extra in
Line 117 & 365: Fast-Plast does not do annotation, only assembly and orientation
Line 122: potentially might be better here
Line 176: It should be a not an
Line 192 & 194: There are extra spaces at some of the < or >
Line 249: I am not sure what you mean known plastomes, but I don’t think it is needed here
Line 264: I think it would be useful to include the GitHub link for the script here
Line 272: I don’t understand possible cases without any detection. Could you rephrase this?
Line 349: This should be plastome and you can delete of
Line 415: If you are planning to further develop your software to generate gene alignments I think it would be useful to have the option of getting alignments of homologous non-coding regions as well. I don’t think this is needed for the paper but I thought it would be worth mentioning 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: ECUADOR—EASY CURATION OF ANGIOSPERM DUPLICATED ORGANELLAR REGIONS, A TOOL FOR CLEANING AND CURATING PLASTOMES ASSEMBLED FROM NEXT GENERATION SEQUENCING PIPELINES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors present the revision of Ecuador a program to facilitate the preparation of plastome data for downstream analyses. They have done a good job addressing all comments from the first round of review and I believe this will be a useful tool in the ever-growing field of plastome phylogenetics.
Experimental design: I don't see any issues in the Experimental design
Validity of the findings: All findings appear valid.
Additional comments: At several places in the paper you have the “r” in SSC-Irb-LSC-IRa in lowercase, I believe it should be uppercase. And at line 226 you have Ira instead of Ira.


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: ECUADOR—EASY CURATION OF ANGIOSPERM DUPLICATED ORGANELLAR REGIONS, A TOOL FOR CLEANING AND CURATING PLASTOMES ASSEMBLED FROM NEXT GENERATION SEQUENCING PIPELINES
Review round: 2
Reviewer: 3

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: I believe the authors have largely addressed the reviewer comments. I only have minor comments.

Lines 73-75: This is a very minor point but how was the number of plastomes on genbank accessed? I am assuming it was through the ncbi ftp database but it would be good to confirm.

Lines 97-99: The following sentence didn't make sense to me and might be worth rewording.
"However, despite such rearrangements are less likely to happen at small taxonomic scale (e.g. at intrageneric level, or in species-complexes), most of studies at various scales use only coding regions."

Line 110: delete repeated word "in"

Line 126: "for a trained worker in the Biodiversity Genomics Team (BGT) lab". Perhaps I have misunderstood but could this equally read "for a trained bioinformatician"?

Line 349: Change "platome" to "plastome"

Figure 3: cite that you used PhyMl software in the figure heading for clarity. The quality of the figure may not be of sufficient for publication given the number of tips. In particular the support values will not be legible, but there are other ways of representing support values on a tree, for example colouring the nodes to represent support.


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: MODELING THE INFLUENCE OF TEMPERATURE AND WATER POTENTIAL ON SEED GERMINATION OF ALLIUM TENUISSIMUM L.
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is overall easy to read and to understand. However, there are still some minor typos that could be fixed. For instance in the abstract the authors say "Our study aimed to characterize seed germination of A. tenuissimum under various temperature (11, 15, 20, 24 and 28 °C)"; it should be under various temperatures (plural instead of singular) or even "under various temperature regimes".
This is only an example but it occurs multiple times throughout the text. However, I consider this to be minor, and can be easily fixed.

Figures and tables are fine but I missed the statistical information on them. For instance, post-hoc tests can be easily incorporated on Figure 1 (also on the text). F values and d.f are missing in the text.

Literature references can be largely improved (as well as the Discussion). Several articles also found 15C and 20C to be the best temperature for seed germination. See for instance:
https://www.ingentaconnect.com/content/ista/sst/2016/00000044/00000001/art00017
https://www.researchgate.net/profile/David_Draper_Munt/publication/233883683_Germination_behaviour_of_seven_mediterranean_grassland_species/links/0deec52e9227a54a59000000.pdf
https://www.cambridge.org/core/journals/seed-science-research/article/seed-germination-and-longevity-of-autumnflowering-and-autumnseed-producing-mediterranean-geophytes/D6BFFAB2EA18201F722DA29998EFCA76
https://www.researchgate.net/publication/227123219_Seed_germination_of_high_mountain_Mediterranean_species_Altitudinal_interpopulation_and_interannual_variability
Experimental design: Models need to be explained with more details, as well as germination tests. For instance:
- were seeds dried before used?
- they were kept in a sealed glass. That is not a good procedure to store seeds as it usually led to the loss of viability. How has this affected the results?
- were seeds kept under light? What about during germination?
- I assumed that results of germination are the mean and SE (or SD?) of the four replicates. This needs to be explained.
- Was seed viability of non-germinated seeds checked?
- Final germination in % usually needs an arc-sin transformation before statistical analysis. Was this done?
Validity of the findings: Overall conclusions seem to be well stated but there is some confusion between the  parameters measured. This needs to be better explained, although it probably might not affect conclusions.
As a major point, the results found and the discussion need to be better explained in light of previous results.
Additional comments: A main objective of this paper was to characterize seed germination under variable temperatures and water potential to define parameters that can be used in industrialized production. How exactly can the industry implement these findings in crop production?


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: MODELING THE INFLUENCE OF TEMPERATURE AND WATER POTENTIAL ON SEED GERMINATION OF ALLIUM TENUISSIMUM L.
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Modeling the influence of temperature and water potential on seed germination of Allium tenuissimum” has noval information about model germination by hydrotime and hydrothermal time analysis. Under different temperatures and water potential the growth or seed germination was most suitable in cold and drought conditions is 20.5 °C. I suggest this manuscript should be publish in Peer J after Some major concerns.
Experimental design: The design looks satisfactory although I have some objections?
The Author try to find a suitable temperature for the growth of Allium tenuissimum and used various temperatures (11, 15, 20, 23 24 and 28 °C) is these temperatures are very low? Why should you not tried with 35 °C or may be 40 °C. Although, this temperature is common in various part of the world. Please explain it briefly?
Validity of the findings: The results from the current study is meaningful.
Additional comments: Before accept, several changes needed to be done.
1.	Throughout your manuscript you just used “Allium tenuissimum” Even in the title you did not mentioned it Author name? please write its common name also even in text or provide taxonomic name.
2.	L31 in abstract do not provide space in (50 %).
3.	Introduction L 42 can you cite latest reference?
4.	 Introduction L 45-46 references looks too old for this important line.
5.	What is the relationship between water potential and supra-optimal temperature ranges?
6.	Introduction L 54-55 references again looks too old.
7.	Introduction L 62. Better to write the refences with the crop name. then it will be easy to understand.
8.	Introduction L 68: please make sure that Allium tenuissimum’s roots are fibrous?
9.	“has economic importance due to its distinctive and tasty flavour” cite reference here
10.	 “exploration of its unique volatile compounds” difficult to understand.
11.	 I suggest you to write your objectives in more effective ways. Like In this study we aimed to focus on Allium tenuissimum under different environment condition (i), (ii) and (ii). Please make points and write it again then it will be clearer to understand your study.
12.	Materials & methods L 84: unable to understand the meaning of m a.s.l. remove it or define it.
13.	Materials & methods L 85: Author has mentioned “Germination assays were conducted in a growth chamber located at the Grassland Science Department, China Agricultural University, Beijing, China with an 8 h 87 light/16 h dark day/night lighting pattern”. Here I suggest you to add some more information. Like about growth chamber? What about humidity factor? And temperature was constant?
14.	 “Seeds were sterilized for 5 min with 10 % NaClO and then washed with distilled water” please cite some reference.
15.	“Seeds were sterilized for 5 min with 10 % NaClO and then washed with distilled water” Also can mention mature seeds?
16.	 Please add some detail about filter paper?
17.	 Why used two layers of filter paper?
18.	 “solutions were transferred to fresh solutions every 2 d” to also avoid fungal attack?
19.	 “Germination was scored daily by observing radicle protrusion”. When seedlings get germinated or should mention the definition of seed germination. Normally Seeds were germinated when radicle was more than 2 mm. As mention in this article. doi:10.3390/plants8120545. Please verify from this article and cite in this sentence.
20.	L 194: Reference looks too old.
21.	L206-207: Please write your reference with crop names as earlier mention.
22.	“yet there was a poor fit at 28 °C” difficult to understand.
23.	L 223: Same comment, write the crop names?
24.	 At 0 MPa Germination was inhibited? Any logic or reference?
25.	 I just suggest you please write your conclusions simply as you can. Means it should be general.


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: MODELING THE INFLUENCE OF TEMPERATURE AND WATER POTENTIAL ON SEED GERMINATION OF ALLIUM TENUISSIMUM L.
Review round: 2
Reviewer: 1

Output:
Basic reporting: The Author have made the changes according to the given suggestions. However, I request to Author please cite the following articles in the introduction section: https://doi.org/10.1016/j.chemosphere.2020.126032
https://doi.org/10.1016/j.ecoenv.2019.109915
https://doi.org/10.1016/j.jenvman.2019.109994
Experimental design: No comments
Validity of the findings: No comments
Additional comments: No comments


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

Input:
Title: A HALO OF REDUCED DINOFLAGELLATE ABUNDANCES IN AND AROUND EELGRASS BEDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Line 36 - missing info in place of ?
Line 137 REFREF CITE, c’mon yall
What is happening in lines 237-242, manuscript needs careful attention - I appreciate the R markdown effort but gotta check it over.
Experimental design: I should note I have seen an earlier version of the Jacobs-Palmer paper at another journal; I thought it was sound at the time and required only minor revisions, which the authors provided. I also think this is interesting and useful, and by generating questions will be well cited.

A question on line 67 - you use a fragment of COI and it is “broadly used in ecological applications” but given the focus of this paper, should clarify how those studies have been applied phylogenetically, in other words that those ecological applications have themselves validated use of this marker across the taxonomic breadth considered here, in terms or representing abundance rather than amplicon success.

I greatly appreciate the cross-contamination controls.

Line 154 but surely your data are more comprehensive than known lists of Dinoflagellates in the north Pacific?
Validity of the findings: I believe the findings to be useful and well supported, statistically appropriate. Conclusions are well stated.
Additional comments: No additional comments


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

Input:
Title: A HALO OF REDUCED DINOFLAGELLATE ABUNDANCES IN AND AROUND EELGRASS BEDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: -Peer J does not have a requirement in reference style, but there are still some guidelines that should be followed. As a personal preference, I would suggest having the references written on the text, not numbered. Nevertheless, if the authors decide to use numbers on text for the references, then the numbers should go in order, starting at 1, then 2, etc.  The first reference cited can not be 22, followed by 8, 18, etc.

-Multiples examples in the text show that the manuscript has not been reviewed properly prior to submission and evince lack of attention to detail: Line 36 “.. ‘Habitat Area of Particular Concern’ (see ? ),  line 124, 137, : [REFREF6 CITE],

-The first time an acronym is used, it should be explained in the text. Ex: line 55:” … eelgrass communities can suppress HAB56roducing”. You have never explained the meaning of HAB in the text.

-The introduction has five paragraphs, but only 2 are actually Introduction. Starting at line 44, the authors present the results (which should be moved to Results session, not introduction), and starting at 47 they discuss the results (which should be moved to discussion). The introduction should be rewritten, and only contain information relevant to the introduction.

-The introduction is very short  (also 3 out of 5 paragraphs are not introduction-see other comments). The introduction needs more detail and more references to literature. For example, can you expand on the ‘reduced exposure to pathogens”? that is a very relevant topic in your study.

-The whole manuscript would benefit from a review and the rewrite of multiple sessions, to improve language and transitions. Some examples to highlight: lines 33, 34, 244, etc.

-The order of elements in figure caption should be consistent among the different figures. Ex- Fig 2 order is Al, Eg, Ba, while the order in the figure legend is Al, Ba, Eg; Fig3 figure caption is Eg-Ba, etc..

-Figure 1 would benefit from having another figure showing where in the US or world that region on the map is located.

-Figure 2 should be improved. The grey background and the lines make it harder to see the colors, the numbers are very big and are on top of each other. It is also not clear how the triangles were made. If the numbers (or letters) in each edge of the triangle is always the same, I wonder what is the point of having them on the graph. The legend should have a title as such, not FILL. The color brown is not present in the graph but it is present in the legend and in the caption. The legend could actually state what Al, Ba, and Eg mean instead of only having the acronyms.

-Figure 3: The grey background makes it harder to see the colors, also, lines are very tick, and points quite big. I think it would make it more pleasant to see and easier to understand the results if lines were thinners, points smaller, grey background and lines were eliminated.

-Figure 4: The grey background and the lines make it harder to see the colors. I would also eliminate the lines connecting the points. The figure already shows that there is an increase in abundance of dinoflagellates when seagrass is not present, no need for the lines to show the trend for each point.

-Figure 5: the meaning of colors should be stated in each figure. The grey background and the lines make it harder to see the colors, also, lines are very tick, and points quite big. There is no explanation of what areas “a” and “b” mean in the figure.

-What is p11 and p 10, psi? (line 128)

-“Magallana (née Crassostrea) gigas…” what does the neé mean here, I have not seen this notation in scientific papers before.

-“As others had begun to suggest, then…”(337) could you please rephrase this?
Experimental design: -Figure 2 highlights how replicates are variables. Could you explain more why there is only one replicate taken above eelgrass and one from the bare substrate?

-Could the authors describe how the water was collected, and the methodology to guarantee that the samples were not contaminated? Ex- what container was used? Was it acid-washed? Bleached? It is a normal procedure in eDNA sampling to carry a container with distilled/ miliwater as a control to the field, did you have control? How long were the samples kept on ice until processing? Hours? Days? Were they kept in the dark? (time before filtering and amount of light could induce algae growth/ reproduction and alter your results).

-Could you explain better your negative control and the use of kangaroo and ostrich?

-It would be interesting to have replicates bottles for each sampling point and location. It would confirm the results and would show how much a single sampling is actually representative of the diversity richness of the site.  Is there a reason why you only collected one little of water in each location per sampling?

-Was there any difference in diversity in the 3 months time point sampled? Do you think that “month” could have affected the results?
Validity of the findings: -I think the study is quite interesting and there is value in reporting the findings of the observational study.

-I like that the authors provided many hypotheses to justify the results (273-296). I would like to see justifications for those hypotheses from the literature. Have other studies suggested some of these hypotheses?

-Fig 3: I assume the points represent the number of identified samples from each one of the categories described in the graph. If that is the case, I find it very suspicions that there is such a clear delimitation where each taxon is only found at a specific distance from eelgrass.
Additional comments: I think the study provides an interesting insight into the effects of eelgrass on dinoflagellates. The manuscript needs some major editing and revision as there are many errors and inconsistencies in the structure and organization of the writing, figures, references in text, etc, which could be an indication of lack of attention by authors and raise concerns about the results.


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 HALO OF REDUCED DINOFLAGELLATE ABUNDANCES IN AND AROUND EELGRASS BEDS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Generally very well put together.  I took this paper on not realising how much of a technical eDNA paper it was but very interested in the idea that eelgrass beds may demonstrate positive environmental impacts.  Hopefully the other refree/s is/are technical experts.

One suggestion - on the figures in supplemental tables and figures, it would be easier if the legend for figure S3 was under the first figure and the legend fully explained all aspects of the graph such as the initial representing locations.
Experimental design: I can find few flaws in the technical aspects of this paper (possibly because of ignorance!) and its a really interesting idea that eelgrass beds might have such strong impacts on the water column above them.  There has obviously been very close attention to detail in the preparation of this paper the methods and analytical approaches taken appear to be very well described.

Did you sample at a consistent time of day?  Was there a long time period between samples on given days?

You measure tidal height in feet???  For consistency I suggest you give this figure in cm or m.

I'd be interested to see time, tidal cycle point, temp, salinity etc also presented in Table S1 and to know that these had been discounted as possible influences,
Validity of the findings: I have a few fundamental questions about this paper (my apologies if they are ridiculous, I'm not expert in the technical aspects of eDNA).

1)  You have demonstrated a difference in the presence/absence of dinoflagellate eDNA along transects over eelgrass and bare substrates.  Surely it would be much simpler (and cheaper) just to take plankton samples?  This would, even if just done for 1 site, validate your results. Could there be some obscure mechanism (e.g. sampling bias) by which eDNA levels are different to abundance of dinoflagellates.

2) Dinoflagellates are true plankton - unable to move against prevailing currents.  Current speeds in Puget sound are, apparently, 0.1-0.6 Kts
(https://www.starpath.com/ebooksamples/9780914025160_sample.pdf). I am puzzled as to how any action by dinoflagellates could fight against this so that you have heterogenous distributions over a few meters that can be explained by the presence/absence of eelgrass.  I would expect to see patchy distributions whatever the habitats beneath.

3) If the above can be explained and current speed is not a issue, how can you discount the possibility that the difference in distribution is not just a reflection of habitat suitability rather tahn some sort of active exclusion process?  Areas above bare sediment may just suit dinoflagellates more than eelgrass habitat and vice versa.

The ordination plots in Supplemental figures generally show that Ba and Eg are closer to each other (CI-May, CI-Aug, PG-May, PG-Aug, Sk-Jul, WB-May, ) than they are to the various points along the transects.  In CI-Jul transect points 1,3,6 and 10 are more consistent with Eg than Ba.  The high degree of within sample variance surely suggests a lot of noise that could be obscuring any other pattern.
Additional comments: No additional comments


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

Input:
Title: A HALO OF REDUCED DINOFLAGELLATE ABUNDANCES IN AND AROUND EELGRASS BEDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: *Introduction is still too short. I think you could expand more into some topics as the conflict of eelgrass conservation and/or aquaculture, or the services the oyster may depend on provided by the seagrass (line 47) and/or reduced exposure to pathogens.

The authors say: “We have amended the introduction, although it remains brief and contains some results. It was our aim this section to survey the positive effects of seagrass habitats, introduce our study design(including the dominant local seagrass species), and use a very brief statement of results to motivate readers to engage with the study, as appropriate (we did not begin this work expecting to see such strong patterns in Dinoflagellata with respect to eelgrass, and do not wish to write the introduction as such, but do believe that a brief understanding of these patterns will help readers to assess the relevance of the study to their own work). “

I would argue that an introduction should not have the results and that the abstract fits the role the authors are talking about: it is short and provides a very brief statement of results to motivate readers to engage with the study. The introduction should not be short or provide results, it should provide all background information relevant to the study, present results from relevant articles, present your hypothesis and motivation for the study. It should provide more in-depth information on the background of the subject matter. It should also explain your hypothesis, what you attempted to discover, or issues that you wanted to resolve. The introduction will also explain if and why your study is new in the subject field and why it is important.

*I appreciate that the authors re-organized the legends and figures for clarity and consistency

*I think some of the explanation of why using the kangaroo and ostrich sequences could be added to the text

*The authors say” We have added references to two additional studies, including those already mentioned in the text for the first and last hypotheses: “A second plausible mechanism is that slower flow-rates cause plankton deposition within eelgrass beds, but not outside (in parallel with sediment deposition (e.g. Potouroglou 2017)” and “A third possibility is that predatory taxa exist in greater abundance within eelgrass beds and thereby consume dinoflagellates in larger quantities within this habitat (e.g. benthic macrofauna in Hosack 2006).”
 It would be interesting to see a discussion of those, as those hypotheses are opposite as one explains why there would be more dinoflagellates within the eelgrass beds, and the other explain why there would be fewer dinoflagellates in the eelgrass beds (they would have been eaten by the predator).
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors did a great job addressing the comments made by the reviewers. I added just a few more comments.


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

Input:
Title: THE ROLE AND PROTEOMIC ANALYSIS OF ETHYLENE IN HYDROGEN GAS-INDUCED ADVENTITIOUS ROOTING DEVELOPMENT IN CUCUMBER (CUCUMIS SATIVUS L.) EXPLANTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some places exist formatting, gramma and other mistakes. It needs an improvement, Please polish it.
Experimental design: methods: The details (manufacturer, city, country) of the Kit used in RNA isolation should be added. The instrument and reagent used in real time PCR experiments should be stated.
How identified proteins can be “functional classification and distribution” ? please add the related methods.
Validity of the findings: no comment
Additional comments: The manuscript entitled: “The involvement of ethylene in hydrogen gas-induced adventitious rooting development in cucumber (Cucumis sativus L.) explants and its proteomic analysis” stated an interesting work on the role of hydrogen gas and ethylene in cucumber adventitious rooting. However, there are some concerns regard to this work.

1.	Some places exist formatting, gramma and other mistakes. It needs an improvement, Please polish it.
2.	Introduction:
the role of ethylene on adventitious root formation is not well stated in the introduction section. Some relevant work are suggested been introduced. Meanwhile, studies with AVG as a inhibitor can be introduced.
The research hypothesis should be clearly described in the end of the introduction section.
3.	Materials and methods:
The full name of some abbreviations should be added where it first appearance, for example, TCA, PVPP, CHAPS, IPG, etc.
The details (manufacturer, city, country) of the Kit used in RNA isolation should be added. The instrument and reagent used in real time PCR experiments should be stated.
How identified proteins can be “functional classification and distribution” ? please add the related methods.
The unit “litre” should be uniformed to “L” or “l”.
4.	Results:
Line 231, the quotation mark “” should be match.
Line 232, Figs. 8, 9.
5.	Discussion:
A deepgoing discussion should be considered. What is the mechanism of H2 regulating ETH signal? How can H2 regulating identified 6 proteins expression? They all are in a ETH-dependent manner?
Line 306, “Considerably more work will need to be done to determine that the change in OEE1 protein abundance is dependent on whether high hydrogen or hypoxia.” Authors stated that the negative effects of H2 on OEE1 maybe duo to the high hydrogen or hypoxia, but the title is refer to “hydrogen gas”, so the further results of the hypoxia effects or additional controls should be given. The stress response-related protein (CAPX) was identified in HRW response, it also suggested to investigate the effects of hypoxia. In addition, is CAPX plays a role in development? If so, please discuss.
6.	Acknowledgments:
No information? Please check.
7.	References:
Some references are not in the correct format. For example: Line 367, 268-271. Line 374: Synechococcus sp. PCC 7002, please pay attention to the italicized. Line 390, lack of doi. Line 421, (Tagetes erecta L.), pay attention to the italicized. Similar mistakes also fund in Line 440, 454, 490, 492, 493, 504, etc.
8.	Table 1: The reference gene Actin should be added as well as its sequnces.
9.	Table 2: the meaning of the symbols “↑”, “↓” should be added. The A, B, C, and D in footnote should be appeared in the table (boxhead), or “A”, “B”, “C”, and “D” is suggested to be deleted.
10.	Table 3: added the meaning of “↑”and “↓”. Why there have blanks in the rank of ETH?
11.	Figure 1: the quality of Fig. 1 should be improved, and the scale bar in the picture should be added. Some pictures in Fig. 1B seems not typically selected as corresponding to the data in Fig. 1A.
12.	Figure 2: there are difference at the adventitious rooting phenomenon (especially in root length) in Fig. 1 and Fig. 2, thus it seems that HRW is beneficial for AR length and ETH helps increase the number of AR? What is the mechanism behind the phenomenon? Please added the scale bar.
13.	Figure 3 and 4: i think the dose effect of AVG and AgNO3 on adventitious rooting are not the key results in this paper, also, it lock of novelty. It suggested that been transfered this two figures to supplemental materials.
14.	Figure 5: delet “and NaNO3 “ in the legend. The HRW, Ethephon in Fig. 5A should be consistent to that of “H2” , “ETH”in Fig. 5B, as well as the whole MS. Please added the scale bar.
15.	Figure 8: Three items correspond to two ordinates, and what is their correspondence? It suggested that add the number of proteins on the top of each column.
16.	Figure 9: figures in Fig. 9 needs to be revised, as the characters are too small to read.


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 ROLE AND PROTEOMIC ANALYSIS OF ETHYLENE IN HYDROGEN GAS-INDUCED ADVENTITIOUS ROOTING DEVELOPMENT IN CUCUMBER (CUCUMIS SATIVUS L.) EXPLANTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1)	I suggest that the article still need an English editing.
2)	The Introduction section is too simple to provide sufficient field background. The authors should tell more researches that has been made about the role of ETH and H2 during AR formation.
Experimental design: 3)	Line 114: Which part of the explants did the samples take or all the explants were used?
Validity of the findings: 4)	I think the current result cannot draw a conclusion that ETH is the downstream signaling molecule in adventitious rooting induced by H2 unless the author can tell us the variation of ETH related gene expression after H2 treatment.
Additional comments: 5)	Lines 253 – 277: I suggest that the author can adjust the order of the sentences to make it more logical.
6)	The author can simplify the context sometimes.
            a.	Lines 286 – 290: I suggest to delete the sentence “In a proteomic study of sorghum leaves under salt stress, Rubisco activity was enhanced in response to salt stress (Ngara et al. 2012). Andersson et al. (2008) reported that Rubisco expression could influence plant growth and lead to complex changes. Previous study showed that Rubisco gene grown at high-CO2 concentration might result in a decline in Rubisco activity in pea (Xu et al. 1994)”
            b.	Lines 291 – 293: These two sentences are almost identical.
7)	I suggest the author to turn all English numbers into Arabic numerals including abstract section, lines 105, 108, 209 – 213, 222 – 227, 233 – 237.
8)	It is unclear to me that how could you divide the 48 proteins into 8 groups in lines 220 – 222, Fig 7. Which database did you use?
9)	The Fig 8 display the result of GO annotation but the legend is COG.
10)	Lines 232 – 236: The result is inconsistent with the presentation of Fig 8. There were 4,3,6 categories in biological progress, molecular function and cellular component in Fig 8, respectively, but the author said 9,4,7 categories in the context.
11)	Table 2: I suggest to divide the proteins into 4 groups according to your treatments.
12)	Table 2: The legend is unclear to me. What is A, B, C D? And I suggest to explain the meaning of score, MW, PI in the legend.
13)	Abstract: “photosynthesis -related proteins” should be “photosynthesis-related proteins”
14)	Line 66: “hydrogen sulfide (H2S; Lin et al. 2012) hydrogen (H2;Lin et al. 2014)” should be “hydrogen sulfide (H2S; Lin et al. 2012), hydrogen (H2;Lin et al. 2014)”.
15)	Line 75: Using the English comma.
16)	Line 231: ‘biological process” should be “biological process”
17)	 Lines 322 -325: APX should be CAPX.