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: ONYX DISPARAMPHIS SP. N. (NEMATODA, DESMODORIDA) FROM SOUTH KOREA WITH A TAXONOMIC REVIEW OF THE GENUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Manuscript is devoted to the description of a new species of free living marine nematode belonging to the family Desmodoridae. In present time ex[anding of our knowledge in marine biodiversity is one of the important and actual field of scientific investigations. So, manuscript must be published in PeerJ journal.
I am not a native English speaker and cannot evaluate this aspect of manuscript.
Manuscript is written in professional language with appropriate structure, qualitative figures and tables.
However I have some remarks.
General remark - check all the figures. Number of figures in the text does not correspond with figures itself.
Line 96. Check the author of subfamily. Should be Chitwood, 1936
Line 113. Must be Shahina instead of "Shabina"
Line 210-212 "There are 14 distinct annules within 10 μm at the level of the middle of the long amphideal branch and about 18 less distinctly discernible annules within 10 μm at the midbody" What is this sentence about? Please, emplane and show in figures.
Line 219. "The second, posterior, less irregular crown of eight subcephalic setae located slightly posterior to the middle point of the long amphideal branch". I do not agree with this. There is second circle of subcefalic setae not far from the first one (fig 3BC, 6B), whereas another circle of setae in the middle of amphid are also there, I would call them cervical setae.
Line 223. Please, show this pores either on light microscopy or SEM photos.
Line 243. There are instead of "there a"
Line 311. Must be amphidial fovea instead of "amphids fovea"
Table 1. Check the body length, is it really 7503-1014?
Figure 4A - where are setae?
Figure 5. What is scale bar for C-D? Is it 1 mkm?
Figure 6. What the scale?
Experimental design: no comments
Validity of the findings: no comments
Additional comments: No additional comments


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

Input:
Title: ONYX DISPARAMPHIS SP. N. (NEMATODA, DESMODORIDA) FROM SOUTH KOREA WITH A TAXONOMIC REVIEW OF THE GENUS
Review round: 1
Reviewer: 3

Output:
Basic reporting: see attached document
Experimental design: see attached document
Validity of the findings: see attached document
Additional comments: see attached document


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

Input:
Title: A SNP VARIATION IN AN EXPANSIN (EGEXP4) GENE AFFECTS HEIGHT IN OIL PALM
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript describes the identification of a SNP of a height gene, namely EgExp4, that recorded significant association with height trait in oil palm. The structure of the manuscript adheres to the journal format. However, the manuscript requires major English language editing.
In the introduction, the authors can also include previous efforts in improving height in oil palm via conventional breeding method. In my knowledge, Elaeis odora does not exist anymore. The citations are relevant but the authors can also cite more recent publications. Figures and tables and their legends are appropriate.
Experimental design: I have specific comments indicated below
1. Are the trees included in categories tall and short the same for HT1, HT2, HT3 and HT4? Palms included in the research were planted at several locations. The height differences observed could be due to palms that are grown at different locations.  This should be investigated further. There could be a requirement to normalized the height measured on all palms across different location and time of measurement.
2. Are the differences  between the means of the tall and short categories for HT1, HT2, HT3 and HT4 significant?
Validity of the findings: 1. In TASSEL analysis, the authors should explain how p value is determined
2. In Table 2, variant change was reported based on some short/tall palms but not all short/tall palms. This lead to a question whether the variant change is truly associated with height. If the change is truly due to height, it should be observed in all short/tall palms.
Additional comments: No additional comments


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

Input:
Title: A SNP VARIATION IN AN EXPANSIN (EGEXP4) GENE AFFECTS HEIGHT IN OIL PALM
Review round: 1
Reviewer: 2

Output:
Basic reporting: Somyong et al. reported the discovery of the SNP variation in expansin (EgExp4) gene that may affect height in oil palm. The story that the author presented is complete, and the biological experiment and in-silico studies were sufficiently designed and performed. Although the authors did not point out the functions of the EgExp4 gene and the contributions of the SNPs to the height, they talked about the potential relationship between cell wall protein expansin and plant growth, inspired by their gene study results.

The English writing is clear and the literature references were well-cited with consistent formatting. The figures and articles were well-made, although the resolutions of the main text figures are low with some of the axis labels being not visible.

Nevertheless, I believe the results presented by the authors will inspire researchers to pay attention to the expansin protein and the EgExp4 gene for breeding semi-dwarf oil palms. I therefore recommend it to be published in PeerJ after making necessary revisions and clarifications (please see my comments in the "additional comments" section).
Experimental design: Please see my comments in the "additional comments" section.
Validity of the findings: Please see my comments in the "additional comments" section.
Additional comments: (1) Why is there a high-intensity band in one of the sample loading wells on the last gel of Figure S3?

(2) Will lowering the height of oil palm affects the growth and in turn, affects the production of oil?

(3) In the sentence “the full-lengths of these genes were successfully amplified by the M13-tagged EgDELLA1-P1, EgGRF1-P2, EgGA20ox1-P2, EgAPG1-P1 and EgExp4-P1 primers (Table S2) in the 1st round of PCR with expected sizes of 3015 bp, 2516 bp, 2759 bp, 2917 bp and 2586 bp, respectively”, how did the author know the exact size of each amplified product? If the result of the 1st round of PCR was evaluated by gel electrophoresis, the authors may want to say something like “with bands close to the expected sizes of 3015 bp, 2516 bp, 2759 bp, 2917 bp and 2586 bp”. This also applies to the sentence “The results of amplification by M13-tagged primers and barcode-tagged M13 primers also had expected sizes of 3015 bp, 2516 bp, 2759 bp, 2917 bp and 2586 bp respectively, as shown in Fig. S3”.

(4) Line 94: For the sentence “concentration and quality of DNA were evaluated by agarose gel electrophoresis and a NanoDrop™ 1000 Spectrophotometer”, the authors may want to flip the order of “concentration” and “quality” since gel electrophoresis is not commonly used for DNA concentration determination.

(5) In the abstract, “by two rounds of PCR amplification” may be used instead of “by two PCR round amplification”.

(6) Line 137: “Python” should be used instead of “Phyton”.

(7) Line 325: Please uncapitalize “T” in “Twelve”.


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

Input:
Title: A SNP VARIATION IN AN EXPANSIN (EGEXP4) GENE AFFECTS HEIGHT IN OIL PALM
Review round: 1
Reviewer: 3

Output:
Basic reporting: More interpretation of figures and tables should be included.
Experimental design: More detailed validation design, validation results, and validation discussion should be included.
Validity of the findings: 1.	In Table 3, the results of previous work and this work were listed. And the previous work confirmed that EgDELLA1 and EgGRF1 was height-associated genes in the GT population, which was mentioned in introduction. However, in this study, only four SNP variations were confirmed in the GT population. And only mEgExp4_SNP118 was significantly associated with height in the oil palm plants. Is there any discussion about those?
2.	Is there any other reported data to support the results of this study?
3.	What is the innovation of this 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: A SNP VARIATION IN AN EXPANSIN (EGEXP4) GENE AFFECTS HEIGHT IN OIL PALM
Review round: 2
Reviewer: 1

Output:
Basic reporting: After reading the revised manuscript, I think that the manuscript still requires major English language editing.

Elaeis odora does not exist. In Elaeis genus, there are only 2 species, Elaeis guineensis and Elaeis oleifera. The plant previously known as Elaeis odora was re- classified as Barcella odora.
Experimental design: Generally the approach taken to sequence full length candidate genes linked to height using PAC Bio is a good approach and authors have deposited the data in the public databases. However, authors need to explain in a  bit more on the  parameters used to determine quality of the sequences and steps taken to avoid wrong calls for SNPs or indels. Also a figure showing the match identity of the candidate genes (likely via  blast search  for example) would be helpful.

Did the authors include the parental palms in sequencing of the candidate genes? This could have indicated the inheritance of the alleles either from the dura or pisifera palms.
Validity of the findings: The authors should justify the p-value applied to determine significance of marker-trait association, as only limited number of markers/individual palms are used in association analysis (4 markers across 40 samples). These obviously limit the power of statistical analysis.

In TASSEL analysis, can the authors present results from MLM model, as this model is available in TASSEL and is more stringent than GLM? The authors can consider presenting the Manhattan plots showing the significant markers in the manuscript which can be easily generate using TASSEL.
Also can the authors explain why only two time points for height measurement- T1 and T4 showed significant association of the allele with height and not T2 and T3? Is there a possibility that the SNPs are linked to other unmeasured traits in the two segregated groups and association with height was incidental?
Additional comments: In the authors previous publications in 2019 and 2020, they found Della and GRF genes. However, in this study the SNPs linked to height was the Exp gene. Three different studies possibly on the same population uncovers three different genes influencing height, and some explanation will help. Although authors explain that height is a quantitative trait, influenced by several genes but a discussion on why different approaches identified three separate genes influencing the same trait in the same population, would be helpful.

Did the authors attempt to link the INDELS to the trait?


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

Input:
Title: A SNP VARIATION IN AN EXPANSIN (EGEXP4) GENE AFFECTS HEIGHT IN OIL PALM
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have made necessary revisions based on the reviewer's suggestions. The resolution of Figure 1 has improved and the axis labels have been provided. I recommend this manuscript to be published in PeerJ, with a few comments left:

(1) Since the authors have now provided the explanation that "Height correlation with FFB (fresh fruit bunch yield) and BN (Bunch number) was weak to moderately positive in both the GT (r = 0.254-0.499) and KU (r = 0.335-405) oil palm populations. This suggests that semi-dwarf palm may contribute to high yield in oil palm", they may consider including this in the main text to emphasize the importance of selecting semi-dwarf palms, and inform readers that lowering the height of oil palm will not significantly affect the growth and the production of oil.

(2) It might be worth adding the explanation that the authors have provided to the Figure S3 caption, so readers do not get confused with the high-intensity band in the sample loading well on the last gel.
Experimental design: No comment
Validity of the findings: The authors have better addressed the impact and novelty of their work in the Discussion section of the revised manuscript.
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: A SNP VARIATION IN AN EXPANSIN (EGEXP4) GENE AFFECTS HEIGHT IN OIL PALM
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
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: SKIN MICROBIOTA DIVERSITY AMONG GENETICALLY UNRELATED INDIVIDUALS OF INDIAN ORIGIN
Review round: 1
Reviewer: 1

Output:
Basic reporting: English language is good.
Some important literature is missing in the manuscript
The figures could have been better, fi. the bacterial composition in each region.
Experimental design: Number of samples is rather low.
more info - see below
Validity of the findings: see below
Additional comments: Authors described the underarm bacterial microbiome of Indian people in 3 distinct locations of India. Using amplicon sequencing of the V3-4 region of 16S rRNA. The author found clear associations with location. No impact of age or diet were found.

Comments

Minor comments
There have been other papers describing the underarm microbiome (eg. Callewaert et al., 2013). It would be meaningful to compare your results with previous results.

Would there be overlap between environmental bacteria and the skin microbiota? Is this a driving factor in the differences seen per location? Can authors hypothesize on this?

What is the Impact of gender? It is not reported in the manuscript, although in the discussion section, it was mentioned as not significantly associated?
Can authors include gender in Table 1 (or another table)?

Can the authors describe the characteristics of Pune, Ahmednagar, and Nashik? Are these big cities? How many inhabitants? I had to go look it up online: Pune (3000000), Ahmednagar (350000), and Nashik (1500000). How is rainfall, temperature, humidity etc different in each city? It is generally discussed in the discussion, but reader has no clue on the differences for each city.
Does skin diversity associate with city size (inhabitant number)?

Can the authors describe more about urbanization grade in each location? How are Pune, Ahmednagar, and Nashik different from each other? Urbanization paper: McCall et al, 2020

Age: no correlation found; but do the authors see pattern with sebum / apocrine sweat production (which is the highest at ~16-30y)? Are there more lipophilic bacteria around that age?

N=58 might be low. Did the authors perform a power calculation to verify if the number of samples is sufficient for each location?

Major comments
The V3-V4 region of the 16S rRNA region was amplified. It is known that this region gives an underrepresentation of the Propionibacterium/Cutibacterium genus (Meisel et al., 2016 JID); which are relatively important taxa on the human skin and in the underarms.

Cutibacterium is not mentioned once. Propionibacterium has been renamed to Cutibacterium recently (Scholz et al., 2016 Int. J. Syst. Evol. Microbiol).

Figure 1 should be a bargraph of the underarm microbiome of the 3 different locations. The current log scale makes it really difficult to interprete. Also, what are the other taxa?? This is not the best way to represent the bacterial composition.

Figure 2 is of poor quality. Can the author add/visualize statistics to the plots?

Figure 4 is again of poor quality.
How have the 10 taxa been chosen; is this the top 10 most abundant taxa, and then ranked according alphabet? I guess not, because Corynebacterium is missing.
Suggest to rank according abundance: so Staph first. Now it seems as if Tuberibacillus is the most important genus in the underarms of Indian people. Which I highly doubt, and to be honest, I cannot even know what are the most abundant taxa; because it is not reported ) (except the top 4 most abundant in Fig 1).

I would argue that Nashik, Ahmednagar and Pune are not so distinct from each other. They are actually geographically quite close to each other. In the same province / mid-West of India. What is the reasoning behind the choice for these 3 areas?
I suggest authors describe in much more detail why these 3 cities are so different from each other.

All in all an interesting paper, but it’s really hard to comprehend and interpret the results. No clear figure on bacterial composition; unclear why the 3 locations were chosen, which gender the volunteers were, etc.


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: SKIN MICROBIOTA DIVERSITY AMONG GENETICALLY UNRELATED INDIVIDUALS OF INDIAN ORIGIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: Line 62: "The commensal....pathogens...immunities" requires citation

Lines 202-203 Indicate that analysis is available on github; however the provided link is not accessible.
Experimental design: Impact of diet on skin microbiome is less explored albeit an important area of exploration. The diets should be defined in more depth to make meaningful interpretation. Additionally, since the authors do review the literature in significant depth, it would be useful to discuss findings from other relevant studies as well.

In sample collection, authors’ state:
“To avoid the effect of geographical location and related climatic differences, the axillary sweat samples were collected within 1 to 2 days from all the volunteers.” It is unclear as a reader, what does “within 1-2 days of what reference point?” mean.

While the representation of figures in the microbiome field is always up to debate, it is always useful to have pictorial/graphical depictions of data wherever possible. It would be useful to have some of the tables, particularly Tables 3 and 4 as graphs. Further, some of the figures are of lower resolution, making them hard to read.
Validity of the findings: Please refer to general comments for detailed points.
Additional comments: The manuscript by Potbhare, et al (2021) is an important manuscript in the context of the Indian subcontinent as there are not many studies that investigate the composition of the skin microbiome in the defined population. The study samples 58 volunteers, of which 36 belong to Pune, making the data from Ahmednagar (n=11) and Nashik (n=11), slightly over-interpretative due to small sample sizes. Generally, the manuscript is written clearly, and the literature reviewed is appropriate in the context of skin microbiome. Since the first description of skin microbiome using culture-independent technique, several advances have been made in the field. The authors use 16s rRNA profiling to sequence the samples, which is fairly adequate in the context of this manuscript. However since then studies performing whole-metagenomic sequencing [Oh, et al., 2016] have described the microbiome at a deeper resolution. It is important to discuss these findings as well. The other issue, I have as a reviewer is that the authors’ only sample the axillary sweat region. The axillary sweat region consists of hair follicles, sebaceous glands and is rich in sweat glands as such making it a completely unique microenvironment that is not exactly representative of the skin microbiome. While the authors’ discuss the differences in body site, this important point is not discussed in great depth. As of current, the axillary microbiome is given its own unique position [Refer to: Li, et al.(2019), Taylor, et al (2003), Council, et al. (2016), Callewaert, et al. (2013)]. Therefore, this reviewer thinks that it would be more valuable and insightful to discuss the findings specifically in the context of the axillary microbiome.


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: SKIN MICROBIOTA DIVERSITY AMONG GENETICALLY UNRELATED INDIVIDUALS OF INDIAN ORIGIN
Review round: 2
Reviewer: 1

Output:
Basic reporting: Please rename Propionibacterium to Cutibacterium throughout the manuscript. Why rename it in the table and not text?

The weather of Ahmednagar is comparatively hot and dry than Pune --> Please rephrase.

317 The average humidity and rainfall received in Nashik are more (67%, ~99.3mm) than in Pune (60.3%, ~67.9 mm) and in Ahmednagar (59%, ~46.3 mm). --> please rephrase.
Please check English grammar!

Among all three cities, the population and urbanization are more in Pune (~ 31 lacks), followed by Nashik (~ 16 lacks) and Ahmednagar (~ 3 lacks). --> please rephrase to proper English.
What is "lacks"? 100 000? Please report in millions or report the full number.

Age: the alpha diversity for ppl aged 41-59 is half as much as those aged 16-40y. Are you sure there is no impact seen for age? The only reason for me to see no sign difference is the low sample size.

254 Staphylococcus and Corynebacterium were abundant on the skin of individuals from the Pune district (Figure 6I-J). --> These results resemble the most with the results found in Europe (Callewert et al 2013) and USA (Council et al 2016; Grice et al 2010). Pune is also the most urbanized location of the three; a city with many urbanization elements. I think the authors should discuss this better in the discussion section. For instance on this location:
325 individuals residing in Pune are inhabitants of a metropolis, adapted to the western lifestyle, developed industrial area and infrastructure.

Did the authors ask the participants for deodorant use? Did the people in Pune use deodorants more frequently as compared to the other 2 locations? I believe deodorant use is more common in Pune, which correlates to more Staph and Coryne presence in that city.

Figure 2: I appreciate the effort, but it still remains very difficult to interpret these results. I would really urge the authors to make a bargraph, as done in f.i. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3337431/ Fig 3. It would be great to see the individual bargraphs per person, and clustered per location. That is the only good way to represent the bacterial composition.

Also: Staphylococcus abundance in Fig 2 is around 1-1.5%, while in Table 3 this is 23.2%. Where does the difference come from?

Figure 6: please add statistics in the figures.
I would suggest to mention the y-axis as done in panel I. (no scientific number - just a comma number, for easy interpretation)
Can the authors also put the most abundant one in panel A, followed by the second abundant one in panel B, etc?

Figure 4: CST nr 1, 2, 3. Can the authors say what locations are 1, 2 and 3? Actually, I don't understand the CSTs, I'm afraid. I also did understand after looking it up in the manuscript. Can the authors explain more on this?

Figures: I would suggest to combine a couple of figures into multipanel figures.

288 Bangalore (India) detected dominance of four phyla viz., Actinobacteria, Firmicute, --> FirmicuteS

335 Callewaert et al., in 2013 studying interpersonal axillary diversity, did not observe a significant correlation with geographical location --> This study was done in Belgium, which is a small country and entirely urbanized. So it is difficult to say something about geographical location here.

Can I ask the authors to provide the track change word document next time? It was hard to find all the differences in the manuscript as compared to previous version.
Experimental design: /
Validity of the findings: /
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 POTENTIAL BIOMARKERS OF VASCULAR CALCIFICATION USING BIOINFORMATICS ANALYSIS AND VALIDATION IN VIVO
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article entitled “Identification of potential biomarkers of vascular calcification using bioinformatics analysis and validation in vivo” by Chen et al. has demonstrated the potential prognostic biomarker and promising therapeutic targets for vascular calcification (VC) using bioinformatics analysis based on RNAseq data from GEO. In addition, the expression levels of candidate hub genes were verified in mouse tissues. They finally concluded that these genes might be a potential prognostic biomarker and promising therapeutic targets for VC. However, several improvements can be made to the manuscript to improve basic reporting and overall usefulness.
Experimental design: In order to strengthen the manuscript, I suggest the following experiments:
- The authors have used only a small RNAseq dataset from GEO. Please explain why you used this dataset despite the accessibility of many more RNAseq and microarray data from other public databases.
- To confirm the reliability of the analyzed results, the expression levels of all candidate genes in VC using different datasets and different databases have to be further examined.
- The immunohistochemistry or ELISA in human samples must be used to confirm the bioinformatics finding.
Validity of the findings: no comment
Additional comments: Overall, the manuscript has been written in a coherent manner. However, the author needs to edit/rewrite some part of the manuscript. In addition, the authors need to perform several extra experiments.


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 POTENTIAL BIOMARKERS OF VASCULAR CALCIFICATION USING BIOINFORMATICS ANALYSIS AND VALIDATION IN VIVO
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the manuscript was written using clear and unambiguous professional English throughout.  Citations were used correctly.  The article structure complies with peerJ and raw data are provided.

Minor point: please check some suggestions provided in the attached PDF, regarding subtitle formating and recommended subtite change.
Experimental design: The manuscript attempts to address the lack of biomarkers for vacular calcification.  By using public data from GEO dataset together with self-generated data, this scientific question was answered, with 5 key hub genes identified as the central biomakers associated with VC progression.

Methods were discussed adequately, although the details regarding the source of data for each section in the result section can be further revised to improve clarity.
Validity of the findings: In general, all claims have been backed up with evidence although some figures were incorrectly cited (refer to attached PDF, specifically figure 7A and B).

Specific points
1) Regarding GSEA analysis, the authors are recommended to use NES score to compare the significance between the different hallmark gene sets

2) On the reporting of the 16 potential drugs, the findings do not address how each drug will affect the expression of these 5 hub genes.  To serve as therapy for VC, the regimen must function, to the minumum, in the opposite direction from the change in expression between VC model and the control group. Authors are recommended to apply connectivity mapping together with the clue.io database for this analysis.

3) This analysis were performed on rat model.  The authors are advised to discuss how consistent these results will be when performing in human specimens.

4) Specifically for the miRNA-transcriptional factors-mRNA analysis, it is very challenging to determine the validity of these results, and how they are unique from other diseases.
Additional comments: The result section can perhaps be revised to address which of the dataset were used for each analysis.  In addition to the discovery of the 5  hub genes, the authors are advised to discuss how the identified GO terms from GSEA and KEGG are relevant to the VC progression.  Currently, some of the GO terms were not easily relatable to the VC progression.


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 POTENTIAL BIOMARKERS OF VASCULAR CALCIFICATION USING BIOINFORMATICS ANALYSIS AND VALIDATION IN VIVO
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English is good but it can be better with English editors.
Experimental design: 1.	Because you used the genes from public dataset (GSE146638), so please contrast how different the previous study with your finding.
2.	Is there any public VC dataset performed experiment in human? In the present manuscript relied only in rat (both public data and the animal model).
3.	In my opinion, the |log2FC| > 1 is too soft cut off. I would like to see the stricter cut off result.
4.	How to make sure that the rat in the experiments got VC?
5. The number of passed genes from each step and the number of gene used in each experiment are not clear. I suggest to rewrite the experimental design in the Method section.
Validity of the findings: 1.	Add more discussion on the drugs that routinely used and compare to the presented drug in the manuscript.
2.	In figure 2, why the cut point for Fig2A is different from that of fig2B.
3.	Please correct the word GESE to GSEA in the legend of figure 3.
Additional comments: The manuscript by Chen C. et al showed very interesting results of genes associated with vascular calcification (VC) and the possible targets for treatments. I really appreciate the experimental design which combines bioinformatics from public data and the animal experiment. However, I have some questions and comments on the manuscript.


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

Input:
Title: IDENTIFICATION OF POTENTIAL BIOMARKERS OF VASCULAR CALCIFICATION USING BIOINFORMATICS ANALYSIS AND VALIDATION IN VIVO
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made writing improvement to address issues on validity of the study using rat instead of human, by stating that further experiment on human cell model is still necessary.  Previously identified annotations were corrected. Literature references were corrected cited.  Raw data were shared.
Experimental design: Experimental method sections were adequate to follow and repeat the work.
Validity of the findings: 1 Regarding the connections of the identified gene ontology terms on VC progression, the authors attempted to justify the connections of some of the identified gene sets by referring to prior literature.  While this sounds plausible, we cannot really be certain on the connection of these gene sets to actual biology, apart from serving as hypothetical inference.
2 Like what was discussed in the previous review process,  the identified 16 drugs were taken from DGIdb database, but there is no evidence that these drugs will be candidate therapeutic agents for VC at all in this study.  Providing the structures of these compounds alone show no connection to their ability to serve as therapeutic agents, but may somewhat mislead the audience in understanding that they are drug candidates. The authors are recommended to justify how these 16 drugs are linked to VC progression.  Statement like '...enriching the options for precisely targeted therapy of VC. Our study can provide some help for targeted genomic therapy for VC in the future.' in the conclusion holds no concrete evidence from this study to support this claim yet.
Additional comments: From the rebuttal letter, authors claimed that the NES score was already performed and gave a similar result to the old approach, and claimed that there is no need to alter the picture.  Without seeing this actual result, we cannot confirm this claim.  In fact, the authors added statement in the Methods section about the NES score, but did not include this result at all.

Based on the response from the authors regarding their miRNA and transcription factors association, it is still impossible for us to determine the validity of these findings and how they are different from other diseases.  The authors referred to 'numerous articles using similar approach' but the former question was asked about how to connect this result to VC progression, and its validity.


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 POTENTIAL BIOMARKERS OF VASCULAR CALCIFICATION USING BIOINFORMATICS ANALYSIS AND VALIDATION IN VIVO
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is much better than the previous one.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I appreciate the improvement of the current manuscript. The authors answered and corrected the manuscript as suggested.


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: CIALIGN: A HIGHLY CUSTOMISABLE COMMAND LINE TOOL TO CLEAN, INTERPRET AND VISUALISE MULTIPLE SEQUENCE ALIGNMENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The architects of CIAlign present a tool to improve the quality of
Multiple Sequence Alignments (MSAs) by removing badly aligned elements
and to elucidate the meaning of these cleaned MSAs. CIAlign does not
align unaligned sequences from scratch but takes initial MSAs (for
nucleotide or protein sequences) as input.  The manuscript is well
written and contains many good elements. The ability to deal with
fragments, in my opinion, is particularly important. The authors state
the time complexity of their algorithm but do not present any
supporting data. They also give real-world resource requirements for
the down-stream analyses but not for CIAlign itself.  Using simulated
and real data, the authors demonstrate the effect of their tool
through its effect on the down-stream analyses (phylogeny
reconstruction).  The authors provide unit tests for their software,
which is extremely laudable!

- I believe that the effect of CIAlign, putting it simplistically, is
  to remove rows and columns from an MSA and not to re-align residues,
  that is, to change the relative alignment of residues and gaps, as
  in an 'iterative' aligner, like Muscle. If this is true, then this
  should be said (more) explicitly.

- The authors should clearly say in their manuscript if the
  application is parallelised/multi-threaded or not.

- The authors should mention in their manuscript resource requirements
  (time/memory) for their application and present a (rudimentary)
  scalability analysis.

- I would like to see which (initial) alignment program benefits
  most/least from the effect of CIAlign. The more different
  aligners/methodologies (Consistency (T-Coffee), HMM (Clustal-Omega),
  Iteration (Muscle), Partial Order (POA), Secondary Structure
  (Decipher), etc), the better. In particular, I would like to know
  how CIAlign deals with MSAs from a phylogeny-aware aligner like
  Prank (Loytynoja, Phylogeny-aware alignment with PRANK. Methods Mol
  Biol, 2014). Prank alignments are usually more 'gappy', as opposed
  to, for example, Clustal-Omega alignments.

- The purpose of an MSA (usually) is to be used in a down-stream
  analysis, so, demonstrating CIAlign's effect on phylogenetic
  analyses is apt. However, I would like to know if CIAlign could also
  be evaluated using sum-of-pairs analysis for real (not simulated)
  data.  I do not believe that Pfam seed alignments or simulated
  sequences can be treated (or are intended) as reference
  alignments. I would therefore like to see traditional benchmarks
  like BAliBASE, Prefab or OXBench being used. I suspect, BAliBASE
  (Thompson et al, BAliBASE 3.0: latest developments of the multiple
  sequence alignment benchmark. Proteins, 2005) may not work, as
  removing sequences or columns will not agree with Balibase's scoring
  program. However, HomFam (Sievers et al, Making automated multiple
  alignments of very large numbers of protein sequences,
  Bioinformatics, 2013) might be amenable, and it would be
  particularly useful for complexity analysis.

- Another down-stream benchmark methodology that can be used to
  evaluate alignments is to use MSAs to construct a secondary
  structure prediction (Sievers et al, QuanTest2: benchmarking multiple
  sequence alignments using secondary structure prediction,
  Bioinformatics, 2019). This would further validate the effect of
  CIAlign.


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: CIALIGN: A HIGHLY CUSTOMISABLE COMMAND LINE TOOL TO CLEAN, INTERPRET AND VISUALISE MULTIPLE SEQUENCE ALIGNMENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, I thought it was well written and easy to understand the functionality of the created software tool CIAlign
Experimental design: The individual functions of CIAlign were clearly described, and tested with a range of simulated and real data sets. The GitHub page provides clear install instructions and guidelines for use.
Validity of the findings: no comment
Additional comments: This paper describes a new software tool called CIAlign – which is capable of filtering and cleaning sequence alignments, as well as providing a number of nice functions like making a consensus, sequence logos etc. Overall, I think it is a nice tool, and should be accepted for publication in PeerJ, many of us have similar scripts lying about that do some of these functions, but the authors have bought all of these functions into a single software tool that is easy to use, readily available and well documented.

I have only minor comments, these are more likely to be more for future functionality rather than problems hindering publication.

CIAlign appears to revolve around determining divergence/differences from within the alignment – could this be altered to be based on a single references sequence? perhaps designating a sequence within the alignment as the reference. Then decisions such as removal of insertions, divergence, 5’ and 3’ trimming could be made with respect to that reference.

At first I found Figure 1C a little confusing – why is the insertion only present in seq 5 not removed during the remove insertions functionality? Digging deeper, seems that it is by design and due to the flanking parameter, which the user can change, and in addition that particular insertion would be dealt with if you also ran crop ends. On the GitHub page (and/or on the CIAalign help function) – could you give the range of acceptable parameters? Would setting the insertion_min_flank to 0 remove it? And is 0 an acceptable value there.

3 sequences – a couple of times, CIAlign, and sequence alignments in general, were referred to as involving 3 or more sequences – why not 2 or more? Whilst obviously many of the functions will be redundant (as they probably are for 3 seqs as well), some will still be useful for cases of 2 seqs alignment – does CIAlign work with 2 seq alignments? This is an incredibly minor/trivial point.

I managed to get CIAlgin installed very easily, and working just as easily. But struggled getting some of the graphical functions working on larger alignments – this is not an issue for publication – more a query. Obviously, the world is slightly dominated by SARS-CoV-2 at the moment, so I tried an alignment of 1000 CoV-2 seqs of length 29903, but it struggled/crashed (this could easily be me not adapting the params correctly, so again this is not an issue for publication). I was more wondering if the authors expected the graphical components of CIAlign to work easily for long alignments such as that. In particular, I was interested in creating a sequence logo of a particular region only within the alignment (not of the whole alignment) – is that possible current with CIAlign (to focus the SeqLogo on a region).

Typos:

94 – at the either the – two the’s
Intro – paragraphs 123 and 133 – both start with Finally


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: CIALIGN: A HIGHLY CUSTOMISABLE COMMAND LINE TOOL TO CLEAN, INTERPRET AND VISUALISE MULTIPLE SEQUENCE ALIGNMENTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: See review below.
Experimental design: See review below.
Validity of the findings: See review below.
Additional comments: Users of multiple sequence alignment software often provide input sequences that are difficult to align, incomplete, or non-homologous. In some situations it might be useful to automatically remove poor quality sequences from the alignment. The authors describe a program, CIAlign, they created to mask problematic regions of an alignment. They also provide graphics to show how the alignment looks before and after trimming, highlighting regions that were removed. Although this appears useful, the utility of the program beyond creating more aesthetically pleasing alignments was not clear to me as a user. To better demonstrate the utility of CIAlign, I have the following suggestions:

MAJOR COMMENTS

1) The Introduction provides insufficient background about the utility (or lack thereof) of masking multiple sequence alignments. It has previously been shown that masking rarely improves accuracy in downstream applications, and often by only a small amount. Previous approaches to masking (e.g., PMC3260272) are also insufficiently covered (although they are briefly in the Discussion section). In general, the Introduction only weakly motivates the problem statement and instead focuses on the program. Is the goal of CIAlign to remove artifacts, accelerate downstream analyses, or improve the accuracy of downstream results? I recommend reworking the Introduction to focus on existing programs and their known impact on downstream applications, then it should be possible to clearly state how CIAlign fills an existing niche.

2) The impact of CIAlign is not clear from Figure 6. It looks like there was basically no impact from different degrees of masking on RF or Quartet measures of tree distance (histograms in panel 6B). I cannot see a difference between these histograms. I suspect many users would want the program to improve the accuracy of downstream analyses by masking problematic regions. Figure 7 does not make the case for CIAlign because, of course, removal of highly divergent sequences removes long branches from the resulting tree. While the alignment before/after CIAlign looks substantially different, it does not mean the meaningful information derived from the alignment has changed substantially.

3) CIAlign was not benchmarked against other similar programs. I believe it is necessary to perform a characterization of CIAlign relative to competitors.

MINOR COMMENTS

1) Figure 1 could be made more clear by keeping the same sequence number across panels. For example, it is unclear that sequence #1 is removed from panel A to make panel B, because #1 is still the name of a sequence in panel B.

2) The output of a multiple sequence alignment program is often dependent on which input sequences are included. So masking an alignment might not be as successful as removing problematic sequences and then realigning the remaining sequences. The authors may wish to discuss this or try it as an alternative approach.


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: CIALIGN: A HIGHLY CUSTOMISABLE COMMAND LINE TOOL TO CLEAN, INTERPRET AND VISUALISE MULTIPLE SEQUENCE ALIGNMENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have implemented all necessary changes. I was very impressed by the variety of alignment programs and benchmarks included. Thank you especially for including Prank (however only supplemental) and Decipher.
Experimental design: Adequate
Validity of the findings: Convincing
Additional comments: double 'and' in line 102 ...full columns and and full or partial...


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: CIALIGN: A HIGHLY CUSTOMISABLE COMMAND LINE TOOL TO CLEAN, INTERPRET AND VISUALISE MULTIPLE SEQUENCE ALIGNMENTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: See additional comments.
Experimental design: See additional comments.
Validity of the findings: See additional comments.
Additional comments: As previous reviewer #3, I commend the authors for their thorough revisions to reviewer comments on the initial manuscript.  I think CIAlign is now presented as a useful tool addressing the important issue of manual curation of sequence alignments.  This is reminiscent of an article:
Morrison DA. Why Would Phylogeneticists Ignore Computerized Sequence Alignment? Systematic Biology. 2009;58(1):150-8.

MAJOR

In this revision, the authors added an analysis of multiple sequence alignments per program using simulated sequences (Fig. S3). I found this somewhat difficult to interpret. Broadly, the indel-aware alignment programs have a higher proportion of residues removed, both correct and total.  Most other aligners have a similar proportion of residues removed, except DECIPHER.  The authors say this is because DECIPHER adds fewer gaps, but Table S3 shows it as being similar to Clustal Omega and some others.

Isn't the goal to remove the fewest correct pairs per residue remove? I would have preferred to see a graph of correct residues removed versus total residues removed and a similar graph for incorrect residues removed. Plotting "Median % Correct Pairs Removed" (y-axis) versus ""Median % Nucleotides Removed" (x-axis) gives a slope of around 3 under "Moderate" CIAlign stringency. This means that every 3% signal+noise I remove I take away 1% of my true signal, right? This slope seems to be higher for relaxed and lower for stringent, suggesting relaxed is better. It would be nice to see these kinds of analyses added to the manuscript.

In general, the trade-off between true positives gained versus true positives lost using CIAlign seems to still be lacking from the manuscript. For example, QuantTest2 showed more correctly predicted residues (76% versus 68%). However, how many residues were lost to make this improvement? That is, for every benchmark the gain in true signal should be placed into perspective of the loss in total signal. This can also be compared across different CIAlign settings (e.g., insertion only versus ends, stringent versus relaxed, etc.).

MINOR

Please report results to a reasonable number of digits that reflects accuracy of the measurement (e.g., 76% or 76.0% instead of 75.99%). I doubt any of these tests were large enough to offer four significant digits of precision.

If I understood correctly, the nucleotide alignments used here are DNA and not non-coding RNA. If the DNA is coding then far better alignments are usually achieved by aligning the codons or translating-aligning-reverse translating. Some programs can do this automatically. If the alignments are non-coding RNA then many of the aligners will handle them differently. I think it is important to show best practices if the goal is to make better alignments. So it would be good to say somewhere that using the aligner correctly is the first step toward good analyses, and CIAlign is for taking the best possible alignment and cleaning it up.


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: DIEGOAELURUS, A NEW MACHAEROIDINE (OXYAENIDAE) FROM THE SANTIAGO FORMATION (LATE UINTAN) OF SOUTHERN CALIFORNIA AND THE RELATIONSHIPS OF MACHAEROIDINAE, THE OLDEST GROUP OF SABERTOOTH MAMMALS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This short paper about a new sabertooth creodont is a pleasure to read. For a group that is so poorly known, even a single lower jaw represents a significant increase of our knowledge. The specimen described in this paper is such an occasion. The paper is a straight forward description and phylogeny, plus some modest discussion about ecomorphology. I find the paper well written, well-illustrated, and the taxon well analyzed and I have little reservations.
Experimental design: I see no problem.
Validity of the findings: A valid new genus and species is named and it is thoroughly documented.
Additional comments: My main suggestion is to add another photo. Since the holotype SDSNH 38343 has a well-developed flange, I suggest that the authors provide an anterior view of the mandible showing the morphology of this structure. As the authors are aware, a flange frequently develops in various sabertooths within Carnivora and often useful as a taxonomic or functional character. Therefore, a more detailed documentation of the flange, in the form of an anterior view, seems warranted, which is often done by sabertooth paleontologists.

I also see a minor problem in the Phylogenetic Results section. The sentence “monophyly of Machaeroides is supported by a single unambiguous synapomorphy, presence of a double-rooted p1 (character 8, state 0)” seems quite implausible to me. Double-rooted p1 is coded as primitive, which I agree, but if so, unless you are trying to demonstrate a character reversal, how could this be a synapomorphy? Merely a shared primitive character, and leaving Machaeroides without a synapomorphy?

Institutional abbreviation: SDSNH suggest adding San Diego Society of Natural History before San Diego Natural History Museum to mitigate the disconnect between SDSNH and the museum.

In general, the ms is well written and quite clean (but I did not have time to read it word for word). It does have an occasional typo, such as line 559, delete “flange” in more ventrally expansive flange.

Xiaoming Wang


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: DIEGOAELURUS, A NEW MACHAEROIDINE (OXYAENIDAE) FROM THE SANTIAGO FORMATION (LATE UINTAN) OF SOUTHERN CALIFORNIA AND THE RELATIONSHIPS OF MACHAEROIDINAE, THE OLDEST GROUP OF SABERTOOTH MAMMALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The text and figures meet all these criteria, although the text could be better written. Commas are sometimes placed rather haphazardly and the modern 'American' English practice of omitting prepositions (especially 'that') makes it sometimes hard to follow what is referred to in comparative descriptions. An overhaul of the text designed to enhance readability would be welcome.
Experimental design: No issues here and no experiments per se.
Validity of the findings: The paper is an important contribution because machaeoroidine fossils are (almost) as rare as hen's teeth. The results are certainly novel and broadly robust (but see comments below).
Additional comments: I have some specific comments and one more general one that it would be well to address. Specific comments first:

Line 88: I am sure the specimen was measured to the nearest hundredth of a mm, but the accuracy of such measurements using digital calipers is no better than one tenth of a mm (at best). This should be noted to avoid the impression of spurious accuracy.

Line 109: 'more accurate branch lengths' This is completely meaningless. If characters are not parsimony informative they have no impact on a parsimony analysis and should be removed.

Line 124ff: The matrix is small enough that it should be analysed using the exhaustive search function of TnT (it would only take a second or two). That way the shortest tree is guaranteed to be found. In all likelihood it already has been found, but the New Technology algorithms do not guarantee this.

Lines 260, 261, 263: The first to lines include 'paraconid' twice. It is clear from context that the second use must be in error for 'protoconid'. However, on line 263 'paraconid' returns and in this case it is not clear whether 'paraconid' or 'protoconid' is intended.

Line 336: Could the lack of serrations on the lower canine of M. eothen be due to wear? In felid sabertooths serrations tend to be worn away quite rapidly.

Line 369: Could the loss of p1 be caused by the shorter postcanine diastema in D. vanvalkenburghae?

Line 386ff: The synapomorphic state of two-rooted p1 is directly connected to the choice of outgroups. These may or may not be representative of the plesiomorphic state. A change from one to two roots in a lineage where anterior teeth are being reduced seems rather unlikely. Could other outgroups be sought (as the author notes in line 435)? This possibility could be stated earlier.

Now the more general comment: The size and shape of the lower canine is quite unique among sabertooths, particularly forms with  a mandibular flange that is as large as than seen in D. vanvalkenburghae. This is the feature that is of most interest in the morphology of the specimen. It's mesiodistal to buccolingual length ratio is very close to that of the clouded leopard (which in turn isn't all that different from any member of Felinae). It contrasts with the condition in Machaeroides and Apataelurus, at least based on how their lower canine alveoli are illustrated in Denison (1938, fig. 5), showing them to be more compressed. This suggests some oddities in the functional apparatus of D. vanvalkenburghae that should be highlighted and discussed.


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: DIEGOAELURUS, A NEW MACHAEROIDINE (OXYAENIDAE) FROM THE SANTIAGO FORMATION (LATE UINTAN) OF SOUTHERN CALIFORNIA AND THE RELATIONSHIPS OF MACHAEROIDINAE, THE OLDEST GROUP OF SABERTOOTH MAMMALS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall, the article is an important contribution in the larger effort to understand the evolution of carnivore faunas in North America. I am especially pleased to see the authors have chosen to honor Dr. Blair Van Valkenburg with this particular taxon, which so perfectly captures her field-defining work. The article is well-organized and it appropriately delineates the descriptive, comparative, and phylogenetic results. The table of machaeroidine material is very useful as are the annotations on Figure 2.

Data - In the Materials and Methods section, the authors describe “the 3D imaging” which is not shown in the figures, but is shown as a media image in the MorphoBank project. These models are “available for viewing and download on the NAT Website.” A permanent link (ideally a DOI link) would be useful to include here, or at least a URL for “NAT,” an abbreviation that is not in the Intuitional Abbreviations section. MorphoSource might be a useful resource for accessioning the photogrammetry images and files, and the 3D models that were produced, as MorphoSource provides DOIs for 3D projects.

The MorphoBank project is important, especially the images of the specimens used for the analysis. Given the limited scope of the phylogenetic analysis, I think it would also be useful to include the character-taxon matrix in the Supplemental Data document as well, since it already contains the character list.
Experimental design: Overall, the methods are appropriate for this study. However, it is not clear why the authors generated a novel character-taxon matrix, rather than expanding on the larger matrix published by the lead author, and listed in the References as Zack 2019a. An explanation for this decision in the Materials and Methods section would be appropriate, especially because the authors repeatedly mention a character describing the roots of p1 may be mispolarized, a hypothesis that could be tested by analyzing Machaeroidinae with a substantially larger outgroup sample.

I should also note that I initially assumed the authors had expanded on Zack 2019a when I scanned the manuscript and saw Figure 6, which includes branches for “other Oxyaenidae” and “Hyaenodontidae.” I thought this meant all oxyaenids and hyaenodonts from Zack 2019a were in the analysis. As a lazy researcher, I will often go straight to the temporally calibrated tree figure to get temporal and phylogenetic data simultaneously, but here, that would have mislead me about the breadth of the analysis. Figure 6 should limit its scope to the tree resolved as part of this study, with only the in-group set in temporal context, or it should include only the outgroup OTUs and not their larger clades.
Validity of the findings: The primary finding of this manuscript is that a new genus and species of machaeroidine was discovered in the late Uintan Santiago Formation of California, expanding the presence of the clade beyond the Rocky Mountain Interior. The descriptions and comparisons convince me that this is a new genus and species and that it is appropriately placed in a phylogenetic context, given the selected OTUs, characters, and character scoring. I have a couple of notes (below) on how some of the results are discussed, but this does not detract from the larger conclusions.
Additional comments: Title – Is there a PeerJ stylistic prohibition on putting the genus name and species in the title or the abstract? If not, I think it would be appropriate to include it in both. If the authors would prefer to not put Diegoaelurus vanvalkenburghae in the title, it should at least be included in the abstract. “A new genus” also suggests that this is not a new species, and the specimen was previously described as a new species lumped into a different genus.

Title – The phrase “the relationships of the first sabertooth mammals” does not make it clear that the analysis is limited to machaeroidines. Perhaps, “Diegoaelurus, a new machaeroidine (Oxyaenidae) from the Santiago Formation (middle Eocene) of southern California, and the phylogeny of Machaeroidinae, the oldest group of sabertooth mammals.” Again, because I was familiar with Zack 2019a, I first interpreted the title to mean the analysis would sample nimravids along with machaeroidines.

Line 27 – Insert “taxonomically” between “a” and “small.” At present it reads as if macharoidines are physically small.

Line 28 – Delete “Documented.” As it reads, it sounds like there is an unpublished trove of machaeroidine diversity that the authors are aware of.

Line 34 – Rather than “the only previously named member of the group” perhaps, “the only near-contemporaneous member of the group.” As it reads, the abstract gives the impression Apataelurus kayi is the only named machaeroidine.

Line 72 – Restructure this sentence, so it does not sound like Zack 2019a was written in the late Uintan. Perhaps, “Zack (2019a) described a skeletal association from the Myton Member of the Uinta Formation (late Uintan).” Then start a new sentence with “Wagner (1999) briefly reported…” As structured, the “while” makes it seem like the twenty year gap between the authors was occurring at the same time.

Line 87 – Because Zack (2019b) does not include the detailed dental diagram and description, but instead further refers to Rana et al. 2015 with suggested modification, for clarity and ease of use for future researchers, I suggest changing the reference to Zack (2019b) to “Dental terminology follows Rana et al. (2015) with modifications from Zack (2019b).”

Line 88 – I suggest starting a new paragraph with the details of the photogrammetry process to distinguish these methods from the dental description methods. I also suggest inserting the DOI or URL for the model and photogrammetry materials at the end of the paragraph. Even better would be to deposit the model and images on a site like MorphoSource.

Line 93 – I may have missed it, but what is the “NAT Website”? A URL and defining the abbreviation in the institutional abbreviations would be useful.

Line 98 – See my comments in Experimental Design on adding justification for not using or modifying Zack 2019a in this section.

Line 109 – Branch lengths are not part of a parsimony analysis, and this justification made me think the authors used likelihood or Bayesian methods in addition to parsimony. The phrase “to provide more accurate branch lengths and” should be removed. The justification that the additional characters will help future investigations is sufficient.

Line 113 – It would be helpful to include the character-taxon matrix in the Supplemental Information as well, especially because the matrix is relatively small.

Line 176 – Delete “both”

Line 186 – Perhaps “mental surface” rather than “anterior aspect”? At first I interpreted this as there being no foramina on the distal section of the dentary (including the buccal surface). But maybe there’s no easy way to describe a portion of the jaw that is so rarely illustrated.

Line 224 – Consider replacing “agrees” with “is shared”

Line 225 – Consider inserting a colon after “preserved”

Line 312 – Just a note to again the compliment the authors on clear, vivid description of the material.

Line 313 – In the Reviewer PDF, these section leaders – “Comparisons” and “Phylogenetic Results” – are in the same font and style as the rest of the text. In a scan through the document, they are lost and setting them off more clearly would be useful for the reader. Will PeerJ be formatting these perhaps with a bold type setting or a small section break?

Line 326 – Move the note that known lower molars of M. simpsoni are fragmentary before the statement “m2 does appear to…” When I first read through, I wondered why the authors wouldn’t just measure relative molar size, then was reminded that the record for M. simpsoni is fragmentary. Perhaps, “Known lower molars of M. simpsoni are fragmentary, but the relative molar sizes in the two species do appear to be similar, though m2 does appear to be proportionally larger in D. vanvalkenburghae.”

Line 360 – Delete “North American.” As written is reads as if there is more than one type species for Apataelurus.

Line 380 – Will this results section be more clearly offset in the final publication?

Line 432 – The authors seem to be suggesting it is surprising that they did not recover an anagenetic sequence in the fossil record. If Machaeroides is a genus that has been identified based on shared characters among species, it doesn’t seem surprising that it would be resolved as a clade. The authors repeatedly emphasize the scanty nature of the machaeroidine fossil record. Recovering a simple march through time and taxa would be more extraordinary than the result that was recovered, no? I think this section could be shortened to just report that the results align with stratigraphy and Machaeroides is recovered as a clade.

Line 458 – The concluding sentence of this paragraph summarizes the state of the record succinctly. The proceeding paragraphs could be tightened to the most remarkable observations or important points.

Line 523 – Move the “suite of craniodental features typical of saber-tooth carnivores” forward in this section, from the paragraph beginning at Line 540 to this introductory paragraph. As structured, these key features that are discussed at length later in the section, are buried after a line that appears to narrow its focus to M. eothen.

Line 551 – How are these “advanced saber-tooth specializations” different from specializations to hypercarnivory? The key features for advanced saber-tooth specialization seem to be the height of the coronoid process, the lowered glenoid fossa, and large mandibular flanges. Consider revising or simply deleting the “simplified lower molars” and “enlarged carnassial blade” sections.

Line 560 – This comparative section would benefit from a few direct anatomical comparisons to nimravids and more familiar machairodonine cats. There is the brief mention of Hoplophoneus, but consider more detailed comparisons to support the statement that Apataelurus “show evidence of more advanced saber-tooth specialization.”

Line 608 – Again, “surprisingly” does not seem like the right adverb for describing a clade that was already a genus. It would be more surprising if different Machaeroides species were sister taxa of different Apataelurus species, or if Diegoaulurus was the sister taxon to all other machaeroidines. Consider making the observation that the genus was recovered as a clade, unless there is a body of literature that strenuously makes the argument that Machaeroides is paraphyletic. If so, add that to the introduction and discussion.

Line 630 – The authors thank the staff involved in the excavation of Jeff’s Discovery, but there is no more description of the locality or its history offered in the manuscript. Consider inserting a couple of sentences in the introduction that describe the locality and the efforts made by these staff to document the fauna at SDSNH locality 3276.

Figure 1 – This figure could be used to communicate more detailed information. As it stands, there is a lot of blank space. Consider using color to highlight the early versus middle Eocene sites and to illustrate the rough size and shape of the different basins. There is also room to place the names of the taxa from each basin on the map. Alternatively, is there more detail on the Santiago Formation? A stratigraphic column showing the geological context for the holotype would also be useful to include as part of this graphic.

Figure 6 – See comments above. In essence, I suggest limiting this graphic to in-group (Machaeroidinae). This would also help the reader figure out why only some of the OTUs are connected to the tree.


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: DIEGOAELURUS, A NEW MACHAEROIDINE (OXYAENIDAE) FROM THE SANTIAGO FORMATION (LATE UINTAN) OF SOUTHERN CALIFORNIA AND THE RELATIONSHIPS OF MACHAEROIDINAE, THE OLDEST GROUP OF SABERTOOTH MAMMALS
Review round: 2
Reviewer: 1

Output:
Basic reporting: My minor comments on this score have been addressed.
Experimental design: The analysis has been modified to reflect comments that I had in the previous round.
Validity of the findings: No issues here.
Additional comments: After reading the revised text and rebuttal letter I find this manuscript ready to be accepted.


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: DIEGOAELURUS, A NEW MACHAEROIDINE (OXYAENIDAE) FROM THE SANTIAGO FORMATION (LATE UINTAN) OF SOUTHERN CALIFORNIA AND THE RELATIONSHIPS OF MACHAEROIDINAE, THE OLDEST GROUP OF SABERTOOTH MAMMALS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors took thoughtful care in addressing the reviewers' comments. Clarifications in the text all flow, and make this manuscript an easy read. The addition of Figure 2 is really helpful, the included links and MorphoSource project, along with the modification of Figures 1 and 7, and the tweak of title all contribute to an even more robust manuscript. I look forward to utilizing this study as we try to sort out the evolution of carnivorous mammals!
Experimental design: Adding the matrix to the supplemental materials and providing the links to specimens are much appreciated.
Validity of the findings: The discussion and conclusions are all consistent with the data. Great work!
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: FREEZE-DRYING CAN REPLACE COLD-CHAINS FOR TRANSPORT AND STORAGE OF FECAL MICROBIOME SAMPLES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This interesting study looks at the effect of freezedrying vs freezing on gut microbiome composition, using 16S amplicon sequencing. The authors apply 20 samples collected from Damaraland molerats, and split each sample into two treatments, generating 40 samples. The aims and language are straightforward and clear, and the overall manuscript is relatively short and to the point, which I appreciate. Since freeze-drying isn’t a standard methodology in the field, the study provides useful information on this method. Data and code are available.

My overall impression was that whilst the study is generally sound, a few more details would improve the usefulness of the paper. Given this is a PeerJ submission which focuses on analysis integrity, I also have quite a few nit-picky comments about some of the details of the analysis which should be clarified.

Conclusions/interpretations: I would suggest having more detail on how microbiome results differ between the two methods in the abstract and conclusions, rather than strongly focusing on the similarities. I think it is important to note that whilst there may be no consistent bias using one method or the other, this is not the same as no/little effect. No effect would mean no consistent bias AND that diversity metrics are perfectly correlated at the sample level. It is perfectly possible for there to be no difference between treatment groups, yet also no correlation in composition at the sample level between the two treatment groups, which would suggest that differences are random yet still potentially serious for results.

Background/Context: Whilst I think a concise introduction and discussion is generally good, I did find it rather light on details. Whilst there are only few studies looking at the effect of freezedrying on composition, there are possibly hundreds looking at how different storage methods affect measured results. I think briefly summarising what is known about this field (~one paragraph) would provide useful context. For example, it is my understanding that overall microbiome measurements seem to be rather robust to various storage methods, with things like DNA extraction kits and primer choice being more important. For someone reading this paper who is looking to decide how to store their samples, or indeed for someone who is completely new to the field, some broad context would be interesting and useful.

Different methods will always generate slightly different results, and even where effect sizes are small it is good to know exactly how they differ. Whilst there appears to be no consistent bias in a given direction by using a certain method, it is clear from the figures that at the sample level differences are generated between the two methods, but this is hardly mentioned and perhaps could give the impression that different storage methods have no effect on composition as the sample level. It seems like there are differences at the sample level, but this isn’t consistently reported for all analyses. In fact, Figure 3a shows quite large differences at the sample level, which I find rather surprising. Summarising and quantifying these differences, and whether they are due to differences in abundant/rare taxa, or perhaps specific taxonomies, would be invaluable. I have more suggestions on how to do this in the next sections.
Experimental design: Bioinformatics reporting: It would be good to see a few more details about what percentage of reads/ASVs were filtered at each major step. Particularly important is having more details on how the authors filtered contaminants and how many were filtered out, which currently is very vague. It is possible to have reverse contamination, where ‘real’ ASVs get into the negative controls. R packages like ‘decontam’ are made make this process a bit more reproduceable, but do depend on having multiple negative controls. Filtering out contaminants can be tricky and in my experience lab contaminants shouldn’t really account for more that ~0.1% of reads. Either way, these steps and how many ASVs were filtered out should be reported. In addition, I think how many ASVs are filtered at rarefying should also be reported.

Alpha diversity analysis: The counts were rarefied, which I don’t have a problem with, but it is more than possible that even after rarefaction there is a signal of sequencing depth, since rarefying is sometimes not enough to completely control for this bias. This should be reported, and if the effect is still there, controlled for.

Alpha diversity analysis: The study reports the effect of treatment on alpha diversity, but not the effect of sample ID, which would also be useful to know (this should effectively represent the correlation between the two treatments at the sample level, and is represented by the model ICC). This can be easily extracted from the GLMM using performance::icc(model, by_group = TRUE). Personally I would prefer Fig. 1 to show this correlation (opposed to a boxplot), with linear trends for the two treatment groups. I think this would be a more effective way of showing the effects at both the treatment level and the sample level together. If there is a low correlation, this would be a bit worrying, but it’s hard to tell from the data presented. Depending on this correlation, it may be required to rethink the subtitle “Preservation treatment did not affect alpha diversity”. As mentioned, there may be no consistent bias, but that does not mean there is no effect.

Beta diversity analyses: The normalization protocol for the beta diversity analyses is not completely clear, and there is perhaps some confusion regarding differences in transformations, dissimilarity indices, ordinations, and permanovas, which is quite common as it can be quite confusing. Just to be clear, the first two beta diversity analyses perform count transformations, but it is not clear which dissimilarity index is used to generate the distance matrix. In vegan/phyloseq, I think Bray-Curtis is the default index if not specified. Hellinger can be a transformation (sqrt of relative abundance) or a dissimilarity index, confusingly, but I dont think it is offered by vegan. I assume in this case it is a transformation. In the second two beta diversity analyses, the two dissimilarity indices are reported (Weighted and Unweighted Unifrac), but it is not clear how the data were normalised/transformed (rarefied? CLR? Hellinger?). On top of this, for visualising beta diversity, the first two plots use PCA (data reduction into n-1 axes), whilst the first two plots use NMDS (which presumably generates 2 axes, although it is not explicitly reported). As such, beta analyses 1 and 2 differ in so many ways from analyses 3 and 4 that it is difficult to gauge where any differences stem from. Perhaps some careful consideration is needed regarding what exactly the authors want to test, then change one thing (either transformation, dissimilarity index, type of ordination) and compare. The type of ordination makes no difference to the PERMANOVA, since this is of course based on the dissimilarity matrix and not the ordination, but it will make a difference to the plots. Overall, each analyses needs a clear description of 1) the data transformation, and 2) the dissimilarity index used to generate the distance matrix.

Adonis: The authors do not explicitly state how they set up the PERMANOVA, which is sensitive to variable order unless the ‘by’ argument is stated as “marginal”. If the authors are measuring marginal effects, this should be reported. If they are not, then the order of the terms can be important, and worth checking whether entering the terms in a different order affects anything. In addition, please include the effect size (F) in Table 1. The R2 and F represent different things, and R2 isn’t exactly the effect size but rather explanatory power (effect size in this case = how far centroids are from one another).

Permanova results: That sample ID only explains ~60% of variation in one analysis is surprising. This means there is nearly 35-40% unexplained variation between the same sample when using the two different methods. For me this is quite a lot. A key question for me is whether this is variation in rare taxa or abundant taxa? Can it be explained by differences in sequencing depth? This is worth adding to the model too to see if it can mop up any of that unexplained variation. Might be worth also testing out different normalisation methods for this analysis, since one ideally wants a normalisation method that maximises the effect of sample identity.

Differential abundance analysis: how were the data normalised for this analysis?

Overall the experimental design can answer the main questions. My one query was why the length of time a sample had been freeze-dried or frozen for was not incorporated into analyses, at least as a check. I wonder whether samples that had been freezedried for longer tended to be more different from their frozen counterparts than samples that were more recently freezedried, given these may be expected to be more degraded. Is there any evidence for this?
Validity of the findings: Please see comments above about summarising the sample-level differences rather than just the treatment-level differences.
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: FREEZE-DRYING CAN REPLACE COLD-CHAINS FOR TRANSPORT AND STORAGE OF FECAL MICROBIOME SAMPLES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The submitted manuscript is professionally written, with a very clear and understandable message. It was a pleasure reviewing such a well-structured and nicely flowing paper. The authors have done a fantastic job highlighting the research gap and putting their work into context of the existing literature. The presented work is an important step towards enabling future microbiome studies in remote regions and has the potential to be expand to other animals as well as humans. In particular, the authors have performed a very thorough data and statistical analysis. The only technical limitation is the sample storage in a super freezer prior to freeze drying, which is exactly the requirement the authors attempted to eliminate in this study. This is likely due to the remote study location and hopefully can be overcome in future studies.

As required by the journal (“Uncommon abbreviations should be spelled out at first use”), please spell out ASV in the abstract. Although this is becoming a more commonly used abbreviation, it is not yet universally used outside of the field.

All raw data files are depicted appropriately and are accessible within the manuscript
submission files or the respective data repositories. However, the deposition information is not noted in the manuscript. Please rectify this by inserting a data deposition and availability statement within the Methods section to comply with the requirements of the journal. When doing so please include the specific sample accession numbers for ease of the reader. Otherwise, it appears difficult to access the described samples without having to search through hundreds of samples in the BioProject PRJNA781121.

Line 64-67: “Although freeze-drying has rarely been used in microbiome studies (Gavriliuc et al., 2021), it offers considerable advantages; no chemicals are added and the samples can be stored and transported at ambient temperature, eliminating the need for cold chain logistics and hazardous material handling.” Instead of such a complex, long sentence, I suggest removing the semicolon and simply splitting this statement into two sentences.

Typically, abbreviations as part of figure headers and legends are spelled out again at first mentioned in order for the reader to understand the figure as a stand-alone without the main manuscript text.

As required by the journal (“When citing use the abbreviation 'Fig.'. When starting a sentence with a citation, use 'Figure 1'.”) please adjust your figure reference to adhere to the guidelines. Furthermore, the figures are not sorted according to their numbering in the text. Your first figure reference is Figure 3 (Line 163 in text) prior to Figure 1 (Line 197). Please either relabel the figures accordingly or switch the figures. Please also adapt all figure titles and legends to adhere to the journal’s instructions, which are for example for Figure 1 as follows: Figure 1: Alpha diversity measures of freeze-dried and frozen fecal samples. A) ASV richness (number of ASVs), B) Shannon index, C) Faith’s PD. Lines between points connect sample replicates of the same original sample identity and point color represent the two sample preservation treatments: yellow freeze-dried; blue-grey frozen.
Experimental design: An appropriate ethical approval statement is provided by the authors as part of their Methods section.

Line 40: “Super freezers require a reliable source of electricity and are expensive to purchase and maintain.” Although this statement is factual correct, freeze driers advocated as the superior piece of equipment, also require a reliable source of electricity. How feasible is it to integrate this requirement considering the remote location of the field site? The manuscript would benefit from including a section on the feasibility of this requirement.

Lines 47-50: "Transportation of frozen samples hence introduces the risk of thawing and incurs additional costs of cold chain logistics. This is especially problematic when the sampling location is in a remote area or in another country than the laboratory facility and commercial couriers are needed.” This section appears a little repetitive and could likely be reduced to one sentence with a more concise message.

Line 79: In the methods section the field work time frame is stated from May to October 2019, however the online metadata sheet states April as a collection date. Please explain this discrepancy and adjust accordingly.

Line 79: Seeing that the samples were collected over a time frame from April/May to October 2019 what impact do seasonal food sources have on the gut microbiome? Has this factor been investigated previously or could you comment on food availability in this region? Further, since animals from the same families have been used for this study, have comparisons between and within families been performed? This factor can have implications on the microbiome analysis as the vaginal microbiota impacts overall animal health from birth.

Lines 85-87: “Freeze-dried samples were then stored and transported at room temperature while frozen replicates were transported on dry ice with a commercial courier service to Sweden.” It is stated that freeze dried samples have been stored at room temperature which is likely incorrect due to temperature variations between the African climate, the cold air during shipping, and storage at final destination. Ambient temperature would likely be more appropriate to use in this instance. Could you highlight how changes in temperature can impact sample composition?

Lines 82-83: “Each sample was split into two replicate tubes with 1-3 fecal pellets per tube and stored in a -80°C freezer at the field site”. Please comment on the fact that even though this study is focused on replacing the requirement for super freezers the samples were stored in a super freezer prior to freeze drying. This statement implies that there is still a need for super freezer immediately upon sample collection and also a potential impact of freezing with subsequent freeze drying on the fecal microbiome composition. This study limitation should be highlighted more clearly in the discussion section with suggestion on how to overcome it where appropriate.

Line 83: “After all samples had been collected...” Could you please elaborate on the study timepoints and include approximate timeframes in your manuscript (e.g. a flowchart)? If some samples were collected on the 1st of May, were they stored in a super freezer until October (last collection day) before being freeze dried? And how would this impact the microbiome composition? Has a comparison been made between samples stored for months prior to freeze drying and those freeze dried immediately?

Lines 140-141: “We combined reads of samples on plates that had been sequenced twice (plate 2 and 3).” Could you please clarify and add if all samples were sequenced in duplicate or only a fraction and if so, why were not all sequenced twice?

Figure 2: Can you comment on the clustering effect seen in Figure 2? Are these animals from the same region or family for example?

Metadata: For ease of the reader and to support open access practices such as replication, it would be beneficial to include the metadata file for all animals in this study as part of the supplementary files. Furthermore, the metadata should be described in more detail to enable interpretation and replication of the results e.g. (currently missing) collection date, transport and storage timeframe, DNA extraction date, family number, longitude, latitude etc.).
Validity of the findings: Line 279: Please elaborate on the cause of significant differences between plates. Typically, technical batch effects are seen across sequencing runs, personnel, or kits (Wang & LêCao 2019, https://doi.org/10.1093/bib/bbz105). Assuming the same protocol, reagents and personnel were used for sample preparation, differences between 96-well plates are not expected and might warrant discussion in the discussion section of the manuscript.
Additional comments: Lines 161-162: “figure S1… Figure S2” Please be consistent with wording and referencing of figures throughout the manuscript according to the journal guidelines.

Raw data files: From the raw data files, it appears that the samples used are part of a larger study. The sample numbers indicate a spread over hundreds of samples. Could you please comment on and include the selection criteria for the here used samples within the manuscript as this might cause potential sample bias? Were these selected randomly from the overarching study, according to a different criterion e.g., first 20 animals caught, age, family etc. or in retrospect after sequencing?

Table 1: “The stars indicate level of statistical significance.” Please include the significance levels in the table legend seeing that p-value levels can differ between publications and to aid the reader’s understanding.

Table S1: The supplementary table is missing a table header as well as appropriate description. Please include these.


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: FREEZE-DRYING CAN REPLACE COLD-CHAINS FOR TRANSPORT AND STORAGE OF FECAL MICROBIOME SAMPLES
Review round: 1
Reviewer: 3

Output:
Basic reporting: ·	L65: Though it was noted that freeze-drying has not been widely used, there have been a handful of studies that appear to have used this approach (at least within the past year, for example https://doi.org/10.1016/j.medmic.2021.100044 or10.1080/19490976.2020.1759489 ). To be comprehensive, I think it would be helpful to briefly mention these in the introduction or whether they support/contradict findings in the discussion.
·	L95: I accessed the SRA data from the larger microbiome study, but I am not sure which samples were used for the present study. Can a list be provided either in-text or as part of some supplementary material?
·	L96: Can the authors provide some information on the storage length for each sample? Are storage times among these samples evenly represented between 4 - 10 months or were most samples stored closer to 4/10 months, either in the text or in supplementary material? Also, can or have the authors tested for the proportion of variance explained by storage duration? I am wondering if samples stored for say the maximum 10 months show differences from those stored at 4 etc (I do not think this needs to be changed in the manuscript, just a confirmation in the author response).
·	L124 - 131: It was said that the Ampliseq workflow was followed. Were there any other steps between running cutadapt and the denoising step? And were all steps/commands run with the same parameters as the workflow/all default parameters?  Please clarify.
·	L160 - 163: Please report the stress value of the NMDS before and after removing the outlier samples as well.
·	L183: How was this comparison done? I am assuming with a t-test? If not described in the methods please indicate.
Experimental design: ·	L83: I am not familiar with fecal samples from mole rats. Could the sample have been split into multiple aliquots for each treatment to test for variation within a sample and treatment? It is too late to change this now, but my thinking is that some of the variation we could be seeing between treatments could be from variation within a treatment.
Validity of the findings: ·	L83 - 87: My main concern is that, similar to other studies that have compared storage methods, there was no comparison of the stored samples versus samples that were immediately extracted. As such, there is no way to confirm that the different treatments reflect the microbial community from when the samples were obtained, and therefore the only conclusions that can be drawn are that the community structure did not differ between treatments. While this may have been the intended goal of the present study, I think it is important to briefly address this limitation somewhere in the text.
·	L195 - L196: The authors mention “preservation treatment of sample replicates did not significantly affect ASV richness”. Though this is supported by the LMM, and I agree with these conclusions, one sample in Figure 1A has approximately 100 ASVs when freeze-dried and approximately double that when frozen. Can the authors provide a possible explanation?
Additional comments: The following are minor comments pertaining to grammar and punctuation that the authors may consider for the improvement of their manuscript.
·	L56 - L57: Consider changing “and has been suggested to preserve the microbial community the best” to “and has been shown to be the most effective for preserving microbiome community structure”
·	L144 - 145: Change “were calculated on subsampled and rarefied ASV counts to the minimum library size” to “were calculated on subsampled ASV counts rarefied to the minimum library size”
·	L147: Change “analyzed with Linear mixed models” to “analyzed using linear mixed models”
·	L154 - 155: Change “Hellinger transformation” to “The Hellinger transformation” and change “while CLR” to “while the CLR transformation”
·	L157 - L158: Add the word distances to “we calculated weight and unweighted UniFrac distances”
·	L161 (and throughout): italicize taxonomic names like Proteobacteria
·	L167 - 168: “as strata argument” to “as the strata argument”
·	L211 (and throughout): I believe “table 1” should be capitalized to “Table 1”?
·	L213 - 214: change “did not significantly explain any of the variation” to something along the lines of “the proportion of variation explained by plate number was low/negligible and was not significant”
·	L271: Change to “explained more of the variation in Hellinger-transformed data than in CLR-transformed data”.
·	L274 - 275: Change to “were the only distance measures with a significant effect by treatment”.
·	L278 - L279: Comma after “Compared to the Euclidean distance measures”
·	L294: Remove comma after “likely”
·	L307: change to “be exported over long distances”
·	L311: change to “stored at ambient temperatures”
·	Figure 2: Consider putting  the color of the treatment in parentheses at the end of the caption. “freeze-dried (yellow); frozen (blue-grey)”.


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: FREEZE-DRYING CAN REPLACE COLD-CHAINS FOR TRANSPORT AND STORAGE OF FECAL MICROBIOME SAMPLES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all my previous comments and I am happy for the paper to be published. Please note that Fig1 d-f do not appear to be referenced in the main text.
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: FREEZE-DRYING CAN REPLACE COLD-CHAINS FOR TRANSPORT AND STORAGE OF FECAL MICROBIOME SAMPLES
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 addressed all previous comments thoroughly and extensively in their rebuttal, which has increased the manuscript's quality to meet all journal criteria. Therefore, I recommend this article to be accepted for publication.