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: CRYSTAL STRUCTURE OF HUMAN ACINUS RNA RECOGNITION MOTIF DOMAIN
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well-written to present the structure of the Acinus RRM domain. There are a few points which could be improved:

Line 33:  "includes the RRM domain" - This makes it sound as though the RRM is part of p17 and not vice versa.

Line 36: location of the caspase-3 target sequence - the text in parenthesis - typically, the references are made starting from the N-terminus and going towards the C-terminus. Also, the current description with 4 AA apart is a little non-scientific.

Line 46: Eponymous - please simplify to ensure international audience can easily understand the text

Lines 76-94: The introduction section gives a through background on Acinus and is extremely well-cited. But lines 76-94 come across as lines of facts stated, which are not particularly adding to the background. In my opinion, some of the references could be combined together to make it sound like a story. And, maybe even include some background on the C-terminal helix (Optional).

Line 182: Artifact spelling

Line 260: "right in the middle" - it is a little colloquial

Figure 2 Legend last line - it should be disulfide "bond"

I would like to add that the figures are well made and the consistency in the color scheme is definitely helpful to the reader. The color for the RNA strand in Figure 6B could be changed to better distinguish it (optional).
Experimental design: No comment.
Validity of the findings: Line 215-232: The section on "comparison with other RRM structures" - the manuscript presents a good comparison of Acinus RRM with other existing structures. While, the article discusses the findings from overlaying with RNA15 and U1A (basically figure 4 and 6), the discussion for figure 5 is missing. Rest of the figures have a clear finding that adds to the story and conclusions of the paper. But, what is being learnt from figure 5 that gets added to the conclusion of the entire paper?

I would like to add that the comparison with RNA15 and U1A are well presented.
Though for figure 4A : the PDB for RNA15 shows the presence of a C-terminal helix, which is missing in the figure.
Additional comments: I would like to commend the authors on successfully solving the structure of Acinus RRM. Overall, the manuscript is well written and conveys the relevance of the work.


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

Input:
Title: CRYSTAL STRUCTURE OF HUMAN ACINUS RNA RECOGNITION MOTIF DOMAIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is written in a clear language and is easy to read.
The introduction is well written and serves to place the research into the structural and biology context. Nevertheless I found the sentence in line 71 a bit confusing because it is not clear to me if serine phosphorylation refers to caspase-3 residues or to acinus (they seem to be very far from the RRM). It is also unclear to me if caspase-3 also causes cleavage at N-terminus of Acinus p17 form
Figures are of high quality, although it is advisable to include labels for secondary structure elements for easy following descriptions in the text. In particular when comparing with other RRM structures.
References are well chosen in general. Perhaps the example of Csft64 (Perez-Canadillas and Varani 2003, EMBO J) should be mentioned in line 255 as an early case of a C-terminal helix blocking the RNA binding interface.
The PDB code (6G6S) of AcRRM should be reported in the article and in Figure 2

The PDB validation report has been provided. The structural data is of good quality.
Experimental design: Molecular biology and crystallographic methods are described in good detail.

I noticed this RRM domain has not been automatically annotated in the protein database, perhaps due to non-canonical RNP1 and RNP2 motifs. Because no experimental binding data has been presented, discussion about the RNA recognition properties should be based in a thorough bioinformatics analysis. However I believe that it is poorly presented in this case. For instance in figure 3 B a conservation map is shown over the structure, however it is not clear which Acinus homologues have been chosen to build this map. A figure with a sequence alignment of acinus homologues from different species should be provided at least as supplementary data.
I have check (blast) that Acinus RRM is very similar to Fox1 with sequence identity much higher (around 31%) than for UA1 and RNA15. Structural comparison with Fox1 should be included in the analysis (lines 200 to 208)
Validity of the findings: The article describes the structure of human Acinus RNA recognition motif that contain a basic RRM-like fold with an extra C-terminus helix. Although there are similar structures with this extra secondary element in similar configuration, the novelty of Acinus RRM is that the C-terminal helix contains the cleavage site of caspase-3, which is being proposed by the authors as a possible regulator of RNA binding activity. Nevertheless such hypothesis is only presented at theoretical level and there is not experimental validation. The article would benefit from doing simple binding experiments (e.g. EMSA with Acinus RRM and C-terminal truncated mutants).
The protein crystalizes as a disulphide crosslinked dimer (Figure 2). In my opinion, authors overlook the potential biological significance of this finding. They mention cysteine 1083 is conserved across species but at the same time claim that the disulphide bridge is probably a crystallization artefact. It should be noticed that the RNA binding properties of a dimer would be dramatically different to the monomer. Hence a more detailed investigation of the oligomerization state of this protein in solutions seems mandatory to me. I would suggest simple gel filtration experiments comparing wild type and C1083 mutant (e.g. to Ser) behaviour under oxidizing and reducing conditions.

In my opinion some parts of the article are of little relevance and describe vague concepts:
Lines between 209 and 213 describe two very small insertions in loops, which I think of little significance giving the high variability of loops across the different RRMs. I recommend removing this paragraph.
Lines 269-278 discuss some common themes in RRM RNA recognition, suggesting that Acinus RRM might follow similar strategies. But there are no real evidences/clues/hints in this work that support any of these mechanisms, apart that the RRM fold. I regard this part as superfluous
Additional comments: At the light of the structure, the potential biological significance of AcRRM dimerization should be experimentally addressed or at least discussed in comparison with other RRM homodimers

Comparative band shift experiments with wild-type and a C-terminal truncated version (at caspase site) should be performed to provide some experimental foundations for 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: CRYSTAL STRUCTURE OF HUMAN ACINUS RNA RECOGNITION MOTIF DOMAIN
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well written for the most part, but there are several grammatical errors which make sentences confusing, as well as several instances where words appear to be missing. The use of commas needs to be improved throughout the manuscript.

The figures are well put together, but there are some issues with clarity.

The following issues should be addressed with the manuscript and figures before publication...

1)	Figure 4A and 4B. Change the colors of the other RRM’s to be different from one another. Having both be light grey and dark grey gives the impression that you are showing two different conformations of the same protein. Additionally, the left most black arrow in 4A is pointing to a different spot in 4B. My understanding is that they should both be pointing to the same place.
2)	Figure 5C-5E. The light grey is very hard to see. Change this to a more visible color. Additionally, what is the purple highlighting representing in these figures? I did not see this stated in the caption for 5B-E, but it was mentioned for 5A. Is it the same thing for B-E? Please clarify.
3)	Figure 5 Caption. Silver-Blue is not the best way to describe the color. Purple would be better.
4)	Figure 5A. Why is RNP2 presented first, when throughout the manuscript and in the caption RNP1 is mentioned before RNP2? Move the RNP1 alignment to before the RNP2 alignment, unless this was for a specific reason.
5)	Figure 2 Caption. Change “bound” to “bond” and change “artefact” to “artifact”. Also, clarify in the caption that the disulfide bond is between C1083 from different chains.
6)	Capitalize the letters in all presented PDB codes (i.e. PDB code 5FJ4).
7)	Line 124. Add “a” before HiTrap.
8)	Change all mentioned amino acid residues to one letter code and number throughout the manuscript. On line 68, for example, the residue is listed as aspartic acid 1093, but D1093 is more appropriate.
9)	In the Crystallization portion of the methods, please provide the vendor for the Index Screen. This is especially important as this screen gave the crystal used for data collection.
10)	Line 171. Change “restrains” to “restraints”
11)	Line 182. Change “artefact” to “artifact”
12)	In the Results section, Figure 2A is never referred to.
13)	Line 195. Change “extents” to “extends”
14)	Line 212. Change “the loop 5” to “loop 5”
15)	Line 242. Change “few” to “several”
16)	Please revise the sentence starting at line 244 and ending at 246. It is a bit unclear in its current form.
17)	Line 267. Point (ii) is very unclear. Please revise.
18)	Line 63-64. Please add commas to this sentence. In its current form it seems to be a run-on sentence and is a bit unclear. Commas in the appropriate places would help the clarity.
Experimental design: No comment.
Validity of the findings: The structure reported is novel, and seems to be the first reported structure of the human Acinus RRM domain, but the authors never explicitly state this fact. A cursory search of the PDB returns no other published structures of this protein. If it is indeed the first time this protein structure is being reported, this fact should be added to the manuscript.
Additional comments: I would recommend this manuscript for publication, provided all issues are addressed.


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: IDENTIFICATION OF SNPS POTENTIALLY RELATED TO IMMUNE RESPONSES AND GROWTH PERFORMANCE IN LITOPENAEUS VANNAMEI BY RNA-SEQ ANALYSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: More and precise details of how SNPs were identified are needed, especially, the minimum depth coverage, min number of reads calling SNPs, MAF. It is not clear why max missing 0.5 was used.

The reviewer found this manuscript very lengthy (especially in the results and discussion part), and I strongly suggest that the authors should make it short and concise.

The manuscript could benefit from discussing and citing calculating the allelic imbalances, in addition, the exist/does not exist of SNPs studies in rainbow trout.
Experimental design: A small number of fish was used especially in the disease part. The RIN  quality value of the RNA was 1.8-2.2, I am not sure if this a typo or not because this is not an acceptable value.
Validity of the findings: The major problem of this study is that none of the SNPs were validated, at least 10 SNPs or so should be validated by sequencing, or by any other platform, outbred populations should be tested as well.  Otherwise, it is hard to make sure these SNPs are high-quality true SNPs.
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: IDENTIFICATION OF SNPS POTENTIALLY RELATED TO IMMUNE RESPONSES AND GROWTH PERFORMANCE IN LITOPENAEUS VANNAMEI BY RNA-SEQ ANALYSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: In this manuscript, Camilla A. Santos and colleagues employ RNA-seq analysis to explore the SNPs related to L. vannamei growth performance and innate immune responses. L. vannamei is of great economic importance in worldwide aquaculture. Shrimp growth and immunity are of great interest for aquaculture activity. The results reported in this study enriches the genomic databases available for shrimp. And also, may be potentially applied in high-density chips and high-density linkage maps for Genome Wide Association Studies, association analysis between complex traits genotypes and phenotypes, and allelic-specific expression studies. This study is of interest. However, the following questions should be addressed before recommendation for publication.

Line 54-61, the introduction of shrimp immune system is not precise and comprehensive, e.g. “crustaceans do not own a real immune system”. Shrimp does not have the adaptive immune system but has a powerful innate immune system. Shrimp Toll-like receptors/IMD-NF-kB signaling pathways (PMID: 21827783; PMID: 19232438) have an equivalent role to mammalian TLRs/TNF- NF-kB cascades. Nucleic acid-induced antiviral immunity (PMID: 23773856) and RNAi-, phagocytosis-, and apoptosis-mediated innate immune responses should be extensively described in this part (PMID: 24886688). Otherwise, this part will mislead the readers.

Line 111, “Shrimps exposed to WSSV (WSSV group)”. The sample size is too small, four samples may not representative enough for SNP study (line 117-118). Can the authors explain why they choose muscle and hepatopancreas for cDNA library construction but hemolymph for WSSV-qPCR test? The primers and detailed testing method should be given for testing WSSV.

Line 333, “Shrimps exposed to WSSV (WSSV group)”. The authors report that inhibitor of apoptosis has SNPs in L. vannamei exposed to WSSV. Shrimp IAP1-3 has been shown to play roles in host defense against WSSV (PMID: 23967321), the potential mechanism, such as blocking apoptosis against WSSV infection, should be discussed.

It is amazing that all the polymorphisms are in coding regions. Can the authors give a rational explanation? Are similar findings reported by other publication?


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

Input:
Title: IDENTIFICATION OF SNPS POTENTIALLY RELATED TO IMMUNE RESPONSES AND GROWTH PERFORMANCE IN LITOPENAEUS VANNAMEI BY RNA-SEQ ANALYSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have improved the work significantly during revision. I recommend its publication now.


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: SPECIATION AMONG SYMPATRIC LINEAGES IN THE GENUS PALYTHOA (CNIDARIA: ANTHOZOA: ZOANTHARIA) REVEALED BY MORPHOLOGICAL COMPARISON, PHYLOGENETIC ANALYSES AND INVESTIGATION OF SPAWNING PERIOD
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study is deserve to be published in Peer J after minor revision.
Experimental design: Experimental design is good enough to be published. However, I suggest the authors to add extra experiments and analyses in the future study if authors hope to do them.
Validity of the findings: Finding is deserve to be published after minor revision.
Additional comments: This study, entitled “Speciation among sympatric lineages in the genus Palythoa (Cnidaria: Anthozoa: Zoantharia) revealed by morphological comparison, phylogenetic analyses and investigation of spawning period”, attempted to delineate Palythoa species boundaries and to clarify the relationship among four species groups by multiple independent criteria. The manuscript is clearly written and unambiguous language. And the data presented in this study are enough to indicate the complexity of speciation with hybridization, and I believe the results by this study are very informative for speciation study not only about Cnidaria but also other organisms. In my opinion, I agree with author’s conclusion, the sentence in abstract as follows; “the morphological and reproductive results suggest these lineages are four separate species, and that incomplete genetic lineage sorting may prevent the accurate phylogenetic detection of distinct species with the DNA markers utilized in this study, demonstrating the value of morphological and reproductive data when examining closely related lineages”.
	I have two suggestions for your future study; first, you may try to observe “real” spawning about your targeted four species group to confirm the extent of pre-zygotic reproductive isolation. There are some papers about sexual reproduction of Palytoa tuberculosa including spawning time (i.e. Hirose et al. 2011; Polak et al. 2011), so it may not be difficult for you to try it. And if possible, you may try heterospecific crossing to clear whether stronger pre-zygotic reproductive isolation is occurred or not.
Second, you may try haplotype network analysis using exon region and structure analysis using neutral region such as MS and/or SNP. Of course, these analyses will not be able to distinguish between hybridization on this time and incomplete lineate sorting, however, you may take outline of some extent of genetic introgression among species groups.

Minor points

Line 77: tuberculosa and P. mutuki. (Fig.1, Table 1).
    Change “Fig. 1” to “Fig. 2”.

Line 122-135
    Did you check ovary only? You should indicate the reason why that you did not check testis in addition to that these Palythoa are hermaphrodite or gonochoric.


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: SPECIATION AMONG SYMPATRIC LINEAGES IN THE GENUS PALYTHOA (CNIDARIA: ANTHOZOA: ZOANTHARIA) REVEALED BY MORPHOLOGICAL COMPARISON, PHYLOGENETIC ANALYSES AND INVESTIGATION OF SPAWNING PERIOD
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Speciation among sympatric lineages in the genus Palythoa (Cnidaria: Anthozoa: Zoantharia) revealed by morphological comparison, phylogenetic analyses and investigation of spawning period” submitted to PeerJ was evaluated. This manuscript is quite dense with several very relevant approaches. The authors attempt to reconstruct, based on molecular data and patterns of gonadal development, the evolutionary history of the species recognized for the study area. The manuscript in general is very good and the authors were very careful in the elaboration of the materials and methods, something increasingly rare these days. Anyway, I have some issues that should be addressed before the final acceptance of the manuscript.

One point that bothered me a bit was the text that justified the non-inclusion of other ALG11 sequences. The authors state “…except for ALG11, as no closely related sequences (e.g. Anthozoa) were included in the dataset”. This statement is not correct, as there are a few dozen anthozoan sequences of ALG11 on Genbank, including Zoantharia sequences. In this way, I suggest to the authors the reformulation of this definition (I do not think it is useful to include sequences in this analysis), since it passes incorrect information.

Another point that I think deserves attention is the phylogenetic trees presented. I think that deserve to be in isolated figures. The authors presented the reconstructions in separate topics for each marker, so I think that trees must be independent figures, because that can help in reading the text. I assume I was quite confused with the figure many times to be able to recognize each pattern.

Still on the figures, I suggest for authors to add the time scale in figure 7 (Species Tree). This is quite simple and can greatly help for future studies in biogeography.
Experimental design: The use of unusual molecular markers can be a very difficult factor. However, the authors addressed this issue very well. Anyway, I do not totally agree with the adoption of filtering methods in a very short sequences dataset. In this way, I suggest to the authors the inclusion (maybe as supplementary material) of an alignment via MAFTT (without any filtering) and new phylogenetic reconstructions (perhaps only ML). In a reconstruction at the genomic level (transcriptomics, etc.) the use of filtering is acceptable (even recommended), but in very short sequences the loss of information may be too high. The inclusion of these data may help to verify variations caused by the phylogenetic reconstruction method and may also be useful for some future studies on the group.
Validity of the findings: no comment
Additional comments: After these modifications, fairly simple, the manuscript can be accepted for publication. The manuscript brings together very relevant data for publication and the discussion has enormous value not only for the group, but for understanding some evolutionary patterns in reef environments.


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: SPECIATION AMONG SYMPATRIC LINEAGES IN THE GENUS PALYTHOA (CNIDARIA: ANTHOZOA: ZOANTHARIA) REVEALED BY MORPHOLOGICAL COMPARISON, PHYLOGENETIC ANALYSES AND INVESTIGATION OF SPAWNING PERIOD
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript is generally written in a clear manner. Occasionally the authors use ambiguous sentence structure and terms, which are noted below. References are sufficient for the reader to understand how the work fits into the broader field. The structure of the article is clear and easy to follow. The figures are of good quality overall, however, additional clarity is needed in figure 2. It would benefit the reader to clearly label each species within the figure to highlight morphological differences between the species. The raw sequence data is freely available. However, it is unclear when reading the methods section that additional data from genbank was used in this study, this should be more apparent to the reader. The description on lines 185-187 obscures this, as the number of sequences generated in this study is reported and then they are aligned with sequences in genbank. It would be much easier to follow if that paragraph ended with the total combined number of sequences for each locus.
Experimental design: The authors of this study are attempting to define a set of morphological and molecular markers that discern between closely related lineages in the genus Palythoa. The authors first collected individuals belonging to each lineage based on their morphological features and the local habitat. The authors then attempted to differentiate individuals from the four groups based on morphology (tentacle number, cnidae, and timing of reproduction) and using molecular tools including a newly developed nuclear marker (ALG11). For the most part, the methods are reproducible, although, as I describe below, could have gone further to address the questions at hand.
Validity of the findings: The conclusions of this paper based on the data the authors collected could have gone further to disentangle whether these lineages are species or populations.
1.	These species are highly morphologically variable (lines 365-371) and the authors suggest that this contributes to difficulty in classifying species (lines 58-59). Furthermore, these species all occupy different “microenvironments” but are also found sympatrically (lines 73-77). The analysis would greatly benefit from careful statistical control to ensure that environmental variation is not contributing to statistically significant signals of phenotypic differences between these species. Especially given the fact that the molecular data (Fig. 6) do not overwhelmingly support differentiating these 4 lineages into species, it is crucial to control for environmental variation in the morphological differences the authors describe.
2.	The molecular dataset does not seem to support the 4 lineages and the discussion should address this issue. First, the mitochondrial genes appear to be inadequate to address species-level differences. Slow rates of mitochondrial evolution are well known in cnidarians (e.g. Daly et al. 2008), which means that these markers are likely inadequate in discerning the relationships of recently diverged species. Second, if P. sp. yoron actually comes from a hybridization event, one would not expect it to form a monophyletic clade with P. tuberculosa in the ITS gene tree. Third, it is true that the ALG11 gene tree supports incomplete lineage sorting as well as hybridization between these groups. However, it seems difficult to rule out the possibility that the ALG11 gene tree reflects the fact that these lineages are all the same species. The most obvious signal in the molecular data appears to be that there are two groups (P. mutuki  and P. aff. mutuki; and P. tuberculosa and P. sp. yoron). The discussion should address if these lineages should actually be divided, in light of the molecular data and taking into consideration that patterns in the morphological data could be due to environmentally driven phenotypic plasticity.
3.	It is concerning that some loci are entirely missing from some species, including the ITS locus which appears to be more informative about relationships than the mitochondrial loci. Although the authors supplemented the dataset with sequences from genbank, the manuscript would be greatly improved if the individuals that were collected for the purposes of this study were the source of the genetic data. This is especially crucial given that the study is aiming to find morphological and molecular differences between these lineages. The inference would be strengthened, for example, if the authors could definitively state that the individuals that they used in the morphological portion of the study fell within the clades shown in Figure 6.
4.	The statistical analysis of the morphological differences between the species would be improved by using a model that accounted for the replication within and between colonies. The sample size appears to be high in the table reporting these differences but they come from sampling 7-13 colonies of each species. The authors should consider using a statistical test that accounts for the replication within colonies when testing for differences between the species.
5.	The data that the authors present here could provide evidence for whether Palythoa sp. yoron is the result of a hybrid speciation event. The authors cite previous work (Reimer et al. 2007a) that this species appears to have increased levels of variation (“shared additive patterns”) due to the hybridization event. This hypothesis could be further tested with the addition of another nuclear marker (ALG11). However, the authors should be cautious if they use this pattern to argue for hybrid speciation. As the authors point out, shared variation can also arise from incomplete lineage sorting, and increased nucleotide diversity could arise from the populations experiencing different demographic histories. It will be difficult to differentiate between these alternatives with so few loci.
6.	The estimated divergence time is reported as 147,000 ybp or 113,000 ybp depending on the model. Uncertainty estimates around these numbers would make these easier to interpret for the reader and would strengthen the argument that divergence was relatively recent.
Additional comments: Line 68: Consider removing “apparently”
Line 77: Do you mean to refer to Fig. 2 instead of 1?
Line 107-108: The final sentence of the paragraph is redundant, please consider removing it.
Ling 123: Remove “in”
Line 134-135: Final sentence is awkward, maybe consider changing to “The end of the estimated spawning period was defined as the point where the number of developed/developing ovaries reached 0%.”
Line 148-152: This sentence is confusing, why is the marker better for resolving relationships if it has no introns?
Line 203: change “codons” to “codon positions”
Line 204: The phrase “because of homogenizing substitution rates between loci” is confusing, did you do this to avoid homogenizing substitution rates because they are known to vary by codon position?
Line 268-271: This sentence is confusing. It is difficult to determine when small holotrichs were present and absent based on the construction of the sentence.
Line 422: “three first” is awkward, is it the three first species? It might be clearer to write: “all species or species groups were reciprocally monophyletic with the exception of P. tuberculosa”
Line 437: Should “within” be “between?”
Line 445: The phrase “hierarchy of timing” is used multiple times (e.g. Lines 465-466) and it is unclear what that means, please consider adding a brief explanation the first time you use it to guide the reader.
Line 450: This statement is likely not only true in marine lineages, reproductive isolation can be a driving force in sympatric speciation in terrestrial lineages too. Please consider adding a reference to reflect this.
Figure 6 caption: Please consider revising “highest posterior density interval,” explain what the interval means.


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: SPECIATION AMONG SYMPATRIC LINEAGES IN THE GENUS PALYTHOA (CNIDARIA: ANTHOZOA: ZOANTHARIA) REVEALED BY MORPHOLOGICAL COMPARISON, PHYLOGENETIC ANALYSES AND INVESTIGATION OF SPAWNING PERIOD
Review round: 2
Reviewer: 1

Output:
Basic reporting: Basic reporting is fine, the phrasing and paragraph structure that was previously unclear is much improved. However, there was one sentence that is unclear on lines 340-346. The purpose appears to be to show how and why the P. mutuki group is not monophyletic but it is very difficult to parse. Although the paragraph would be less compact, it would be easier to read if that sentence was broken up into smaller sentences.
Experimental design: The experimental design is within the aims and scope of the journal. The questions are well defined and the approach to addressing them is clear throughout the paper.
Validity of the findings: The findings are valid and the statistical analyses are sound. The conclusions are well-stated and are reasonable given the patterns in the data.
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: MOLECULAR DYNAMICS ENSEMBLE REFINEMENT OF THE HETEROGENEOUS NATIVE STATE OF NCBD USING CHEMICAL SHIFTS AND NOES
Review round: 1
Reviewer: 1

Output:
Basic reporting: I would better detail the simulated annealing protocol (see comments below).
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The work by Papaleo and coworkers uses experimental restraints (typically
averaged on long times and multiple conformations) to force,
in an average sense, molecular dynamics simulations to be consistent with them.
Average restraints allow the system to explore
conformational space thus providing realistic simulations.
The approach is assessed by comparing the results with experimental data
not used as restraints in simulation.
The results show that the approach is valid and they also further support the validity
of the refined force field charmm22*.

The work is well written and arranged in a very logical way.

I find only the methods section not very detailed. I imagine readers
will like to implement the same protocol in their own simulations.
To this end the protocol should be detailed and, possibly, tools made
available.
In particular the SA protocol should be detailed:
"In the simulations, each replica was evolved through a series of simulated
annealing (SA) cycles between 304 and 454K for a total duration of 0.6 ns per
cycle. We only used structures from the 304K portions of the simulations for
our analyses."
it is not clear exactly what is the protocol, is the temperature dropped
linearly or non-linearly at each step, or in discrete steps?
The cycles at higher temperature start from the lowest temperature structure
from the previous cycle?
Could the authors detail better the protocol in order to make the simulation
fully repeatable by the readers?

- Why  are the Cbetas of glutamines not considered?

- I do not understand the phrase "averaged over the replicas of the replicas"

Although this is reported in the cited publication, due to the important role
in the simulations I would also report the major differences of charmm22* with
respect to charmm22 with the cmap correction.


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: MOLECULAR DYNAMICS ENSEMBLE REFINEMENT OF THE HETEROGENEOUS NATIVE STATE OF NCBD USING CHEMICAL SHIFTS AND NOES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is well-written. The cited literature is accurate.

In Figure 3 , the blue dots are described in the legend as "CS-NOE-C22*", but according to Table S1 it should be "CS-NOE-4-C22". Indeed the CHARM22 ff is used for these simulations.
Experimental design: This research fits within Aims and Scope of PeerJ. The article provides sufficient information for others to adopt the same methodological approach for different protein systems.

NMR chemical shifts have a measurable T-dependence, especially for amides (see Baxter, N.J. & Williamson, M.P. J Biomol NMR (1997) 9: 359. https://doi.org/10.1023/A:1018334207887). I presume that CamShift does not take this into account, so that all chemical shift predictions during the MD runs are effectively at 304 K (or 298 K, which is not relevant within the uncertainty of the prediction). The authors may want to mention this explicitly.
Validity of the findings: No comment
Additional comments: Simulations with 4 replicas appear to reproduce the data better than those with 8 replicas. Can the authors comment on this? It could be useful to researchers who would like to apply this methodology to other proteins.


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: MOLECULAR DYNAMICS ENSEMBLE REFINEMENT OF THE HETEROGENEOUS NATIVE STATE OF NCBD USING CHEMICAL SHIFTS AND NOES
Review round: 2
Reviewer: 1

Output:
Basic reporting: All my comments have been completely addressed.
Experimental design: All my comments have been completely addressed.
Validity of the findings: All my comments have been completely addressed.
Additional comments: All my comments have been completely addressed.


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: MOLECULAR DYNAMICS ENSEMBLE REFINEMENT OF THE HETEROGENEOUS NATIVE STATE OF NCBD USING CHEMICAL SHIFTS AND NOES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed the points raised by the reviewers in a satisfactory manner.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: PRELIMINARY STUDY ON PHOSPHATE SOLUBILIZING BACILLUS SUBTILIS STRAIN Q3 AND PAENIBACILLUS SP. STRAIN Q6 FOR IMPROVING COTTON GROWTH UNDER ALKALINE CONDITIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Suggest to include "Preliminary study" on the title.
Line 205. I think it will be better to place "%" unit in each of the value.
Experimental design: Line 95. Please include details of the general-purpose agar used. Eg. manufacturer etc.
Line 124-126. Please include the time used to shake the root tips on an orbital shaker.
Line 134-146. What experimental design were used in the in-vitro experiment? How many replicates? Where was the experiment conducted? Growth chamber model?
Line 142. What modifications were done on the Hoagland's solution? Please state.
Line 143. What rock phosphate was used?
Line 160-172. Please consider to eliminate or to redo the phylogenetic tree. A phylogenetic tree is used to show the relationship between the strains and a ref strain of choice, and is therefore shown in only one tree/figure.

Table 2,3,4. It will be better to include the grouping information in the tables. If the treatments did not showed any significant differences, please indicate in the tables.
Validity of the findings: This research has it's merit as a preliminary paper. This paper will be significantly improved if the authors can include a pot study of the Bacillus subtilis strain Q3 and Paenibacillus sp. strain Q6 on cotton, possible a short 1 month study so that a correlation between the parameters can be drawn.
There are improvements that can be made on the data, as mentioned in the experimental design comment section.
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: PRELIMINARY STUDY ON PHOSPHATE SOLUBILIZING BACILLUS SUBTILIS STRAIN Q3 AND PAENIBACILLUS SP. STRAIN Q6 FOR IMPROVING COTTON GROWTH UNDER ALKALINE CONDITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I only suggest  a  reference for introduction (see general comments).
Experimental design: This work is original and very interesting because shown some easy potential strategies to capture bacteria that could increase the growth of cotton. However, in my opinion the information presented is very preliminary yet, especially in experiments in association to seeds and plants. Several aspects of the article are not enough explained, for example there are not figure legends, replica numbers are not showed, methodology should be described more detailed in some cases like in osmoadaptation assays, where authors should mention what they measured in this experiment (optical density?). The problem with optical density is that bacteria under saline stress could express genes in different form and produce minor quantity of secondary metabilites and therefore diminish the value, not nessesary these diminution is linked to bacterial growth.
Validity of the findings: In my opinion the information presented is very preliminary yet, especially in experiments in association to seeds and plants. The number of replicates for plant experiments apparently is very low. Authors in the excel file shown only three replicates for parameters measured, therefore results are not enough for made a conclusion. For bacterial numbers authors apparently assay only two times for colonization or maybe only one for germination and they did not do a corroboration of inoculated strains. This point is essential because today we known that bacteria could be living inside the seeds, therefore is important corroborate that colonization observed is due to inoculated strain and not due to natural population stimulated by inoculated strain. Authors not shown dry weight data which is essential to reinforce conclusions for growth promotion results. Therefore, authors should complete the work before submit again this work for publication.
Additional comments: Dear Michael LaMontagne
Member of the Editorial Board
Of Journal PeerJ

I carefully reviewed the manuscript ID 24744 entitled “Phosphate solubilizing Bacillus subtilis strain Q3 and Paenibacillus sp. strain Q6: High potential candidates for promoting plant growth in cotton under alkaline conditions”. Authors of the present work carried out the isolation of 20 strains from cotton rhizosphere, these strains were screened in some characteristics linked to plant growth promotion like catalase activity, ammonia production and phosphate solubilization, growth under alkaline pH. Seventh strains were selected because they showed all the explored characteristics and were used to explore their ability to colonize and promote the growth of cotton plants. Two strains were the most promising to be used as possible plant growth-promoting bacteria in alkaline fields to increase yield production of cotton. Those strains were partially characterized using molecular tools (16S rDNA sequencing) as Paenibacillus sp. and Bacillus subtillis.
This work is original and very interesting because shown some easy potential strategies to capture bacteria that could increase the growth of cotton. However, in my opinion the information presented is very preliminary yet, especially in experiments in association to seeds and plants. First, Several aspects of the article are not enough explained, for example there are not figure legends, replica numbers are not showed, methodology should be described more detailed in some cases. Second, the number of replicates for plant experiments apparently is very low. Authors in the excel file shown only three replicates for parameters measured, therefore results are not enough for made a conclusion. Third, for bacterial numbers authors apparently assay only two times for colonization or maybe only one for germination and they did not do a corroboration of inoculated strains. This point is essential because today we known that bacteria could be living inside the seeds, therefore is important corroborate that colonization observed is due to inoculated strain and not due to natural population stimulated by inoculated strain. Fourth, authors not shown dry weight data which is essential to reinforce conclusions for growth promotion results. Therefore, authors should complete the work before submit again this work for publication.
Other minor questions and suggestions.
1)	In all the paper authors say “16S rRNA sequencing”, but they carried out the amplification of DNA and they did not made a RT-PCR. Therefore this should be substituted per “16S rDNA sequencing”
2)	Introduction section. Authors wrote “…(4) production of phytohormones such as indole-3-acetic acid (IAA) (Gupta et al., 2000; Fernando et al., 2006). PGPR are also able to produce plant growth regulators like indole acetic acid, gibberellic acid, cytokinins and ethylene as well as change their concentrations in the rhizosphere (Beneduzi et al., 2008).” Please increase the quality of the text.
3)	Introduction. I suggest a reference in the phrase: “When selecting the best-suited PGPR to a given site, a combination of two or more traits is a much more promising approach compared to the use of a single character (Zahir et al., 2003; Ahmad et al., 2008; Baez-Rogelio et al., 2017).”
Baez-Rogelio A., Morales-García Y. E., Quintero-Hernández V., Muñoz-Rojas J. 2017. Next generation of microbial inoculants for agriculture and bioremediation. Microbial Biotechnology 10(1): 19-21.
4)	Material and methods, section Isolation of rhizobacteria from cotton rhizosphere. What does mean general-purpose agar plates? Please clarify.
5)	What did authors measure in the osmoadaptation assays experiment? Please clarify.
6)	Material and methods, section Growth of PGPR at different pH levels. Use international unities, therefore, change “24 hrs” per “24 h”
7)	Why the bacterial inoculation time was different for “Root colonization assay” (10 minutes) than that for “Evaluation of phosphate solubilizing PGPR under axenic conditions” (5 minutes). Please clarify.
8)	Material and methods, section “Evaluation of phosphate solubilizing PGPR under axenic conditions”. Authors should mention the parameters used to measure the growth of plants, additionally is important corroborate the identity of bacteria quantified in colonization assays.
9)	Results, section “Characterization”. In the phrase “Twenty strains were isolated from the rhizosphere of cotton. These were characterized for plant growth promoting traits (Table 1, Pictures 1-4). All of the strains showed positive results for catalase activity by producing gas bubbles;”, I suggest eliminate Pictures 1-4 and “…by producing gas bubbles”, that information is not relevant. Also eliminate the file Picture 1-4.
10)	Results, section Identification of selected strains through 16S rRNA sequencing. In my opinion the strain called Bacillus subtilis shod be changed by Bacillus sp. with base to rules of polyphasic taxonomy (16S rDNA is not enough to assignee the bacterial species).
11)	Discussion. After the first paragraph authors should explain why they use the catalase assay. By the way, two times the word catalase was bad redacted “catalyse positive”.


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: PRELIMINARY STUDY ON PHOSPHATE SOLUBILIZING BACILLUS SUBTILIS STRAIN Q3 AND PAENIBACILLUS SP. STRAIN Q6 FOR IMPROVING COTTON GROWTH UNDER ALKALINE CONDITIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The author has addressed the previously suggested improvements.


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: PRELIMINARY STUDY ON PHOSPHATE SOLUBILIZING BACILLUS SUBTILIS STRAIN Q3 AND PAENIBACILLUS SP. STRAIN Q6 FOR IMPROVING COTTON GROWTH UNDER ALKALINE CONDITIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Dear Michael LaMontagne
Academic Editor
Peer J
In my opinion all questions were answered satisfactorily and this version of the manuscript was increased in quality and good preliminary results. I have not more comments.


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

Input:
Title: VOLATILE PROFILING IN RHUS CORIARIA FRUIT (SUMAC) FROM THREE DIFFERENT GEOGRAPHICAL ORIGINS AND UPON ROASTING AS ANALYZED VIA SOLID-PHASE MICROEXTRACTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Dear authors, congratulations for the theme and the work. They have enormous scientific and technological potential. The manuscript is well written, making clear the objectives and results found.

Literature references are pertinent to the content and are well updated, presenting very recent work.

Professional article structure and formatting, apparently conform to the journal's standards. But I suggest that before the definitive publication is reviewed.
Experimental design: In item 2.1 of the Material and Methods, mention that they purchased the fruits in trade and were taken to the department. However, immediately afterwards the following sentence is added: "Cold beverage was prepared by percolating 10 g of powdered fruits in 100 ml distilled water for 10 minutes kept at 25 ° C, then filtered to remove debris".
Thus, it is implied that powdered fruits are purchased fruits that have been made into powder. However, it is not specified in the article how these fruits have reached this stage. I therefore suggest that you add in the Material and Methods how they obtained the powder of the studied fruit.
Validity of the findings: Congratulations on the innovation and originality of the work.

I suggest that the discussion be improved by adding the role of each compound found, where possible, in the quality of the fruit. Not only the nutritional importance, but also the technological importance, especially if this fruit is used as an ingredient in the preparation of some product.

Would this fruit be indicated to be used in some industrialized product to replace synthetic ingredients?
Additional comments: I suggest putting the cat number of the chemicals and reagents used.

I also suggest that in future work the technological potential of this fruit as a natural ingredient in some products is verified.


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: VOLATILE PROFILING IN RHUS CORIARIA FRUIT (SUMAC) FROM THREE DIFFERENT GEOGRAPHICAL ORIGINS AND UPON ROASTING AS ANALYZED VIA SOLID-PHASE MICROEXTRACTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The title does not reflect the actual work that was performed in this study, the title as is gives the impression that the 3 sumac fruits from different origin was roasted. Suggested modification: “Volatiles profiling in Rhus coriaria fruit (Sumac) from 3 different geographical origins as analyzed via solid-phase microextraction”.

In the abstract section the word flavor was used in the wrong way, once this word is used to describe compounds responsible for taste and for odors, and in the present study it was only performed a volatile profile of the Sumac fruit, not an assessment on its taste.
In the introduction section some comments:
Line 34: how does this fruit is used as flavoring agent in food? As the whole fruit, processed as a powder or essential oil, for example or other form? This was not clear in the text.
Line 35: the reference is very old, Pojero 1891.
Line 49: why the name of the compound cembrene was whiten in quotation marks?
Line 55: the abbreviation for gas chromatography mass spectrometry appears in the text differently, (-) or (/), the authors must check the official/ scientific form of abbreviation.
In general, an improvement/ uptade in the background literature regarding the subject of study is necessary.
In figures 1 and 2 the chromatograms starts with 6 min of analysis, once the analysis was performed by SPME there is no need for solvent delay, so where are the initial compounds. In figure 1, chromatograms of the sumac fruits from Jordan and Egypt show poor resolution; it could be an indication that the SPME conditions could be optimized.
In figure 2, some compounds identified in table 1 as present were not marked in the chromatogram of roasted fruit. For example, alpha-pinene that was identified in the fresh fruit, should have the same retention time in the roasted and was not identified. In addition, as in figure 1 the initial compounds are missing.
Experimental design: The study, especially in the introduction, does not make clear how the knowledge gap is filled. The description of the experimental part is lacking important information, such as:
Line 67: the authors did not explain how the fruits were collected. Once depending on the form species collection, this can influence the sampling and consequently the results. For example, how many units of the species were collected to form the Palestine sample, and how the maturities of the fruits were assessed? Besides the geographical region, degree of maturity is an important factor that could influence the results.
Line 68: the samples from Egypt and Jordan were commercial, this raises the question of how to be sure of the integrity of the collection and degree of maturation of the samples.
Lines 71 and 72: it is not clear how the cold beverage, that latter in the article is called cold tea, was obtained; it was obtained from the fruit powder? How this powder was obtained? What kind of filter was used, with what specifications? And how the filtration was performed, under vacuum?
Line 73: it is not clear how the roasting process was performed? The fruits were whole? And only the Palestine fruits were roasted, this was not clear in the text.
Line 80: in the volatile analysis section, the SPME conditions were not exactly as described in the Farag’s article. In the article cited, the authors used 100 mg of allium sample and a internal standard was used, which did not occur in the present study.
Line 85: the chromatographic conditions were not present.
Validity of the findings: The results as stated need to be revise, and further explanation with literature support is necessary. For example, line 111, alpha-pinene was not the majority compound for Jordan sumac as stated, the majority compound was (Z,Z)-alpha-farnesene according to table 1.
In the results section some errors are present, such as:
Line 103: the word sesquiterpene was misspelled.
Lines 108 and 109: the result on roasted fruit is misplaced between the results for fresh fruit. This sentence should be moved before line 118.

The most serious problem with the results presented has relation to:
-	The authors use the GC-MS for assessing relative percentage and this detector has known ionization discrimination, thus the response for different volatile class can vary. The best detector for relative percentage is the flame ionization (FID), which responds to carbon and hydrogen bond or improve analytical care, for example using internal standard to correct response. The authors did not use this FID detector or internal standard.
-	Also, the sampling of the fruits from different origin was performed in triplicate (as stated in the experimental section). However, this sampling could be an important point that is making the results not robust enough or statically sound.
-	Both of these issues are reflected in the results presented in table 1, several compounds show very high standard deviation, in some cases this deviation is over 50% of the average value, thus making the results not reliable.

In lines 119 and 120, the concentration of some volatile class did not decrease as stated, as shown in table 2, roasting increase the relative percentage of alcohols, although this was not significantly once taking into account the standard deviation. Also, ethers and ester increase, the latter the S.D. is also very high.
In line 133, the PCA analysis is explained wrongly, the sum of both PC, 1 and 2, which is accounted for 60%, not only PC1. Figure 3 has very low significance, once with the sum of PC1 and PC2 only 60% of the data is accounted for.
In the discussion section further comments regarding the reason for the volatile changes in the roasting process is missing, also which are the climate conditions of the fruits from the geographical regions  studied, what are their differences, or similarities, also in the other different studies cited.
The authors comment in line 181 that furfural and nonanal could be markers of degradation, what compounds in the fruit are the precursors? This is not discussed. Also, in line 182 the authors state that storage conditions can cause degradation of these fruits, but they do not explain witch conditions, heating, oxygen content, light and etc.
In the conclusion with the errors of the results presented the statement in line 208 is not true.
I wonder the relevance of presenting figure 4, once it brings the results for the fresh fruits from the three geographical origin, together with the roasted from one single origin. The figure 5, presents similar results than figure 4, I suggest exclusion of figure 4.
Additional comments: In general, there are several issues with the data presented by the authors; high standard deviation shows lack of robustness. Experimental conditions are not fully presented. The type of detector chosen should be revised, maybe other experiments or more samples should be analyzed should be conduct to improve data. The background literature of the sumac fruits should be updated and some figures are not needed.


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: VOLATILE PROFILING IN RHUS CORIARIA FRUIT (SUMAC) FROM THREE DIFFERENT GEOGRAPHICAL ORIGINS AND UPON ROASTING AS ANALYZED VIA SOLID-PHASE MICROEXTRACTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript presents the volatile profile of Rhus coriaria (Sumac) fruit from three different countries, and compares the effect of roasting on the volatile profile of this fruit. The experiment has a good experimental design and was carried out properly. I recommend that this manuscript should be published after major revisions that were identified among this document, but especially after a language revision from an English native speaker.

The English language should be improved, to increase the quality of the manuscript and ensure a clear comprehension. Some lines present unclear language, including the abstract.
I suggest that you get in touch with some native speaker to correct the English.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Title: I suggest a few changes in the title of the paper. For example, change the number “3” to the word “three” and also change “upon roasting” that I don’t think is so common to use in the title. One example that I suggest is:

“Volatiles profiling of fresh and roasted Rhus coriaria fruit (Sumac) from three different geographical origins analyzed via solid-phase microextraction”

Keywords: I suggest that you remove the two first ones, since they already appear in the title. Change for the name of the family, or some other popular name of the fruit.

Abstract

Line 23. Standardize the way you express %, first it is close to the number, then the second time it is after a space. Put the first one close to the number as well.

Lines 24-27. Another example of the unclear language, you should try to express the information in a fluid and clear way.

Line 27. “Such knowledge is expected to be the key for understanding the olfactory (…)”. This sentence is vague, which “such knowledge” is that? This is your abstract, you should be very clear and direct in this part, even more when you said that this knowledge is the KEY for understanding something.

Introduction

Lines 33-36. Other example of language that should be improved, the sentences when you introduce the fruit seems truncated.

Lines 36-39. You start both phrases with the same expression “In terms of”. Change one of them.

Line 49. Correct the first “ of the word cembrene.

Line 51. Remove the “or”.

Line 52. Start a new paragraph here, with the objectives of your study.

Line 53. Here and among the manuscript I suggest you to change the word “accessions” to make clearer your meaning.

Lines 53-54. Remove “further” and avoid use “its”, here and among the text because it is not always evident what “its” means. You can change it for “the fruit”or “Sumac” for example.

Lines 54-55. You should write the full names and in the front the acronyms.

Line 56. What do you mean by “untargeted manner”?

Materials and methods

2.1 Plant material – The fruits from Palestine were collected in the full ripe stage, and the fruits from Jordan and Egypt? Describe they stage as well, the ideal is to compare them all in the same stage. Here and among all the manuscript standardize the way you express the temperature, sometimes you write the number together with ºC and sometimes with a space between them.

Line 69. Delete “are”.

Line 71. “in 100 mL OF distilled water”.

Line 72. Add a comma after minutes.

Line 73. “the fruits”

Line 80. Change the / for “and”

Lines 83-84. Remove the ( ).

Results

I suggest you to write the tables and figures already at the beginning of the paragraphs were you discussed them (example: XXX analysis is presented in Table X, Figure X), so the reader can check them and follow the paragraph knowing what they need to see.

Lines 138-141. In this sentence you discussed a very interesting result of your study, but the whole sentence is very confusing. I think that once you get a native English speaker to read and help to improve the language problems you could increase the quality of the description and discussion of the results.

Line 142. Here you could do the suggestion I wrote before, to introduce the figure and what the reader is supposed to find there. It is confusing to read the results in this way.

Discussion

I think that in the first paragraph of the discussion you explained the objectives of your study better than on introduction. I suggest you to insert this part on the end of the introduction and to resume your objective here; since it is the discussion and the reader already saw then on introduction and follow all your manuscript until here.

Line 161. Change “or” for “and”.

Conclusion

Line 209. Delete “or”.

Tables

Standardize the title of the tables. And do not put the description with the title, like you did in Table 1.

Figures

In the figures 3 and 4 you made change that could be very confusing for the reader. In figure 3 you choose the colors from the 3 different regions, the readers saw and possibly spent some time analyzing this figure. Then in figure 4 you change the colors of the already displayed different regions… You should standardize these 3 colors that you already showed to the reader, and change only the additional legends. The fruit from Egypt were green, from Jordan blue and from Palestine red, then on figure 4 Egypt is blue and Jordan yellow, just Palestine that remained red, it could be very confusing for the reader.


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: VOLATILE PROFILING IN RHUS CORIARIA FRUIT (SUMAC) FROM THREE DIFFERENT GEOGRAPHICAL ORIGINS AND UPON ROASTING AS ANALYZED VIA SOLID-PHASE MICROEXTRACTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The suggested changes were performed and the questions answered, thus the manuscript is ready for publication.
Experimental design: The suggested changes were performed and the questions answered, thus the manuscript is ready for publication.
Validity of the findings: The suggested changes were performed and the questions answered, thus the manuscript is ready for publication.
Additional comments: The suggested changes were performed and the questions answered, thus the manuscript is ready for publication.


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

Input:
Title: VOLATILE PROFILING IN RHUS CORIARIA FRUIT (SUMAC) FROM THREE DIFFERENT GEOGRAPHICAL ORIGINS AND UPON ROASTING AS ANALYZED VIA SOLID-PHASE MICROEXTRACTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors performed several modifications along the article.
Altought the authors explain the statistical possibility of high values of SD presented in table 01. However, some compounds still have a high standard deviation, to cite a few for example, beta-linalool from Jordan (1.84 ± 1.60); alpha-pinene  (2.01 ± 1.81), alpha-fenchene (0.04 ± 0.04), beta-myrcene (0,17 ± 0.12) from roasted fruit. The atuhors stated that they use the present data to compare the samples using multivariate data analyses in nature form and between the roasting process, thus it is important that these SD are as low as possible, even if these SD are consider statistically ok, we as scientists should have a critical assessement of our work and express this concern.
The authors are comparing fruits from different origin based on these values found and in some cases, because of the high SD there are no statistical difference, thus the authors shoul point this as a critic point of their work, and suggest alternatives to improve these results, by using other detector, such as FID, for example, in the end of the discussion. This statement does not invalidate the work.
Also, the authors stated "Considering our interest in volatile terpenoids and hydrocarbons, 5 minutes is appropriate for this study" this is not clear in any part of the article, and it could be important to mention this.


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: CORSEQ: FAST AND EFFICIENT IDENTIFICATION OF FAVOURED CODONS FROM NEXT GENERATION SEQUENCING READS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. A more thorough overview of what you are trying to do and how corseq accomplishes that (similar to what is in the discussion) placed before the "software implementation" section would be helpful for the reader
2. You focus on "optimizing" codons. It might be worth noting that there are various other studies claiming that sub-optimal codons at specific positions are important in protein formation to facilitate co-translational folding.
3. Figure 1's layout is a little confusing
4. Some minor grammatical/language issues throughout
Experimental design: Why do you use only the top and bottom 2% of kmers? Why not 1% or 3%? Add some reasoning here in the "software implementation" section.
Validity of the findings: No Comment
Additional comments: I think there might be a minor error in "The effect of the provided number of reads" section. Based on your other calculations your reads are 150bp, meaning that the bp coverage from 25,000 reads should be 3,750,000bp and not 3,500,000bp.


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: CORSEQ: FAST AND EFFICIENT IDENTIFICATION OF FAVOURED CODONS FROM NEXT GENERATION SEQUENCING READS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper presents a method to identify codons that are preferably used in highly expressed genes in a given organism, given as input RNA sequence fragments and amino acid sequences of proteins from this organism.

The paper is generally clear, except for its methods section, which needs additional information. It is not clear how the chi square test is performed, on which values, and how the results are interpreted. Equations would help to document the methods used. Currently it is not possible to reproduce the research with the information provided. The "conventional expression method (CEM)" is not described in sufficiently detail, and references to tools are used to substitute to descriptions of the method itself. It is also unclear to me why the Pearson correlation coefficient value is used instead of the corresponding p-value, which would be significantly more informative.

English could be improved with some proofreading, to catch small errors like "were" instead of "was" on line 118, the use of the word "elaboration" throughout the manuscript, etc.

References and background are sufficient and support the premise of the paper, as well as the objectives. The structure, figures, and tables of the article seem sufficiently professional. I could not find though any raw data, the only supplemental material I could locate was a manual for the corseq software. The article is mostly self contained, except for the methods' descriptions, as mentioned above. The results are relevant to the hypothesis.
Experimental design: In my opinion the primary research of this article falls within the aims and scope of the journal. The research question is well defined and meaningful, and solving the stated problem would be useful to researchers in the field of heterologous expression.

The proposed solution is interesting, but the technical part is dominated by empiricism instead of analytical methods. K-mer size for example is determined by experimenting with seemingly random values of k, settling on the largest one that seems to reduce the number of k-mers shared between over- and under-expressed genes. Why not a higher value then? And why the 2% of the most frequent and least (not "less") frequent sequences are selected from the k-mers? How is this 2% value determined? All this information would help determine better the technical soundness of the methodology used. As presented currently, it is hard if not impossible to replicate the findings.
Validity of the findings: The findings of this paper are interesting and the conclusion is well stated. It may still be difficult to determine and even further quantify the effect this method's preferential codon selections would have on gene expression vs other methods, but that is probably outside the scope of this research.
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: CORSEQ: FAST AND EFFICIENT IDENTIFICATION OF FAVOURED CODONS FROM NEXT GENERATION SEQUENCING READS
Review round: 1
Reviewer: 3

Output:
Basic reporting: See attached
Experimental design: See Attached
Validity of the findings: See attached
Additional comments: See attached


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: CORSEQ: FAST AND EFFICIENT IDENTIFICATION OF FAVOURED CODONS FROM NEXT GENERATION SEQUENCING READS
Review round: 2
Reviewer: 1

Output:
Basic reporting: My main critique is a few very minor grammatical errors that remain:
line 31: "to infer the" to "the inference of"
line 193: "design" to "designing"
line 202: I think you mean something like "for the determination of" instead of "to investigate"
line 222: "In facts" to "In fact"
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The paper was much more clear and readable following the revisions. My concerns from the first round of review were addressed. The only revisions I would recommend are the few minor grammar issues noted above.


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

Input:
Title: CORSEQ: FAST AND EFFICIENT IDENTIFICATION OF FAVOURED CODONS FROM NEXT GENERATION SEQUENCING READS
Review round: 2
Reviewer: 2

Output:
Basic reporting: While the authors should be commended for addressing my previous comments and those of the other
reviewers, I do have one major concern, which should be easy to address, and a number of more minor
corrections that prevent me from being able to recommend this paper from being published in its current
form.
    As a result of the authors responses and my re-reading of the paper, I have a fundamental problem
with their use of the term ’optimal’. In the codon usage world, ’optimal’ usually refers to the one codon
out of a family of synonyms most favored by natural selection. Here the authors apply the term to any
codon whose usage increases between the low and high expression sets. This is inconsistent with previous
usage and inaccurate. The authors’ comparisons allow them to identify ’selectively favored’ or ’adaptive’
codons and not necessarily all of them since as we illustrate and discuss in our work the authors now cite
(e.g. Shah and Gilchrist 2011 and Gilchrist et al 2015), for amino acids with 3 or more codons, we can see
non-linear changes in a codon’s frequency. Specifically, the frequency of a codon can actually increase and
then decrease with protein production rate. For example, in Fig 6 from Gilchrist et al (2015, attached), we
can see how the frequency of Ile’s ATT initially increases and then decreases with protein production rate,
φ. This is because ATT is selectively favored over the pessimal codon ATA and mutationally favored over
the optimal codon ATC. As a result, ATT frequency can initially increase with φ because it is more fit than
ATA which is initially used with non-zero frequencies. In turn, both ATT and ATC frequency increases with
φ at the expense of ATA. However, once gene expression gets moderately high (around ln(φ) = 0.75, ATA
is so selectively disadvantageous that it is no longer used and only ATT and ATC are competing with one
another. As a result, when increasing ln(φ) above 0.75, we see ATC frequency increase at the expense of
ATT. If selection was efficacious enough and/or protein translation of the genes with the highest rates, we’d
only see ATC, but that’s not the case in yeast and not likely the case for codon families with 3 or more
members other organisms. Thus, I’m going to insist that the authors use a different term than ’optimal’,
such as ’selectively advantageous’ or ’favored’ or ’adaptive’.
    In a future study, the authors might want to see how well our code works with the k-mer data they are
using. I suspect if they can get 50-100 codons for about 1000 genes, they might be able to estimate our
model parameters ∆M and ∆η, especially if they can use the density of reads per gene per codon as proxies
for φ.

Minor Criticisms
  1. The language is overall good, but there are numerous typos and inconsistencies. Here are a few
     examples from the .docx file with tracked changes
      (a) line (l) 113: vice versa is two words. (as is de novo l 69)
      (b) pseudo-Orf (l 222) vs pseudo-ORF (l 158).
      (c) l 83 lists only two, not three, fungal species.
      (d) Not sure what the authors mean by ’elaboration time’ on line 183.
  2. The significance column Supplemental File 3 is not defined. Are the numbers − log10 (p)?
  3. I still think the Venn diagrams are essentially uninformative. Why not just have a graph with how the
     frequency of concordance changes with sample size?
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: No comment


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

Input:
Title: DISTRIBUTION AND ECOLOGICAL SEGREGATION ON REGIONAL AND MICROGEOGRAPHIC SCALES OF THE DIPLOID CENTAUREA ASPERA L., THE TETRAPLOID C. SERIDIS L., AND THEIR TRIPLOID HYBRIDS (COMPOSITAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, the manuscript reads well, it is well written, but it should be checked by a native English speaker because there are some hardly understandable sentences (lines 157-158, lines 267-268, among others).
The introduction is very well structured, but it would be necessary to add some reference on general polyploidy studies carried out by I.J. Leitch et al.
Authors should avoid redundancies between tables and text.
Experimental design: I think that the main weakness of this manuscript lies in the experimental design. After reading the previous papers of the authors and taking into account their expertise, I think that they can determine the cytotypes according to their morphological characters, but it would have been better to use the flow cytometry to determine the ploidy level of each individual included in this study.
Validity of the findings: Data have been well analyzed and the results correctly interpreted.
Additional comments: The authors should improve the conclusions section in order to avoid redundancies with the results section.
The manuscript is well structured and well written and it deserves to be published after minor revisions that are detailed in the preceding sections.


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: DISTRIBUTION AND ECOLOGICAL SEGREGATION ON REGIONAL AND MICROGEOGRAPHIC SCALES OF THE DIPLOID CENTAUREA ASPERA L., THE TETRAPLOID C. SERIDIS L., AND THEIR TRIPLOID HYBRIDS (COMPOSITAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: I have reviewed manuscript #26915 entitled ‘Cytotype distribution and ecological segregation on regional and microgeographic scales in Centaurea aspera L.-C. seridis L. (Compositae)’, co-authored by Garmendia and colleagues. The manuscript represents an interesting case-study aiming at getting further understanding of the potential role of ecological variables in shaping species distribution. I believe this manuscript is sound and fluently written, and as far as I understand, the statistical approach has been carefully designed.
However, my main criticism lays on the somewhat misleading understanding of the ‘cytotype’ concept in this manuscript. I am surprised that no methodological approach (e.g. cytotype screening by flow cytometry or chromosome counting) is taken to identify cytotype occurrence beyond a rather morphological description to allocate ploidies (if based on previous findings, then it should be referred accordingly with further details). In fact, on M&M, l153-onwards it is stated that cytotypes are attributed based on morphological traits solely. Bearing this in mind, one assumes that all C. aspera are (2n=20, 22), C. seridis (2n=44) and C. x subdecurrens (2n =?). If that’s the case, then I am wondering why using cytotype distribution in the title and not just ‘Distribution of C. aspera complex’? I would recommend the authors to clarify this point and address this issue on the introduction to avoid further misunderstandings along the manuscript. It seems that across the species complex there is some sort of chromosomal stability, but this point needs to be proven.
In addition to that, is it likely that tetraploid cytotypes of C. aspera could occur in the W Iberian Peninsula? The authors reported their presence on Morocco, and I see on the CCBD (http://ccdb.tau.ac.il/search/) that Humphries et al. also reported it previously. Could this eventual WGD occur elsewhere along the distribution area?
Concerning the triploid cytotypes (or C. x subdecurrens). So far I believe this cytotype has been only recorded by means of DNA ploidy allocation, but no chromosome work has been reported so far to either (i) understand its meiotic behaviour, or (ii) to confirm the chromosome number and get insights into the cytogenetics complement of the parental donors (e.g. 2n=20 + 44/2n= 22 +44). Is there any chance that 3x individuals could overcome sterility through production of unreduced gametes and therefore hexaploid cytotypes might exist, even if they could be extremely rare? Even if this has not found yet, one would appreciate some discussion on the text. Spontaneous and induced (auto)polyploidization is something reported in plants as a mechanism to overcome sterility in triploid cytotypes. Obviously, natural recovery of fertility is rare, but worth exploring in detail on those 3x-ploidy allocated populations. I understand this might fall outside the main scope of this work, but a detailed cytogenetic exploration of 3x-populations could shed light into the ongoing mechanisms shaping (or preventing) its current distribution.
In my opinion, Figures 7 and 8 would benefit from adding further details on the caption. As they currently state, I find them difficult to interpret (e.g. I assume that filled/empty/crossed data points are cytotyes, but cannot differentiate among them beyond the triploid (assumed x)).
Experimental design: See my basic reporting section. I believe the analytical approach is sound and robust.
Validity of the findings: See my comments on the basic reporting section
Additional comments: See my comments on the basic reporting section


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: DISTRIBUTION AND ECOLOGICAL SEGREGATION ON REGIONAL AND MICROGEOGRAPHIC SCALES OF THE DIPLOID CENTAUREA ASPERA L., THE TETRAPLOID C. SERIDIS L., AND THEIR TRIPLOID HYBRIDS (COMPOSITAE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: Word choice and syntax throughout this manuscript are often unusual and occasionally make the work hard to follow. I would suggest the authors reread this with an eye for clarity and potentially enlist someone for whom English is their first language. I have made specific comments on particular phrases in the line edits, but these should not be considered comprehensive. A general revision of the English would greatly improve the manuscript’s clarity and ease of reading.

In general, the literature is sufficiently cited, though I made comments to authors where some additional reading may be necessary.

The figures which are present are neat and clear, but some figures and tables are either unnecessary or could be pared down from their current sizes.

The manuscript is self-contained and internally consistent, though the distributions of cytotypes reported here aren't that novel given previous work by these authors.
Experimental design: The primary concern I have with this study is the treatment of density of each cytotype relative to the others. The method used here, coarse binning into three categories followed by chi-squared tests, is not described in sufficient detail for its biological relevance to be determined. There are no citations given in the methods or discussion linking this method to anything in previous literature, leaving me with the impression that it is of the authors’ own design. If that is not the case, it needs justification based on the literature. If it is the case that this is a novel way to describe relative density of pairs of cytotypes, it is on the authors to establish its biological relevance. Given that the three plots used for microspatial analysis differ in density by an order to magnitude in some cases, the authors do not successfully make the case that equal-area bins of relative density as described here are relevant to competition between cytotypes. Either further literature review needs to be conducted and cited to justify their approach, or they need a new approach. It seems that an adaptation of neighborhood analysis (along the lines of Silander and Pacala, 1985 or Sanderson et al., 2016, Ecology and Evolution—subbing in cytotype in place of sex) might allow for finer description of relative densities of each cytotype and allow for direct comparison between populations. To my knowledge, such an application to a polyploid complex would be quite novel if the authors chose to pursue it, and it looks like they have the data to do so. Related but a bit tangential, I don’t find Figure 5 particularly informative, as it basically recapitulates what is shown in the fine-scale mapping in Figure 2.

While the treatment of local density is my primary concern, I also wonder why the authors focus on the relatively broad life-form categories when they have species-level data that actually shows some significant differences. Apologies if this is a question from ignorance, but could the NMDS analyses be re-done using these species-level associations? It is possible that the authors have done so but not reported it here, but if not, perhaps worth considering?

Lastly for major concerns of the paper, I feel that the interpretations of the close-proximity of diploid and triploid individuals is not fully realized in light of what is known about polyploid complexes and interploid reproduction. The association of triploids with diploid moms would be highly unusual if one did not know that tetraploids are largely autogamous. Typically crosses between diploids and tetraploids fare better when the maternal plant is tetraploid, and it might be worth exploring why the opposite is true here. Not merely due to tetraploid autogamy: the abundance of triploids suggests that these crosses are relatively successful, when they are often not in other systems! Also, while the authors go into detail about seed dispersal distance and how short dispersal distances may facilitate clumping and therefore foster cytotype coexist, they say very little about pollination or pollinator behavior. If diploids are at risk of being swamped out by tetraploid or sterile triploids, distances over which pollinators travel can matter greatly, and that isn’t addressed or even referenced in citation here.
Validity of the findings: Given that novelty is not assessed and negative or inconclusive results are allowed, I have no comments on that aspect of the work.

The data presented are robust and statistically sound.

The conclusion is well-stated, though it could be improved with stronger connections to the existing interploid reproduction literature.
Additional comments: All line comments are as follows. While these are largely grammatical, some are non-trivial.

Line comments:

Line 43-45: There are a number of recent phylogenomic papers that could (and I think should) be included in a discussion of the overall success or failure of polyploidy. Mayrose et al., 2011 and Soltis et al., 2014 would be good places to start.

Line 46: Arrigo and Barker is not the best citation for this idea and is only a secondary concept of that paper. The Levin or Petit, Bretagnolle, and Felber papers cited therein would be better, as would some of Husband’s work in Chamerion angustifolium.

Line 53: confer onto them, not confer them

Line 56-57: Zozomová-Lihová is the correct citation, yet you make this two people five words later. In general, citations throughout the manuscript are rather sloppy.

Line 63-64:
“even in the separation of cytotypes into formations of a different physiognomy” This phrase is lifted almost word-for-word from Hülber et al., 2015. Revise.

Line 67: vegetative reproduction isn’t adaptive?? Either cite something that specifically goes to this point or revise.

Line 72-73: Herben, Trávníček, and Chrtek (2016) might be worth reading and citing here.

Line 75: fewer, not less

Line 79-80: “polyploids used to be separated from their diploid ancestors” I’m not sure what you mean here, and this might just be a wording issue. Neopolyploids almost invariably start out sympatric to their diploid progenitors given how they usually form. If you feel this phrase is accurate, its meaning is unclear to the reader.

Line 84: No space in McAllister

Line 89-90: Odd phrasing: “can allow to infer”. Try “can allow one to infer” or “can allow inference of”

Line 108-109: That’s quite a few outlying sites if you want to make the case that C. seridis has a more limited distribution. Perhaps give a sense from these studies how common it is in these locations. Are we talking single populations? Also, do you mean Albania, not Albany?

Line 110: Generally the –ern ending is used on directional adjectives (i.e. eastern, western). Using east and west as done here is understandable and should be fine, but be consistent throughout the manuscript.

Line 122-124: I’m a bit dubious on this as an example of assortative mating that would preserve cytotypes. Yes, the tetraploids are expending fewer resources than the diploid maternal plants are, but pollen expenditure is still a cost.

Line 147: “Here the Mediterranean coast is particularly stressed as it is the typical habitat of C.
seridis” This phrasing is unclear. What do you mean by stressed? And it sounds as if it is stressed because it is the habitat of C. seridis. Consider revising.

Line 151-153: Do the salt-marshes prevent formation of these dune structures or simply fail to occur where dunes are present? The phrase “avoid the presence of” makes this unclear.

Line 171: For North American publications, 2.400 should be 2,400.

Line 175: Orientation is somewhat unclear to me. By orientation, do you mean slope aspect, or something else?

Line 177: This may be overly pedantic, but how does one take the central quadrat from a 100-m2 area (or what I assume is a 10-quadrat x 10-quadrat area?). It sounded like, based on the methods thus far, that the quadrats were already laid out, so did this “quadrat” span four existing ones at the center of each area? Again, this may be overthinking.

Line 193: Did you mean sand here, and not soil? If so, how was this retain (I assume following the final sieving?). This might be worth briefly pointing out for the uninitiated.

Line 199-217: I’m not sure listing the software used to prepare the document is necessary, and writing out all software used at the beginning of the section makes it hard for the reader to follow. I recommend cutting all or most of this paragraph and citing each software package as it comes up in the text, which you’ve already done to a large extent.

Lines 249-263: Unless this is the first reporting of some of the details (overall distribution of each species, which is a dune specialist, etc), these details belong in methods under a “study system” heading or something similar.

Line 349: conclusive, not concluding

Line 376: find, not found

Line 395, Figure 2: It would really be helpful if, on the fine-scale maps, the authors could indicate the locations of pathways used to access the beach. It would make it easier to fully grasp their effect on C. x. subdecurrens and C. seridis.

Line 436: Both Kolář and Trávníček need to be corrected.

Line 448: for should be over

Line 465: “could have been facilitated” does not work here in the sentence. Perhaps, “the transport of tetraploid propagules could have been facilitated by movement on pets and human belongings”

Line 469: two “nots” in a row

Tables 3, 4, 5: I’m not sure why we’re being shown uncorrected P-values. Just show us the Bonferroni-corrected values.


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: AGE OF ONSET OF CANNABIS USE AND DECISION MAKING UNDER UNCERTAINTY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The research is interesting, but the paper is hard to follow at times, particularly in the discussion.

Be consistent with tense.

There are several grammatical errors throughout the paper.

A lot of sentences have the same structure and makes it difficult to follow (e.g. a lot of sentences start with “this” and “these”).

Please put what the conditions on the x axis stand for in the figure caption.

In the intro and discussion, there is mention of previous research that looked at addiction. The research in this paper did not examine addiction. I understand the inclusion of addiction research, but it was a bit confusing at times how it relates to you research. Cannabis use, regardless of onset, does not equal addition to cannabis. Also, when discussing addiction in the intro, please discuss what the participants were addicted to in these studies.

Line 52: Do not use the word “proved” the researchers “suggested” or “found”

Line 60: what substances?

Line 86: “hat”

Line 118: why is this an exploratory study? Is it not a study? Exploratory suggests that that the task was not designed to address a question, but you are just exploring other results. Is that the case in this study?

Lines 134-137: informed consent was repeated 3 times

Line 289: “In No significant…” ?
Experimental design: I think the primary research is within the aims and scope of the journal.

I have a few comments on the Method section

Did you control for last use to ensure you are looking at residual use and not acute?

Line 162: “participants without addictive problems” what does this refer to?

I commend the authors for the way they controlled for length of use.

Line 195-196: Did you correct for multiple comparisons when using multiple t-tests (e.g. Bonferroni)? Why not use an ANOVA?

Line 197: a repeated measures analysis of variance (ANOVA)?
Validity of the findings: Results
Lines 200-205: Why start by clamming that control group made more advantageous choices than late onset group when the difference was not significant?

Line 226: Be consistent in spacing when reporting stats

Line 263: why not run some stats to see if the scores between normal and inverse were different?

Line 275-276: Shouldn’t be doing a Bonferroni if the ANOVA was not significant.

Line 289: “In No significant…” ?

Discussion

Line 314: “probably due to their inability to use emotional signals…” seems like a bold clam, did you test this?

Line 322: Who has problems? do not rely on pronouns as much throughout the paper

Lines 330-333: Expand on this

I would like to see a discussion of the limitations of the current study.
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: AGE OF ONSET OF CANNABIS USE AND DECISION MAKING UNDER UNCERTAINTY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is well written and taken care of with an adequate level of English language, clear and unambiguous.

However, there are aspects that can be improved. For example, in the explanation of the PVL model the formulas are redundant, and are already exposed in various papers, which can be accessed, and which are duly referenced. It would be better to explain the parameters, the logic of the model and omit superficial aspects “here”.

The references to the papers of Alloy, seem a little forced, since these works are based on very different tasks to igt and administered to a different clinical group.

In Table 4 & Table 5 the column “range” must be resized.

Finally, in the final section, lines 394-404, I think it should be rewritten, since it includes proposals related to this work, the age of initiation to cannabis use, with others more related to the task itself, which I think do not fit now. . In addition, the proposal that makes use of economic models of dual response difficult to fit with the IGT as it is currently configured.

The structure complies with the PeerJ standards.

The figures are adequate and quality. They are duly referenced in the text and properly titled

The raw data supplied are those used
Experimental design: This paper is within the scope of the magazine. The research objective is well defined, and is relevant, in addition, provides data in a field little studied. Research carried out according to a high technical and ethical standard. Described methods with sufficient detail and information to replicate.

Only note that the authors choose to report the percentage of elections and not the IG, although it is true that they are parallel data, which would not change the results, the usual in these works is to take into account the IG (ig = ( c + d) -a + b)). On the other hand, it would be interesting to consider alternative proposals, such as analyzing separately the elections associated with frequent losses (c-a) against the elections associated with occasional losses (d-b)
Validity of the findings: The data is relevant, statistically well worked and controlled. They abound on an interesting topic such as cannabis use and its repercussions on brain development and the dangerous trivialization of cannabis use and the importance of the development of intervention programs to cancel or delay their consumption as much as possible. The authors are also aware of the circular relationship that is established between substance use and decision making, which can be cause and effect at the same time. However the conclusions are well expressed, linked to the stated objective.
Additional comments: I agree with that Cannabis consumption in adolescence should be considered as a public health problem, therefore, it is important to have jobs that objectively show the consequences of their use, especially in adolescents, where their consequences may be permanent in terms of cognitive functions.

For all these reasons, I consider that this paper is highly recommended for publication in PeerJ, and I propose that the authors take into consideration the proposals made, since they involve making minor changes.


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: AGE OF ONSET OF CANNABIS USE AND DECISION MAKING UNDER UNCERTAINTY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Satisfactory
Experimental design: Satisfactory
Validity of the findings: Satisfactory
Additional comments: In line 314 you stated “probably due to their inability to use emotional signals…” Your rebuttal was not satisfactory. The word "inability" suggests that cannabis users CANNOT use emotional signals. Please change "inability" to "difficulty" which more accurately reflects the results.