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: TRICHOCERA MACULIPENNIS (DIPTERA)—AN INVASIVE SPECIES IN MARITIME ANTARCTICA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is clearly structured and appropriately referenced. My main concern is with the quality of the English language used, and this goes beyond suggesting a few sentences for specific editorial attention - basically the entire text needs to be very carefully edited by a first language English speaker, preferably with an ecological science background.
Experimental design: This is a largeyl descriptive study, so comments on experimental design are not appropriate. The subject of the paper is clear and well defined, and the importance both of the general research area (biological invasions in Antarctica) and of the specific example being reported are clear. The data being reported are appropriately described.
Validity of the findings: As an observational study, statistical analyses are not included or necessary, but the results are robust and clear. Some comments are included below about what I perceive as an area to include the 'balance' of the conclusion of the importance of sewage tanks in the dispersal/distribution of this species on King George Island as distinct from its possible/probable presence in the natural environment, from which it then opportunistically colonises these sewage tanks.
Additional comments: This is a very important report of a significant increase in distribution of one of the few known invading species to date in Antarctica, and of a species that may have a fundamentally new ecological role in the ecosystems it occupies, as well as one that looks extremely unlikely to have any possibility of control or eradication, other than through the application of strict and effective biosecurity measures by the national operators using King George Island and the South Shetland Islands. I would even suggest a sentence such as the previous would be appropriate to include in both the Abstract and as a conclusion of the paper. Also, although not part of the reviewing process, I would strongly urge the authors to report this new finding as an Antarctic Treaty paper - probably too late to do so at the imminent and abbreviated Buenos Aires meetings, so perhaps for the 2019 meeting in the Czech republic.

Introductory text: one general point that does not come through clearly to me at this stage in the ms is that in the original report of this species (Volonterio et al 2013) the suggestion was made explicitly that the species was actually established in the natural environment on KGI and from there recolonised the sewage facility at Artigas Station a couple of years after the first eradication. This is mentioned quite late on in the Discussion, but I think the earlier study should be credited with this earlier in the paper (as early as mention of this study at l68-71). Realising this also changes the balance between the new colonisation event being possibly related to direct human movement (eg through eggs on boots contained in rotting debris) and natural dispersal (eg a wind blown gravid individual - the answer will never be known, but I don't think the balance is quite right in the current text. It would perhaps also be appropriate to mention that, as far as I am aware, none of the operators (national operators, I believe tour operators mostly apply boot washing around landings) on KGI apply any biosecurity measures around personnel and cargo movement at present, despite several years' knowledge of this species' presence on the island. I am also not aware of any detailed surveys of occurrence/distribution in the wider ice free areas accessible from the airport at Frei, other than those carried out in the vicinity of Artigas Station a few years ago.

l48 - the most recent estimate of this (Burton-Johnston et al 2016) reduces this estimate to c. 0.2%

l160 - such discrepancies are not unusual, particularly in studies of invertebrates that may have a diapause stage in the natural environment

l186 - the form of any attempt to survey beyond the station ('outside') is not actually described?

Line 308:  The eradication of Lycoriella was successful at Rothera, but not at Casey

Line 271-273:  The first half of the sentence could be clearer (it wasn’t the biomass of the whole island, rather in the specific location where Eretmoptera occurs!)

l289 - the airport is clearly such a risk, but should also be noted that most cargo (including fresh food) arrives for the many operators on KGI and in the S Shetlands by ship, and again the transit time from southern S America is a small number of days, so provides another high risk area of operation.

l293 - the comment about not being resistant to higher temperature contradicts that a few lines above about the species being distributed in Europe as far south as the Mediterranean.

Peter Convey
British Antarctic Survey


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: TRICHOCERA MACULIPENNIS (DIPTERA)—AN INVASIVE SPECIES IN MARITIME ANTARCTICA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript is improved on the first version but there are still many problems with it and it is not suitable (yet) for publication.  I note the problems below in order (roughly) of priority.

1.	As requested, a detailed plan for eradication is now included but it omits improving quarantine regulations.  There is no point in eradicating the fly if fresh root vegetables are still being imported.  There will just be continuous invasions. Anyone who has the privilege of visiting the Antarctic must expect to be deprived of certain comforts that they enjoy in Europe.  If these expeditioners are not prepared to live without fresh fruits and vegetables, then they should not accept a visit to the South Shetlands or any other Antarctic region.  The Polish authorities should not put the desires of Polish scientists above that of the health of the environment.  They should follow the lead of other European and nations elsewhere and ban the entry of fresh food to this region.  I would suggest this must be an obvious recommendation included in this paper.
2.	Why have the authors ignored the directions of referees as to correcting formatting?  Hyphens are still being used when n dashes are the correct form.  Moreover, hyphens have even been used when an m dash should be.  It is quite annoying for referees who have spent some hours reviewing a mss to have their corrections ignored.
3.	Although the English style has improved, there are still numerous instances of poor writing.  I have again had to make corrections (see attached pdf).
4.	 I do not believe it is true that this is the first time an alien species has been shown to be transported within the Antarctic region.  There are wind trap records both set on land and also towed behind aeroplanes in the region, that are published. None is referenced here.
5.	The authors have cut some text and inserted what was asked for but there is still much repetition and the whole paper is far too long.  The authors seem to be suffering what I can only describe as “verbal diarrhoea”.  I repeat that for a fairly minor mss of this type, repeating information which is readily available on line or in other recent publications on a similar topic is not helpful for a reader not economical for the journal. There is also considerable repetition even within the text itself.
6.	I have not checked the references and will not do so until the formatting is corrected.
7.	Figure 4 is unnecessary and the morphological variation could be kept until the sequence data is available.
8.	The authorities for species have still not been included the first time it is mentioned.
9.	More discussion on why the attempts to eradicate the fly at Casey Station failed could be included i.e. what are the risks of the same failure on King George Island?
10.	The authors do not say if there are any alien plants on the island.  If so they could be a refuge for alien insects.  Just a statement saying there are none would be sufficient.
Experimental design: n.a.
Validity of the findings: Adequate
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: TRICHOCERA MACULIPENNIS (DIPTERA)—AN INVASIVE SPECIES IN MARITIME ANTARCTICA
Review round: 2
Reviewer: 2

Output:
Basic reporting: My concern here with the revision remains as previously that in some parts the language is still not up to the formal standard required by the journal, although it is much improved on the initial submission - even a cursory read of the abstract identifies several syntax errors that will require editorial correction, although this seems to be less of a problem with the rest of the ms.
Experimental design: As before, this is an observational study so experimental design is not relevant. The revised ms has been extensively altered from the original submission, in particular with several runs of text being deleted, especially following the suggestions of the other reviewer. This has improved the structure and flow of the ms.
Validity of the findings: Again, as before, this is an observational study of a novel and important observation, in the context of Antarctic biological invasions, and threats to native biodiversity. The revised ms is more focused and has improved logical flow.
Additional comments: a few remaining detailed comments:
l64 - spelling of Fildes Peninsula
l87/8 - were any found in these other locations?
l118 - it would be interesting to know the most northern 'natural' occurences of the species, as well as those that are synanthropic (and hence I assume in some sense 'introduced')?
l156 - I agree this knowledge is important, but perhaps even before that is the knowledge of where the species occurs - it is one thing to eradicate a species from within an enclosed sewage tank, but if it occurs naturally in the outside habitat the challenge becomes much greater if not impossible.
l161 - perhaps worth noting this is a very typical temperature range within the entire maritime Antarctic region - without pushing for self citation, see the detailed microclimate data presented and discussed in Convey et al 2018, Polar Biol. from several sites across the region, and also the simple distribution modelling for the also invasive species Eretmoptera murphyi presented by Hughes et al 2013 Biol Conserv.
l185 - is the species recorded from Spitsbergen as well as Bear Island? The former is not mentioned in earlier distributional text.
l187- worth noting that Kerguelen is a sub-Antarctic island which, as with all sub-Antarctic islands, faces much reduced seasonal thermal variation, and a much milder winter than more southern locations in the maritime Antarctic.
l193 - spelling of steinenii
l213/4 - the airfield also serves locations in the northern and southern Antarctic Peninsula, not only the South Shetland Islands.
l216/7 - there are wider references to the risks of transfer with cargo that would be appropriate to cite, in particular papers by JE Lee, and M Tsujimoto.
l245 - although the species hasn't been found in the sewage system of Escudero Station (which is a different station to Frei), I have personally caught adults inside windows in the station in January 2015 (if quoted this would be pers comm P Convey & T Contador) (same comment applies at l254)
l251 - 'identified' or 'confirmed' would be better than 'disclosed'
l279 - generally the IAATO biosecurity measures apply to visitor landings (mainly boot washing and kit cleaning), as these vessels do not normally discharge cargo. However, some cruise ships do send 'gifts' of fresh food to the stations they visit, so there may be a risk there.
l318 - the 'end of the 2017/18 season' has now passed, so has this eradication now been attempted, and what are the initial outcomes thought to be?
l354 - this is overstating what is said in the paper - as above, if the species is established in the natural environment, then none of the mentioned protocols will be appropriate to apply, and it is doubtfull whether any form of 'total eradication' is possible. This should be stated.


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: DRUG THERAPY AND OTHER FACTORS ASSOCIATED WITH THE DEVELOPMENT OF ACUTE KIDNEY INJURY IN CRITICALLY ILL PATIENTS: A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an interesting article about acute kidney injury (AKI) in an intensive care unit of a tertiary hospital in a large city in southeast region of Brazil. The main finding was that multi-drug therapy was the main risk factor for AKI development, which often includes nephrotoxic drugs, and physicians must be aware of this risk. The text is well written and the data presented is relevant. I suggest some minor corrections before publication.
Experimental design: The study design requires to be better detailed because it is a little confusing, for example, it was not clear how the authors have distinguished a patient as having AKI or chronic kidney disease, and which criteria was used for considering complete or partial renal fucntion recovery at the time of ICU or hospital discharge. I also wonder if the study can be considered as cross-sectional or longitudinal (if patients were followed until the time of hospital discharge or only at the time of ICU admission or until the time of AKI development). A flowchart describing all the methodological process would clarify this questions, showing all the moments of the study.
Validity of the findings: In the results it would be interesting to provide more details about the drugs that were used in these group of patients (not only the number of drugs). The reader will certainly have the curiosity to know which drugs were most often used, and which were the potential nephrotoxic drugs. Maybe a table showing all nephrotoxic drugs used, with its frequency would add important information in the results section.
In the discussion section I would suggest to include more studies from Brazil because thera are some important studies of AKI in ICU in that country that should be cited and compared with the results of this paper. I also suggest to discuss a little bit more in detail about some specific important nephrotoxic drugs, such as amphotericin B, vancomycin, polymyxin, once they are frequently used in clinical practice and have important impact on AKI development.
Additional comments: The paper is, in general, well written, the findings are interesting and deserves publication. I suggest to give more details about some specific nephrotoxic drugs and try to compare with other studies in Brazil, once there are some studies in this area in this country that should be cited.


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: DRUG THERAPY AND OTHER FACTORS ASSOCIATED WITH THE DEVELOPMENT OF ACUTE KIDNEY INJURY IN CRITICALLY ILL PATIENTS: A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Overall, I found the manuscript to be well-written and the study design and methods were clearly defined.  Some specific comments for consideration are included below:
1.	Line 66, the sentence beginning “However, the period of functional…” needs to be expounded upon.  What is the cutoff period, etc.?
2.	Line 72, not sure that treatments is the correct word to be used.
3.	The final sentence of paragraph 2 of the introduction starting on line 73; the previous statements do not justify this conclusion.  It would probably be better placed after the next paragraph.
4.	Line 82, nephrotoxic or “potentially” nephrotoxic drugs.  Clarify since it would be unwise to give critically ill patients drugs known to cause nephrotoxicity.
5.	Line 121, should be “for” instead of “of”.
6.	Laboratorial is used twice, line 129 and line 141.  This is not the correct usage of this word.  Consider “clinical chemistry” or something along that line instead.
7.	Line 186, how was p<0.25 chosen for testing in logistic regression models since this is not considered a significant value.
8.	Lines 238-239, it would be good to show the ROC curve for the model when mentioned.
9.	Line 304, should be “for” instead of “of”.
10.	For the dataset, the headings for the columns should be defined at the end of the dataset or beginning.
11.	For the dataset, it appears that there are 125 patients but only 122 were used for analysis.  Why is there a discrepancy here?


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

Input:
Title: DRUG THERAPY AND OTHER FACTORS ASSOCIATED WITH THE DEVELOPMENT OF ACUTE KIDNEY INJURY IN CRITICALLY ILL PATIENTS: A CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors report interesting findings about acute kidney injury in an intensive care unit in Brazil. The corrections were made according to reviewers suggestions, and there is no any other correction required.
Experimental design: The research question is well defined and relevant and methods are well written and correctly described.
Validity of the findings: The finding are very interesting and conclusiong are valid.
Additional comments: The manuscript can 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: FOSSIL SNAKES (SQUAMATA, SERPENTES) FROM THE TAR PITS OF VENEZUELA: TAXONOMIC, PALAEOENVIRONMENTAL, AND PALAEOBIOGEOGRAPHICAL IMPLICATIONS FOR THE NORTH OF SOUTH AMERICA DURING THE CENOZOIC/QUATERNARY BOUNDARY
Review round: 1
Reviewer: 1

Output:
Basic reporting: I am not native English speaker, but it seems to me that this manuscript, or at least some parts, needs a language editing by a native speaker. In the present form some sentences seem to be basic translation from Spanish. There are also many errors to check in the manuscript, as line 32 “wielded” instead of “yielded”, line 191 “have an oval shape”, line 192 “On each sides of the”, line 218 “following vertebral features”, line 223 “due to its comparative reduced size”, line 233 “Regarding the intracolumnar variation”, line 304 “zygantrum” etc.

Minor comments
1.	Line 695, much than localities you are speaking about countries?
2.	On your figures, in my opinion, your snake vertebrae are not well positioned in posterior and anterior views. Traditional position would place the postzygapophyse  articulation surfaces above the prezygapophyse ones, like it articulates in a living specimen. But it is only a small detail.
3.	In the figure 8, I did not understand to what are referring the letter that are together with the snake silhouette (A to G). Maybe you can explain it in the caption?
4.	Last small comment, in the whole manuscript I suggest to use mayor caps for “Tar Pit” like in the title.
Experimental design: Done
Validity of the findings: In the discussion and conclusions, there is one argument that I completely disagree when you use the presence of Corallus and Epicrates in the Plio-Pleistocene to argue that there were “none great climatic environmental change affected the North of South America since then”. It completely disagree with what is known from the global Quaternary paleoclimatic evidences. Yes, Venezuela has not had major changes as those registered in North America with important glaciations but the distribution of Epicrates and Corallus may have change owing to minor climate variations inside your study area. Please try to reformulate your argument in a not so definitive way. Because maybe great climate changes really occur in Venezuela during the Quaternary, but your snakes may have survived in small refugia and dispersed again after.
Additional comments: First of all I want to state that I am not aware of the Venezuelan snake fauna, nevertheless I read carefully the manuscript entitled “Fossil snakes (Squamata: Serpentes) from the Tar Pits of Venezuela: Taxonomical, palaeoenvironmental, and palaeobiogeographical implications”. In my opinion descriptions of the fossil vertebrae sound very well and this manuscript represents an interesting contribution to the knowledge of south American fossil snakes, even if most of attribution are made at genus level only. I strongly support its publication besides a few 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: FOSSIL SNAKES (SQUAMATA, SERPENTES) FROM THE TAR PITS OF VENEZUELA: TAXONOMIC, PALAEOENVIRONMENTAL, AND PALAEOBIOGEOGRAPHICAL IMPLICATIONS FOR THE NORTH OF SOUTH AMERICA DURING THE CENOZOIC/QUATERNARY BOUNDARY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The general organization of the manuscript is good, but, except in ‘Introduction’ and ‘Geological Settings’, English is poor (including many basic mistakes). I think that English is the main problem in this manuscript.
I think that the palaeoenvironmental and palaeobiogeographical contexts should be outlined in the Introduction (what was the knowledge for the concerned time interval before the present study ?). The Introduction deals only with the fossiliferous localities that yielded the studied fossils, and with similar sites, which is not the core of the study.
Descriptions are too detailed (because they are not descriptions of new taxa), parts of them are not useful.
In the Discussion, please clearly distinguish what is confirmation of previous studies and what is new. The Discussion appears to be too long and somewhat muddled. I suggest to shorten it (for example, the report of all fossil snakes from Venezuela is largely out of the scope of the paper) and to clearly state results (e.g., what are the taxonomic contents (for snakes) of the two reported waves that entered South America). The Abstract is more informative than the text itself, which is a question of drafting.
Literature appears to be as complete as possible.
Figures are relevant and of quality. A remark about captions: Anatomical abbreviations are in caption of Fig. 2, not of Fig. 3 as is stated in Fig. 3.
Experimental design: This is an original research that is within the scope of the journal. It may fill a gap (e.g. about elapids), but this should be more clearly stated.
Validity of the findings: There are novelties but they do not readily appear within the provided information (information also includes confirmations of previous works).
Additional comments: Aside from English, which should be improved, results would be more accessible to readers if parts of the text were shortened.


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: FOSSIL SNAKES (SQUAMATA, SERPENTES) FROM THE TAR PITS OF VENEZUELA: TAXONOMIC, PALAEOENVIRONMENTAL, AND PALAEOBIOGEOGRAPHICAL IMPLICATIONS FOR THE NORTH OF SOUTH AMERICA DURING THE CENOZOIC/QUATERNARY BOUNDARY
Review round: 1
Reviewer: 3

Output:
Basic reporting: There are numerous minor instances of idiomatic or grammatical errors in English, most of which do not create ambiguity and are trivial to correct; many but not all of these are marked in the annotated copy.

Background review and literature references appear fully adequate. Zaher et al. (2009) should be cited at first use of Colubroides/Colubroidea, as this is relatively new usage and may be confusing.

Structure of the ms and presentation of data is fine.
Experimental design: The ms reports original work based on previously unreported specimens, much increasing knowledge of the history of snakes in northern South America; it shows a relatively high degree of rigor in description, comparison and diagnosis.
Validity of the findings: Positively stated conclusions are justified by relevant data and arguments; the authors are appropriately cautious in discussing taxonomic assignment of specimens and implications for historical biogeography.
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: FOSSIL SNAKES (SQUAMATA, SERPENTES) FROM THE TAR PITS OF VENEZUELA: TAXONOMIC, PALAEOENVIRONMENTAL, AND PALAEOBIOGEOGRAPHICAL IMPLICATIONS FOR THE NORTH OF SOUTH AMERICA DURING THE CENOZOIC/QUATERNARY BOUNDARY
Review round: 2
Reviewer: 1

Output:
Basic reporting: I have now re-review this manuscript and feel that it is much clearer than the first version. I strongly recommend it for publication, besides I saw a few erratas in the text, but I think they can be solve during proofing.

Not all Tar Pits have been capitalized in the main text. And I think that for JC Rage it must be (in memoriam) and no memorian in the Acknowledgment section.
Experimental design: ok
Validity of the findings: ok
Additional comments: no more comments


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

Input:
Title: FOSSIL SNAKES (SQUAMATA, SERPENTES) FROM THE TAR PITS OF VENEZUELA: TAXONOMIC, PALAEOENVIRONMENTAL, AND PALAEOBIOGEOGRAPHICAL IMPLICATIONS FOR THE NORTH OF SOUTH AMERICA DURING THE CENOZOIC/QUATERNARY BOUNDARY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The English grammar is still very poor in most of the text, and again I have marked (highlight, strikethrough or notes on pdf) many instances that are jarringly incorrect. I rate this as 'minor' in the context of the whole article, but not yet acceptable.
Experimental design: no comment
Validity of the findings: The faulty extrapolation noted by reviewers in the original version has been corrected, and the conclusions are now more robust.
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: MULTILOCUS MOLECULAR SYSTEMATICS OF THE CIRCUMTROPICAL REEF-FISH GENUS ABUDEFDUF (POMACENTRIDAE): HISTORY, GEOGRAPHY AND ECOLOGY OF SPECIATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript examines Abudefduf, a pantropical genus of damselfishes and important component of virtually all coral reefs and tropical rocky shore fish assemblages. The authors estimate a new phylogenetic hypothesis and a time-calibrated molecular phylogeny for the group based on a large dataset of genome-wide data (UCEs) and complete taxonomic sampling. The authors also use this new phylogenetic framework to explore hypothesis regarding how biogeographical, historical and ecological factors affected diversification among Abudefduf major lineages. The phylogenetic results are robust and specially interesting, as the inferences here are made based in an independent dataset from that used for previous molecular studies of the evolution of pomacentrids.

1- In general, the manuscript is well written and uses a technically correct English. However, there are few structure issues. For instance, the introductory paragraph is too long and somewhat confusing. Presenting previous phylogenetic hypothesis along with biogeographical, behavioral, morphological, and feeding ecology information does not seem ideal. I would suggest splitting long paragraphs in shorter text components dealing with particular points.

2- Additionally, I strongly recommend adding a figure comparing the most relevant competing phylogenetic hypothesis to date, the figure can also summarize layers of additional ecological and biogeographical information. This would make easier to understand conflicting and congruent ideas.
Experimental design: 3- I thank the authors for providing sequence data, however I suggest the authors to also add RAxML and Beast tree files to the supplementary data.

4- Fossil calibration scheme is poorly detailed. I suggest indicating the used calibration points in the time-calibrated tree figure. Information about constrained node ages' priors, including lower and upper bounds, are missing. Additionally, there are no fossil placement justifications. If the authors used clade ages recovered by Santini (BMC, 2009) as secondary calibrations, it should be specified in the main text.
Validity of the findings: 5- Habitat shifts have been discussed as a source of ecological release and subsequent diversification in several groups of both fresh water and marine fishes. Here, the authors extensively relied in this general pattern as an explanation to the asymmetric distribution of species richness in Abudefduf. Although sister-clade comparisons suggest that the adoption of planktivory have promoted differential accumulation of species between Clade C and the other two Abudefduf major lineages, it is concerning that there is no clear testing of hypotheses. It is understandable that the small sample size (number of tips) reduces the possibilities of approaching the problem in a hypothesis testing framework, however it also limits possible explanations to plausibility. While speculation is welcome, it should be identified as such.

6- Is Clade C exceptionally diverse? The authors addressed this question using an exploratory approach—BAMM. BAMM is a popular macroevolutionary tool that estimate rates of diversification and infers shifts in diversification-regimes across clades in a phylogenetic tree. Moore et al (PNAS, 2016) have raised important concerns about critical errors in BAMM's the implementation and likelihood function. I was surprised to not see this study mentioned here.

7- Even assuming that there are no issues in BAMM's implementation, the presented results do not support a significant shift in diversification rates in the branch leading to Clade C. The authors argue that the lack of support reflects low statistical power of the test due to small sample sizes. Based in the non-parametric sister-clade comparisons, I suspect that authors' conclusions are generally correct. However, the conclusions are again based only on plausibility and I strongly recommend the authors not to reject a null hypothesis that the observed differences in the number of species in clades of equal age could arise from random variation in speciation/extinction rates.

8- The cause-and-effect relationship between the evolution of planktivory and asymmetric distribution of species richness in Abudefduf is also problematic due to the lack of evolutionary repetition (see de Queiroz, Syst. Biol., 2002). In Abudefduf, planktivory has apparently evolved once (twice, if you consider the recently diverging Abudefduf notatus) and although it might be a recurrent pattern in Pomacentridae, the lack of repetition limits inferences regarding the role of planktivory in spurring taxonomic diversification at genus level.
Additional comments: In general, I find the questions asked here and the results presented fully suitable for PeerJ. I hope you find the comments useful.


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: MULTILOCUS MOLECULAR SYSTEMATICS OF THE CIRCUMTROPICAL REEF-FISH GENUS ABUDEFDUF (POMACENTRIDAE): HISTORY, GEOGRAPHY AND ECOLOGY OF SPECIATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This report covers a lot of ground, from speciation mechanisms and diversification rates to cyptic species and biogeography. For the most part it is clearly written and adheres to scientific standards of economy (a few exceptions indicated below) but is still a long read. The Discussion could be trimmed without loss of information or interpretation.
Experimental design: This is a robust study design with all 19 known Abudefduf species, and multiple sampling across the range of widespread species. Most phylogenetic inferences are based on 100+ loci, providing the nearly definitive statement on sergeant-major relationships. Overall study design is excellent.
Validity of the findings: Findings are robust and very well documented with extensive supplemental materials. One minor point is that starting at 249, ages are presented to two decimal points, which implies much greater precision than is actually available. I recommend rounding to nearest MY.
Additional comments: The Discussion section on local endemics (554 – 628) is interesting but speculative and hangs on a thin premise: Half of planktivores are local endemics, but only a third of benthivores are local endemics. Is that a real difference? I think so, but recommend that this section be trimmed.

618 – 626 Another case of early speciation – coloration divergence was recently published: Whitney et al. 2018.

652: Hobbs & Allen is appropriate to back this statement about hybridization.

MINOR POINTS
Typos: 33, 230, 447, 534, 653, 744, 750, 765, 799, 801, 820, 825, 831, 857, 872, 918, 921, 922, 956, 963, 971

167-168 Some software uses the word ‘version’ and some don’t. Either way, please be consistent.
202 Be consistent in format for describing sample size.
344 Delete ‘should be considered to be’.
365-366 Not sure what this sentence means. Please reword for clarity.
391-392 Please reword.
522 ‘Speciation’ echo. Please reword.
540 Maybe use ‘phylogenetic’ instead of ‘geographic’ here, because this passage is comparing two studies, one population genetics, and the present study which lacks the sample sizes to evaluate population structure.
574 “Fish’ should be ‘fishes’.

Figure 1 caption. Indicate what that light color for the A. cf. vaigiensis Kiritimati lineage represents

Range maps – Should these be listed as supplementary figures?

Potentially useful references:
Hobbs J-PA, Allen GR. 2014. Hybridization among coral reef fishes at Christmas Island and Cocos (Keeling) Islands. Raffles Bulletin of Zoology S30:220 – 226.

Whitney JL, Bowen BW, Karl SA 2018. Flickers of speciation: sympatric color morphs of the Arc-eye Hawkfish, Paracirrhites arcatus, Molecular Ecology Online Early


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: MULTILOCUS MOLECULAR SYSTEMATICS OF THE CIRCUMTROPICAL REEF-FISH GENUS ABUDEFDUF (POMACENTRIDAE): HISTORY, GEOGRAPHY AND ECOLOGY OF SPECIATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have substantially improved their contribution and have addressed all reviewers' comments. I would recommend this manuscript to publication with no further consideration.
Experimental design: The authors have addressed all reviewers' comments
Validity of the findings: The authors have addressed all reviewers' comments
Additional comments: No additional comments


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

Input:
Title: VARIATION IN THE LEAF AND ROOT MICROBIOME OF SUGAR MAPLE (ACER SACCHARUM) AT AN ELEVATIONAL RANGE LIMIT
Review round: 1
Reviewer: 1

Output:
Basic reporting: I really enjoyed the ecological context introduced in the second paragraph of the introduction but I think it could be better integrated with the objectives of the experiment. For example, why is it ‘expected’ that the plant-associated microbiota will be affected along an environmental gradient (L78-79)? Is it because the microbial species pool will be changing, host phenotype/genotype will be changing, environmental constraints imposed on potential microbial colonists, some combination of explanations? Can you provide some recent work that demonstrates any of these mechanisms? A number of recent publications show how exposure to drought modifies the root microbiome, likely indirectly through changes in host phenotypes (Naylor et al. 2017 ISME, Santos-Medillin 2017 mBIO, Fitzpatrick et al. 2018 PNAS). Could a similar mechanism be operating here?

The authors also allude to the possibility that ‘biotic interactions’ may be contributing to sugar maple range expansion to higher elevation (L82-83). This is very intriguing but I’m craving more here. What biotic interactions? Plant-plant, plant-herbivore, plant-pathogen, plant-mutualist? Are you hinting that plant microbiota may be facilitating host range expansion? How? Are associations with particular microbes beneficial under stressful environments? Again, the papers listed above seem to suggest that under drought plant roots associate with a particular lineage of bacteria, with potential beneficial effects. And how might changes in plant-microbe interactions have widespread ecological and economical effects (L85-86)? Will these altered interactions increase growth, cause evolutionary change, facilitate interactions with other plant species, herbivores, pathogens?

Why were fungal communities only characterized in root endosphere compartments? Throughout the manuscript the fungal portion of the project feels a bit under-explained/justified, an add-on.

Sample preparation and DNA extraction (L130-151). Readers will need more detail either in the main text or the supplement to effectively understand these methods.

L133: ‘aboveground surface of the plant’ – does this include stem, floral, petiole etc? Phyllosphere typically means foliar tissue, excluding the rest of the aboveground tissues.

L135-141: I don’t understand how rhizosphere and phylosphere compartments were separated here. Did you remove all leaves from an individual and vortex these to get the phyllosphere? And then remove all roots and vortex to get the rhizosphere? Did you standardize by # of leaves or roots, or biomass of leaves or roots? Given that there may have been differences in growth between seedlings growing at edge versus within sites was this taken into account at the sampling stage or analytical stage, using biomass as a covariate?

L142-151: I don’t understand the sequence of events in this protocol. I think the authors took samples after epiphyte collection and performed an EtOH wash with water rinses, then finely section them before putting them into DNA extraction tubes. This isn’t clear as written though. Do you have a citation for this particular method of surface sterilizing? Same comment as above, did you standardize the plant biomass going into each extraction across tissues, sites? Also why a 45 min. bead beating stage? Did the authors use a vortex for this step or a tissue homogenizer? 45 mins. in a high powered homogenizer would likely shear all DNA.

Biomarker analysis (L227-237): Given that the LEfSe results are so central to your study I would like to see more description of the modeling process here or in the supplement. A brief description of LDA would be helpful for unfamiliar readers and a lucid description of what ‘effect size’, how it is calculated and statistically tested is needed. With 100s-1000s of microbial taxa tested in analyses like these multiple testing can be problematic. Can the authors comment on how this was controlled for?

L 350: Is there any ecological significance of Mortierella. Given its high abundance maybe it has been documented previously as an important endophyte, mutualist or pathogen?
Experimental design: I’m not familiar with forest ecology in Quebec but stating that 100 m is an elevational gradient is a stretch in any system. The authors state that sugar maple’s elevational range is from 600 – 1000 m, why was more an effort not made to capture the extent of this range? Even 400m seems unlikely to result in any large environmental variation but at least it is biologically relevant for the host. Additionally, given the lack of replication of within and edge populations it’s near impossible to attribute any of the differences observed here to elevation, they could be due to confounding spatial variation. This is a major limitation that needs to be addressed much more openly throughout the manuscript.

Why were soil or other environmental samples accompanying each site not collected? This would have been very informative regarding whether host-associated shifts were caused by a shift in the microbial species pool occurring across sites.

Given that all of the sampling occurred on plant seedlings is there any reason to suspect that results may differ across developmental stages of the plant?

Would it be useful to have an additional biomarker analysis which compares epi- and endophytes separately for roots and leaves? The biomarker analysis using the comparison of endophytes to epiphytes pooled across above and belowground tissues may be missing a number of taxa that differentiate epi and endo habitats in each particular tissue.

One analysis that I would like to see is whether or not measures of diversity and composition covary among epiphytic and endophytic compartments across and within root and leaf compartments, and possibly across elevation. Simple correlation for measures of diversity and a Mantel test for measures of composition could do this.

Figure 4 seems to indicate that the variance in community composition seems to decrease at edge sites in root microbiota. Testing for unequal variance might be informative.
Validity of the findings: L326: According to figure 5, only root endophytic compartments exhibited altered diversity across range. Though Table 5 and figure 5 conflict with respect to sig. of rhizosphere compartments.

L 354: I don’t agree with the biological interpretation of these results. To say that Proteobacteria are more associated with leaves than roots just doesn’t make sense. There are members of the Proteobacteria that are very strongly and exclusively associated with roots and at high abundance (e.g. Rhizobiales). Speaking in terms of relative abundance may be more appropriate here and in other instances regarding the LEfSe results.

L357: I’m not following the logic here. How do greater taxonomic associations found in roots lead to greater effect of the environment on belowground host microbiota?

The authors state in L 360 – 363 (and elsewhere: L381, 385-387,  that their results confirm a shift in microbial community structure in leaves and roots across the natural range of sugar maple to its edge. This is much too strongly worded given the inability to actually detect elevation-specific differences in the dataset (see comment in Experimental Design above). Qualifying that the elevational and not geographic range is being considered here is also important as this statement suggests a much larger sampling design than exists (also at L427). Finally, though I appreciate the speculation, that these shifts may be linked to host plant fitness and function is just unfounded. I think if this section were expanded with relevant citations, which demonstrate how shifts in associated microbial communities may be linked to host function, than this would be ok.

In the discussion I was hoping to see more links back to the concepts from the introduction. If we assume that the differences observed here really reflect altered host microbiota across an elevational range, what does this tell us? That the microbial species pool from which host microbiota are assembled has changed? That host phenotype or genotype has changed, thus precipitating a shift in the associated microbiota? Are these changes in host genotype or phenotype being driven by environmental differences across the elevational range, in turn shaping microbial communities (e.g. GXE shown in Edwards et al. 2015 PNAS, Wagner et al. 2016 Nat Comm)? How might changes in associated microbial communities be altering host plant survival or growth across an elevation change?

L404-405: This is misleading. According to figure 5 only root endophytic compartments exhibited statistically different diversity across range.

Fig. 2: What do the light shaded green and red fans mean? Something to do with monophly of association? Why do some clades (e.g. Actinobacteria in panel a) have a fan of one color but individual nodes with a different color?

Fig. 5: no legend for boxplot colors.
Additional comments: Wallace et al. investigate variation in the diversity and composition foliar and root microbiota of Acer saccharum across an elevational gradient. First, the authors should be commended for taking the study of host-associated microbiota out of the lab and into the field. Characterization of host microbiota in natural populations and across environmental gradients is needed for a complete understanding of their ecological importance.

L23-24: Here it sounds like the authors characterized bacterial and fungal communities for each compartment but from reading Table S2, fungal communities were only characterized from the root endophytic compartment? Please clarify.

L52-53: Completely optional, but I’m wondering if the distinction between epi- and endophytic communities should come in now? Here the authors say on the leaves but the study focuses on both in/on leaves and roots.

L62: I completely agree that this is an important question in host microbiome research but I would like to see a bit more explanation here. From an evolutionary perspective, measuring the genetic covariance across traits is important because it can tell us whether pleiotropy and adaptive constraints may exist. I think the same reasoning goes for microbial community structure if you imagine communities across different tissues as ‘traits’, are they controlled through independent mechanisms?

L66: What about sugar maple is ecologically important? Just biomass or something more?

L96: I don’t quite understand how this statement is connected to the current research. Are you suggesting that genetic/phenotypic differences in sugar maple at the expansion front may cause differences in associated microbiota?

L119: Are these trees from an experimental plot or have they recruited to these sites naturally?

Fig S1: The informational content of Figure S1 could be improved. Maybe a Google earth map adjacent to the existing figure that shows the spatial structure of the collection sites? I would like to see information about the spatial separation between sites included in the Specimen Collection section.

L152: Were negative controls used during PCR? Any indication of contamination during the extraction or amplification stage?

L172: Two-step PCR for 16S sequencing seems like it might introduce (even more) bias into the final amplicon pool. Can the authors comment on this?

L202: Just curious why 20 cycles were used for 16S amplification but 35 cycles for ITS amplification? Is this simply due to there being lower fungal DNA yields after extraction?

L265: How were taxa defined as core here? For example, core rhizosphere taxa = found in every rhizosphere, whereas core plant taxa = found in every compartment of every plant?

L331: is the word ‘by’ a typo here?

L360: I don’t understand the ‘associations with planting’ here.

L374-377: This model of microbiome assembly has been proposed. Who knows when it was originally proposed but I usually attribute it to Bulgarelli et al. 2013 AREES.

L421: Here and throughout I think the authors can do a better job drawing from recent advances in host (and particularly plant) microbiome research. We now know that plant immune phenotypes can alter associated microbial communities, that resource availability can trigger immune responses, in turn shaping associated microbial communities (Lebeis et al. 2015 Science, Castrillo et al. 2017 Nature, Hacquard et al. 2017 CHOM). The root microbiome can dynamically respond to abiotic stress, potentially mediating negative effects (Naylor et al. 2017 ISME, Santos-Medillin 2017 mBIO, Fitzpatrick et al. 2018 PNAS).


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: VARIATION IN THE LEAF AND ROOT MICROBIOME OF SUGAR MAPLE (ACER SACCHARUM) AT AN ELEVATIONAL RANGE LIMIT
Review round: 1
Reviewer: 2

Output:
Basic reporting: -	The R code available from FigShare appears to be missing the code to perform the PERMANOVA as described in the text at L251. Please add this code to the file.
-	The raw data (CSV files, R code, sequences) are available through FigShare, though not referred to in the text or as a section of the manuscript.
Experimental design: -	L184, It is unclear why the fungal community sampling and analysis was only done for endophytic roots, when the bacterial sampling was done for four different compartments? Why only sample one component for the fungi, but then make a claim to be broadly comparing bacterial and fungal microbial communities?
-	L264-7, I would not consider classes/phyla to be ‘core microbiome’, but rather lower taxonomic levels, such as species or strains. Additionally, for the Biomarker analysis/ISA (L234), it is not clear why only the phyla level was examined. Illumina sequencing produces only amplicon abundances or short genomic regions, it would be worth adding a qualification for the value of this analysis given that finer taxonomic resolution is not being presented and the discussion of variability of groups across elevational ranges and plant compartments does not reflect specific species counts but sequencing reads.
Validity of the findings: No Comments.
Additional comments: Introduction
-	L66-101, The two paragraphs beginning at L66 (through to L101), the information regarding the specific biology of Acer saccharum and the more general ecological properties of shifting range limits under climate change with abiotic and biotic interactions are too mixed together. I would suggest a paragraph discussing the more general properties of climate change on species distributions and biotic interactions with the microbiome, and then a more specific paragraph on A. saccharum to increase clarity.
-	L83-4, can you describe what this reference found a little more? It seems very relevant and is currently too vaguely stated.

Materials & Methods
-	L143-5, it is unclear how this step in obtaining endophytic microbiota is different, (or another step in the process?), from those steps described at L146-9. What is meant by “the interior samples”?
-	L186, do you mean ITS2 primer, or ITS2 region. Please clarify. Similarly, at L192, do you mean ITS1 and ITS2 primers, or both regions of the ITS?
-	L236, it is confusing to switch between the terms ‘interior’/endophytic vs. ‘exterior’/epiphytic. Please clearly define interior/exterior in the context of compartments earlier in the Methods.

Results
-	L276, This header does not match with the terminology that was used previously, “indicator species analysis” needs to be defined. Previously, the terms ‘indicator taxonomic groups’ and ‘biomarkers’ were used (L227-9).
-	L320, I think you mean Fig. 4e
-	L333-5, The results presented in this line match the corresponding figure 5, but do not make sense in light of the statement presented at L327. How are these results different, does the bacterial diversity differ among the elevations for the rhizosphere or not?

Discussion
-	L351, this might
-	L390-4, But how would this model of experimentation be linked to the effects of the microbiota on plant fitness rather than just the effects of the abiotic environment (re-climate change) on plant fitness?
-	L406-9, You can’t say this for all fungal communities, but only root endophytes which is all that you measured. Please adjust text to indicate this qualification.

Figures
-	The description for Figure 2 is not very clear, please revise to make it clearer that each figure shows a dichotomy in how the different taxa respond to endo/epi or leaf/root.
-	Fig.2B has the opposing color scheme from Fig.3 for root vs. leaf.


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: VARIATION IN THE LEAF AND ROOT MICROBIOME OF SUGAR MAPLE (ACER SACCHARUM) AT AN ELEVATIONAL RANGE LIMIT
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a well-written manuscript describing a study on plant-associated bacterial and fungal communities associated with Acer saccharum (sugar maple) within and at the edge of its elevational range in Quebec. The authors use a high-throughput culture-free MiSeq-based approach to quantify microbial community composition and diversity. They compare the bacterial communities between leaf surface and interiors, root surfaces and interiors, and the fungal communities from within roots, between two sites within and two sites at the upper edge of the elevational range. They find differences between the plant compartments and between plants sampled within and at the edge of the elevational range.

The literature is, for the most part, appropriately used to provide context and is cited appropriately. I was surprised not to see mention of the Fierer et al. study from 2011 (published in Ecology) on elevational ranges of phyllosphere microbial communities, since it seems quite relevant. I've included the citation below. The figures and tables are professionally done, except for a few omissions related to the captions or legends (detailed below).

The raw sequence data are available, however they may be incomplete. The barcode sequences are not provided for 16S or ITS, nor the metadata necessary to demultiplex the raw fastq files, so the raw sequencing data are currently unusable. A quick grep search of the fastq files suggests that they contain just the sequences themselves, and lack the 16S or ITS primers and the barcodes that would be necessary for de-multiplexing them. This information does not seem to be encoded in the fastq sequence names, either, as far as I can tell.

Additionally, there is no mention in the text of where the raw data will be deposited (even a placeholder), so it is not clear that they will be placed into an appropriate publicly available sequence repository (e.g. NCBI SRA) upon publication.

There were a number of minor technical errors in the methods that I point out below.
Experimental design: In my opinion, the research questions inherent in this study (the extent to which microbial communities vary within plants and within and at thresholds of a plant's elevational range) are important ones where additional work is welcome. I agree with the authors that only a few (although the number is growing) plant microbiome studies simultaneously assess the communities in multiple plant compartments. I also think that the question of whether the microbial communities associated with plant hosts change at the edge of the host's elevational range is an interesting one and is worth pursuing.

I would have liked to see some information about the geographic proximity of the two within and two edge sites. My understanding of how the statistical analyses were presented in the paper is that all of the samples were treated independently. However, because there were two sites in each of the major classes (within and at the edge) there may be pseudoreplication within a given site that is structuring the data and needs to be addressed.

The other study design concern I had was in relation to the age of the saplings. From looking at the metadata table provided as a supplement, it looks like all of the saplings sampled from the range edge sites are two years old, while the ones from within the range varied from 2-7 years. How can you be sure that the effects you observed are due to the differences in sapling location (within vs edge) and not just due to differences in plant age? It seems like an additional test on the within range data specifically focusing on sapling age could be used to test for an effect. If there is none, then this should be stated in the results.

The authors incorrectly state that they used the ITS1 primer for their fungal sequencing. The primer they list out fully in the text is ITS1F, the fungal-specific version of ITS1 that was first presented in Gardes and Bruns 1993. These are different primers; the F isn't just for 'forward'.

I liked that the field sampling permit was included.
Validity of the findings: While I have confidence for the most part in the wet lab components of this project (aside from no mention of a negative extraction or PCR controls, and no citation for the effectiveness of the surface sterilization method used -- most studies I've read use a dilute bleach solution in addition to ethanol to remove surface microbes and degrade their DNA), I have several fairly major concerns with the statistical analyses that will definitely need to be adressed before acceptance. The most important one is that I am not convinced that the permutational p values that were presented in association with the PERMANOVA tests are meaningful. In many of the cases where the authors are making comparisons between plant compartments or sites, they would need to block their permutations to only permute within a given plot, compartment, or elevation. Otherwise, treating all samples as independent ignores the structure in the sampling design and could serve to inappropriately lower the p-values derived from the calculations. The authors mention on line 293 that communities within a given elevation (within vs edge) did not show significant between-plot differences, but it is unclear which samples they are talking about here (which compartment, bacteria vs fungi, etc). To analyze differences in compartments, the permutations should be blocked by elevation, otherwise you risk confounding elevation and compartment effects. To analyze differences in elevation, permutation of samples should be blocked by compartment.

These nested permutation designs can be specified with the permute package in R, and then these restricted permutations can be applied to tests such as the PERMANOVA (via the adonis or adonis2 functions in the vegan package). However, these restricted permutations require a balanced experimental design, so in order to use this approach, the authors would need to subsample their data to the least number of samples in a given category, and this will lower their statistical power. It may be that after recalculating the proper p values, the results all remain valid, but I think it is important to correct this before the manuscript is accepted for publication. They should also test for multivariate homoskedasticity, as big differences in multivariate dispersion (such as those shown in the NMDS ordination plots) can influence the conclusions from a PERMANOVA analysis. The authors should also check and verify that the assumptions of the parametric ANOVA (independence, homoskedasticity, normality of the residuals) are met for their Shannon diversity comparisons, or else switch to a test that is robust to non-parametric data.

While I appreciate the authors including the R script they used for their analysis, it seems to be incomplete -- there is no portion of the code that runs the PERMANOVA tests and so it is impossible to assess if these were done correctly or not.

On a similar note, I think it would be appropriate to include the commands and parameters used for the PEAR and QIIME processing of the data. These bioinformatic pipelines have many parameters, each of which can exert a large influence on the final outcome and conclusions of a study and are not otherwise mentioned in the methods as described.

I appreciate the inclusion of the OTU, taxonomy, and metadata tables as supplementary info, but I think the metadata tables in particular could be improved with the addition of the units used in each of the numeric columns and some sort of key (perhaps in a README) to interpret the codes used in the categorical ones.
Additional comments: The wording in the Methods section of the abstract is imprecise. You did not quantify 'bacterial and fungal community structure in four plant compartments' -- you only did this for the bacteria. I'd suggest revising to make this more clear. This is also true in the Results -- you don't show that the communties  differ in all four compartments for both bacteria and fungi, only bacterial data are presented in all four plant compartments. You don't have the data to show that 'the fungal genus Mortierella was also very abundant in all compartments'. Upon reading these I thought that perhaps I was misunderstanding the study design, but table S2 clearly suggests that the only compartment you sampled for fungi was the root interior.

I am not clear why only the fungi from the root interior were sequenced. I think the rationale should be included -- this is particularly confusing because in several places in the abstract, there is mention of fungi being sampled in all plant compartments. I think the bacterial aspects of the work could stand on their own, but I also think it's valuable to have the additional fungal data out in the world. So I would suggest leaving them in and just being a little more comprehensive when explaining the rationale for just focusing on fungi in the one compartment.

Specific comments:

line 183: ITS is not a gene.
lines 210-211: The sequences were removed if the window score dropped below five? Perhaps you mean they were trimmed?
line 220: Why this number of sequences?
line 223: Perhaps this is a typo? It would make sense if it were 280,000
line 239: This is a reasonable cutoff, but then your species accumulation curves are much less informative if they are drawn without these rare OTUs.
line 322: In this paragraph and elsewhere, I think it would be helpful to include the degrees of freedom used for the ANOVA F-test. Since there are so many comparisons being made between and within categories, having the degrees of freedom listed would help to clarify the number of groups and the number of samples being compared.
line 335: Did or did not? Most people generally consider alpha = 0.05 to be a reasonable cutoff, not alpha = 0.1.
line 362: How can you be sure that the effect you observe is due to the range edge and not just simply an increase in elevation (or some other abiotic or biotic factor)?
line 363: But might there also be associational gains? In many cases the diversity is not significantly different even if the community composition is.
line 386: I think this is a bit too strong of a statement. You don't show any evidence that indicates that the microbial communities could be having a causal effect on range edge dynamics.

Figure 3: I would suggest including the legend on this figure -- it would make it easier to interpret at a glance.
Figure 5: It isn't stated in the caption or on the figure which of each pair is within vs edge.

Table 3: I think these p-values should be recalculated with proper restricted permutations. That won't affect the R^2 value, so I imagine the primary trends will be robust, but I do not think that the p values are likely to be accurate.
Table 5: Are these the mean of overall values? vs the means of each of the two elevational classes (within vs edge)? What are the degrees of freedom being used here?

Figure S1: I think a map of the study area would be useful in addition to this diagram. That would help the reader evaluate the proximity of the sites to one another.

Table S1: I think this table could be augmented with some of the data in the 'metadata' table provided on figshare. I'd also be curious about the other factors that could be driving your results -- temperature, surrounding species, substrate changes, etc.

Sources cited:
Fierer, N., C. M. McCain, P. Meir, M. Zimmermann, J. M. Rapp, M. R. Silman, and R. Knight. 2011. Microbes do not follow the elevational diversity patterns of plants and animals. Ecology 92:797-804.


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: VARIATION IN THE LEAF AND ROOT MICROBIOME OF SUGAR MAPLE (ACER SACCHARUM) AT AN ELEVATIONAL RANGE LIMIT
Review round: 2
Reviewer: 1

Output:
Basic reporting: Meets all standards.
Experimental design: Meets all standards.
Validity of the findings: Meets all standards.
Additional comments: A much improved manuscript! Very clearly written and results nicely presented.

Three minor things to fix:

Is it 'Acer saccharum Marsh' or 'Acer saccharum Marshall'? They are both used at different places in the manuscript.

The manuscript switches back and forth between ectophytes and epiphytes. I would stick with epiphytes throughout, since it's the term I think more people will be familiar with.

The ordination figure captions say they are PCoA ordinations, but the axis labels on the figures (and the methods section) say NMDS. Which is it?


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

Input:
Title: A MINIMAL SET OF INTERNAL CONTROL GENES FOR GENE EXPRESSION STUDIES IN HEAD AND NECK SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please check at line 115 the term "previously"
Experimental design: The weaknesses of the study are well described from line 169 to line 188.
In particular, the use of the tissue adjacent to the tumor as a control could represent a strong bias; the authors could add a histological characterization of this tissue in order to confirm its non-tumor origin.
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: A MINIMAL SET OF INTERNAL CONTROL GENES FOR GENE EXPRESSION STUDIES IN HEAD AND NECK SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: the article is clearly written and well structured.
Experimental design: Although the topic of reference gene selection for RT-qPCR experiments is not novel, the experimental set-up by which the authors first screen microarray and sequencing data to come up with candidate reference genes makes this paper stand out.
However, I have one major concern about the method used for comparing and selecting the most stable reference genes. The authors do not sufficiently disclose which software package was used to ompare the reference genes. This is important as some applications (such as Reffinder) do not take the PCR efficiencies into account. And this can impact the ranking of the genes, see De Spiegelaere et al. (Plos One,      https://doi.org/10.1371/journal.pone.0122515)

The authors should use the original software packages for Genorm, Normfinder and Bestkeeper or they should use open source packages (such as packages in the R environment) that allow us to validate whether the algorithms are appropriately used.

Second, the Reffinder tool also performs a combined analysis of reference genes, but here again, the methodology is not based on statistically sound data but a mere summing up of four tools, of which the last (the delta CT) is not even a frequently used method to explore reference gene stability. Hence I would not recommend the authors to use this 'combined analysis' do describe their research results.

Hence, I would strongly recommend that the authors step away from the reffinder tool and use peer-evaluated software packages to reperform their analysis. Or, in case they did not use Reffinder as the sole sourse, then they should clearly write this out.
Validity of the findings: The validity of the finding stands and falls with the use of Reffinder, as small differences in gene ranking may occur when PCR efficiencies are not taken into account.
Additional comments: No additional comments


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

Input:
Title: A MINIMAL SET OF INTERNAL CONTROL GENES FOR GENE EXPRESSION STUDIES IN HEAD AND NECK SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Well written manuscript that is addressing the internal control genes for gene expression studies in head and neck squamous cell carcinoma. However, some point of major and minor criticism should be considered.
Experimental design: This is an original primary research within Aims and Scope of PeerJ. Research question well defined, relevant and meaningful. Presented study performed to a high technical and ethical standard. Methods described with sufficient detail and information to replicate.
Validity of the findings: The study has a limitation related to the small sample size used for validation. However, to select a preliminary list of genes, the authors used data obtained  using different platforms (sequencing, microarrays and qPCR), which significantly reduces the probability of error.
Additional comments: Well written manuscript that is addressing the internal control genes for gene expression studies in head and neck squamous cell carcinoma. However, some point of major and minor criticism should be considered.
Major points
1.	In the comparative  Ct method the authors used 2^  Ct values (also see the difference of designation in table 1B and Figure 2B), but the classic comparative  Ct method operates with values of Mean Standard Deviation (MeanStdDev) of  Ct for each genes in pairs with each other genes from author's discovery set (see ref. Silver et al., 2006). Comparative  Ct method is similar to geNorm methods but uses only Ct values and do not uses amplification efficiency as geNorm does. Authors should add the MeanStdDev values to the manuscript and change the Results and Discussion section accordingly.
2.	Dissociation curves are not enough for evidence of specific amplification. Analysis of PCR products by gel electrophoresis using DNA markers should be done.
3.	According to the results provided by the authors the RPL30 and RPL27 genes show the most stable expression indeed. However, choosing the genes as a minimal set of reference genes has an important limitation that both genes belong to the same metabolic pathway. Therefore, the authors should propose at least one more gene from the other metabolic pathway to exclude an error due to a mutual expression alteration of the ribosomal genes.
Minor points
1.	On Supplementary Figures 1 and 2 provide the diagrams in the same order for reader convenience.
2.	It should be provided the exact values of amplification efficiency for each gene based on the amplification efficiency graphs on Supplementary Figures 1.
3.	In Table 1B delete the "1" from "Normfinder 1" column name or explain what does in mean.
4.	On Figure 2B all axis Y should be named or none of them.
5.	Correct the typo in word "Quantitiatve" in line 106.

References
(Silver et al., 2006) Silver, N., Best, S., Jiang, J., & Thein, S. L. (2006). Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC molecular biology, 7(1), 33.


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

Input:
Title: A MINIMAL SET OF INTERNAL CONTROL GENES FOR GENE EXPRESSION STUDIES IN HEAD AND NECK SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: In my opinion, the manuscript is clearly written, well structured and the references are appropriate
Experimental design: The authors have answered all the points discussed by the reviewers, and the experimental design of the manuscript has now been improved
Validity of the findings: Conclusions are well stated, and this manuscript can be very useful for gene expression studies in head and neck squamous cell carcinoma
Additional comments: The authors have answered all the points discussed by the reviewers, in this way the manuscript has now been improved


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

Input:
Title: A MINIMAL SET OF INTERNAL CONTROL GENES FOR GENE EXPRESSION STUDIES IN HEAD AND NECK SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article is well structured and clear
Experimental design: The experimental design is good
Validity of the findings: The validity of the findings is supported by the results.
Additional comments: I have no major comments on this article. However, since the authors cite the papers from Bustin et al. on the MIQE guidelines I would urge them to provide all data. This means showing all replicates of the standard curves in supp Fig 2, and providing at least a list of all Cq values or preferably provide the rdml files of the qPCR runs.


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

Input:
Title: A MINIMAL SET OF INTERNAL CONTROL GENES FOR GENE EXPRESSION STUDIES IN HEAD AND NECK SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 3

Output:
Basic reporting: Well written manuscript. There are no other comments.
Experimental design: There are no other comments.
Validity of the findings: There are no other comments.
Additional comments: After the corrections performed by authors I have no other comments and I would recommend the manuscript for publication.


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

Input:
Title: A NOVEL LONG NON-CODING RNA, AC012456.4, AS A VALUABLE AND INDEPENDENT PROGNOSTIC BIOMARKER OF SURVIVAL IN ORAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some minor grammar problems.
Experimental design: “A novel long non-coding RNA, AC012456.4, as a valuable and independent prognostic biomarker of survival in oral squamous cell carcinoma” by Hu et al. is  a paper that takes on the task of using the TCGA data on oral cancer and using that do determine lincRNAs associated with patient survival post diagnosis. This is a great idea to use the TCGA data set of OSCC to look for a link between lncRNAs and patient outcomes.
One question is why the authors use Significance Analysis of Microarrays to determine differentially expressed RNAs? This method has largely been replaced by DeSeq2 and a number of other approaches that do not produce the high number of false positives seen with SAM. The differential expression needs to be done with another method besides SAM, a method  which is more reliable.

Line 105 false discovery rate was controlled at approximately 1% -    Please eliminate approximately and should probably put 0.01 not 1%.
Figure 3 What is the y axis- log base 2 or 10? It should be labeled.

There needs to be more explanation of the Gene Set Enrichment Analysis. My understanding is that subjects with short survival were grouped together to find lncRNAs that correlated with that. Out of that lncRNA AC012456.4 was associated with poor survival. If GSEA is then run on the mRNAs that were enriched in the lncRNA AC012456.4 enriched samples in the same dataset then it is really just a search of molecular pathways associated with short survival. To say that it is associated with lncRNA AC012456.4 is misleading and requires a different set of samples, a validation set, to do the GSEA, if I understand how this was done.
Line 270  We found that AC012456.4 expression was significantly down-regulated in OSCC tissue when compared with matched adjacent non-cancerous tissues…		This is redundant as it was just said in line 269.  And since all the RNAs tested for survival correlation were already shown by SAM
 to be differntailly expressed in the dataset it is to some degree circular reasoning. Though it is gratifying that it decreases with OSCC.
This paper would be much better if 100 samples could be set aside to use as a validation set for the survival analysis. Otherwise it is quite speculative. If that is not possible and the paper is accepted I see no value in the GSEA done the way it was done unless a second dataset is used or another approach is used.

Table 3 Univariate and Multivariate Cox regression analysis …
What is the rational of doing age specific analysis of Stage and tumor size? I think there may need to be more validation of why this was done.
Validity of the findings: Due to lack of validation set some of the findings are speculative.
Additional comments: No additional comments


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

Input:
Title: A NOVEL LONG NON-CODING RNA, AC012456.4, AS A VALUABLE AND INDEPENDENT PROGNOSTIC BIOMARKER OF SURVIVAL IN ORAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Method is poorly written and should be re-rewritten and has to include the algorithm used for differential expression analysis.
the followings are examples method that should be revised:
“Download the R and SAM software paceages from the Stanford University website, then install and import into Microsoft Excel. Then, unzip the original data paceage into TXT text file format. Open this in Microsoft Excel and sort the data into the format required for analysis as instructed for the SAM software.”
“lncRNA data for tumor and normal tissue samples with fluorescence signal values of 0 must first be removed.”

What did the authors mean by the following sentence? What is the exact level for FDR? What they mean by saying “the change in fold was controlled more than twice?”
“The false discovery rate (FDR) was controlled at approximately 1%, and the change in fold value was controlled more than twice.”

The majority of discussion is the repetition of the results and does not provide any perspective on the methodology and findings and significance of the findings in relation with other studies.
Experimental design: The authors should provide the information about the definition of each variable. For example, how alcohol habit has been defined?
Smoking is correlated with Head and neck cancer. Authors should justify why they did not include smoking in their univariate and multivariable analysis.
How the author defined the cut-off for expression of lncRNA at 2? Did the author perform sensitivity analysis to check for the other cut-off and possible misclassification?
Table 3: Which variables were included in the multivariable analysis. Those variables and their definition should be mentioned under the table.
Table 5: What was the cut-off value for the category of “decreased number” and “non-decreased number”.
Validity of the findings: NA
Additional comments: English and grammar should be improved


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

Input:
Title: A NOVEL LONG NON-CODING RNA, AC012456.4, AS A VALUABLE AND INDEPENDENT PROGNOSTIC BIOMARKER OF SURVIVAL IN ORAL SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors addressed some of the concerns regarding the manuscript, however, several issues are still remains. Authors stated in the revision : "The false discovery rate (FDR) was controlled at 0.01 (the fold change>2)." FDR is completely independent of fold change. what the author means by this sentence. The authors defined the stagings in the cover letter, however, they  should also include that under the tables that classify patients based on staging. The authors stated that they defined the cut-off expression of lnc-RNA at 2 based on the publication of Sun et al (PMID: 27651312), however, they should find the cut-off based on sensitivity/specificity analysis derived from their own data to classify the patients.
Experimental design: NA
Validity of the findings: The authors stated that they defined the cut-off expression of lnc-RNA at 2 based on the publication of Sun et al (PMID: 27651312), however, they should find the cut-off based on sensitivity/specificity analysis derived from their own data to classify the patients.
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: EXPRESSION AND PURIFICATION OF HUMAN DIACYLGLYCEROL KINASE Α FROM BACULOVIRUS-INFECTED INSECT CELLS FOR STRUCTURAL STUDIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Takahashi and Sakane have used a mix of experiments to assess the production of diacylglycerol kinaseα (DGKα), which is considered a potential target for drug discovery in cancer. Their investigation is especially focused on the purification of the enzyme from baculovirus-infected insect cells. This study might represent a promising approach to obtain the three-dimensional structure of the protein. The authors conclude that the protein is enzymatically active, soluble and homogeneous.

The manuscript is clearly written with the introduction and the references relevant to the study. However, I would like to suggest some editing of the manuscript, where I found some mistypos. For example, in several sentences the word "was" should be changed in "where" as plural is used. Furthermore, the language could be slightly improved in lines 94, 157, 316-317.

The figures are relevant to the work presented and very well labelled and described. However, Figures 1C and E, S1a and b, S2A could be improved to better show the homogeneity in the purification steps (i.e. through a TCA/acetone precipitation or Silver staining before performing the SDS-page analysis).

I thank you for providing the raw data, however I noticed that the data relative to Fig 4. (Inhibitory activity of CU-3) represent a single experiment and reported in Relative activity (%) and not as raw data. It would be useful to carry out an assay in duplicates or triplicates. Furthermore, the future readers would benefit in having the raw data from the kinetic assay and the transformed ones for comparison.
Experimental design: The methods are well described and meaningful.
However, with regard to the kinetic and inhibitory assays,  adding the negative controls (with no DGK and no DGK in presence of EGTA) to the figures 2B, which are missing, will strongly support the statement about the enzymatic activity of DGK. The kinetic assay with CU-3 should be carrying out also with the negative controls (with no DGK) to exclude any side reaction with the ADP-glo kit (most important issue).
Validity of the findings: 1.  The authors assert that they obtained the protein to near-homogeneity and in a soluble form, and it can be concentrated without any loss of the protein.
To confirm this statement, the loaded fractions in the Figures 1C and 1E should be concentrated, using the Silver staining or the TCA/acetone or Methanol/Chloroform precipitation approach, as the protein bands appear too thin. Furthermore, as the full-length DGK contains 12 Tryptophans, 21 Tyrosines and 25 Phenylalanines, it is not clear why in the figure 1D, 2A and S3, the absorbance at 280 nm is too low. As the authors claim that the protein could be concentrated and it is not aggregating during this step, they should perform the gel filtration chromatographies with concentrated samples, which will clarify the homogeneity and help the future readers for the crystallization purpose.

2. The authors suggest that DGK with a molecular weight of ~ 85kDa is around 77 kDa when performing gel filtration analysis. In order to have better resolution and consistent data, I would advise to perform an analytical gel filtration instead.

3. Figure 2B is very nice, however I would suggest adding the negative controls, as ADP-glo assay is a coupled assay.

4. For the kinetic assay and to easy read the results, I would propose adding a table with the Km (in mM for both ATP and DG), Kcat and Vmax, with the relative standard errors, which are missing in the manuscript.

5. For the inhibitory activity of CU-3, the experiment should be implemented at least in duplicate, as it is reported as single experiment.
IC50 value should be reported with its standard error.
Most importantly, the negative control is missing. The assay in presence of CU-3 should be carrying out without DGK, to exclude that CU-3 is not a PAINs compound (Baell J and Walters MA, Nature 2014). The compound has phenol-sulphamide and iso-thiazolone moieties, which might be both redox cycler and covalent modifier. For the reactions involved in ADP-glo assay kit, it is kindly recommended to check the inhibitory activity of this compound against DGK.
Additional comments: I commend the authors for their extensive work in finding the conditions for the production and purification of a challenging protein.
Soluble proteins have also been obtained from bacterial expression. As an extensive work was done, the expression could be optimised with an IPTG titration or auto induction approach.

Conclusions are well stated and linked to the original research question.


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

Input:
Title: EXPRESSION AND PURIFICATION OF HUMAN DIACYLGLYCEROL KINASE Α FROM BACULOVIRUS-INFECTED INSECT CELLS FOR STRUCTURAL STUDIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors provide for the first time an experimental design that allows purification of the full length DGKalpha isoform. Kinetics studies, calcium dependance and inhibitor sensitivity correlate with those reported previously by the group and other laboratories. The article demonstrates the expertise of the teal on this particular area where have contributed with important findings. in this ocasiion, the purification of recombinant DGKalpha opens the possibility for long awaited structual 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: EXPRESSION AND PURIFICATION OF HUMAN DIACYLGLYCEROL KINASE Α FROM BACULOVIRUS-INFECTED INSECT CELLS FOR STRUCTURAL STUDIES
Review round: 1
Reviewer: 3

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

This is an interesting investigation done by Sakane group. Author has done extensive work to optimize the expression and purification of an important human diacylglycerol kinase α enzyme. Several constructs have been made and expression was tested in E. coli and insect cell line expression system. Author makes the point that catalytic domain of the DGKα form soluble oligomer whereas full length enzyme is stable and catalytically active. In vitro assays of the full length DGKα shows that purified enzyme is active and can be used for structural characterizations. The study has been carried out with great care and caution, however, some issues demand proper attention by the authors.

1. Line 285 and 288:  In the enzyme activity assay, it seems author has reported the Km for the ATP and DG but the standard error is not reported.

2. Line298: The IC50 is reported without standard error.

3. Inhibition assay: In the raw data, the DGKα with zero concentration of CU-3 has relative activity of 100% as control. But 0.02, 0.04, 0.08 and 0.16 uM CU-3 treated enzyme has relative activity higher than 100%. I am wondering, what is explanation for this higher activity? If possible, I would encourage the author to repeat the experiment as points on the plot (Fig4) are a bit scattered.

4. No figure legends are provided for supplementary figures.

5. Line 273: correction; To test whether the purified DGKα is biologically active, ……. Should be “To test whether the purified DGKα is catalytically active,………”

Clear and unambiguous; and professional English used throughout in the manuscript. I would recommend this important piece of work for the publication in PeerJ after the raised issues are taken care.


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: EXPRESSION AND PURIFICATION OF HUMAN DIACYLGLYCEROL KINASE Α FROM BACULOVIRUS-INFECTED INSECT CELLS FOR STRUCTURAL STUDIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Takahashi and Sakane performed several experiments for the production of diacylglycerol kinaseα (DGKα), a potential target for drug discovery in cancer. Their investigation, especially focused on the purification of the enzyme from baculovirus-infected insect cells, is a  promising approach for future structural studies. The authors conclude that the protein is enzymatically active, soluble and homogeneous.

The manuscript is clearly written with the introduction and the references relevant to the study. Corrections and new raw data would definitively be beneficial for other scientists.
The addition of standard errors for the activity and inhibition assays is very helpful, as well the table which would be beneficial for other readers.
I commend the authors for their extensive work in finding the conditions for the production and purification of this protein and for the effort made in revising the manuscript.

Conclusions are well stated and linked to the original research question.
Experimental design: The methods are well described and meaningful.

The addition of negative controls to the kinetic and inhibitory assays makes the investigation rigorous and makes the research question well defined.
Validity of the findings: 1. The authors assert  that protein samples from affinity chromatography and size-exclusion chromatography show homogeneity in SDS-page and they decide to retain the Figure 1C and 1E ( and the supplementary one). However, in the gel the bands relative to the enzyme are too thin and some contaminants could be clearly seen below the DDGKα band.
Protein precipitation (i.e TCA/acetone, Ethanol or Methanol/chloroform) protocols are not only used to remove detergents, salts or lipids: they are useful to concentrate a diluted sample. Furthermore, silver staining will definitively show contaminants. For crystallisation purpose,  the protein should be seen as strong band in a SDS-page (which I do not see), and often two chromatography steps are not enough to obtain a pure sample.
Furthermore, as the authors claim the protein could be concentrated, they could have performed a SDS-page with a concentrated protein without using TCA/acetone precipitation approach. This would have clarified the homogeneity and helped the future readers for the crystallisation.

2. The authors affirm their size-exclusion chromatography is clear enough to show the monomeric form of DGKα. The column superdex 200 16/60 is not always able to discriminate between different forms (i.e. dimer/monomer). The reason why I have suggested the use of analytical gel filtration superdex 200 10/300 gl increase relies on the fact that this new generation column allow higher resolution and high-quality data. I do not exclude that their results with GF 16/60 suggest a monomeric state of the protein.
Additional comments: I commend the authors for their extensive work in finding the conditions for the production and purification of this enzyme.
Surely, the expression of this protein could be optimised from the bacterial system described in the manuscript.


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

Input:
Title: EXPRESSION AND PURIFICATION OF HUMAN DIACYLGLYCEROL KINASE Α FROM BACULOVIRUS-INFECTED INSECT CELLS FOR STRUCTURAL STUDIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am happy with the revision authors have made. It is an excellent paper and worthy of publication in PeerJ.