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: INTEIN-MEDIATED BACKBONE CYCLIZATION OF ENTOLIMOD CONFERS ENHANCED RADIOPROTECTIVE ACTIVITY IN MOUSE MODELS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Ye et al. report that backbone cyclization of a truncated flaggelin protein, termed Entolimod, using a split intein from Nostoc punctiforme and showed that the circular form of entolimod has increased in vivo stability and activity than the linear form. This could be hugely advantageous for the clinical application of entolimod, which has anti-cancer activity as well as radioprotective activity.  The article is written logically and understandable, but could still be polished by indicating the important information more clearly. There are several technical issues in the manuscript, which needs to be addressed (see below).
Experimental design: The authors used the split intein to create circularized entolimod to test in vivo activity for the comparison with linear form and produce the circular form by head-to-tail backbone cyclization using protein trans-splicing as well as non-circularized liner entolimod for the comparison. The both forms were used for reporter assays and mice survival experiments to demonstrate the in vivo stability. The authors claim that C-entolimod was purified from the inclusion bodies but no solubilizing conditions is described. It is very unclear how the proteins are purified. This is the crucial point of the experiments.  Moreover, the sequence as entolimod provided by the authors in Fig.1 contains additional StepII-tag and His-tag.
To my knowledge, no protein with His-tag has been currently approved by FDA. Does entolimod currently under development as drug contain the identical sequence?
Validity of the findings: Typically, backbone circularized proteins migrate faster than the liner form in SDS-PAGE, when N-and C-termini are in the proximity, which is not clear from the manuscript. The migration in SDS-PAGE has been used for confirming backbone cyclization because it could migrate slower (increased apparent molecular mass) after proteolytic digestion, which is unusual. However,  Fig.3E shows the identical migration in SDS-PAGE for both forms. This is very unusual for the backbone cyclization. There are also two bands in Fig. 3E. What is the other band? The authors could use different percentages of acrylamide SDS-gel for better separation.
In Fig. 4, the expected molecular weight can be indicated for the clarification. Is this MALDI-TOF spectrum?  In the experimental section, it is written as QE-Mass? Is this spectra convoluted or the original? Why the authors modified cycteins with IAA.  18 Dalton difference is expected for the difference between linear and circular forms. Therefore, the mass spectrum should be obtained by an appropriate mass spectrometer for ionizing and detecting.
Additional comments: 1.	The authors used “L-entolimod” and “C-entolimod” for linear form and Circular form respectively. This nomenclature could be confusion as “L-form” is often used for indicating the chirality.  I recommend to use “l-“ and “c-“ in italic, or “lin-“ and “circ-“.
2.	Line 141, the authors wrote“L-entolimod was purified described previously (inclusion bodies did not require refolding)(Burdelya et al., 2008)”.  I think it is not inclusion body if the protein does not require any refolding but soluble.
3.	Line 93, the authors wrote “C-entolimod is its increased stability”. This is not true.
The authors provided only in vivo stability without anlazying thermodynamical stability. Why didn’t the authors analyze and compare the thermostability? This is a very easy and quick experiment to perform.
4.	The sequence in Fig. 1 should be presented in a clear manner. There are His-tag and StrepII-tag sequences, which, I believe, is not the part of entolimod.  The authors could present the sequence of entolimod clearer,  indicating the part of the flagellin removed.
5.	Section 2.1. in method and material could be simplified so that one could follow them easily.
6.	Circular GFP and increased stability was originally reported in 2001, which should be cited (J. Biol. Chem. 276, 16548-54)
7.	The authors cite several addgene plasmid numbers but did not include the references for these plasmids, which are supposed to be cited.
8.	Line 213, Townend and Tavassoli, 2016 is incorrect reference for this.
9.	Did the authors try to express at a lower temperature to increase soluble proteins? It is mysterious how the protein could be purified without any denaturant from inclusion bodies.
10.	In my opinion, SCICLOPPS is a bad jargon created.


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: INTEIN-MEDIATED BACKBONE CYCLIZATION OF ENTOLIMOD CONFERS ENHANCED RADIOPROTECTIVE ACTIVITY IN MOUSE MODELS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is written in clear professional language and technically correct. The introduction and background are sufficient to identify a problem, emphasize the significance of the work and impact on the field. The manuscript is supported by a sufficient number of references possessing both quality and relevance. However, in the introduction and also the abstract, the authors write about the flu-like syndrome induced by entolimod (lines 20 and 48), but the references in the introduction do not describe these observations which most likely were drawn from Phase I safety clinical trials. If entolimod was indeed used in the clinic for radioprotective and anticancer clinical application when the “flu-like syndrome” was observed, appropriate references must be provided. In both the abstract (line 24) and the introduction (line 86), the authors state that different strategies were used to purify linear and cyclized entolimod. The methods of purification should be provided and described or not mentioned at all if they are unimportant.
Overall, the structure of the article is very good with complete presentation of material that is sufficiently described and supported by raw data. The authors sought to create a circular version of entolimod hoping to improve its biological properties.  The results of the study support the hypothesis.
Experimental design: The experimental work was conducted with rigor and to a high technical standard. There is concern that C-entolimod could contain LPS from the E.coli purification. As the authors show that C-entolimod is similar or better then linear entolimod with regard to NF-кB activation and also radioprotection, it is important to confirm that it is mediated by TLR5 and not TLR4. One possible approach to do this is to add data showing that C-entolimod does not activate NF-кB in the cells expressing TLR4, but not TLR5.
Validity of the findings: The authors clearly present their conclusions and validate them with their supporting results. This is an interesting approach to increase the activity of entolimod while reducing its undesirable properties. Although the degree of improvement in tissue protection and reduction in toxicity and immunogenicity must now be determined for this new variant of entolimod, the current manuscript describes the steps to obtain a cyclic version of entolimod and shows the increased NF-кB activating capacity as compared to the parental linear version. The existence of a cyclic version of entolimod with improved tissue protecting capacity would enable further investigation of the mechanisms responsible for this activity and possible development of the drug for prevention of radiation toxicity in hematopoietic and gastrointestinal tissues.
Additional comments: Minor issues:
1. In figure 6, statistical differences are described in the figure legend, but not shown in the plots making up the figure.
2. lines 274 and 277: it would be more appropriate to use term “cause or prevent lethality” rather than “killed”
3.	line 133: remove “an”


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: UNUSUALLY LOW GENETIC DIVERGENCE AT COI BARCODE LOCUS BETWEEN TWO SPECIES OF INTERTIDAL THALASSAPHORURA (COLLEMBOLA: ONYCHIURIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: a.	There is some room for improvement concerning style, prose, grammar, and structure of the article.
i.	In general, the methods sections seems out of order. For example, the discussion of preparing specimen vouchers that were recovered post-DNA extraction before the sections on molecular analysis is confusing. Consider presenting the methods in a more logical/sequential order to improve flow of the section.
ii.	The methods also includes sub headers which are redundant, and provide slightly contrasting information. The “Molecular analysis sub section suggests that molecular analysis follows Porco et al. (which only describes recovery of vouchered specimens following DNA extraction) while the DNA Extraction sub section describes in full (and correct) detail the actual methods of molecular analysis. I suggest collapsing these two sections together and revising for clarity and cohesiveness.
iii.	The Data analysis sub section of the methods also requires some editing, as it lacks cohesion and is difficult for the reader to follow.
iv.	The description of DNA barcoding in the introduction seems a bit pedestrian. More discussion on the gene region that is commonly used, and the strengths and weaknesses of the method would be helpful to the reader.
v.	Overall, some sections are very well written while others use colloquial language and are unpolished. Below are several suggestions for correcting grammar or improving style, but is by no means an exhaustive list.

Line1: suggested edit to title “Unusually low” rather than “Unusual low”
Line 21: suggested edit: “combined morphology and DNA barcode data”
Lines 25-29: the use of the word “weaker” suggests, strength of signal rather than size of divergence. Suggested edit: “much smaller than previously reported in Collembola”. These two sentences could be further strengthened if edited to be more concise.
Line 44: suggested edit: “large numbers” or “in very high abundance”.
Line 78: suggested edit: “firmly setting” rather than “setting fimly”
Line84: “DNA barcoding” rather than “DNA barcode”
Line 86: “is at pain” this seems rather odd terminology. I suggest rephrasing
Line 113: “strictly restricted” is redundant
Line 140: “Dewaard” should be spelled “deWaard”
Line 400: “less than 2% of intra-population divergence” –remove the “of” from the sentence.
Line 422: I think you mean “strip” rather than “stripe”
Line 427: use “genetic” rather than “genetics”
Line 428-430: I don’t really understand what you mean here
Experimental design: a.	Overall I think the experimental design is valid and well expressed. There are a few questions raised which could be clarified.
i.	Line 127: how many specimens were sent for DNA barcode analysis in total? Did you send all specimens for DNA barcode analysis, or did you choose some for barcode and some for morphology separately?
ii.	I’m not sure if this is a community standard, but there doesn’t appear to be any description of the re-examination of type material in the methods section. Is that normal for manuscripts which revise species descriptions?
Validity of the findings: a.	I would like the authors to consider biological reasons why the two species in question have smaller interspecific divergences than were expected. It is important to consider the age of the species (perhaps these are two young sister-species?), the rate of accumulation of mutation (could these taxa be evolving more slowly at COI for some reason?). I think it’s important to place the sequence divergence results more clearly into biological context.
b.	Use of the work “weak” in reference to your barcode results is disheartening. The results clearly discriminate both species, with very low intraspecific divergence detected. Using the word weak suggests to the reader that the relationship is weak, but it is rather clear in your results. I would highlight this, and replace the work weak with something else. Perhaps the word small. You should place more emphasis on the fact that a barcode gap was clearly detected.
c.	Figure 9 – this figure is not really providing a whole lot to the reader. It might be more useful to provide the typical barcode gap graph which depicts the maximum intraspecific divergence on the x axis and the minimum intraspecific divergence on the y axis. This will allow you to highlight the taxa which have, smaller/larger barcode gaps in a more intuitive way.
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: UNUSUALLY LOW GENETIC DIVERGENCE AT COI BARCODE LOCUS BETWEEN TWO SPECIES OF INTERTIDAL THALASSAPHORURA (COLLEMBOLA: ONYCHIURIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors did well in the language and structure of the text. However, I found that the background might not be sufficiently introduced. I suggest adding a brief introduction to the taxonomic difficulty of the objective group.
Experimental design: no comment
Validity of the findings: The main aim of the study is to clarify that T. thalassophila and T. debilis are independent species. The results showed that neither morphological nor molecular divergence is pronounced between the two forms, therefore only the combination of them can perhaps justify the two species. However, in the text the two parts are treated separately, which weakened the validity of evidences. I suggest an expanding of the beginning of the 4th paragraph in the discussion section and raise it up to the beginning of discussion, because it is basic and most important that the morphological and molecular evidences are congruent.
Additional comments: Some comments are made in the text. In general, some parts of the text need to be rearranged to achieve a logical coherence.


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: UNUSUALLY LOW GENETIC DIVERGENCE AT COI BARCODE LOCUS BETWEEN TWO SPECIES OF INTERTIDAL THALASSAPHORURA (COLLEMBOLA: ONYCHIURIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: a. There is some room for improvement concerning prose and grammar
i. Overall, some sections are very well written while others use colloquial language and are unpolished. Below are several suggestions for correcting grammar and improving clarity

Line 47: “in intertidal environment” suggest “in the intertidal environment”
Line 97: “are regarded powerful tools” suggest “are regarded as powerful tools”
Line 103: “or Lepidoptera” suggest “and Lepidoptera”
Line 104-105: “High percentage divergence in DNA sequences provides…” Suggest “Large divergences (>5%) in DNA barcode sequences provides..”
Line 107 – 110 “Insects usually have lower divergence than non-winged arthropods, and percentage divergence is 11.5% in Hymenoptera and 9.3% in Diptera (Hebert, Ratnasingham & deWaard, 2003), 13.9% in Ephemeroptera of North America (Webb et al., 2012) and 7–8% in holarctic Lepidoptera (Hebert 110 & Landry, 2010, Hausmann et al., 2011).”
Suggest “Insects usually have lower interspecific divergences than non-winged arthropods. For example, average DNA barcode distances between congeneric species range from 7–8% in holarctic Lepidoptera (Hebert 110 & Landry, 2010, Hausmann et al., 2011) and 9.3% in Diptera (Hebert, Ratnasingham & deWaard, 2003), to 11.5% in Hymenoptera and 13.9% in North American Ephemeroptera (Webb et al., 2012).
Line 115 – 118: “Furthermore, recent molecular studies amongst Collembolan lineages have revealed divergences almost as deep as amongst congeneric morphological species (Cicconardi, Fanciulli & Emerson, 2013; Emerson et al., 2011; Frati et al., 2000; Katz, Giordano & Soto-Adames, 2015; Porco et al., 118 2012b; Soto-Adames, 2002).” It is not clear here what you mean by Collembolan lineages – do you mean divergences within species?
Line 392:  “clear distinct…” suggest “clearly distinct…”
Line 393: “not synonymys roposed by Fjellberg” suggest “not synonymous as proposed by…”
Line 400: “The remaining specimens were damaged during the extraction” suggest adding “… damaged during DNA extraction and therefore morphologically uninformative”
Line 401: “A small barcoding gap was observed at K2P distances of around 0.02 (Fig. 8).” I understand the visual you are trying to express here, but the bars should be different colors to show how the intra-specific divergences are distributed in comparison with the inter-specific divergences, as illustrated in Figure 2 of Meyer and Paulay 2005 - http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.0030422). In fact – this particular type of figure is far less in formative than if you showed a scatter plot of the maximum intraspecific divergence on the x axis and minimum distance to the nearest congeneric species on the y-axis of each species, similar to figure 2 in Blagoev et al 2016 (https://onlinelibrary.wiley.com/doi/full/10.1111/1755-0998.12444).
Line 444: “have divergence level” suggest “have divergences similar…”
Line 458: “which diverged from conspecific MOTUs from 11.33 to 21.47%” suggest “..MOTUs by 11.33 to 21.47%”
Line 463: “We observed similar high levels” suggest “… similarly high levels”
Line 467: “but did not diverge genetically for COI sequences” suggest “but did not show genetic divergence at COI”
Experimental design: Overall I think the experimental design is valid and well expressed. No comments here.
Validity of the findings: The research is integrated into the literature well and attempts to explore biological reasons for the unexpectedly low divergence between these two inertial species.
Additional comments: Good job addressing the previous concerns, This paper is much closer to a final form that could be published.


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: UNUSUALLY LOW GENETIC DIVERGENCE AT COI BARCODE LOCUS BETWEEN TWO SPECIES OF INTERTIDAL THALASSAPHORURA (COLLEMBOLA: ONYCHIURIDAE)
Review round: 2
Reviewer: 2

Output:
Basic reporting: Additional background has been added in the introduction, no more comment
Experimental design: no comment
Validity of the findings: Changes have been made, no more comment
Additional comments: The authors made some changes in the arrangement and expression of the text except some part of the description. They explained the reason and I find there is no problem that they insist on their own style. No more 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: AVAILABILITY OF NANOPORE SEQUENCES IN THE GENOME TAXONOMY FOR VIBRIONACEAE SYSTEMATICS: RUMOIENSIS CLADE SPECIES AS A TEST CASE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written and structured. The background provided is correct as well as the references employed. Tablas and Figures are adequate.
Experimental design: The aims and objectives are clear, as is the rationale of the experimental design.
The methods are in general described with enough detail. Some minor points or doubts are: 1) why the ZoBell 2216E broth was prepared with natural seawater? That means that the salt concentration was doubled?. 2) More detail is needed within the heading "Phylogenetic reconstruction and tree comparison"; it is a MLSA? Which aim?. 3) It is no needed to define the acronyms more than once.
The investigation carried out was carried out to a high standard, and the comparison of the differente NGS methods is rigurous and complete.
Validity of the findings: The data presented are robust and the conclusion well based on the findings. The validity of nanopore-only assemblies for taxonomic purposes may have an impact in this field within Microbiology.
Additional comments: The manusctipt constitutes an interesting piece of work, than may have an impact in microbial  taxonomy.


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

Input:
Title: AVAILABILITY OF NANOPORE SEQUENCES IN THE GENOME TAXONOMY FOR VIBRIONACEAE SYSTEMATICS: RUMOIENSIS CLADE SPECIES AS A TEST CASE
Review round: 1
Reviewer: 2

Output:
Basic reporting: There is no reason to believe nanopore provides error free sequences. And some previous studies have pointed out error rates>30%! Although the authors mentioned in their paper that nanopore is highly accurate and make very very few reading errors, I am not convinced. The authors list in Table 4 mismatches due to nanopore errors, the reader really needs more details about the %error, types of most frequent errors (A, T, G, C?), comparison with previsou Sanger sequences (eg MLSA), which the authors have abundant databases(eg  Sawabe et al. 2007; JBac).
Experimental design: include Sanger sequences in the comparison.
Validity of the findings: requires evaluation.
Additional comments: I have major doubts about the validity and reliability of nanopore for taxonomic and ecogenomic applications. The authors failed to properly prove the relevance of nanopore in this version.


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: AVAILABILITY OF NANOPORE SEQUENCES IN THE GENOME TAXONOMY FOR VIBRIONACEAE SYSTEMATICS: RUMOIENSIS CLADE SPECIES AS A TEST CASE
Review round: 1
Reviewer: 3

Output:
Basic reporting: This a well-written manuscript, The rational of this work is convincingly spelled out, background is  sufficiently defined, relevant illustrative material to support the authors' statements  is provided.
Experimental design: The evaluation of  advanced genome sequencing technique is timely. In the context of this manuscript I would suggest to clearly define the aims of this work in the last paragraph of the Introduction.
Validity of the findings: Indeed, the novelty  aspect should be highlighted. In my view the manuscript will benefit from comparative analysis of advantages and disadvantages of the three current approaches for whole genome sequencing. This could be useful and could be included in the Abstract.
It appeared that in silico DDH values are not matching. Since this is one of most important characteristics for species discrimination, the authors should highlight this outcome, comment on the possible reasons and perhaps future work to overcome this drawback.
Additional comments: Please revise Table 2 by minor re-arrangement, e.g., removing the two rows with same words. Consider to improving Table 4 as well.

Please provide the source of the type strains used for the sequencing.Since some data retrieved from previous sequencing, the authors should double check if the same type strains obtained from the same sources have been used. These maybe the reason for some discrepancies in sequencing results.


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

Input:
Title: AVAILABILITY OF NANOPORE SEQUENCES IN THE GENOME TAXONOMY FOR VIBRIONACEAE SYSTEMATICS: RUMOIENSIS CLADE SPECIES AS A TEST CASE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have responded to my main concern and so I have no more questions. Nanopore can be indeed an excellent tool for vibrio taxonomy as demonstrated by the authors in this test case of rumoensis.
Experimental design: ok.
Validity of the findings: ok
Additional comments: ok


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: COMPARISON OF CRYPTOBENTHIC REEF FISH COMMUNITIES AMONG MICROHABITATS IN THE RED SEA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript presents a study on the distribution of cryptobenthic fishes among three microhabitats on a single reef in the central eastern Red Sea. While these fishes are poorly studied, particularly in the Red Sea, and the manuscript is quite well presented, I have some serious reservations as to the rigour and technical standard of the study. These concerns are presented in the sections below.

1. The language throughout could be improved a little. It has a tendency to be too wordy and formal. Phrases like "have been found to be" and "are shown to be" should be avoided (e.g. lines 46, 54-55, 81-82 etc.). Instead, just state the finding of the paper and cite it (e.g. instead of "bananas were found to be yellow [Bloggs 1997]", write "bananas are/may be yellow [Bloggs 1997]"). A thorough proof read throughout would also help to improve a lot of little typos.

2. Paragraphs also tend to be very long (e.g. paragraph 1 of the introduction is 21 lines long and paragraph 2 is 29 lines long). Paragraphs need to present 1 idea, and as a basic rule of thumb, you should aim for most being around 10 lines long.

3. In the abstract, stating the number of fishes collected is of no use as you do not state the sampling regime. Presenting the data per unit area or similar would be more intuitive.

4. The authors mention the potential impacts of coral reef degradation in a few places in the introduction (lines 61-63, 108-109) but do not mention this idea in the discussion at all. If you are introducing this concept (particularly with regards to coral>rubble>sand being a pathway for degradation) you should consider the potential implications for reef communities in the discussion (or alternatively remove it from the introduction).

5. As a related comment the writing in lines 61-63 is very unclear. This sentence needs to be restructured. Maybe make a complete paragraph about degradation that compiles lines 61-63 and 108-109 so this concept isn't 'tacked-on' to other paragraphs with other themes.

6. In line 77-78 you make it sound like the 2 studies you cite do not account for cryptobenthic reef fishes, when they are examples of the few studies that do. Be careful with your wording and tone (you don't want to annoy other authors inadvertently).

7. Line 89-90 - Depczynski and Bellwood 2004 does not measure home ranges, you should look at Luckhurst and Luckhurst 1978a cited in D&B and draw your own conclusions on whether their home range estimates are reliable.

8. Paragraph 1 of the discussion needs to be restructured to state the findings of the study and put them in context. at the moment the 4 sentences do not link with each other at all.

9. Lines 285-onward. Be very careful with your discussion of sediment vs. detritus (these are not the same thing). Increased sediment decreases the availability of detritus. The current explanation seems to indicate a limited understanding of this concept and there are some mis-citations (Goatley et al. 2016 suggests sediments are a bad thing for cryptobenthic fishes). Perhaps think more about surface area and refuge availability in different microhabitats.

10. Line 297-298,. Thankfully the coral microhabitats (that you selected because they were dominated by corals) were dominated by corals! I don't think it's really necessary to make this point.

11. Line 316-320. What about sand dwelling gobies such as shrimp gobies and even things like Istigobius spp.? Would it be worth mentioning how far the sand was from other microhabitats/shelter?

12. Lines 327-329. If you're discussing the impacts of sediment size it would be worth including references surely? As a gratuitous cite-me request you could look at work by S. Gordon and/or S. Tebbett that consider the impacts of sediment size on feeding in fishes.

13. Line 341, do you mean a biodiversity hotspot?
Experimental design: The research is clearly in the aims and scope of PeerJ, and the aims/question are well stated in the introduction.

14. The description and rigour of the methods, however is currently very poor. The data are collected from 5 replicate samples from each of 3 microhabitats from 1 site on 1 reef. This experimental design means that the patterns observed cannot even be stated to be representative of even the leeward side of the reef (which appears to be ~10km long), let alone the entire central Red Sea. To accurately assess whether the patterns are representative of an area larger than the site studied, you really need to conduct sampling at multiple sites and include site as an extra, random factor in your analyses. If you do not find differences among sites, then you can begin to discuss the likelihood of the findings being representative of a larger area. Unfortunately, without this level of replication I cannot see how this manuscript is suitable for publication, as your data do not support your aims and conclusions.

15. I am also worried about your methods regarding sampling tabulate acroporids as your 'coral' microhabitats. The issue with tabulate corals (as I present in Goatley and Bellwood 2011) is that there is stuff underneath them (clearly visible in Fig. 1B), and your 'coral' microhabitats are therefore a composite microhabitat of the coral and whatever is beneath them. From your data I would posit that the predominant benthic cover beneath your coral canopies was rubble, which would explain why your rubble communities appear as a subset of your coral communities in Fig. 3A (i.e. why the rubble polygon is enveloped in the coral polygon). Unfortunately as you have overlap in your microhabitats this makes analyses/interpretation very difficult and if the benthos beneath the canopies differs among your samples it would be even more challenging to assess . Luckily you took photographs of each quadrat so you may be able toi account for this post-hoc. For the future I would recommend looking at 'reef' microhabitats (i.e. coral growing on matrix) so your microhabitats actually differ from each other and do not overlap.

16. Regarding the methods, you do not state what depth the samples were taken from. This is potentially very important as an environmental factor, as it relates to not only depth but also hydrodynamics, light and nutrients.

17. I really struggled to understand why you would use slow and expensive genetic based identification before morphological techniques. It seems very backwards to me. Why would you not identify your fishes to the best of your abilities, then barcode some of them as a tool to verify your findings???

18. Line 227-228. Explain which statistical tools you and why you used them in the appropriate places rather than just saying which stats you used at the end of the paragraph. As it stands, I have no idea why or what you used these techniques for.
Validity of the findings: While the study is essentially a low replication repeat of Depczynski & Bellwood 2004 conducted in the Red Sea. The authors repeatedly point out that little work has been conducted on cryptobenthic fishes in the Red Sea, and highlight the potential of finding new and important results from this study. I agree with these statements. However, the study design means that little useful information can be gleaned from the data.

19. As mentioned above, the level of replication is simply not sufficient to draw any conclusions about general trends of microhabitat utilisation. However, there are also potential statistical problems with the data, probably associated with low replication rates within the microhabitats. One of the (and pretty much the only) assumptions of PERMANOVAs is homogeneity of dispersion of the data. As PERMANOVAs consider relative positions of multivariate data clouds, not the point density in the clouds, you can have problems with your findings if the clouds are different sizes with the centroids in different positions. Using PRIMER you would do a PERMDISP to test these assumptions. Eyeballing Fig. 3A I think you may well have problems with this as the rubble polygon is much smaller than the coral one, but completely enveloped in it.

20. Also, due to the small number of data points the stress values on your MDS plots are very high. Usually 0.15 is a cut-off for whether the findings are reliable.

21. Finally in your conclusion I don;t think you can state that these findings are a "baseline" as they are really only representative of one small part of one reef.
Additional comments: I really like the subject of this manuscript, and think it has the potential to be important. Unfortunately I very strongly believe that the authors need to collect more data to make the findings useful. Hopefully they can do this as I would like to see this manuscript published in the future.


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

Input:
Title: COMPARISON OF CRYPTOBENTHIC REEF FISH COMMUNITIES AMONG MICROHABITATS IN THE RED SEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article cites most of the relevant literature, but I have made suggestions on the pdf where additional literature could and should be cited.  I have also made many comments/questions on various parts of the manuscript where additional analyses and discussion would greatly improve the study.  Overall, once the authors address the comments I made on the PDF, the paper should be suitable for publication.
Experimental design: I have suggested that the study add a dendrogram showing the bray curtis distances between all sites in the study, as well as a SIMPER analysis to highlight key species in each microhabitat.  I have also commented on the use of rotenone without a plastic tarp over the quadrat, because several coral-obligates were collected on sand or rubble. Clearly they weren't there when the net when down, but instead almost certainly came off of neighboring coral heads.  This should be noted and accounted for.
Validity of the findings: The findings appear to be valid, but based on very limited sample sizes.  I think it needs to be shown with species accumulation curves that 5 samples per microhabitat are only scratching the surface.
Additional comments: Very happy to see more work being done on cryptobenthic fishes.  The Red Sea is a big question mark in cryto knowledge (albeit there were some great works by Herler et al), and its encouraging to know folks are studying them at the detail that they deserve.  I am looking forward to more great projects coming out of KAUST.  Keep up the great work.


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

Input:
Title: COMPARISON OF CRYPTOBENTHIC REEF FISH COMMUNITIES AMONG MICROHABITATS IN THE RED SEA
Review round: 2
Reviewer: 1

Output:
Basic reporting: This revised version  of the manuscript is much improved over the initial submission. There are a few grammatical errors throughout (e.g. species' in the first sentence of the introduction). While the manuscript could do with a thorough proof read, I see little need for any major changes.
Experimental design: As mentioned in my previous review, i would like to see some discussion of both the relative surface areas among microhabitats (i.e. the fact that coral and rubble have large habitat areas than sand), and in particular the effects of benthic habitats beneath table corals (I appreciate the authors have made mention of this in the introduction). As an example of the importance of this, the Istigobius in live coral is just as incongruous as the Gobiodon over sand.
Validity of the findings: Apart from the experimental design issues reiterated from my previous review (mentioned above) the data seem relatively robust. The authors have made considerable efforts to rein back their interpretation and done a good job of revising the manuscript.
Additional comments: Well done with the reviews. I'd like to see a few more caveats/explanations of your findings in the discussion and a good proof read looking for grammatical issues, but apart from this your manuscript is very nearly ready to go.


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: IS PERFECTIONISM ASSOCIATED WITH ACADEMIC BURNOUT THROUGH REPETITIVE NEGATIVE THINKING?
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The manuscript describes the direct and indirect relationships between the two higher-order dimensions of perfectionism (perfectionistic concerns and perfectionistic strivings) and three elements of academic burnout (exhaustion, inadequacy, and cynicism). Using a measure of repetitive negative thinking, the association between the constructs was also explored by employing path analysis. The results indicate that there is a relation between perfectionistic concerns and academic burnout, and that there was an indirect path through repetitive negative thinking. Overall, the manuscript is well-written, clear, and coherent, and the design and method used seem relevant for the type of research that has been performed. The authors should also be applauded for the fact that they used a non-binary response to gender when recruiting their sample. In sum, the manuscript makes an interesting contribution to the field of perfectionism and academic burnout, however, a few limitations need to be addressed before being considered eligible for publication in the journal PeerJ.

1)	The sample used in the study is considered appropriate for its aims, but a major concern is that there might be selections bias due to the large number 89 (41.4%) of missing values. If the authors have any information on those who did not complete all of the measures, this should be reported, such as testing the difference between completers and non-completers. If not, this issue should receive more attention when discussing the limitations.
2)	Validated measures of perfectionism, repetitive negative thinking, and academic burnout were used, but it would be useful for the reader to know how the levels on these instruments relate to the population as a whole. Did this particular sample have higher than average levels of, for instance, perfectionism? One concern is that the may be a restriction in range when employing a non-clinical, self-recruited, student sample as the one in the study.
3)	Is there any additional information to describe the sample? For example, is the specific university used in the study representative for students in Australia in general? Or did it only include students within a particular discipline, e.g., engineering?
4)	At what stage of their education were the students in the sample? One concern is that the relationship between perfectionistic concerns, repetitive negative thinking, and academic burnout could be explained by the level of experience the individuals had in terms of their academic career. It is plausible that novice students are more concerned about making mistakes and thus more prone to academic burnout, while more experienced students have already “learned the ropes” and are less susceptible for negative thinking. The same argument could be used for at what occasion during the semester the study was conducted – could the responses to the measures fluctuate depending on how much the students had to do in their studies?


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: IS PERFECTIONISM ASSOCIATED WITH ACADEMIC BURNOUT THROUGH REPETITIVE NEGATIVE THINKING?
Review round: 1
Reviewer: 2

Output:
Basic reporting: The reporting is clear and unambiguous. There are minor issues with inconsistency with regard to the use of capital letters and whether the subscales of Perfectionistic Concerns and Strivings are considered as singular or plural.

Although the literature appears adequate, there is a lack of discussion of the role of depression. I am unclear if there is anything specific to Repetitive Negative Thinking or whether the construct of depression could explain the findings equally well. Similarly, it would be helpful to consider the literature on universal school-based programs by Wade and colleagues (Nehmy, T. J., & Wade, T. D. (2015). Reducing the onset of negative affect in adolescents: Evaluation of a perfectionism program in a universal prevention setting. Behaviour research and therapy, 67, 55-63) rather than only the clinical literature since it is more relevant to this study.

The article is professional, raw data are shared, results are relevant to the hypotheses.
Experimental design: This is original primary research within the Aims and Scope of the journal.

The research question is well defined and has the potential to fill an identified knowledge gap. However, there are significant methodological limitations that include:

1) Sample of undergraduate students (many of whom are likely to have been studying psychology). Academic burnout in psychology undergraduates may not generalise to other samples. Information about the representativeness of the sample would be helpful, particularly since the mean age of 23 seems relatively high for undergraduates.
2) No measure of depression despite the very close relationship between repetitive negative thinking and depression
3) Relatively high drop-out; only completer analyses presented. It would be improved with intent-to-treat analysis.
Validity of the findings: The authors themselves highlight concerns with the psychometric properties of the inadequacy subscale of the School Burnout Inventory which calls the reliability and validity of the findings of this subscale into question. The study is sufficiently well described that it would be straightforward to replicate, and the data appear sound. The conclusions are well-stated although there are places (marked on the manuscript) where it is clear the authors are not familiar with the clinical interventions. Suggesting that it might be an idea to try to tackle concern over mistakes and Perfectionistic Concerns rather than Perfectionistic Strivings is not novel, and is a real focus of current therapeutic strategies.
Additional comments: This is a well-written manuscript and study in an important area that has the potential to contribute to the literature. It would benefit from having a clinical expert in the field contribute (such as Sarah Egan who is based at your University) and from more information about the sample to ascertain generalisability; intent-to-treat analyses would be welcome


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: ECOTOXICITY OF DISINFECTANT BENZALKONIUM CHLORIDE AND ITS MIXTURE WITH ANTINEOPLASTIC DRUG 5-FLUOROURACIL TOWARDS ALGA PSEUDOKIRCHNERIELLA SUBCAPITATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article text is supported by the appropriate literature, however some citations need to be corrected:
(a)	paper by Abel (1974) is not good citation for membrane-QAC interaction. I would recommend to change it. It may be e.g. Tischer, M., Pradel, G., Ohlsen, K., & Holzgrabe, U. (2012). Quaternary ammonium salts and their antimicrobial potential: targets or nonspecific interactions?. ChemMedChem, 7(1), 22-31 (or something similar);
(b)	Kosjek and Heath (2011) is incorrectly cited in the text as “Kosjek et al.”;
(c)	Kuemmerer and Ruck (2014) is incorrectly cited in the text as “Kuemmerer et al.”;
(d)	Altenburger and Greco (2009) is incorrectly cited in the text as “Altenburger et al.”;
(e)	Kreuzinger et al. (2007) is incorrectly cited in the text as “Kreuzineger et al.”.

Figures
(a)	Figure 1 – right photo (electron microscope) was already published in Authors paper (2016) but it is not stated in the figure caption. In my opinion this figure is not essential for publication so it may be removed. Otherwise it should be described as reprinted from the other paper;
(b)	Figure 2 – please, give the information how many replicates did you use to calculate mean values.

Tables. In Table 1 description it should be stated that 5-FU toxicity was investigated earlier and described in the previous Authors paper. There is a misspelling in the word “benzalconium”.
Experimental design: It should be clearly stated in the “Toxicity studies – algal growth inhibition assay” section, that 5-FU toxicity was investigated earlier and described in the previous Authors paper (it is only mentioned in Discussion and in Fig. 3).
Validity of the findings: No negative comments.
Additional comments: The problem presented in the manuscript is very interesting and environmentally important. The results could be considered as a valuable broadening and supplementing of the previous Authors’ works. The manuscript is well prepared and needs only minor improvement.


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

Input:
Title: ECOTOXICITY OF DISINFECTANT BENZALKONIUM CHLORIDE AND ITS MIXTURE WITH ANTINEOPLASTIC DRUG 5-FLUOROURACIL TOWARDS ALGA PSEUDOKIRCHNERIELLA SUBCAPITATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Article could use minor revisions for wording for professional English throughout.

Introduction (general): Introduction needs more citations for the points the authors make. QACs are common pollutants in wastewaters (Lines 53-54); pharmaceuticals are commonly found in municipal wastewaters (70-71); they enter the environment via effluents… (71-74).
Experimental design: Line 102: Did authors use a solvent control to control for possible effects of DMSO on algal growth?
Line 116-119, and Table 1 and 2: The concentrations of BAC used in this study need clarification. Based on the text, authors chose their concentrations for the mixture toxicity based on previous work by other authors. However, the concentrations listed as used for BAC are exactly half the relevant ECX values shown in Table 2. So were the concentrations chosen based on previous studies, or this study, or did the previous studies result in exactly the same point estimates as the current one?
Line 118: Why was 5-FU not retested on its own in the current study? Authors are relying on data from a 15 year old study (Cleuvers 2003) performed by a different laboratory?
Line 135: How many replicates were used for each treatment?
Validity of the findings: Line 225: It is unclear what the authors mean by "we observed heterogeneity". Please reword.
Lines 263-273: Too much info from the previous study. Not necessary.
Additional comments: Line 26: Calculated EC50 was 0.255, not 0.3. No reason to round up in the abstract.
Lines 52-53: Units of t versus kt. Change one of these to be consistent.
Line 81: Needs rewording, or another comma. "…even at low". Also, what is low here?
Line 88: Is 5-FU an antincancer drug or an antineoplastic?
Table 1: Typo in caption "benzalconium". Third column in the table is unnecessary
Table 2: If BAC concentrations used in Table 1 are based on ECX values from Table 2, it stands that the results in Table 2 should be discussed first, and thus the two tables should switch spots.
Table 3: Are there statistics associated with these CI values? Or confidence limits of any sort?
Figure 1: Photos are too dark. Scale bar not visible on left photo.


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

Input:
Title: CHARACTERIZATION OF THE ATPASE FLAI OF THE MOTOR COMPLEX OF THE PYROCOCCUS FURIOSUS ARCHAELLUM AND ITS INTERACTIONS BETWEEN THE ATP-BINDING PROTEIN FLAH
Review round: 1
Reviewer: 1

Output:
Basic reporting: This group has been working on Archaellar flagella for years and in this paper they analysed the motor components FlaH and FlaI. Authors have already established a method to overproduce and purify these proteins and examined interaction between the two. Data presented here to show the direct interaction and effects on ATPase activity are convincing, and the description in the text is well written.
I have some questions below:

1. Fig.1A is not necessary to understand research backgrounds, because authors have not discussed any details of the atomic structures.
Instead, it will be nice to give a cartoon figure to show the relative locations of FlaH, FlaI, and FlaX at the base of archaellum.
Fig.1B is also not necessary in the present form. How about adding Walker motifs in the figure?

2. There is redundant usage of two words: previously and remarkably. Authors can write text (especially Discussion) without using them.

3. I do not see a logical connection between the last sentence (275-277) and the preceeding sentences.

4. Place a space in "aFlaH"(364).
Experimental design: Experimental design is conventional and easy to understand.
Validity of the findings: Not a big discovery but important to know to elucidate the mystrious motor function of Archaeal flagella.
Additional comments: I do not find any reasons to reject this paper.


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

Input:
Title: CHARACTERIZATION OF THE ATPASE FLAI OF THE MOTOR COMPLEX OF THE PYROCOCCUS FURIOSUS ARCHAELLUM AND ITS INTERACTIONS BETWEEN THE ATP-BINDING PROTEIN FLAH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript describes the use of various methods for characterization of the Pyrococcus furiosus ATPase FlaI and its interactions with ATP-binding protein FlaHs and is a logical continuation of the experiments with the same objects described earlier (Chaudhury et al., Mol Microbiol (2016) 99: 674). The publication is fully justified. I have only minor remarks and recommendations to improve the manuscript.
Experimental design: The experimental design is clearly defined and performed at a high technical standard. The methods used are adequate and well described in detail.
Validity of the findings: The data obtained are novel and the presented information is valuable.  The results are adequately documented with tables and figures.
Additional comments: The paper of Chaudhury et al. “Characterization of the interactions between the ATP-binding protein FlaH and the ATPase FlaI of the motor complex of the Pyrococcus furiosus archaellum” deals with an interesting problem of protein-protein interaction providing formation and function of the archaellum. This paper contains original experimental results and can be recommended for publication after certain minor changes. Some errors found in the text relate mainly to the correct arrangement of the literature cited.

Minor points.

1. Most of the article describes experiments on the characterization of the FlaI itself, which is not reflected in the title. It would be,  the next  title is more comprehensive “Characterization of the ATPase FlaI of the motor complex of the Pyrococcus furiosus archaellum and its interactions between the ATP-binding protein FlaH”.
2. Line 17. There should be "P. furiosus" instead of "P.fusiosus".
3. Line 20-21. It is indicated that Interactions between the N-terminal domains of FlaI were not observed previously/ But in “Discussion” (line 326-328 the authors claim that similar interaction was observed for traffic ATPase of the Helicobacter pylori. It will be more correct to say that such interactions were not observed for the crenarchaeal FlaI.
4. Lines 23-24. It should be add that a 1:1 stoichiometry suggests that hexameric PfFlaI interacts with hexameric PfFlaH.
5. Lines 47-48. Three references must be enclosed in one parenthesis.
6. Lines 54-56. It would be more correct to say about the "three unique subunit conformations".
7. Lines 61-63. 5 references must be enclosed in one parenthesis. Reference (Kenneth A. Satyshur…)  should be presented as required.
8. Lines 102-105. Is there any information about the presence of homologues of the FlaCDE or FlaX in thaumarchaeota and nanoarchaeota?
9. Line 110. It would be more correct to say about the "the archaellar basal body".
10. Line 117. There should be "T. kodakaraensis" instead of "T. kodakaensis”.
11. Line 174. “Hill” should be written with a capital letter.
12. Lines 189-190. Three references must be enclosed in one parenthesis.
13. Lines 212-217. The Authors present the data on the PfFlaI ATPase activity at different temperatures. Are there any similar data for the SaFlaI?  If yes, they must be compared.
14. Lines 224-226. See comment #7.
15. Lines 280-281; 284; 285-286; 288; 293-294). 47-48. The references should be presented as required.
16. In the “Discussion” (lines 333-343) the Authors discuss in detail the interaction of the SaFlaH and SaFlaI with SaFlaX, but do not make any assumptions about the interaction of the PfFlaH and PfFlaI with FlaX analogues, which may be FlaCDE. The P. furiosus does not have a chemotaxis system, and in this case the corresponding proteins should not be related to the CheY signal transduction cascade, similar to what the Authors write in the introduction (106-109).
17. Line 370. Apparently, "and" should stand between “gel filtration” and “SDS-PAGE”.
18. Lines 406-406. The name of the journal (Mol Microbiol, 99: 674-85) is not specified.
19. Lines 416, 423, 446, 468. Names of the organisms must be indicated in italics.
20. Lines 437-440. Reference (Kenneth A. Satyshur…)  should be presented as required.
21. Line 480. The journal name should be written with a capital letter.


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

Input:
Title: CHARACTERIZATION OF THE ATPASE FLAI OF THE MOTOR COMPLEX OF THE PYROCOCCUS FURIOSUS ARCHAELLUM AND ITS INTERACTIONS BETWEEN THE ATP-BINDING PROTEIN FLAH
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article of Chaudhury et al. describes a focused and clearcut biochemical analysis of cytoplasmic archaeallum components from Pyrococcus furiosus. The authors describe their goals and their focus on this less studied model, which  appears to offer advantages for biochemical analysis.  The paper is clearly and succinctly written, while providing sufficient information in the Introduction and Discussion that place their study in a broader context.
The authors cite most of the recent and relevant literature.
I have two minor comments:

1. Line 210. It is not clear what the authors mean by "three unique subunits". Presumably there are three different conformations of monomers in the crystal, but this should be better explained.

2. Multiple references in the text and the list are incomplete or need reformatting. The authors should also group their references, when successive, together in a single pair of brackets. Examples include lines 47, 62, 224 and all references in the Discussion.  Several references have parts missing or are misformatted in the list (e.g. lines 394, 404, 406, 421, 433, 437, 475, 480).
Experimental design: The authors describe purification and biochemical analysis of FlaI, the hexameric ATPase and the main motor of the system, and FlaH, an ATP-binding protein of the RecA family. The quality of this work meets the high standards and is supported by quantitative approach.

The authors show that Pyrococcus FlaI can be purified to homogeneity and behaves as a hexamer, even in the absence of ATP or analogues. unlike Sulfolobus FlaI used as a control. The authors further show that Pf FlaI binds ATP in vitro at a 3:1 molar ratio, suggesting that only two ATP binding sites are accessible in the hexamer and leading the authors to deduce a two-fold symmetry of the hexamer. Using the purified FlaI Pf the authors analyze the kinetic constants, optimum temperature and pH for its ATPase activity.

The two domains of FliI were produced separately and their multimeric state was tested by SEC. These experiments show that the N-terminal domain alone behaves as a hexamer whereas the C-terminal domain was monomeric. When the two domains were mixed, the C-domain co-eluted with the N-domain multimer, and this interaction was required for reconstitution of ATPase activity.
Next, the ATP binding protein FlaH was purified and its ability to stimulate FlaI ATPase activity was demonstrated in vitro. This stimulation required intact walker A and walker B boxes of FlaH. When FlaH was added to FlaI variant defective for ATPase activity, no ATP hydrolysis was detected, demonstrating that FlaH is unable to hydrolyse ATP, even in the presence of FlaI.

Finally, using microscale thermophoresis, the authors determined FliH binding affinities of to the N- and C-terminal domains of FlaI.  These experiments showed higher affinity of FliH to the C-terminal domain, which was even higher than that observed for the FlaI hexamer. The C-terminal binding and 1:1 stoichiometry is consistent with positioning of FlaH hexamer underneath the FlaI hexamer at the base of the archaellum.
Validity of the findings: Overall, the experiments are well executed and support the conclusions. Proper controls are included in the experiments and statistical analysis is applied.

The Discussion highlights differences between Sulfolobus and Pyrococcus FlaI and the role of N-domain oligomerization in ATPase activity and in the cooperativity of the process. The results are placed in the context of recent structural data obtained by crystallography and cryo-tomography in different species.
Additional comments: None


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

Input:
Title: CHARACTERIZATION OF THE ATPASE FLAI OF THE MOTOR COMPLEX OF THE PYROCOCCUS FURIOSUS ARCHAELLUM AND ITS INTERACTIONS BETWEEN THE ATP-BINDING PROTEIN FLAH
Review round: 2
Reviewer: 1

Output:
Basic reporting: All my comments have been taken into account. The resulting manuscript has become more perfect. It meets the PeerJ criteria and should be accepted.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: All my comments have been taken into account. The resulting manuscript has become more perfect.


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: PRIMATES IN PERIL: THE SIGNIFICANCE OF BRAZIL, MADAGASCAR, INDONESIA AND THE DEMOCRATIC REPUBLIC OF THE CONGO FOR GLOBAL PRIMATE CONSERVATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper represents an excellent review of the current situation that leads to the endangerment of the primates in Brazil, Democratic Republic of Congo (DRC), Madagascar, and Indonesia.  It primarily relies on presenting current statistics of the situation, which is extremely valuable.  This will be very useful to conservation biologists and individuals seeking to set conservation policy.

	I have few comments as there seems little point in addressing or questioning specific statistics; however, I would like to see 2 major issues addressed
	First, the authors rarely question the validity or strength of the data that they are relying on to make there arguments, and while I do not see any current alternatives to use these data, I think that pointing out where data is weak would help researchers in the future as they could focus on strengthening these areas.  I see two particular areas. The IUCN species categorizations area typically all we have to assess endangerment, but in some places (particularly the DRC) these estimates have to be considered poor at best and improved efforts to survey over multiple time periods are desperately needed. Second, we know frightening little about how tropical forests and primates will respond to climate change and this relies on long-term research which is extremely hard to fund and more and more long-term primate sites are closing.
	Second, the authors make many many claims of what needs to be done, but almost totally shy away from stating the likelihood that these challenges will be met.  This is particularly the case in the conclusions. If these challenges cannot likely be met (and to me this is likely often to be the case), the authors should make the appropriate speculation that they will not be met – the authors are in a good situation to make these statements, or at least better than most readers.  Without doing so, borders on misleading uninformed readers.

Minor comments
Abstract – the authors give nice information on protected areas in the four countries, but fail to state that many of these areas are ineffective at protecting primates, thus the statement borders on misleading.

Lines 250 and thereabouts: In the section of expanding agriculture, the authors really need to mention that growing human population and consumption patterns favoring meat are driving this expansion. This is presented later, but a line to this effect would be useful here.

Line 325: A mention of the fact should be made that that where oil is found, these governments will likely exploit it even if the oil is found in protected areas.  This will occur if there is good or poor governance.

Line 491: I think a mention should be made of the fact that the logistics of administering vaccinations to great apes is extremely challenging to say the least, particularly since these animals are hunted.

Reintroductions: I think that the authors should mention the costs of these reintroductions. In a world where conservation funds are unlimited, this could be a priority, but since more primate populations are unprotected and are declining, these efforts should be seen as a costly option, relative to other options.

Conclusions and Key Challenges Ahead: I think that this section could be reduced and repetition to what has already been stated could be eliminated so that this section could have a stronger impact.
Experimental design: Not Applicable
Validity of the findings: Great
Additional comments: See 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: PRIMATES IN PERIL: THE SIGNIFICANCE OF BRAZIL, MADAGASCAR, INDONESIA AND THE DEMOCRATIC REPUBLIC OF THE CONGO FOR GLOBAL PRIMATE CONSERVATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The manuscript entitled “Primates in peril: the significance of Brazil, Madagascar, Indonesia and the Democratic Republic of the Congo for global primate conservation” by Estrada et al. provides a comprehensive review of primate populations in these countries, as well as threats and anthropogenic pressures that these populations are facing. The authors also predict how the distribution would be affected by agriculture expansion in the future under business-as-usual scenario. The manuscript is generally well-written and could contribute to understanding of primate populations and threats to their survivals. I have some recommendations to improve the current presentation of the manuscript.

Line 96-97: I am not sure if the effectiveness of governance is really asses in the manuscript, as it would require a thorough causal analysis (with counterfactuals) to determine whether the current primate situation is attributed by good/poor governance (see Baylis et al. 2016). I would suggest moderating the language in the link between governance and conservation efforts.

Line 182-253: The section on “Large-scale encroachment and loss of primate habitats” perhaps can be divided into two sub-headings: (1) historical and (2) projected. The historical component would encompass the current “Forest loss” and “Expansion of agricultural land”. The projected component would encompass the “Modelling agricultural expansion and primate range contraction in the 21st century”.

Line 521-549: The impact of climate change on forest and primate populations through wildfire is not well mentioned. Mitigating climate change impact on fire is critical for the survival of primates in Indonesia (especially in Borneo and Sumatra where large areas of peatland have been degraded and vulnerable to fire, which could spread to adjacent forest).

Line 602-608: It may worth to assess how the Gini index (disparity in wealth) in each country based on the UN data. In some countries, the Gini index might have increased over time, which indicates increased disparity between the rich and the poor. So the average HDI index masks the actual distribution of poverty.

Line 745-776: There has been some recent progress on research in community forestry which can be worth to discuss in this section. Examples include: Rasolofoson et al. (2015, 2017) that shows how community forest management in Madagascar have successfully reduced deforestation and also improved local community wellbeing, Santika et al. (2017) that shows the impact of community forestry on reducing deforestation in Indonesia, and other studies from South America. Perhaps could also look at Bowler et al. (2012) and Burivalova et al. (2017) for recent review of community forestry and reduced impact logging and forest certification scheme.

References:

Baylis, K., Honey‐Rosés, J., Börner, J., Corbera, E., Ezzine‐de‐Blas, D., Ferraro, P.J., Lapeyre, R., Persson, U.M., Pfaff, A. and Wunder, S., 2016. Mainstreaming impact evaluation in nature conservation. Conservation Letters, 9(1), pp.58-64.

Bowler, D.E., Buyung-Ali, L.M., Healey, J.R., Jones, J.P., Knight, T.M. and Pullin, A.S., 2012. Does community forest management provide global environmental benefits and improve local welfare?. Frontiers in Ecology and the Environment, 10(1), pp.29-36.

Burivalova, Z., Hua, F., Koh, L.P., Garcia, C. and Putz, F., 2017. A critical comparison of conventional, certified, and community management of tropical forests for timber in terms of environmental, economic, and social variables. Conservation Letters, 10(1), pp.4-14.

Rasolofoson, R.A., Ferraro, P.J., Jenkins, C.N. and Jones, J.P., 2015. Effectiveness of community forest management at reducing deforestation in Madagascar. Biological Conservation, 184, pp.271-277.

Rasolofoson, R.A., Ferraro, P.J., Ruta, G., Rasamoelina, M.S., Randriankolona, P.L., Larsen, H.O. and Jones, J.P., 2017. Impacts of Community Forest Management on Human Economic Well‐Being across Madagascar. Conservation Letters, 10(3), pp.346-353.

Santika, T., Meijaard, E., Budiharta, S., Law, E.A., Kusworo, A., Hutabarat, J.A., Indrawan, T.P., Struebig, M., Raharjo, S., Huda, I. and Ekaputri, A.D., 2017. Community forest management in Indonesia: Avoided deforestation in the context of anthropogenic and climate complexities. Global Environmental Change, 46, pp.60-71.


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: MLSTAR: AUTOMATIC MULTILOCUS SEQUENCE TYPING OF BACTERIAL GENOMES IN R
Review round: 1
Reviewer: 1

Output:
Basic reporting: Excellent standard of English and a good literature review.

It would be nice to include a sentance or 2 in the introduction motivating why MLST is so useful, such as outbreak investigaitons, survellance etc...

You might find the following preprint useful:

GrapeTree: Visualization of core genomic relationships among 100,000 bacterial pathogens
https://www.biorxiv.org/content/early/2017/11/09/216788
Experimental design: Whilst Minimum spanning trees are fast, the quality of tree isnt the best, particularly from alleles. You can get better resolution using the underlying sequences (so based on SNPs) and ML based methods.

The performance comparisons are weak. Just testing it on C. coli doesnt tell us much, particularly when nearly 100 of the samples have the same ST, and the actual alleles of the rest are are drawn from a tiny pool. Try testing it on a few different organisms, with diversity, for example the 10 most common pathogens.

You should also add in controls so that you can calculate specificity and sensitivity. There are 10 different campy MLST schemes, does your software falsely return an ST if you test your 400 genomes against the other 9 schemes?

The numbers in Table 1 need to be double checked. The accuracy of the ST should be less than or equal to the lowest accuracy for the individual alleles, with a lower bound of multiplying each of those numbers (~97%).
Validity of the findings: The software appears to call STs when there are only partial matches. For MLST to be called properly there must be a 100% match in all alleles.

Whilst extended MLST schemes are mentioned in the title, there is no validation or results in the paper itself. Does it actually work on extended schemes or is this theoretical? Either include some results from an extended scheme or remove this claim.

Some MLST schemes have alleles completely missing, how does your method deal with this?

How does your software compare against the data in this MLST comparison paper (which you have cited)?
http://mgen.microbiologyresearch.org/content/journal/mgen/10.1099/mgen.0.000124
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: MLSTAR: AUTOMATIC MULTILOCUS SEQUENCE TYPING OF BACTERIAL GENOMES IN R
Review round: 1
Reviewer: 2

Output:
Basic reporting: MLSTar software is tested and validated to carry out MLST (~7 housekeeping genes) so discussion about cgMLST should be removed from the abstract.

README.md document should state that script works only on the Unix-based operating system.
Experimental design: 'no comment'
Validity of the findings: please see attached document
Additional comments: Does MLSTar software carry out cgMLST ? Would there be any future development for MLSTar to carry out cgMLST?


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: MLSTAR: AUTOMATIC MULTILOCUS SEQUENCE TYPING OF BACTERIAL GENOMES IN R
Review round: 2
Reviewer: 1

Output:
Basic reporting: Excellent
Experimental design: Excellent
Validity of the findings: Thank you for including much more comprehensive experiments.
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: HORIZONTALLY TRANSFERRED GENES IN THE CTENOPHORE MNEMIOPSIS LEIDYI
Review round: 1
Reviewer: 1

Output:
Basic reporting: English and structure are ok (in particular, raw data, including scripts and command lines are all already freely available on-line), and the manuscript represents a clear unit of publication.

Literature should be improved in order to give the reader proper and fair explanation about the phylogenetic position hypotheses in deep animal phylogeny. Also, few sentence rephrasing or clarification are needed (please see the pdf annotated file).
Experimental design: Methods are really well thought and applied for automatic detection of HGTs, including multiple approaches (BLAST, phylogeny) and criteria (intron/exon structure, neighboring genes in scaffolds, multiple ctenophore data sources) to filter possible false positives.

The main concern I have is that alignments and phylogenies for the 9 bona fide HGTs could have been done with a much broader taxon sampling to gain resolution. For example, if most ctenophores have the homolog as Mnemiopsis, why not including them? Also, adding more bacteria and eukaryotes could improve the topology and statistical support. In fact, I'd like to see the topologies in supplementary material (not a lot of work to do), and the raw newick/nexus files for the best trees. To be fair, it would be great to have contrasting topology using bayesian inference. The reason of all this is that ctenophore sequences are long-branch and extra care should be taken.

Regarding the expression analysis, even if I'm not an expert on the matter, I don't think that the extra replicates can be compared directly with the other data due to methodological biases (not real replicates from 14-19?). I would suggest authors explicit more details instead of just citing a previous study, to facilitate reader comprehension. Finally, some concerns about the results are in the annotated pdf.
Validity of the findings: 'no comment'
Additional comments: This manuscript aims to find specific horitzontal gene transfer at the onset of ctenophors by using rigorous methods of sequence similarity and phelogenetics, including expression data for Mnemiopsis leidyi. The manuscript is suitable for publication after few minor revisions.


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: HORIZONTALLY TRANSFERRED GENES IN THE CTENOPHORE MNEMIOPSIS LEIDYI
Review round: 1
Reviewer: 2

Output:
Basic reporting: The reporting is clear, with very few and very minor grammatical issues.
line 40; not 'less' but 'fewer'
line 106 should state Woods Hole not Woodshole
line 116 should include: "the following fully sequenced organisms: " after 'from'
line 173 should use 'after the method of' not 'by'
see line 276. I am not sure how to correct it.
line 286: ...that can contaminate preparations during...

The authors should carefully examine their references.
Hontzeas and Willerslev et al are not in the body of the manuscript
Wybouw et al. 2016, Haegeman et al. 2011, Eyres et al. 2015; Conaco et al. 2016 are not in the references
There may be others; these are just ones I happened to pick up because they looked out of place.

In the Lit Cited, there are minor formatting issues; see:
Boto
Hejnol
Moroz
Experimental design: The manuscript meets high quality of scientific design. The concept of alien index is well established. There are areas that I am uncertain of; this could be because of my limitations with the methods chosen.

in line 104, the authors state that they examined neighboring genes to the putative HGTs; I may have missed this, but how did they do this?

in line 119, I am not sure to what the authors refer when they say 'the above criteria' - could they please clarify?

When testing between the SOWHAT and nonparametic methods, what are they seeking?

line 144: How was CONSEL used specifically?
Validity of the findings: I am only concerned about what appears to be the primary test for HGTs: intron structure.

line 176: Group I and Group II introns are indeed seen in bacteria, archaea  and viruses:  (see Mobile DNA 5:8, J Bact 187:5437, Nuc. Acids Res22:2532). Perhaps the authors need to more closely examine to determine whether the introns they see are all actively handled by splicosomes since those are only eukaryotic. How precisely to do this is not clear to this reviewer, but there may be a method.

I am not a professional molecular statistician so many of the arguments, which I can follow technically but which certainly have implication beyond my broader experience, may answer this type of question.

Otherwise the work appears to be mathematically solid and the entire question and conclusion well stated and properly connected together.
Additional comments: At 157 it was not made clear why the additional times were taken, but the figures showed that there were important events occurring during that time period that used these specific putative HGTs. What are these events? what sorts of tissue/cellular reorganization are occurring? this is to this reader the most exciting area of your findings.

Were putative Archaean HGTs discovered at all? looks like not...I am surprised that they are not mentioned. Perhaps I have missed something in the paper.

I have included all comments so that they can be seen by the authors.


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: HORIZONTALLY TRANSFERRED GENES IN THE CTENOPHORE MNEMIOPSIS LEIDYI
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript shows perfect and fluent English writing, with technical descriptors well formulated and inserted in the text. The literature cited help to understand the context of the problem, horizontal gene transfer (HGT) detection, and their application in new genome studies. References to other HGTs studies and their transcription activity would be useful (see general comments). Article structure, figures and tables are well formatted and almost ready to go online. I would suggest the implementation of one table (see general comments). Also, I like to thanks the authors for having all necessary materials ( i.e.  programs and databases available in github) to reproduce the results and even apply the methodology to other cases. I think it is the way to go in today’s science communications in order to present relevant results.
Experimental design: The question and challenges of HGT in new sequenced species was well defined and their implementation in the ctenophore M. leidyi was executed nicely, combining homology search methods along with phylogeny reconstruction and confirmation. The methodology explained, with excellent detail, would be useful for future analysis and detection of HGT in non-model organism.
Although I find incomplete execution and lack of details at the developmental expression profile methods and results interpretation. I understand this part in an implementation of previous research by Levin et al. 2016, where they look (among other things) into gene expression levels at the developmental transition at embryo level in different species representing different phyla, but I don’t fully understand their inclusion in this study and the way it has been addressed. First, I would like to see more details explaining data collection (RNA extraction, mapping reads, analysis, etc), along with a brief description of the embryology of the species (M. leidyi). Why look into early stages, first 20 hours, and not some adult stage too?; so you could compare transcription values (early vs. late). If someone wants to point some gene/s are expressed at a specific state, I think first they should compared the designated estate with “other” state, and second, I think it should be usefull the use of some control candidate (housekeeping gene?) in order to normalize all transcriptional environment. I understand that the manuscript just imply their HGTs “are expressed during early development”, and not “more expressed”; though still it is rather confusing not to have something to compare with.
Please, let me put this into another perspective: if instead of early stage development, we only have RNA-seq data from late state, adults from their final live state, and a few HTGs show transcription at them, then the title would change to “expressed during late development”. This statement belongs to the title, no just a sentence in the discussion trying to bring some hypothesis for future testing. I think it is great these HGTs showed transcripts, but I think we should take this with more descriptors, analysis and comparisons, and maybe formulating other hypothesis and/or points of view. Otherwise, maybe a better formulation would be: HGTs showed transcription at the stages were RNA-seq data was available (early stages).
Validity of the findings: The authors have investigated horizontal gene transfer in the ctenophore M. leidyi, and although it’s not at a massive scale in its genome, the authors have validated each of one and have proposed a exhaustive method to validate their findings, by implementing BLAST-based scores for detection of HGTs and hypothesis test with phylogeny analysis. In general, the conclusions are well stated in the horizontal gene transfer investigation.
I think the robustness and validity of transcriptional data are more than fair for today’s standards, though some extra steps should be taken care of (reference points, further analysis explanation). But I kindly disagree with their conclusions and their link to the original question, horizontal transfer detection and validation. Under my perspective, I address horizontal transfer as the main question, since it fulfill the mayor part of the manuscript, where HGTs expression fill up 3 and a half lines in the Results section.
At the same time, I disagree with the inclusion of the term “enzyme”, specially in the title; the authors described genes and their transcript levels, and yes, they find they have homology with other proteins (UniProt database), with a function more or less characterize.
Additional comments: In the manuscript submitted “Horizontally transferred genes in the ctenophore Mnemiopsis leidyi encode enzymes and are expressed during early development” the authors described a methodology to identify horizontal gene transfer events in animals, and they develop it in the unexplored Ctenophora. They have found and validate several candidates by different means in the sequenced genome of M.  leidyi. Their approach could be easily translated to other new sequenced species of metazoans, which could benefice the scientist community to discern contaminants from potential HGT candidates. All the procedure is quite well outline in the manuscript (text and supplemental material), along with external material (scripts and databases).
I think it would make a worthwhile contribution to PeerJ, but I have a few suggestion (as pointed in Experimental design and Validity of the findings), along with some other points.

Moderately important points:
Title: I think including in the title the statements of “expressed during early development” and “encode enzymes” is going too far given the nature of the analysis and discussion performed. It might mislead the reader initially with the scope of the article (see  Experimental design and Validity of the findings). I think a good example can be found in the paper Hespeels et al. 2015 “Against all odds: trehalose-6-phosphate synthase and trehalase genes in the bdelloid rotifer Adineta vaga were acquired by horizontal gene transfer and are upregulated during desiccation”. The authors here dissect apart a specific group of horizontal transferred genes, where it is stated not only the RNA-seq screenings but a deeper description with comparison with other genes, implementing a metabolic context with their results, and just hypothesizing  that these GENE COPIES (not enzymes) might be functional.

Figures and tables were easily followed in the text. But it was quite hard for me to keep track of the candidate genes in each of the steps. I think the reader would appreciate having a table like Table S2 in which it is described which genes are candidates and which one is confirmed, but including like which are phylogenetically confirmed (7 out of 13), the ones that have no animal Blast (6 of them), the removed genes (4), highlight the ones plotted in Fig1….along with the intron presence (maybe include numer of introns?).

Using a custom database to automate the generation of alien index. I understand the use of custom db in order to minimize the inclusion of contaminants which has been wrongfully assassinated. But I think the custom db could have more curated species, why is it not included plants, green or red algae?

Minor suggestions
Line 17. Please, replace “appear to perform enzymatic activities” with “transcriptional active”.
Line 90. Author should include what is the query (M. leidyi gene models) used in this section, and maybe describe it a little; how many genes etc…
Line 93. Authors used alien_index_db version 0.01, but at the github repository it is listed version 0.02 (January 21, 2016). Why don’t use the latest one? Is there any big differences in number of sequences/taxa?
Line 97. The database included 11 animals, but in table S1 there are 12. Please, clarify.
Line 105. Confirmation of HGTs in 7 other ctenophores. What’s the query and parameters used in that search? How many M. l. genes?
Line 110. Why is it not included the species included in the family Mertensiidae (Moroz et al 2014)?
Lines 122-126. Could the authors specify what is the E-value with animal sequences for those genes; how much (%) is the query cover (HSP).
Line 155. It should be mentioned something about the Levin experiment, like we take the 3 replicas (GEO accession number ###). And then, the additional time points (include GEO ##). It would help the comprehension of this analysis.
Line 156. It should be explained the methods used in these replicas; material (flash frozen?) , RNA extraction, sequencing, etc. The methods outlines in Levin et al. 2016 are several. It should benefit the comprehension of the text and clarification of the results obtained.
Line 159. Authors used 100 or more reads to classified a gene as expressed; please explain why choose this value. As I mentioned above, it’s hard to say without a referernce point. Question: are any of these HGT genes assigned as dynamic expressed genes (as in Levin et al 2016)?
Line 172. I would like to see Table S1 with Alien Index values for each gene too.
Line 179. Would agree the authors with me that representing GC (%) content in genes and scaffolds could also validate these findings? And if so, would it be useful to state their value in the table?
Line 186. So Fig 1 has 9 genes (HGT candidates). But at this point is hard to figure out why are only represented 9, which is resolved later in the text. An implementation of table S2 is highly recommended with maybe some text explanation at this point. Also, I think Figure 1 could have more information, like instead of only colored squared, indicate the % identity among them. I am really curious about the % identity (or divergence) between the Wood Hole and the Florida individuals.
Line 217. What Blast E-values gave the 4 genes removed?
Line 231. Please rephrase like” all HGTs except one showed homology to known characterized enzymes”.
Line 238. 73% (X of 37 candidates).
Line 259. ….”disproportionately enzymes”? Sorry, I don’t understand the term.
Lines 268-271. Speculation is welcome, but saying that ML02771a is highly expressed I think is not accurate. Maybe just say “it is expressed”.
Line 281. No, genes are not enzymes. Please, rephrase.
Line 290. “, lack evolutionary perspective” I think there is something missing in the text to make sense. Please check.
Figure 2. Please check the assignment of trees in the text, (F) is showed twice.
Figure 4. The Y axis should have all the same scale for comparisons. I wonder how are the profiles of the three genes not included (but included in table 2); maybe it would make a better example of the claims assured for RNA-seq values in these stages for HGTs.


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: HORIZONTALLY TRANSFERRED GENES IN THE CTENOPHORE MNEMIOPSIS LEIDYI
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: First, thanks for all the corrections and explanations that, in my opinion, have substantially improved your manuscript. Congratulations for your work. I've marked the recommendation as minor revisions for a couple of small points (see below), but a part from that, it could be accepted as it is.

Sorry to insist but, in lines 427-428, I'd like you to cite a recent non-animal holozoan phylogeny (you already cited one but there are more recent papers, whatever fits you) and say "Choanoflagellatea or Ichthyosporea (two of the closests protistan lineages to animals)". The reason is that another protistan lineage is closer to animals than Ichthyosporea, the Filasterea (although ncbi database may not include it, you must use state-of-the-art taxonomy, and I mean since more than 10 years ago; for further information contact the multicellgenome lab in Barcelona).

Also, please be aware that taxonomic sampling could have been wider for protistan lineages (several genomes and transcriptomes not present in ncbi but publicly available) which might challenge your results, such in line 485-486, where the Amoebozoan origin could be simply an ancestral gene from both ctenophore and amoebozoan lineages that was certainly lost in fungi, choanoflagellates, etc., but still present in closely related species not comprised in your database.


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: HORIZONTALLY TRANSFERRED GENES IN THE CTENOPHORE MNEMIOPSIS LEIDYI
Review round: 2
Reviewer: 2

Output:
Basic reporting: Thank you for the opportunity to rereview the manuscript by Hernandez and Ryan entitled “Horizontally transferred genes in the ctenophore Mnemiopsis leidyi”. The authors have extensively addressed the previous concerns of the reviewers. This is a much improved manuscript. I have a few minor changes and suggestions.
Again, the manuscript shows a fluent English writing, with better technical descriptors. The literature has been improved since the last version. The article is structured as a comprehensive unit, with figures and tables and supplemental material correctly placed.
Experimental design: HGT phylogeny analysis is better formulated with more accurate descriptors.
Questions addressing data collection ((RNA extraction, mapping reads, analysis) have been addressed. Though I have a few comments:
-Line 109
The authors have included the query source, M. leidyi gene models (ML2.2). Could it be possible to have its reference, how the gene model was created? And if there’s no reference, can the authors explain how they get it (methodology)? Maybe it can be included in the supplementary material.
-Line 185
HGT developmental expression profiles
This section was clearly improve, but it has raised me a few more question:
-Line 197 “Since the CEL-Seq protocol considers only the 3’ end of transcripts”
Just a quick check on that protocol, it states that is “Strongly 3’ biased”. I’d suggest to change the manuscript sentence with the term “biased”. Question: have the authors check their reads “bias” along the transcripts where they are mapped?
-Line 198
I am confused with the term used by the authors in order to calculate their reads per million, no considering the transcript length. They use tpm (transcripts per million), but if you look in the literature, you find TPM (transcripts per million) which takes the transcript length into the equation (eg. Wagner GP, Kin K, Lynch VJ. Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples. Theory Biosci. 2012). I would suggest to use the term and acronym reads per million (rpm); which meaning and calculation is pretty straight forward.
-Line 206
Sorry, but it’s still not clear to me how the tpm (or rpm) are calculated giving the replicas. So, after counting the reads per transcript (htseq-count),the authors calculate the tpm (or rpm) for each replica? Is that correct? And then the median tpm values (for all the replicas in each gene?) are summed? I’d appreciated if they can rephrase it to facilitate reader comprehension.
Validity of the findings: 'no comment'
Additional comments: Minor suggestions
- Figure 1
The authors have included Figure 1 in order to show the pipeline in each scenario, with the number of genes included. This has help a lot in the understanding of the manuscript. But since the genes IDs are not included, the reader (at least me) still needs to go back and forth  all the time to follow each step and what genes are represented. I originally suggested to implement this on a table S2, but since the authors designed Fig. 1, could it be possible to correlate Fig. 1 with table S2; like include in table S2 some coding (color, A-B-C...) to refer in which step/steps is included each gene?
- Figure 3
The boxes with “Confirmed” and “Unconfirmed” are missing, like in the previous version and in Fig S3. Please, let me know if it’s intentional or if not, it can be fixed.
-Line 66
It will be worth to include here some literature regarding false negatives/positives in BLAST searches, as addressed in the discussion (eg. Longo 2011, Merchant 2014)
-Line 270
It is not clear to me if the scaffold with geneID ML49231a has other genes at all, or the genes nearby don’t have any introns.