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: DEFINITION AND REVIEW ON A CATEGORY OF LONG NON-CODING RNA: ATHEROSCLEROSIS-ASSOCIATED CIRCULATING LNCRNA (ASCLNCRNA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. Language is well.
2. Literature well referenced & relevant.
3. This field (Atherosclerosis-associated circulating lncRNA) was not been reviewed recently.
Experimental design: 1. Article content is within the Aims and Scope of the journal.
2. This review organized logically into coherent subsections.
Validity of the findings: No comment.
Additional comments: Atherosclerosis (AS) is one of the most common reasons of cardio-cerebral-vascular system diseases, which threaten human health in modern society. Considering the difficulty of detection on such subtle changes in vivo, potential biomarkers during the complex pathological progress of AS is of great significance for the prevention and treatment of AS. Long noncoding RNAs (lncRNAs) are defined as transcripts >200 bp which commonly have no protein coding potential. LncRNAs can be secreted into the circulatory system through exosomes, microvesicles and apoptotic bodies. The lncRNAs in blood are expected to be new non-invasive diagnostic markers for monitoring the progression of AS. In this manuscript, Dr. Shanshan Lu and colleagues briefly reviewed the previously reported lncRNA transcripts which related to AS development and were detectable in circulating blood. Although the current article is interesting, there are some minor concerns that need to be addressed before consideration of publication.

Minor comments:
1. To avoid confusion caused by the name. All LncRNAs should have uniform code from NCBI (https://www.ncbi.nlm.nih.gov) or Ensembl (http://asia.ensembl.org/index.html).
2. Whether the mentioned LncRNAs interact with each other or have synergistic effects on some signaling pathways. It is suggested that the molecular mechanism of lncRNA should be included in the schematic diagram.
3. Can the diagnostic efficacy of the mentioned LncRNAs be compared in a table?


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: DEFINITION AND REVIEW ON A CATEGORY OF LONG NON-CODING RNA: ATHEROSCLEROSIS-ASSOCIATED CIRCULATING LNCRNA (ASCLNCRNA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The review's introduction has not been adequately introduced. Additional information on the criticality of AS, it's incidence & mortality rates, etc. may be elaborated upon. The authors have also introduced the term "circulating lncRNAs" (line 87) but have not clarified on its origin at the juncture. Additional review of the available literature is recommended to draft a more explanative introduction section.

Citation not provided in many parts of the manuscript. The authors must revise their article with proper references added wherever necessary. Some examples of missing citations include Lines 106-109, 110, 128, 135.

What do the authors mean by "Circulating blood"?
Experimental design: In the search terms used, why was CAD included when it is only a further complication of AS? If so, why were other terms like stroke and HF not included?

What was the time-span of the search? Papers published from which year until which year were included in this review?
Validity of the findings: The authors have collated information on "circulatory lncRNAs" involved in AS through literature mining on PubMed. The findings have not been summarized in a simple, cohesive manner.

The role of ncRNAs in AS has been reviewed and published quite recently in an elaborate manner (Refer: https://doi.org/10.1093/cvr/cvz203). However, this paper hasn't even been cited in this review. The only novelty in this manuscript is the focus on "circulating" lncRNAs - however, the literature survey done by the authors seems inadequate and the manuscript is also very poorly written. Other recent literature on AS & lncRNAs which are notable in their exclusion include PMIDs 30384894, 30300747 and 31212708 to name a few.

The authors also suggest clustering the lncRNAs mentioned in this paper as ASCLncRNAs - however, there does not seem to be a specific need for this classification. In addition, some of the possible members like ANRIL and LIPCAR are also associated with other CVDs and cancers. Therefore, the classification seems redundant. The authors may perhaps choose to create an online repository (or) portal for the collection of such AS related circulating lncRNAs and this would serve a better purpose for the researchers in this domain.
Additional comments: This manuscript by Lu et al. presents a distinct set of lncRNAs related to atherosclerosis (ASCLncRNA). The work has been presented well. However, there are a few parameters which the authors may further fine-tune as suggested here.

The manuscript will greatly benefit from being reviewed by a native English speaker or a language editor. While small sections of the article have been written well, most parts seem repetitive/redundant and need to be further worked upon.

The authors may kindly provide the statistics about how many articles they retrieved from PubMed using the search terms - and info regarding any further filtration & inclusion/exclusion criteria. A simple graph depicting the growth of publications in this field may also be included.

Figure 1 may be amended to use a variation of colours - some of the arrows inside the cells and the text (like MALAT1) are not clearly visible. It would be better if the labelling was done externally.


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: DEFINITION AND REVIEW ON A CATEGORY OF LONG NON-CODING RNA: ATHEROSCLEROSIS-ASSOCIATED CIRCULATING LNCRNA (ASCLNCRNA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Atherosclerosis (AS) is one of the most common reasons of cardio-cerebral-vascular system diseases, which threaten human health in modern society. Considering the difficulty of detection on such subtle changes in vivo, potential biomarkers during the complex pathological progress of AS is of great significance for the prevention and treatment of AS. Long noncoding RNAs (lncRNAs) are defined as transcripts >200 bp which commonly have no protein coding potential. LncRNAs can be secreted into the circulatory system through exosomes, microvesicles and apoptotic bodies. The lncRNAs in blood are expected to be new non-invasive diagnostic markers for monitoring the progression of AS. In this manuscript, Dr. Shanshan Lu and colleagues briefly reviewed the previously reported lncRNA transcripts which related to AS development and were detectable in circulating blood. The paper is improved and most concerned raised by the reviewer have been addressed. I think it is might suitable for publication at this version of revised 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: DEFINITION AND REVIEW ON A CATEGORY OF LONG NON-CODING RNA: ATHEROSCLEROSIS-ASSOCIATED CIRCULATING LNCRNA (ASCLNCRNA)
Review round: 2
Reviewer: 2

Output:
Basic reporting: Line 82: AS is being compared to tumors, but the context here is not explained clearly.

Line 94: Kindly add a relevant reference for this statement.

Line 95-96: The authors mention the extreme temp. resistance qualities of lncRNA. However, they do not explain it's relevance in this context.

Line 119-122: The intended meaning is lost within the long sentence. The authors may kindly look into it and perhaps break it down into shorter, comprehensible sentences.

Line 144: "firstly discovered" might indicate it being found there first and later at a different location. Kindly make sure to fix this issue.

Lines 156-158, 190-191, 248-250, 377-378: Citation not provided.

Line 208: Plasma?
Experimental design: Line 111: The authors can add the points mentioned in the rebuttal letter about no. of manuscripts considered (285) and how full-texts were taken, in this main article as well. It would be a valuable addition to information.

The authors are also requested to kindly go through their paper in-depth and add citations wherever applicable - it is missing in many places!

The efforts of the authors to create the unified table is laudable. It would be more beneficial if they also add a note on how specificity/sensitivity percentages were calculated.
Validity of the findings: Line 165-166: Since the role of ANRIL in AS is not clear to date, does it ratify inclusion in this list?

In the case of some lncRNAs mentioned here, the authors have tried to combine findings from multiple studies, to propose a possible role for the lncRNAs - i.e., a lncRNA with a presence in circulation and having a separate role in disease progression. While the approach is commendable, the hypothesis will bear more credence when associated with some experimental proof. Therefore, these results seem hypothetical at best. And, it is for this reason, that it seems premature to me to classify these lncRNAs as ASCLncRNAs as suggested when sufficient experimental proof has not been put forth.
Additional comments: This manuscript by Lu and colleagues is a much-improved version of the previous draft. However, as mentioned above, there still remain some concerns to be addressed, as mentioned above. The authors and the manuscript would also benefit greatly from being reviewed once by a copy-editor or a native speaker after they complete making changes to it, to remove some grammatical errors.

The authors can add the note on circulating blood, as specified in the rebuttal, into the main manuscript to enable new readers to easily understand the paper better.


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 COMPLETE MITOCHONDRIAL GENOME OF GYPS COPROTHERES (AVES, ACCIPITRIDAE, ACCIPITRIFORMES): PHYLOGENETIC ANALYSIS OF MITOGENOME AMONG RAPTORS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Emmanuel et al. explored the complete mitochondrial genome of Gyps coprotheres and investigated the correlation between phylogeny and susceptibility to diclofenac toxicity among raptors. The manuscript is interesting and fitted for Peer J. however there is some problems for example we did not found the table and figure Legends, and some new references did not cite. In addition, I suggested the authors may improve in introduction and discussion part to make more concise. Some minor and specific suggestion is attached in the manuscript.
Experimental design: suggested to  add new GenBank accession numbers:KY594709 into your analysis
Validity of the findings: meet the standards
Additional comments: Emmanuel et al. explored the complete mitochondrial genome of Gyps coprotheres and investigated the correlation between phylogeny and susceptibility to diclofenac toxicity among raptors. The manuscript is interesting and fitted for Peer J. however there is some problems for example we did not found the table and figure Legends, and some new references did not cite. In addition, I suggested the authors may improve in introduction and discussion part to make more concise. Some minor and specific suggestion is attached in 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: THE COMPLETE MITOCHONDRIAL GENOME OF GYPS COPROTHERES (AVES, ACCIPITRIDAE, ACCIPITRIFORMES): PHYLOGENETIC ANALYSIS OF MITOGENOME AMONG RAPTORS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is professional (with a few exceptions, see below), as is the article structure.

However, the results presented are not suitable for testing the hypothesis.  The authors’ aim is to test whether diclofenac toxicity is correlated with relatedness amongst raptor species (with particular emphasis on new and old world vultures).  However, the authors do not provide information on diclofenac susceptibility in all 32 species included in their phylogeny.  Instead, they review diclofenac toxicity in eight species (by my count).  It is unclear whether this information was just omitted from the manuscript (in which case it should be included in a table in the next draft) or does not exist.  If diclofenac susceptibility is only well understood for eight species, I do not believe that any meaningful conclusions can be reached.

Minor comments:

Line 37: “Rejuvenating” is not an appropriate word here.

Line 87: Which three species are you referring to, explicitly?

Lines 287-288: The authors argue that “all old world vultures are vulnerable to the lethal consequences of diclofenac”.  If this is true, I would provide a table with each of the 32 species in the phylogeny and a citation describing diclofenac toxicity.

Lines 261-283: The authors should review their results in the context of what is already known regarding raptor phylogenetics.  For example, you might refer to Nittinger et al. 2005, Helbig et al. 1994, Wink et al. 2004 and Doyle et al. 2018 when discussing relatedness amongst falcon species.

References:

Nittinger F, Haring E, Pinsker W, Wink M, Gamauf A. Out of Africa? Phylogenetic relationships between Falco biarmkus and the other Hierofalcons (Aves: Falconidae). J Zool Syst Evol Res. 2005;43:321–31.

Helbig AJ, Seibold I, Bednarek W, Gaucher P, Ristow D, Scharlau W, Schmidl D, Wink M. 1994. Phylogenetic relationships among Falcon species (genus Falco) according to DNA sequence variation of the cytochrome b gene. In: Meyburg B-U, Chancellor RC, editors. Raptor Conservation Today. Berlin: World Working Group Birds of Prey and Pica Press; 1994. p. 593-599.

Wink M, Sauer-Gurth H, Ellis D, Kenward R. Phylogenetic relationships in the Hierofalco complex (Saker-, Gyr-, Lanner-, Laggar Falcon). In: Raptors Worldwide; 2004. p. 499–504.

Doyle JM, Bell DA, Bloom PH, Emmons G, Fesnock A, Katzner TE, LaPre L, Leonard K, SanMiguel P, Westerman R, DeWoody JA. 2018. New insights into the phylogenetics and population structure of the prairie falcon (Falco mexicanus).  BMC Genomics 19:233.
Experimental design: The research question is relevant and meaningful.  However, the investigation is not rigorous.  As noted above, the authors would need data on diclofenac toxicity in a much larger number of species.  Although it is not my area of expertise, it also seems appropriate to use some form of phylogenetic/character mapping.

Minor comments:

Lines 177-180: Can you describe the library preparation?

Lines 187-190: I’m not following you here.  You generated multiple assemblies of the mitochondrial genome?  What is the “representative mitochondrial genome queried”?

Line 202 Italicize Gyps coprotheres?

Line 214: Why generate three different phylogenetic trees rather than a single tree using the complete mitogenomes?
Validity of the findings: I do not believe the analysis to be robust (see above).  If the comments above cannot be addressed, I believe it might be best to revise the manuscript as a note for Mitochondrial DNA Part B, without the emphasis on diclofenac toxicity.

Minor comments:

Figure 1 appears distorted?
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 COMPLETE MITOCHONDRIAL GENOME OF GYPS COPROTHERES (AVES, ACCIPITRIDAE, ACCIPITRIFORMES): PHYLOGENETIC ANALYSIS OF MITOGENOME AMONG RAPTORS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: i only have minor suggestion see the attach 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: THE COMPLETE MITOCHONDRIAL GENOME OF GYPS COPROTHERES (AVES, ACCIPITRIDAE, ACCIPITRIFORMES): PHYLOGENETIC ANALYSIS OF MITOGENOME AMONG RAPTORS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Lines 132-135: Might revise to: “As CYP enzymes share an evolutionary link, we speculate that species susceptible to toxicity are closely related.  This might be visualized using a phylogeny derived from complete mitogenomes.”  The line “For this study the complete mitogenomes were used as surrogates for these comparisons” (lines 135-136) could then be removed.

The authors noted in their rebuttal letter that they revised the phylogenetic tree to make use only of complete mitogenomes, but there are a number of references to the trees built with mitochondrial genes built into the manuscript (e.g., Lines 163-165: “In addition, blood samples were collected from six unrelated Cape vulture in polycarbonated heparinized tubes for PCR amplification of COX1, COX3 and NAD3 genes.” The COX1 phylogeny, etc. are also still included in the supplemental files and Table 2 seems unnecessary).

Line 250: Please revise to correct grammar: “While the Gyps+Aegypius+Spilornis and Nisaetus+Aquila genera forming a clade."

Lines 271-283: There are a number of grammatical errors throughout this paragraph (e.g., double period on line 273, missing parentheses in lines 282-283).  I would review the entire paragraph and edit appropriately.

Figure 2: There appear to be several preliminary notes associated with Figure 2 that were left in my mistake (e.g., a note that the common name for Falco sparverius needs to be included in the figure).
Experimental design: The updated Table 1 indicates that the authors have susceptible/not susceptible statuses for 8 species (with citations).  Only a single species is listed as being not susceptible.  In a number of cases, the authors state that a species is “suspected” of being susceptible to diclofenac toxicity but there are no citations for this information included and I’m not sure where these arguments are coming from.  If diclofenac susceptibility is only well understood for eight species, I do not believe that the authors can show a “correlation between phylogeny and susceptibility to diclofenac toxicity” (as argued in the rebuttal letter... and the title, which is concerning), particularly given that only a single species is described as being not susceptible!  Personally, I do not believe this meets the journal’s requirement of “Rigorous investigation performed to a high technical & ethical standard”.  That being said, the editor would know better than I the requirements of the journal, so I’ll leave it up to them.
Validity of the findings: I believe it might be best to revise the manuscript as a note for Mitochondrial DNA Part B, without the emphasis on diclofenac toxicity (see comments above).
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 COMPLETE MITOCHONDRIAL GENOME OF GYPS COPROTHERES (AVES, ACCIPITRIDAE, ACCIPITRIFORMES): PHYLOGENETIC ANALYSIS OF MITOGENOME AMONG RAPTORS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The literature review and background/context seem sufficient and the article structure is professional.  The mitogenome has been archived with GenBank, although it is not clear to me if the raw sequencing reads have been archived and/or if that is required by the journal.

There are some problems with punctuation/grammar throughout the manuscript, which seem to be associated with the most recent revisions.  I am listing a few below but I think it would be useful if a PeerJ editor worked with the authors to polish the manuscript.

Lines 42-44: Revise to read: "Based on this, we speculate that other species could also be sensitive to the toxic effects of diclofenac, which warrants further investigation."

Lines 92-95: Please revise this line for clarity/grammar: "From work in other species, this limitations tends to be an evolutionary link in the Cytochrome P450 enzyme network, which is responsible for xenobiotic metabolism."

Lines 94-95: Revise to read: "As the CYP enzyme of the vulture has yet to be identified..."

Line 129: A period is needed at the end of the sentence.

Lines 170-171: Citation formatting for Jiang et al., 2015 and Jiang et al 2019 is not consistent, please revise to meet the specifications of the journal.

Line 177: Please revise to read: "Also the tree indicates...."

Lines 192: Please revise to read: "... it is possible that these closely-related bird species...."

Lines 205-206: Please revise to read: "These enzymes are important in the detoxification of environmental pollutants..."
Experimental design: The methods are described with sufficient detail, with the exception that I would include the following information included in the rebuttal letter but not in the manuscript (as far as I can tell): "The preference model GTR+G+I was derived using the Molecular Evolutionary Genetic Analysis (MEGA) software by conducting DNA/Protein models analysis to determine the Disparity Index Test Pattern of heterogeneity of the aligned nucleotide sequences used for the construction of a maximum likelihood phylogenetic tree."

Otherwise, I believe the experimental design meets the requirements of the journal.
Validity of the findings: I believe the findings/conclusions meet the requirements of the journal.
Additional comments: No additional comments


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

Input:
Title: FIRE WEATHER EFFECTS ON FLAMMABILITY OF INDIGENOUS AND INVASIVE ALIEN PLANTS IN COASTAL FYNBOS AND THICKET SHRUBLANDS (CAPE FLORISTIC REGION)
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment, the manuscript complies with the standards.
Experimental design: Methods require a few clarifications. There are some relevant issues:
- Moisture content variation after sampling and its relation with weather;
- The FWI as a suitable indicator of weather influence on flammability;
- Fire intensity as a temperature;
- Fuel samples for moisture content determination exposed to a temperature lower than desirable;
- Fuel moisture content expressed wrongly.
Details are provided in the general comments section.
Validity of the findings: The findings suffer from the problems identified above. More generally, flammability experiments are known to be poor surrogates of "real" fire conditions. Any finding from an experiment of this type is always subject to strong doubts about its validity.
Additional comments: Flammability experiments are a difficult topic, because they tend to be subjective and the researchers involved tend to have poor understanding of fire processes. I did identify a number of issues in the manuscript:
L59-63. Some confusion here between weather and climate. Individual fires are never climate-driven, the fire regime is.
L75-76. This sentence does not make sense. The only reason why RH and temperature are used in fire danger rating is because they determine fuel moisture content.
L118. Live fuel moisture? Because fire weather determines dead fuel moisture.
L153-154. "subsequently spreading fire to other plant structures" is not really accurate nor relevant. Better to just say that fine fuels such as plant shoots are the primary carriers/vectors of fire spread.
L159. What defined a sample? One stem?
L164. The experiments deal with live fuels. Live fuels lose moisture rapidly after collection. So, either you ensured the samples maintained their moisture status or you let the moisture vary after collection. In one case or another the moisture content is not expected to correlate with the current fire weather at the time of the burning. Were the experiments carried out outdoors?
L166. A Canadian reference should be used, e.g. Van Wagner (1987). As the study deals with live fuels the authors should have used other indices of the Canadian system which should be better correlated with live fuel moisture content, namely the Drought Code (DC) or the Buildup Index (BUI), as the FWI integrates this plus the atmospheric influences. Better yet, the authors could have tried to separate the effect of the atmosphere (by using the vapour pressure deficit, currently used as a predictor of live fuel moisture content) and the effect of drought.
L187. Although unfortunately found quite often in the fire ecology literature, this is extremely wrong: maximum temperature does not reflect fire intensity or the heat flux produced by combustion, as all flames attain the same temperature. Temperatures measured in fires are not the temperature of the fire, which is meaningless by definition, but the temperature of the measuring device. Using the measurements of the study, fire intensity would be expressed by the product of time-to-ignition (assuming it is an acceptable proxy for fire spread rate) and the proportion of burnt fuel, i.e. following the fire intensity concept of Byram (1959).
L200. Current knowledge indicates that drying temperatures lower than 100ºC (Matthews in the Int. J. Wildland Fire) underestimate fuel moisture content.
L201-202. Do you mean that it was calculated on a wet weight basis? The standard is to refer fuel moisture content always on a dry basis ((wet-dry)*dry*100).
L203. Make the sentence more accurate: fire intensity is directly proportional to fuel load. Keeley (2009) is not the right ref. here and you should cite Byram (1959).
L205. So you had % fuel consumed and fuel load, which enables calculation of fuel consumption (in actual weight) from which fire intensity is calculated.
L203. Reference to running GLMM appears here a 2nd time.
L293. Also in response to drought (water availability).
L301-302. Very unlikely! There are other motives but note that the fuel samples were pre-heated and this would totally override any effect that temperature could have had.
L311-312. Flammability experiments are microscale experiments that do not warrant this type of inference.
L317-323. Basically you are undermining the results here by acknowledging that they are representative of real world conditions.
L350-354. This is totally unwarranted. Burn completeness as an outcome of microscale experiments cannot be compared with that resulting from a wildfire. Under wildfire conditions the amount of residual fuel is mostly a function of fuel size (particle diameter or thickness) and condition (moisture).
L373. A more relevant motive is that dead fuel moisture content is much lower under conditions that allow fire spread.


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

Input:
Title: FIRE WEATHER EFFECTS ON FLAMMABILITY OF INDIGENOUS AND INVASIVE ALIEN PLANTS IN COASTAL FYNBOS AND THICKET SHRUBLANDS (CAPE FLORISTIC REGION)
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: All good. There should be mention in the discussion of how accurate the method was.
Validity of the findings: There is some confusion about the aims and the conclusions.
Additional comments: This study examines the flammability of three different vegetation types in the Cape region of South Africa and how flammability responds to fire weather. It is an important contribution and in general the methods are appropriate, it is well written with appropriate references. However, I think some of the logic was loose, which left me confused about what the actual hypothesis and conclusions were. In particular, there seems to be no distinction made between live fuel moisture and dead fuel moisture.

Specific points.

Line 60. I think ‘fires’ should be ‘fire regimes’. An individual fire is weather-driven, not climate driven.

Line 60-63. I think this could be clearer. For example, Bradstock 2010 explains in arid areas, dryness limits fuel and fires follow episodic rain, whereas in temperate forests, fuels are not limiting, and fire follows drying of those fuels. Bradstock, R (2010) A biogeographic model of fire regimes in Australia: current and future implications. Global Ecology and Biogeography 19, 145-158.

Line 66. Replace ‘would’ with ‘may’. This is only a theory.

Line 87. Add ‘and’ before ‘changes’.

Line 97. I think here you need to add something about litter fuels. Fires are often an interaction between litter and live fuels. For example, in eucalypt forests, it is generally thought that you cannot have a canopy fire without a corresponding surface fire. If this is the case in your system, then you are not measuring everything you need to about the flammability of these vegetation types.  If litter fuels are not important, then say so.

Line 111. Add ‘it’ after ‘showed’.

Line 117. These aims are not very clear. Surely comparing vegetation types is one of the main aims. Why pose those hypotheses, when broadly speaking, they have been established in previous studies?

L146. What form are these invasive species? Presumably small trees, but still taller than the natives? Do they co-occur with the natives, or replace them?

L160. It is important to describe how soon the burning occurred after sampling. E.g. if they were all sampled at once and then burned one by one during the day, then some would have dried out more than others.

Line 204. How was this dry mass calculated? Using the ratios found in the subsequent study of dried plant samples?

Line 287. This is not true. Dead fuel moisture (e.g. in leaf litter) is responsive to weather. Live fuel moisture responds to drought (as your 2-week drying shows). Those fire danger indices account for dead fuel moisture. See for example Nolan et al 2016. Here is a quote from there: “The first major novel outcome of this study was the formal demonstration that dynamic transformations in fuel moisture associated with major wildfires can occur rapidly for dead fuels and within several weeks to months for live fuels”. Nolan, RH, Boer, MM, de Dios, VR, Caccamo, G, Bradstock, RA (2016) Large-scale, dynamic transformations in fuel moisture drive wildfire activity across southeastern Australia. Geophysical Research Letters 43, 4229-4238.

Line 293. Similarly, you would not expect live fuel moisture to be related to fire weather.

Line 308. I don’t think anyone argues the latter.

Line 310. I disagree. They are very useful for tracking fire risk through drought, and as you explain later, the 2017 fires were partially caused by drought. They are not a component of hourly or daily fire weather, but they are a component of weekly or monthly flammability fluctuations.

Line 324. The reason that fuel load is negatively related to ignitability is probably to do with leaf traits like thickness and surface area to volume ratio. This has been studied in some detail. This is not my area of research so I don’t know the studies, but I suggest you look one or two up.

Line 333. It strikes me that your method can be a source of variation and you should acknowledge that. For example, each time you put a branch on the apparatus, a different amount is touching the plate. Can you show this variation, e.g. in graphs of the actual data?

Line 372. I like that you are considering this, but it is complicated. Most (but not all) dead leaves will drop to become litter, and litter has different flammability properties than leaves on the tree (probably a lot less flammable due to packing).

Figures. It would be nice to have a study area figure, either a map, or photos of the vegetation types or both.


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

Input:
Title: FIRE WEATHER EFFECTS ON FLAMMABILITY OF INDIGENOUS AND INVASIVE ALIEN PLANTS IN COASTAL FYNBOS AND THICKET SHRUBLANDS (CAPE FLORISTIC REGION)
Review round: 2
Reviewer: 1

Output:
Basic reporting: No problems.
Experimental design: Ok.
Validity of the findings: The authors improved the manuscript, although the basic conceptual problems persist. As usual in this subject of study, the paper will likely please flammability people and will be ignored by fire behaviour people.
Additional comments: No additional comments


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

Input:
Title: FIRE WEATHER EFFECTS ON FLAMMABILITY OF INDIGENOUS AND INVASIVE ALIEN PLANTS IN COASTAL FYNBOS AND THICKET SHRUBLANDS (CAPE FLORISTIC REGION)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The paper is clear, the methods appropriate and the use of literature is good.
Experimental design: The minor issues I raised in the first review have been addressed
Validity of the findings: The minor issues I raised in the first review have been addressed
Additional comments: The authors have done a thorough job of responding to the reviews.


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: CHEMICAL ANALYSIS OF CALLUS EXTRACTS FROM TOXIC AND NON-TOXIC VARIETIES OF JATROPHA CURCAS L.
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an interesting work and adds some scientific information to this important medicinal plant species. However, authors need to connect the story as the whole MS is missing the flow. The English language is Ok. The chosen title is not the best one.
Experimental design: Jatropha is a widely studied plant and has important commercial value. I suggest enriching the introduction and discussion part on the basis of current findings. What is the originality of your work? Why have you chosen toxic and non-toxic varieties? are there any similarities or differences in the chemical profiles on your varieties and other varieties reported by other authors?
Validity of the findings: I think the conclusion past can be improved. It would be nice to summarize all findings in the chemical profile of different parts and varieties in a table. This will help the readers to understand the findings quickly. Some figures have poor quality (i.e., fig 6, fig 4). As a reader, I could not find the importance of this work. Please highlight and specify your findings. Why did you plant this work? do you have worthy findings?
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: CHEMICAL ANALYSIS OF CALLUS EXTRACTS FROM TOXIC AND NON-TOXIC VARIETIES OF JATROPHA CURCAS L.
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.1- English is correct.
1.2- Sufficient and relevant prior literature is cited
1.3- Quality of Figures and Table is sufficient
1.4- Results are in part relevant to the hypothesis (see validity of the findings)
Experimental design: 2.1- This work fall into the aims and scope of the journal
2.2- Research question is well defined
2.3- Methods have to be improved for publication: quantification of the different compounds (at least relative) is strongly needed to support the conclusions
2.4- Methods section has to be more clearly described. In particular, US apparatus as well as US extraction parameters are not described in details. The choice of the extraction parameters is not justified. The description of the US apparatus did respect the conventional description. The authors will find these standards in the paper “Ultrasonically assisted extraction (UAE) of natural products some guidelines for good practice and reporting” published in Ultrasonics Sonochemistry, 25 (2015) 94-95.
The reference cited by the auhtors is not related (ie, on podophyllotoxin extraction from another plant source!)
Validity of the findings: 3.1- impact and novelty are basic
3.2- no quantification therefore no statistical analysis, this is a drawback for the present work.
3.3- conclusions have to be rewritten to answer the main questions of the present work
Additional comments: 1. The need of in vitro culture initiation is not described by the auhtors
2. Quantification (at least relative) is vital to present firm conclusion
3. Title is not correct. Callus are not stabilized so "during callus initiation" should be used instead
4. Difference between toxic and non-toxic is unclear in the conclusion (again quantification and statistical analysis are required)
5. Objectives of the present work are not clearly exposed


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: CHEMICAL ANALYSIS OF CALLUS EXTRACTS FROM TOXIC AND NON-TOXIC VARIETIES OF JATROPHA CURCAS L.
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors do not refer to the occurrence of J. curcas in other countries to compare their toxicity. In these aspects, the author should introduce some aspects broader out of the local place studying. Its relevance should be mentioned because of the chemotype variety is not discussed to justify the work. The article should include sufficient information if or not chemotypes are one of the important aspects of the presence or absence of the aim metabolite in the introduction, and relate it to J. curcas toxicity.

   Figure 2, it is not so clear, it must be sufficient clearness to show the reference standard. When the extract amount is applied as spot the band appears as spots when applied as a horizontal band (like on electrophoresed gel), it should appear in this paper in the horizontal band form, the results present in this article are not so conventional. It needs an explanation.

   Besides, it is important to explain the TLC variation in the volume application to justify the not uniform profiles amounts.
Inline 208, the statement is not appropriate, because TLC does not identify, It does detect the aim metabolite, groups or class. it is important to review.
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: CHEMICAL ANALYSIS OF CALLUS EXTRACTS FROM TOXIC AND NON-TOXIC VARIETIES OF JATROPHA CURCAS L.
Review round: 1
Reviewer: 4

Output:
Basic reporting: 1-Fair
2-Fair
3-Fair
4-Fair
Experimental design: 1-Fair
2-Fair
3-Fair
4-Fair
Validity of the findings: 1-Fair
2-Fair
3-Fair
4-Fair
Additional comments: The information provided in this article may aid in enhancing awareness of Jatropha plants and its products with respect to the potential toxicity of Jatropha.
1-	Generally, the paper needs English editing for some spelling and grammar mistakes along with many unclear sentences.
2-	What is the main clear idea(s) we get from this paper? Please clarify in conclusion section.
3-	Add photo for seeds and leaves of toxic and non toxic Jatropha varieties at the initial stage of cultivation.
4-	About 61 references include only five references raised last five years, please update with recent references.
5-	You are kindly requested to follow the corrections made within attachment 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: CHEMICAL ANALYSIS OF CALLUS EXTRACTS FROM TOXIC AND NON-TOXIC VARIETIES OF JATROPHA CURCAS L.
Review round: 2
Reviewer: 1

Output:
Basic reporting: English is acceptable
References have been updated
The figure and table have been modified accordingly
Experimental design: All the questions and suggestions have been incorporated  in the revised version by the authors
Validity of the findings: All the questions and suggestions have been incorporated  in the revised version by the authors
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: CHEMICAL ANALYSIS OF CALLUS EXTRACTS FROM TOXIC AND NON-TOXIC VARIETIES OF JATROPHA CURCAS L.
Review round: 2
Reviewer: 2

Output:
Basic reporting: see General comments for the author
Experimental design: see General comments for the author
Validity of the findings: see General comments for the author
Additional comments: The Authors have perfectly answered to all my queries.
From my point of view, this work is now acceptable for publication.


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

Input:
Title: VARIATION IN FEMALE REPRODUCTIVE TRACT MORPHOLOGY ACROSS THE REPRODUCTIVE CYCLE IN THE ZEBRA FINCH
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are a few minor editorial changes required, as well as some more methodological detail. The title and abstract do not provide a totally accurate reflection of the results so require re-writing. There is too much focus on the estradiol part of the study (which ultimately didn't work) in the abstract and title.

The introduction would benefit with some more explanation of and justification for the estradiol question (and why other key reproductive hormones were not considered).

Table 1 could arguably be a supplementary file/appendix. It also needs some more information to be useful in the context it is intended (specifically, information on when sampling was carried out in each study is important for comparison with the current study). Figure 3 requires a clearer legend and there are some conceptual issues with Figure 1.

I'm not convinced the data obtained allow the hypotheses to be satisfactorily tested.

These points are all expanded upon in my comments to authors.
Experimental design: Further data are required to fully answer the questions posed by this study. If this is not possible, the study's aims/scope need to be adjusted and interpretation of the results tempered.

More detail is required in parts of the methods.

These points are all expanded upon in my comments to authors.
Validity of the findings: Further data are required to fully answer the questions posed by this study. If this is not possible, the study's aims/scope need to be adjusted and interpretation of the results tempered.

These points are all expanded upon in my comments to authors.
Additional comments: This manuscript presents the interesting question of how the oviduct changes throughout a reproductive cycle in an opportunistically breeding bird, the zebra finch. Our current understanding of how the reproductive tissues of birds changes from non-breeding to breeding state is largely limited to seasonal breeders such as temperate songbirds. In seasonal breeders, the oviduct in females (and testes in males) grows at the start of the breeding season in response to hormonal changes triggered by increasing day length, and regresses significantly after breeding, presumably as a cost-saving mechanism. This growth and regression take place over a relatively predictable and stable timeframe, allowing the timing of reproduction to be matched with environmental conditions. Opportunistic breeders, however, tend to breed in unpredictable environments, responding quickly to optimal breeding conditions. How they achieve this is poorly understood, despite the fact that in captivity, opportunistically breeding species like the zebra finch have been recorded to produce eggs less than a week after pairing with a male. How this rapid response happens at the mechanistic level, and what the costs might be, are fascinating questions. I was pleased to see a study focused on them.

The manuscript is generally well written and straightforward, but I have some concerns about the methods and interpretation/presentation of the results. I also have some additional questions/ideas that I would like the authors to consider. The key issues are listed in order of importance here, but see also the specific comments afterwards for more minor comments and suggestions.

Key concerns

1. The study doesn't explicitly measure 'non-breeding' state, so we do not know what the baseline values are for this species. The earliest sampling period is 'pre-breeding', which is before a nest site and material was provided. However, if I understand the methods correctly, females were already paired with a male that they had produced a clutch with previously, before they were moved to the experimental cage. It is therefore not clear that these females were in a 'baseline' reproductive condition (especially since some females of this species will lay on the cage floor if nesting sites are not available). The authors note this at the end of the discussion, but do not sufficiently deal with the implications of this in terms of their comparisons with seasonal breeders and their interpretation of patterns in Figure 1 (see specific comments below). It would be useful to have some measure of oviduct size from a group of females that haven't been paired, so that a true baseline state (and variation within) can be inferred.

2. The study explicitly aims to quantify how oviduct size changes throughout a reproductive cycle in an opportunistic breeder, for comparison with what is known about seasonal breeders. In this study, however, opportunistic breeding is confounded with domestication and captivity. It seems likely that the costs of maintaining reproductive tissue would be lower for captive birds with ad lib food etc, than it would be for wild birds. Moreover, domesticated strains may have been selected for high reproductive output and therefore more likely to be 'productive' (e.g. domestic chickens are an extreme example of this). These factors will surely influence oviduct condition (just as they do body condition), so it would have been ideal to have some wild comparison data (even just at one non-breeding and one breeding time point) to see if the patterns in captive birds are generally representative of wild birds. If it's not possible to add this, the possible effects of captivity/domestication should at least be discussed in the manuscript.

3. The estradiol part of the study largely failed. Because of this, the data obtained for this part of the study are very few and potentially not reliable. Yet, the estradiol 'result' is made to be a focal point of the manuscript via its inclusion in the title and coverage in the abstract (without any indication of the test failure).  The emphasis needs to be taken off of the estradiol part of the study in the title and abstract. I support including information about the attempted trial and failures (as well as ideally some attempt to explain why), because it is useful for other researchers to be aware of potential methodological issues in the future. However, I do not think very much at all should be inferred from the data obtained. More generally, I also wonder why estradiol was focused on exclusively (especially given the poor performance of this test), in preference to other important reproductive hormones such as FSH or LH?

In my opinion, a revision of this manuscript should either (a) be rewritten to focus primarily on the oviduct size data alone, framed more simply and with the interpretation of the results somewhat tempered (i.e. taking into consideration the lack of a true 'baseline' measure and the confounding issue of captivity/domestication) OR (ideally) (b) include additional data to resolve these issues. While option (b) is obviously more time consuming and dependent on the availability of funds for further work, it would ultimately make this paper far more useful and interesting.

Below are some more specific comments on the manuscript (line numbers referred to), including some developments on the key points made above. I hope the authors find these comments useful when revising their manuscript.

Abstract

line 20 (and throughout MS): use of "recrudescence". This is a somewhat obscure word that I've never heard used in this context. When I looked it up, it seems to be most commonly used to describe disease relapse. I would personally prefer a more straightforward word, perhaps simply "growth" or "re-growth" (as is typically used in similar studies).

line 27: ‘Physiological readiness’ rather than ‘physiologically readiness’

line 33-35: the estradiol part of this study didn't really work and we can't infer much from the data obtained. But in the abstract it is presented as a straightforward result. I would remove this from the abstract (and remove the reference to estradiol from the title).

line 35-37: I think the abstract should include something about the regression being less dramatic in the zebra finch than has been previously found for seasonal breeders. To me, this is probably the most interesting and illuminating result (as you nicely explain at the end of your discussion). The caveat to this is, of course, if this is truly a consequence of being an opportunistic breeder, rather than just a consequence of captivity/domestication.

Introduction

line 69-70: "average" and "mean" are used interchangeably here, which is a bit confusing and unclear. Presumably "average" means "mean"? If so, use "mean" consistently

line 72: remove the bracket around (Perfito et al. 2007) to read Perfito et al. (2007) since the study in question is being directly referred to.

line 76: it is not clear what "maintained partially developed gonads when not breeding" means – partially developed compared to what? If all non-reproductive females have gonads that appear to be at the same level of development, presumably this is their baseline (i.e. undeveloped) state? Perhaps opportunistic breeders baseline is simply more developed than seasonal breeders (or to flip it round, seasonal breeders regress more dramatically than opportunistic breeders)? This also has implications for how the situation is depicted in Figure 1C (see later comment on this).

line 76: remove the bracket around (Prior et al. 2013) to read Prior et al. (2013)

line 80-81: (linked to previous comment) could there be something else about seasonal breeders (i.e. the environments they are typically found) that makes them need to regress more dramatically than opportunistic breeders would? I.e. it's not so much that opportunistic breeders are 'hanging on' to their reproductive capacity, but rather that seasonal breeders have to substantially pare back in order to survive.

line 101-103: the way this is written makes it sound like individuals will be followed through the reproductive cycle, which is slightly misleading. Could be reworded e.g. "no study to date has quantified variation in reproductive tract morphology at different stages throughout the reproductive cycle in an opportunistically breeding bird."

line 111: the estradiol question feels a bit tagged on at the end of the introduction. If this is retained this as a focal part of the study (which it perhaps shouldn't be; see general comments), the introduction would benefit from a little bit more coverage of the role of estradiol earlier on e.g. when introducing about how tract growth and regression typically occur in seasonal breeders, the role of estradiol could be mentioned, and then as the manuscript goes on to introduce opportunistic breeders, it could highlight the fact that we don't really understand how estradiol works in this type of reproductive mode (which then sets up the question/hypothesis about estradiol and better integrates it into the overall biological background). Also, why just estradiol and not other important reproductive hormones such as FSH and LH?

Methods

line 121: "thirty-sex" = "thirty-six"?

line 121: "force paired" meaning may not be clear to someone with no experience of bird breeding. Suggest replacing with "were established in individual cages to prevent pairing or copulations with other birds" (or similar)

line 121-122 "at least one clutch" – how much variation in prior breeding/laying experience was there between females? Might we expect oviduct size/flexibility to change with age and/or reproductive experience? I also wonder if there could be some sort of anticipatory effect here - if you have produced 3 clutches in a row are the perceived odds that you will be able to lay another higher? Could this influence retention of reproductive tissue?

line 132 when is "pre-breeding"? (precisely)

line 132 how is "nesting" defined? (precisely) - is this nest-building? Also more generally, it would be helpful to use an easily differentiated word for either nesting or nestling to avoid confusion.

line 138: how often to females lay on the floor in this population (both non-breeders and those in pairs)? To produce an egg, females must have a functioning (i.e. developed) oviduct, and it seems this is quite common in captive populations of zebra finches even without the typical cues that induce breeding condition (i.e. pairing up with a male). Could this potential variation influence the interpretation of your results?

line 145-146: were the oviducts stripped of connective tissue and blotted first, before weighing? Can you provide a bit more explanation about how follicle counts were done from photos? The ovary is three-dimensional so some follicles would be hidden in a two-dimensional photo (evident from Figure 2)? Wouldn't it have been easier to preserve these and count directly?

line 168: why mixed models? What was the random effect? Each female provides just one data point, right?

Results

line 186-187: this first sentence is redundant, suggest omitting

line 193-194: isn't it obvious that follicle number and ovary mass will decline during this period? Not because of ovary 'regression', but simply because ova are being released? During and after laying follicles are releasing or have released their ova, respectively... not sure why these changes in the ovaries are particularly biologically interesting. The oviduct mass data seems more useful.

line 200: do you have any idea what went wrong with the estradiol trials? Was it more likely a technical problem (if so, details of this might be important from a methodological perspective for other researchers) or could it be that the birds actually have very low estradiol levels? If the latter, could opportunistic breeders be fundamentally different to seasonal breeders in terms of their reproductive hormonal profiles? I.e. different proximate mechanisms for triggering oviduct growth and ovulation? Is anything known about this?

Discussion

line 211-212: over what timeframe do the changes in oviduct size occur in seasonal breeders previously studied? Does it take a much longer time? To me, this seems to be the interesting biological question - can opportunistic breeders do this far more quickly than seasonal breeders, and if so, how?

line 217: it would be interesting to know if between-female variation depends on other maternal factors e.g. age/experience (see my similar earlier comment)

line 246-248: this is a good point (and links to the above comment) – was there any variation like this within the study cohort e.g. previous breeding experience? E.g. as mentioned previously, the methods specifically state that birds were allowed to lay "at least one" clutch (implying that some laid more). Is there any variation in this respect that could be informative?

line 254: how repeatable and reliable are these tests? Presumably not very, given the high failure rate. Are the detectable measures definitely reliable?

line 275: it would have been interesting to sample a random set of females before or very soon (i.e. one/two days) after pairing, to get an idea of what their baseline state is and therefore what the rate of oviduct regrowth is. I wonder if, under captive conditions, females are able to maintain their oviduct tissue more readily than a wild bird would be able to because living conditions are 'easy'? In this context, how representative are domesticated captive zebra finches likely to be of their wild counterparts? It may simply be easier to maintain oviduct tissue in captivity, so the effect we see here is actually a captivity effect, not an opportunistic breeder effect. Ideally, data from truly non-breeding females (to get real baseline data) and/or some comparison data from wild birds (even just non-breeding vs breeding time-points to get a rough idea of how comparable captive birds are to wild birds) would be added here to complete the puzzle. Currently, the existing data only really tell us is how the oviduct changes from after pairing (by which time changes may have already happened) through to chick-rearing, in a domesticated captive bird with (probably) limited costs associated with maintaining reproductive function outside of breeding.

line 277: definitions of what "pre-breeding" and "nesting" phases actually are need to be included in the methods, not just at the end of the discussion

line 278: change ‘destinction’ to ‘distinction’

Fig 1C - this figure may be misleading. The data collected in this study don't allow us to infer reproductive tissue state outside of the breeding period (i.e. completely before and after the breeding cycle). This figure makes it look like the opportunistic breeder baseline is the same as it is in seasonal breeders outside the breeding season. However, it is possible that it never regresses to the same extent.

Figure 3: figure legend could be clarified by changing ‘quantified variation in zebra finch female reproductive tract across and within…’ to ‘quantified variation in zebra finch reproductive tract tissue across and within…’

Table 1: while it's interesting to see these data and what is already known, it is not clear at what point in the reproductive cycle these traits were measured in other studies, which makes it difficult to draw comparisons with the current study. I also think this table probably isn't needed in the main text and should instead be in supplementary material.


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: VARIATION IN FEMALE REPRODUCTIVE TRACT MORPHOLOGY ACROSS THE REPRODUCTIVE CYCLE IN THE ZEBRA FINCH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written and easy to follow with appropriate figures. Data are shared.

Overall, the the literature is well referenced. However, I do have a couple of suggestions for improvement in this regard.

In general, I think the characterization of our current understanding of opportunistic/flexible breeders is a bit of an oversimplification. For example, Ln 66 - I would disagree with the interpretation of the Hahn 1998 data as shown "little annual variation". Fig 2 shows females with an ovary score of 2 in late fall, which the Methods indicates reflects an absence of apparent follicles.

Related to the comment above, it isn't clear how data were selected for inclusion in this Table 1, as it does not appear to be comprehensive. For example, the Hahn data mentioned above are not included and it appears that only some species in the Wikelski et al. 2003 study are included.

Seasonal breeding is oversimplified in places as well. In particular, ln 48 - I suggest deleting "much" from the statement, ln 52 - "inflexible" is an overstatement as fine-tuning certainly occurs.

Minor comments

Provide full references for studies in Table 1.  I couldn't locate these.
Experimental design: Overall methods experimental design and methods are well defined and meaningful. However, further detail is needed in a few places.

1) More information is needed on how the "pre-breeding" period was defined and the state of those birds.. The authors note some difficulties with this category in the Discussion (and provide necessary caveats for interpreting the data), but it would be useful for readers to have more information about this categorization in the Methods. Is this defined purely on the fact that they did not have a nest-box and nesting materials? Was this period immediately following the 2 week acclimation? How long had it been since these birds had produced their last clutch?

2) It is stated that mixed models are used for statistical analysis, but it isn't clear what is the random term(s) in the models

3) No information is provided on priors used for Bayesian modeling. Were additional diagnostics performed for this modeling?

Minor comments
 ln 144 - is "deep anesthesia" really what is meant here given that birds are euthanized?
Validity of the findings: Overall results and conclusions are well supported. But see comments below.

1) Ln 191 - the statement about a significant decrease between laying and incubation does not match statistical results presented in Table 2.

2) ln 286 - the conclusion that this reflects a lack of photo refractoriness is a bit of a leap. Perhaps this could be better supported with use of literature?

3) ln 288 - reword to make clear this is a hypothesis (e.g., change "can" to "might")

Minor comments

Figure 4 legend use of the term "bar" to describe both the black line showing mean and the boxes for counts is confusing.

Figure 3. Is it possible to include letter labeling to indicate significant pairwise differences (from Table 2)?
Additional comments: Other minor suggestions

ln 22 - "seasonal" and "environmental" cues are not necessarily alternatives. I suggest rewording.

ln 48 - delete "much"
ln 221 - define what is meant by the resting phase.

ln 285 - should "nesting" be "nestling"?


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: EMERGING ROLES OF APLN AND APELA IN THE PHYSIOLOGY AND PATHOLOGY OF THE FEMALE REPRODUCTIVE SYSTEM
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: Here there are some suggestions related to the sources cited, the topics and paragraphs considered.

Line 96: delete “(1996)”. The year is already write in the citation.
Line 134: please, check the reference “Chun et al., 2010” and verify that it is correct in this position. Information described in the previous sentences (From line 129 to line 134: Human APLN gene is located on …..  …. generating pyroglutamate-APLN-13 (Pyr1-APLN-13) cannot be found in Chun et al., 2010.
Line 138. Since the review takes into account the entire female reproductive system and not just the ovaries, the oviducts should also be added in the following sentence: "To date, APLN is widely distributed in the female reproductive system such as the ovary, uterus and placenta". Please, consider: Mercati F, Scocco P, Maranesi M, et al. Apelin system detection in the reproductive apparatus of ewes grazing on semi-natural pasture. Theriogenology. 2019;139:156-166. doi:10.1016/j.theriogenology.2019.08.012"
Line 142. It would be better to insert at least another citation describing not only apelin system in energy metabolism but also its role in GnRH, gonadotropins and steroids through hypothalamo-pituitary-gonadal axis; for example, Yang N, Li T, Cheng J, Tuo Q, Shen J. Role of apelin/APJ system in hypothalamic-pituitary axis. Clin Chim Acta. 2019;499:149-153. doi:10.1016/j.cca.2019.09.011.
Line 228. Please, check the reference “Kwak et al., 2019”. Information regarding “APLN as a steroidogenic regulator in ovaries” are not included in this article.
In chapter 5 "Roles of APLN and APELA in the ovary", sheep must also be mentioned among the species in which apelin and apelin receptor were detected at the follicle and corpus luteum level, as described in “Mercati et al. Theriogenology. 2019;139:156-166. doi:10.1016/j.theriogenology.2019.08.012"
Since this review is intended to describe the apelin system in the female reproductive apparatus, all information existing in the scientific literature must be considered. Apelin and apelin receptor have been described in the ewe oviduct with a higher level of apelin during the luteal phase than in the anestrous phase of the ovarian cycle. A role of the apelin system in the regulation of gamete transport and nutrition and support along the oviduct has been hypothesized. I suggest that the data on the oviduct are taken into account and are described in a dedicated paragraph. Please, see this publication: “Mercati F, et al. Theriogenology. 2019;139:156-166. doi:10.1016/j.theriogenology.2019.08.012
Validity of the findings: No comment.
Additional comments: Dear Authors,
This review deals with a very interesting and current topic. As the reviewer’s knowledge, to date there are not papers that describe apelin system on all organs and structures of female genital system as it is showed in this review.
The manuscript is clearly written and English style is fluent. A number of articles have been taken into account by Authors. Each section is accurately described and well detailed. However, a paragraph describing apelin on oviduct should be added.
I have made some corrections in order to improve the clarity of the text and enrich information where necessary.
All consideration are listed below:

Line 91: “HG11” should be mentioned among receptor known names.
Line 96: delete “(1996)”. The year is already write in the citation.
Line 134: please, check the reference “Chun et al., 2010” and verify that it is correct in this position. Information described in the previous sentences (From line 129 to line 134: Human APLN gene is located on …..  …. generating pyroglutamate-APLN-13 (Pyr1-APLN-13) cannot be found in Chun et al., 2010.
Line 138. Since the review takes into account the entire female reproductive system and not just the ovaries, oviducts should also be added in the following sentence: "To date, APLN is widely distributed in the female reproductive system such as the ovary, uterus and placenta". Please, consider Mercati F, Scocco P, Maranesi M, et al. Apelin system detection in the reproductive apparatus of ewes grazing on semi-natural pasture. Theriogenology. 2019;139:156-166. doi:10.1016/j.theriogenology.2019.08.012"
Line 142. It would be better to insert at least another citation describing not only apelin system in energy metabolism but also its role in GnRH, gonadotropins and steroids through hypothalamo-pituitary-gonadal axis; for example, Yang N, Li T, Cheng J, Tuo Q, Shen J. Role of apelin/APJ system in hypothalamic-pituitary axis. Clin Chim Acta. 2019;499:149-153. doi:10.1016/j.cca.2019.09.011.
Line 172. Delete a parenthesis: “(Ulitsky et al., 2011))”
Figure 2. In reviewer’ opinion Apela is (in yellow) and not (in orange).
Line 195. Figure 2 C should be mentioned after figure 2 B that is at line 203.
Line 228. Please, check the reference “Kwak et al., 2019”. Information regarding “APLN as a steroidogenic regulator in ovaries” are not included in this article.
In chapter 5 "Roles of APLN and APELA in the ovary", sheep must also be mentioned among the species in which apelin and apelin receptor were detected at the follicle and corpus luteum level, as described in “Mercati F, Scocco P, Maranesi M, et al. Apelin system detection in the reproductive apparatus of ewes grazing on semi-natural pasture. Theriogenology. 2019;139:156-166. doi:10.1016/j.theriogenology.2019.08.012"
Since this review is intended to describe the apelin system in the female reproductive apparatus, all information existing in the scientific literature must be considered. Apelin and apelin receptor have been described in the ewe oviduct with a higher level of apelin during the luteal phase than in the anestrous phase of the ovarian cycle. A role of the apelin system in the regulation of gamete transport and nutrition / support along the oviduct has been hypothesized. I suggest that you consider the data on the oviduct described in this publication: “Mercati F, Scocco P, Maranesi M, et al. Apelin system detection in the reproductive apparatus of ewes grazing on semi-natural pasture. Theriogenology. 2019;139:156-166. doi:10.1016/j.theriogenology.2019.08.012
Line 322: replace “adema” with “edema”.
Line 432: replace “marine” with “murine”.
Table 1. In the title of the table, it would be better to write the full name of disease type than their abbreviation.
Figure 1. In the caption, please, explain the significance of the arrows contained in textboxes.
Please, check English language.


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: EMERGING ROLES OF APLN AND APELA IN THE PHYSIOLOGY AND PATHOLOGY OF THE FEMALE REPRODUCTIVE SYSTEM
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Apelin and Apela are two endogenous ligands of APLNR, and composing the apelinergic axis with APLNR. The apelinergic pathway has been generating increasing interest in the past few years for its potential as a therapeutic target in heart failure, pulmonary arterial hypertension, atherosclerosis, but also type 2 diabetes, and preeclampsia. This review summarizes and evaluates the current role of apelinergic system in female reproductive system at both physiological and pathological conditions. suggesting its potential prospect as a diagnostic biomarker and/or therapeutic intervention in the polycystic ovary syndrome, ovarian cancer, preeclampsia and gestational diabetes mellitus. This work is of significance to Gynaecology and Obstetrics.

1、Recent published paper in J Matern Fetal Neonatal Med (PMID: 32008387) should be cited in this review.
2、The description of APLNR in PE should be added.

3、The expression level of APLA in patients with late-onset PE is controversial (Ho et al., 2017 ,Wang et al., 2019; Zhou et al., 2019, Canfield et al., 2019) , while that of ELA in patients with early-onset PE is consistent (Pritchard et al., 2018; Villie et al., 2019; Zhou et al., 2019; Panaitescu et al., 2020), Therefore, table1 should distinguish early-onset PE from late-onset PE.

4、Please, add corresponding reference(s) the following sentences in 3.2: ……reduction of cyclic adenosine monophosphate (cAMP) and activation of phospholipase C-β (PLC-β), respectively (Fig 2 A).
Gαi/o activates PI3K/Akt dependent manner which is crucial for cell survival and nitric oxide (NO) induced vasodilation.


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: EMERGING ROLES OF APLN AND APELA IN THE PHYSIOLOGY AND PATHOLOGY OF THE FEMALE REPRODUCTIVE SYSTEM
Review round: 2
Reviewer: 1

Output:
Basic reporting: English revision is suggested.
Experimental design: No comment
Validity of the findings: No comments
Additional comments: The manuscript entitled “Emerging roles of APLN and APELA in physiology and pathology of female reproductive system” was edited as suggested and a point by point response to my comments were provide. However, I have a few other considerations to address.

Figure 2: The figure and the text were changed as suggested but the caption were not changed. Please, reverse B and C description.
Line 101: Change “and” with “&” in this reference and other references in the text.
Line 234-235: Since Kwak reference was delete, also “mouse” should be deleted by the sentence. Otherwise add another reference regarding mouse species.
Line 488: Please, change “another two groups” with “two other groups”.
Line 501: “Guo et al., 2020” is missing in the references chapter.
Line 637: Please, do not delete “92(6):431-440.”
I suggest to revise citations in the text and in the chapter of References where several references were cancelled even if they are cited in the text. For example: Fagerberg et al., 2014; Lee et al., 2000; Than et al., 2014; Wang et al., 2019;


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: EMERGING ROLES OF APLN AND APELA IN THE PHYSIOLOGY AND PATHOLOGY OF THE FEMALE REPRODUCTIVE SYSTEM
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The author answered all my questions and I agreed to accept. however, there is one point that should be corrected by the authors.
In table 1, Zhou et al. also measured ELA concentrations in EOPE (n=15) and LOPE (n=22) using plasma samples, and the sample size used was larger than that of placental tissue. Therefore, the results are more convincing and should be reflected in Table 1.


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: EMERGING ROLES OF APLN AND APELA IN THE PHYSIOLOGY AND PATHOLOGY OF THE FEMALE REPRODUCTIVE SYSTEM
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript has been edited and improved from the initial version.
I have no other comments to add except that one of the suggested changes was not made as stated in the rebuttal letter. Probably due to an oversight of the authors. Please, note that the noun “mouse” was not deleted at line 234.


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: EMERGING ROLES OF APLN AND APELA IN THE PHYSIOLOGY AND PATHOLOGY OF THE FEMALE REPRODUCTIVE SYSTEM
Review round: 3
Reviewer: 2

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: IMPROVING DECOLORIZATION OF DYES BY LACCASE FROM BACILLUS LICHENIFORMIS BY RANDOM AND SITE-DIRECTED MUTAGENESIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the abstract section, I suggest giving information about the origin of the enzyme, at least mentioning the organism.

Line 27. It is not necessary to write that it has been repeatedly verified.

In the introduction section.
I suggest a bit more information on the origin of the enzyme, there is very little mention of the organism from which the enzyme comes from and although it is not the objective of the investigation, I consider it important to add information so that the audience knows the organism that produces said enzyme.

Line 66. The organism has not been mentioned before, write the full name.

I consider that line 10 of the abstract can be developed a little more in the introduction section; This will give the study more impact.
Experimental design: Line 77: acid, acid??

Line 79: Eco RI

Line 93-98: Method reference

Line 112-113: Method reference or if it is from this study, I suggest describing it a bit more, for reproducibility.

Line 127. You can save the full name of the IPTG (it was already described above in the text)
Validity of the findings: Figure 1, I suggest placing it in the supplementary material.

Figs 12 and 13. Mention in the legend of the figure, the development method (coomassie or silver).

Lines 231 and 232: Here are the repeated data, I suggest removing them, as they are the same from the previous lines.

Fig 6.
The legend in figure 6 is confusing. I suggest Reorder the indications to observe and analyze the figure.

Line: 254. There are no results in this table, instead are the primers for the PCR. Please, check this part.

In Figure 8. Specifically D500G, you know the location of the amino acids, so it would be helpful if in this figure you could point them out, at least with a box and observe that possible changes; showed in fig 9.

Line: 285.  Bacillus licheniformis   B. licheniformis.

In the supplementary material (specifically: optimum copper ion concentration) you should switch to the English language.

In the conclusion section. I suggest not to expand too much and not to put data that was previously shown and discussed in the text.
Additional comments: Only if you have images of the dyes with the respective enzymes, wild type and mutant; They would be of great help in observing the improvement in discoloration and the laccase activities. It is just a suggestion and if you have the pictures you could include them in the supplementary material.


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: IMPROVING DECOLORIZATION OF DYES BY LACCASE FROM BACILLUS LICHENIFORMIS BY RANDOM AND SITE-DIRECTED MUTAGENESIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: This is an interesting study in the field of environmental biotechnology. The paper is generally well structured and underlines the usefulness of random and site-directed mutagenesis in improving enzyme activity, stability, catalytic efficiency and dye decolorization ability of bacterial laccases. However, in my opinion this paper has some shortcomings in regards to some data analyses. Below I have provided numerous remarks.
Key critical points are : a) a lack of a discussion of magnetic and spectral properties of these true or laccase-like multi-copper oxidases compared to other bacterial laccases, b) a lack of zymograpgy data revealing enzyme activity and dye decolorization potential, c)  a lack of statistical optimization approach using experimental designs for dye decolourization process, d) the authors should give proper and more justification for their claim that the mutant enzyme has a high degradation capacity for the dyestuffs. For this, in-silico, in-vitro or in-vivo toxicity tests of dye degradation products should be given. e) the activity of mutant enzyme at acidic pHs could limit its application in dye wastewater treatment particularly in textile industry, and f) finally, many typos and grammer check up is also needed (lines 52, 293, 306, ..).
Given these shortcomings the manuscript requires major revisions.


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: IMPROVING DECOLORIZATION OF DYES BY LACCASE FROM BACILLUS LICHENIFORMIS BY RANDOM AND SITE-DIRECTED MUTAGENESIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you very much for attending to the proposed suggestions.
Experimental design: Thank you very much for attending to the proposed suggestions.
Validity of the findings: Thank you very much for attending to the proposed suggestions.
Additional comments: Thank you very much for attending to the proposed suggestions. I have no other observation.


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: FORELIMB JOINTS CONTRIBUTE TO LOCOMOTOR PERFORMANCE IN REINDEER (RANGIFER TARANDUS) BY MAINTAINING STABILITY AND STORING ENERGY
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, the writing was clear. There are some word choices that should be changed. For examples a careful proof reading will find many of these:
Abstract Line 18: “…vertical GRF were increased as the locomotor…” The word increased is more important than enhanced.
Introduction, Line 54: use ‘terrain’ instead of “landforms”
Line 116: “…tested reindeer were in healthy condition and…”
Line 118: “…outdoor fenced area (2 x 10…”
Discussion, Line 312: why use the word “permanently” here?
Discussion, Line 320: “work” not “works”

The review of literature in the introduction accounts for previous work in the field. The paper is generally well referenced, however there are papers that are cited which contain more information relevant to the study than indicated. For example, the paper by Dutto et al (2004) has joint angle, moment, and power during trotting in horses and it is not in the references. The focus on comparison with other ungulates is relevant, particularly with regard to the morphology of the forelimb and associated function. Comparing morphology and mechanics of the reindeer with other, similar quadrupeds would provide a good analysis. That is only done peripherally as part of the overall paper.

Dutto, D. J., Hoyt, D. F., Cogger, E. A., & Wickler, S. J. (2004). Ground reaction forces in horses trotting up an incline and on the level over a range of speeds. Journal of Experimental Biology, 207(20), 3507-3514.

The paper follows the format guidelines and standards of PeerJ.

Figures are generally of high quality. Here some issues:
Figure 2. It was unclear where the dotted line in Fig 2C is located to indicate the distal foot. Is it the partial square? It did not come through very clear in the copy I received.
Figures 4, 5, 6,7,8: It is helpful to have all plots within a figure to have the same scale. This allows for an easier visual comparison of each joint/condition.
Figure 7 and 9. The caption is cut off so that some of the text is missing.

Finally, there is raw data supplied. However, I was not able to open all of the data files, as I got an error when attempting to do so.
Experimental design: Determining the forelimb kinetics of a cursorial, quadrupedal, ungulate is relatively novel. It appears that the research questions being addressed are:
1.	“Is the function of reindeer limb joints different from other ungulates?”
2.	“Do reindeer adopt different strategies across different gaits and speeds?”

These are good questions. However, neither one is answered well in the context of the paper. Some of the confusion might be with the wording of the question. For example, if the second question was “Does the contribution of work of the forelimb change with gait or speed?”, it would provide more clarity and focus on what was being discussed. Additionally, the comparison with other ungulates is relevant due to the type of terrain over which these animals must traverse. Providing more in-depth comparison with previously published work would help to illustrate the function of the limb in the animal pertinent to the environment.

The methods, in general, are appropriate for an investigation of this type. Creating a space to safely test animals such as these can be challenging, especially given the tricky nature of capturing both video and ground reaction force. The set-up is sound. With regards to the video and reconstruction of three-dimensional coordinates, there is no information given on the calibration of the capture space. Also, since just three cameras were used, obtaining accurate reconstruction of the marker trajectories can be tricky. Did the investigators validate their set-up? Were the cameras synchronized? How was the video synchronized with the pressure plate data?

It indicates that “each reindeer completed at least five groups of tests”. Does this mean that the results provided are an average of 5 trials at each gait condition for an animal? How were data combined if so? Or was just one trial used for each animal?

With regard to pressure plate, how was the center of pressure validated? How was this combined with the kinematic data? How was calibration of the entire set-up (force and kinematics) done to insure accuracy? Small discrepancies in COP location relative to marker locations can have a significant effect on joint moment calculations.

For both the kinematic and kinetic data, did any data processing/reduction occur? Were the data smoothed or resampled in any way? There is a fair amount of noise in the power data, but not in the moment or presented angle data.

How were the speed bins chosen for trot 1 and trot 2?

The sample size is very small, which is not uncommon with these types of data collection. In order to confidently indicate that differences were present, statistical analysis is required. Further, consistent indication of variability (such as standard deviation) is relevant. Using the questions posed in the introduction, perform specific statistical assessments on your data for comparison. This will strengthen any conclusions you have to make.
Validity of the findings: The observations of the study are relatively novel. Similar results have been observed from trotting in horses, dogs, goats, and other animals. However, the apparent structure of the distal limb (foot) lends import to the observations. A more detailed comparison with previous literature on quadrupeds would strengthen the findings. In terms of whether energy savings are occurring, understanding function of the proximal joints, and the general metabolic cost of transport will help to provide relevance. One other consideration is that previous work has found that the forelimb acts more for support and less for propulsion during locomotion (Dutto, et al 2006, Payne et al, 2005). I suspect that this is similar for the reindeer. The relative energy absorption of the MCP is helping to control the vertical motion. Combining these results with what is occurring mechanistically in the hindlimb, will give a better overall picture of the changes associated with gait and how similar or dissimilar these animals are from other ungulates.
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: FORELIMB JOINTS CONTRIBUTE TO LOCOMOTOR PERFORMANCE IN REINDEER (RANGIFER TARANDUS) BY MAINTAINING STABILITY AND STORING ENERGY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The grammar and writing are acceptable. There are a few instances where corrections are advisable and these are noted in my comments. The authors have met criteria for references, background, professional article structure, figures and tables. They have also provided the raw data. The results are self-contained and relevant to the introduction in the article. I would only ask that they make their hypotheses or expectations explicit in the introduction (though they somewhat implicit; see General Comments).
Experimental design: This is original primary research that is within the aims and scope of the journal. The research questions are somewhat well defined (though I would like to see explicit hypotheses/expectations; see General Comments), relevant and meaningful. The authors state how this study fills a gap in the literature. The authors provided a rigorous investigation of reindeer forelimb mechanics using inverse dynamics and appear to have met institutional ethical requirements. The methods have been described sufficiently to allow for replication.
Validity of the findings: The findings in this study are valid, robust, and are sound. The authors used statistics but did not indicate what tests were used (see General Comments). The authors conclusions are well stated, though not linked to well defined hypotheses/expectations setup in the introduction. There are a few instances of speculation that need to be identified as the authors novel hypothesis or require elaboration (see General Comments).
Additional comments: Overall, this is a well done study of forelimb mechanics in reindeer. However, I do have some questions and suggestions:

I think the title needs to be more specific and relay the what the authors did in the study or what their findings were. The current title ‘Forelimb joints contributing to locomotor performance in reindeer’ is a statement that could be made without the contents of this manuscript and should be ‘Forelimb joints contribute to locomotor performance in reindeer’. In what way do they contribute?

The abstract would benefit from a conclusion statement or discussion point. This is just a summary of methods and results.

The introduction is comprehensive, but it took a long time to get to your question. I think a statement of the question/problem early on would help frame the introduction and background necessary to understand what we know and what we don’t know.

Did you collect data from the female reindeer and then exclude them because they showed differences compared to males or did you not collect data on them? Are there expectations that female reindeer forelimb mechanics will differ from males other than body size effects?

Is there a reason why you didn’t calculate joint work for the shoulder to characterize joint work/power for the entire forelimb?

In Fig 9, the authors present statistical results, but don’t indicate what statistical test was used. Please put this in methods.

Overall, I think this paper would benefit from some predictions or hypotheses. The introduction is comprehensive and following a discussion of previous findings in horses and other ungulates, the authors could setup a few expectations that they could use as a comparison in the results or discussion section. This could just be a couple of paragraphs in total. As is, the manuscript is fine and provides a sufficient description of their findings. However, I think the overarching conclusion that reindeer use different strategies, presumably to moderate energy costs at different speeds (which should be the null hypothesis) needs to be expanded on a little bit. The headings the authors used in the discussion (e.g., ‘Wrist joints as a stabilizer and pusher’) would be perfectly suitable as predictions that could be setup in the introduction.

Figure 2B is a little challenging to interpret. It might be easier to follow if you labeled the circles in the figure so you don’t have to keep referencing the figure legend. One option is to put labels like FL (front left) in the circle.

Figure 8. I assume Walk is (A), Trot 1 is (B) and Trot 2 is (C), but these should be listed in the figure legend.

[LINE 111] The more appropriate term is ‘sex’ in reindeer. Gender refers to identity.

[LINE 139] How did you decide to separate trot speeds into 2 categories? Were speeds bimodal? Was it arbitrary?

[LINE 141] I suggest defining what a, b, c, d, and e represent or at least reference Figure 2A here

[LINE 162] In the sentence beginning ‘Relationship between GRF and time’ should begin as ‘The relationship between GRF and time’

[LINE 164] Please include vendor information for Origin

[LINE 166] This is the first time that I see that you have referred to the ‘toe joint’. Please define it here using terms you used previously or use terminology from Figure 2.

[LINE 173] “a positive joint moment supports the weight in the normal standing state” needs to be rephrased to be an accurate statement (and I think you want to say ‘body weight’ instead of ‘the weight). I think you’re just trying to suggest that in a lot of species, a positive joint moment as an extensor moment, but that is not the case for reindeer at every joint. I would also suggest that while Vereecke et al 2008 [36] is a fine paper, I’m not sure it’s the most appropriate for citing generalities about extensor moments. That said, I think it would be reasonable to just delete the sentence on line 173 and just state which joints have positive moments for extension and for flexion.

[LINE 205] The sentence beginning “The data of speed” should probably be “Speed data” or “Animal velocity”. Additionally, “normalized by the Froude number and the nondimensionalized speed u” should probably be “normalized by Froude number” since Froude number is a nondimensionalized speed. How did you measure speed? Did you use a joint marker? Was this a mean value from TD to LO?

[LINE 234] Wouldn’t you expect these variables to differ in a walk vs a trot and at different speeds? Is
this different than what you would predict based on other ungulates?

[LINE 312] “Reindeer have permanently enhanced MCP joints” – Relative to what?

[LINE 319] Do you present data on contact time to substantiate this?

[LINE 320] ‘…positive and negative works’ should be ‘positive and negative work’

[LINE 322] Does improved elastic energy storage mean increased?

[LINE 332] Is the hypothesis that increased ROM in reindeer toes is an ecological adaptation a result of the findings of this paper or an existing hypothesis?

[LINE 352] “…for stability and propulsion through other forms”. Can you elaborate on what you mean by forms?

[LINE 388-389] If the authors are going to say that these results underlie bionic foot design of extreme environment robots, I think you will need to elaborate on this. Or remove this statement.

[LINE 360, 366, 367] There are some unnecessary placements of “the” – ‘the foot’ ‘the horse knees’

[LINE 362] I’m not sure that you need to include a discussion of human foot mechanics. I think it would be fine to just discuss mechanics in horses. But, if the authors feel it is important, then they can keep it in.


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: FORELIMB JOINTS CONTRIBUTE TO LOCOMOTOR PERFORMANCE IN REINDEER (RANGIFER TARANDUS) BY MAINTAINING STABILITY AND STORING ENERGY
Review round: 2
Reviewer: 1

Output:
Basic reporting: There remain a few wording issues throughout the manuscript.

For example:

Line 99. I would change the question to something like "Does the work done by or on the forelimb change with speed?"

Line 319. A suggestion would be to change this line to "Locomotor strategies change depending on the speed and/or gait adopted by the animal." Trying to avoid using 'different' twice in the sentence. This appears a couple of other times in the manuscript. It is a small issue, but relates to the readability of the doc.

I would have still liked to see the same axis scale (maximum, minimum and intervals) used in Figures 6,7, and 8. This would allow for a more visual comparison of the presented data in addition to the interpretation as to the values.
Experimental design: Improved and sufficient.
Validity of the findings: Explanation is sufficient.
Additional comments: Thank you to the authors for the extensive revisions to the document. I appreciate the time and effort that are associated with doing these revisions.