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: EVALUATION OF FULL-LENGTH NANOPORE 16S SEQUENCING FOR DETECTION OF PATHOGENS IN MICROBIAL KERATITIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript concerns the proof of concept application of Nanopore 16S amplicon sequencing as an alternative, quick, accessible, and inexpensive diagnosis tool for bacterial keratitis. The authors identify two important steps to explore and optimize 1) sampling instrument 2) bioinformatic pipeline for classification of the 16S data. The authors find that sampling tool has a big impact on the amount of DNA that is obtained. The authors also find that processing of mapping results and database clustering impacts the apparent classification of the data.
In general, I am a big fan of this type of proof of concept and I believe this type of application of Nanopore sequencing in a clinical setting is possible and extremely valuable. What the authors are doing is important work. However, using 16S for classification is not straight forward and is extremely contextual. On-top of this the high raw error rate complicates this further. I have tried to point out where in the paper I think this needs to be addressed.

1.	A key misunderstanding is that 16S can be used for classification below genus level. There are many examples of different species with the same full length 16S sequences. Hence, you cannot in general do species level classification with 16S. Furthermore, in general it is accepted that two 16S sequences that are less than 97% identical usually originate from different species. The average read identity for Nanopore data is 95%, so it does not make sense to talk about species level classification in this respect either – your reads are almost always so different from the reference that they are considered a different species. So, on a fundamental theoretical level species level classification is not possible.

So why do the authors of this article as well as many others seem to observe decent species level classification of raw Nanopore 16S reads? We have had a bioinformatics PhD student investigating this for more than year and the factors seem to be many and these factors interact in complex ways. The primary factor is the reference database; if the reference database contains sequences from the bacteria in your sample, raw Nanopore reads tend to map to the correct references or closely related references (which can be different species!). So yes, you can observe correct species level assignments if the database you map to only or primarily contain sequences that match the species in your sample. But imagine that your sample contains a species not in the database, when you map to the database your reads will find the next best reference, which is a different species. In worse cases this can be different genus or even family! If this database only contains one closely related sequence most of the reads will map to this closely related sequence making it seem like a strong signal, even though it is wrong. The other factor is how to perform sensible mapping filtering. Nanopore data today has a median accuracy of around 95%, but the range is still 80 – 99%, where the low-quality reads are the real issue, as these reads tend to map more randomly, and hit many different species if you are unlucky.

Does it make sense to do 16S Nanopore sequencing then? It really depends on the use case and how the pitfalls above are addressed. In the case presented in this paper I think it might make sense. A way to do this would be for the authors must identify the species known to be relevant to keratitis and make sure these species are correctly represented in the databases. When doing analysis, only mappings to these species should be considered, as there is too much uncertainty about other mappings in general. The other mappings can be used qualitatively but not in a diagnostic setting before they have been validated. If a substantial amount of reads map to a specific genera known to cause keratitis this can be used as a crude indicator of infection and can likely be used as basis for early treatments if these treatments can be chosen based on only knowing the bacterial genera e.g. if you know all Bacillus infections can be treated a certain way. I think something like the might work, but it needs to be stated as a premise in the beginning with accompanying arguments and the manuscript needs to be purged of considerations of species level classification. It also needs to be backed up by empirical data, and here two patients simply isn’t enough.

2.	In my opinion the introduction contains most of the relevant considerations for this topic, but the flow and argumentation is a bit messy and difficult to follow and many important points are first introduced in the discussion.

-	Line 89: I am missing a transition from culture dependent to culture independent techniques and a very short mention of culture independent options and why sequencing is better than or a relevant alternative to qPCR (Only feasible to look for a couple of specific species with qPCR?).
-	Line 89: I am also missing a sentence presenting the two sequence options: `metagenomic sequencing` and `targeted sequencing`.
-	Line 89 – 94: Here you provide some really good examples of the strength of metagenomic sequencing, but I would like you to say it outright – A completely untargeted approach allows you to discover unexpected pathogens.
-	Line 93: I a missing a bit more background information as to why the running cost is high, which you mention in line 116-117. I would state it here: pathogen DNA usually makes up << 0.1% and host DNA the rest (please find ref and check the numbers) of the sample DNA and hence requires deep sequencing only possible at specialized centers. Optional: You could add there are efforts pushing for solving this problem but they are not “production ready” yet and not compatible with a generic method that can be used everywhere (PMID: 31235920).
-	Line 96: I would introduce the concept of marker genes and state that 16S rRNA is by far the most well established for bacteria, and hence the most obvious to use if you want a general overview of bacteria profiles in a sample. Also explain it is targeted by PCR which enables us to avoid background and sequence smaller parts of the genome, which drastically reduces sequencing depth requirements and therefor price.
-	Line 98: The mention of OTUs in this sentence is weirdly specific. I think I would just say there are several examples of 16S being used to identify bacteria in clinical samples. Add more references than 1.
-	Line 99: Here the authors appropriately include an overview of variables regions used for clinical studies (maybe add refs?), but I am missing some context here, which I think is key. Marker genes can be used but the resolution is generally lower than for `metagenomic` approaches (in some contexts the resolution goes down to genus level for 16S), which is a very important point to make here. The resolution is further reduced by use of only a fraction of the 16S gene. Use this space to qualify what primers you have used in your study. How much diversity do they cover or why this is not important as they cover the important genera for the disease (PMID: 22933715)? How big part of the 16S do they cover? etc. It would also be good to point out that databases has a huge impact on the `resolution` as well, which is why there are many specialized 16S databases out there (http://www.homd.org/).
-	Line 104 -112: I like the points you are making here, but maybe add some stats to make the contrast to the aforementioned techniques/platforms more clear: How many 16S samples in how short time?
-	Line 105 – 108: I would omit these sentences as these details are irrelevant to the points you are trying to make – Yes it can read 6 million bp reads, but your focus is sequencing 1500 bp.
-	Line 114 – 119: I think I would move most of the examples up to when you introduce sequencing as a strategy, because they are universal problems irrespective of platform (ineffective sampling, low pathogen count/high host background and contamination, standardization) and where some are specific for the approach you are advocating (Nanopore error rate and how to handle it bioinformatically), which should stay here. I guess host DNA/low pathogen count is “solved” by using a targeted approach? Reagent contamination can be handled by using appropriate protocols, which you could mention, as your experiments does not address this specifically.
-	Line 119: The key issue for using Nanopore for 16S sequencing is the error rate, which means  conventional 16S pipelines cannot be used. There are several other papers (quick google shows > 20 papers e.g. PMID: 30815250 , PMID: 31980229 – find the best ones) that have dealt with this problem and you should cite and discuss their findings, so you can clearly show how your pipeline differs or if it is the same as some other pipelines give them credit and explain why you chose that particular pipeline.
-	Line 120: I would state more directly in the first line that you focused on optimization of DNA extraction and bioinformatics pipeline to make 16S nanopore sequencing feasible.

3.	Regarding sampling and DNA extraction I recommend using more `space` in the introduction discussing these steps as they are absolutely critical to the final results - as the authors do point out - and many researchers don’t consider this often enough. I would like to see some references to articles showing impact of sampling and DNA extraction to really underline this. This will nicely support your emphasis on the sampling experiment and support why you have chosen ZymoBIOMICS DNA Miniprep kit for DNA extraction, which uses the absolutely critical step of bead beating to obtain hard to lyse bacteria.
Experimental design: 1.	I am missing a clear definition of the success criteria for this study.
-	Sampling: The criteria seems to be high DNA recovery. This can be misinterpreted, as most of this DNA will be host DNA anyway. Technically you are interested in higher 16S bacterial copy numbers. But I see what you mean and because you prove copy number is a function of DNA concentration, DNA concentration can be used as a proxy measurement.
-	16S sequencing: This is unclear. Is it simply to detect specific genera? What about false positives and false negatives? This is essential information to evaluate your results.

2.	The patient cohort is too small. I know this is a proof of concept study, but even these can be too small. I would like to see at least 10 patients samples, and >20 would be better so you can estimate a crude success rate and convince to the community that this a valuable tool.

3.	Line 157: How did you decide how many CFU to add to Ex vivo porcine model of microbial keratitis? Can you provide references that describe this model or support this amount with empirical data?
4.	Line 164: I would like a better overview of the sampling scheme here. How many sample replicates were taken with each method. Was it 6? What does “sampling order was varied” mean? Hopefully, the same eye wasn’t sampled with multiple methods?
5.	Line 183: Add a short line describing the purpose of these qPCRs.
6.	Line 218: Some might wonder why you chose 27F-AGAGTTTGATCMTGGCTCAG; 1492R-CGGTTACCTTGTTACGACTT primers as they are not the best full length primers for capturing diversity (PMID: 22933715). Maybe add an explanation.
7.	Line 240: How many patients?
8.	Line 248: Can you provide a reference to a standard protocol for this “routine clinical practice”? My clinical knowledge is limited, but this might be different around the world.
9.	Line 263: What protocol was used for 16S amplicon prep and sequencing? Is it the references by the end of the paragraph?
Validity of the findings: 1.	The bioinformatics pipeline is not novel and does not warrant the emphasis that it gets. The “filtering” performed on the mapping results is not filtering but rather basic clean up of mapping results – removing unmapped, secondary mappings and supplementary mappings is default in these types of workflows. Comparing “unfiltered” versus “filtered” is therefore not a relevant comparison. For filtering you would look at reference coverage (how much of the reference does my read cover) and reference/query identity (how different is my read from the reference). These are essential checks and cutoffs need to be qualified.

2.	There are some major issues with the mock community validation. Several times there is talk of “species level assignments” (which is not possible with 16S), but the results from the mock data clearly shows that several other species and genera are targeted and the implications of this is for early diagnostics of keratitis is not addressed properly. Line 280-281: I would not expect this few reads to map with the mapping settings you used (only ~14% !!). This indicates that something is wrong. Maybe you need to do adapter trimming (e.g. porechop) for the mapping to work better, or the error rate is not what you think it is (estimate error rate for your data), or there is something else in your library that is not 16S or is concatenated 16S amplicons (check your read size distribution).

3.	I am missing an explanation of why database clustering was considered as a strategy (Line 288). I actually think it is a good idea since it makes the mapping faster and species level assignments are not possible anyway and genera level assignments seem to be decent. But the reason presented is that it improves species level classification, which cannot be done in the first place. Clustering will lump similar sequences belonging to different species together, so of course clustering at lower identities will result in poorer “classification” – you risk removing your reference, which actually seems to be the case for Klebsiella pneumoniae in the SILVA database (Table 3). Clustering actually creates the problem I talk about in comment 1, where your data will map to the next best thing if your actual reference is not present in the database.

Line 276: Please provide us some basic data statistics and QC. Number of raw reads produced and an estimate of error rate (estimate from mapping data directly to the correct references and using cigars to calculate identity), aswell as a raw read length histogram.
Line 288-297: It is unclear what is going on here. Did you cluster the reference databases and then tried to classify them afterwards or are you comparing read classification with reference databases clustered at different identities? The last sentence ends with “… and 297 align them to the NCBI 16S RefSeq database at 100% similarity.” With a 5% error rate there is not going to be any reads that are 100% similar.
Line 312: I am not following this statement. From the 16S Ct values in supplementary table 4 the highest Ct is 23.7, which in my experience usually generates fine 16S amplicons and can be quantified with qPCR. Can you elaborate on the cut off you used?
Line 342: I don’t understand the meaning of “… Minimap2 provided robust alignments that were closely aligned to their mock community taxa”. As far as I can see from the Urban reference, minimap succeeded in mapping most reads from a mock community dataset. Does this mean it is better or is it simply because Minimap mapping settings are more lax?
Line 426: This line seems broken “primers amplifying genome for genus”
Additional comments: Line 102: Many people regularly sequence up to 500 bp amplicons on the MiSeq, so maybe use that number.


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: EVALUATION OF FULL-LENGTH NANOPORE 16S SEQUENCING FOR DETECTION OF PATHOGENS IN MICROBIAL KERATITIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed most of the major concerns I had raised in my initial review, and I have do not have any major remaining concerns at this time, and I am happy to recommend this manuscript for publication.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: N/A


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: COVID-19 INCIDENCE AND MORTALITY IN NIGERIA: GENDER BASED ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: NO Comment
Experimental design: NO Comment
Validity of the findings: Comments

Manuscript Lines 64 to 67
“Of the 118,145 deceased patients, 53% were males, while some other countries such as Peru, India, Burkina Faso, Guatemaia, Kenya, Pakistan, Afghanistan, Thailand, Bangladesh and Nepal had mortality rates of males as high as 70%, 73%, 74%, 74%, 74%, 74%, 76%, 76%, 77% and 81% respectively (UNWOMEN, 2020).”
Reviewer’s comment:
The gender skew heretofore reported and the subject of present study is based on UNWOMEN. (2020). Emerging Gender Data and Why It Matters, Available from: https://data.unwomen.org/resources/covid-19-emerging-gender-data-and-why-it-matters,(accessed September 6, 2020)

The relevant website mentions prominently the following

"Source: Data submitted to NCOVmart reported through the global surveillance system of WHO, as of 14 July 2020.
Notes: Data cleaning are ongoing and in progress. All numbers should be interpreted with caution. As of 14 July 2020, 12,880,565 cases were reported. Data presented here, therefore, represent only 37% of all reported cases."

It is not denied that there may be a gender skew but the reliability of data or relative lack thereof that forms the basis of such conclusion has to be mentioned prominently in the manuscript.

Moreover there are reports of massive underreporting of COVID19 cases globally “Lau H, Khosrawipour T, Kocbach P, Ichii H, Bania J, Khosrawipour V. Evaluating the massive underreporting and undertesting of COVID-19 cases in multiple global epicentres.. Pulmonology. 2020;S2531-0437(20)30129-X. doi:10.1016/j.pulmoe.2020.05.015”

Therefore the extent of underreporting in different countries has to be mentioned and taken into consideration even if the authors speculate that underreporting is more or less equal for male and female members in a given population.

The authors of the present study do admit to the uncertainty of the nature of data the analysis is based on by noting in Lines 254 and 255 that “This study is limited by dearth of information on mortality cases by gender and daily population in Nigeria.”
However in a manuscript with rigorous statistical methods employed such as in the present study, the authors are expected to quantify the extent of underreporting in a way similar to the one carried out in “Lau H, Khosrawipour T, Kocbach P, Ichii H, Bania J, Khosrawipour V. Evaluating the massive underreporting and undertesting of COVID-19 cases in multiple global epicentres.. Pulmonology. 2020;S2531-0437(20)30129-X. doi:10.1016/j.pulmoe.2020.05.015”

Reviewer’s Conclusion: The authors ought to mention the degree or uncertainty of the data that forms the basis of analysis. The said nature of data ought to be mentioned in statistical terms as opposed to subjective and qualitative.
Additional comments: NO Comment


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

Input:
Title: COVID-19 INCIDENCE AND MORTALITY IN NIGERIA: GENDER BASED ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript provides a good overview of what its goal is, namely to highlight the gender differences in COVID-19 in Nigeria. It provides sufficient references in the introduction but has no discussion section. The latter leaves a lot to be desired. Differences in COVID-19 incidence by sex have been noted worldwide, but none of that literature is cited.
Figures could be presented much better, with axes not labelled. In fact, Figure 4 does not have a title. The figures need to be strengthened to make it easy for the reader to understand the message. Tables 1 and 2 are not labelled.
Overall, the paper reads like a draft and needs much revision. Grammar and spelling errors abound.
Experimental design: The methods section does a good job of displaying all the tests that are employed but is not exhaustive. For example, Augmented Dickey-Fuller test is not mentioned in the methods but in the results for the first time.
Validity of the findings: The data provided offer scope for replication. I do not have the specialization to fully assess the veracity of the statistics employed in this manuscript.
Limitations of the methods employed are not described in the discussion section, as is typically expected.
Additional comments: Overall, this manuscript is off to a good start and requires significant revision. Grammar and editing for clarity is much needed. All tests performed should be first mentioned in the methods section. I recommend separating the results and discussion sections- once that is done, more relevant literature ought to be cited in the discussion.


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: GUT BACTERIAL COMMUNITIES IN THE FRESHWATER SNAIL PLANORBELLA TRIVOLVIS AND THEIR MODIFICATION BY A NON-HERBIVOROUS DIET
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript suffers from a lack of clarity due to poor use of English.
Experimental design: I outline the issues that I detected here in the general comments below.
Validity of the findings: No comment.
Additional comments: The manuscript by Hu et al. describes the intestinal microbiota of the freshwater snail Planorbella trivolvis and its modification by an artificial diet. There is merit in the data as there is still scarce information about snail microbiota. Snail intestinal microbiota is of interest not only for basic scientific knowledge but also because it harbors bacteria that can process plant biomass and digest cellulose and lignocellulose, and the enzymes of these bacteria could have biotechnological potential in a variety of industrial processes. The authors used Illumina sequencing of the V3-V4 region of the 16S rRNA gene to study the taxonomic composition of the intestinal microbiota and analyzed it with established methodologies. I think that the work could be published as long as the authors address several issues that I will outline below:

General comments:
The manuscript suffers greatly from a lack of clarity due to many mistakes in the use of the English language. The authors should send their work to an expert editing service to substantially improve readability and clarity.

Specific comments:

Introduction:

1- Please do not refer to mollusks in a scientific paper as shellfish that is a generic term used for a variety of different animals. Call snails, snails, and slugs, slugs, for example.
2- On line 94, the authors mention recent studies that investigated the intestinal bacterial communities of freshwater snails but do not provide a reference.
3-The authors did not give a specific reason for analyzing the effects of a modified diet on the intestinal microbiota. Is there a specific reason for this or a proposed hypothesis?

Materials and methods:

1-Where were the snails obtained? Were they collected in natural environments? Were they part of an established colony?
2-What do you mean by three repetitions? (line 106)
3-Market purchased feed is not clear (line 108). What is the brand? What kind of feed is it? Is it for fish feeding?
4-I understand the need for pooling the intestines but why did the authors only used 5 pooled samples (that would account for 25 snail intestines in total, 5 intestines per analyzed sample). The authors should have a really good reason for not analyzing more samples. At least 10 samples for each experimental condition would be the minimum number for a proper analysis. That would only require 50 snails in total for each condition. Illumina sequencing would allow the simultaneous analysis of dozens of samples so there were no limitations regarding the sequencing run.
5-Please describe the primers used for the V3-V4 region and give a suitable reference or describe how they were designed if this was done by the authors themselves. Please indicate the size of the amplified product.
6-How many PCR reactions were performed for each sample?
7-I do not understand the need for a library prep kit (line 142) as the 16S rRNA amplicons are usually directly sequenced. Please explain.
8- Please supply the R scripts used for the statistical analysis with the vegan package as supplementary material.

Results:

1-OTUs were consistently misspelled as OUT.
2-On line 256, the authors state that phylogenetic analysis showed that genera that were present in a higher proportion in the HV group were closely related but they did not provide the data or figure.
3-On line 269, the authors describe LEfSe analysis but there is no description on how it was done in the materials and methods section. Please provide this information.
4-From line 309 to the end of the paragraph the text is of poor clarity and it needs to be improved to increase readability.
5-As it was done for enzymes related to plant degradation it would be of interest to perform this analysis for enzymes related to protein degradation such as proteases and peptidases.


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: GUT BACTERIAL COMMUNITIES IN THE FRESHWATER SNAIL PLANORBELLA TRIVOLVIS AND THEIR MODIFICATION BY A NON-HERBIVOROUS DIET
Review round: 1
Reviewer: 2

Output:
Basic reporting: The MS by Hu and co-workers describes the differences in the microbiota of one species of freshwater snail (Planorbella trivolvis) in relation to diet (plant-based vs. commercial pellet). In this context, authors carried out a targeted amplicon-sequencing of 16S rRNA bacterial genes to delineate differences in composition, richness and diversity of bacterial communities in the gut of the two groups of snails and also studied the predicted metagenome by using PICRUST. Authors found some differences in the studied variables but results are confusedly presented and discussed. In my opinion the MS is not suitable for publication in its current form. The introduction and discussion sections are week and contains many flaws and confusing terms.

The use of English language is limited and careless. In my opinion, the MS needs a thoughtful language edition by someone with a full proficiency in English. The MS contains many colloquial expressions, confuse terms and speculative statements. Overall, the MS needs a thoughtful revision to improve in clarity and readability.
Experimental design: Despite the research is within the scope of the journal, the research question is not well defined and authors need to highlight why it is important the study of the microbiota of this snail species.

The experimental design has flaws and many details are missing in the Material and Methods section (too numerous to cite here but see some in the general comments section). Some sections of the M&M are especially troublesome and confuse.
Validity of the findings: Authors found some differences in the studied variables and also in the predicted abundance of some genes encoding cellulolytic enzymes. Despite these findings, results are confusedly presented and discussed. Since many methodological details are missing the statistical significance of the findings is difficult to assess.
Additional comments: The term “intestinal (or gut) flora” is outdated and should be replaced by “intestinal (or gut) microbiota” throughout the MS.

Some specific comments are highlighted below:

L26: High compared to what? To NHV snails? How high is high?
L28: intestinal flora is an outdated term, I suggest using “intestinal micorbiota” throughout the MS
L28: herbivorous gut of what type of animals? mammals? other?
L31: These are phylums… this sentence provides no information… of course there will be differences in members of these large phyla. Be more precise. By the way, Bacteroidetes is misspelled.
L37-39: Well, ok but why it is important to study the gut microbiota of these snails? Authors should sell better their story to highlight the importance of their findings.
L41: Keywords is misspelled
L412: the last two keywords are not keywords. Please, delete
L46: I guess that citing the genus here is dispensable.
L47: Species names should be written in italics and in small caps for the species designation (Planorbella trivolvis)
L50: Ribeiroia ondatrae should be written in italics
L57: “a gut microbiome specialized in the…”
L58: Maybe a reference is needed here to support this argument
L59: I suggest rewritting this final sentence here. Write more concisely.
L45: “Planorbella trivolvis belongs to…. I suggest writing more concisely.
L51: “huge ecosystem of bacteria”? Do you mean a huge bacterial diversity in the intestinal tract of animals…?
L65: Again, I suggest getting rid of this outdated term throughout the MS (flora)
L67: “…to efficiiently digest…”
L71: “no dominant phenomenon of certain strains”? What does this means? Please, be precise when writing
L72: replace “flora” by “microbiota”
L79: “Are relatively more” what? Please, be precise when writing.
L83: “functional intestinal microorganisms”? What is that? Again, be precise when writing
L84: Excavation? I’m lost...
L85: delete “snail” as adjective for “metagenomic study”
L87: GH? Please, define
L89: As simply as that? Again, be precise when writing.
L90: Replace “flora” by “microbiota”
L91: A single gene or various? Please, rewrite.
L92: See above comments on the use of “flora”
L93: developing enzymes??
L94: If there are recent studies, please cite them
L99: under conditions of oblivion? Again, I’m lost.
L100: to represent?
L105: many key details are missing: How many animals in total? How many by group?
L112: This part is specially confusing. Also, it is written in present tense instead of past tense.
L122. This section needs explanation.
L129: All together? By individual?
L130: Authors extracted all DNA not only bacterial genomes.
L131: There are many DNA extraction kits by MP-Bio, please, be more precise.
L132: OD? Plese, define
L133: Add reference to the Company.
L134: which primers?
L149: Reference to this software?
L152: Please, cite references by Edgar as author of UCHIME and Uparse, not the web address.
L153-154: Cte QIIME authors.
L163: performed is misspelled.
L165: Analysis of alfa diversity indicators is not statistics but analysis of sequence datasets. Also, please, cite Schloss and co-workers when citing Mothur.
L204-205: 307+90 = 397… but authors stated that they delineated 525 OTUs, so, where are the other 228 OTUs?
...


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: GUT BACTERIAL COMMUNITIES IN THE FRESHWATER SNAIL PLANORBELLA TRIVOLVIS AND THEIR MODIFICATION BY A NON-HERBIVOROUS DIET
Review round: 1
Reviewer: 3

Output:
Basic reporting: Clarity and quality of the writing needs to be improved. Data has been shared with NCBI. See general comments.
Experimental design: Original research. The knowledge gap could be more clearly defined. Overall, seems to be of high quality, but more details on methods needed. See general comments.
Validity of the findings: Findings seem to be generally valid. Conclusions could be more effectively summarized. More detail on data collection is needed.
Additional comments: Summary – Hu et al. describe an experiment where freshwater snails (Planorbella trivolvis) were fed either herbivorous (HV) or non-herbivorous (NHV) feed. Their main focus was on comparing the microbial community of snails fed the different diets. They also examined differences in snail growth and intestinal morphology, as well as potential differences in metabolic functioning; they found that diet had clear effects on bacterial communities and snail traits.

General comments
The study provides new insights into how diet affects the gut microbiome of these snails and other effects of diet. However, the quality and clarity of the writing needs to be improved. There are many typos and grammatical errors, particularly in the Introduction and Discussion, which in some places make it difficult to understand what the authors are trying to communicate. I point out a few examples below, but there are many more. Careful copyediting is thus needed. The introduction should also be revised to more clearly set up the objectives of the paper, and the discussion could be more focused on discussing rather than rehashing the results.  I also think some important methodological details are currently lacking from the Methods (see specific comments below).

Specific comments

Abstract
Line 12 – “invasive” depends on the context? In China they are invasive? They are native many other places.
Line 13 – “in aquarium of ornamental fish market” – this phrasing doesn’t make sense. This is one of a number of sentences that need to be rewritten. I point this out as an example, but there are many others with incorrect or confusing phrasing.
Line 13-14– Unclear what “strong adaptability to food” means. Also, the subject “Planorbella trivolvis is singular, but the verb “feed” is plural. Again, this is one example of an error that occurs repeatedly – subject-verb agreement needs to be checked throughout the paper.
Line 15 – Another example - “which allows them to cross the oversea to China by Ornamental fish trade” – not grammatically correct and confusingly written. Also, “ornamental” does not need to be capitalized (see, e.g., lines 298-306 for other words that are capitalized but don’t need to be).
Line 19 – “The variation of intestinal flora from herbivorous feed (HV)” – again, confusing phrasing. As phrased, the text indicates the focus was on the flora in the feed. It needs to specify that in fact the authors examined the flora in the snails fed different diets.
Line 21-22 – I’m not sure this level of detail is needed in the abstract – the previous sentence already indicated that the main treatment was that snails were fed different diets.
Line 34 – “shifted to” is not really correct. Perhaps, “contrasted with”?
Line 35 – “Functional prediction” – unclear what this means. Also, it would help to explain what COG functions refers to.

Intro
Line 45 – “Planorbella trivolvis is taxonomic belonging to the family Plamorbidae” – unclear
Line 47, 50 (and elsewhere) – Species and genus not italicized
Line 49 – “Trematode” misspelled. This is one of several examples (e.g., perofrmed in line 163, sanils in line 203, miacrobial in line 327) that would be caught by a spell-check.
Lines 61-93– These paragraph could be more focused to outline specifically which gaps occur in knowledge of animal gut microbiomes, which would motivate the work done for this study.
Line 66 – Seems to be redundant with line 58.
Line 94-96– Unclear what the point of this paragraph is.
Line 98-99– “which has shown remarkable food adaptation and survival under conditions of oblivion” – unclear
Line 100 – “represent” is wrong word here
Line 97-102 – This paragraph seems to be explaining the main objectives of the study. Would an additional objective be examining effects of diet on snail growth and intestinal morphology? Also to examine functional differences in the microbiome of snails fed different diets? I think it needs to be clear later on in the paper that those were also objectives, or else it is unclear why those analyses were done.

Materials and Methods
Lines 104-111– Details are needed. What was the origin of the snails used? Were they reared from eggs? If not, what were they fed prior to the study? Were snails that were ultimately used for different samples housed separately? If not, then there may be an issue of pseudoreplication (e.g., if all the NHV group were housed in the same aquarium, then different snails would not be truly independent from each other). What is pellet feed composed of?
Lines 114-117– Need to be in past tense. Phrased as instructions, rather than describing what was done.
Line 122-127– It’s odd to just list these formulas without context. A sentence or two is needed introducing why these formula are being used and what they are.
Line 129 – It’s unclear what the mucosal samples are and why the intestinal contents were mixed with them.
Line 130 – Rephrase to clarify the MP BIO kit was used to do the extraction according to the kit instructions. As written, it doesn’t actually say that the kit was used, just that the instructions were followed.
Line 134 – What specifically were the primers? Is there a citation? Also, the tense here is wrong.
Line 149 – Is a reference needed for Trimmomatic?
Line 158 – Unclear what PICRUSt is – a brief explanation may help.
Line 170-177 – It seems like this belongs earlier, maybe before explaining the molecular work. As I note above, it would be good to explain why intestinal morphology was examined in the Introduction – how does it fit into the overall objectives of the paper?

Results
Lines 180-191- Did the authors test if the initial size/mass of the snails in each treatment group differed at the beginning of experiment? It would be good to know that initial size did not differ.
Lines 192-197– The authors haven’t given much context for these results. It should be clear why the histology was done, and what the differences between treatment groups were, and what those differences mean.
Line 206-207– Rather than “belong to”, I would say the OTUS “include members of” the different taxonomic groups listed. “All of the alpha-diversity indices” – would be good to list them here.
Lines 210-213 - “the rarefaction curves showed clear asymptotes . . . further sequencing would be unlikely to significantly increase the observed microbial diversity detected” – I disagree – they clearly don’t actually fully level off, and the number of OTUS would continue to increase with more samples. But, I don’t think it really matters. What matters is that, for a given level of sampling effort, there are clear differences between the treatment groups. But I don’t think this sentence is accurate as written.
Lines 226-251: It seems like this could be more succinct and summarize the key patterns that are shown in Fig. 3. Overall, I think there are several places in the Results that just seem to be listing taxa (e.g., line 287-292) or other information (e.g., functions in lines 298-303)  that is already effectively summarized in a figure.

Discussion
Lines 340-342: “it was found in experimental procedures that the intestines of NHV snails became easily broken” – where is this reported in the Results?
Lines 342-344: “Second, HE staining of intestinal sections of the NHV group showed that
the fat droplets in the epithelial columnar cells had increased, and striated border
detached.” – which is a measure of intestinal health? and is different that the HV snails?
Line 345-353 – seems to be redundant with the Results. x
Line 361 – “the regulaton of feed” – unclear what this means
Line 362 – “through the feed treatment experiment” – this clause isn’t needed
Line 367-368 – “A large number of Proteobacteria, Bacteroidetes and Acidobacteria” – a large number of bacteria? Or is this referring to high relative abundance? The phrasing is unclear.
Lines 373-379 – Again, seems to be just restating rather than discussing the Results. Also lines 404-408
Line 379-380 – “Due to the limitation of determination methods, many previous studies have reported less at the genus level.” – unclear
Line 384-387 – “Many of the genera in our study were not reported in previous studies, indicating that the intestinal flora composition of Planorbella trivolvis differs from that of other terrestrial or aquatic gastropods.” – this is an overstatement. Since different methods were used, it is important to be cautious in comparing across studies.
Lines 388-403 – The flow of this paragraph, and the links between the different pieces of information, need to be improved.
Lines 397-399 – unclear what this is saying.
Lines 410-419 – Here and elsewhere, reports of results of other studies are described, but it is not clear how findings from these other studies relate to the results of this study.
Line 421 – the reference to “Termitomyces association” is confusing – what is this referring to?
Lines 421-451 – This paragraph could be more concise
Line 466 – “across the phylogenetic diversity of the Planorbella trivolvis” – unclear phrasing
Table 1 – The title of the table only says alfalfa, but both food items are shown. Where did the data on the composition of the feed come from? From the manufacturer?

Table 2 – I suggest putting the letters (a or b) in a different column, or just note that the treatment groups differed for all metrics.

Table 3 – “Indices” rather than “Indexes”. Are p-values from a t-test?


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: GUT BACTERIAL COMMUNITIES IN THE FRESHWATER SNAIL PLANORBELLA TRIVOLVIS AND THEIR MODIFICATION BY A NON-HERBIVOROUS DIET
Review round: 2
Reviewer: 1

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


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

Input:
Title: GUT BACTERIAL COMMUNITIES IN THE FRESHWATER SNAIL PLANORBELLA TRIVOLVIS AND THEIR MODIFICATION BY A NON-HERBIVOROUS DIET
Review round: 2
Reviewer: 2

Output:
Basic reporting: See general comments. Some of the writing could be improved for clarity and to better explain how this study fits into the literature.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: General Comments
Overall, most of my previous concerns have been addressed, but the writing could still be improved. Results could be more contextualized in existing literature to spell out how this study fits in and fills an existing knowledge gap. Information from other studies in the Intro and Discussion is provided without context; the outside literature should be more effectively integrated into the paper. Generally, the flow and clarity of the writing could still be improved as well.

Specific comments
Title - I suggest removing “and their functional prediction” from the title. It’s not clear what “functional prediction” refers to.

Line 15-16: It’s unclear what “without specific feeding” means. I suggest removing or revising.

Lines 21 – I would revise this sentence to move or remove “After 60 days of rearing” – that clause does not modify “the results”, so should go elsewhere in the sentence.

Line 35 – “different” should be “difference”

Line 48 – “Trivolvis” should not be capitalized.

Line 51 – “Echinostome” should be “echinostomes”

Lines 46-57 – The writing here and elsewhere in the paper (particularly portions of the Intro and Discussion) is choppy. For example, “Snails are common in ponds, lakes and marshes. Snails feed on algae, aquatic plants, the fallen leaves of terrestrial plants and various types of detritus.” Writing like that is listing information without connecting the ideas. Use of transition words or other revisions could help improve the flow between those two (and other) sentences. I suggest revising the text throughout the manuscript to improve the flow of the writing.

Line 74 – “Two articles examined the gut microbiota of freshwater snails.” – Why did the authors choose to focus on these two articles? Are they the only two prior studies on this topic? I suggest revising to clarify.

Line 74-100 – I’m not sure the details from these other studies are necessary to include or useful to the reader. I think what is important is to spell out specifically what knowledge is missing in the literature, and the gaps that the current study sought to address.

Line 101 – I would either revise or add a topic sentence that introduces the focus of this paragraph. I think the specific goals of this study merits its own paragraph.

Line 117 – “When sufficient number” should be “When a sufficient number”

Line 118 – “newborn snails were prepared”  - confusing phrasing. Prepared for what?

Line 117-120 – I would revise this sentence – it is awkwardly phrased.

Line 127 – Awkward sentence.

Line 132-33 – “The shells were carefully destroyed and disassembled from the snail body.” – Destroyed? Is that the right word. It’s unclear what that means.

Line 213 – I suggest changing “Growth of snail” to “Snail growth”
Line 215-217: I think the numbers in the text aren’t necessary, since they are provided in the table.

Line 233 – Missing a period somewhere.

Line 234 – “in this image” – in what image? Should a figure be indicated?

Line 237 – “These differences in the results” – Which differences specifically? Phrasing is vague.

Line 253 – “good’s” should be capitalized

Line 281 – missing a space “groups.Others”. Noticed at least a couple other times in the manuscript (e.g., line 288, 384), so would check throughout.

Lines 292 – It’s unclear what “Closer analyses” means. I would revise to clarify.

Line 350 – “This may be affected” Awkward phrasing.

Line 366-69 – It’s unclear why the authors decided to compare snails with bison. Is there a reason to compare these two taxa? If so, the reasoning should be clarified.

Line 370-71 - “Core microbes were considered to be diet generalists under the condition of high variation of diets.” It’s unclear what this sentence is saying. I suggest revising.

Line 372-77 – It’s very unclear what the point of the comparison with these other studies is or what the author’s point here is.

Line 378-79 – Unclear what “in the water environment” means? Free-living in the pond water itself? Or in aquarium water?

Line 401-403 “In this study, the intestinal microbiota of freshwater snails was modified by diet, as well as other animals, such as mammals (Muegge et al., 2011) and fish
403 (Kokou et al., 2019).” – Confusingly written. As written, suggests other animals were included in this study. I know that’s not what the authors actually mean, but the text needs to be revised to actually reflect what the authors are trying to say.

“A similar result reported” – A result reported? I think there needs to be another subject to this sentence.

Line 418 – “snail” should be “snails”

Line 429 – “this will be help” – needs to be revised for grammar

Line 446 – “better adaptation” – adaptation is the wrong word in this context


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: RNA SEQUENCING-BASED EXPLORATION OF THE EFFECTS OF FAR-RED LIGHT ON LNCRNAS INVOLVED IN THE SHADE-AVOIDANCE RESPONSE OF D. OFFICINALE
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: Your introduction on Materials & Methods needs more detail, especially on Plant materials. How often the plantlets were subcultured was not introduced in this paper?I don’t think they were stayed in one bottle for 120 days. And I suggest that you give us a picture of the seedling used in the experiment before the treatments.
Validity of the findings: No comment
Additional comments: In the manuscript ,the authors used high-throughput sequencing technology to identify putative lncRNAs and  investigated their expression profiles in Dendrobium  under different illumination patterns. By comparing and analyzing the sequence data of the treatment group and the control group, the specific lncRNAs were studied for their involvement in the shade-avoidance response of D. officinale caused by far-red light. While, there are still some points , which need to be improved and corrected in the manuscript.
There are other issues that need to be revised:

1) The species and genus names of the experimental materials are confused in the manuscript. Please unify D. candidum and D. officinale.

2) Fig 1, I suggest that you give us a picture of all the seedling(all the different treatment) used in the experiment. There should be a scale as the reference in Fig 1.

3)Line 113 “200 μmol/m2/s” is wrong, please correct it. I think it should be  200 µmol·m-2·s-1.

4) The author made a serious mistake in the introduction of experimental materials in “RNA sequencing (RNA-seq) library construction and sequencing”. Line 124, “According to the phenotypic differences of D. officinale, three treatments-CK, FR1 and experimental group 5”, while we just can just see the results CK, FR1 and FR4. The authors are very careless.

5)In the section INTRODUCTION, the author mentioned that lncRNA has been reported in many plants, but the number of lncRNA identified in this study is far less than that of other species. What is the reason? Is the test data reliable?

6)The logic was weaker in RESULT. Please give us more evidence for the goal about the experiment. In the manuscript, after identified the lncRNA by RNA-seq, qRT-PCR and bioinformatics prediction are the main means for the author to obtain the experimental results. I think you need more evidence to support your results and conclusion.

7) Fig. 5 is very obscure.


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: RNA SEQUENCING-BASED EXPLORATION OF THE EFFECTS OF FAR-RED LIGHT ON LNCRNAS INVOLVED IN THE SHADE-AVOIDANCE RESPONSE OF D. OFFICINALE
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The present manuscript describes the analysis of lncRNA differential expression under different red:far-red light conditions in D. officinale.
Even though the article is well orientated I believe that it should be restructured and some extra analysis included. Some comments and suggestions are included below:

Line 175-180 Results are delivered without any context and statistical significances of differences in plant height, stem length, etc are not reported. The number of biological replications is not clear. I suggest the authors include this information.

Line 195 There is little context to support the study of lncRNA over, for example, gene expression. I recommend the authors to briefly introduce their hypothesis.

The article will benefit from the inclusion of a PC analysis on the structure of the sequencing of the biological replications.

Line 205. I wonder why authors avoid differentially expressed genes. What proportion of all the reads mapped to the genome are mapped in lncRNA? What proportion of genes is affected? Is there any of this lncRNA associated with differentially expressed genes? Can you summarize lncRNAs and putative gene target? Are there any retroelements between these lncRNAs?

Line 290. The authors should consider restructuring this section to help the reader to follow the text. The mere description of a big number of genes and pathways is very hard to follow. Positive or negative correlations should be supported by statistical differences.

The Discussion section goes deep in the analysis of important genes related to these responses but does not mention if these were differentially regulated in the present study.

The inclusion of gene pathways and the annotation of new roles in those should be avoided. The authors should mention the limitations of the current study in assigning roles and functions with only differential expression data.


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: RNA SEQUENCING-BASED EXPLORATION OF THE EFFECTS OF FAR-RED LIGHT ON LNCRNAS INVOLVED IN THE SHADE-AVOIDANCE RESPONSE OF D. OFFICINALE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The current study is well-organized and discussed with good scientific quality. However, the authors should incorporate proposed comments and issues to improve the presentation of their work.
Experimental design: Check the below comments.
Validity of the findings: Check the below comments.
Additional comments: Material and methods
	Line 127 and 132, 12 h/d, 6 g/L. Please use standard style throughout the text, i.e., 12 h d-1, 6 g L-1. Check the entire manuscript and fix this issue.
	Line 177, add a full stop at the end of the sentence (Fan et al. (2017).
	Line 182-187, authors must include the qRT-PCR ingredients profile and volume.
	Provide the developer name and location of SPSS and GraphPad Prism.

Results and discussion
	Please use standard style throughout the text, i.e., mg g-1, g L-1. Check the text, figures, and tables, and fix this issue. E.g., Figure 1. Line 207, 208, 210,
	In table S2-S7, remove dot between (mg·g -1 DW). It should be mg g-1. Further, replace u with the micro symbol in table S6 and S7. Also, check other tables and text for this error.
	Line 249-266, mention the GO IDs along with terms.
	The caption of figure 4D “The green color indicates that the comparison contains this pathway, and the yellow color indicates that the comparison does not contain this pathway”. Please note that there is no yellow color in the figure. It should be green. Please check and modify the caption or color in the figure accordingly.
	Line 304, add the Cytoscape version used in this study.
	In figure 5, authors should add an expression heatmap scale for both figures, indicating the highest and lowest values, to make it self-explanatory.
	Line 303-326, the authors constructed an interaction network of lncRNA-mRNA, which is based on the cis roles of lncRNAs involved in their action on neighboring target genes. Further, the authors searched the coding genes that were 10 kb/100 kb upstream/downstream of lncRNAs and then subsequently analyzed their function. However, I cannot see the validity of this interaction network. The authors must have considered a correlation analysis like the Pearson coefficient correlation analysis to analyze the strength of lncRNA-mRNA interaction in terms of positive or negative interaction/correlation.
	Line 306-326, please cite suitable supporting data, i.e., table S8 or S9 to support your results.
	Line 406, what is the meaning of “29”? Is there was any reference? Please check and add the appropriate reference.
	I suggest the authors cite suitable figure or table in the discussion part as well.
	In figure 10, please explain the abbreviations in the caption.
	Define the abbreviations the first time they appeared in the text. Check the entire document and fix this error, if any.
	Authors must include a separate section of “conclusion”.

Please make sure all the cited references are given in the list and vice versa. Further, prepared references according to the authors guidelines.


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: RNA SEQUENCING-BASED EXPLORATION OF THE EFFECTS OF FAR-RED LIGHT ON LNCRNAS INVOLVED IN THE SHADE-AVOIDANCE RESPONSE OF D. OFFICINALE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: 1.	The authors detected the metabolite contents in D. officinale under different light treatments, such as flavonoid, alkaloid and carotenoid  contents . But why not detect the contents of polysaccharide ? As we known, polysaccharide is the main active component of D. officinale.
2.	The title of Figure 7 is not appropriate, please revise it. qPCR is the abbreviation of quantitative real-time PCR, so you need delete “quantitative”. And the Y axis in the Fig.7 should be relative expression level.


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: RNA SEQUENCING-BASED EXPLORATION OF THE EFFECTS OF FAR-RED LIGHT ON LNCRNAS INVOLVED IN THE SHADE-AVOIDANCE RESPONSE OF D. OFFICINALE
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors addressed the comments and suggestions made in the previous review.


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

Input:
Title: RNA SEQUENCING-BASED EXPLORATION OF THE EFFECTS OF FAR-RED LIGHT ON LNCRNAS INVOLVED IN THE SHADE-AVOIDANCE RESPONSE OF D. OFFICINALE
Review round: 2
Reviewer: 3

Output:
Basic reporting: Dear Authors,
Thank you for revising the MS according to the proposed comments and suggestions. Notably, MS has been improved. I have only one suggestion before acceptance.
Please define PIF in the introduction on the first appearance. phytochrome- interacting bHLH factor (PIF).
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: No comments.


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

Input:
Title: THE EFFECT OF ETHANOL CONCENTRATION ON THE MORPHOLOGICAL AND MOLECULAR PRESERVATION OF INSECTS FOR BIODIVERSITY STUDIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: Seven species were investigated by the authors. I noticed that some of them are the ones whose individual integrity are more sensitive to the concentration of ethanol in preservation. That means the results and conclusion are highly affected by which representative species are studied.
Validity of the findings: With just seven species representing four orders, I don't think a proper general conclusion for insects can be made. Therefore, I recommand the authors to limit the conclusion of the positive results to "at least n percent of insect species". For example, Miridae is the largest family in Heteroptera and its species account for about 1/4 of all true bugs species. Besides, no moth or butterfly species was included in this study. No taxonomist will use the alcohol kept specimens for morphological studies of Lepidoptera, no matter what concentration of alcohol is. Therefore, the finding of "trade-off" is partially right, and the authors are responsible to make the readers be aware of such situation.
Additional comments: No additional comments


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

Input:
Title: THE EFFECT OF ETHANOL CONCENTRATION ON THE MORPHOLOGICAL AND MOLECULAR PRESERVATION OF INSECTS FOR BIODIVERSITY STUDIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is very well written and clear throughout. The context is explained well and is properly documented with respect to the literature. It seems like a long manuscript for what is otherwise a mostly straight-forward report: it could probably be shortened, although I would leave it as is if there is any risk of losing readability.

     It is easier to amplify extracted DNA from dried specimens than from ethanol-preserved specimens, as has been documented frequently in the many studies trying to sequence DNA from older museum specimens. There are other, more important reasons specimens for DNA study are often stored in ethanol, but then there is also the mistaken belief that ethanol-preservation is best. The sentence on lines 58-61 is over-stated and needs to be revised in order not to promote this false notion.
Experimental design: The research questions are clearly presented and tested. The four experiments are clearly presented and the application of spike-in standards for amplicon quantification method in is particularly interesting.
Validity of the findings: The findings quantifiably support the commonly held belief that specimen and DNA integrity are inversely affected by ethanol preservative concentration. The findings are all reasonably supported.

     The conclusions of the paper suggest trying to find a compromise given the needs of the researcher and the taxonomy of the insect specimens. However, the authors neglect another possibility, that is, of transferring material between ethanol concentrations for different purposes. For example, my standard practice is to collect field material into 70% for transport, transfer it to 95% for long term storage in the lab, but then to re-transfer it back to 70% whenever I need to manipulate or transport the specimen. This increases handling time, of course, but it also seeks to have the best of both worlds regarding specimen and DNA integrity.
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: PROGNOSTIC IMPLICATIONS OF CELL DIVISION CYCLE PROTEIN 45 EXPRESSION IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors conclude that low expression of CDC45 has a poor prognosis for HCC, but the mainstream paper is that high expression is associated with poor prognosis in most cancers. If HCC concludes that low expression of CDC45 has a poor prognosis, further confirmation by WB or PCR is required.
Experimental design: .
Validity of the findings: .
Additional comments: Data should be collected more carefully if it claims the opposite of what is pointed out in most cancers.


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: PROGNOSTIC IMPLICATIONS OF CELL DIVISION CYCLE PROTEIN 45 EXPRESSION IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: English language needs attention throughout the manuscript. There are a significant number of grammatical and logical errors so I suggest that the article is subject to English language editing. A few examples are list as below:

Line 27: Reword the sentence to ‘The overall prognosis of HCC is poor’. Also, exploring prognostic biomarkers is to monitor the progression/prognosis of HCC, rather than ameliorating the disease

Line 29: Delete ‘gradually’

Line 34: Revise the sentence to ‘analyzed how clinical features are related to HCC prognosis

Line 36: to describe the DEGs between HCC patients and normal control, and the related pathways.

Line 43: Reword the sentence to ‘CDC45 expression was positively correlated with OS rates among HCC patients’

Line 59: Prognostic biomarkers can only provide information/a prediction of disease progression, not treatment
Experimental design: Line 99-110: Please provide manufacturer information of all the reagents, solvents, and equipment

Line 141: How did you pair the samples? What statistical method did you use to compare the frequency of HCC stages?
Validity of the findings: Line 73: CDC45 was associated with poor prognosis in non-small cell lung cancer. However, you reported a positive association between CDC45 and HCC prognosis. Please discuss such discrepancy in the discussion section

Line 153: CDC45 will be exported out of the cell nucleus after DNA replication, as a method of preventing DNA re-replication. So the ratio of nuclear/cytoplasmic CDC45 is important. Please provide such information.

Figure 1B: The figure showed that the CDC45 score between HCC and normal control was not significantly different (the upper 25% SD bar of the ‘tumor group’ was higher than the 75% SD bar of the ‘control group’). However, the authors are claiming that the P-value was less than 0.0001. Please provide the original data.

Line 171-173: Please use another model to adjust for potential confounders (such as age, HCC stage, tumor size, etc.) in the multivariate Cox regression
Additional comments: The authors investigated the role of CDC45 in HCC prognosis and provided evidence that a lower level of CDC45 was associated with poor HCC prognosis. However, the authors need to improve scientific writing and provide extra information to validify their findings.


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: PROGNOSTIC IMPLICATIONS OF CELL DIVISION CYCLE PROTEIN 45 EXPRESSION IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: •	As a reviewer I could find the translated medical ethics approval document
•	I was not able to find raw deidentified data for Table1.
Experimental design: •	All the 321 patients were not considered for Table1. Here Authors have to detail why all the 321 patients were not been included for each of the clinical pathological variables (ex: Tumor size etc). If they do not have data on some of them then they might have to include another section called “NA” in each of the variables.

•	Similarly, inclusion and exclusion criteria were not described for survival cohort. As a reviewer I see that entire cohort is about 321 patients and in survival graph from figure 1C, i could only see 190 patients. Authors have to describe which set of patients were considered for this figure by describing inclusion & exclusion criteria.

•	Survival graph description in manuscript should not be Fig 3c but it should be Fig 1c (line 166).
Validity of the findings: •	Authors have described in study limitation that they could not do expression levels of CDC45 in transcriptome due to lack of samples but they haven’t explained why they have not considered doing a survival plot of CDC45 expression utilizing publicly available dataset such as GSE 76427. It would serve as validation of survival plot using protein expression and make the study robust overall.
Additional comments: I appreciate authors for putting great deal of effort into this study that details of CDC45 in HCC. This manuscript details a combination of ex vivo studies using tumor, normal tissues and bioinformatics analysis of publicly available dataset to discuss novel findings of CDC45 linked to poor prognosis of HCC.


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: PROGNOSTIC IMPLICATIONS OF CELL DIVISION CYCLE PROTEIN 45 EXPRESSION IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 4

Output:
Basic reporting: a) This paper is well-organized with good background material. I found no significant grammar issues.

b)  Although there are no studies exactly investigating CDC45 as a prognostic marker in HCC, can authors add a couple of studies (PMID: 19136905) that use proteomic analysis for biomarker detection in HCC?

c). The authors have done a good job of organizing the paper in a professional manner. Publicly available raw data is clearly mentioned throughout the paper.

d) Authors should expand more on the results of figure-3  or move the figure to supplementary. I do not think this figure adds much to the study.
Experimental design: The article Fits well within the Aims and Scope of the journal. The following minor improvements should be made to the manuscript :

a) I am worried a little looking at 95% CI HC for CDC45. Can the authors try cross-validation to see if the standard error for CDC45 is stable in both univariate and multivariate cox regression?
Validity of the findings: The authors have done a good job of providing all underlying data needed to replicate the study. The tables and figures are sufficiently clear to understand the results. The following improvements can be made to the manuscript :

1)  I am curious why did authors specifically study CDC45 besides it is reported in the literature since TMA can be performed on multiple genes at once. The authors should add a line expanding on this.

2) Inline 226-27, the claim that " correlation between CDC45 and HCC is related to these functions". I don't think GO and KEGG analysis warrants such speculation.
Additional comments: I think the authors have done a good job of presenting the prognostic value of CDC45 in this paper.
The author's studied the prognostic implications of CDC45 expression using IHC and TMA in HCC. Overall, results and methodology was presented in a clear and concise manner and are of interest to a broader set of audiences. I am curious why did authors specifically study CDC45 besides it is reported in the literature since TMA can be performed on multiple genes at once. (authors should add a line expanding on this)
Since the author's claim that "CDC45 expression is lower in Tumor cells" compared to previous studies, I am wondering whether this can be attributed to comparisons to adjacent normal tissues instead of healthy controls? The authors should discuss this issue/limitation in the discussion section.

  While I appreciate the effort, if authors could do some below minor revisions, that would greatly improve the paper.
1) Expansion of discussion of results of figure-3 or removal if needed.
2) Check standard error stability by cross-validation for CDC45
3) Add a statement about why authors specifically chose CDC45.


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: PROGNOSTIC IMPLICATIONS OF CELL DIVISION CYCLE PROTEIN 45 EXPRESSION IN HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Unfortunately, this result cannot be approved unless the relationship with prognosis can be proved by WB or PCR using HCC tissue.
Experimental design: Unfortunately, this result cannot be approved unless the relationship with prognosis can be proved by WB or PCR using HCC tissue.
Validity of the findings: Unfortunately, this result cannot be approved unless the relationship with prognosis can be proved by WB or PCR using HCC tissue.
Additional comments: Unfortunately, this result cannot be approved unless the relationship with prognosis can be proved by WB or PCR using HCC tissue.


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: PROGNOSTIC IMPLICATIONS OF CELL DIVISION CYCLE PROTEIN 45 EXPRESSION IN HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: English writing has been improved.
Experimental design: Please provide the passage number of the cells and the density of cell seeding.
Validity of the findings: No comment
Additional comments: The authors have responded to the questions well. The manuscript has been greatly 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: PROGNOSTIC IMPLICATIONS OF CELL DIVISION CYCLE PROTEIN 45 EXPRESSION IN HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 3

Output:
Basic reporting: na
Experimental design: na
Validity of the findings: na
Additional comments: The authors have addressed my comments and made the required modifications.


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: PROGNOSTIC IMPLICATIONS OF CELL DIVISION CYCLE PROTEIN 45 EXPRESSION IN HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 4

Output:
Basic reporting: This paper is well-organized with good background material. The authors have addressed all my previous comments
Experimental design: The article Fits well within the Aims and Scope of the journal. The authors did address my comment about 95% CI and performed a lasso regression
Validity of the findings: The authors have done a good job of providing all underlying data needed to replicate the study. The tables and figures are sufficiently clear to understand the results
Additional comments: I think the authors have greatly improved the paper by addressing concerns by the reviewers


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

Input:
Title: EFFECTS OF PHARMACEUTICALLY ACTIVE COMPOUNDS (PHACS) ON FISH BODY AND SCALE SHAPE IN NATURAL WATERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript provides interesting data and analyses that clearly fit the standards of the journal. The quality of the English language seems to be high, however, I am not a native speaker.
Experimental design: Data collection and data analyses are well designed. Research questions are well defined with an identified knowledge gap. Investigations were performed in the high technical and ethical standard.
Validity of the findings: In the first view, the analyses looked a little bit like the data dredging, but the explained possible mechanism how pharmaceutically active compounds can influence the fish body shape or/and scale shape eliminated this feeling. Conclusions are well stated. I have one issue related to the statistical analyses. Authors collected 20 fish specimen per site, so data are spatially replicated, so there could be not the real effect of a significant variable. E.g. just speculation, water velocity can influence body shape, but it also can more intensively flush away the compound. I would suggest including analyses where this issue can be eliminated. Is it possible to test this effect by some mixed models that can eliminate site-specific patterns, e.g. just compare the body and scale shape among sites using some alternative of PERMANOVA or interaction between variables? In the case of non-significant results, the changes in body or scale shape can be caused by pharmaceutically active compounds.
Additional comments: The overuse of abbreviations, e.g. abbreviation of pharmaceutically active compounds is quite disturbing during the reading, especially when the reader is not familiar with the abbreviations (like me). I would suggest changing these abbreviations with full names.


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: EFFECTS OF PHARMACEUTICALLY ACTIVE COMPOUNDS (PHACS) ON FISH BODY AND SCALE SHAPE IN NATURAL WATERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the paper is well written, but there are a few sentences where the English could be edited to improve readability (e.g. lines 66, 207, 273).

References seem appropriate

Article structure is professional and raw data are provided.
Experimental design: This is original primary research with a well defined and approached research question.

Methods are described with sufficient detail & information to replicate.
Validity of the findings: see general comments
Additional comments: In their manuscript, Stazny et al investigate whether exposure to PhACs could possibly play a role in altering the phenotypic characteristics scale and body morphology of three low land fish species.  To this extend river water samples were analyzed from the sites where fish collections took place.  The water analyses are extensive comprising general water parameters, ion measurements as well as a suite of PhACs.  The authors provide references describing that, to date, certain water chemistry parameters are known to have effects on body and scale shapes whereas little is known to this effect for PhACs.
The approach taken is interesting and if thoroughly validated, could certainly be a valuable addition to existing risk assessment approaches.  It is a shame that there is not a single river where the authors could catch all three species which would really have strengthened the paper.
Whilst the discussion has an appropriate length, I feel the introduction could benefit from a bit more detail (e.g. a brief description on current knowledge of effects of water chemist parameters on phenotypes which is discussed in detail in the discussion.  Overall, the paper is well written, but there are a few sentences where the English could be edited to improve readability (e.g. lines 66, 207, 273).

Specific comments:
I am missing some information on the age groups and sex (it has been determined, line 158) of the sampled fish as both measured endpoints (body and scale shape) could possibly differ at different age or sex.  The latter is especially the case depending on when sampling occurred as if fish were sampled during the spawning season, females are definitely rounder in body shape compared to times outside of the breading season.
Are any sampling sites considered as controls, i.e. not impacted by the presence of PhACs?  A classic approach would be sampling upstream and downstream of WWTWs as described for instance by Jobling et al, Env Health Persp 2006 or Tyler & Jobling, BioScience 2008).  As there were different sampling sites along different rivers, it might be the case that fish were sampled up- and downstream of WWTWs, but at the moment this is not clear in the paper.

Was a comparison of body/scale shape performed within one species across the different sampling sites to exclude possible population differences?  A few words to the effect would be beneficial in the paper.

Lines 39- 40	Although E1, E2, EE2 and E3 are common abbreviations, they should be defined at first mention.  Also, what is aE2 (seeing that data for bE2 are also provided in table 3, this cannot me a typo)

Line 47		be more specific where the first PhACs where detected.  It is probably meant in aquatic ecosystems, the aquatic environment or similar.

Lines 83 – 84	Be more specific with regards to ‘relevant national and international guidelines concerning the care and welfare of fish’.  This is a very generic statement and should be supported by references.

Lines 101-135	The methods description for chemical analysis is sufficiently detailed which is nice to see, but I am missing the mention of limits of detection for the different variables measured, especially the PhACs (this could even be included in Table 3.

Line 158	I would question the accuracy of wet weight determined after freezing and defrosting.  It has been shown that post-thaw wet weight differed from pre-storage wet weight (Crane et al 2016, Fisheries Research).  Has any correcting factor been applied?

Line 164/Figure2	Are scale and body shape of roach and chub comparable to gibel carp?  It would be helpful for the reader to show images for all three species in figure 2.

Lines 182-187	It is not entirely clear how the PNECs and especially the assessment factor AF were derived.

Lines 191-192	the log10 transformation is unclear.  Also, how can this be applied to landscape variables which are not numerical if I understand correctly.

Lines 213-222	There could be a little more detail/description explaining the results presented in the corresponding tables.

Lines 213-222	The results described here refer to figures which are informative, but these figures only show certain sampling sites for each species and not data for all sites where the corresponding species were sampled.  What is the reason for omitting some sampling sites?
Lines 229-234	The overall largest joint effect presented in table 10 is for chub on body shape (8%), but is not reported at all in this results section whilst joint effects of 1%? On roach and gibel carp are.  Please add or explain why this has been omitted.

Lines 242-249	It is good to see a cautious statement on possible effects of PhACs on body and scale shapes of wild fish and a mention of possible underlying reasons.  I would argue that there are a probably a variety of other factors too, including genetics and adaptation that, over years/decades, could have led to slight differences in body shape of different, independent populations of the same species inhabiting different rivers/streams that are not connected and that PhACs play a minor role in this.  As mentioned above, phenotypic plasticity, genetics and adaptation are the most likely factors underlying the differences in shape.  Therefore, as suggested, it would be beneficial, to also draw a comparison between fish of the same species collected at different river systems.

Lines 270-290	Nice discussion how certain ions/PhACs could result in different body/scale shape and nicely supported by relevant literature.

Lines 323-325	Good conclusion making the point that more studies of this nature and with larger sample sizes are required.  A critical point to be added here though would be of ethical nature, i.e. the use of too many animals.  Therefore, a comparison between different river systems as suggested might be a first step.

Table 3		Is there a reason that some compounds in table 3 are written in bold, others in italics and others in normal?  If there is a reason, this needs ort be explained in the table legend.

Table 3		What is the difference between aE2 and bE2?  This needs to be defined.


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: EFFECTS OF PHARMACEUTICALLY ACTIVE COMPOUNDS (PHACS) ON FISH BODY AND SCALE SHAPE IN NATURAL WATERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Interesting data that are well presented in relation to existing literature.
Experimental design: The experiment is well designed and unclarities have been well addressed in the revised version.
The only small criticism would be that that age of the fish has not been determined. Having scales to hand would have easily allowed to determine the age of the sampled fish which might have been a useful factor in the statistical analyses, especially seeing the size variation for roach and giebel carp reported in table S1. However, the process is time consuming and possibly not feasible in the time the revision was due.
Validity of the findings: Interesting data and a novel approach that can add to existing risk assessment approaches.  Raw data have all been provided.
Additional comments: More out of interest, in the revised version, it is mentioned that the scale shape of roach at one sampling site differed from most other sites.  Do the authors have any idea why this could be?  IT is well known that roach can interbreed with other cyprinid species such as rudd (Scardinius erytrophthalmus) or bream (Abramis brama) (Wyatt et al., 2006, J. Fish Biol., 69, 52–71. https://doi.org/10.1111/j.1095‐8649.2006.01104.x) and it woudl be interesting if any of these species inhabited this particular stream?


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: FRESHWATER SPONGE HOSTS AND THEIR GREEN ALGAE SYMBIONTS: A TRACTABLE MODEL TO UNDERSTAND INTRACELLULAR SYMBIOSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors performed successful infection experiments using a freshwater sponge and their native symbiotic Chlorella algae as a model organism to test hypothesis of interactions and specificity. The authors analyzed the host transcriptomic data to identify key genetic pathways in response to the established symbiosis. In the analysis they used symbiont-free sponges as the control group for the differential expression analysis. The research question is well defined, and the study is relevant to a broad range of scientific community.
Experimental design: General remarks:
The study is missing a key information on the genetic response of the algal symbiont. The authors mentioned that they did not assemble the algae transcriptomic data due to lack of genome references to this particular symbiotic strain. It would be beneficial to the study to perform de novo transcriptome assembly of the symbiont and perform the genes expression comparison between the free-living cultured strain and the symbiotic strain. The AUGUSTUS software can be used to predict genes from protein homology information when mapped to other available Chlorella genomes.
Minor points:
Please specify the method/ kit that was used for preparing the RNA libraries by the LC sciences
Line 222 says that you sequenced with NovaSeq 6000 sequencing system LC Sciences, but line 268-269 says that libraries were sequenced on Illumina HiSeq 4000 platform. Please fix this.
Line 237-238 the sentence is not completed.
In the results the authors are encouraged to include a table with all upregulated and downregulated genes.
The discussion is very lengthy and can be shorten for at least the first few pages, some sections can be moved to the introduction.
Validity of the findings: The authors draw their conclusions based on comparing  gene expression values between symbiont-free sponge and sponge re-supplied with cultured algae, 24 hours post-infection.  The TEM provide evidence that symbionts colonized the host (Figure 4), however, the authors did not provide a supporting data or information that this 24 hours time point is critical for detecting a genetic response associated with the established symbiosis. The findings in this study would be more supported if gene expression data were evaluated at different time points.
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: FRESHWATER SPONGE HOSTS AND THEIR GREEN ALGAE SYMBIONTS: A TRACTABLE MODEL TO UNDERSTAND INTRACELLULAR SYMBIOSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article entitled "Freshwater sponge hosts and their green algae symbionts: a tractable model to understand intracellular symbiosis" is well written, interesting, and novel transcriptome study shedding light on sponge-Chlorella photosymbiosis.

As pioneers in the transcriptomics of this attractive symbiotic system, I suggest that the authors provide an extra figure(s) with a general model with cellular/metabolic pathways that depict their DEGs/main findings. This will make a stronger point when proposing this photosymbiosis model system.

The figures, specifically the microscopy and pictures provided are excellent. However, figures related to the differential expression analyses need bigger fonts, bigger figures, and self-explanatory labeling.

Finally, the authors should change the Symbiodiniaceae nomenclature at different parts of the paper (see LaJeunesse, et al 2018, Current Biology and the reviewed pdf version).
Experimental design: The authors claimed at line 283: “We do not have a genome sequence available for the sponge-derived Chlorella-like algae and thus were not able to map algal transcripts from the RNASeq data set at this time”. This explanation is insufficient to explain the lack of algal transcriptome analysis, the authors could do a de novo transcriptome assembly and map the algal reads to this assembly. It is understandable that in this paper the authors are not focusing on the algal portion, but this should not be the full explanation, perhaps the authors could solve this issue by simply removing this sentence.
Validity of the findings: The discussion section could be improved when considering the following points:

1. Line 598: Do the authors lookup for glutamate synthase, and ammonium transporters differential gene expression? Include information when possible.

2. Line 600: This part of the discussion needs to be enriched, and the function of asparagine synthetase and its link with nitrogen assimilation/metabolism should be clearly stated.

3. Line 603-611: This part of the discussion could be enriched with the perspectives and future experiments, such as direct manipulation
of the nitrogen sources (amendments) and or carbohydrates such as maltose, to get deeper insights on this.
Do the authors found a carbohydrate/ nitrogen transporter differentially expressed among apo vs. symbiotic sponges?
Could the authors provide a broader comparative approach by using the Hydra- Chlorella genes (carbohydrate/nitrate/ammonium transporters) from Hamada et al., 2018 (or others), and assess their expression in this sponge-Chlorella study? This will only enrich this study and will shed light on the gene expression conservation (if any) when comparing diverse taxa establishing symbioses with this green algae.
Additional comments: The authors will find comments and revisions throughout the pdf file.


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

Input:
Title: TAXONOMIC REVISION OF THE THREATENED AFRICAN GENUS PSEUDOHYDROSME ENGL. (ARACEAE), WITH P. EBO, A NEW, CRITICALLY ENDANGERED SPECIES FROM EBO, CAMEROON
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment beyond minor matters in the attached line-by-line feedback.
Experimental design: The paper is not experimental, so no comment.
Validity of the findings: No comment
Additional comments: The discovery of Pseudohydrosme ebo is an important find and a significant range extension for this highly endangered and very small tropical West African genus. The new species is clearly very closely related to, but sufficiently morphologically and biogeographically distinct from, Pseudohydrosme gabunensis. The contrast drawn between the leaflet venation of P. gabunensis and P. ebo appears to be overstated, but the species are nevertheless distinguishable on inflorescence characteristics, supported by the marked geographic disjunction. Pseudohydrosme itself is *extremely* closely allied to the genus Anchomanes, and likely to eventually be subsumed into it. The authors have taken a prudent, conservative approach by describing the new species in Pseudohydrosme, but it would also be open to them to describe the new species in, and transfer the already recognised two species into, Anchomanes, though that could perhaps await a better sampled molecular analysis than is currently available. I think this should be acknowledged in the paper. One fairly significant matter is that the authors stress that Pseudohydrosme and Anchomales are sister genera, but this has not actually been demonstrated in the molecular phylogenetic analyses cited, wherein each genus has been represented by only one species with the consequence that it is impossible to discern if Pseudohydrosme is sister to, or nested within, Anchomanes. This is rather cryptically acknowledged in the paper, yet conflicts with the bald statement that the genera have a sister relationship. I have itemised some further minor errors and quibbles in the attached line-by-line review.


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

Input:
Title: TAXONOMIC REVISION OF THE THREATENED AFRICAN GENUS PSEUDOHYDROSME ENGL. (ARACEAE), WITH P. EBO, A NEW, CRITICALLY ENDANGERED SPECIES FROM EBO, CAMEROON
Review round: 1
Reviewer: 2

Output:
Basic reporting: Authors are reporting a new species P. ebo. Authors provide all the detail very effectively and the study is reproducible in all aspect. However, authors did not performed some important analyses.
Experimental design: The study is well explained and reproducible up to the data authors provided in the manuscript. However, I have some concerns as authors did not perform pollen and molecular related studies.
Validity of the findings: The addition of pollen grain study and molecular markers can make the study completely valid.
Additional comments: Dear authors,
I appreciate your efforts and you have provided a lot of detail.
I have some suggestions.
1. You have mentioned the use of microscope in methodology but the reader can not get your point from methodology that which kind of characters you examine. Please improve it.
2. You need to provide complete detail of the characters of the three species of pseudohydrosme in a table instead of mentioning 2 species only. This will be helpful to provide all relevant information to the readers.  Please shift details related to the genus pseudohydrosme, its sections, and of two other species into supplementary. I think these information can make the reader confusing. You need to just provide a short summary of the characters of the genus and its section as well as each species instead of providing the details which is already well documented. Then, you can provide complete detail of P. ebo. You can also follow Cusimano et al. and can provide a list of character as descriptors which you have focused on  pseudohydrosme and its related genera in the methodology. This will be good to make the methodology further clearer.
3. I think you have not provide data about the morphology of pollen. I think the electron microscopy of the pollen analyses will be great to get further insight into the similarities and variations among the species of pseudohydrosme.
4. You can see the molecular data is already reported for the genera of Araceae. You have already cited those article therefor I am not going to mentioned those articles. I think the generation of the molecular data of even few loci will be better to provide molecular level evidence. Please avoid the used of trnH-psbA and matK as the recent study show the shifting of these genes from LSC to IR in Anchomanes. Moreover, the recent study also show the effect of rate Heterotachy.
https://www.sciencedirect.com/science/article/pii/S0888754319308420
https://link.springer.com/article/10.1007/s00239-020-09958-w
The phylogeny can be constructed in comparison to previous data. You just need to sequence 3-4 loci per species for only species of pseudohydrosme.
5. Please improve the last lines of discussions. You are providing some suggestions but I feel the language can be improved by rewriting and by shuffling information of those lines.


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: TAXONOMIC REVISION OF THE THREATENED AFRICAN GENUS PSEUDOHYDROSME ENGL. (ARACEAE), WITH P. EBO, A NEW, CRITICALLY ENDANGERED SPECIES FROM EBO, CAMEROON
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Referees comments and corrections have been appropriately addressed, and I recommend acceptance subject to correction of some typos (see below)

The author should check that all references in the body of text are italicised and include a comma before the date: there are some inconsistencies.

I draw particular attention to a typo in line 576 where the population increase is surely 20-fold not 200-fold as stated!

Line 216: African
258: delete 2nd comma after fornicate, and slash before very
407: full stop after clade
438: Sasaf et al., 2005
497: comma after 4–6
516: 2–5 [i.e. en-dash not hyphen]
518: 3 mm [i.e. add space]
530: en-dash, not hyphen, after Sept.
576: 20, not 200
670: locular
699: fonts for half and three quarters are different
707: comma after cylindrical, and close up 50 and en-dash
714: 2–5 [i.e. en-dash not hyphen]
717: pores


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: RAPID RELATIVE INCREASE OF CRUSTOSE CORALLINE ALGAE FOLLOWING HERBIVORE EXCLUSION IN A REEF OF EL SALVADOR
Review round: 1
Reviewer: 1

Output:
Basic reporting: Elías Ilosvay and colleagues perform a fish exclusion experiment and survey benthic cover. The methods are appropriate, and the results are clear. The responses to reviewers are complete. I really see no problems. I recommend accepting the manuscript.
Experimental design: Appropriate
Validity of the findings: Strong
Additional comments: No additional comments