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: GENOME-WIDE ANALYSIS OF LECTIN RECEPTOR-LIKE KINASES FAMILY FROM POTATO (SOLANUM TUBEROSUM L.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Manuscript should be modified in some parts and English improved, fgure are not clear and not well labeled
Experimental design: The work can give a good contrbiture to the research area. However statements made should be carefully revised.  It is important descrive how was made  data collection  to understand if genomes  have the same coverage, similar annotation etc
Validity of the findings: Comparative Analysis should not be performed with data realesed from other authors. The analysis should be performed with omegeneus datasets
Additional comments: Zhang and Quin have submitted a manuscript entitled ‘Genome-wide analysis of lectin receptor-like kinases family from potato (Solanum tuberosum L.)’ to be considered for publication in J Peer.  Authors annotated LecRLKs members in potato investigating their diversification. The expansion of family, occurred mainly by tandem duplication, and chromosome regions containing LecRLK members were highlighted. Transcriptomic investigations of potato infected with bacteria and fungi revealed that LecRLKs  belonging to L-type, G-I, G-IV and G-V are high responsive. In particular qRT-PCR profiles of potato infected with Fusarium sulphureum (Fs) or Phytophthora infestans (Pi),  indicated the activation of two common members.  The effort made to analyze this gene family was useful.  However, some important issues should be resolved before paper can be considered for publication.   I suggest to revise carefully data obtained and to improve result presentation and discussion
Abstract
 The sentences “Based on phylogenetic analysis of LecRLKs from potato, Arabidopsis thaliana, apple (Malus domestica), rice (Oryza sativa) and tomato (Solanum lycopersicon), distinct evolutional process of LecRLKs were found” should be deleted since data came from literature and they are original data produced in this paper .
Substitute 7 clusters with 7 clades
Based on qRT-PCR for potato incubated . It better rewording in Based on qRT-PCR for potato infected

Change:  genes for functional analysis and further resistant breeding in genes for functional analysis and breeding.
Introduction
From line 24 to line 26. It is not clear why you highlight the function of C type if you not provide evidence in your work of their involvement in innate immune response.
Line 35 The LecRLKs   are believed to be involved in plant immune receptors. It is not clear what does it means
Please delete from line 40 to 43 and try to provide more general information since potato is cultivated wordwide
Delete from line 46-50
 From line 51 to line 56 The goals of work  shoud be better delineated
Modifiy Methods in Material and methods
From line 59 to line 66 Provide clear information about data downloaded, proteome versions used and  matching repository
67 change were obtained as method in following the method
79	Please better define what you consider a cluster since this is a very tricky point
You can use a window approach or a gap approach (Richly et al. 2002; Luo et al 2012; Di Donato et al 2017)
92 transformed may be is transferred
Results
116 Rise may be is Rice
Please comment table 1 or delete it
118 Proteome were downloaded from different repositories and we have not information on version used as well as sequencing and assembling methods. So you should be wise in presenting data. Moreover, you cannot compare data form literature obtained with different procedures. Please modify description according to this point
From line 135 to 138  Too many details
From 142 to 143 I’ don’t think  that data support such finding
Line 174 It is not clear
Line 176 Like above three proteins located in the same position of potato genome and only one of these was visualized on chromosome.  Proteins on chromosomes ????
Figure Legends should be better detailed

Discussion should be carefully revised.
For instance, you cannot write at line 326 the proteins could be used for  further resistant breeding.
You just demonstrated that is active during plant-pathogen interaction


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: GENOME-WIDE ANALYSIS OF LECTIN RECEPTOR-LIKE KINASES FAMILY FROM POTATO (SOLANUM TUBEROSUM L.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The work of Zhang and Qin presents an in silico genome-wide analysis of the lectin receptor-like kinases in the crop plant Solanum tuberosum. Authors follow up by looking at the expression of selected members of the family in plants challenged with bacterial and fungal infection to identify candidates for further studies. I find  the work both interesting and relevant. The aims are clearly explained. As stated above experimental strategy is well described and follows an established route.

Nevertheless I find that the manuscript could be further improved.

Major revisions

1. The introduction would benefit from referring to the closely related and published work of  Nazarian-Firouzabadi F et al., 2019 (doi: 10.1007/s11033-019-04951-z); Li et al., 2018 (doi: 10.3390/cells7090120); Dezhsetan S. 2018 (10.1007/s12298-017-0471-6). Along similar lines I suggest that authors discuss how their work compares to the published studies (see above) dealing with in silico identification of receptor-like kinases in Solanum tuberosum.

2. The English language should be improved. However, the text is understandable; it suffers from grammatical and stylistic errors. For instance, and starting with the first sentence of the abstract “commonly discovered” would be better phrased as “evolutionary conserved in the plant lineage,” the verb is missing before “involved in.” There are multiple examples throughout the text, including statements like the one in line 119 /120. Moss is also a plant; although non-vascular.

3. I thank the authors for providing the raw data. However, they would greatly benefit from a better description. As it stands now, I found it challenging to extract information. For instance, both the “q-RT-PCR terminal data” and “LEC-Zhang” files lack column headers.

Minor revisions

Figures 5 and 6: Could the authors refer to publication and/or depository from where they extracted the data?
Lines 195 and 201. It would be great if the authors could specify how they define differential expression.
Line 226. Could the authors justify selection of these particular 14 genes?
Experimental design: As stated above experimental strategy is well described and follows an established route. However I find an issue with an experimental design.

Major point

4. Infection experiments (Figure 7 and Figure 8) were performed three times; this is how I understand the meaning of three bio-reps (lines 236 and 246). Could the authors provide more information? Do they refer to the three completely independent experiments or for example, separate leaves/tubers in one experiment? It is not clear to me why having three independent biological experiments; the authors decided to merge the material and rely on three technical measurements of the same cDNA sample (Lines 108 and 109). I gather that the bar plots on figures 7 and 8 represent mean ± SD. Am I correct? This information should be in the legend. Performing statistical analysis and discussing significance, having exclusively technical replication is highly problematic. I suggest that the authors repeat the expression analysis and provide data for the three biological replications.
Validity of the findings: Please see my comment to the experimental design.
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: GENOME-WIDE ANALYSIS OF LECTIN RECEPTOR-LIKE KINASES FAMILY FROM POTATO (SOLANUM TUBEROSUM L.)
Review round: 2
Reviewer: 1

Output:
Basic reporting: See in the General comments for the author.
Experimental design: See in the General comments for the author.
Validity of the findings: See in the General comments for the author.
Additional comments: I find the manuscript of Zhang and Qin improved. Nevertheless, there are still points that need addressing.

Authors did not refer to the suggested literature, which is fine, if they explained why rather than writing “We have revised them in the manuscript” when they did not.

For the English editing, it could be done better as I can still find plenty of grammatical, stylistic, and punctuation errors. For instance, the second sentence in the abstract should be “largely” and not “large.”

Authors used “an absolute value of log2 ratio ≥ 1 as thresholds to judge the significance of differences in gene expression” (lines 88 and 89). Why use a fold change cut-off rather than a statistical test? Are reported changes truly significant?

I am grateful to the authors for explaining replication. An extra line (or two) in Materials in Methods explaining what constitutes a biological replica is necessarry (also for the readers to know).

I am grateful to the authors for providing headers in the Tables S1 to S4. Now I can understand the data, I have few questions to table S3. Why are the raw Ct values given for either mock or treatment, but never both? Why did the authors decide to use fold change rather than deltaCt values to calculate TTEST?  I am a bit surprised that actin Ct values are always so close between three replicas of the same time point/treatment. For the measurements done on independent cDNA preparation, I would expect random distribution of the actin values, rather than rep 1 to 3 clustering together. Could the authors think of the undelaying reason? Small comment, there are spelling errors in the headers, please correct. For instance, in Table S3 should be “with” rather than “ith”.


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: FIRE SEVERITY EFFECTS ON RESPROUTING OF SUBTROPICAL DUNE THICKET OF THE CAPE FLORISTIC REGION
Review round: 1
Reviewer: 1

Output:
Basic reporting: All good.
Experimental design: All good.
Validity of the findings: All good. See review for further comments.
Additional comments: Review of Strydom et al: Fire intensity is not a major determinant…….
The paper provides valuable data on resprouting of woody shrubs in a system, thicket, whose ecology has been neglected in southern Africa (and elsewhere in the world). Snapshot surveys such as this are very useful in indicating resprouting response to an extreme event, such as the fires in this region. The study seems competent with field variables collected that seem appropriate to the questions being addressed.  I’m a bit skeptical as to your ability to record small thicket plants after high severity fires which may bias the survival probability figures upwards for this size class. The analyses look good. But please add units for figures and tables recording canopy volume.
An important feature of these fires is that all the sprouting seems to have been basal since no epicormic sprouting is reported. This means that the thicket has to recover its structure from the ground up. This would allow browsing mammals greater access to the resprouts, and should also allow much more light to reach the soil layer where fynbos plants could potentially establish. Did they? Were there sufficient gaps between the thicket shrubs for colonization by shade-intolerant grasses and shrubs?
It might be worth  referencing thicket responses to herbivory and fire from southern African  savanna studies. As was found here, they have proven more tolerant of fire than forest species (see e.g. Charles-Dominique et al. 2015). Their sprouting behavior has been linked to bark traits, bud insulation and clonal spread below-ground. It would be intriguing to see how traits of eastern Cape thicket species compare in future studies and whether they predict species responses recorded in this study.
Other comments
Title. Too long. Too parochial. Think again for one with wider readership appeal.
Study area: For readers less familiar with the study area, are there any analyses showing differences in fire regime between fynbos and thicket?
p. 61 Replace ‘canopy’ fires with ‘crown’ fire, the more common term.
l.65. Replace coexistence with co-occurrence
l.71. coexist replace with co-occur.
l.101-102. Its quite common to have bimodal distribution of resprouting with high mortality in small plants and in old, mature plants but good survival and resprouting in intermediate ages/sizes.
l.106. How does two fires provide a wide range of fire intensities? Presumably both fires had variable fire intensity while the two regions test the geographical generality of any findings
l.124. replace ‘an excess’ with ‘in excess’.
l.131-132. What does Mucina and Rutherford’s volume on Veg of Sth Africa map the vegetation as? Cite that too.
l.150d. How many belt transects?
Figure 4. What are the units for volume in Figure 4?
Figure 2. You have to wonder if probability of survival response under high intensity was not affected by complete consumption of the smaller plants by fire – i.e. small plans were burnt out hence not sampled.
l.275-276. I think you don’t mean ‘accordingly’ but ‘in contrast’?
l.307-308. Fynbos vs thicket soils have similar chemistry e.g. in terms of OM and N. Really? I don’t think this is right. Check e.g. Figure 4 in Cowling and Potts where thicket has much higher nitrogen, Phosphorus, exchangeable cations and Carbon than fynbos. The situation seems similar to Cramer et al and Coetsee et al with feedbacks between vegetation and soil nutrients causing soil differences reinforcing veg differences supported by divergent fire regimes.
l.343. Not only fynbos. What is the effect of frequent fires on thicket expansions? Not part of your study so your recommendation on frequent (how frequent), medium intensity burns to maintain the fynbos-thicket mosaic  is a reasonable guess but not really based on any data?
l.348-350. I’m puzzled by this ‘consistent’ positive effects on survival and vigour. Figure 4b from Knysna shows that shrub resprouting volume declined with increasing intensity. So not a positive effect?
A comment on shoot number versus measures of regrowth such as volume:
Shoot count = measure of the bud bank and bud survival, volume of regrowth reflects CHO reserves surviving fire obscured by, depending on the timing of the census,  vigor of subsequent growth. Knysna plants showed more shoots than CSF and shoots increased from low intensity to high intensity. So high fire intensity released more buds.
Shoot volume showed different responses with high intensity fires causing reduced regrowth in Knysna relative to low intensity. So tissues may have been killed in high intensity fires resulting in less CHO reserves to resprout? The difference between the sites is apparently due to different times since burn with CSF having had longer to recover than Knysna so the link with pre-burn reserves is clouded by post-burn regrowth. Or am I mis-reading Figure 4?


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: FIRE SEVERITY EFFECTS ON RESPROUTING OF SUBTROPICAL DUNE THICKET OF THE CAPE FLORISTIC REGION
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment.
Experimental design: This article shows that thicket species are i) fire tolerant and ii) that this simplifies local fire management. Two issues I have relate to independence. Firstly, is fire intensity independent of plant size. The same fire could damage a small plant more than a big plant but that this would look like variable intensity. Show some analysis of this. Secondly, numbers of resprouts could be correlated with plant size and therefore number of resprouts is not an independent measure, say of plant response to fire. Show some analysis of this. Finally, I could not make out where the line is drawn between ramets and genets. Line 156 says stems > 5 cm were surveyed. Was the biggest stem per individual used or were all stems > 5 cm used and how was an individual defined. Line 223 says 85% of stems…again is this of all stems? Some stems may have died but at the individual level was there any mortality at all? This seems to be avoided in the discussion. Similarly, the value judgement that more resprouts is a positive response needs some discussion. A mild fire may have more canopy/stem survival and therefore lower basal resprouting on a particular species and this lower degree of resprouting could be a positive response to fire (i.e. greater stem survival).
Specific comments
Line 59 fire-avoiding- meaning?
Line 264 how much greater?
Line 392 reference incomplete?
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: FIRE SEVERITY EFFECTS ON RESPROUTING OF SUBTROPICAL DUNE THICKET OF THE CAPE FLORISTIC REGION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors have a clear and very structured writing style, I have made specific comments where there is ambiguity or grammatical errors.

The literature references and context are generally well covered, I have added specific comments below where I believe clarification is needed.

The article structure, figures and tables are presented professionally. I have made specific comments about figures below.

This is a self contained publication unit.
Experimental design: The research is original and relevant.

Research question is well defined, and the knowledge gap is clearly stated.

The investigation has been conducted with rigor. I have an issue with the data collection and subsequent analysis, I have detailed this below.

The methods are very detailed and reproducible.
Validity of the findings: The impact, novelty and management implications of this work have clearly been assessed by the authors.

The underlying data have been provided.

Conclusions are well stated and linked to the original question.
Additional comments: Broad Comments:

1) Supp Material S4 does not seem to open correctly on my device. I am not sure what the values in column 4 represent..? Did you count 808080808 resprouts on a single individual?

2) Some species in Table 1 are only sampled once or twice at the different sites. Have these species been excluded from the survival analysis? A survival rate of 100% that is calculated from one individual does not seem like a defensible assumption.

3) It would be helpful to the reader to have representative photographs of the different levels of fire intensity. This is a subjective measure, so I believe some photos in the Supp Materials would aid in reproducing this study.

4) Was the site comparison done by species? I think it would be difficult to be sure that site had no effect unless you are comparing individual species between sites. Otherwise, the community composition would have an influence on the overall survival and resprouting values.

5) The number of buds an individual has available to resprout from should increase with size. Therefore prefire-size and number of resprouts should be related. It would be interesting to see the relationship between the number of resprouts per unit of tree (controlled for size), for the different fire intensities. This would give you some relative measure of the number of active buds vs killed buds.

Specific Comments:

Line 81: Are you absolutely sure there is no research on the fire ecology? Midgley and Cowling, 1993 seems to deal with an aspect of fire ecology.

Line 174-175: See broad comment 2 above. Some species in Table 1 have one or two sampled individuals for each species. A threshold should be set to exclude species which were not sampled enough to have confidence in their resprouting response.

Line 228-229: See Charles-Dominique et al (2015) Bud protection: a key trait for species sorting in a forest–savanna mosaic.

Line 233-235: Knysna had serious drought before the fires, there may be an interaction between drought and fire intensity effects on survival and resprouting i.e. The knysna species were already stressed. (Addressed in the discussion).

Line 239-240: I would be hesitant about drawing this conclusion based on the the confidence intervals on the final panel of Figure 2.

Line 294-298: Linked to the comment above about the Charles-Dominique et al 2015 paper. As the authors state, there are considerable differences in survival rates between species. There should be considerable differences in the post-fire resprouting ability which is related to the level of insulation of the buds.

Figure 1: What does orange represent in the bottom panels?

Figure 2: A slight separation of the panels from the two sites might make this figure easier to interpret at speed.(This is a personal preference).


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: FIRE SEVERITY EFFECTS ON RESPROUTING OF SUBTROPICAL DUNE THICKET OF THE CAPE FLORISTIC REGION
Review round: 1
Reviewer: 4

Output:
Basic reporting: This paper is well written and clearly organised. The introduction provides sufficient and reasonable background to the study and how it fits into the broader field of knowledge.
The results provided are all relevant to the hypotheses, and thus this submission is self-contained. The figures and tables presented are clear, comprehensive and appropriately described and labelled. For figure 1, I would suggest adding a legend to the satellite images to be clear that they are showing the extent of the fires rather than the extent of the vegetation type.

My only major comment is regarding the use of the terms fire intensity vs fire severity. See Keeley JE (2009) Fire intensity, fire severity and burn severity: a brief review and suggested usage. IJWF 18: 116-126. What the authors have measured here is technically fire severity (i.e. vegetation damage) rather than fire intensity (e.g. energy output, fire temperatures). Of course, these two fire characteristics are often correlated, but for accuracy, I would suggest considering if fire severity is the more appropriate term to use in many places throughout this manuscript (including in the title).
Experimental design: This research fits well within the Aims and Scope of PeerJ. The research questions are clear and meaningful and identify a fundamental knowledge gap. The experimental design is excellent and described clearly. The investigation appears to have been conducted rigorously and to a high standard.
Validity of the findings: The raw data have been made available in the supplementary material. I suggest adding metadata for these raw data, especially units for the size and volume measurements.
The conclusions stated are appropriate and clearly supported by the results presented. A few comments below:

Lines 332-334: Presumably, small fynbos shrubs are also vulnerable to frequent fires –resprouters that are too small to survive and obligate seeders that have not yet produced sufficient seed. Therefore, a frequent fire regime may have unintended consequences for fynbos shrub species. It is probably worth commenting on the comparative survival/reproductive effort of small shrubs of fynbos vs thicket species and what this could mean for their response to frequent fires – even if this is unknown, it is worth mentioning that.

Lines 341-342: What is the justification for suggesting a moderate intensity fire regime? Presumably this is because fynbos vegetation is fire dependent, i.e. many species require fire to promote seedling recruitment, and therefore a low intensity fire regime may not provide germination/seed release cues. Maybe worth briefly mentioning this reason at the end of this sentence, e.g. “... and provide fire regimes appropriate for fire-dependent fynbos species.” Also relevant to this sentence and my previous comment – you should include at least a rough estimate of what frequent fire regimes are, e.g. 5-10 years?
Additional comments: This is a well presented and well written manuscript that I found easy to read. It clearly helps develop key ecological knowledge of the thicket-fynbos ecotone, with clear implications for fire and land management. Some minor comments below:
Lines 56 – 58: to help with the flow of this sentence, consider moving “of both fire-dependent and fire-avoiding biomes” before “in the same mesoclimate and geology”
Lines 105 – 106: suggest adding “and under different weather and climatic conditions” after “that burnt at different times”
Line 118: Should this refer to Table S1 rather than Fig. S1?
Line 131: add a comma after ‘classification’
Line 136-137: Where species names are mentioned, I would suggest including plant families for the benefit of an international audience
Line 138: remove comma after ‘mesophyllous’
Line 153: add closing bracket after ‘Cape St Francis site’
Lines 155-157: This sentence is a bit awkward. Consider splitting it in two, i.e. “Within transects, we measured each shrub with a stem diameter > 5 cm. Measurements included...” Also clarify if this is basal stem diameter (10 cm above the ground).
Lines 157-161: I would suggest explicitly stating that this measurement is called Firebase. This will help the reader to easily interpret the figures presented.
Line 188: weigh -> weight

Also, check for spelling errors in the reference list, e.g.:
Line 355: statstical -> statistical; indentification -> identification
Line 364: reguirments -> requirements
Line 379: persistance -> persistence
Line 380: disperesed -> dispersed
Line 382: succesion -> succession


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: FIRE SEVERITY EFFECTS ON RESPROUTING OF SUBTROPICAL DUNE THICKET OF THE CAPE FLORISTIC REGION
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 addressed the reviewers' comments sufficiently.


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: DISCOSNP-RAD: DE NOVO DETECTION OF SMALL VARIANTS FOR RAD-SEQ POPULATION GENOMICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Literature cited and background in the introduction are sufficient. There are some minor grammar issues throughout, which I note in the comments section below. As an example, the caption for figure 1 states: "The leftmost bubble contains two symmetrically crossroads.", yet I believe this should be 'symmetrically branching crossroads'. Another pass through the manuscript with an eye for things like this will be beneficial.

I rarely find pseudo-code to be informative, so I would recommend removing Algorithm 1.

Throughout the manuscript the sequence data for any given sample is often referred to as a 'dataset', which is pretty non-standard. For example (line 179): "...allele read counts contingency table over all possible pairs of datasets…". In my mind a dataset is the totality of all data for a given project, i.e. all the sequence data for all the samples.

Line 184: Are there other things besides "approximate repeats" that might generate a low rank value for real variants, which might thus be excluded unnecessarily?
Experimental design: The research is within the aims and scope of the journal, and it is well defined and relevant. Certainly providing a high-performance method to extract variants from raw RADSeq data is a worthy target. Overall, the structure of the materials and methods section looks good and makes sense, though there are some sections where critical information is missing, which I highlight below.

Section 2.4 Application to real data from Chiastocheta species - This section doesn't provide sufficient information to reproduce many parts of the analysis, especially the stacks/ipyrad assemblies and structure analyses.

For paired end data, if R1 and R2 are non-overlapping then they will be treated as independent reads, correct? This discards valuable information about linkage between snps from R1 and R2.

Line 78: Stacks2 makes use of de Bruijn graphs as well, so it might be nice to highlight the differences of this method versus stacks2.

Line 136: "Although this prevents the detection of variants as close as 3 bp…." If I'm not mistaken, this means that "loci" with variants in the last 3bp are discarded. Is there some other way this could be handled w/o discarding these real variants?

Line 162: "(data not shown)". Please show this data, or at least indicate what constitutes the threshold for significant change in recall.

Line 179: The explanation of the rank value could be clarified a bit. I understand what it's doing now, but I had to look at the code, look up Phi coefficient, and read this paragraph several times before I got it.

Line 197: "... each bubble of this locus should share one k −1-mer…", but what if they do not? "Should" is not a very strong condition, and it would seem that if the bubbles from the same locus do not share a k-1-mer then they will be treated as independent loci, if I'm not wrong.

Line 227: The 1% simulated sequencing error is substantially higher than is typical of current Illumina instruments, which tend more toward 0.1%.

Line 229: The evaluation protocol should specify whether multiple snps from a given locus are identified as coming from the same locus, or if they come from different loci. It's true that the precision is quite good, but if the snps are being split up into individual loci per snp then it's losing information about linkage.

Line 242: It's not clear why the denovo tags/loci from stacks/ipyrad were mapped to the D. melanogaster genome in different ways.

Line 249: The "custom script" here is not provided in the github repository.

Line 332: Recovering phylogeographic patterns - It's more typical to run STRUCTURE analysis for various values of K and to assess the likelihood of each K value. It's well known that the current methods of evaluating likelihoods of K values are unable to evaluate the likelihood of K=1, so choosing K=2 here seems somewhat arbitrary, especially given the very small ancestral components assigned to the few Eastern samples.
Validity of the findings: One of the big selling points of this method appears to be the ability to run without tuning parameters, yet in practice I found that `run_discoSnpRad.sh` has many parameters that are configurable, and that changes in these have marked impacts on the resulting outputs. It's also not clear how many of the parameters of the component parts (dbgh5, kissnp2, kissreads2, fasta_and_cluster_to_filtered_vcf.py) are configurable and how many are simply hard coded. It seems like a mix of these, as far as I can tell. Line 294: "A major advantage of DiscoSnp-RAD stands in the fact that it does not require fine parameter tuning." In examining the code in the file run_discoSnpRad.sh (provided in the github repository), in the section under DEFAULT VALUES, it appears there are at least 10 parameters that roughly correspond to very similar parameters in ipyrad and stacks (e.g. min and max coverage, max number of snps per bubble, etc). I would say, rather than not requiring fine tuning, that DiscoSnp-RAD tries to choose sensible defaults. Figure 3 shows somewhat similar results for variation of the k-mer size parameter, but in order to make the general claim that discoSnpRad doesn't need parameter tuning I think a little more work needs to be done to show that the various parameters do not change the results.

Line 325: Unless I'm mistaken, the topology of the RAxML tree in figure 4 and the topology of the ML tree genreated from RADSeq data in Suchan et al 2017 (Figure 1b) actually do _not_ agree. In the manuscript (C. macropyga, (C. Trolli, C. dentifera)) is reciprocally monophyletic with respect to (C. lophota, (C. setifera, C. inermella)), but in Suchan this is not true.
Additional comments: Line 29: "... due to his completely different principle…", I would say "its" instead of "his".

Line 56: Add or update citations to include the most recent versions of stacks and ipyrad:

	Rochette, N. C., Rivera‐Colón, A. G., & Catchen, J. M. (2019). Stacks 2: Analytical methods for paired‐end sequencing improve RADseq‐based population genomics. Molecular ecology, 28(21), 4737-4754.

	Eaton, D. A., & Overcast, I. (2020). ipyrad: Interactive assembly and analysis of RADseq datasets. Bioinformatics.

Line 71: "arbitrarily"

Line 82: "Chiastocheta species". Chiastocheta is the genus, correct?

Line 95: "Graph" is not a proper noun, so it should not be capitalized, in my view. "de Bruijn graph" is better. This is a small thing. It's correct on line 78.

Line ~105: Figure 1 caption: "symmetrically" should be "symmetrical".

Line 108: "... filtering out kmers with a too low abundance in the read sets, and by limiting the type or number of branching nodes along the two paths." These seem like parameters that could take different values based on characteristics of the specific dataset in hand. How are "Low abundance" and "type or number of branches allowed" defined by DiscoSnp++? Are these exposed to the user? If not, how are the defaults chosen?

Line 159: "We limit the maximal number of traversals of SBCs per bubble to 5." This is another example of a parameter that is simply hard coded, rather than allowed to be adjusted by the user based on their understanding of the data. It is later stated that "This value has been chosen after tests showing that larger values lead to longer computation…" and so on, but there is no indication of the datasets used for these tests. Were they from simulated or real data? If from real data, what was the taxonomic scope of investigation? Within species genetic diversity could have a drastic impact on setting of this parameter.

Line 167: "...allowing up to 10 substitutions...", again, this seems to be an arbitrarily chosen and fixed value. Stacks and ipyrad get a lot of attention in the introduction based on their parameterizations, yet this method seems to be simply making these parameter decisions for the end user.

Line 175: "...deriving from coalescent events." I would prefer "alleles sampled from non-homologous loci", or something like this. If the alleles are paralagous, they would certainly share a common ancestor, just deeper in time.

Line 185: "2.1.3 Clustering variants per loci", should be 'locus'.

Lines 173-184: There is a lot in this paragraph that is unclear. For example, "approximated repeats" are not well defined. "...read counts contingency table over all possible pairs of datasets…", it's not clear which pairs of datasets are referred to here.

Line 203: "Clusters containing more than 150 variants are discarded…." This is another hard coded parameter of the model.

Line 240: ipyrad clustering values tested on the simulated data appear not to have been included, "... composed of 100 samples (ie. )...". Also, how final stacks `-M` and ipyrad clustering threshold values were chosen is not clear.

Line 262: STRUCTURE is not simulation based.

Line 318: This is a somewhat informal description of how STRUCTURE works, it's also more "methods-y", in my mind and so doesn't belong in the results. I would move to the methods and make it more formal.

Line 382-384: It is not true that Stacks or ipyrad only use R2 mainly "to remove PCR duplicates". It's also not true that "Properly recovery of loci is therefore not possible with read stacking approaches."

I installed using the conda package and `run_discoSnpRad.sh` did not work out-of-the-box. I had to edit clustering_scripts/discoRAD_clustering.sh and change #!/bin/sh to #!/bin/bash, otherwise `run_discoSnpRad.sh -r fof.txt -S` would crash. This is on a stock Ubuntu linux machine, nothing fancy.

It's good practice to ship working example data with the code to make it easier for people to try out. I tried running the test data (https://github.com/GATB/DiscoSnp/tree/master/test) with discoSnpRad defaults and it returned 3 snps in the vcf. A 3 locus dataset with 1 snp per locus isn't close enough to biological reality to give people a good sense of what's going on. I generated my own simulated dataset (which assembled fine in both stacks and ipyrad), very clean 1000 loci with a reasonable level of variation (nucleotide diversity ~0.002), and discoSnpRAD returned 0 snps in the vcf. Update: I figured out what's going on, the fq.gz files need to be demultiplexed to samples. I think it's important to mention this in the manuscript and in the documentation because it led me to an extreme amount of confusion for a while.

Once it started working it finished fine and claimed to place the results in a file which doesn't exist:
***********************************
Short read connector finished
results in:
   discoRad_k_31_c_3_D_0_P_5_m_5_raw_filtered_simpler.txt
Contact: pierre.peterlongo@inria.fr
***********************************


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: DISCOSNP-RAD: DE NOVO DETECTION OF SMALL VARIANTS FOR RAD-SEQ POPULATION GENOMICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In their manuscript ‘DiscoSnp-RAD: de novo detection of small variants for population genomics’, Gauthier and coauthors present a novel method for RADseq data analysis. The method adapts the DiscoSNP++ (Peterlongo et al. 2017) reference-free SNP discovery and genotyping approach to the specificities of RADseq data, thus allowing its application to a data type that is widely used, and often so in systems where no reference genome is available. They compare the performance of the method against two methods commonly used for RADseq analysis, finding that their method performs similarly to existing ones in terms of recall and precision of SNPs and very favorably in terms of computational requirements. They then use it to re-examine a real insect dataset (Chiastocheta flies; Ronikier & Alvarez 2017).

The presented work is relevant and of interest to the community, the analyses appear to be well executed, and the manuscript is generally clear and well written.

I nevertheless have comments on specific elements of the manuscript, listed below:

1. The authors state on multiple occasion (L28, L87, L293, L367, etc.) that their method successfully bypasses the need for any similarity parameter. Since this is a core aspect of the method, I believe the authors should develop their current discussion of this property (L369 ‘[parameter choices] impact the number and type of detected variants’), that they should further explain how this property arises eg. in the introduction, and that they should discuss the drawbacks of the approach.

2. Figure 3: it would be very desirable to add a panel showing how k-mer size influences some general statistic(s)  for the real dataset, in addition to the results given for the simulation dataset.

3. It is repeatedly affirmed that clustering in Stacks and ipyrad require ‘all-versus-all read alignments’ and thus that the computational requirements ‘increase quadratically’. This is wrong; both methods use k-mer-based heuristics. Please review the relevant literature and update the text in all relevant places.

4. The most recent Stacks and ipyrad publications should be cited (respectively 2019 and 2020). Also, for Uricaru et al. the final citation appears to be 2015.

5. For simulations, the authors should justify why they compared the results of the different methods to ‘'the standard protocol of DiscoSnp++ when a reference genome is provided’ (L230) rather than to the actual simulated genotypes, especially as this has the potential to bias the results in favor of DiscoSnp-RAD.

6. L262 Authors should state that they have explored a relevant range of values of K and that K=7 was the most biologically meaningful (cf. Novembre, 2016).

7. L276 Variant calling fundamentally has two components: the discovery of polymorphisms, and their genotyping in each individual. The authors should justify why they report recall and precision for SNPs but not for individual genotypes (or give the figures for genotypes).

8. L332 ‘The STRUCTURE analysis of this dataset tended to identify two populations and assigned 31 samples to one of them and 9 to the other (Fig.4)’ I disagree with this statement; all samples are primarily associated with the same component. The signal the authors refer to may exist, but STRUCTURE does not seem to be the right approach to describe it. The authors may want to perform a PCA and consider the PC(s) most correlated with longitude and update figure 4.

9.  Authors should make their custom scripts available, especially those related to benchmarking and simulations.

Minor comments:

10. L20 ‘DiscoSnp-RAD […] avoids this pitfall since variants are detected by exploring the De Bruijn Graph built from all the read datasets.’ Phrasing could be improved for non-specialist readers

11. L47 ‘The sequencing output is thus composed of hundreds of thousands of reads’ Millions?

12. L52 ‘mainly’ is unwarranted

13. L79 ‘We propose an adaptation of the DiscoSnp++ approach to [RAD-Seq data]. After validation tests […]’ Authors could insert a short description of how their method differs from the previously described DiscoSNP++ implementations (cf. Methods).

14. L88 Please proofread

15. L91-93: Please proofread

16. L130 Please clarify whether ‘variants’ comprises SNPs, indels, or both.

17. L170 The default minimum per-allele depth threshold of 3 seems quite high; this may create bias against heterozygous genotypes. This is also redundant with the binomial likelihoods approach, which is more relevantly combined with a filter on total coverage.

18. L192-200 The definition of edges (and thus of loci) could be clarified, especially as this functionality is going to be of particular interest to users.

19. L220 ‘SNPs were randomly generated at a rate of 0.01%’ Please confirm that this is not 0.01/1%

20. L235 Authors should state why they did not compare against dDocent (Puritz2014).

21. L240 Please proofread

22. L296 “between samples” is confusing and should be removed; samples are typically diploid.

23. L310 Which of the filtering criteria is the primary cause of the removal of 95% of SNPs?

24. L329 ‘The same run of DiscoSnp-RAD obtained with the 259 samples was used, only the vcf was limited to the 40 sample columns of this species and the same filters were applied on this reduced dataset’ Please improve this sentence.

25. L383 Please clarify whether Disco-SNP itself is able to remove PCR duplicates, as for this purpose alignments to a reference are usually needed.

26. L391 Please clarify why DiscoSNP-RAD is more relevant than DiscoSNP++ for sequence capture datasets


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: DISCOSNP-RAD: DE NOVO DETECTION OF SMALL VARIANTS FOR RAD-SEQ POPULATION GENOMICS
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: Line 253. Despite both the main text and the documentation provided in the github repository may suffice to the average experienced bioinformatician, I think maybe some beginners could find it a little hard to replicate the results when they reach the filtering part, after the execution of DiscoSnp-RAD itself. That’s because even the scripts to do so are available on github, there aren’t any instructions on which and how or when to use each one of them, besides the texts inside the scripts themselves. I understand that they aren’t part of DiscoSnp-RAD itself, and even that one could use any other tools provided around the internet to perform their tasks, but I think your pipeline could have a greater reach if even a brief explanation around the scripts was given either in the paper or in the software documentation.
Validity of the findings: Line 327. It’s great to see that DiscoSnp-RAD is capable of detecting such a shallow, intraspecific signal, proving the other positive results aren’t just coming from greater differences between lineages. As far as I could find on the original paper by Suchan et al 2017, it seems to be an unpublished result regarding C. lophota, and if it doesn’t it has to be pointed out more clearly. But considering it is indeed a new result, it wasn’t clear to me whether the intention was suggest DiscoSnp-RAD can be more sensitive than PyRAD (the software used on that work) or if you ran STRUCTURE only with the SNPs got from DiscoSnp-RAD, unlike what was done in the previous section in the paper (line 309).
Additional comments: I was able to successfully replicate something similar to your results on my own data, when comparing results between DiscoSNP-RAD and Pyrad. I’d like to say it was great, and even in a quite small dataset the processing time with DiscoSnp-RAD was twice as fast as Pyrad’s. Moreover, I could take a close look on what the users will find when they start using this great new tool, and I have a couple suggestions.

- I think you should consider incorporating  short_read_connector (or an equivalent) into DiscoSnp-RAD. It’s was not very difficult for me to make everything work, but I have the impression that it could discourage more inexperienced users the way it is now, due to issues with dependencies;
- At first I couldn’t use the clustering option (-S flag), and just after looking into the code I realized it HAS to come first in arguments order, when running the DiscoSnp-RAD. I didn’t find any information regarding that in the documentation. Either way, isn’t hard to make the position of arguments flexible, what certainly could save users some trouble.
- I think you could give DiscoSnp-RAD its own exclusive user’s manual and expand it a little, including not only the equivalent to what you have in DiscoSnp++’s part but also the information I talked about in other topic. Many researchers – myself included – will make use of the RAD part only.


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: DISCOSNP-RAD: DE NOVO DETECTION OF SMALL VARIANTS FOR RAD-SEQ POPULATION GENOMICS
Review round: 2
Reviewer: 1

Output:
Basic reporting: L194: It's not clear what 'binary variables' are being referred to.
Experimental design: Are there options for producing haplotypes rather than just snps (i.e. recovering the full RAD locus information)? It seems not, but this should be clarified. Some downstream tools require haplotype data.

I would recommend including in the manuscript information about how R1 and R2 are handled for paired-end data. I understand that if R1 and R2 are overlapping the linkage information will be retained, but am somewhat fuzzy on how many bp they must overlap by for this to be true. Also, if they are not overlapping are they treated as independent? In response to reviewers it is stated that they "create independent connected components in the de Bruijn graph", but it's not clear to me if this means the linkage information is retained. In particular, "independent connected" does not clarify this for me.

I thank the authors for clarifying the Phi coefficient and 'rank' in the manuscript, and I appreciate the information given in response to Reviewer 1s question about low rank variants, which I find interesting, but which I do not find in the revised manuscript. In particular, I would recommend discussing why "'heterozygous in all' variants should be very rare", and what might be the downstream ramifications of removing these variants.

The text introduced to address reviewer 1s comment on Line 136 is still ambiguous. If you "constrain the last 3-mer of both paths to be identical" it is still not clear how this "does not prevent to detect loci containing such variants." Instead of "we also constrain the last 3-mer of both paths to be identical" I think it would be more accurate to say "the last 3-mer of both paths are excluded from the analysis.", or something like this.
Validity of the findings: RADSeq datasets are notorious for their heterogeneity along multiple axes: missingness, read depth per locus within samples, sample depth per locus, not to mention nucleotide diversity and other aspects of the genomic architecture of the focal species, and the peculiarities of any given library prep protocol, etc, etc. There is a reason other assembly tools have parameters and this reason is because many RADSeq datasets are peculiar. In truth of fact, Figure 3 shows that changes in parameter values actually do impact the results for DiscoSnp-RAD, contrary to what the authors insist on. The asymptotic behavior of precision and recall for each parameter shown in Figure 3 are relevant only to THIS ONE simulated dataset that is tested. Heterogeneity in real datasets will require tuning DiscoSnp-RAD parameters in much the same way as other RADSeq assemblers, in order to optimize results. I think a lot more work would need to be done to demonstrate this is not the case, for example analysis of simulated data generated under a wider array of conditions. It would also be nice to see something like figure 3 for the empirical data, varying parameters and plotting number of SNPs/loci recovered. This would go a long way toward strengthening their claims, if the recovered SNPs do not change with varying parameters for the real data. My previous recommendation was to emphasize that "rather than not requiring fine tuning, DiscoSnp-RAD tries to choose sensible defaults," and I stand behind this recommendation, given the current state of the manuscript.

For the comparison to other tools it occurs to me that the 1% sequencing error in the simulations is more than just unrealistic. It is also probably what's causing the significant difference in performance measured in STACKS and ipyrad. This insight is supported by the results described in the "Breakthrough in running time" section which shows that on real data STACKS and ipyrad perform significantly more similarly to DiscoSnp-RAD than with the simulated data. Rather than redo the simulations, I recommend adding something to the discussion about the choice of sequencing error rate for the simulated data and how this might impact performance of the various assemblers.

The manuscript still states: "The phylogeny realized with RAxML on the 4,364 SNPs obtained after filtering, is congruent with the one obtained by Suchan and colleagues [25] (Fig.4).", which the authors agree is actually false. This sentence literally says that the RAxML trees are congruent between studies. If this is not the case this wording should be adjusted accordingly.

L407: Proper handling of GBS data involves accounting for the fact that reads from any given locus can come from either the forward or the reverse strands. It's not clear this method considers strandedness when building the DBG. I am not a DBG expert, so perhaps reverse complement matching consideration is made during this process, and if so I apologize. If revcomp is not considered then I would remove the reference to GBS here.
Additional comments: During the course of my second review I find that the authors satisfactorily addressed many of my comments from the first review. I also must emphasize that DiscoSnp-RAD actually is quite fast, and in independent experiments I never found a case where STACKS or ipyrad were faster, this is totally true. However, I have several comments of secondary concern enumerated below, and my primary concern remains the insistence on "Robustness with respect to parameters", which I elaborate on in the "Validity of findings" section below.


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: DISCOSNP-RAD: DE NOVO DETECTION OF SMALL VARIANTS FOR RAD-SEQ POPULATION GENOMICS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have honestly and thoroughly addressed the concerns raised by me and the other reviewers. In particular, the expansion of figure 3 and the work on the Methods section are welcome additions. I believe that they present a strong manuscript and that their method DiscoSNP-RAD will be very valuable to the community.


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: SUCCESSION OF BACTERIAL COMMUNITIES ON CARRION IS INDEPENDENT OF VERTEBRATE SCAVENGERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some small editorial comments: authors' use the word "utilize" incorrectly (ex line 93; utilize = a strategy is put to practical advantage or a chemical or nutrient is being taken up and used effectively; see https://www.quickanddirtytips.com/education/grammar/use-versus-utilize?page=1); there are double periods at the ends of some sentences while others are lacking punctuation (ex lines 46 and 101); wording is ambiguous, vague, or sometimes wrong maybe to avoid sounding too much like author's original wording? (ex lines 51-54); common names are not capitalized unless they are a proper noun (ex line 83; lines 211-215)

I think the paper is at some times insufficient in it's literature review, lacking citations of recent research in the field and leaving out citations entirely (ex lines 47-50; lines 51-62; lines 70-75; line 93-96; lines 337-346).
Experimental design: There doesn't appear to be any control or discussion of a control: Were any carcasses used a s a control? (Such as caged as to exclude as much vertebrate scavengers as possible?) If not, was a baseline of microbial diversity established at t=0? so like at the time of placement? That could also serve as a control of sorts. Or a soil sample? I think this should be addressed at least.

Lines 226-230: this method alone is insufficient to determine if the scavenger activity was the reason for lack of diversity. See comment about controls.

Can't make the conclusion in line 381-382. You didn't test any other factor but scavenging. Insects could be homogenizing the microbial community....
Validity of the findings: Data analyzed appropriately, analysis is recreateable, methods robust, figures appropriate, figure captions appropriate.

Some speculation is made but not tempered as such:

Lines 226-230: this method alone is insufficient to determine if the scavenger activity was the reason for lack of diversity. See comment about controls.

Can't make the conclusion in line 381-382. You didn't test any other factor but scavenging. Insects could be homogenizing the microbial community....
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: SUCCESSION OF BACTERIAL COMMUNITIES ON CARRION IS INDEPENDENT OF VERTEBRATE SCAVENGERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The concept of identifying the microbes in combination with vertebrate scavenging is excellent. However, the lack of clarity in this manuscript made it cumbersome to read and difficult to understand the logic and justification of the experimental design. Understandable, carrion research is often difficult to perform due to logistic and financial constraints, but the lack of replication in this manuscript is a source of concern.

Major Concerns:

•	I am concerned about the sampling regime to make broad statements about the results for the microbial portion. It is under-described, ambiguous, and not well justified and thus a major weak point of the manuscript.  It is unclear if specimens form only two of the seven carcasses were collected at each of the five days? where they different carcasses each of the five days? Please add more details to clarify if the samples carcasses were sampled each day, if not, why?

•	There is an entire section the “bacterial taxa associated with macroinvertebrates” but nowhere in the methods is there a section described about how inverts were collected, processed, identified, etc. The assumption that they are insect based without collecting and identifying bacteria from the insect is a weak section of the manuscript.

•	How did the temperature relate to the decomposition? Four days for fresh is a long time, unless the carcasses were not thawed prior to field deposition and the ambient temperature was closer to 4C.

•	The authors describe the bacterial community composition at broad scales (phyla, order, family), with 16S data why not delve into pattern differences at the genus or OTU/ESV levels?

•	In the discussion, the authors highlight the novelty of excluding vertebrate scavengers in their study design, but many of the other carrion-microbe studies focus on the insect activity and have anti-vertebrate scavenging cages placed on top of the carcasses to reduce scavenging pressures from vertebrates (works by Weiss et al, Metcalf et al., Hyde et al., Pechal et al., Weatherbee et al., etc.).

•	The comment about vertebrates “could be important vectors” bacteria is an interesting one (and one in which I agree about potential for bacterial movement), but there is no data to support the potential for this type of activity in the current manuscript. 16S is detecting DNA and not viability, and there did not appear to be any analyses concerning actual transfer from carcass to vertebrate even based on a reduction of bacterial taxa post-scavenging events.
Experimental design: See above section.
Validity of the findings: See above section.
Additional comments: The authors should review the manuscript carefully, and check for typos, spacing errors, and punctuation errors. Below are more specific comments (minor concerns) that the authors may want to consider addressing:

Line 51: Payne had six stages in terrestrial systems
Line 71: Provide a scale for “quickly” and are you referring to exogenous or endogenous microbes?
Line 77: Please expand on “harsh conditions present in their hindgut”.
Line 80: change “has” to “have”
Line 83: “Burying Beetles” is not capitalized, and “spp.” is not italicized.
Line 84: Need citations to support the sentence.
Line 99-100: Citations after “Scavengers act as a vector of
100 dispersal for many microbes…”
Line 101: End of sentence needs only one period.
Line 101: Beginning sentence, is this true across all ecosystems (e.g., aquatic and terrestrial)?
Line 103: Is this really the use of “eDNA” in the classical sense?
Line 112: Justification behind the regular sampling intervals? What were they?
Line 115: Were the carcasses thawed prior to field deposition? How long were carcasses kept frozen? We know that long-term freezing will impact the microbial communities. Also, thawing bodies will impact the microbial succession (Pechal et al 2018 JFS) of human bodies, and thus one may assume would also impact the successional patterns of other vertebrates.
Line 116: were all carcasses placed into the field on a single day?
Line 123: I would recommend moving this information closer to line 112; also, please justify why these intervals were chosen.
Line 136: Why the muscle tissue? How far were the biopsies made? Was a punch used or a standardized volume? Was the depth of muscle tissue sampled for the microbes? Again, how was this standardized?
Line 136: why not all carcasses? Also, were these soil samples taken prior to carcass placement? What does “Directly adjacent mean” in terms of distance from the carcass? Why not sample from all the carcasses each time?
Line 138: Again, it is confusing about the sampling regime given that tissue samples were only
Line 142: What was the length of the amplicons?
Line 152: Remove the second parenthesis after the citation.
Line 161: Are the data presented for the lack of correlation? If not, it would be great to see in the supplemental material.
Line 193: Again, the results from the comparisons would be a good addition to the supplemental material.
Line 194: if samples were only collected from two carcasses during each day, and one carcass had to be removed, how many sampling days have an n of 1?
Line 222: Data/results/figures to support no statistical difference?
Line 228: Remove “using edgeR”, as it is in the methods.
Line 233: How were “macroinvertebrate-associated genera” identified? What host species were used? Did this overlap with the insects found during decomposition? How do you know if they are insect related if no insects were collected?
Line 237: Unnecessary line.
Line 299: “this is the first…” may want to add in a terrestrial system given Burkepile’s work. Also, a new study by Blandford et al. 2019 that you may want mentioned in the discussion.


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

Input:
Title: SUCCESSION OF BACTERIAL COMMUNITIES ON CARRION IS INDEPENDENT OF VERTEBRATE SCAVENGERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Where will the analytical code be deposited?
Experimental design: Please further clarify the justification for the soil sampling in the main text.
Validity of the findings: No comment.
Additional comments: Review the document for plural agreement and word choice.

Review document for use and appropriateness of adverbs.

Payne 1965 stages were not properly addressed in the revisions (Line 51)

“Postmortem time intervals” is not abbreviated “PMI” (Line 160).

Review line 236 for clarity.

Despite the comment that “Soil sampling was added during the study and was not executed entirely consistently across all carcasses, as explained in the text”. Where in the revision, is the fact explained to readers that are not familiar with experimental design/justification? One cannot assume the justification from the details provided. Please further clarify in line 283 why the soils were only collected adjacent (<1 m) from 2 of the 7 carcasses on day one, and then at 5 m the remaining sites during day 4.

My apologies for not inquiring in the previous version, but is the code for all analyses going to be made publicly available, if so where?

In the alpha-diversity results section, please provide quantities for how much diversity increased/decreased.

In line 400, I would recommend being more forthright about the fact that these 39 OTUs were only detected in a single carcass (as indicated in the discussion).

Line 59, due to the data collected in this study, you can perform a post-hoc power analyses to determine how many carcasses would be needed to detected differences. Further, in the discussion section about no consistent patterns of bacterial community change after scavenging, there is not discussion about the potential temporal aspects of when sampling occurred relative to the scavenging event. This factor too could be important in identifying transient scavenger derived taxa.

Finally, I do not believe there is an adequate discussion about the sample size of the study and the power/effect size of their results. Carrion work in general can be difficult to obtain sufficient independent, replicates. The authors may consider discussing how their sample size impacted their results.


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

Input:
Title: MINING OF CANDIDATE GENES INVOLVED IN THE BIOSYNTHESIS OF DEXTROROTATORY BORNEOL IN CINNAMOMUM BURMANNII BY TRANSCRIPTOMIC ANALYSIS ON THREE CHEMOTYPES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript would benefit from english editing, this is based on the following examples:
Line 34: "When applied orally administration"
Line 66: "prenyl diphosphatesare" should read "prenyl diphosphates are"
Line 69: "under the catalysis" is not the right term
Line 193: "chmotyps"
Line 197: "statistics were concluded"
Line 248: "exhibit more actively"
Line 299: "proved by"
.......

The cited literature is sufficient; however it favors citation of Chinese researchers.

The article structure, figures and tables are largely professional, except for Figure1a I do not think that it is necessary to include a picture of crystalline D-borneol in the article. Figure 2b the chromatogram looks like a screenshot and the line indicating the retention time of D-borneol makes the peaks hard to see. Moreover, from the legend it is not clear if c is a spectrum of D-borneol in a sample or from the NIST-library. Figure 6: the legend speaks about black and grey bars, however the bars are pink and blue.
The raw data is shared, however the link to the transcriptomics data supplied with the review material is not found in the manuscript itself. In line 204 of the results the authors talk about a reference transcriptome for mapping, but in the Material and Methods no reference transcriptome is specified.

The largest drawback of this research is that the core goal to identify a D-borneol synthase is purely hypothetical. While they claim to have identified two terpene synthases that are good candidates, I disagree. First of all, these enzymes were not characterized at all and terpene synthases are notorious for producing multiple products, therefore while these enzymes might produce D-borneol this might be only one terpene among many and not even the major product. This would prevent using this enzyme for biotechnological production of d-borneol without further protein engineering near impossible. The best way to improve the manuscript and support the claim that these enzymes could be used for biotechnological approaches, would be to express the two enzymes in a well-established heterologous host like E. coli and either use a metabolic engineering approach or purify protein for in vitro assays.
Experimental design: The research tries to fill a knowledge gap, however it falls short as stated above in supporting the conclusion that this research and the investigated genes will help in the intended biotechnological approach for the production of d-borneol.
Validity of the findings: Again the conclusions overstate the findings, as the two suggested genes were not characterized and might not be the searched for D-borneol synthase.
Additional comments: The authors identified GPPS and their expression pattern (Figure 5), which enzyme was used for anotation? The previously identified GPPS in Arabidopsis was later identified to produce longer chain prenyl diphosphates (Hsieh FL, Chang TH, Ko TP, Wang AHJ (2011) Structure and mechanism of an Arabidopsis Medium/long-chain-length prenyl pyrophosphate synthase. Plant Physiol 155(3):1079–1090) and instead a heteromeric GGPPS is producing GPP (Chen Q, Fan D, Wang G (2015) Heteromeric Geranyl(geranyl) diphosphate synthase is involved in monoterpene biosynthesis in Arabidopsis Flowers. Mol Plant.).


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: MINING OF CANDIDATE GENES INVOLVED IN THE BIOSYNTHESIS OF DEXTROROTATORY BORNEOL IN CINNAMOMUM BURMANNII BY TRANSCRIPTOMIC ANALYSIS ON THREE CHEMOTYPES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Mining of candidate genes involved in the biosynthesis of dextrorotatory borneol in Cinnamomum burmanni by transcriptomic analysis on three chemotypes” by Zerui Yang et al. identified three structural genes and seven transcription factors involved in the D-borneol biosynthesis in Cinnamomum burmanni. The manuscript is overall well-structured and focused. I think the major conclusions are well supported. The findings are interesting and will be utilized in the field. I have only minor concerns:

Line 32: Replace “administration” with “administrated”.
Line 193: Spell check for chemotypes.
Please refer Caihui Chen et al. (doi: 10.1186/s12864-018-4941-1) in introduction
Line207: replace blast with BLAST
Line 256: replace “partiality” with “partial”
Figure 5: In figure legend replace factorsa with factors
Experimental design: Experiments are well designed and executed.
Validity of the findings: Findings are novel, interesting and useful for future studies within field.
Additional comments: Manuscript is suitable for publication in PeerJ after minor 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: MINING OF CANDIDATE GENES INVOLVED IN THE BIOSYNTHESIS OF DEXTROROTATORY BORNEOL IN CINNAMOMUM BURMANNII BY TRANSCRIPTOMIC ANALYSIS ON THREE CHEMOTYPES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article meets the Journal's standards.
Experimental design: The experimental is well designed.
Validity of the findings: More DEGs should be conformed by the QRT-PCR. For the DEGs that be discussed in the discussion section. For examples, For example, candidate transcription factors related to D-borneol accumulation.
Additional comments: 1. What are the means of HBT, LBT and BFT groups? They should be well defined in the M&M section.
2. Line 214-216 should be moved to the M&M section.
3. To date, many transcriptome analyses have been performed in Cinnamomum plants. However, no one has been mentioned in the Introduction.
4. The differences in the genes related to the synthesis of terpenes should be compared with the previous published Cinnamomum transcriptomes. Are there any differences among them? More unigenes have been identified in the present study?
5. The LBT, HBT and BFT are three different chemotypes of C. burmanni, though they exhibit similar morphology. Are the sequences of key genes involved in synthesis of terpenes also similar?
As I saw, three transcriptomes from LBT, HBT and BFT were assembled together. I suggested that three transcriptomes should be assembled separately. Then, the differences in gene sequences should be compared and analysis. Maybe, this is the reason of the difference in borneol content.
6. For the QRT-PCR, more DEGs should be validated. For example, candidate transcription factors related to D-borneol accumulation.
7. For monoterpene biosynthesis, many TFs have been reported to be involved. In the discussion section, the novel insights on the TFs involved in monoterpene biosynthesis should be highlighted.


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: MINING OF CANDIDATE GENES INVOLVED IN THE BIOSYNTHESIS OF DEXTROROTATORY BORNEOL IN CINNAMOMUM BURMANNII BY TRANSCRIPTOMIC ANALYSIS ON THREE CHEMOTYPES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The Manuscript has significantly improved from the previous version. I still think that inlcuding biochemical data would have made this manuscript way stronger in its impact, but it should also not preventing it from being published.
Experimental design: no additional comments
Validity of the findings: no additional comments
Additional comments: In the rebutal you shared SDS-PAGE pictures that show the TPS being in inclusion bodies, the reason for this might be that you used a Vector with N-terminal His-Tag on a truncated plastid protein. In this case mostly c-terminal Tags were used, as the are not as detrimental for folding as an N-terminal Tag.


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: MINING OF CANDIDATE GENES INVOLVED IN THE BIOSYNTHESIS OF DEXTROROTATORY BORNEOL IN CINNAMOMUM BURMANNII BY TRANSCRIPTOMIC ANALYSIS ON THREE CHEMOTYPES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript has improved a lot after revision.
Experimental design: no comments
Validity of the findings: Authors have incorporated all my suggestions
Additional comments: I think the manuscript can now be considered for publication in PeerJ.


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: RELATIONSHIP BETWEEN THE AVERAGE SLOPE IN THE ACTIVE COMMUTING TO AND FROM SCHOOL AND FITNESS IN ADOLESCENTS: THE MEDIATOR ROLE OF FATNESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper tests an interesting subject in the kinesiology field and many insights can be drawn from this article.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: My concerns about the study are:
1) The article needs to go through a review of the English language, as the text is often difficult to interpret.
2) In studies that concern physical fitness indicators in children and adolescents, biological maturation is a variable that should be considered. The process of growth and development at this stage of life interferes with health-related behaviors and interferes with indicators of physical fitness.
3) What are the sample size calculation parameters? What is the statistical power of the sample employed in this research? Such information should be described.
4) The description of the method is repetitive at many times - example: information on the use of the equation of Slaughter et al appears twice throughout the method, which is not very objective.
5) It is important to describe the technical measurement error of skinfold evaluators.
6) The authors proposed a mediation analysis for each physical fitness (strength and VO2max) variable separately (i.e., Figure 2). This analysis proposal does not contemplate reality, because the indicators of physical fitness manifest simultaneously. Although separate model analysis is important for understanding initial assumptions about a particular topic, it has no practical applications. Thus, it is best to make a model for mediation testing that takes into account both the strength variable and the VO2max variable. In this sense, my suggestion is that the authors test a single mediation model that presents the physical fitness indicators simultaneously. To do this, authors must make use of path analysis (Path Analysis is a special case of structural equation modeling) and test this mediation.


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: RELATIONSHIP BETWEEN THE AVERAGE SLOPE IN THE ACTIVE COMMUTING TO AND FROM SCHOOL AND FITNESS IN ADOLESCENTS: THE MEDIATOR ROLE OF FATNESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors use a clear language of English and a correct article structure.

Major comments:
1)TITLE
The title does not express the real topic of the manuscript. I suggest adding the Google Earth software in the title.
2)INTRODUCTION
It is very large. In general, the introduction have an extension of 2 pages maximum.
Also, I suggest reorganizing the introduction. Start by defining the general problem, and then finish with the specific issue, which is active commuting to school. It has not the opposite way.
The ideas are mixed and unstructured, and the speech is incoherent.
The authors use a  lot of references but some of them are not updates.

Minor comments:
1)INTRODUCTION
Line 43: authors use “active school transportation” and in other parts "active transportation to school". I would suggest homogenizing using always “active commuting to school”.
Line 58,75: I suggest updating references.
Line 63: It is not clear if the minutes are per day or per week.
Experimental design: Major comments:
1)METHOD
The title contains "High School" but this word does not appear in the text.
The method is unstructured. I recommend including several information about: numbers of schools in the sample description and recruitment process (contact process, convenience or random schools, all by telephone or how...).
I don't see clear the program that authors applied. The article does not shows information about duration, process or what is it.
The inclusion criteria are very important and in this article does not exist. I recommend including several information about inclusion criteria. Do exist a minimum of informed consent per school?
The author uses somes references to categorize active commuting to school but there are several errors. Firts, I suggest adding all active modes and not only on foot. Then, I recommend searching references because they exist to find another way to categorize active commuting to school.
The author classified "mixed modes" such as: the number of weekly trips was ≤ 4, and the time spent on the trip was < 15 minutes. With this type of classification.. What Do happen with children that commuting to school every day but spent less than 15 minutes because they live near to school? Are not active commuters?
I recommend categorizing active or passive modes only by number of trips and I also suggest categorizing travel time between home and school by reviewing the current bibliography.

Minor comments:
1)METHOD
Line 119: See active commuting p.8 and not page 7.
Line 137: I suggested adding a reference.
Figure 1: I suggest changing the name of the figure. For example: Figure 1. Flow chart of participants according to the mode of travel. Also, I recommend making it more visual. The numbers of trips and time spent is not totally clear.
Validity of the findings: Major comments:
1)CONCLUSIONS
I would suggest including a new paragraph including the future directions of both researching and public health policies.

Minor comments:
Line 315: I suggest adding the means of "AC" during all the text.
Additional comments: Dear authors,

The manuscript does not provide anything new to the literature, since there is a similar work (that it is referenced)  and the new inputs (Google Earth software) are weak, not well analysed and organized. Furthermore, there are some major mistakes in the method that are relevant. My opinion is to reject the manuscript.  Under my opinion, it does not contribute to the existing literature except an objective measurement of active commuting. However, I provide major and minor comments.

Yours sincerely,