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: CAS9/GRNA-MEDIATED GENOME EDITING OF YEAST MITOCHONDRIA AND CHLAMYDOMONAS CHLOROPLASTS
Review round: 4
Reviewer: 1

Output:
Basic reporting: Data supporting genome editing in yeast mitochondria is significantly strengthened in the revised manuscript. While in the previous manuscript only a single event was reported, in the current manuscript editing was detected in 15/15 samples when Cas9 is present, and 0/15 samples when Cas9 is absent. Because genome editing is reproducible in yeast mitochondria, the data are suitable for publication. However, manuscript should be improved to make it acceptable.

1) It would be helpful to explain in more detail what constitutes a sample in the results and document the data.

2) When preparing the manuscript, please, omit all data and reference to CRSPR/Cas9 mediated gene insertion in Chlamydomonas chloroplasts, because the chloroplast CRISPR/Cas9 constructs are not functional.

3). Authors correctly suggest that the chloroplast CERISPR/Cas9 construct is probably inactive when write: “The results indicate that the SNPs detected at the AvaII site in experimental lines and control lines are statistically similar and not a consequence of Cas9- induced SNPs. Enrichment of mutations at the AvaII recognition site is not due to Cas9 activity since they are found in similar frequency in the control lines without Cas9 or sgRNA. They likely represent enrichment of preexisting native polymorphisms or PCR errors at the AvaII site by our strategy for enriching the AvaII resistant clones. Based on our data, we did not find evidence for NHEJ DNA repair in chloroplasts.”

NHEJ is not the only mechanism of double-strand break repair. Double-strand DNA breaks in chloroplasts, when occur, frequently leave behind telltale signs, such as deletions repaired by microhomology-mediated end joining (seed Odom et al, cited).  Because such deletions were absent in the present experiments, the simple explanation is that no double-strand breaks formed, because the CRISPR/Cas9 constructs is inactive.

 4) Inactive CRISPR/Cas9 constructs explain the lack of statistical support  for CRISPR/Cas9 mediated insertion in chloroplasts: 2 events in the YP13 pool of 20; and 0 events in the control pool of 32 clones (YP21 and YP23). The two events could be the products of rare homologous recombination.

Minor points:
5) In Figure 2, double underlining is not visible.
6) For information on linker design which yield fluorescent gfp fusion proteins see Zhou et al. (2011) A recombineering‐based gene tagging system for Arabidopsis. The Plant Journal 66, 712–723
Experimental design: Innovative.
Validity of the findings: Conclusions are acceptable in case of yeast mitochondria. Unacceptable in case of Chlamydomonas chloroplasts.
Additional comments: Manuscript should be revised to contain data only on yeast mitochondria.


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: CAS9/GRNA-MEDIATED GENOME EDITING OF YEAST MITOCHONDRIA AND CHLAMYDOMONAS CHLOROPLASTS
Review round: 4
Reviewer: 2

Output:
Basic reporting: See general comments.
Experimental design: See general comments.
Validity of the findings: See general comments.
Additional comments: The authors have adequately responded to the reviewer's 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: OLA1 IS RESPONSIBLE FOR NORMAL SPINDLE ASSEMBLY AND SAC ACTIVATION IN MOUSE OOCYTES
Review round: 1
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: The manuscript entitled ‘OLA1 is responsible for normal spindle assembly and SAC activation in mouse oocytes’ by Xie et al., has investigated the role of OLA1 in meiosis. This is an interesting manuscript that shows a novel role for OLA1 in meiosis.

Overall this manuscript requires significant language editing. There are some concepts that are difficult to interpret and some language is incorrect.

Introduction:

The introduction is brief and could do with some addition information to help guide the reader. For example, little is given about the spindle assembly checkpoint and why this is so important. While it could be a language issue, there is an implication that this is a single event rather than a multi protein effort to ensure correct progression.

The authors mention in the introduction that this GTPase is actually a better ATPase. This is not the case. It is from a family from GTPases (Obg-like) but has evolved to use an alternative nucleotide. OLA1 is mentioned briefly but its importance is not fully defined. It would be beneficial to include additional information about this protein and why the authors decided to study it. Much work has been done to look at the role of OLA1 in centrosome regulation. It has been shown that OLA1 is involved in centrosome focusing and microtuble aster formation (Matsuzawa et al., 2014 Mol Cell 9:101-14), Several sentences are directly taken from the above manuscript, for example: ‘results in the centrosome amplification and the activation of microtubule aster formation’. The authors should re-word the published data and fully explain the importance of this protein. The authors also mention BRCA1 (a known oncogene) but fail to fully address its importance in the context of this study.

Additionally, OLA1 has been shown to be involved in protein synthesis (Chen et al., 2015 Scientific Reps, 5:13241). OLA1 has also been shown to regulate p21 (Ding et al., 2016, Mol and Cell Biol, 36:2568-82). This could be very important as p21 has been shown to inhibit CDK1 (Boufraqech et al., 2106, Oncotarget, 7:29023-35) and be involved in spindle positioning and orientation through p21-activated kinase 4 (Bompard and Morin, 2012, Bioarchitecture, 2:130-33)

Specific points:
Line 68 – GVBD not defined in main body of text.
Line 71 – SAC not defined in main body of text.
Line 81 – NEBD acronym not required and not used elsewhere in the manuscript.

Results:

The results are clearly laid out in a logical order. Line 162 the authors say that localization to spindle means that it is involved in spindle formation – this is not necessarily the case.

Fig 1B (line 170) – The authors state in the main text that OLA1 is consistently expressed throughout meiotic progression, however the blot (representative?) presented shows that there is a 50% reduction over time.

In Fig 1C the authors say that there is a redistribution of OLA1 in cells, however from the images that are presented, this cannot be determined.

Fig 2A/B – The OLA1 siRNA shows that there is a 50% decrease in the amount of protein. This is still a significant amount. Additionally, one assumes that this is a population and therefore some may have a small reduction and others more substantial. This does make interpreting the data difficult. As the protein is still present, perhaps authors should use a reporter based system (i.e. miRNA with bicistronic GFP expression) and cell sort for knockdown cells. This would then allow the authors to be confident of their results and may generate more significant differences.

Fig 2C/D the rates presented are decreased in the RNAi treated cells, however is this a block or just a delay? It is not clear from the data presented.

Authors should explain the importance of the BubR1 (Line 219) experiments.

Can the siRNA phenotype be rescued with reexpression of OLA1?

The authors conclude that OLA1 works in a BRCA1-mediated pathway but there is no data to support this. It would be useful to determine if BRCA1 co-localize. Additionally, is OLA1 localization dependent upon BRCA1?

Discussion/Conclusions:

The data presented here clearly shows that OLA1 is involved in spindle regulation and that in its absence there is faster progression through the cell cycle.

There are several open questions that could be addressed.

One would expect the same results be seen in cells expression E168Q point mutant. While this may not be experimentally determined, it should be addressed in the discussion. Given that this is a breast cancer associated mutation, it would be interesting to see if this would be the same in meiosis.

It should be made clearer that OLA1 may be part of the SAC. Given the additional information mentioned above, it may influence spindle dynamics rather than specifically be part of the SAC.

Overall, this is an interesting study but requires more work to reach some of the conclusions highlighted by the authors. At least, the authors should look at BRCA1 localization and whether OLA1 localization is dependent upon BRCA1.


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: OLA1 IS RESPONSIBLE FOR NORMAL SPINDLE ASSEMBLY AND SAC ACTIVATION IN MOUSE OOCYTES
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, Xie et al report the role of OLA1, a member of the GTPase protein family in oocyte meiosis. The authors show that OLA1 can regulate germinal vesicle breakdown (GVBD), spindle assembly and spindle assembly checkpoint (SAC) activation during oocyte meiosis. Transient siRNA knockdown of OLA1 is associated with defects in spindle assembly and SAC inactivation during meiosis. While the authors validate their findings in a clear manner, a few points of concern are mentioned below:
Overall the results support the hypothesis put forward by the authors, but the manuscript needs careful proofreading. The authors report the role of OLA1 in oocyte meiosis. However, there is not sufficient mention or explanation of the oocyte meiotic cycle in the Introduction section. A brief description of the oocyte cycle should be described, explaining the process of GVBD and polar body extrusion (PBE). Sufficient references and background context is provided otherwise.
Earlier studies have already shown that OLA1 is implicated in centrosome amplification and astral microtubule formation during mitosis, associated with spindle defects. The role of OLA1 in meiosis, exhibiting spindle assembly defects would have been predictable to some extent. Thus, in terms of novelty, this manuscript might require additional mechanisms to strengthen their observations.
There are several grammatical errors throughout the manuscript which if corrected will enhance the quality of this article considerably.
Line 59 “Mammalian gametes are yield” is incorrect grammar.
Line 66 “normal progress of oocyte meiosis” should be rephrased as “normal progression of oocyte meiosis”.
Line 71, 216, 263: “premature of chromosome segregation” is again incorrect, should be rephrased as “premature chromosome segregation”.
Line 73 use “established instead of “amended”.
The transition from line 76 to line 77 has no connecting link. The authors might want to atleast write a sentence highlighting why they were interested to study the role of OLA1 in meiosis after describing meiosis in line 76.
Lines 80-84 is directly copied from the reference Matsuzawa et al 2014. The authors need to rephrase the observation in their own language if they are citing a reference.
Line 87, 189, 255: “interactive factor/protein” is not sounding correct. The author means to say OLA1 is an interacting partner, not interactive.
Line 91: “whether OLA1 involve in meiosis” is again grammatically incorrect. It should be “whether OLA is involved in meiosis”
Line 108: “mice firstly intraperitoneally injected” should be “mice were intraperitoneally injected”.
Line 116: “wild type” instead of “wide type”
Line 117: “To reassemble” instead of “To reassembly”
Line 167: “oocytes were thoroughly washed out of nocodazole” is not the correct representation of the sentence. It should be “nocodazole was washed out”.
In the Results section, where the authors describe that silencing OLA1 abrogated GVBD and subsequently PBE, they should describe how did they assess the GVBD and PBE rate?
The transition from line 213 to the subsequent section describing SAC inactivation upon OLA1 knockdown is missing. The authors should begin this section by mentioning why are they looking at SAC activation and its relevance to the context.
Line 228: “processes” instead of “progresses”
Line 232: “cell cycle symbols” should be replaced with some appropriate word. Maybe “GVBD and PBE are characteristic features/hallmarks of meiotic progression”.
Line 244: “During the time spindle assembly” should be “During the time of spindle assembly”.
Line 250-251 needs grammatical error correction and rephrasing.
Line 263: “mouse oocyte” instead of “muse oocyte”
Line 264: “Taken” instead of “Token”.
Line 266-267 needs rephrasing.
The Discussion section of the article is redundant and is simply states the observations reported in the Results section. An appropriate explanation of the phenotypes reported needs to be provided.
The raw data showing the Western blots of OLA1 expression during meiosis needs labelling of what the lanes correspond to.
Experimental design: The authors validate their hypothesis by relevant experiments. They report several significant phenotypes that are characteristic of defective spindle assembly during meiosis. The images shown are representative of the defects mentioned in the article. A few suggestions that the authors might consider are mentioned below:
The authors may want to conduct some colocalization studies with known spindle proteins in mouse oocytes during meiosis. This would further validate that OLA1 colocalizes with the meiotic spindle (Figure 1A).
The blot showing OLA1 expression (Figure 1B) during meiosis should be probed with stage-speficic meiosis markers to validate that particle stage. For example, SCYP3 antibody can be used as a marker for meiosis. Other proteins that are expressed during GV, GVBD, MI and MII should be probed along with OLA1 to validate the corresponding stage of meiosis.
The authors should mention the experimental procedure to measure the GVBD and PBE rate (Figure2).
This article shows several phenotypic defects on spindle assembly upon OLA1 knockdown. However, the authors do not show any rescue experiments by expressing OLA1 to show that the defects can be restored.
The authors show multipolar spindles and chromosome misalignment as a readout of aberrant spindle assembly (Figure 3). They might want to provide some insight into spindle orientation upon OLA1 knockdown by measuring the spindle angle relative to the substratum. Typically, in control cells, the spindle angle should be close to zero, as they are aligned parallel to the substrate. However, during misorientation, there is an increase in the spindle angle.
In the experiment showing anaphase inset in mouse oocytes, was the population synchronized? (Figure 4) It is better to synchronize the cells and then study the meiotic progression to get uniform data set. A flow cytometric analysis showing the progression of a synchronized population of mouse oocytes should generate data that describes anaphase onset better.
Another general question that the authors may want to comment on would be whether the function of OLA1 in maintaining spindle assembly is dependent on the BARD1/BRCA1 complex or it acts independently? It has been shown that OLA1 binds to BARD1/BRCA1 complex to regulate centrosome amplification during mitosis. Mutants of OLA1 that are unable to bind to this complex do not act efficiently to control centrosome number. Thus, it may be worthwhile for the authors to explore the mechanism of action of OLA1 that underlies the defects observed during meiotic spindle assembly.
Validity of the findings: The authors show convincing defects in spindle assembly during meiosis upon silencing OLA1. It is recommended to conduct rescue experiments to check if the observed phenotypic defects can be restored upon OLA1 expression. This will help in complementing he data further.
Also, it would be good if the authors explained the mechanism of action of OLA1. Since it has been reported that OLA1 functions together with BARD1/BRCA1 to control centrosome number, the authors could shed light on whether OLA1 acts cooperatively with this complex to drive spindle assembly. Also, it would be interesting to check if mutants that are not able to bind BARD1/BRCA1 can still exhibit these phenotypic defects.
Additional comments: This data shown in this manuscript is convincing. The authors report interesting data, validating the role of OLA1 in spindle assembly. The experiments performed are relevant and support the hypothesis well. However, some additional experiments, like the rescue of the observed defects in spindle assembly upon OLA1 restoration might be important. Some sections of the manuscript particularly the Discussion seemed redundant and can be rephrased to explain the results better. The Introduction section should provide some background on the meiotic cycle in oocytes. Careful proofreading is required before the final submission as this manuscript has several typos and grammatical errors.


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: OLA1 IS RESPONSIBLE FOR NORMAL SPINDLE ASSEMBLY AND SAC ACTIVATION IN MOUSE OOCYTES
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript by Xie et al., investigated the functions of OLA1 in mouse oocyte meiosis. The authors found that OLA1 distributed in the cytoplasm at GV stage and OLA1 co-localized with spindles after GVBD. Knockdown of OLA1 impaired the germinal vesicle breakdown progression. The authors reported that knockdown of OLA1 led to abnormal spindle assembly and loss of OLA1 resulted in the premature segregation of homologous chromosomes. The authors also found that OLA1 knockdown induced premature anaphase onset was due to the precocious inactivation of SAC. The manuscript is well written and provided evidence to support that OLA1 plays important roles in oocyte meiosis. There are some concerns to be addressed.
Comments:
1)The decreased PBE rate in OLA1-knockdowed oocytes resulted from partially inhibited GVBD. Strictly speaking, the phenotype of OLA1 knockdown is impaired GVBD not decreased PBE.
2)The authors could check if aneuploidy exists in OLA1-knockdowed MII oocytes because of premature segregation of homologues.
3)For BubR1 staining, oocytes in stages earlier than pre-MI (6.5h) should be checked to make sure that BubR1 was failed to be loaded or it could be loaded and removed prematurely.
4)The mechanisms of impaired GVBD in OLA1-knockdowed oocytes in further study.


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: OLA1 IS RESPONSIBLE FOR NORMAL SPINDLE ASSEMBLY AND SAC ACTIVATION IN MOUSE OOCYTES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all the grammatical errors, pointed out in my previous review comments and the manuscript has been proofread carefully. The manucript reads well after incorporating the amendments suggested in my review comments. However, the authors did not modify the Discussion section to provide a better explanation of the results in the revised draft.
Experimental design: The authors did not include the Western Blot showing the different meiotic stage markers to validate their data. However, they provided an explanation stating that most of the studies use time point to validate the corresponding meiotic stage. The authors have not added experiments that rescue the phenotypic defect caused by OLA1 knockdown and have cited references that have not showed rescue experiments. However, the authors could have tried to conduct a rescue experiment. The authors also have not incorporated any statistical analysis to shed light on the spindle misorientation caused by OLA1 knockdown, as suggested in my review comments. Overall, the authors have not conducted any of the experiments suggested in the review comments. Nevertheless, they have provided references to cite that similar studies have published their results and have provided explanation for not incorporating any additional results.Although the manuscript could have been better if the experiments suggested earlier were done in the revised version but the authors have done a good job in addressing the comments and have provided explanation for the same. They have modified the text which definitely reads better now and is acceptable for publication.
Validity of the findings: See above.
Additional comments: See above


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

Input:
Title: OLA1 IS RESPONSIBLE FOR NORMAL SPINDLE ASSEMBLY AND SAC ACTIVATION IN MOUSE OOCYTES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed my concerns.
Experimental design: The authors have addressed my concerns.
Validity of the findings: The authors have addressed my concerns.
Additional comments: The authors have addressed my concerns.


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: PHYLOGEOGRAPHY OF THE RARE AND ENDANGERED LYCOPHYTE ISOETES YUNGUIENSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English is sufficient, but it can be improved.

Literature references are good enough, even though some additional suggestions have been made.

Structure, figures, tables are good.

Results are adequately discussed and logical conclusions are related to aims as set out in the Introduction.
Experimental design: This paper deals with an interesting topic, within the scope of the journal.

The aims are clearly stated and introduced, the results and facts presented clearly and sufficiently fully and separate from interpretations. There is an appropriate statistical analysis of data.
Validity of the findings: Findings are interesting with general implications.
Additional comments: Some general issues:
1) Manuscript has to be revised to make it more coherent: see what is written in the abstract  ("genetic variation between populations was significantly higher than that within populations") and in the Results ("lower genetic variation between populations than within populations").
2) the term "fern" (in the title, introduction etc.) seems not correct (see attached file).
3) Manuscript has to be revised to make it more fluid for English;
4) Some additional references have been suggested to improve discussion (about dispersal, and comparison with the genetic structure of other Isoetes spesies).

Additional notes in the attached file.


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

Input:
Title: PHYLOGEOGRAPHY OF THE RARE AND ENDANGERED LYCOPHYTE ISOETES YUNGUIENSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Summary – This manuscript focus on phylogeography of a rare and endagered plants (Isoetes yunguiensis) in China. The purpose was to identify gene-flow and genetic structure and dynamic of this species and use these finding to identify population suitable for conservation. The manuscript need some revision but provide interesting results concerning genetic diversity and dispersal within the fern allies clade.
The writing is clear.
Introduction - The taxonomy of I. yunguiensis is well described and the context of the manuscript (in terms of gap of knowledge and usefulness of the finding) clear. Considering the focus of the paper (i.e. Phylogeography), phylogeographic or biogeographic information is missing in the introduction. I will encourage authors to improve this section with the addition of relevant informations concerning the actual knowledge for the studied region and for analysis of similar species in other geographical context. Finally, I will suggest to formulate more explicit hypotheses.
Matherial and Method – line 81 : authors cite a collection of water samples, but no additional details are provided in the text (samples were used for some analysis, stored for other use?). If these samples were used for pH analysis (reported at line 310) why only pH was analyzed considering that one of the conservation issue could be related by eutrophic state?
On the PCR description I would like to have a reference citing the primer composition and choice, in addition brand providing reagent is missing, as well as annealing temperature. No informations about PCR purification are provided.
I will suggest to authors to improve data analysis paragraph. To improve understanding can be subdivided in two small paragraph (species distirbution prediction and DNA analysis).
Statistical part need improvement (e.g., informations concerning how indels are treated in the analysis are missing (e.g., 5th state?), if gene were used concatenated or not, how alignment was performed, ...). Moste of theses informations occour within the Results section, but this setting make understanding of the metholody more difficult.
I will also expand the distirbution/range section: for example, adding details on the bioclimatic variables included, if checked and corrected for correlation and their biological relevance on predicting Isoetes range, which setting were used, and so on. I’m not familiar with MaxEnt software, but I would also be interested in knowing if a 1km resolution is suitable for predicting species occourence in case of plants with this size, and additional information concerning number of herbarium datapoints.
Discussion -  I appreciate the final opening with a practical-conservation oriented discussion. Therfore I think is too much considering the phylogeographic focus of the paper and need to be reduced (it take half of the length of discussion).

Figures – Fig. 1 I will remove color from background to improve the readibility of the image. I will try to make bigger pie chart and/or to reduce colors of haplotypes (e.g., selecting 3-4 main colors for the principles groups, can be assigned the same used in Fig. 8) in order to make more clear the main pattern.
Fig. 8 in captation for self-explanation I will add the origin of data (cpDNA), I will specify what the number represent (bootstrap or probability) and that only values higher than 60 are shown. Outgroup instead of out group.
Fig. 9 as already marked, in the main text is described as a figure composed by several panels , but only the network is displayed.
General remark : I will add more details on the legends of graphs in order to make each one self explaining (e.g., origin of genetic material is often missing).

Tables – In tab. 1 on line n.6 (BSH) altitude is probably wrong (134m, when all the other sites are > 1200m). Longitude need uppercase.
In tab. 2 and tab. 3 only mean is reported, standard deviation should be added.
For Tab. 4 check also Fst (which is in % but in Tab. 6 is not).
Tab. 4 I will add an additional column for F statistiscs and associated p-value. Then for self-explanation I will add on note the explanation of Va, Vb, and F-stat; and the number of permutation used to obtain the associated p-value. The same for Tab. 6. In Tab. 6 I will also add the origin of genes. Finally check in the text : Tab. 6 is not cited (I assume that probably is a mistake in line n. 194).

Supplementary material – DNA file can be opened correctly, and numbers are correct in according to what reported in the text. Each DNA sequences is labelled using the same code used within the text. Unfortunately no additional metadata are added in order to make the supplementary material self-explaining (e.g., information related to the different genes sequeced, additional informations on origin of the DNA material). In addition data are provided only for chloroplast DNA and no raw data for EST-SSR are provided.
Finally no information within method section are provided on where data will be available (e.g., genbank repository, Supplementary material, ...).
Experimental design: Probably limited by costs and material quality. But the adding of additional population better covering the region can be an improvement for understanding haplotype connections and biogeographical pattern. Why author not used herbarium samples cited on lines 122-23?

Explanation of statistical analyses need improvement (reshaping MM section as suggested before). Methods used are appropriates.
Validity of the findings: Results provided support the findings of authors. I think the strong genetic homogeneity is interesting and surprising considering the deep relationship within water for spore dispersal .
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: PHYLOGEOGRAPHY OF THE RARE AND ENDANGERED LYCOPHYTE ISOETES YUNGUIENSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is now clearer (but see notes about introduction, in the attached file).
Experimental design: The experimental design is now better explained.
Validity of the findings: The findings are interesting also for other cases. Discussion has been improved (but see attached file about IUCN category).
Additional comments: The manuscript has been improved, following my previous suggestions, but I ask you to change something (in the introduction and in the conservation strategies).


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: PHYLOGEOGRAPHY OF THE RARE AND ENDANGERED LYCOPHYTE ISOETES YUNGUIENSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have made the requested changes and answered the doubts raised above.
The text, tables and images are currently clearer.
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: THE INFLATED MITOCHONDRIAL GENOMES OF SIPHONOUS GREEN ALGAE REFLECT PROCESSES DRIVING EXPANSION OF NONCODING DNA AND PROLIFERATION OF INTRONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well prepared and interesting manuscript. I am sure that it deserves to published and it would certainly be a useful addition to curent knowledge of algal genome evolution.
Experimental design: no comment
Validity of the findings: The key finding seems to be that in Ostreobium the compressed chloroplast genome is not accompanied by a similarly compressed mitochondrial genome. Apparently, this limits plausibility of the "low-light selection" hypothesis that was used as the likely explanation of the plastid genome patterns in this sciaphilic organism. The authors provide a number of alternative explanations; however, given the very limited number of available bryopsidalean mitochondrial genomes (just 2), the explanatory power of their interpretations is necessarily limited. It might be useful to expand the scope of the "Discussion" to other chlorophytan green algae with available mitochondrial and plastid genome data, such as other members of Ulvophyceae and even other UTC-clade lineages. This is also suggested in individual points to particular statements and paragrpahs of the text (see below).
Additional comments: In general, I think that the manuscript should be accepted for the journal after a minor revision and, possibly, addition of a more general context ivolving other Chlorophyta into the discussion of the data. This might make an even more interesting and probably more frequently cited publication.

1) It should be mentioned in the "Abstract" that only one of the studied mitochondrial genomes has actually been newly published as part of the study (Ostreobium quekettii).

2) l. 59: "approximately 60% noncoding DNA in smaller lineages"
It is unclear what you mean by "smaller lineages". Smaller by relatively lower number of described taxa, available genome data, or even smaller body sizes? Subsequent sentences that discuss genomes of Volvox carteri keep open all these possibilities.

3) l. 63: "the large non-coding DNA content in V. carteri genomes supports the mutational-hazard hypothesis (MHH)"
The MHH hypothesis presumes that non-coding DNA accumulates in taxa with slow mutation rates (e.g., slow reproduction modes) and low population numbers. However, what data do you have to support your notion that V. carteri has smaller population numbers (or reproduction modes) than other phototrophic eukaryotes? The population numbers do not depend just on the number of cells in colonies but also on niche breadth, distribution area, etc. In my view, your statement is a pure speculation and it should be stated as such.

4) l. 70-71: "There is expected to be a large reduction in..." < "A large reduction in ... ... is expected."

5) l. 190: ENA #

6) l. 285-286: "the largest green algal mitochondrial genome sequenced thus far, even larger than that of C. lentillifera."
This would perhaps better fit into Discussion.
In addition, it might be interesting to compare the size of the genome with streptophytan lineages, too.

7) l. 295-301: The GC content
How usual is it that the GC content of the mitochondrial genome vastly exceeds the GC content of the plastid genome of the same organism? It might be useful to menation a few examples.

8) l. 383-384: "ribosomal protein genes ... that surprisingly appear to be missing from the mitochondrial genome of C. lentillifera"
What about in other members of the UTC clade with known mitochondrial genome data?

9) l. 484: "Sexual reproduction has not been observed in O. quekettii..."
It would be quite surprising if it turned out to be a truly asexual genus/species. Are there any known members of Bryopsidales with reasonable evidence of purely asexual way of life?

10) l. 494-495: "The differing configurations of the two organellar genomes in Caulerpa lentillifera and Ostreobium quekettii..."
I understand that with just two members of the Bryopsidales any interpretation of different patterns of bryopsidalean plastid and mitochondrial genomes is limited. However, maybe you could provide more comparison with other members of the Ulvophyceae and even the UTC clade. Of course, these algae are even more distant but the estimated divergence time of Ostreobium and Caulerpa in early Ordovician makes eventual correlated patterns between the two taxa less probable, anyway.


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

Input:
Title: THE INFLATED MITOCHONDRIAL GENOMES OF SIPHONOUS GREEN ALGAE REFLECT PROCESSES DRIVING EXPANSION OF NONCODING DNA AND PROLIFERATION OF INTRONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is definitely interesting and worthy of publishing - it presents new data, thoughtfully and thoroughly analyzed; it is well written and pitched to a broad audience with interest ranging from algal genomes to evolutionary theory.

The article is appropriately structured and the data are disclosed - it appears that the genbank number MN514984 has not yet been released, so that should be done before the article is published. I also ask in my general comments if the authors could submit their intron alignment in a more usable format - like plain text or fasta.
Experimental design: On the whole, the manuscript's methods are sound and appropriately described. I only have one comment regarding one particular part of the methods:

Assessing homology in highly variable sequences like introns is quite tricky. So, I generally view any alignment and phylogeny of introns with a level of skepticism. I appreciate that the authors acknowledged that in most cases the homology was not demonstrable and therefore the data not phylogenetically analyzable. However, more information on the analysis could be useful. Were only the conserved regions of the introns analyzed? Or their entire sequences? Focusing the analysis on a small, yet 'cleaner' subset of the data might have been insightful.
Validity of the findings: All conclusions are justified, and even though much of the evolutionary interpretation comes with great uncertainty, the authors adequately discuss the possible explanations and applicable hypotheses described in previous literature.
Additional comments: I have a few specific comments on the text, figures and data that I think could use some clarification, but they are all rather minor:

L54 - The term "protist" is rather nebulous, and I think it would be better to be specific. So, not just in green algae, but also in other photosynthetic (or also non-photosynthetic?) eukaryotes? Evolutionarily the term protist doesn't have much meaning, so I think in an evolutionary-slanted paper it would be better to be precise.

L90 and beyond
Some of the pieces of evolutionary theory are explained very clearly and extensively, so it is surprising that hitchhiking and background selection are terms just tossed in with the rest without further explanation. I realize that these are all related to the same topic/phenomenon, but not every reader might know this connection right away - so I think as the rest of the theory is well explained, these terms should be elaborated on adequately too.
Further on in the same paragraph, if I understand it correctly, it says that regions with lower recombination rates have more and bigger introns - but then a later statement says that introns tend to increase recombination rates. These two seem contradictory to me, even though I think I understand how both can be true. Does it mean that low-recombination regions tend to get invaded by introns more, but in turn those introns themselves can then serve as recombination sites?
It would be good to have a bit more clarification on how these two statements are connected.

L106 - "therefore differing Ne" - this could use a more explicit explanation. It is explained above how multicellularity and Ne are connected, but not complexity or diversity of cell/tissue types.

At times, in the discussion especially, it gets a bit murky whether the authors are talking about populations (and population sizes) of the organisms, or populations of the organelles and their genomes. Given that there can be numerous organelles per cell, and they may not always be identical genetically, this could use some extra specificity in the text.

Maybe this is just the review pdf, but Fig. 1 is not of very good quality, quite fuzzy.
I like Fig.1, it is very informative, but I'm not sure how length of repeats is shown in the innermost circle - it doesn't seem discernible. Is it the shade of the lines that represents the length?

In Fig 2 the one LAGLIDADG2 reverse transcriptase being so far from other LAGLIDADGs - does this mean that the LAGLIDADGs are not monophyletic? or that the one (brown square) has acquired a different domain in addition to the LAGLIDADG motif? I find this quite intriguing; maybe a bit more explanation would be good in the text.

Supplements: Alignments in Fig S2 are appreciated, but not very usable in pdf form. Could the authors also submit a plain-text version of these?

The supplementary tables (especially those that are in word docs) should have some sort of captions too, so that the reader doesn't have to look up what they represent.


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: POPULATION GENETIC STRUCTURE AND VARIABILITY IN LINDERA GLAUCA (LAURACEAE) INDICATES LOW LEVELS OF GENETIC DIVERSITY AND SKEWED SEX RATIOS IN NATURAL POPULATIONS IN MAINLAND CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Xiong et al. use a tree species with mixed mating system to explore the influence of mating system on levels of genetic diversity. By using a combination of Chloroplast and nuclear microsatellites they reveal overall low genetic diversity and skewed sex ratio matching the pattern detected in Japan. Overall, I am satisfied with the main conclusions but have some major suggestions for the representation of the methods, the motivation behind doing some analyses and the interpretation of results.

The introduction is very restrictive to the species or at best the genera investigated here. Implications for genetic diversity and population structure across other apomictic species and species with mixed mating systems, such as Taraxcum (Majesky et al. 2012), Capsella (Wright et al), Arabidopsis, Plantego and Silene (McCauley 1997) would be useful to broader audience.  Either the introduction or the discussion should also provide citations of other studies that have investigated correlations between reproductive strategy and climatic conditions (e.g: Preite et al. 2015; Van Dijk 2003). For instance, why is selfing/apomixis favoured in certain populations despite it causing reduction in genetic diversity.

 How does the pattern of diversity they found and the sex ratio relate to the study in Japan on the same species that was also apomictic.

Line 284 to 286: The authors indicate that the two types of markers provided similar results, but in the next sentence they refer to higher differentiation in the nuclear markers. These sentences are contradictory to each other. Please clarify and rewrite.

Highlight the relationship between FIS values, sex ratio skewness and genetic diversity. Do pops with more negative FIS tend to have low genetic diversity.

Highlight the comparison between apomictic and non-apomictic populations, since that is the primary purpose of this work.
Experimental design: I am a little confused on why pollen and seed migration rates were used, specifically because all the pops examined here had no males?

I like the idea of using several methods to detect bottleneck but these approaches need to be justified. For instance, how are they different from each other. A bigger problem here is why did the authors want to access bottleneck to begin with. No-where in the introduction or the methods do the authors reference anything about the demographic history of the species, so the estimation of bottleneck comes out of left field. I would suggest the authors to elaborate on why understanding past demographic history would be useful.

The authors also use several different mutation models in their estimation of Ne, the bearing of these on their inference of bottleneck needs to be explained in the discussion.

The estimation of “r” relies of different modes of inheritance for chloroplast and nuclear markers. This needs to be mentioned somewhere either in the introduction or just before “r” is estimated in methods.

How were the mutation rates for estimating Ne obtained?
Validity of the findings: In line 175-176 & 238 the authors mention about multi-locus pattern and mutli-locus test. This is very ambiguous.

Please clarify what “robust maternal lineage” means and how the authors concluded that it is different from the haplotype found in Japan. In the absence of an analysis done across the two countries, I don’t think the statement listed on line 251-252 is valid and neither does it add any value to the primary purpose of the paper.

It is not clear what the authors are trying to convey via line 261 to 263. Please add more details and reword this sentence.
Additional comments: Line 67: Please indicate year after Dupont and change “reproducing” to “reproduce”.
Line 69: Change “equality” to “equal”
Figure 1: If the green indicates the geographical range of the species this should be mentioned in the figure caption or in the legend.
Line 138 is redundant with line 135.
Line 142 to 144: This sentence is written awkwardly. I would suggest rewording it to emphasise that IBD and pollen/seed migration ratio were assessed to determine whether gene flow was limited between populations.
Line 236 & 240: Change “exited” to “existed”
Line 280: change “pollen that was vitality” to “pollen that was vital”
Table 3: Please complete the caption sentence “were considered for”
Figure 1: Please complete the caption sentence


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: POPULATION GENETIC STRUCTURE AND VARIABILITY IN LINDERA GLAUCA (LAURACEAE) INDICATES LOW LEVELS OF GENETIC DIVERSITY AND SKEWED SEX RATIOS IN NATURAL POPULATIONS IN MAINLAND CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript presents relevant analyses to describe the effect of asexual reproduction through apomixis on structure, differentiation and genetic diversity of a perennial bush throughout a partial distribution range, based on the variation of nuclear and organelle genetic markers. Sampling is appropriate for the study and the applied methods are useful to answer the questions posed by the researchers. However, some aspects could be reviewed to improve the quality of the manuscript and the interpretation of the results. As well, most of the sections are written in an understandable way however I detected some errors and some paragraphs are susceptible to be improved. Tables and figures are presented properly, although it is suggested to the authors to look for a public database to present the raw data

I find that the manuscript is based on two assumptions that should be reconsidered or discussed more broadly. The first assumption is that the determination of the sex of the individuals of this species has exclusively a genetic basis. A contradiction arises when the plant is described as dioecious, even though both in the introduction and discussion sections it is mentioned that according to the observations in the field the population seems to present gynomonoecious individuals. A gynomonoecious system suggests absence of sex chromosomes or incipient chromosomal differentiation. Therefore, it is possible that the formation of pistillated or staminated flowers is determined by environmental factors This must be considered both in the interpretation of the data in the discussion section and in the conclusions and implications for conservation. The second assumption is related to the mention that in years previous to the study, the presence of staminate flowers has been reported. Further considering that the species under study is a perennial shrub, it should not be assumed that the absence of trees with staminated flowers in a period of tens of years will have a detectable effect on the genetic variation and the structure of the current populations. This means that the results presented can also be interpreted as a result of ancestral genetic flow.

Abstract:
r.13-14. … a tree of economic and ecological significance with a mixed reproductive system with sexual and asexual reproduction by means of apomictic seeds.
r. 17 … Sex ratio analysis is just mentioned in the text but is not directly analyzed in this work.
r. 24 … Estimates of structure obtained with nuclear and chloroplast markers were similar

Introduction:
The introduction should focus mainly on the possible effects of clonality and particularly apomixis, on the genetic structure and diversity of plant populations.
It would be important to mention in more detail the mechanisms involved in the movement of pollen and seeds.
Experimental design: Materials and Methods and Results sections
Please give more information about cultivated samples, i. e. if there some information about the origin of these artificial populations (r. 94-95).

Give more information about the inference of the parsimony network for chloroplast haplotypes (r. 140-141). Considering that this network is not resolved maybe the analysis could be improved. Moreover, if the options for data were the appropriate to infer this network, maybe authors could resolve some loops using the criteria suggested in Crandall and Templeton (1993; https://www.genetics.org/content/genetics/134/3/959.full.pdf).

r. 155-157 It is recommendable to use 2Nu to estimate Ne for haploid markers. Authors should mention why this mutation rate was assumed for both type of markers.

It is convenient that the authors do not interpret the values of the fixation indices as levels of differentiation. This applies to the results as well as to the discussion and the table 3. For more information you can review Jost et al 2018 (https://onlinelibrary.wiley.com/doi/full/10.1111/eva.12590)
Validity of the findings: Suggestions for discussion section

r.239-246. Negative Fis values are commonly interpreted as the individuals in the population are less related than expected under a model of random mating. Therefore, the conclusion that Fis negative values are result of apomictic reproduction is not fully supported. Maybe authors could find more information in other systems to support this statement.
r. 246-249 The most common haplotype is commonly interpreted as the ancestral one, therefore this interpretation should be reconsidered. What is a “robust maternal lineage”

It is notable that populations of green cluster tend to have greater observed heterozygosis than expected (although according to their analysis they do not show deviations from the HWE), can the authors discuss this?

Please consider these questions as well
The two groups detected in STRUCTURE are related with a geographical pattern?
considering that greater movement of seeds was detected, the divergence between the groups can be associated with the migratory patterns of the birds responsible for the dispersion of seeds?
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: POPULATION GENETIC STRUCTURE AND VARIABILITY IN LINDERA GLAUCA (LAURACEAE) INDICATES LOW LEVELS OF GENETIC DIVERSITY AND SKEWED SEX RATIOS IN NATURAL POPULATIONS IN MAINLAND CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a version of the article where the theoretical framework and interpretation of the results show an improvement, however this version shows more flaws in written English.
Although interpretations have been redirected to contrast the effect of sexual reproduction through pollen dispersion with asexual reproduction through apomixis, the assumption about the existence of a male and female genetic lineages, without an evidence for a genetic system of determination of sex in this species, causes a bias in the interpretation of the results.
Experimental design: Authors accepted most of the suggestions and made significant improvements in the article, however it is not yet clear how the effective size of the populations was estimated, as it is unlikely that the effective population size for cytoplastic markers is higher than for nuclear ones. For this purpose, it is necessary to clarify whether the authors show values of the scaled effective population size (theta: 4Nu and 2Nu) or whether they are directly calculating the effective population size (Ne).
According to table 2 It should be noted that populations with larger effective sizes for chloroplast show a single haplotype, which may indicate that there is a failure in the calculation of effective population size.
Validity of the findings: Although the results of the article are valuable, the effective population sizes obtained should be reviewed, so the overall interpretation of the patterns found may need to be remade.
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: POPULATION GENETIC STRUCTURE AND VARIABILITY IN LINDERA GLAUCA (LAURACEAE) INDICATES LOW LEVELS OF GENETIC DIVERSITY AND SKEWED SEX RATIOS IN NATURAL POPULATIONS IN MAINLAND CHINA
Review round: 3
Reviewer: 1

Output:
Basic reporting: There are still several places where the sentences needs to be re-written. These include: 297, 316 & 357.

I am happy with the incorporation of more context in the introduction and the discussion.
Experimental design: Several of the points I had noted under the experimental design are yet to be addressed. Only in the discussion do the authors bring up issues related with using the equation for "r" and the various ways of interpreting it  based on the reproduction mode.

The other points have not been addressed.
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: POPULATION GENETIC STRUCTURE AND VARIABILITY IN LINDERA GLAUCA (LAURACEAE) INDICATES LOW LEVELS OF GENETIC DIVERSITY AND SKEWED SEX RATIOS IN NATURAL POPULATIONS IN MAINLAND CHINA
Review round: 3
Reviewer: 2

Output:
Basic reporting: I still think that the authors assume, without evidence, a genetic system of sex determination, which biases the approach of the article and the interpretation of the results. In spermatophytes the dominant phase is the sporophyte, while the gametophyte is a microscopic tissue contained in the flower or pollen. Sex chromosomes have been found only in less than 1% of the plants studied so far. Moreover, in different plants has been proven that production of staminate or pistillate flowers could be related to environmental conditions.Therefore, it is advisable to avoid calling individuals as males or females, since probably the sporophyte is not sexually determined and not assume that there is a male or female lineage. Examples of this are found through the document, review particularly rows: 81-83, 104, 125, 303, 315, 377. This assumption is taken to the extreme in results interpretation by assuming that the most abundant haplotype with the most connections in the haplotype network is linked to the recent apomixis (rows 333-334), and not to historical demographic patterns.

The reason why I worry about estimates of effective population sizes, is that under the drift-mutation model it is expected that populations with greater genetic diversity (He or number of haplotypes) have the largest effective sizes, which is not satisfied in these results. I should maintain that a review of the analysis is desirable.

I must insist that written English is deficient, and the document requires a professional review, The following are examples of phrases that need to be reviewed:

r. 17 “a package genetic analysis” should say “a package of genetic analyses”
r. 56 “Adventitious embryony ARE widely distributed in nature, and gametophytic apomixis ARE reported in few families” should say “Adventitious embryony is widely distributed in nature, and gametophytic apomixis is reported in few families”
r. 105 “whether” is used when two alternatives are exposed, for example “whether natural populations experienced a bottleneck or not”
r. 247 “Detaily”
r. 264 “Another explanation is establishment”, should say “Another explanation is that” or “Another explanation could be that”

Moreover, some sentences must be revised, as they seem incomplete or without sufficient support:
r. 38 “The genetic basis for these abilities is sex”… asexual organisms can not adapt?,
r. 39 sexual reproduction is predominant only in eukaryotes
r. 106-108 This is not tested in this work, and natural selection as well could reduce effective population size
r. 323-324 “According to known research… “ , this sentence must say what is according to known research
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: POPULATION GENETIC STRUCTURE AND VARIABILITY IN LINDERA GLAUCA (LAURACEAE) INDICATES LOW LEVELS OF GENETIC DIVERSITY AND SKEWED SEX RATIOS IN NATURAL POPULATIONS IN MAINLAND CHINA
Review round: 4
Reviewer: 1

Output:
Basic reporting: I don't have much to add with regard to major comments, just a couple more editorial changes that need to be incorporated are listed below:

Line 14: add is after "it"
Line 28 : Please change "In additional, and some" to "In addition, some populations"
Line 81 to 82 need to be rewritten: remove "few" in line 81 and change to "limiting gene flow" and make sure the parenthesis and capitalisation is in the correct place.
Line 109: Should be removed because it is misleading to the readers as to why pollen and seed gene flow was calculated. Please remove or move to the discussion where the presence of vital pollen is mentioned from female flowers.
Line 149: Change to "Considering that isolation by distance (IBD) can be a key factor keeping populations apart by limiting gene flow..."
Line 239: Change "limited" to "limitation".
Line 250: Please elaborate what "clone corrected data" means and how this could alter any results presented here.
Line 247: Please complete "involve seed instead..."

It is still not clear what the authors mean by" robust maternal lineage", this is mentioned in line 308-310. I suggest either moving it up to line 288.
Experimental design: None
Validity of the findings: None
Additional comments: None


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: POPULATION GENETIC STRUCTURE AND VARIABILITY IN LINDERA GLAUCA (LAURACEAE) INDICATES LOW LEVELS OF GENETIC DIVERSITY AND SKEWED SEX RATIOS IN NATURAL POPULATIONS IN MAINLAND CHINA
Review round: 5
Reviewer: 1

Output:
Basic reporting: Although the manuscript has improved in this latest version I have found numerous typographical errors, grammatically incorrect sentences and unclear concepts.
I suggest to make an exhaustive and objective review of the text again.

Authors mention that the species has a dioecious reproductive system, but that there are bisexual flowers with viable pollen therefore it could be an androdioecious reproductive system. I have questioned the authors on different occasions about evidence regarding what mechanism of sex determination they assume, however I note that they make no mention of it. This assumption influences the interpretation of the results and must be supported. Dioecy and sex ratio are commonly influenced by environmental and population factors, if this is the case, it is incorrect to assume that there is a male lineage and a female one, since probably under different conditions the same genotype can produce staminate or pistillate flowers. In the same way it would be useful for the discussion, to mention the evidence that exists regarding the genetic basis of apomixis.

In the attached file you will find some observations over the document. I suggest an exhaustive and objective review of the text again.
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: POPULATION GENETIC STRUCTURE AND VARIABILITY IN LINDERA GLAUCA (LAURACEAE) INDICATES LOW LEVELS OF GENETIC DIVERSITY AND SKEWED SEX RATIOS IN NATURAL POPULATIONS IN MAINLAND CHINA
Review round: 6
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this version of the manuscript I still find multiple writing errors, many of which I think had already been pointed out in previous versions. I have reviewed this article multiple times and I consider that the quality of writing and understanding of concepts is not enough for publication. However, I do not want to affect the authors and I would like them to reconsider the option of having the manuscript reviewed by an editorial service. Attached you will find the PDF with observations made to this version.


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: POPULATION GENETIC STRUCTURE AND VARIABILITY IN LINDERA GLAUCA (LAURACEAE) INDICATES LOW LEVELS OF GENETIC DIVERSITY AND SKEWED SEX RATIOS IN NATURAL POPULATIONS IN MAINLAND CHINA
Review round: 7
Reviewer: 1

Output:
Basic reporting: The study " Population genetic structure and variability in Lindera glauca (Lauraceae) indicates low levels of genetic diversity and skewed sex ratios in natural populations in mainland China", by Xiong, Zhang, Dong, and Zhang, provides an insight into genetic diversity and population structure of Lindera glauca, a tree species growing in China. The species reproduces sexually and asexually via apomictic seeds. This study is adding new knowledge that may assist in sustainable utilization of the species and in the conservation of its genetic resources.
The manuscript is globally well written, although it will require some editorial work. The introduction shows well the context of the study. However, some additional background information should be provided. For instance, I think it would be important to indicate that in angiosperms maternal inheritance is assumed for chloroplast DNA. I do not think that it has been tested for Lindera glauca and the authors take for granted that all the readers know that maternal inheritance is assumed. On line 85, the authors state that dioecious L. glauca has bisexual or functionally unisexual flowers. Dioecious species have distinct male and female individual. Thus, I am not sure that strict dioecy applies to that species otherwise more background information would be welcome.
The structure of the article is fine and tables as well as figures are well designed and of good quality.
Experimental design: The objectives of the study are clearly stated and define the research question. However, I have some concerns that I describe below.
The importance of this work does not seem to me to be well reflected in the sampling design. For instance, the sample size is quite small in more than half of the populations; below the standards of population genetics analyses. The initial average sample size is 13.6 trees/population. Some populations end up with extremely low sample sizes (such as n=10, 8, 5), rendering the corresponding results hard to interpret due to the potential confounding effect of small sample size in the observed genetic parameter values.
Thus, it is not possible to determine if the small sampling size of many populations was because the number of individual making up these natural populations was very small or because the authors did not have the resources to collect more samples, or have access to other larger populations. Thus, more details would be required including the average distance between sampled individuals. There is no background information on the natural population size.
I also wonder why samples of two cultivated populations were included in this study. No arguments are given by the authors about the importance to include these populations in their study. In the aims of the study, they indicate they want to detect genetic variation within and differentiation among natural populations, assess the relative importance of pollen and seeds as agents of gene flow, and determine whether natural populations experienced a decline in size (bottleneck effect). Does it mean that data of the two cultivated populations were not used, because one cannot consider them as natural populations?
Hardy-Weinberg equilibrium tests were carried out to test for the presence of null alleles, as indicated on line 130. However, it would be interesting to know what the frequency of the null alleles was. Such frequencies could be obtained using the Micro-Checker software, for instance.

I have also  the following minor points to share.
L. 216: You have stated: “Adopting a P-value of 0.001, no significant correlation between pairwise genetic distance [FST/(1 − FST)] and geographic distance (in kilometers) was found using the Mantel test …”. What s the justification of the P-value level? Why not P-value < 0.01?
L. 244: Are you referring to the Zhang et al. 2016 paper?
L. 262-264: This sentence is not clear. Please rewrite.
L. 277: How did you determine that r was biased?
Validity of the findings: With regard to the validity of the findings, the results appears to be quite novel for Lindera glauca. However, my my main concern is with the potential confounding effect of small sample size in the observed genetic parameter values, as indicated in the previous section.  Although the number of populations as well as the number of sampled individuals are larger than that of a previous paper on the same species (Zhu et al. 2016, if I am not wrong), I cannot rule out the possibility that the lower within population diversity observed in the current study is only due the small number of individuals sampled in many populations. We can see on Table 2, that almost all the populations represented by at least 20 individuals have the highest percentages of polymorphic loci and the largest number of alleles for nuclear markers.

The authors have indicated that they have taken measures to avoid sampling clones but they nevertheless ended up with presence of few clones, and copies did not seem to have been removed for the analyses. This should have been done at least in an additional analysis to evaluate the impact of the presence of clones in the results obtained.
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: POPULATION GENETIC STRUCTURE AND VARIABILITY IN LINDERA GLAUCA (LAURACEAE) INDICATES LOW LEVELS OF GENETIC DIVERSITY AND SKEWED SEX RATIOS IN NATURAL POPULATIONS IN MAINLAND CHINA
Review round: 8
Reviewer: 1

Output:
Basic reporting: I have no additional comments.
Experimental design: I have no additional comments.
Validity of the findings: I have no additional comments.
Additional comments: I still think that the sample size of most of the populations is under the standards for a population genetics study (at least for nuclear markers). Moreover, I still do not see the added-value of including the two cultivated populations in this study. I will let the Editor make his decision, as I do not know the minimum standards required by PeerJ.


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

Input:
Title: IDENTIFICATION AND VALIDATION OF A THREE-GENE SIGNATURE AS A CANDIDATE PROGNOSTIC BIOMARKER FOR LOWER GRADE GLIOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.1.	Language
1.1.1.	Line 24: please give the full form of WGCNA, since it is first mentioned here in the Abstract.
1.1.2.	Line 47: As we known -> As we know
1.1.3.	Line 52: clinically indicators -> clinical indicators; targeted therapeutic targets -> therapeutic targets
1.1.4.	Line 73-75: This sentence is directly extracted from the abstract of the rbsurv paper by Cho et al. I would suggest rewriting the second half “and separates training and validation sets of samples for robustness” to avoid confusion on the terminology of training and validation. Something to the effect of “… and adopts a cross-validation approach for robustness”, for example.
1.1.5.	Line 132: showed -> is shown
1.1.6.	Line 166: Go -> GO
1.1.7.	Line 176: Oocyte -> oocyte
1.1.8.	Line 205: adopt to -> adopted in

1.2.	Literature reference
1.2.1.	Line 47-51: suggest adding references for molecular markers used in clinical tests of glioblastoma.
1.2.2.	Line 56: Please put a citation for UCSC Xena (https://doi.org/10.1101/326470) and for CGGA (https://www.nature.com/articles/sdata201724).
1.2.3.	Line 65: Please add appropriate citation or acknowledgement of TCGA. See https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga/using-tcga/citing-tcga
1.2.4.	Line 83: Work of Ouyang et al. (2019) is cited here without much context. I would suggest changing the citation at the end of this line to a separate sentence, “The same approach was used to identify gene signatures for endometrial carcinoma (Ouyang et al. 2019).”
1.2.5.	Line 85: The citation “(Wang, Wang et al. 2019)” seems out of place. Was it meant to go with “Ouyang et al. 2019” in the previous sentence, or with the “prognostic risk score” at line 88?
1.2.6.	Line 107: Please cite the WGCNA R package paper (https://doi.org/10.1186/1471-2105-9-559)
1.2.7.	Line 116: Please cite Cytoscape properly.
1.2.8.       Line 118: Which tool did you use to perform the GO enrichment analysis? Please give a citation, and also indicate the multiple test correction method for the reported adjusted p-values.
1.2.9.	Line 129: If the authors want to give a citation for “risk score”, it should come in the Methods section where “risk score” is first mentioned.
1.2.10.	Line 248-250: please reference appropriate work at the end of this sentence.

1.3.	Article structure, figures tables, and raw data:
1.3.1.	Figure 1: I find the color scheme a little confusing or distracting. Is the purple color intended for the training process or for analyses done on the TCGA data? Does the blue line indicate chronicle order of the analyses rather than a step-wise dependency between analyses? From the figure, it is not clear which dataset the WGCNA was performed on.
1.3.2.	Figure 3: (B) Please indicate whether subjects are ordered by risk score in the figure legend. (C) Which expression level metric is used in this heatmap?
1.3.3.	Line 158-165: (1) This is a paragraph in the Results section, but it is written in a way that like it belongs to “Methods”. Please include more detailed results, such as the total number of modules detected, number of modules associated with vital status, and number of genes in each of these modules. It seems that a paragraph in the Discussion section (line 214-219) contains this information and should have been placed in the Results section. Please address this issue. (2) Please add figure title and legend for the supplemental figure.
1.3.4.	The supplemental file “peerj-41271-TCGA-LGG_clinicalMatrix.tar.gz” and file “peerj-41271-TCGA-RNAseq-HiSeqV2.tar.gz” appear to be only gzipped rather than tarballs. Please match the file name suffix with the format.
1.3.5.	Supplemental code:
1.3.5.1.	Comments in the R code were written in Chinese. I would suggest adding comments in English to serve the general readers.
1.3.5.2.	Code file peerj-41271-lastcodeCGGA.R
1.3.5.2.1.	Line 65, it appears that the first column contains CGGI subject ID, but it’s renamed to “Gene_Name” here.
1.3.5.2.2.	Line 92 and line 105, the data is saved to “TCGA...Rdata” instead of “CGGA...Rdata”.
1.3.5.3.	Code file peerj-41271-TCGAcode.R line 165, where in the code was this file “TCGA-LGG-rbsurv_result.txt” generated? I was able to run the code up to this line, but not beyond.
Experimental design: 2.1.	In Table 1 (referenced at line 69), suggest adding a p-value column to show the composition similarity/difference between the training and validation sets by individual clinical variables
2.2.	Line 107: Please give more detailed criteria for filtering genes and samples and give the final number of genes and samples that WGCNA was performed on.
2.3.	Line 125-126: “We further reduced the dimensionality of these high- dimensional data.” Which analysis does this sentence refer to?
2.4.	By “modules associated with the three genes” (line 115) and “genes interacted with those three genes” (line 162-163), do you mean the modules that contain the 3 genes as members and genes belong to such modules? If this is the case, please explicitly state so, since “associated” and “interact” are quite vague descriptions.
2.5.	For the Gene Ontology analysis, are the 3 co-expression gene modules (yellow, green, black) enriched with similar or different GO terms?
Validity of the findings: 3.1.	 Line 178-186: Which dataset was the model comparison performed on?  It would be key to know whether there is data leakage in this comparison.
Additional comments: No additional comments


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

Input:
Title: IDENTIFICATION AND VALIDATION OF A THREE-GENE SIGNATURE AS A CANDIDATE PROGNOSTIC BIOMARKER FOR LOWER GRADE GLIOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are typos and some format issues in the text. Please correct them.
Experimental design: - Why do you need to reference Wang 2019 when describing the risk score on Line 129 in the first paragraph of Results? Isn't this the new results reported here? Using the estimated regression coefficients may not be the most accurate. You could build a classifier just using the 3 genes, and use the coefficients from the classifier.
Validity of the findings: - In Figures 4E, F and G, the p-values need to be adjusted for multiple hypothesis testing.
- When reporting significant results, p-values should also be included in the text in Results. e.g. Line 147.
Additional comments: No additional comments


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

Input:
Title: IDENTIFICATION AND VALIDATION OF A THREE-GENE SIGNATURE AS A CANDIDATE PROGNOSTIC BIOMARKER FOR LOWER GRADE GLIOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this manuscript, the authors give a three-gene signature for predicting the prognosis of patients with LGG and show that the signature outperformed two other gene signatures. The authors should provide more details about the methods for the identification of survival-related genes and the comparison with other models.
Experimental design: The authors should provide more details about the methods for the identification of survival-related genes. How to deal with missing data? How about the normalization of gene expression levels? What are the parameters used in the R packages?
Validity of the findings: More details should be provided about the comparison with other models. What genes were used in the models? Were DNA methylation biomarkers used?
Additional comments: 1.	Abstract background: the main ?? of our study
2.	Abstract methods: what is WGCNA
3.	Line 65, full name of TCGA is needed.
4.	Table 1, some information cannot be shown correctly, such as age group and sex.
5.	Line 65, the data level of the TCGA data should be provided.
6.	Line 71, it is unclear how missing data are handled.
7.	Line 128, the author should clarify if the three genes have been reported in the literature.


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 AND VALIDATION OF A THREE-GENE SIGNATURE AS A CANDIDATE PROGNOSTIC BIOMARKER FOR LOWER GRADE GLIOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed my previous comments. One minor typo on line 259: To compared -> To compare.
Experimental design: no comment
Validity of the findings: Line 259-267: I am concerned about the conclusion drawn from this comparison.

First of all, although the AUC by the model from this work is higher, I suspect the difference is insignificant. I would suggest adding a confidence interval to each AUC.

More importantly, the 3-gene-signature model developed in this work was trained on the TCGA dataset. The comparison with other models was also performed on this training set. The slightly better performance may be a result of overfitting. I strongly suggest the authors (also) report the comparison using the CGGA data. Otherwise, I think the authors should absolutely tone down their conclusion that their “3-gene model had a better efficiency” and make a note that the comparison was done using the training data.
Additional comments: No additional comments


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

Input:
Title: IDENTIFICATION AND VALIDATION OF A THREE-GENE SIGNATURE AS A CANDIDATE PROGNOSTIC BIOMARKER FOR LOWER GRADE GLIOMA
Review round: 2
Reviewer: 2

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: IDENTIFICATION AND VALIDATION OF A THREE-GENE SIGNATURE AS A CANDIDATE PROGNOSTIC BIOMARKER FOR LOWER GRADE GLIOMA
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have addressed my concerns in the revised version.

Minor comments:

Some symbols in the captions of Figures 5, 6, and 7 cannot be shown correctly.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No comment.


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

Input:
Title: IDENTIFICATION AND VALIDATION OF A THREE-GENE SIGNATURE AS A CANDIDATE PROGNOSTIC BIOMARKER FOR LOWER GRADE GLIOMA
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I appreciate the authors' efforts addressing my previous comments. My concerns have been appropriately addressed.

A minor comment: the AUC confidence intervals in Figure 4 B&D are not mentioned in the text or the figure legend; please add details regarding the CIs in the figure legend.


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 AND VALIDATION OF A THREE-GENE SIGNATURE AS A CANDIDATE PROGNOSTIC BIOMARKER FOR LOWER GRADE GLIOMA
Review round: 3
Reviewer: 2

Output:
Basic reporting: The authors have addressed my concerns in the revised version. The current version of the  manuscript can be accepted.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: The authors have addressed my concerns in the revised version. The current version of the  manuscript can 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: SURVIVAL BETWEEN SYNCHRONOUS AND NON-SYNCHRONOUS MULTIPLE PRIMARY CUTANEOUS MELANOMAS—A SEER DATABASE ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript needs a linguistic revision
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The distinction among MPM patients between synchronous and non-synchronous disease onset is definitely a valuable scientific target above all when such a distinction is used to assess and test a differential in survival rates. In other words, the detection of synchrony in MPM could have significant implications in clinical practice. Although the Authors used a reliable database (1975-2016 SEER 18 program), applied reasonable inclusion/exclusion criteria (about 32,000 with at least a 5-year follow-up time after diagnosis) and performed appropriate statistical analyses, some remarks have been highlighted to improve the paper. In particular, extensions, modifications and better explanations of statistical methods are suggested. In this way, the information conveyed by this study can reach a larger scientific audience, namely not only researchers with an advanced statistical proficiency but also biomedical investigators and clinical professionals.


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: SURVIVAL BETWEEN SYNCHRONOUS AND NON-SYNCHRONOUS MULTIPLE PRIMARY CUTANEOUS MELANOMAS—A SEER DATABASE ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, Authors focused their attention in the development of a criterion to distinguish synchronous and non-synchronous multiple primary cutaneous melanomas (MPCM) and, based on the result obtained, performed survivals analyses between synchronous and non-synchronous MPCM.

The article includes sufficient background related to the unclear and arbitrary time interval considered to define synchronous and non-synchronous MPCM, however literatures related to the different survival of these patients in relation to their multiple primary CM should be added.
Figure 1: Authors stated: “The numbers in the brackets are intercepts and slopes of the regression lines”. It is not so clear to me the intercept of the solid line. The number is 7.9 but the intercept should be more than 8.
Figure 2: X axis labeled is missing.
Fig 3: Results about the inferior overall survival should be added here and in the text.
Experimental design: The Research question raised by the authors is well defined and the size of investigated features is considerable to address the scope of the study. However, overall the methods utilized by the authors should be more clearly defined. For example (and this is just an example) the algorithm utilized to optimize the cut points of the distribution is not specified.
Validity of the findings: The high number of patients analyzed contributes to give value to the obtained results; however, as correctly pointed out by the authors, the investigation of molecular (i.e., genetic and epigenetic) characteristics of the different lesions could add important value to the findings obtained. In addition, information on the possible adjuvant therapy made by patients who developed a subsequent primary CM, as well as information about the pathological staging of the primary lesion are not mentioned. These additional clinical data could indeed affect patients' survival.
Additional comments: The authors have well addressed the question raised about the time interval required to define a second primary tumor as synchronous or non-synchronous with the first one. However, no pathologic, genetic or epigenetic information about these lesions are described. Although the authors pointed out these limitations in the discussion, such data would have improved the novelty of the work.


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

Input:
Title: SURVIVAL BETWEEN SYNCHRONOUS AND NON-SYNCHRONOUS MULTIPLE PRIMARY CUTANEOUS MELANOMAS—A SEER DATABASE ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript needs some minor English revision.
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: SURVIVAL BETWEEN SYNCHRONOUS AND NON-SYNCHRONOUS MULTIPLE PRIMARY CUTANEOUS MELANOMAS—A SEER DATABASE ANALYSIS
Review round: 2
Reviewer: 1

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


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

Input:
Title: SURVIVAL BETWEEN SYNCHRONOUS AND NON-SYNCHRONOUS MULTIPLE PRIMARY CUTANEOUS MELANOMAS—A SEER DATABASE ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: In this manuscript, Authors focused their attention in the development of a criterion to distinguish synchronous and non-synchronous multiple primary cutaneous melanomas (MPCM) and, based on the result obtained, performed survivals analyses between synchronous and non-synchronous MPCM.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Authors did a good job of incorporating important review elements.


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: OBESITY IN THE BALINESE IS ASSOCIATED WITH FTO RS9939609 AND RS1421085 SINGLE NUCLEOTIDE POLYMORPHISMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Priliani and collaborators examined the associations of FTO rs9939609 and rs1421085 SNPs with obesity in the 612 unrelated Balinese subjects living in urban and rural areas. They found several significant associations with obesity anthropometric parameters (BMI, WC and WHtR) as also with high BMI (overweight/obesity), high WC and high WHtR.
The paper is interesting and well written. However, some results are hard to understand.
Experimental design: Methods described with sufficient detail & information to replicate.
Validity of the findings: The statistical analysis was done using linear regression, with adjustments for age, gender (male/female) and population (urban/rural). In Results section (L143-145) the authors claim that “Of the non-genetic parameters, age was shown to influence BMI, and being male increased BMI and WC, while living in an urban setting increased all obesity parameters” (data shown in Table 2). Also Tables 3, 5 and 6 shows that urban setting increase most obesity parameters in a statistically significant way. However, Table 4 shows that significant results were obtained for BMI in rural population, and the authors claim in Abstract that “The significant associations were particularly found in … rural population.” Thus, some kind of contradiction seems to exist between the obtained data. Or the covariates across all tables should be referred as gender and population (instead of male and urban, respectively)?
Additional comments: Minor points:

Change “females” by “women” and “males” by “men” along the manuscript.

Tables 2-6 report data obtained by linear and logistic regression. Hence, both models should be referred below the tables.

L52: “increased” rather than “increases”

L124: Consider “0.05/2” rather than “0.050/two SNPs”

L201: Consider rephrasing “that having the FTO rs9939609 and rs1421085 SNPs” to “that having the FTO rs9939609 and rs1421085 risk alleles”

L223-225: “Our results indicated the modulation by FTO rs9939609 and rs1421085 in maintaining higher BMI in older subjects, in contrast to the Balinese general characteristics where BMI tend to decrease by age.” Consider rephrasing this sentence since it seems confuse…

L228: “more” instead “mroe”

L229: Consider rephrasing “obsese students” by "students having obesity"


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: OBESITY IN THE BALINESE IS ASSOCIATED WITH FTO RS9939609 AND RS1421085 SINGLE NUCLEOTIDE POLYMORPHISMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: It was clear and well structured.
Experimental design: The design was good. I have some notes about details for information that is being provided in the general comments.
Validity of the findings: No comment
Additional comments: Review PeerJ
Overall, this is an interesting study, well written and advanced statistical analysis.

Introduction
It will be interesting to put the prevalence of obesity in Bali compare to national data based on RISKESDAS 2018.

Tourism might play a role in speeding up a modernity in a population. And this is not exclusive to Bali. It might be important that author highlight the impact of urban development on obesity (which Jakarta being the important case), however repeating role of tourism might at  the introduction might lead to an assumption that tourism is to be blame for increasing rate of obesity in Bali. My suggestion is that Author can suggest economic development induces obesity without necessarily point finger that tourism industry is the sole factor.

Methods
Can author describe the inclusion and exclusion criteria for subjects? What is the maximum age for subjects to be involved.

Results
To me, it is unclear why in the title and context, author used obesity as an outcome, while in the analysis authors used “high BMI” which means BMI higher than 25, rather than the actual cut off for obesity.

Table 4.
When looking at the unadjusted estimates and odds ratio, the correlation between FTO and high BMI was some significant in rural rather than in rural. Is there any explanation. What is the differences between rural and urban adults in Bali, in respect to their diet/physical activity or other obesity-related factors?

The role of age.
Authors divide the group based on the mean age of the population. How old is the oldest subject? Can author describe the distribution of the number of subjects in each group?

Discussion
The author needs to be careful when mentioning "older subjects” as in this study it meant “subjects whose age higher than the mean age of study population”. The terminology of older adults is not that clear, however, WHO suggested anyone above 60 years old (https://www.who.int/healthinfo/survey/ageingdefnolder/en/). The author also might need to explain that BMI 30 in adults and older adults is different because the older we are, the higher fat percentage we have in our body (and less muscle). This issue can be avoided by measuring body fat.

The differential effect of FTO on obesity parameters between urban and rural is indeed the unique point of this study. It is unfortunate that Authors did not have records on lifestyle factors within those study populations. However, Authors might need to explain more about the difference in lifestyle between Balinese who live in rural and urban, based on previous findings by other studies. This could help the reader understand the context of gene-environment interaction that might be involved in this study.

It is important that the author also explains the mechanism of action of FTO on obesity. One aspect that was not really explained in this paper was the influence of FTO on appetite regulation. This was confirmed by appetite parameter or hunger/satiety hormones. Which might be the difference between gender/ or this influence might be modified by the environment.

Can the author suggest the implication of this study for developing personalized nutrition advise for the case of obesity?

Line 228 “more”


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: OBESITY IN THE BALINESE IS ASSOCIATED WITH FTO RS9939609 AND RS1421085 SINGLE NUCLEOTIDE POLYMORPHISMS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is clear and well-written. Appropriate references are cited.
Experimental design: Well-defined research question and clear methods and experimental approach.
Validity of the findings: Findings appear to be robust. Clear conclusions based on the findings.
Additional comments: The goal of this study was to examine the association between two SNPs in the FTO gene and markers of adiposity in a Balinese population. The study is well-designed and the manuscript is clear and very well-written. The authors used appropriate methods and analyses and the findings show significant associations between FTO genotype and various parameters of adiposity across different demographics. I only have the following minor comments for the authors to consider:
1.	In Figure 1, it is not clear how the top 3 figures differ from the bottom 3 figures. This should be marked in the figure legend and perhaps use letters to denote each panel (eg Panel A-C on top and panels D-F on the bottom).
2.	Tables 5-7 show the results of haplotype analyses, however, it is not clear what the reference/comparison group is here. A clearer description of the analyses conducted here would be helpful. The table should clearly describe what the haplotype refers to. For example, rs9939609/rs1421085 should be noted in the column heading so that readers can clearly see that AC refers to nucleotides of the respective SNPs. Are these true haplotypes (ie nucleotides on the same strand), or are they combined genotypes? What exactly does AC represent? Does it mean they are carriers of the A and C alleles of the respective SNPs, or are they homozygotes for those SNPs (ie AA/CC)? Is there a TT haplotype? If the authors choose to remove the haplotype analyses (since the two SNPs are highly linked), I don’t think that would diminish their findings.


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

Input:
Title: OBESITY IN THE BALINESE IS ASSOCIATED WITH FTO RS9939609 AND RS1421085 SINGLE NUCLEOTIDE POLYMORPHISMS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I have no further 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: CLINICAL OUTCOME OF ENDONASAL ENDOSCOPIC PRELACRIMAL APPROACH IN MANAGING DIFFERENT MAXILLARY PATHOLOGIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: English should be polished by professionals.
Experimental design: Methods, Line 197
“EPLA was performed before the other procedures for patients  scheduled to receive endoscopic prelacrimal recess approach for maxillary sinus lesions.” Could you clarify what were “ the other procedures”? Did the surgeons performed middle meatal antrostomy as well as EPLA procedure?
Validity of the findings: Results, line 232 to line 234
According to the manuscript, 21 patients (16 males and 5 females) with 22 lesion sites received surgical procedures utilizing the endonasal endoscopic prelacrimcal recess approach (EPLA). Was there one patient undergoing endoscopic surgery via EPLA bilaterally during the surgery? What was the nature of the bilateral maxillary sinus disease? Or, was there one patient undergoing EPLA for two times? What was the indication for revised endoscopic surgery via EPLA?
Results, line 245 to line 255
According to the manuscript, 11 patient had neoplastic lesions, including 9 papillomas and a organized hematoma, and a cavernous hemangioma. However, on line253, one lacrimal duct adenoid cystic carcinoma and one recurrent buccal cancer were also mentioned. It would be better for a more clarified descriptions of the nature and disease entity of maxillary sinus diseases.

Line262 to line 263
What was the hypothesis to compare the VAS ratings and SNOT-22 scores between patients with and without papillomas? Why didn’t compare the subjective symptoms scores between neoplastic or non-neoplastic group? Why didn’t compare the scores between unilateral and bilateral diseases?
Additional comments: In general, the authors described their own experience in doing EPLA without obvious novel findings. The length of discussion section needs to be shortened.
The percentage of complication is too high compared to reference which should be explained.