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: GENERATION AND CHARACTERIZATION OF CROSS NEUTRALIZING HUMAN MONOCLONAL ANTIBODY AGAINST 4 SEROTYPES OF DENGUE VIRUS WITHOUT ENHANCING ACTIVITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript was improved, but it is still no publication material. Even though they included necessary information sometimes ill positioned in the manuscript, English is still not at a level for publication. For example, and just one example of many throughout the manuscript, cells do not "contain" a receptor, cells express a receptor. Scientifically the paper is ok, but the written manuscript  is poorly explanatory, with many grammatical mistakes, many sentences without any sense.
I still need a more thoroughly  revision of the literature about Glycosylation of antibodies and how this affects their function. Also, why the phage displayed was used for the detection of the antibody specificity.
In the results section, the subtitles have to show a finding, not merely described what they have done to achieve this finding.
Negative controls are intended to be part of the figures. In the IFA figure, the negative control of mock infected cells detected by the antibodies are not included.
ADE was not originally described by Schmidt, but by Halstead. Please revise. It is Ab dependent enhancement. please revise through all the MS.
I still think that the paper is publicable, but has to be improved.
Experimental design: I think that some of the experimental design is lacking.
For example to include the negative controls in the figures.
I do think that they need to show still Fc binding of both antibodies and if they eliminate the glycosilation of the original one, do they obtained the same results as the new one?
When something is Similar...it has to be similar to another thing. a lot of results are just described as "similar"...to what?
I do not agree in just using a predictive software for glycosilation, or lack of it. But if they would give more emphasis in showing that just this point mutation is sufficient for an antibody to loose its glycosilation, then I can accept it.
Regarding their DNA PCR product electrophoresis for IgG subtyping, how can you differentiate between a band of 211 bp for IgG1 and 207 bp for IgG3?
Some trivial Methods are described extensively, and most important methods are not. As a virologist, moi description is mandatory for a FRNT test.
Validity of the findings: The results are ok. Data is ok, conclusions can be more extensive and emphatic.
But their English level do not allowed the reviewer or the reader to understand everything thoroughly. One examplae of many: "To determine NT and ADE activity in Fc receptor bearing K562 cell, B3B9 rIgG was able to neutralize DENV2 and 3, but not DENV1 and 4".There is no sense in this sentence.
Additional comments: Please give the manuscript to an English native speaker for revision. In such a state at it is know, publication is not possible.


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: GENERATION AND CHARACTERIZATION OF CROSS NEUTRALIZING HUMAN MONOCLONAL ANTIBODY AGAINST 4 SEROTYPES OF DENGUE VIRUS WITHOUT ENHANCING ACTIVITY
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors claim that the manuscript was proofed by a native speaker and by a professional service. The manuscript has improved, but there are still some mistakes and poor choices of words (I marked everything that needs to be changed in the enclosed document)
Results are still  relevant and can be published.
Experimental design: There are some experimental details that need to be stated. Please take a look in the document attached.
Validity of the findings: The last sentence is a bit too out far reaching stating that the antibody produced  by the authors can be used in therapy. I would say that it can be used in models testing for a possible anti dengue therapy.
Additional comments: Manuscript has improved, but needs still  some polishing before 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: GENERATION AND CHARACTERIZATION OF CROSS NEUTRALIZING HUMAN MONOCLONAL ANTIBODY AGAINST 4 SEROTYPES OF DENGUE VIRUS WITHOUT ENHANCING ACTIVITY
Review round: 4
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: Lines 460-463, the authors should indicate that these are in vitro results and it is a proof of concept.
Additionally, the authors should be clear on potential "problem" of a human anti-mouse antibody response. The sentence should be reworded for clarity and good scientific presentation.
Lines 464-468, the authors should be clear on the conclusion. Try to include the phrase "with further studies on...." and emphasize on the potential utility of the antibody.
In the abstract, please remove the word "fortunately".
Additional comments: Generally the manuscript is in good order, the authors would need to do a thorough proof-reading, with a native English speaker. There are some minor grammar mistakes throughout the manuscript, eg line 396 (balancing vs balance); 472 (advisement vs advise).


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: VEGETATION STRUCTURE OF PLANTAIN-BASED AGROSYSTEMS DETERMINES NUMERICAL DOMINANCE IN COMMUNITY OF GROUND-DWELLING ANTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is generally well written and the data have been subjected to sound statistical analysis. There are some textual flaws and I have annotated these on the manuscript.
I am not entirely comfortable with the use of the term 'plantain based agroecosystem'. If cocoa or coffee is present then aren't such farms likely to be cocoa or coffee farms with some plantain interplanted? In other words, is plantain always the main crop in the farms that were studied?
What concerns me is the lack of ant identifications to species level, with one exception. I suspect that the Phedole app. are members of the megacephala group. As presented, the reader is unsure whether this study represents a set of native ants or whether the fields are colonised by 'weedy', cosmopolitan ant species. I would have thought that this was essential information.
Experimental design: Since the ultimate thrust of this work is to see if farms harbour useful biological control ant agents, this study has missed an important opportunity to include the sampling of arboreal ants. Baits could easily have been placed in the plantain, although I doubt if plants would occur in each of the 25 subplots. This would therefore demand a somewhat different analysis.
As this manuscript stands, the are copious implications about arboreal ants and control of tree pests, without actually having looked at the arboreal system. Maybe this has been done in the thesis, but if so surely some data could be brought into this manuscript.
Validity of the findings: The findings presented here are valid but not as profound as they could be due to a) lack of species-level identifications and b) lack of data on arboreal ants.
Additional comments: lines 129-133. It is not clear how you qualified dominant versus subdominant species. I realise that this is embedded in the text here, but you really need a separate sentence for definition of dominant species and another for subdominant species.
Figure 1. This should be in colour as the grey tones are not well differentiated.
Line 201. Refers to functional groups, yet nothing is said about this in the Methods nor in the results.
Lines 231-232 and elsewhere in the Discussion. You refer to species dominating the area, yet you have previously dismissed most of these species as being dominants. Change terminology to ''predominating in the area' or something else that does not clash with the word dominant.
Many other editorial matters are marked up on the PDF.


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

Input:
Title: VEGETATION STRUCTURE OF PLANTAIN-BASED AGROSYSTEMS DETERMINES NUMERICAL DOMINANCE IN COMMUNITY OF GROUND-DWELLING ANTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a manuscript about how vegetation densities at high and intermediate strata can change the numerically dominant ground foraging ant at a local scale in plantain polycultures. For the most part, this manuscript uses clear, unambiguous, and professional language. The Figures are relevant and layout is fine. Raw data is included.

However, the manuscript could be improved by considering the following:
Line 27: instead for “cropped in association” you may want to consider using the terms “intercropping” or “grown in a polyculture” (also applicable for line 78)
Lines 47-49: It would be clearer if this were rephrased to “Ecological factors, which are the focus of the present study, can include both ecological interactions (e.g. foraging interference) and habitat-related factors (e.g. nesting sites).
In general it would improve readability and make the manuscript more succinct if the study examples were restructured to include the citation at the end. For example Lines 57-60: This sentence would be clearer if rephrased to something like “For example, in South American savannas tall grass cover has been shown to reduce the incidence of the dominant ant species, Solenopsis substituta (Santschi) (Vasconcelos et al. 2008).
Line 73: change “proved” to “showed” or “provided evidence”
Line 76: change “understorey” to “understory”
Line 122: Add a period after “dominance”
Line 127: Remove “of”
Line 133: change “genus” to “taxa”
Line 134: remove “species”
Line 135: change “genus” to “taxa”
Line 154: change “(Table 1)..” to “(Table 1).”
Line 155: change “genus” to “taxa”
Line 158: change “species or genus” to “taxa” and remove “as”
Line 177: should be “Abera-Kalibata et al. (2007) found that two morphospecies of Pheidole were among the most abundant”
Line 183: change “with a recruitments” to “with worker recruitment”
Line 219: change “genera” to “genus”
Line 239, 240, and 243: change “habits” to “strategies”
Experimental design: The manuscript provides original primary research within the aims and scope of the journal, however there are several areas where the methodology needs clarification:

Lines 117-120: Only half of the ant taxa you report are identified to morphospecies in your results, the rest are identified to genus. Your methods should be fixed to reflect this, and explain your reasoning (why not identify them all to morphospecies?).
Line 124: Your abstract (Line 29), indicates that you deliberately excluded less abundant ant species (which would explain what appears to be surprisingly low diversity in your reported ant taxa). Please clarify if that is the case and include it in the methods if it is.
Lines 127-134: Baccaro et al. (2010) used somewhat different dominance parameters than those used in this ms ( >5% bait presence, >25% bait control, and a minimum mean abundance score of 4 compared to your >10% bait presence, >25% bait control, and a minimum mean abundance score of 3.5). Thus with the methods in Baccaro et al. (2010) none of your ant taxa would be “dominant” (Pheidole spp. has a mean abundance of 3.76).   However, if you had lowered the bait control parameter instead of the mean abundance score parameter, the Axinidris sp. would have be “dominant”. I think it would improve the manuscript if you provide an explanation for your reasoning in changing these parameters from the original methods and why this measure of dominance is meaningful in general.
Line 121: What do you consider “control” of a bait to be for this study?
Validity of the findings: Validity of the findings:
Many of the findings appear sound, however I have some concerns about the following:
(1)	Pheidole is a large genus, and it’s possible that you have a large number of rare species masking as a single abundant taxa, I think this is important to address.
(2)	Ant biocontrol of pests occurs at the level of the plant leaves, rather than on the ground, and ant dominance will often be quite different on plants than on the ground, this is also important to address in your discussion section.
(3)	“Dominance” has so many meanings, several of which you use throughout your manuscript.  I think it would be useful to clarify why you chose to use your two different measures of dominance (rather than just numerical dominance at the local scale and overall abundance at the broadest scale) or conversely why you didn’t consider bait monopolization at the local scale in your vegetation strata analysis.
Additional comments: All general comments are above


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

Input:
Title: VEGETATION STRUCTURE OF PLANTAIN-BASED AGROSYSTEMS DETERMINES NUMERICAL DOMINANCE IN COMMUNITY OF GROUND-DWELLING ANTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Gbéblonoudo et al. have accomplished one manuscripts deals on an interesting topic, with a great effort to collect vegetal information in different strata in plantain crops, however I have some concerns about ant datasets, analyses and description of the results.

I think that english must to be check.
Experimental design: The design is ok but I'm not sure if evaluate dominance of ant species using only genera is the most correct way.i'ts
Validity of the findings: Important results can be found but i think that a the ant taxonomy to specie level shoul be revised.
Additional comments: Gbéblonoudo et al. have accomplished one manuscripts deals on an interesting topic, with a great effort to collect vegetal information in different strata in plantain crops, however I have some concerns about ant datasets, analyses and description of the results.

Line 39: All your Key Words are present in the tittle. I would suggest that you can make better use of this part to give more ideas of your work and results.
Par exemple: Cameroon, Formicidae, density, abundance, baits, vegetation strata, Dominant, subdominant, and subordinate ants, Musa etc, etc……

Introduction
Line 47: humidity and temperature are environmental factors not physiological. Maybe you can say that these factors affects ant physiology…. please rewrite this idea to be consistent.

Line 47: To be more direct this phrase can be re write as …..  “and ecological factors (Philpott and Armbrecht, 2006), that include ecological interactions (e.g., foraging interference) and habitat-related factors (e.g. nesting sites), which are the focus of the present study.
And please if you can, include some references for each factor mentioned.
Line 56: (hymenoptera: Formicidae) can be deleted
Line 60: Idem
Line 62: The phrase: “but that dominance by ants in the Dolichoderinae was higher in pastures” have not sense…..  maybe you want to say that: ‘…..but dominance of Dolichoderinae ants was higher in pastures”….

Line 79: Please include a reference at the end of the phrase.

M&M
Line 67: I’m pretty sure that the phrase is ‘ant diet varies….’ instead ‘Ant diets vary…’ Please check the English.
Line 116: Here you speak about species and morphospecies but you don’t give information of them.
Line 119: delete “the”
Line 122: Please you should include one dot before “We”
Line 118: Rewrite as follow: according to Fisher and Bolton (2016)…..
Line 127 to 129: you say that you follow Baccaro et al. 2010 and Carval et al. 2016, but they use the specie and morphospecies level in theirs analysis. Why do you decide to use genera level? Can you explain your decision of use genera instead of specie level?
As far as I know, is important to take in account the variation inside the genera because one genera can have many species with different diets, behavior, etc, for example inside the genera Pheidole, different species can have marked differences in terms of resource exploitation, (one sp. can be more dominant than others) and in my opinion this is an important factor to take in mind.
Also, in some place you should state how many baits did you put on the field, or say the exact number of subplots and say that in each one your put a bait, it’s seem simple but this information is really important.
Results

You must to begin with a general phrase saying the total abundance of species (genera in your case). Also the number maximum the genera par bait or the minumun… etc… this information is also relevant.
Line 154: Please delete one of two dots between (Table 1) and Pheidole…..

Line 154 and 155: ‘Pheidole spp.were identified as the dominant genus’…. Maybe this could be due to the fact that is the most diverse genus?

Line 153 to 158: You should give the data information of how much each one controlled the bait. And know that this in the table 1 too but you can also give data about the number of baits or the number of workers in each bait, etc….

Line 160: “frecuency” instead “frecuencies” please check the English.

Line 164: I’m not sure that the word collected is the most appropriate maybe is ‘recorded’…

Discussion

Line 178: please use a contrast connector between ‘Uganda’ and ‘We’ …. Juste to differentiate two ideas.

Line 184: Please use “on baits” instead “at baits”

Line 191 to 197: And how you explain those results… less/more resources to nest, or to feed, etc????

Line 213: “…..in our study, negatively related to the density of arboreal habitats”… I think that this is due to the sampling method used, this is expected when you use soil baits, but if you use arboreal baits? Or if you follow your soil baits in the time…. In those cases the results can show that different species can visit the bait…. Its important to make the difference….

Line 232: Axinidris sp. dominate the area (Vasconcelos et al., 2008)… I’m not sure that this work can say something about Axinidris (an African ant), when they mainly  works in Amazonian landscapes.

Line 235 to 236: See how is important the specie level? You cannot make a generalization in term of diets or trophic level with certain ant genera, as Pheidole.

I like so much the figure 2, its very informative but I think that you show explain it better in the text.

A general conclusion is missing.

Please check also these references, that can be useful:

-	Achury et al 2012. Psyque. “Effects of the Heterogeneity of the Landscape and the Abundance of……….”
-	LeBrun et al 2007. Ecology. An experimental study of competition between fire ants and argentine ants in their native range


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: VEGETATION STRUCTURE OF PLANTAIN-BASED AGROSYSTEMS DETERMINES NUMERICAL DOMINANCE IN COMMUNITY OF GROUND-DWELLING ANTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have attempted to address the comments of the reviewers. It is now a serviceable piece of work, although a few edits still need to be attended to, including one uncited reference. I have made comments directly on the manuscript, which appear in blue.

I am still not overly excited about the study for the reasons I already mentioned, namely the lack of sampling of vegetation and the lumping of species. This latter has, to an extent, been addressed, but the analysis and figures still treats the species within a genus as lumped together.
Experimental design: Adequate
Validity of the findings: Adequate
Additional comments: The authors have attempted to address the comments of the reviewers. It is now a serviceable piece of work, although a few edits still need to be attended to, including one uncited reference. I have made comments directly on the manuscript, which appear in blue.

I am still not overly excited about the study for the reasons I already mentioned, namely the lack of sampling of vegetation and the lumping of species. This latter has, to an extent, been addressed, but the analysis and figures still treats the species within a genus as lumped together.


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: OVERLAND MOVEMENT IN AFRICAN CLAWED FROGS (XENOPUS LAEVIS): EMPIRICAL DISPERSAL DATA FROM WITHIN THEIR NATIVE RANGE
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are no doubt differences between South African English and US English, but I found the following to be noteworthy enough to include:

Make a clear distinction between dispersion and migration

Pp. 61, 62: no reason to cite a reference twice in the same sentence.

p. 82: Poyton should have a date - a full citation

p. 93: delete “When it occurs …

Line 183: Why add the word “event” to movement?  Not necessary, besides, “occurrence” may be a better term

Line 218: This truth of this sentence depends on capture-recapture techniques used.  If using drift fence/pitfall trap arrays, this statement is false.

Line 240: Replace “to” with “from.”

Line 272: How about “burrowed animals” or “buried animals?”  This phrasing sounds microbial.

Line 304: replace “further” with “farther.”  Farther is the term relating to dostance.

Lines 316-317: How can sites receive movement?  Animals move.

Line 324: Remove “do”

Line 333: Move this to the bottom of the paragraph – the least important conclusion.

Line 345: Delete “would like to”

Also, much of the North American literature on migration, dispersal, and population dynamics is left out.  There is one citation, though, you must incorporate: Smith, M. A., and D. M. Green. 2005. Dispersal and the metapopulation paradigm in amphibian ecology and conservation: Are all amphibian populations metapopulations? Ecography 28:110–128.

You should also make a clear distinction between dispersion, migration, and perhaps habitat abandonment or some such other term.
Experimental design: Very good.  I have no issues.
Validity of the findings: High, plus these findings are important.
Additional comments: Nice design, important study, clunky word usage and at least one NA citation should be 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: OVERLAND MOVEMENT IN AFRICAN CLAWED FROGS (XENOPUS LAEVIS): EMPIRICAL DISPERSAL DATA FROM WITHIN THEIR NATIVE RANGE
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: I have some major concerns regarding on the statistical analyses used here.
Additional comments: This manuscript examines the dispersal of a native population of Xenopus laevis. Most of individuals moved less than 200 m, with a maximal distance reported of more than 2 km. No sex-biased dispersal have been identified in this study. This is an interesting paper, for which the dispersal data of this invasive species might be very useful in models.

The manuscript is well-written. I have some major concerns regarding on the introduction and the statistical analyses used here. I recommend the acceptance of this publication but after several corrections. I have detailed my major and minor comments below, and I hope that they will contribute to improving this manuscript.

Introduction

Page 6, Line 47. I suggest to change “Locomotion” to “Movement”. In my point of view, the term “locomotion” is commonly used related to the way to move (for instance S-shaped movement in salamander species), whereas the movement is linked to both the migration and dispersal processes (with dispersal kernels, dispersal propensity and distance moved).

Page 6, Line 49: Dispersal is a multi-causal process, which could be measured using two methods: either by tracking animals in the field (using capture-recapture for example) or, because dispersal is a movement which could induce gene flow, molecular tools are also used to determine the dispersal ability of a given species. Consequently, dispersal is not only estimated by the observed distance moved by individuals. Doing so, I suggest to change “whereas dispersal represents the observed distance dispersed” to “whereas dispersal represents the distance moved”. More details about dispersal are needed in this part.

Page 6, Line 50. Of course, performance abilities might be correlated to the mobility capacities in a species. I am surprised to not see the papers from Herrel and Bonneaud here. Herrel and Bonneaud (2012) and Herrel et al. (2014) demonstrated several relationships between locomotion abilities and performances in a sister species Xenopus laevis.

Herrel A, Bonneaud C (2012) Trade-offs between burst performance and maximal exertion capacity in a wild amphibian, Xenopus tropicalis. J Exp Biol 215:3106–3111

Herrel A, Vasilopoulou-Kampitsi M, Bonneaud C. (2014) Jumping performance in the highly aquatic frog, Xenopus tropicalis: sex-specific relationships between morphology and performance. PeerJ 2:e661

Page 7, Line 95. You did not define the difference between dispersal (a movement which could induce gene flow) and migration. Maybe you could add few lines about this difference (from line 49 for instance) that should help for the understanding of the paper.

Materials & Methods

Page 8, Line 113. Do you have more information about the permanent and temperature water-bodies? Temperature of water, width, depth, or if other amphibian species are living within?

Page 8, Line 121. How many tracking session? How many days between them?

Page 9, Line 128. Did you applicate a protocol to avoid the dissemination of Bd (Batrachochytrium dendrobatidis) after each tracking session?

Page 9, Line 135. Did you anesthetize individuals before pit-tagging? You should be, please details the method used.

Page 9, Line 145. Please give the p-value of the Shapiro test for testing the normality of data.

Page 9, Line 149. Please explain why you perform performance tests additionally to the capture-mark-recapture session.

Page 10, Line 166. Why did you chose the longest distance performed in the shortest time in your analyses? You can keep the 3 distances by individuals and run mixed-effect models by adding the individual as random factor in your models. By taking into account the intra-individual variation of the distance performed in controlled conditions, this statistical method would increase the robustness of your data.

Page 10, Line 179. You could give the results using the complete dataset for the tests related to the size.

Page 10, Line 184. Your analysis tacking into account both the dry and wet season is interesting. But it could be very pertinent to have the humidity conditions recorded at each capture session in order to relate these data with the movement events.

Results

Page 11, Line 203. Please, give the range of sex ratio in percent.

Page 11, Line 213. Remove “as is usual in amphibian dispersal” (which is more appropriate in the Discussion part).

Page 12, Line 225. “The maximum dispersal distance observed was a X. laevis female, which travelled 2 420 m” is a repetition of the line 214. Please add details about this long-dispersal event at line 214. Also, give the distance in km or in meters throughout the manuscript.

Discussion

Discussion could become reduced by at least 30% without losing essential information.

Page 14, Line 294. In fact, sex-biased dispersal found in amphibians have been revealed using genetics, which suggest that juveniles (individuals that we cannot tag in most of amphibian species) are responsible for most of dispersal events (inducing gene flow).

Line 341. Do you envisage performing a radio-tracking study in this native population?


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: FOLIAR MICROBIOME TRANSPLANTS CONFER DISEASE RESISTANCE IN A CRITICALLY-ENDANGERED PLANT
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a good manuscript.
Experimental design: No problem. See General Comments.
Validity of the findings: No problem. See General Comments.
Additional comments: This is a well written manuscript on interesting results. I don’t see any issues with design and I think that the findings are valid.  So, I’ll proceed with more general comments to hopefully help the authors find ways to improve the manuscript.  I think that the context could be deepened somewhat here and there.  A brief background is needed on species of Phyllostegia endemic to Hawaii and other Pacific islands.  Is there any knowledge of what continental plant gave rise in the islands to endemic Phyllostegia, and of when that migration event might have occurred? Is that ancestor a host to this same powdery mildew, at least at the taxon level?  A little more context and background on the powdery mildew might be helpful too. It is considered to be currently cosmopolitan but is there any reason to think that the mildew was recently introduced?  Or does it seem more likely that the mildew has always been there in the islands as diversification in Phyllostegia progressed?  I am not aware of any endophyte study reporting that the “ vast majority of sequences from … were identified as the pathogen”.  Might the authors draw a little more attention to this finding if it truly is unique? The authors report relatively little endophyte diversity, and might emphasize that more.  Another question that should be raised has to do with the presence in the leaf slurries of Ampelomyces quisqualis.  The latter  is a known mycoparasite of powdery mildew. Was the mildew that developed on plants examined microscopically for the presence of pycnidia of Ampelomyces?  Was it examined for the yeast?


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: FOLIAR MICROBIOME TRANSPLANTS CONFER DISEASE RESISTANCE IN A CRITICALLY-ENDANGERED PLANT
Review round: 1
Reviewer: 2

Output:
Basic reporting: It was difficult to get hooked into the manuscript details.

There was no indication of name of the plant species until Line 65 or the causal agent of the disease in Line 70. I suggest these organisms be identified in the title and abstract to keep the reader engaged.

Why did the authors choose this pathosystem to study? What is significant about this plant other than it is endangered, many plants species have this status.

The continued use of the generic terms 'pathogen' and 'plant' is vague. The presented research focuses on specific plant species, one specific powdery mildew pathogen species, a specific and potentially important yeast biocontrol agent and a few named fungal taxa in the discussion. The research has potential implications for the conservation of highly imperiled plant species. To sustain the reader's interest, it may be best to relate the pertinent information using clear communication of the taxa involved throughout the manuscript.

In Line 63, the use of the term 'pathogen mortality' is not clear. Does the pathogen die or does the host plant die as a result of colonization by the pathogen?

In Lines 61, 128, 130 and 165 the authors use quotation marks. When one is unsure of the correct term, it may be preferred to choose words that convey the intended meaning to facilitate comprehension by the reader rather than highlight perceived indecisiveness with quotation marks. If these terms are in fact quotations from another source, then a citation will clarify the use of quotation marks.

In Lines 97-98, 116, 146, 147-148, 158, 159, 161, 172, 199, 227, 251-252 and 265 the authors use parentheses. This is unconventional and discredits the strength of the statements.
Experimental design: Lines 139-140, the authors discuss 'pathogen exposure and pathogen introduction'. Is this in reference to N. galeopsidis?

Continuing on in Lines 140-142 the authors mention the air intake system in the greenhouse. Are you claiming to inoculate your experiment through random chance of air dispersal through the greenhouse ventilation system? If so, this unusual inoculation method will be best supported with citations of its use in other peer-reviewed publications.

Were negative controls used in this experimental design? If so, it will be important to report the data collected for comparison to the inoculated plants.
Validity of the findings: The research presented claims to be preliminarily successful in the use of a microbiome inoculation on the leaf tissue of Phyllostegia hirsuta to protect the plant from disease after out-planting. This is an important finding, if there is evidence the pathogen is present at the out-planting site to support the claim the slurry inoculation technique shows true efficacy.

I strongly urge the authors to become familiar with the biology of the powdery mildew organism and its relationship with a host plant. Powdery mildew infections are almost always host specific. Therefore, P. hirsuta is in all likelihood able to defend against successful colonization by Neoerysiphe galeopsidis even though its close relative, P. kaalaensis is deemed to be less resistant to infection by this pathogen. Moreover, powdery mildew conidia burst in free water e.g., sterile water slurry. The OTU counts of N. galeopsidis the authors obtained in the sequencing effort using the P. hirsuta leaf tissue may represent dead cells of N. galeopsidis rather than viable cells. As the manuscript reads now, the analysis of the experimental results does not give a convincing argument for the underlying biology of the N. galeopsidis and its interaction with P. hirsuta.
Additional comments: One interesting finding is the potential biocontrol interaction between Pseudozyma aphidis and N. galeopsidis. This yeast has been shown to be an effective biocontrol agent against Podosphaera xanthii on cucumber. Gafni et al. (Frontiers in Plant Science, 2015, doi:10.3389/fpls.2015.00132).


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: FOLIAR MICROBIOME TRANSPLANTS CONFER DISEASE RESISTANCE IN A CRITICALLY-ENDANGERED PLANT
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Zahn and Amend describes the efficacy of using foliar microbiome extracts on the endangered Phyllostegia spp. as biocontrol agents against the foliar pathogen N. galeopsidis in Hawai’i. The manuscript examines two endangered species. P. kaalaensis and P. mollis, that received inoculations of either fungal endophyte spores or filtered leaf extracts prior to inoculation with the pathogen. The foliar fungal community is monitored at three intervals with NGS amplicon sequencing of the ITS rDNA locus.

The manuscript is written clearly and the authors provide sufficient background and context to understand the rationale for the study, the methods, and the results.  The figures are professional and easy convey the results.  The raw data are shared in an appropriate manner.
Experimental design: The research question is well defined and meaningful.  In general the methods are clearly described in sufficient detail for the reader to replicate the study.  I have noted below one area where additional detail on methodology is necessary to replicate the results.
Validity of the findings: The experimental methods and data analysis are experimentally robust and the author's conclusions are well stated and supported by the results of the study.
Additional comments: I have some general comments and suggestions.

Line 22.  ‘Throughput’ is misspelled.
Line 38. To help clarify the meaning of this sentence change ‘and are often defined negatively’’ to ‘but are often defined negatively’.
Line 125.  Please provide citations for primers use in the study (ITS1F and TW-13).
Line 129.  What method was used for blending?  Please describe in greater detail.


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

Input:
Title: HOW CAN WE RELIABLY IDENTIFY A TAXON BASED ON HUMERAL MORPHOLOGY? COMPARATIVE MORPHOLOGY OF DESMOSTYLIAN HUMERI
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, this is a well-researched article with useful scientific information for the study of desmostylians. The use of English in this article is mostly clear and unambiguous in its presentation. Some sentences were unclear in their sentence structure or word usage. The author may benefit from having a native English speaker review the article again before submitting the final manuscript to ensure clarity to an international readership.

The section that needs the most attention is the Conclusion. This section needs to be elaborated more or simply removed entirely. One single sentence does not help to summarize the important details of this important paper for desmostylian research. Perhaps quickly summarize the differences in all desmostylian humeri and how it could help future research.

Below are sections of the article that need revision:

1.	Line 35: Barboza et al., 2017 mentions evidence of Desmostylia from the early late Hemphillian Oso Member of the Capistrano Formation, which may extend the known range of Desmostylia on the eastern Pacific Coast if the material is not reworked.

2.	Line 44: You may want to mention Santos et al., 2017 as the authors provide an updated ontogenetic sequence for Desmostylus as well as features diagnostic of advanced age specimens based on mandibular morphology. Additionally, the authors synonymized Vanderhoofius with Desmostylus.

3.	Line 67: The additional paragraph does not seem needed. This sentence might work best being added to the end of the previous paragraph.

4.	Line 75: “desmostylian” is misspelled as “desmosdtyian”

5.	Line 75: This sentence is a little confusing as the author states they “examined morphology. . . based on direct examinations of specimens or literature review.” This wording is odd with the two uses of examine. Perhaps exchange “examined” with “analyzed” or something similar.

6.	I would suggest moving the Institutional Abbreviation section from line 166 to the end of the Introduction or at the beginning of the Methods and Materials section. This might make it easier for the reader to have the abbreviations first instead of having to go find them below.

7.	Line 138: Perhaps make it clear for readers not familiar with the outgroup relationships that the Sirenia are within Tethytheria.

8.	Line 179: Depending on time for the author, creating figures the compares the stated features between the different taxa might be incredibly useful for the reader, especially those not familiar with them. I don’t believe it is absolutely necessary, but could improve the ease of understanding of this important section of the paper for the reader.

9.	Line 181 and 82: Dugongidae is misspelled as Dugonidae

10.	Line 181 and further mentions: Hydrodamalis is within the Dugongidae family

11.	Line 185: “desmostylian” is misspelled as “desmostylidans”

12.	Line 185-185: The wording within the parentheses is overly complicated and confusing to the reader. Perhaps simplifying the wording to just “Equidae smaller than Merychippus” removing the need to double parentheses.

13.	Line 186-187: The line “the intertubercler groove of Perissodactyla covered by greater tubercle” may be missing a word. The sentence reads odd to me, but perhaps not.

14.	Line 187: “characteristic” is misspelled as “charavteristic”

15.	Line 207: “Behemotops is relatively ompared to other desmostylians.” I believe this sentence may be missing a few words.

16.	Line 213: “extends to more distally” perhaps remove “to”

17.	Line 229: This sentence is confusing to the reader as I am not sure what is trying to be said. “tubercle is not developed nodule-like unlike those in other desmostylians.”

18.	Figures 3-7: These figures are incredibly useful and well-done, but may I suggest improvements to make the figures more useful for readers.

- On figures with multiple specimens, please label which image is which specimen for quick identification and greater use.

- Please provide explanation for the colored coded boxes and, while redundant, the characteristics associated with each number could be listed again for quick reference.

- Please make the numbering larger as some are hard to see.

- Is it possible to provide the actual specimen photos below the illustrated line drawings? This may help future researchers identify the characteristics on real world fossils as drawings may not provide enough detail that is present on the specimens.

19.	Line 294: This single sentence paragraph could incorporated into the beginning of the next paragraph.

20.	Line 303 and 304: This is the first mention of these Trichechus species. It may help the reader to spell out the names fully and provide taxonomic authority for the first instance.

21.	Line 308: “Berta t. al” is missing the “e” in “et al.”

22.	Line 310: “manatees are “walk” on the sea floor and by using their forelimb” this sentence has slightly confusing wording. Perhaps change to “manatees use their forelimb to “walk” on the sea floor” for a little more clarity.

23.	Line 321: “based diagnostic features” perhaps missing “on” between “based” and “diagnostic”

24.	The Conclusion section needs to be elaborated more or simply removed entirely. One single sentence does not help to summarize the important details of this important paper for desmostylian research. Perhaps quickly summarize the differences in all desmostylian humeri and how it could help future research.

25.	Figure 1: Please label which groups are Desmostylia, Perissodactyla, and Tethytheria. And provide more detail on the relationship between the two others to desmostylia. The connected dotted line might be confusing to readers not familiar with desmostylian taxonomy. One solution might be to split the tree into two trees: one showing the relationship to Tethytheria and the other showing relationship to Perissodactyla.
Experimental design: Please see previous comment on suggestions for improvement to figures.
Validity of the findings: No comment.
Additional comments: No additional comments


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

Input:
Title: HOW CAN WE RELIABLY IDENTIFY A TAXON BASED ON HUMERAL MORPHOLOGY? COMPARATIVE MORPHOLOGY OF DESMOSTYLIAN HUMERI
Review round: 1
Reviewer: 2

Output:
Basic reporting: Aside from minor misspellings and inappropriate uses of plurals/singulars, all is in good order. See General comments to authors for further explanation.
Experimental design: The authors have put a sincere effort in justifying the use of each specimen and exploring the impacts of ontogeny on the findings. This is very good for the experimental design of the study, I have no problems with it.
Validity of the findings: The observations that differentiate the taxa are clear and supported by the evidence.
Additional comments: The authors have submitted a manuscript outlining anatomical features that are diagnostic to the humeri of most species of desmostylians (all of those for which humeri of associated skeletons are known). Desmostylians have historically had much of their postcrania ignored in terms of taxonomic utility, and generally even though they are not all the same, many studies on locomotion tend to argue for different modes while using different taxa. The obvious thing is that those arguments could potentially all be correct, if only the authors recognized that they were all referring to different animals. Matsui and colleagues have done a great service to combat this tendency by writing to specifically identify differences among the diversity of desmostylian taxa. For this reason, I support publishing this paper.

The manuscript is not ready for publication yet, but it is only for a collection of minor revisions that are needed that are mostly related to language used. Some are about the scientific content, but others are simply copyediting matters that are not the role I should be playing here. Rather than outline every single one, here is a description of the groupings that they all fall under. It is important for the authors to go through the entire text and look for these; it will make the paper much more effective, clear, and useful:

1) Grammatical – There are some inappropriate uses of plurals and misspellings, such as “Abstracts” (should be abstract, if there is only one) and “mirabilis” (should be mirabilis). The best advice I can give is to ask a colleague to look through it to make those corrections. After seeing a manuscript so many times as an author, it is easy for  one’s eyes to overlook those minor errors and fresh eyes can do a good deal of difference.

2) Clarity of terms – Throughout the text, most especially in the Results, terms such as “weakly developed”, “apparent”, “stronger”, and “robust” are used when describing morphology. Though the author may have a sense for what that means to them, these terms don’t really mean anything precise that easily translates to the same meaning to anyone else. This is a common problem in vertebrate paleontology, and something wise to avoid if you want your writing to be useful to anyone else (which is presumably why you are writing this). For example, does “desmostylians do not have an apparent deltoid tuberosity” mean something different than “desmostylians for not have a deltoid tuberosity”? All that “an apparent” does there is invoke a sense of uncertainty in your observation. If you do not observe a deltoid tuberosity, that’s unnecessary. If someone finds one later on, they can update the observation with that added detail, no harm done.
Terms like stronger/weaker/robust are meaningless is shape terms. As this is a morphological study, perhaps the better terms to use are larger/smaller/longer/shorter/wider/narrower. And, that needs to be comparative (see below).

3) Descriptions need to either be clear descriptions of shape/measurement (such as “circular”, or “5cm long”), or comparative. You have plenty of good examples of how to do that correctly here, but there are also incidences where the text includes statements of something being stronger or robust without a comparison to something else it is stronger than or more robust than. Aside from changing that to explain that the structure is larger (see point 2 above), making sure it is compared to something else is the only way to make that useful.
For example, one could say that Cornwallius has thick enamel on its molars. But thicker than what? A more useful description would state that “Cornwallius has thicker enamel on its molars than do the molars of Anthracobune, but thinner enamel than similar sized molars of Desmostylus”. If you go through the text and look for incidences in which you use a comparative term without comparing it to anything else, you should either revise it or eliminate it. The entire work will be more useful and unambiguous.


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 RESPONSE OF SOIL MICROBIAL COMMUNITIES TO VARIATION IN ANNUAL PRECIPITATION DEPENDS ON SOIL NUTRITIONAL STATUS IN AN OLIGOTROPHIC DESERT
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some text, particularly in the Introduction, needs editing by a native English speaker. Specific suggestions to improve clarity are given below, but there are other areas of text where editing could make meaning more clear while reducing length of text. In some cases, clarity could be improved by separating ideas into two sentences (e.g., lines 143-147).

Literature references are appropriate.

Table and figure structures are professional. Raw data is shared.

The paper's theoretical constructs and organization could be improved to better use the results to support the conclusions. The strength of conclusions, as stated, should be toned down. Please refer to the attached document for details.
Experimental design: The scope is appropriate for the journal.

The research question is relevant, but the approach could be better described through re-organization of the text. See the attached document for details.

Data collection met high technical standards.

Methods are described with sufficient detail.
Validity of the findings: The paper has two primary novel aspects. The authors note, in one case, that such results have not yet been reported.

Data is statistically sound.

Conclusions are a bit overstated. Reorganization of text could improve linkage to research questions. See attached document for details.
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: THE RESPONSE OF SOIL MICROBIAL COMMUNITIES TO VARIATION IN ANNUAL PRECIPITATION DEPENDS ON SOIL NUTRITIONAL STATUS IN AN OLIGOTROPHIC DESERT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors of this research present interesting findings that are well supported by thorough attention to various measures of microbial biogeochemistry (microbial biomass stoichiometry, enzyme activity, soil nutrient pools) and analysis in a desert ecosystem.  The authors demonstrate that desert ecosystems may respond the variability in rainfall in unique ways depending on soil nutrient content (plant cover) (e.g. under wetter conditions an increase in relative production of P-targeting enzymes in RS soils and higher microbial nutrient concentration in G soils) and also in broad overarching ways as well.  These responses are observed both in soil microbial mediated boigeochemsitry as well as bacterial community composition.  I believe that the content that is presented in this manuscript is of interest in building understandings of microbial community function in these ecosystems with increasing pressures of climate change and precip variability.

I do believe that the communication of this study could stand to improve a bit.  I think that the discussion is well related to past studies and prevailing theory, but I think that generally these results are over-interpreted.  For example, when one of the wet years has significantly greater enzyme activity in one cover than another and conclusions are drawn about  mechanism.  I think that the scope of this journal may be okay for this type of study, but I do as the author to include a caveat about the generalizability of their study (being limited).  If the study had a more limited scope in its thesis, I think it could be better communicated.

Title:
Unclear what “vulnerability” refers to.  Can you think of a title that more reflects the central findings: That microbial responses to variation in annual precipitation depend on soil nutrient status in a desert ecosystem.  This is the type of insight that I draw from this study…. And the narrative could stand to improve my having a clear thesis as such.

It appears that this is a discussion of “interannual variation”- so I would specify that in title and after when talking about variation.

Line 39:  Please introduce the acronym earlier in the paragraph with parentheticals before using.

Line 80-81:  This sentence does not make sense “… are highly enzymatic activity..” ??

Line 83:  This sentence does not make sense “In the rainy enzymatic…”  ??

Line 132:  I would just ask the authors to consider that there could be great functional redundancy with these enzymes (many microbes produce them) and that the change in microbial structure does not necessarily relate to adaptation to different variable precipitation, but just the turnover of microbial communities and the fact that many different microbes can produce the enzymes (unless some sort of correlation is shown between precip->microbial community structure->enzyme activity.

Line 181: word choice… perhaps “dynamic” instead of “aspect”

Lines 510-514: Can you move this up from the discussion to frame the hypothesis- that basically high nutrient environments are more resilient to variability in precip?

Line 220:  2011 was sampled in Feb.  Can you make a note that this timepoint was left out of regression analysis- to clarify it is not confounding this results if so.

Lines 292-294:  This can go in results

Lines 286-291:  First please write out J. Craig Venter Insitute (JCVI) if this is what is meant.  Secondly, please include a citation that details the JCVI pipeline methods for sequence processing/analysis.
Experimental design: My other concern is just to ask if the authors could make sure to communicate how they address that other factors other than precipitation change from year to year.  For example, is it clear that the soil moisture content actually reflects the differences in annual precipitation? these two things exist on such different scales and it could be soil moisture driving differences rather than interannual variation in precip, per se.  In the results- moisture seems to be significantly different between years, and moisture itself could control variation in nutrient dyanamics (and is known to affect enzyme activity) on temporal scales that are disjointed from annual precipitation- and yet the regression models are all focused on annual precipitation (year).  pH also changes…  The manuscript could better address how variability is annual precipitation is actually synonymous with year and not confounded by other factors and temporal scales.

I don’t believe the study adequately tests responses variability itself, but rather seeks to understand the relationships of variable precipitation with soil/microbial mediated processes.  There is no control to test variability over the years or a measure of variability itself, but the study rather show correlations between annual precipitation and dissolved nutrients/microbial nutrient ratios etc. over 3 years.  Thus, I believe the manuscript needs to be reframed to communication this and not claim to assess the impact of variability itself (which I think in many sections is okay- but should be attended to). There may be some implications regarding increasing variability in precip with climate change that can be generated from this study…but must be framed as such and not as a study of this in itself.
Validity of the findings: Overall, I think if the scope of the study were more focused on the differences in response to changes in precip across different nutrient regimes, the manuscript would be better communicated.

Please see above in study design for questions of validity in interpretation of the findings

Lines 491-509: No statistics are actually demonstrating any connection between enzyme activity and microbial taxa relative abundances, I think this is a bit too handwavey and should be much more limited if included.
Additional comments: Overall the authors to a great job of collected and analyzing interesting and compelling data regarding nutrient dynamics, microbial activity, and bacterial community composition in desert soils across two different vegetation covers (soil nutrient status).  I think that the manuscript could just be limited in the scope of its findings/interpretation to avoid taking on questions like variability in precip (interannual- which only has 3 years here, etc.)  Rather focus on the interesting differences from year to year within the study's two cover types/nutrient levels.

Best


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 RESPONSE OF SOIL MICROBIAL COMMUNITIES TO VARIATION IN ANNUAL PRECIPITATION DEPENDS ON SOIL NUTRITIONAL STATUS IN AN OLIGOTROPHIC DESERT
Review round: 2
Reviewer: 1

Output:
Basic reporting: All of the criteria are met, with the following exceptions:

Text added to the manuscript by the authors since the original submission needs editing. I provided suggestions in my review.

Figures 1 and 2 were not in the materials I downloaded for review; but they were part of the original submission's supplementary information.
Experimental design: All criteria are met
Validity of the findings: All criteria are met
Additional comments: The authors have adequately addressed the reviewers’ constructive criticisms. Specific comments below are largely related to editing of text to improve clarity, with the exception of line 583 (see details below). This conclusion needs to be put into the context of functional redundancy of ecoenzyme expression by microbial communities with different taxonomic composition, given that the authors included a citation about functional redundancy in the introduction of the manuscript.

Specific comments

Line 51 (abstract): change to “Soil communities within both sites …”

Line 53: “… to face up the wet years” is not a phrase commonly used in English. I would change the sentence to something like the following:

However, the community of the site with lower resources (RS) is well adapted to acquire P resources by ecoenzyme upregulation during years with adequate precipitation, suggesting that this community is resilient after drought occurs.

Line 56: change to:

the soil microbial communities of both sites could be vulnerable to drought through C and P co-limitation and reallocation of resources to physiological acclimatization strategies in order to survive.

Line 95: ‘The’ is not needed at the start of the sentence.

Line 370: change ‘AP’ to ‘PME’

Line 418: Figures were not included in the manuscript I downloaded, so I assumed Figs. 1-2 were moved from the supplement of the original submission.

Line 474: Needs editing. I suggest:

These results suggest that the microbial community in the RS soil, with lower resource availability, must reduce growth as a result of: 1) the physiological cost associated with a low reallocation to P-rich ribosomal RNA, as suggested by the growth rate hypothesis (GRH) (Sinsabaugh & Follstad Shah 2012; Sterner & Elser 2002; Zechmeister-Boltenstern et al. 2015) and 2) the required investment of energy towards the acquisition of P in order to produce ecoenzymes (Evans & Wallenstein 2012; Schimel et al. 2007; Wallenstein & Hall 2012).

Line 537: ‘resilient’ not ‘resilience’. I would add the phrase ‘during times of adequate moisture’ at the end of the sentence.

Line 557: I am not sure this statement is accurate. I would change it to:

‘Our results show how values of TERC:N and TERC:P may shift with respect to variation in annual rainfall and different vegetation cover.’

Line 568: Needs editing. I suggest:

Our results for the dry year (2012) showed that the ecoenzymatic activities associated with C and P acquisition were lowest in the RS and G soils. Values for TERC:N and TERC:P were similar between the RS and G soils, suggesting that both sites may be vulnerable to drought.

Line 575: Needs editing. I suggest:

Similarly, increased ecoenzyme activities associated with P acquisition and elevated TERC:P values when the water is not limiting (2013 and 2014) suggest that the RS soil microbial community is well adapted to acquire P resources via ecoenzyme upregulation post drought.

Line 579: Needs editing. I suggest:

We suggested that, under the scenario proposed by Global Climate Change models for desert ecosystems that predict reduced annual precipitation and increased rainfall variability, the microbial community from both sites could be vulnerable to drought events, but the RS soil microbial communities can make adjustments in order to obtain nutrients in wet years, suggesting that this community is resilient post drought.

Line 583: This result would occur only if functional redundancy is lost through change in community composition.

Line 590-597. Needs editing. I suggest:

Soil communities of both sites (RS and G) may be vulnerable to drought. However, the community at the site with lower resources (RS) may have evolved adaptations, such as rapid ecoenzymatic upregulation, under chronic P limitation. This adaptation confers greater resilience within the community to respond to precipitation events post drought. Under the Global Climate Change scenarios for desert ecosystems that predict reduced annual precipitation and an increased intensity and frequency of torrential rains and drought events, soil microbial communities within both sites could be vulnerable to drought through the combination of C and P co-limitation and reallocation of energy and nutrient resources to physiological acclimatization strategies in order to survive.


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 RESPONSE OF SOIL MICROBIAL COMMUNITIES TO VARIATION IN ANNUAL PRECIPITATION DEPENDS ON SOIL NUTRITIONAL STATUS IN AN OLIGOTROPHIC DESERT
Review round: 2
Reviewer: 2

Output:
Basic reporting: The introduction still has a few English language issues which I tried to edit- In addition, I'm not sure the communication of the study is quite at a level where the manuscript will make the intended impact.  I have included an attached version in which I try to make some edits to the intro to improve the logic/flow of the intro/narrative.  The authors should consider these possible edits.  Edits/comments are in track changes.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The introduction still has a few English language issues which I tried to edit- In addition, I'm not sure the communication of the study is quite at a level where the manuscript will make the intended impact.  I have included an attached version in which I try to make some edits to the intro to improve the logic/flow of the intro/narrative.  The authors should consider these possible edits-


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 RESPONSE OF SOIL MICROBIAL COMMUNITIES TO VARIATION IN ANNUAL PRECIPITATION DEPENDS ON SOIL NUTRITIONAL STATUS IN AN OLIGOTROPHIC DESERT
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have made all of the requested revisions. I believe the manuscript may now be accepted for publication.


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

Input:
Title: THE RESPONSE OF SOIL MICROBIAL COMMUNITIES TO VARIATION IN ANNUAL PRECIPITATION DEPENDS ON SOIL NUTRITIONAL STATUS IN AN OLIGOTROPHIC DESERT
Review round: 3
Reviewer: 2

Output:
Basic reporting: I have no additional substantive comments-  I think that the manuscript could still stand improvement in narrative organization and editing of a few remaining English grammar issues, but to a certain extent this is a matter of preference and relates to the elements of style more than content. As indicated by the editor, further efforts here are not necessary for the publication of this manuscript.  I would encourage the authors to just take one last scan for grammar, in particular, as this may improve paper's impact in terms of readership.

The authors have attended to my previous substantive concerns in the last round of review. Good job- I think the manuscript encapsulates substantial work and interesting results.
Experimental design: no additional comments
Validity of the findings: no additional comments
Additional comments: No additional comments


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

Input:
Title: MOBILE ACOUSTIC TRANSECTS MISS RARE BAT SPECIES: IMPLICATIONS OF SURVEY METHOD AND SPATIO-TEMPORAL SAMPLING FOR MONITORING BATS
Review round: 1
Reviewer: 1

Output:
Basic reporting: It would be great to have a more thorough explanation for why you chose to do this methods comparison. You note that  Whitby et al. 2014 found different results in their methods comparison. Can you explain in your hypotheses why you believed another method comparison was necessary in the ENP? E.g. was this because of the high species richness in the area? The diversity of vegetation, etc?

I really liked your Conclusion section. The rationale for the topic came through very clearly. Can you highlight some of those main questions in your introduction?
Experimental design: Page 4- Can you give a clear explanation for the benefits and issues of using each method type? It would help the reader understand the importance of your later findings to have this background. I would suggest creating a table showing: each method type, advantages/disadvantages, and the prominent literature citing recommendations. If there has not been adequate literature on one method this also highlights the importance for this and future methods studies.  I think such a table would be a very useful/quick reference of what studies have previously suggested and been done.

The reason for testing the 2 methods against each other on Pg. 10 could use a better explanation. You have clearly set up what you aimed to quantify on Pg. 10 (line 203) in your methods. I would like to see this same clarity reflected in your results. Even by using the same subheadings later.

Some clarity on methods and site selection are missing. See my comments below.
Validity of the findings: I think you have found a useful result for researchers and conservation managers. It is nice to see the quantification of conservation methods.
Additional comments: Line 37- add to “document bat population trends…”

Line 69- Hayes 2000 – This ref is not in your citations. Please add.

Line 75 – Loeb 2015- This seems like it should be Loeb et al. 2015. Please change this throughout

Line 132- Be consistent with your use of “Figure 1” and “Fig. 1”

Line 137- I think I would add the make/model of the external microphone in parentheses here and then delete the next sentence starting with: “We conducted mobile transects using….” This just seems to be a bit wordy, but I recognize this is a stylistic recommendation.

Line 143- “We collected data with the three methods during a 40 night sampling period…”
Your methods here are a little bit vague. Can you add the number of hours of recordings per each method type?

Line 164- Can you explain more clearly how you selected your sites? For example, you used water sources, but was this randomly determined based on x distance to a water source? In easiy accessible areas? Near known roosts? Just a bit more detail here might better inform managers that want to follow this method in the future.

Line 177- “…accounting for accessibility…” does this mean locations were randomly selected within, for example, 10 m of a track? Might help to clarify this again.

Line 178 – Can you report the ~height of the painter’s poles?

Line 200- I would delete ‘language’ from this sentence since R is a programming language not a software language as such.

Line 215- Please clarify what ‘this analysis’ refers to.

Line 228- A lot of repetition of the word ‘test’ in line 228 and 229. Can you clean this a bit?

Page 11- Can you mirror the  subheadings to your methods so that it matches the subheadings in your results? E.g. spatial…. Temporal

Page 13- You report the results of species richness based on season and vegetation in Table 1. I realize you are emphasizing the methods comparison, but it would be nice to see the difference in species richness based on these factors highlighted briefly at the end of PP1 on Page 13 and then reference Table 1 .

Line 333-35 – Interesting discovery and provides good info. for managers!

Line 348- please add “mobile” before ‘transect surveys’

Line 385 – “… monitors population trends…” I don’t think your research monitors population trends specifically at this point. Rephrase to rather emphasize the trends in species/community composition.

Line 449- To be more succinct, I would delete: “… some studies report the # of nights required to reach a specified threshold of the estimated species richness.”

Line 473- Can you give specifics on what “slightly more?”

Line 647- "pespective,,.."  – Reference needs fixing

Line 657- "US Geological…"Fill in here.


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: MOBILE ACOUSTIC TRANSECTS MISS RARE BAT SPECIES: IMPLICATIONS OF SURVEY METHOD AND SPATIO-TEMPORAL SAMPLING FOR MONITORING BATS
Review round: 1
Reviewer: 2

Output:
Basic reporting: - adequate
- a few mentions of "population trends", e.g. lines 65, 114 could be removed as this was not the intention of the study
- some difficulty locating sampling stations within Fig. 1 within darkest shading; perhaps larger map of ENP at expense of smaller image of Florida.  More important to the manuscript to denote detail of study area than to know that ENP is in southern Florida.
Experimental design: - question of interest in the study is moderately well-described.  There are a few shortcomings however.  The given metric against which to measure estimates of species richness is derived from range maps.  It is not obvious that range maps are completely accurate; furthermore, because two species cannot be uniquely identified acoustically (Eastern and Seminole), the "true" number of species is different than described by the range maps.  Finally, the range maps, I presume, do not differentiate richness by habitat type.  Consequently, the validity cannot be assessed at the level of habitat type, but rather, at the level of ENP.  That creates a mismatch which makes it awkward for evaluating study design at the habitat level (level of spatial and temporal replication).
- it was not clear what randomisation scheme was used to place the spatial replicates.  Given concern about distance between sampling stations and roads; I would have thought measures might have been taken to place replicate sampling stations perhaps away from roads; perhaps challenging in pineland habitat.
- unclear why there was no attempt to have spatial replication in mangrove habitat--manuscript should make some mention of this
- as noted in marginalia of the submitted PDF, the study could not readily be replicated because of the manner in which sampling locations were chosen and the inclusion criteria of Supplemental S1.
Validity of the findings: - data appear fine; I could replicate most of the analyses presented via the R script provided
- my main concern when species richness is the metric, maximal species richness is small (9) and species identification is auditory is the potential for false positive identification (recording presence of a species not detected).  With so few species in the taxonomic target, only a single false positive might alter the inference of the study. Supplemental Appendix S1 discusses this, but the manuscript ought to highlight mitigation taken to prevent false positives and perhaps tabulate number of files falling into various eliminated (<5 calls; <75% calls with ID match, ...) categories.
- because of the absence of the mangrove habitat type from spatial replicates, this limits the scope of inference of this study, not just to ENP but to pineland and prairies of the park
- I made several comments regarding suitability of confidence interval overlap as an inferential tool as well as whether the R code using a multiplier of "2" on the SE produces the desired "84%CI"
    - there exists a literature suggesting that CI overlap is not an effective means of examining differences.  The "84%" level only resolves this matter when the SEs of the two contrasting estimates are equal, which is not the situation here. a closer reading of the supporting literature cited by Gotelli and Colwell (2011), namely Payton et al. (2003, J. of Insect Science 3:34) states "assuming equal standard errors (k = 1) yields γ = 0.166. In other words, if you wish to use confidence intervals to test equality of two parameters when the standard errors are approximately equal, you would want to use approximately 83% or 84% confidence intervals"
	- Wolfe, R. and Hanley, J. (2002). If we’re so different, why do we keep overlapping? When 1 plus 1 doesn’t make 2. Canadian Medical Association Journal, 166, 65–66.
	- Spanos, A. (2014). Recurring controversies about P values and confidence intervals revisited. Ecology 95: 645–651. doi:10.1890/13-1291.1
	- this is not a large portion of the manuscript, more details included in the marginalia of the PDF, but it should be noted by the authors
- Lines 265-270 cause me concern.  Is my logic correct?  If so, deserves further discussion in the manuscript:
	- line 266: note these two numbers of files are roughly equal (although there were double the number of spatially replicated nights)
	- line 268: this step is unclear to me; <1/3 of files containing bat sequences were identified to species; is that the correct interpretation?
	- line 269: whereas here the 16K to 26K ratio is roughly back into rough parity with the number of detector nights.
	- What this suggests is that the "conservative protocol" eliminated a larger proportion of single strategic files than spatially replicated files.
	-That seems as if it might be a threat to the comparison between the ability of two methods to detect bat species.  If so, should that be addressed in the manuscript?
Additional comments: It is challenging to experiment with design parameters of a monitoring programme, nevertheless, an important component of any monitoring work is to determine whether it is able to achieve its goals.  Hence, the goals of the programme need to be explicit and evaluation of the attainment of the goals is nedessary.


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: MOBILE ACOUSTIC TRANSECTS MISS RARE BAT SPECIES: IMPLICATIONS OF SURVEY METHOD AND SPATIO-TEMPORAL SAMPLING FOR MONITORING BATS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Suggested changes on basic reporting were addressed to my satisfaction or the authors provided logical reasoning for ignoring changes.

I like the addition of Table 1, because it seems like a great resource for methods. Is all information attributable to Loeb et al. 2015?

Check consistency around using ENP and 'Everglades National Park.' For example it is written out on line 455. Would suggest only writing out on first use.
Experimental design: Satisfied with changes addressed to make methods and questions more clear.
Validity of the findings: No Comment.
Additional comments: I am happy with the changes the authors have made to this manuscript. I think progress has been made to clarify some of the confusing aspects of project design, methods and the congruity with results.  I look forward to seeing this paper contribute to the well-quantified studies that use sensory-based conservation/monitoring.


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: HGTSIM: A SIMULATOR FOR HORIZONTAL GENE TRANSFER (HGT) IN MICROBIAL COMMUNITIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Song et al. describe the open-source tool HgtSIM, which was developed to simulate HGT events among microbial community members taking into account user-defined mutation levels. The authors give an illustrative example how HgtSIM works, starting from the insertion of a set of selected genes into a collection of genomes, followed by modification of these genes simulating mutation and adaption. Song et al. then employ the tool GemSIM to simulate metagenomic reads from their simulated dataset, perform assemblies using the assembler IDBA-UD and finally assess the recovery rate of gene transfer events.

HgtSI is not the first tool to simulate HGT in microbial genomes (see HGT options of EvolSimulator by Beiko&Charlebois, 2007, https://doi.org/10.1093/bioinformatics/btm024), but seems to be more straight-forward to use. It could be downloaded from Github together with a comprehensive documentation. HgtSIM is written in python 3 and the code is well readable.

After testing HgtSIM I felt that it could be slightly more flexible in terms of query genome input format and user-specified options seemed to be rather limited.

HgtSIM was successfully used on the provided test dataset. However, gene insertion was only possible on closed genomes (or single contigs), as soon as additional contigs were added (in the same file) the HGT simulation failed. This was somewhat surprising as the tool supposedly was designed to simulate HGTs in metagenomes - many published metagenome assembled genomes are not closed. Thus, it would be great if HgtSIM could also be used on genomes with more than 1 contig.

Flanking sequences are often introduced together with transferred genes. In HgtSIM the user has the option to provide upstream and downstream flanking sequences, which is great. However, it seems that these flanking sequences are then equally used for all inserted genes. I would suggest to implement a feature to automatically generate flanking sequences with varying sequence length and composition (within a given range). This would help to make the simulation result more similar to real world data.

An important feature of HgtSIM is that acquired genes do undergo evolutionary drift and adaptation to the new cellular context. However, in its current form this process felt slightly limited due to the fixed identity value provided by the user. Ideally, HgtSIM would include an optional "mixed mode" -  which would use a range of different identity values randomly to alter the query genes.

Line 119: The here described results imply that recent HGT is most difficult to detect. I was wondering to what extend this observation was affected by the assembler (IDBA-UD) and to what extend a better performing assembler would change the result. An alternative assembler could be SPAdes (or metaSPAdes), it is popular and widely-used and presumably outperforms IDBA-UD considering contig and scaffold lengths (see Vollmers et al., 2017 https://doi.org/10.1371/journal.pone.0169662).

Lastly, the authors evaluated the impacts of read length and insert size on the recovery rate but they did not elaborate on the potential influence of gene length. I would assume that successful detection negatively correlates with gene length. If there is such correlation, then a length distribution plot of inserted genes together with their recovery rate would be a helpful reference for the user.


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: HGTSIM: A SIMULATOR FOR HORIZONTAL GENE TRANSFER (HGT) IN MICROBIAL COMMUNITIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript presents a software for the simulation of genomes, which contain simulated horizontally transferred genes. The authors provide an example application to an artificial microbial community. I agree this is an interesting aspect in metagenomics, which besides strain diversity and limited coverage introduces an additional challenge to metagenome assemblies.

The manuscript is a brief “Applications Note” rather a full research article, as required for PeerJ. It lacks a central research question and suitable, comprehensive experiments to address it. For example, the manuscript does not introduce the challenges of metagenomic assemblies in detail, and does not address the problem of HGT event prediction, or donor/acceptor inference, for which HGT simulations might also be useful. The results on an artificial set of genomes, using one assembler, demonstrate the general utility of HgtSIM and give some general hints for metagenomics assembly. For those who work on shutgun metagenomes, the results are quite expected. However, they don’t resemble any realistic microbial community and thus have no meaning for metagenomic assembly practice.
Experimental design: I have downloaded and tested the software. It is written in Python3 and has only few dependencies, which are easy to fulfil. I did not completely understand the implementation strategy, as parts of the script seem unnecessary (e.g., the genetic code is hardcoded although the script imports Biopython, which already provides more sophisticated genetic code classes and methods). Anyway, I could successfully run a simple test, based on the provided example files.
Validity of the findings: Does not apply.
Additional comments: To become a PeerJ Research Article, the manuscript would require fundamental expansion. A suitable alternative might be a submission to OUP Bioinformatics, as “Applications Note”.


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: HGTSIM: A SIMULATOR FOR HORIZONTAL GENE TRANSFER (HGT) IN MICROBIAL COMMUNITIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors present a simulator for horizontal gene transfer in microbial communities.
The article is written in unambiguous and professional English and only a few typos need to be corrected.
I would suggest to add more background into the introduction and also include a couple of references about more recent tools for detecting HGT, e.g.
https://doi.org/10.1371/journal.pcbi.1004408
https://doi.org/10.1371/journal.pcbi.1004095
https://doi.org/10.1093/bioinformatics/btw423

Figure 1B is hard to read. It is impossible to distinguish some of the labels. Please refine.

In Table 2 it would be useful to add the size of the genomes, as this has an effect on the simulated coverage (see experimental design), which again might have an effect on the results presented in chapters 3.2 and 3.3.

Table 3 is missing a caption which explains the numbers in the table. I guess these are recovery rates (in %) of the HGT genes. Also, the 0% and 5% need to be explained in the caption or table.

The authors do not share the raw data, but at least provide accessions for the genomes used for the simulation. Please provide all details about which parameters were used with the different tools (read simulator and IDBA_UD), so that results are reproducible.
Experimental design: In general, it remains unclear what the goal of the simulation should be. What should be benchmarked with the simulated data? Is it mainly about assembly performance? If so, the authors have to make sure that they are not measuring a different effect when evaluating the assembly (see below). Or is the goal to benchmark tools for detection of HGT? If so, it would be nice to see how one if these tools performs. I understand that a thorough benchmarking of HGT detection tools might not be within the scope of this manuscript, but if there are benchmarks published already, the “best tool” could be applied on the simulated data set.

Lines 54-62: Taking a number of gene sequences as input and inserting them into a number of target genomes seems to be a relative straightforward task. Having an automated way to do so is very useful for simulation and benchmarking. Therefore, the described pipeline is a step into the right direction, if such studies are performed often and in high-throughput. It remains unclear, though, how realistic the chosen model of mutating the horizontally transferred genes is. The authors barely explain the underlying principles of their model. One can immediately argue, that a distinction between one-base (silent or non-silent), two-base and three base mutations is too simplistic. There are examples where two-base or even three-base mutations are silent mutations (e.g. UCU and AGC both code for Serine). There are more sophisticated models available, could they be integrated in the simulation pipeline (e.g. https://doi.org/10.1080/10635150701546231)?

Line 71: Even for the simple model, how would one choose C1:C2:C3:C4? What is the default for the pipeline and why was it chosen this way?

Line 80: Does the recipient genome have to be a finished genome (i.e. no gaps allowed)? Many genomes are permanent draft genomes which cannot be used for a large scale simulation if the pipeline only works for finished genomes.

Line 84: When cutting the genome for inserting the HT genes, this will disrupt genes in the recipient genome? Is this realistic assumption or are insertions into CDSes much less likely? If so, this should be included in the model

Line 100: “The category ratios of “0:0:0:1” and “1:0:1:1” resulted in level of amino acid sequence changes that were similar to the user-defined level of nucleotide mutations” This sentence is unclear. What exactly is the user-defined level of nucleotide mutations? And how does this relate to these ratios?

In general, details about running the pipeline and assembly (parameter setting) are missing, which  makes reproducibility difficult.
Validity of the findings: The authors present a small benchmark data set consisting of 20 genomes, where 100 genes from Alphaproteobacteria get randomly transferred to 10 genomes of Betaproteobacteria. I am not convinced that the effects of missing genes in the subsequent assemblies are actually always the degree of mutations. If we assume that the 20 genomes have a length of 4Mbp on average, the sequencing depth for the simulated data set is only about 12.5x (10 mio x 100 bp / 20 * 4 Mbp = 12.5). This is already a pretty low coverage for assemblies based on long kmers and could explain that for kmer ranges >60 most of the genes are lost. What are the total assembly sizes for these assemblies? I would guess they are rather small. Additionally, a couple of genomes are >5Mbp long, which reduces the coverage for these genomes even further.
Line 120: therefore, the mink parameter has a general effect on the assembly of low coverage genomes and not on HGT detection. Only using show kmers (20) in the kmer range pick up the low coverage genomes and therefore allow HGT detection

Line 131: there are no results shown for longer reads of different input sizes

I would suggest to prepare two different data sets, one without HGT and one including HGT. Comparing these two datasets would elucidate if the missing genes are actually an effect of HGT or just an effect of too low sequence coverage.
Additional comments: The manuscript seems to be very preliminary and needs a more carefully designed experimental setup. It is not clear, if the findings are an effect of HGT or missing sequencing depth. Also, it would be nice to see an HGT detection tool applied on the data set.


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: HGTSIM: A SIMULATOR FOR HORIZONTAL GENE TRANSFER (HGT) IN MICROBIAL COMMUNITIES
Review round: 2
Reviewer: 1

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


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

Input:
Title: HGTSIM: A SIMULATOR FOR HORIZONTAL GENE TRANSFER (HGT) IN MICROBIAL COMMUNITIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revised version has been substantially improved. The authors have carefully addressed the reviewer's comments. As there is not any substantial issue remaining from my side, I thank the authors and recommend acceptance of the manuscript.


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

Input:
Title: HGTSIM: A SIMULATOR FOR HORIZONTAL GENE TRANSFER (HGT) IN MICROBIAL COMMUNITIES
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors switched to metagenome assemblers for the simulation study.
This improved the quality of the simulation study tremendously. However, the findings
are not reproducible, if details of the simulation are not described. This is the case for
many details in the study and need to be addressed (see details below).

Please correct the following typos:
line 29: ...tool offers...
line 31: ...events...
line 49: ...error...
line 124: ...paired-end...
line 144: ...optimal...
line 163: ...read length...

Figure 4A, y-axis: Assemblies
Experimental design: lines 122-124: which mutation model were used for the simulation?
Were flanking regions added to the transfered genes? If so, what sequences, how long?

lines 124-126: does the script introduce sequencing errors?
Please add the resulting sequencing coverage to the main text.

The methods are still not described in detail. The presented results are not reproducible.
Please provide the all input files for the scripts such that the study can be reproduced.
The GitHub repository has all the necessary scripts, but e.g. the abundance.txt file or
the input_genomes_m0 files are missing.

How exactly were recovered genes identified? Alignment against the reference?
Which tool was used? What were the alignment parameters? And what the criteria for
calling a HGT gene recovered?

I found all details in the python scripts on GitHub, but they should be included in the manuscript (Methods section). Also, please include the version numbers of the assemblers.
Validity of the findings: line 135: what happens to the HGT genes? Are they not assembled at all? Do you recover only one copy of the gene?

lines 146-152: First, it is not surprising that the recovery rate is not increasing linearly with sequencing depth. It is well known, that the assembly in terms of recovered genome sequence behaves in such a way (Lander and Waterman 1988).

Very surprising is the fact, that higher sequencing coverage leads to a lower recovery rates. I was convinced that there is a bug in the simulation and analysis pipeline, but the results seem to be correct.

I evaluated the assemblies using metaQUAST and from these results,
the higher coverage data sets seem to produce better assemblies (less contigs, higher genome recovery, higher N50, etc). I would suggest to add these basic statistics to the manuscript, just to show that there are no flaws in the simulation process.

Taking a closer look why the HGT genes get lost in the high coverage data sets, I was able to re-run the analysis using the Assembler_recovered_transfers_iden100.py script  from GitHub and the data provided. It was very surprising for me to see that the flanking regions get lost in the assemblies with higher coverage.

Here is an excerpt from the BLASTN results generated by the script for
the metaspade_m0_k21-125_l100i250_3_million.fasta assembly

AMAC_01196	NODE_75_length_51023_cov_1.46669	100.00	1203
AMAC_02518	NODE_2577_length_11107_cov_1.63078	100.00	1269
AMAC_02914	NODE_5740_length_2456_cov_2.52716	100.00	1908
AMAC_03303	NODE_3040_length_9082_cov_1.74892	100.00	1452
AMAU_01212	NODE_938_length_22333_cov_1.55155	100.00	1530
AMAU_01255	NODE_1152_length_20072_cov_1.53219	100.00	1497
AMAU_02414	NODE_3750_length_6844_cov_1.81192	100.00	1290
AMAU_02488	NODE_4885_length_4137_cov_2.12155	100.00	1518
AMAU_04187	NODE_921_length_22539_cov_1.54172	100.00	1176
AMS_00102	NODE_26_length_64149_cov_1.46827	100.00	1395
AMS_01465	NODE_491_length_29719_cov_1.50854	100.00	1413
AMS_01716	NODE_1033_length_21171_cov_1.56804	100.00	1176
AMS_01785	NODE_3328_length_8142_cov_1.76752	100.00	1194
AMS_02653	NODE_869_length_23152_cov_1.47987	100.00	1062
AMS_02811	NODE_139_length_44053_cov_1.48303	100.00	1410

For the higher coverage data set (assembly metaspade_m0_k21-125_l100i250_20_million.fasta) the BLASTN results look like this:

AMAC_00685	NODE_684_length_1608_cov_19.649	100.00	1608
AMAC_01196	NODE_766_length_1203_cov_20.8583	100.00	1203
AMAC_02518	NODE_752_length_1269_cov_17.6364	100.00	1269
AMAC_02914	NODE_660_length_1908_cov_18.9573	100.00	1908
AMAC_03303	NODE_711_length_1452_cov_17.7535	100.00	1452
AMAU_01212	NODE_697_length_1530_cov_19.0856	100.00	1530
AMAU_01255	NODE_702_length_1497_cov_18.6773	100.00	1497
AMAU_02414	NODE_744_length_1291_cov_18.9364	100.00	1290
AMAU_02488	NODE_700_length_1521_cov_19.2229	100.00	1518
AMAU_04187	NODE_792_length_1102_cov_28.4966	100.00	1101
AMS_00102	NODE_726_length_1395_cov_17.3326	100.00	1395
AMS_01465	NODE_718_length_1414_cov_20.2716	100.00	1413
AMS_01716	NODE_777_length_1178_cov_19.8879	100.00	1176
AMS_01785	NODE_771_length_1194_cov_19.8293	100.00	1194
AMS_02653	NODE_800_length_1062_cov_18.9562	100.00	1062
AMS_02811	NODE_720_length_1410_cov_17.9443	100.00	1410

Basically, all genes are assembled, but only on contigs which have exactly
the size of the HGT gene.
Additional comments: The presented results are very surprising and the authors should elaborate on
the findings and try to interpret these in more detail. The manuscript would improve significantly from digging a little deeper into the results and trying to explain what has been observed.

A way to do this might be to load the metaSPAdes results into a tools like e.g. Bandage (https://rrwick.github.io/Bandage/) and visualize the HGT genes in the assembly graph.

I tried to re-run metaSPAdes, but I failed as some files were missing in the GitHub repository,
which made the presented results not reproducible (see above).