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: EFFECTS OF THE LOSS OF ESTROGEN ON THE HEART’S HYPERTROPHIC RESPONSE TO CHRONIC LEFT VENTRICLE VOLUME OVERLOAD IN RATS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study and data are well presented.  Limitations are addressed by the authors.  Overall well written with sufficient literature review.  I have recommended that they include the effects of EST on cardiac remodeling in males as well, as those studies have been performed by others.
Experimental design: There are several limitations in the design, but the authors have addressed them in the discussion. Details of the ultrasound conditions should be included. I have included these in my comments below.
Validity of the findings: The authors do not overstate their conclusions and give a good discussion as to why their results in the AR model differ from published studies utilizing the ACF VO model.
Additional comments: Couet and colleagues present a study showing the effects of ovariectomy (OVX) and estrogen (EST) replacement on the cardiac function and remodeling response of wistar rats to chronic aortic regurgitation (AR).   The study and data are well presented.  Limitations are addressed by the authors.  They show that OVX reduced the hypertrophic response to AR resulting but did not lead to further dysfunction when compared to intact rats.  Estrogen replacement restored the hypertrophic response.  Measures of ECM, metabolism, and fetal reprogramming were normalized by EST treatment.

Comments:
Define CH in abstract.

Differences in AR severity between the EST treated study and the OVX study make it difficult to compare, but the authors acknowledge this as a limitation.

Has anyone noted a difference in remodeling response between Wistar and Sprague Dawley rats in response to AR of ACF VO?

For Echo method, include a brief description of the anesthetic/sedation and ultrasound equip used.

Table 2: Have others shown a decrease in BW in response to OVX in female wistar?  Were the animals pair fed?

Table 3: what EF do the Sham group have?  The FS is very high.  Also, the strongest effect on CO is due to OVX and not AR.  Do the authors have a theory as to why?  I do not believe that change occurs in Sprague Dawley females.

Fig 1 &2: recommend including x-axis bar labels in the top row as well.

Discussion page 8: define Ocx.

Est has been shown to reduce VO remodeling in male animals.  Authors should include that information.

Fig 4 &5: recommend using hashed bar for OVX (Fig 4) and E2 (Fig 5) groups to aid in gray scale reproduction.


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 DISTRIBUTION AND MITOCHONDRIAL GENOTYPE OF THE HYDROID AGLAOPHENIA LATECARINATA IS CORRELATED WITH ITS PELAGIC SARGASSUM SUBSTRATE TYPE IN THE TROPICAL AND SUBTROPICAL WESTERN ATLANTIC OCEAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language is clear and to the point.
The literature references are complete.
The new sequence data have been assigned Genbank accession numbers.
The voucher specimens, with DNA aliquots, have not been deposited in a museum where they could be accessed by future researchers. Nor is it indicated how these vouchers could be obtained. This situation should be rectified.
The article structure is clear and of professional standard.
The MS is a good publishable unit.
Experimental design: The work is original and entirely worth publishing. It will make a nice contribution from PeerJ. The MS provides evidence that population structure of a hydroid species that colonizes different Sargassum species/forms is likely explained by colonization of the different species/forms in different source regions, with only rare transfer from one Sargassum type to another. The picture that emerges fits in with limited prior information on larval settlement of hydroids. The results are relatively straight forward. The MS includes a quick phylogenetic analysis of mt16S sequences for various species of Aglaopheniidae. They identify a likely misidentification of one specimen/sequence from Brazil. The authors are careful to indicate that it might have been cryptic, but the zero length branch it forms with A. rhynchocarpa suggests a misidentification. For the phylogenetic analysis, the choice to remove all "regions that contained gaps"is probably more conservative than needed. Nonetheless, it is not likely to impact any of the results in a meaningful way. Aglaophenia will still be polyphyletic, but including more data might bring out the population level structuring a bit more in the hypothesized topology.
Validity of the findings: The MS is a well written description of an interesting piece of research. They conclude from the data they present that the settlement of one particular hydroid species associated with Sargassum likely occurs in type – specific Sargassum source regions, and that this can explain observed population structuring of the hydroid species.
Additional comments: It may seem obvious, but it could be nice if the authors were more explicit about how to specifically test their main conclusion. What do we know about the source regions of the Sargassum species/forms? Can one go to them and find and sample A. latecarinata? If these two species of Sargassum are holopelagic, how does A. latecarinata colonize them? From the benthos?


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 DISTRIBUTION AND MITOCHONDRIAL GENOTYPE OF THE HYDROID AGLAOPHENIA LATECARINATA IS CORRELATED WITH ITS PELAGIC SARGASSUM SUBSTRATE TYPE IN THE TROPICAL AND SUBTROPICAL WESTERN ATLANTIC OCEAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: See general comments to authors
Experimental design: See general comments to authors
Validity of the findings: See general comments to authors
Additional comments: The distribution and mitochondrial genotype of the hydroid Aglaophenia latecarinata is correlated with
its Sargassum substrate type in the Sargasso Sea

In this paper the authors produced ~140 sequences of the hydroid Aglaophenia latecarinata from two different forms of Sargassum. They use one single gene and produce what they call “a family level tree” using additional sequences from genbank.
My first concern with the paper is that the 16S gene is not suitable for family level phylogenies, and in fact has rarely (if never) been used with that intent. It is a good barcoding molecule (similar to COI for most organisms), and it does a pretty good job in identifying species boundaries, but it is not very useful for higher-level phylogenies. It is not very good for population studies as well.  The discussion about the fact that A. latecarinata falls outside the genus Aglaophenia, or the monophyly of the genus, is of little value, in my opinion (just look at the bootstrap values and at the posterior probabilities of most of the clades in the tree).
Other claims in the paper are not well substantiated. Line 251 to 254 authors say that their data confirm previously published work that shows A. latecarinata as the dominant hydroid on Sargassum. It is my understanding that the authors did not survey other hydroids, they just collected Aglaophenia. Thus it is not clear how they came to the conclusion that A. latecarinata is the dominant Hydrozoa.
So in summary, what the paper is showing is that using one single gene (which is not ideal for population analysis) there are soma haplotypes that are mainly found on one Sagassum type, and some that are found on the other. The finding is somewhat interesting, but limited in scope. I will let the editor decide whether this finding is sufficient to grant publication in PeerJ.
Other comments:
In the introduction it would be good to have an explicit question that the authors are trying to answer with this paper.
The discussion on the species-specific substrate pattern (starting at Line 261) does not take in consideration asexual reproduction (asexual pieces of the colony can attach themselves to the substrate). It should probably be mentioned in this context.
It would be useful to know (if possible) the substrate of other A. latecarinata in the phylogenetic tree (that were obtained from genbank) to expand the discussion a little.
Species names in the phylogenetic tree should be in italic
In abstract, A. latecarinata is misspelled (A. latecarina)


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

Input:
Title: A SYNOPSIS OF KNOWLEDGE, ZOOGEOGRAPHY AND AN ONLINE INTERACTIVE MAP OF BRAZILIAN MARINE GASTROTRICHS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: no comment
Validity of the findings: no comments
Additional comments: Gastrotricha is one of the less known invertebrate phyla. Even if representatives of this group are numerous and ubiquities in any aquatic and semi-aquatic environments, both marine and freshwater, there are treated truly neglected in meiofaunistic studies. Without a comprehensive, critical review of species distribution, the knowledge develop is about these animals is impossible.
I have the pleasure to review manuscript entitled: "Synopsis of the knowledge, zoogeography and an online interactive map of Brazilian marine gastrotrichs." This is good quality, a comprehensive, complete review of knowledge about Brazilian Gastrotricha. This work does not contain the only summary of knowledge about species distribution in Brazil but also contain new interactive, easy to use tools allowing for analyses each recorded species together with their habitus and details. I cannot found in the text any bigger mismatches. Taking into consideration all questions above-mentioned, I recommend this manuscript with great satisfaction for publication in PeerJ.
I attached to this review a pdf file with my suggestion marked. I have hope that their help in improving this extensive and very needed work. I am also waiting impatiently on the new gastrotrichs researchers who will start their adventure with these animals just from the studying attached interactive map and online tool.
I also attach my best regards for Editor and all Authors of this manuscript,
 Małgorzata Kolicka


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

Input:
Title: A SYNOPSIS OF KNOWLEDGE, ZOOGEOGRAPHY AND AN ONLINE INTERACTIVE MAP OF BRAZILIAN MARINE GASTROTRICHS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript represents a commendable effort to gather and take stock of the faunistic and taxonomic knowledge of marine species of Gastrotricha from Brazil, the study of which has only started systematically in the last few decades.
The study aims at giving an overall view of the diversity of the marine gastrotrich fauna from Brazilian coast, correlating it to the marine ecoregions of the area and discussing the geographical distribution of each species. The study also includes numerous undescribed species. To support their analysis, the authors present an on-line interactive map with a number of parameters related to each species.

-	The Introduction gives an overall picture of the research on marine Gastrotricha over the world, with particular reference to the knowledge of marine species from South America. The context of the work is well described and completed with adequate references.
-	The manuscript’s structure  conforms to PeerJ standards
-	The figures illustrate the text appropriately, are of high quality and adequately labelled.
-	Supplementary material include two detailed lists of marine gastrotrich records throughout Brazilian coast and the world, the first concerning the species formally described and the second one the species undescribed but reported in this analysis.
-	the English language is clear and generally applied correctly.
Experimental design: -	The subject matter falls within the scope of the journal
-	The topic of this study is clearly defined and really integrates the knowledge of gastrotrich fauna with data from Brazil
-	The analysis of data is complete and based on reliable techniques
Validity of the findings: -	Results of this analysis are very detailed and can provide a current picture of the diversity of marine Gastrotricha along Brazilian coasts within a context of global kowledge on this subject.
-	Records of all the species, both named and still unnamed, are listed into two separate lists and  include essential information
-	Conclusions adequately summarize the topics advanced in the Introduction and  covered in the text, and are sufficiently clear
Additional comments: 1.	I consider this work quite valid especially because it provides the first check-up of the marine gastrotrich fauna of Brazil and also  it proposes a interactive map as an useful, more general tool in research on gastrotrichs. That is a strength of this manuscript.

2.	The authors' effort to carry out a careful and detailed analysis of the Brazilian marine species and to outline a reliable picture of the current faunistic situation must be acknowledged: this is a strength of this article, too.

3.	I have some doubts about the extensive use of the unnamed species in the global list of species, even producing a special list for them.   I agree  that a global overview of marine gastrotrich fauna rests on the knowledge of all the species found so far, both named and unnamed, but that can only give a rough idea of the total species diversity and should be only a preliminary step, waiting for the formal description of the unnamed species. Otherwise, the unnamed species constitute a mass of partial information that can be managed only at genus level.
The literature on gastrotrichs is even too rich in formally named  species but incompletely described: if also unnamed species are added, from the taxonomic point of view a 'ballast' is created which is hard to manage in subsequent works and by other researchers.
The suggestion, indeed a reminder, is to describe these new species as soon as possible because they are useful now in the overall counts on the diversity and the distribution of the phylum in Brazil, but may constitute a non-irrelevant taxonomic problem  if they remain unnamed..
All that cannot be considered as a weakness of the manuscript but it is certainly an aspect to be considered and explained in the objectives of the work.

4.	While acknowledging the merit of the authors for having relaunched the research on the marine Gastrotrichs of Brazil, I find that the text in some points underlines this point in an excessive way, risking to seem self-congratulatory (see for example Lines 217-222).

5.	From the point of view of the formal style of the text I notice numerous inaccuracies and misprints both in the text, in the captions of the figures and in the supplementary material.  I corrected or highlighted a number of them for correction through small notes along the text. Among them is the unifomity of the style in reporting bibliographic references according to the journal’s rules.

Overall, in my opinion, the manuscript could be accepted with minor revision especially focused on two points:
1.	To eliminate inaccuracies of the current version:  I found a high number of misprints, oversights and also few errors. Authors should check more carefully the journal’s rules.
2.	to revise some passages of the text that in the current version have tones a bit too emphatic for what should be in a scientific work (I marked these passages with comment notes)

I attach a revised version of the manuscript with a number of corrections and notes, hoping it may be useful to Authors for improving this interesting 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: EFFECTS OF PARTICLE SIZE OF GROUND ALFALFA HAY ON CAECAL BACTERIA AND ARCHAEA POPULATIONS OF RABBITS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Reporting language is clear although English editing is encouraged. The type of corrections that should be made are minor but mandatory. Some examples of corrections I suggest are:

Line 9. “treatment groups” instead of “treatments group”.
Lines 27. “experienced” instead of “experience” and “archaea are” instead of “the archaea is”.
Line 33. “herbivore animal that has” instead of “herbivore animals that have”.
Line 41. “and so on” does not sound very scientific.
Line 63. “In addition, most” instead of “In addition, the most”
Line 67. “in rabbits exist” instead of “in rabbits exists”

More corrections of this type are needed in the rest of the manuscript so I reccomend careful English editing.

Line 75. Animal experimentation permission code should be included.

Table S1. I find the nutritional data redundant because percentages are identical in all four formulations but it is OK to leave it this way since it is supplementary.

Literature references are sufficient. The article is well structured and raw data es adequately shared.
Experimental design: I find the study of interest. The effect of particle size should be explored.
The experimental design is original and perhaps this data combined with previous results of other parameters within the same experiment would be more complete.
Methods. Animal and experiment sample collection. How was the intake of alfafa controlled? Was first feeding always consumed before second feeding was admisnistered? If not, how was second feeding administered? Were animals individually caged? How was slaughter performed? Anesthetics drugs and doses should be detailed.
DNA extraction. Raw data on DNA quality and concentration should be included as supplementary material (a table with the 120 values of concentration and 260/280 ratio) or at least in the results section DNA quality should be mentioned.
PCR amplification, Illumina library
Was control library spiked in amplicon pools in order to improve the unbalanced and biased base composition of the generated 16S rRNA libraries? If so, please report it.
Validity of the findings: Findings are interesting. I would like conclusions to be better fundamented. I don't understand why authors state that archaea are more sensitive to changes thatn bacteria. This is not the picture I get whean I look at Figures 5 and 6 I see more phylum variability among treatment groups in bacteria than in archaea. Am I missing something? Could it be better explained?

I would work on the figures as indicated below:

Figure 4. Results for effect of small particle on bacteria and large particles in archaea seem homogenous. The rest are disparate. This is not really discussed. The authors state that their PCoA analysis reveals that archaea are more sensitive than bacteria. From the PCoA I interpret that intersample variability is greater among archaea when fed with small size particles.

Figure 5. The legend is confusing because there are far more groups in the legend than in the bars. Please adapt.

Figure 6.The same  as Figure 5.
Additional comments: The study reported by Yuan and collaborators on the effect of particl size of ground alfafa hay on caecum bacteria and archaeas populations of rabbits shows interesting results. However, there is some information missing in methodology and conclusions should be better supported. Please address my concerns.


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

Input:
Title: EFFECTS OF PARTICLE SIZE OF GROUND ALFALFA HAY ON CAECAL BACTERIA AND ARCHAEA POPULATIONS OF RABBITS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript presents a novel data in relation to rabbit caecal microbiota (bacteria and archaea) and alfalfa particle size. The article is well structured with enough figures, tables and the raw data are shared. Although some parts are well written using clear and professional English, there are other parts in the manuscript with substantial English mistakes. Although I collect some of them, I would like to suggest to the authors to revise it thoroughly.

L42- “function in animal”, instead “animals”
L62-“specific studied on changes in microbial composition and structure is scarce” “specific studies on changes in microbial composition and structure are scarce”
L63-“the most” instead “most”
L81- “the animal was fed twice daily at 0900 and 01700” instead “the animals were fed twice daily at 09:00 and 17:00”.
L94- “the extracted DNA were detected” instead “was detected”
L99- “total DAN” instead total DNA”
L170- “In total of 2107 OTUs were shared in all of four treatments” instead “A total of 2107..”
L174- “In total of 2246 OTUs were assigned” instead “A total of 2246…”
L186-“bacterial composition is changed when..” instead “bacterial composition changed”
L191-“that the archael composition” instead “archaeal”
L215-“with relative abundance were larger than 1%” instead “with relative abundance larger than..”
L220-“with relative abundance was above 1%” instead “with relative abundance above..”
L244- “Table 2 showed” instead “Table 2 shows”.
L270: “Furthermore, bacteria belongs to the” instead “bacteria belonging to the..”
L279: “animals are related to the higher fiber content of food intake” instead “animals related to the..”
L334: “it was reportd that” instead “it was reported that”.

The cited references are accurate and a sufficient field background is provided. However, in the L31-33 the author state that rabbit meat is important in china. I would like to suggest to include some specific data about rabbit meat production or consumption to put in context the importance of this sector in some countries.

The presented Figures are relevant to the content of the article and appropriately described and labeled. However, since in the rarefaction curve (Figure 1) are 24 curves (samples) included, I would like to suggest representing the OTU mean of each group (10,100,1000,2500) with the SD or SEM of each group. In the Figure 2 there is typo in “OUTs”. It would be helpful also, have a better-quality Figure 6 to be able to read properly the taxonomic groups. In addition, in the Table 1, the used superscripts are confusing. In the Shannon value are some numbers with b superscript at different size and later is stated that the particle size of alfalfa meal was 2500, 1000, 100 and 10 μm, respectively. It is confusing. If the objective of the author is to indicate the significant differences I would suggest mark the p value with an asterix or use any number as superscript with further explanation. As in the Table 1, in the Table 2 the superscripts are very confusing as well. Superscript a and b are refereed to an item and treatments, but they are also used in the given numbers.
Experimental design: The used methods in this study are the appropriate ones to try to answer the stated hypothesis.  The animal experiment has been approved for an animal care committee. However, it would be convenient if the authors refer the specific number or reference of the approved protocol (L72).

In the Line 81 the author say that animals were fed twice daily and later (L83-84) is stated that animals had ad libitum access to the diet. It is confusing, and some clarification of what food was limited or ad libitum is required.
In addition, there is some missing information about experimental design like where the animals were housed (research facility or experimental farm), the sex of the animals, and how they were housed, by groups or individually (specially the animals that were sampled). The information of how animals were slaughtered and if all animals were slaughtered the same day of on groups in consecutive days is missing. Also, the sampling procedure is not thoroughly explained. Since this is a microbiota study it should be highlighted how the cross contamination was avoided and how a representative sample from the whole caecal content was obtained.

Regarding the sequencing I wonder if in the sequencing run any negative or positive control was included. The positive and negative controls help detecting cross contaminations during the library preparation or a quality control for the sequencing process.

Following the recommendations for 16SrRNA sequencing I assume that the authors did include PhiX control in the run. If so, it should be reflected in the manuscript along with the used concentration. In the materials and methods section, singeltons are defined as a “bad amplicons” (in the bioinformatic analysis section), while I would define them more as a sequencing errors as the reference provided.  Qiime version used should be stated as well as Silva database used to assign taxonomy. I addition, I wonder why the author used Mothur software to calculate diversity indexes if it can be done in Qiime. Is there any scientific reason?

In the statistical analysis the author state that they used MIXED procedure in R. It may be a mistake considering that in general mixed procedure is a SAS procedure rather than R (although it could be implemented within some R packages). Anyway, it should be indicated the specific R packaged used for data analysis. Also, the specific statistical analysis implemented for each data comparison (in R or SAS software) should be provided.

The author only presented the analysis of the weighted UniFrac distances that refers to a quantitative analysis. Were the unweighted UniFrac distances analyzed (qualitative analysis)? Is there any reason to do not include the analysis of unweighted UniFrac distances?  If they were analyzed, and no significant differences were detected it should be mention in the manuscript.
Regarding the PCoA representation of the UniFrac distances (Figure 4), I strongly encourage the authors to complement the analysis with a statistical analysis to test whether the observed differences in the plot (clusters) are statistically significant (for example using Permanova test).
Validity of the findings: This study collected a meaningful result. Although some details are lacking in the methodology the obtained data look robust. However, I would like to suggest to the authors avoid using the word “significant differences” (L187) in the results if a p value is not provided.
In addition, in the L-207 is stated that “the archaeal composition structure changed before the bacterial structure”. Since all animals are sampled at the same time the data are not providing enough information to make this statement and it should be removed.
In the manuscript there are some references to anaerobe instead archaea (L157, L194). Anaerobe refers to oxygen requirement and booth archaea and bacteria can be aerobic or anaerobic. Therefore I suggest to change anaerobe by archaea.

Conclusion are well stated and linked to the original research question. The authors attempt to describe the impact of fiber size on the caecal microbiota. However, this manuscript is a complementary study (same experiment) to a previously published study, assessing production performance, digestibility and methane emissions. (Liu, S.et al. 2018. PloS one). Therefore, the authors should take advantage of the available data (although previously published) and link the microbiota results with the production performance (there is only a small reference to methane production in the discussion).

In the discussion there is no any reference to the effect or particle size in microbial diversity. Although generally high diversity has been claimed as beneficial, in the case of ruminants, lower richness of microbiome gene content and taxa was tightly linked to higher feed efficiency (Shabat, et al.The ISME journal 10.12 (2016): 2958). In the present study there are significant differences in the Shanon index between treatments and the authors have published the productive data. Therefore add this point to the discussion would enrich the manuscript content.

In the L-282 states that Proteobacteria “exist as the dominant bacteria of some animals”, please provide a reference.

In the discussion is stated that (L294) “it is speculated that the increased relative abundance of Tenericutes may be attributed by the ultrafine smashing of alfalfa”. I am lacking the information of its impact in rabbit’s performance (it is good or not?) and if there is more literature in this regard.
Lactobacillus is generally present in low abundance in the cecum of rabbits, and the factors affecting the colonization has been studied (ie. Yu, B., et al. 1993. Journal of applied bacteriology, 75(3), 269-275). In the present study data suggest that small fiber particle size could increase its presence. How could the particle size enhance lactobacillus colonization?  Is the presence of Lactobacillus associated with better growth parameters?

I found difficult to understand the following sentences (L209-211):  “ Species annotation and statistics were performed on different taxonomic levels using the effective sequences obtained (Fig. 5). The results showed that the sequences were finally annotated as bacteria (99.87%) and archaea (0.13%)”.
I would like to suggest to the authors to express it in a different way taking into account that the sequencing process is carried out after 16S rRNA amplification (PCR) and is difficult to extrapolate the amount of sequences obtained in bacteria and archaea if they have been amplified (before sequencing) with different primers.
In addition I found difficult to understand L226-230. The author should rephrase it.

Regarding the conclusion section, most of its content is a summary of the obtained results and it should be reflected at the beginning of the paragraph (Summarizing,…..) to do not confuse the readers.

In addition, I would like to suggest to the authors to include in the last part of the conclusion what would be the benefit of manipulate the gastrointestinal microbial population by feeds processing technology (Improve animal health, growing performance, increase farms profit, decrease meat price, etc...).
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: EFFECTS OF PARTICLE SIZE OF GROUND ALFALFA HAY ON CAECAL BACTERIA AND ARCHAEA POPULATIONS OF RABBITS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript submitted by Yuan and coworkers describes the characterization of rabbit cecal microbial communities in response to different particle sizes in their diets using Illumina amplicon sequencing. The authors show that a reduction in particle size leads to changes in microbial community composition.

My main concern is that is unclear how much this study overlaps with another recent study from the same lab: Liu, et al, 2018 PlosOne (as stated in their submission). The authors need to clearly highlight and state potential overlaps and differences between the 2 studies which obviusly seem to have used the same animal trial (and sequencing samples?). Otherwise, I fear double-publication of the same data, without clarification of this issue. The authors should either refer to and discuss their current resutls with those from the 2018 PlosOne paper, or mention why there is no overlap between the 2 studies. Specifcally, are these e.g. the same sequencing data? On the other hand, if these two studies are sufficiently different from eah other, the authors should discuss the results e.g. from methane production in the currently submitted manuscript.

The authors should improve English usage throughout the manuscript. There are a number of minor English errors in grammar (e.g. singular/plural - e.g.: animal-animals, rabbit-rabbits, microoganism-microorganisms, genus-genera). I suggest that the authors have a more experienced English speaker help with English usage.

I would like to encourage the authors to provide more details about the rabbit microbiota in general and rabbit microbiome studies in the introduction section.

It seems that the raw sequencing data have not been submitted to NCBI SRA, in case the data have not been submitted, please submit the sequencing data to NCBI SRA.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Specific comments:
L3: should be "communities"
L33-34: Plese reword and use either singular or plural consistently.
L34-41: Please mention that rabbits are hindgut fermenters.
L57: Please change "Flexibacter-Cytophaga-Bacteroidetes" to: "Bacteroidetes"
L58: Should be "albus"
L99: Should be: "16S rRNA gene", and "DNA"
L113: please change "strip" to "band"
L124-146: Did the authors perform a chimera check?
L133: should be "lead"
L157: Please clarify: do the authors refer to archaea with "anaerobe"?
L158 (and Table 1): Were the samples normalized to the same number of reads for alpha diversity estimations?
L180: Please rephrase this sentence, it is unclear.
L185: Please rephrase "crossed"
L228: Please change "left" to: "remaining 10 were". Should be: "The number of genera with relative..."
L260: Please rephrase "attempted"
L270: Should be "belonging"
L277: please rephrase "community" - Bacteroidetes was e.g. the second most abundant phylum or group. L278-280: Indeed Bacteroidetes is among the most abundant bacterial phyla in animal microbiota (please rephrase "suggested". Please provide some references here. I suggest that the authors expand their discussion here and discuss a potential role of Bacteroidales S24-7/Mutibaculaeae phylotypes here. See e.g. Lagkouvardos et al., 2019 "Sequence and cultivation study of Muribaculaceae reveals novel species, host preference, and functional potential of this yet undescribed family" or Ormerod et al., 2016: "Genomic characterization of the uncultured Bacteroidales family S24-7 inhabiting the guts of homeothermic animals" for more details (please see also L300-302).
L281-282: Please delete "as gram-negative bacteria"
L282: Please reword "dominating" here
L284-286: Were any proteobacterial genera significantly increased in abundance in response to decrease particle size? If so, please briefly discuss.
L288: Please rephrase "declaim"
L307 (also L316): Please rephrase "probiotic" - in this case, Lactobacillus was not added as a probiotic to the diet, I assume. Maybe "potential beneficial"?
L352-356: Please clarify: what do you mean by "limitations" - a reduced/lower methane production? Please disucss the increase of Methanosphaera in more detail. Do the authors claim that an increase of Methanospaera (and a decrease in Methanobrevibacter) would be beneficial because of lower methane production levels?

Fig. 1: I suggest to move Fig. 1 to the supplementary material
Fig. 2: (Figure legend) please correct "OUTs"
Fig. 5: I suggest to either delete panel B or move it to the supplementary material. Because of the dominance of just one phylum, I think this panel is not very informative. Should be "communities"
Fig. 6: Should be "ceacal" and "communities"

Table 2: The title should read: Phyla and genera with different abundance in ceaca of rabbits fed diets with different particle sizes of.... Please clarify: What is shown in the "treatments" columns - mean/median relative abundances?


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: EFFECTS OF PARTICLE SIZE OF GROUND ALFALFA HAY ON CAECAL BACTERIA AND ARCHAEA POPULATIONS OF RABBITS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Reporting language is clear although English editing in the new information that has been added, is encouraged. Authors have correctly addressed concerns. However, minor corrections should be incorporated prior to definetly accepting the paper. These are mainly in the new modified parts of the manuscript and are the following:

Lines 102-103	Rephrase “Once slaughtered, bring sterile gloves to collect cecal 50 g contents to sterile test tubes.” to “Once slaughtered, 50 g of cecal content was collected in sterile conditions.”

Line 112. Eliminate simple form the word buffer. “was eluted from the elution column with TE buﬀer”

Lines 317-18. Verb not in correct tense and author’s name in reference is incorrect.  Please change “There are also studies showed that high diversity of microorganisms was beneficial to animals (Aeeazaria et al., 2016).” to “There are also studies showing that high diversity of microorganisms is beneficial to animals (Arrazuria et al., 2016).

Lines 364-365 Change “It is further confirmed the important role of Bacteroidetes in the cecum of rabbits.” to “This further confirms the important role of Bacteroidetes in the cecum of rabbits.”

Lines 440-441 I suggest changing “However, the archaea is changed earlier than bacteria as alfalfa meal particle size decreased. To “However, these changes in the archaea are produced earlier in time”
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: EFFECTS OF PARTICLE SIZE OF GROUND ALFALFA HAY ON CAECAL BACTERIA AND ARCHAEA POPULATIONS OF RABBITS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript has been improved substantially. However, still some English editions are needed because there are some sentences like “archaea responsed earlier”, “There are also studies showed”, “However, the archaea is changed earlier than bacteria as” etc..

In addition, I would like to suggest to the author separate the sentence in L 66-71 in two sentences, because that long sentence it is difficult to understand. Also, the ADG and ADFI acronyms should be defined in the text.
Experimental design: The author included information about the sex of the animals included in the whole experiment. But it is not clear if it was a sex-selection made for sampling. If the same number of samples from male and females were used for sequencing was there any difference in the microbiota based on the gender that could impact the obtained results?
Have the authors showed any “cage effect” in the microbiota? It has been described in mice and it would be interesting to know at what extent this factor affects rabbits microbiota.
In any case, sex and cage effects should be mention in the discussion, especially when the results are compared with previous studies.

The authors introduce in L 133 that they used emulsion PCR. What was the goal of that PCR? Could you provide more details about it? Afterward, was the electrophoretic band of the emulsion PCR purified?

In addition in the L138, is stated that  TruSeq DNA PCR-Free Sample Prep Kit following manufacturer’s recommendations was used for library preparation. However Illumina recommend this kit for whole genome sequencing and no for amplicon sequencing. What was the reason for using this library preparation kit? It has been previously validated for amplicon sequencing?

https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-dna-pcr-free.html

As in the first review, I would like to suggest again change the term “bad amplicon” referring to the singletons for “sequencing errors” or at least if the author does not consider it is appropriated it should be explained why is considered “bad amplicon”.

Although the formula of the MIXED procedure of SAS has been included, I Would like to suggest to the author to be more specific about the R packages, comparison test and multiple comparisons corrections used for ALL data analysis.

I would like to suggest to the author to include the statistical differences sowed by permanova analysis in the PCoA figure and/or include in the supplementary table 2 what are the analyzed data.
For a complete analysis of the UniFrac distances the weighted as well the unweighted distances should be analyzed in order to understand if the differences are only quantitative or both qualitative and quantitative. I would like to suggest again to include both measurements in order to complement the UniFract distances analysis.

L236-241 I would suggest rephrasing this sentences taking into account that the PCoA does not demonstrate significant differences, they are obtained by Permanova analysis taken all samples. It seems confusing in the text (specially the exceptions).
Validity of the findings: As I stated before the cage effect as well the sex effect could have an impact on the obtained results and they should be mention in the discussion and taken into account when comparing the obtained results with previous studies.
In addition the used coccidiostatic could have an impact on the observed differences with a previous study (stated in L352) and it should be mention.
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: COMBINING SIMULATION MODELING AND STABLE ISOTOPE ANALYSES TO RECONSTRUCT THE LAST KNOWN MOVEMENTS OF ONE OF NATURE’S GIANTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The basic concept is great for this study - a valuable sample and dataset - and a creative combination of tools (simulating expected isotopic patterns) to then attempt and recreate movement for the animal over many years. And with some improved clarification and explanation this will be a great manuscript. Yet in it's current form, it is not yet ready for publication, as far too many assumptions are not fully supported (or clarified) and throughout the paper, there are many inconsistencies that bring up questions about the experimental design. I do not expect a significant amount of work needs to be done in altering the study nor analysis, but rather more care needs to be given to fully and clearly convey to the reader that what has been done is sound in its design.
Experimental design: As noted in the above section, further clarification on assumptions and decisions made will greatly improve this manuscript. To help, many specific suggestions and points are detailed below. But this is not an exhaustive list. Notes and suggestions below should be considered and incorporated throughout the manuscript.

The primary sections needing attention are those related to the baleen stable isotope profiles and assumptions based on these related analyses and its application to the modeled movements. The sections relating to the agent-based movement model, while more complex, are better explained, but still require some attention. As is, the methods are not sufficiently described nor justified to support the assumptions made.

Further details provided in the next section.
Validity of the findings: With regards to the isotope profiles, the following details and notes require attention:

L178 – adjust wording to state that it was assumed that growth of the baleen was constant; the 1 citation is good to have, but this is still an assumption.

L197 – before the behavioral phases are used, as done in this line for the first time, much more explanation on what these phases are, and how they were characterized and divided is needed. It is confusing to the reader to drop this note of “behavioral phase two”, without any prior mention, introduction, of discussion of this idea. This is just one of several parts where this idea is mentioned without the full clarification it deserves. This is also a good example of where the organization of the paper needs some improvement.

L198 – similarly, more clarity and description needs to be provided on how the isotopic periodicity was identified and assigned. It is mentioned here, and references Fig S1 (which also needs improvements for clarity –see below), but without full details. It is in a much later section where the R package and details are provided – all the info on periodicity assignment should be made together. Further, it is never made clear on how the isotope data was used in the Fourier Transformation – was it just the d15N data? Just the d13C data? Both together? And critically – WHY the N or C or N+C was used needs to be explained clearly to the reader.
L346 – again, clarify what data this “strong periodic repetition” was based upon. C? N? C + N? Why?

Related to this, additional detail and clarification would greatly improve Figure S1. As is, it appears that the provided inset values presumably show the periodicity for the different analyses (N full, N phase 2, C full, C phase 2) – but this brings up more questions on why the 13.5cm = 1 year was then selected as the defining periodicity for the rest of the assumptions in the manuscript.
These comments are not to question this assumption or reasoning, but rather to emphasis that further clarification should be provided such that these assumptions and justifications are fully understood by the readers.

Additional notes on Fig. S1 – can a more detailed title for the x-axis be provided? Or at least a description given relating the “Frequency” to cm in the baleen.
Also – why is Phase 1 not shown as a stand-alone, but Phase 2 is? And what about the last section of the baleen (most recent) that is described as outside of Phase 2? As is, this figure is confusing.

L358 – To improve clarity, suggest change the wording of “84 cm from the distal end” to be consistent with the labeling of the data previously described and also presented in Fig 1. It is labeled on the x-axis as “cm from youngest sample”, so references to specific positions in this timeline should follow this direction. --> “26 cm from the gingival end” or “from newest”, or similar. Just needs to be consistent.

Related to the baseline isotope comparisons, further detail should be provided to better support how temporal isotopic fluctuation was accounted for. A primary reason why it has been difficult to model oceanographic isotopic patterns over space and time is because of the highly dynamic nature of this environment and our limited ability to capture the full spectrum of samples/observations to support the scale of data necessary to make these predictions or recreate conditions. The approach here in this paper is a good one, but requires that additional clarification and justification be provided.
For instance, L233-229 needs more detailed explanation on the temporal aspect. Why was the decadal data forced? How, specifically, did the forcing change at given latitudes and why? How was seasonality incorporated? Were time lags considered between physical conditions and biological responses? Why/why not? Etc.
This part is also partially touched upon in ~L248-258; but requires additional explanation into the rational for some key assumptions and additional citations would strengthen these positions as well. i.e. – please provide a citations for the 2-4 month turnover within krill (L256); citation for keratin (L250).

Another main point that needs clarification and improvements for consistency throughout is with regards to the terms being used to categorize the different geographic regions and associated whale behavior/foraging location. At L323-330, when the 3 modeled movement behaviors are described, the later discussions and result analyses would be improved if specific terms were defined here, and used consistently throughout – including with associated figures. Further, in strategy (a) where the residency regions are described, again, key names/terms should be assigned and then used consistently throughout and in figures. For example, Figure S4 shows 5 regions, which don’t exactly match with all the terms used in L323-326; nor in L383-390 (here, each area should be specifically listed out instead of just saying “around the British Isles” or “sub-tropical regions”, etc.)
Further examples -  L401-410, and throughout most of this section where the observed baleen isotopes are compared to the model.

Related to the interpretation and assumptions made – a key point that needs to be clarified is in Fig. 3, why are the d13C scales so different? It is made clear why the absolute d13C permil values are not expected to be the same, this argument was presented well; however this mismatch in scale shown in Fig. 3 is large and requires further explanation.

Related to supporting the assumptions made – suggest presenting the permil amount of amplitudes observed when discussing the modeled movement, as well as the observed isotope patterns. (i.e. L393). This will help readers better appreciate the relative fluctuation each environment/region is expected to experience.

Fig 1 – Suggest marking the “phase” boundaries on the figure. Also, it seems that the d13C and d15N values appear negatively correlated, even in Phase 1. Please explain why this was stated as not being the case.

The rest of the assessments, comparing the baleen isotope values and the modeled movement, follow well and will be better supported with the additional clarification and justification in the earlier sections. The suggested adjustments for consistency and organization outlined above, when made throughout, will greatly improve this manuscript and make it ready for publication.

Misc. suggested edits –
L289 – suggest changing the word “Directly” to “Here,” or “Meaning”, or similar.
L296-299 – this same information was just stated above. Suggest leaving it here and deleting it in the earlier occurrence.
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: COMBINING SIMULATION MODELING AND STABLE ISOTOPE ANALYSES TO RECONSTRUCT THE LAST KNOWN MOVEMENTS OF ONE OF NATURE’S GIANTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Manuscript is clear and concise. Introduction provides background to set research objective into perspective. The modeling component of the methods was a bit difficult to follow. There are a lot of pieces and at times it was difficult to see how they all fit together. How do the authors combine whale isotopes, agent based model, and comparison to the d13C isoscape? For me this only became clear from reviewing the provided code. I commend authors on extensive supplemental materials, easy access to data, and transparent code.

243-245: “Confident” seems like a very strong word when assuming a relative distribution for an isoscape hasn’t changed in the last century. Historical work is difficult and always makes a lot of assumptions; however,“Confident” in a scientific publication implies a degree of certainty generally imparted from statistics. I would recommend making a stronger case for this claim based on literature or clearly noting it as an assumption.

248-251: The assumption of reflecting foraging in the “weeks” prior seems like a best guess. Claim as such or provide literature to support.

253-257: The estimated completion of turn over seems again like a best guess. Please support or claim as such. “Estimate” makes the statement seem based on something concrete.

260-264: Unclear how data is being transformed with respect to trophic level between the isoscape and baleen.

265-267: I think authors go from talking about krill to killer whales? Please clarify.

454-457: Unclear clear how statements on sexual maturity relate to interpretation of data for readers not familiar with blue whale natural history

491-492: Stocks of what were so depleted? Fish? Moved outside of what area?

I recommend using either Hope or NHM blue whale as consistently as possible.
Experimental design: I will qualify that I am not well suited to judge the adequacy of the design in relation to others in the literature. However, my concerns are as follows:

1)	The model appears to have been designed backwards based on the data. The data is split into three time periods based on how the authors interpret seeming changes in the isotopic profile. This adds a subjective layer to the analysis. If the goal is to describe what this one whale did then perhaps it is acceptable however, if the goal is to extrapolate to the population these determinations are problematic. It is unclear how a researcher who wanted to use a similar technique should go about assess isotopic profiles for a larger samples size.
2)	More explanation is need with respect to how the growth rate is determined. It is unclear as to why d15N values cycle annually. Additionally, given d15N is variable across the North Atlantic how is this not confounded by location especially if the whale was migratory for much of her life.
3)	More justification is need for why an agent based model which makes many assumption about movement is acceptable in an analysis to determine the whale’s movement. Analysis seems circular. Additionally, how sensitive is the analysis on changes to parameters in the agent-based model?
Validity of the findings: 1)	I greatly respect the authors’ efforts in attempt to connect isotope data to real world animal behavior such as movement patterns. I think too often practitioners of stable isotopes cannot adequately describe what variation along their isotope axes mean ecologically. Trueman et al. provide potentially strong methods as to how isotope data may be put into a modeling framework and may be useful for specific hypothesis testing scenarios. However, I did not review this manuscript as a methods paper and would not be strongly qualified to do so. Rather I reviewed the paper based on the ability to achieve the stated objective in lines 83-87: “Here we illustrate the potential of this conceptual approach by assessing the relative likelihood of differing, plausible hypotheses of movement behavior for a single individual blue whale based on carbon isotope compositions sampled across a baleen plate” as well as the title “Reconstructing the last known movements of one of Nature's giants”. Based on this, it is I find that the authors did not achieve the stated objective nor were the movements of the blue whale in question reconstructed. In science hypotheses must be falsifiable. For the last period of the whale’s life the observed pattern of isotope values could not be simulated. It did not match the pattern that would be expected for low latitude residency, yet the authors interpret the pattern as low latitude residency and explain the anomaly as a possible pregnancy. While based on my experience in stable isotope ecology, I think this sounds plausible, the fact remains that when data was encounter that did not support the hypothesis that the whale had an extended low latitude residency, the hypothesis was not rejected. Thus, it fails to be falsifiable. Additionally, it leaves me asking: under what scenario would the authors have deemed this conceptual approach incapable of assessing the relative likelihood of differing, plausible hypotheses of movement behavior?
2)	Trueman et al. is attempting something extremely difficult as it relies on biological processes which span all the way from the incorporation of carbon into the biomass of primary production at the base of the food web to the synthesis of keratin by literally the largest known animal to have ever existed. These are complex processes and variation is inherent. For this reason, the model must stack uncertainty upon uncertainty: how accurate is the isoscape?  how accurate is the agent-based model? How accurate is the estimated rate of keratin growth? And finally, how sensitive are results to variation in all of these underlying estimates? As none of these are discussed it is really difficult to interpret how potentially useful this work is.

Along these lines:
Authors claim the Suess effect and trophic fractionation do not impact results as they only use the change in d13C not the absolute value. However, this is only true if we assume the Suess effect and trophic fraction are uniformly impacting the entire study region. The Suess effect impacts low latitudes far more than high latitudes given the much larger pool of dissolved CO2 in cold water. Additionally, trophic enrichment may be variable based on the growth rate of the organism (among other things) which may be related to productivity. I agree that these are unlikely to impact results however I think assumptions (especially for modeling heavy papers) should always be transparent.

The modeled and observed data for Magozzi model are most desperate off the west coast of northern Africa and in polar regions (but in opposite directions) given that these are both important locations for blue whale how might that impact the authors’ ability to model movement?

Lines 505-509: How can you make inferences about what is common in modern blue whales from a single historical sample? This idea could be flush out a bit more and should be noted as a speculation or prediction.

Lastly, in lines 528 to 532 when authors claim: “Our results confirm that sequential sampling of stable isotope compositions in whale baleen, combined with simulation modeling can yield plausible inferences of individual whale movements that are consistent with assumed movement behaviours.” This is only true if the large number of assumptions inherent in their analysis are also true. Authors need to be more explicit about assumptions and potential violations.
Additional comments: I commend the authors for this ambitious analysis. I appreciate the amount of time and effort that was clearly placed into this complex analysis. I struggled as to how to interpret the paper, as either methods paper or scholarly research. It didn’t seem like a methods paper as the focus was clearly the blue whale and there wasn’t any sort of model validation. If the authors deem the methods in the paper sufficiently novel, I would encourage them to focus more on the methods and use the whale as a proof of concept. As it stands, the samples size of one makes this a case study not a research article. Given the detailed code the authors make available I would encourage them to really think about the circumstances in which a potential researcher should or should not use these methods. As I am sure authors are aware new quantitative techniques are emerging using stable isotopes that have awesome potential to answer novel questions and provide estimates of things like diet that were previously only obtainable through substantial effort. However, given the nuance of isotope analysis and propensity for researchers to preform analysis they don’t completely understand I would encourage authors to provide some guidance as to the circumstances in which this sort of analysis would or would not be appropriate.

A few additional comments:

Please provide any additional details as to the circumstances of the stranding, could the whale have been sick/injured etc? The details of the stranding could inform interpretation of last behavioral phase.

187: Remove per mil from C:N ratios

Was the number of simulations limited due to computing constraints? Perhaps justify the number of simulations. 1059 doesn’t seem like a large number given the potential number location histories.

See other line item corrections in attached PDF

Sincerely,
Sam Rossman


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: COMBINING SIMULATION MODELING AND STABLE ISOTOPE ANALYSES TO RECONSTRUCT THE LAST KNOWN MOVEMENTS OF ONE OF NATURE’S GIANTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The re-framing of this MS made a significant improvement. It is now not only a more accurately focused paper, but it is now an extremely useful guide for future studies that hope to utilize SI data and model simulations to infer movement and behavior of migratory marine animals.

Other incrementally grown tissues that are also comparable to this type of study include teeth and bones (i.e. long bones such as humeri); if not directly discussed, both "teeth and bones" should be added to Line 615 at the end.
Experimental design: Good adjustments and added clarifications. The new set up which presents a binary test of expected behavior and associated SI pattern (residency vs seasonal migration) makes this a much more focused study with a clearly testable and realistic hypothesis. The more simplistic approach taken by limiting model complexity is a great adjustment.
Validity of the findings: Much improved after more clearly defining and supporting the assumptions made and process applied.
Additional comments: Overall - the adjustments made greatly improved this MS. Specific line edits/comments are listed below - and the only main overarching comment is to given attention in the Results and Discussion section to clarify, at a final level, the 3 behavioral phases that are identified in the baleen sample and then compared to the simulated results. There is a bit of jumping around during the Results and Discussion section that makes it a bit disjointed, but with a bit of additional clarifying wording, this will be easily resolved. You'll see this theme in many of the comments below.
Again - great refocusing, and this will be a great addition to the spatial / behaviourial stable isotope literature and field.

Misc. suggested edits –
L 25 – should be “relative” not “relatively”
 L 446 - Suggest adding one line at the start of this paragraph to describe what is being done here. i.e. “For each of the three behavioral phases identified within the baleen plate, the modeled simulation results were compared.” (or similar)
 L 457 – add “likely”; “…the observed low δ^13C values likely imply foraging in colder, more northerly latitudes…”
L 460 – to further clarify the connection between these two paragraphs, suggest adding this phasing to the start of L460, “To further examine this, we modeled…
L 461 – deleted the repeated phrase “compared the”
L 480 – this section seems to move through the initial assessment of phase 1 and 2 – but it seems odd that the Discussion section begins without mention of phase 3. Suggest adding a similar descriptions/assessment of phase 3 here, before moving on to the Discussion section.
Adjust the Discussion, then, if/as necessary to eliminate any repetitiveness.
L 494 – says “two distinct phases” – but wasn’t this adjusted to 3 phases? Clarify.
L 507 – this is now describing “phase 3” right? If so, state this here to clarify.
L 528- Here, and overall, there needs to be adjustments made to make the comparisons and discussion of the 3 behavior phrases consistent throughout. Suggest making it explicitly clear to the reader which phase is being discussed/mentioned each time.
i.e. “In summary, our combined isotopic measurements and simulations allow us to propose a movement history for the last 5-6 years of Hope’s life. Behavioural phase one lasting for at least a year most likely reflects residency in sub-tropical waters, potentially in the Cape Verde, Canary current region. Subsequently, behavioural phase two, we infer that Hope conducted three uninterrupted annual latitudinal migrations, wintering in sub-tropical waters and moving to sub-arctic waters during late spring and summer. Finally, in behavioural phase three, we tentatively suggest that Hope may have given birth in the winter of 1889/1890….” Or similar.
L 552 – add a comma after “However”
L 615 – should add teeth and bones (or long bones) to this list


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: COMBINING SIMULATION MODELING AND STABLE ISOTOPE ANALYSES TO RECONSTRUCT THE LAST KNOWN MOVEMENTS OF ONE OF NATURE’S GIANTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Very professional and concise. Flow of ideas is easy to follow and logical. I think the headings provide a useful guide for readers and aid in the clarity of the manuscript.
Experimental design: The details relating to the exp. design, assumptions and limitations of the work are now well described. It's not that I thought the assumptions in the previous  version were unreasonable, I just felt that the needed to be more explicitly stated with the possible implications. Authors do a good job of this in the current version.

In my previous review my recommendation of reject was not really based on the merit, usefulness or quality of the research but rather that I felt it was mostly a case study which is not within the scope of the journal. I think author's have sufficiently reframed the paper with a more methodological focus.
Validity of the findings: The authors do a much better job in this version of separating the which parts of the results are well supported by their analysis and which are more speculative. While the results of this case study are not, by themselves, a huge contribution to the literature given the single individual it is a valuable approach which if adopted by other marine mammologists has to potentially to shed light on threatened and hard-to-study populations.
Additional comments: I commend the authors for a dramatically improved manuscript. I felt the response to previous comments were thoughtful and well articulated.  I feel the manuscript is now very clear, and much easier to follow. In it's current form I believe the manuscript will be a far more useful contribution to the literature as the accessibility of the research has been dramatically improved for researchers less familiar with these sorts of models. Additionally, framing the paper around evaluating support for specific hypothesis turns the focus of the work from something I think is mostly impossible (constructing fairly specific movement patterns) to something useful and very applicable to other organisms.


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 PHYLOGENY OF DESMOSTYLIANS REVISITED: PROPOSAL OF NEW CLADES BASED ON ROBUST PHYLOGENETIC HYPOTHESES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: This study is a welcome and well-executed reevaluation of desmostylian relationships and interrelationships.It usefully incorporates the character sets used by earlier workers, and advances our understanding of the group. It is also well-written, with only very minor corrections of the English needed.

I have made numerous editorial corrections and suggestions on the marked-up copy of the MS. that is attached here, which is to be shared with the authors. These include questions about the definitions of some of the characters used, and some matters of nomenclature (such as the authorship of "Desmostyloidea"). In general, I am not a fan of cladistic classification, or unranked classifications; however, the authors are certainly at liberty to use these procedures.

My most substantive criticism of this MS. is that many of the character-state definitions need more work: specifically, expressions like "smaller than" vs. "much smaller than"; "short" vs. "elongate"; etc. The authors should imagine themselves in the position of an investigator becoming acquainted with desmostylians for the first time, and trying to score a new specimen using definitions like these. They should reconsider all the definitions in the Character List, and expand on such definitions using ratios or other quantifications, positions of a feature relative to other features of a bone, or even illustrations to clarify the distinctions between character states.


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 PHYLOGENY OF DESMOSTYLIANS REVISITED: PROPOSAL OF NEW CLADES BASED ON ROBUST PHYLOGENETIC HYPOTHESES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The usage of English throughout is fairly consistent, though there are several minor typos that should be correctly to improve clarity. These appear to be errors that might have persisted because of errors in the Word dictionary, as they are repeated throughout and often are proper nouns. They include:
Vonderhoofius - should be Vanderhoofius
canie - should be canine
prpteus - should be proteus (this is found in Figure 2)
emlongis - should be emlongi (this is found in Figure 2)
repeninngi - should be repenningi (this is found in Figure 2)

The authors are thorough in their citation of appropriate references. The structure of the article is excellent, data shared, and the results to hypotheses make sense.
Experimental design: The authors have defined a clear question and have done a thorough job of analysis. This is by far the most thorough analysis this group has received, and the outcomes make sense. They have gone above and beyond to make things replicable and test the replicability of odler studies as well.
Validity of the findings: The results make sense and conclusions are the rational outcome of the results. They are fairly consistent with regard to avoiding speculation. This includes their conservative inclusion of D. coalingensis, which other authors have dismissed as a nomen dubium without much clear support. Though I have my doubts about D. coalingensis as a valid taxon, they explain that ascertaining this is not in the scope of the paper and thus do not attempt to argue to their exclusion. This was wise.
Additional comments: I am impressed by the care and detail by which this study was conducted. It doesn't overreach the results and comes to a rational conclusion based on results alone.
I only mark this as minor revisions being needed for the corrections to the typos. Scientific content is sound.


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: ROSIGLITAZONE ACCELERATES WOUND HEALING BY IMPROVING ENDOTHELIAL PRECURSOR CELL FUNCTION AND ANGIOGENESIS IN DB/DB MICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. English writing should be reviewed by a native speaker
2. Legend of some axis should be explained, what does hpf mean? (y axis in figure 3b and c) What does lpf mean? (y axis in figure 4c and d)
3. Figure 2a does not have a scale bar in at least one picture
Experimental design: 1. Why a control group with RSG was not included?
2. In what volume was RSG given to animals
3. How many animals were used in each group?
4. How many animals from each group were used for cell extraction from bones?
5. If VEGF was measured by ELISA in figure 5 why the units in Y axis are fold of control instead of concentration?
Validity of the findings: Experiments are well performed but they are mere correlations.
There are no clear conclusions besides a very general one that indicates that RSG is beneficial for the parameters measured
Additional comments: In this manuscript authors evaluate the effect of Rosiglitazone in wound healing in a mouse diabetic model. Upon reading several concerns have rised:
1. How was determined the dose of RSG administered to mice?
2. On lane 245 authors conclude from the experiment that “RSG protects against wound healing injury. Which results are considered to make this conclusion?
3. Lane 252, authors did not measure the expression (synthesis) of the proteins, they measured the abundance of them (that includes processes of synthesis and degradation)
4. A better characterization of BM-EPC is needed, are these cells insulin resistant? Did the treatment with RSG reduce the resistance?
5. Which is the important factor in the results observed by the administration of RSG?, the reduction in glycemia?, the increase in Insulin?
6. If VEGF or SDF-1 are not present, can RSG improve the process of wound healing, and cell proliferation?


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: ROSIGLITAZONE ACCELERATES WOUND HEALING BY IMPROVING ENDOTHELIAL PRECURSOR CELL FUNCTION AND ANGIOGENESIS IN DB/DB MICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: A work presented for review entitled "Rosiglitazone accelerates wound healing by improving endothelial precursor cell function in db / db mice" is an interesting contribution to the development of basic and clinical sciences. The work is written in the correct language, however, in a few places, the abbreviations that are introduced should be developed. The structure of the article is correct.
Experimental design: The experimental design is correct, however maybe interesting would be to give the drug to non genetic  mice and compare this between group.

I have a few question:

1. Where was the control wound located? Please insert a macroscopic photo of both wounds.
2. The attached photos do not show any stitches on the wound. In what mechanism did the wounds heal? Was it generous by granulation or contraction?
3. Do you measure level of any cytokine. In literature we can find information that RSG showed anti-inflammatory properties (https://www.sciencedirect.com/science/article/pii/S0022202X15371050, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834268/)
4. Have you performed histological tests (review of HE staining) or immunohistochemical tissues taken from the wound? I found in the manuscript that only these stains were made to evaluate angiogenesis. Am I right? I recommended to perform this staining and evaluate tissue responses. It could be semi-quantitative measurements. See this papers: DOI: 10.1177/0885328218801114, https://onlinelibrary.wiley.com/doi/abs/10.1111/wrr.12500
DOI: 10.1177/0963689718807410
5.  You can also measure the thickens of dermal layer.
Validity of the findings: No comment
Additional comments: My general comments are mentioned above.


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

Input:
Title: ROSIGLITAZONE ACCELERATES WOUND HEALING BY IMPROVING ENDOTHELIAL PRECURSOR CELL FUNCTION AND ANGIOGENESIS IN DB/DB MICE
Review round: 1
Reviewer: 3

Output:
Basic reporting: I found the manuscript difficult to read on a first pass as the grammar, spelling, and sentence structure needs improvement. I’ve made a few suggestions below, but I would also advise a thorough read through and check before resubmission.
Line 83: remove “the” before angiogenesis. Hypoxia (reduced oxygen) is a consequence of Ischemia (reduced blood flow), you can use either term, but not both as they elude to the same things
Line 91: “is a kind of…” instead of “as a kind of…”
Line 92: “to treat type 2 DM” instead of “to therapy of type 2 DM”
Line 94: “signalling” instead of “signal”
Line 170: “suspended” instead of “slightly dispersed”
Line 171: “plated” instead of “planted”
Line 174: “ELISA” instead of “Elisa”
Line 305: “role” instead of “partly”
Line 312: “underlying” instead of “underline”

Line 93: In this line you refer to “side effects” of RSG on EPC, do you mean the previously found effect of RSG treatment or result of RSG treatment on EPC? Please clarify the use of the phrase “side effect”
Experimental design: In this manuscript you have used a wound protocol cited by Han et al 2017, this protocol does not use silicone splints to keep the wounds from healing by contraction. For comparison to human skin wounds mouse wounds need to be splinted to cause healing by re-epithelialization (as seen in human skin) rather than healing by skin contraction (as seen in mouse skin). Please refer to Dunn et al, J. Vis. Exp. (75), e50265, (2013), and Wang et al Nature Protocols volume 8, pages 302–309 (2013) for protocols on splinting of wounds in mice. Please discuss these papers and why a protocol without splinting was used. This is very important.

I found the explanation for experimental protocol difficult to follow, it would benefit the reader if you break up the explanation into 3 main components ie: 1) all mice (db/db and C57BL/6) at 8 weeks old began blood glucose monitoring for 3 weeks to determine diabetes, 2) treatment with RSG or vehicle commenced at 11 weeks old for 28 days in diabetic mice, C57BL/6 mice received vehicle only, 3) at 15 weeks old, mice were further divided into 2 cohorts (bone marrow harvest and wound closure experiment). It is unclear which mice were used for bone marrow harvest, are they diabetic with RSG treatment, diabetic with vehicle treatment or C57BL/6 with vehicle treatment? Please clarify. The use of graphical timeline, as in figure 1, is beneficial, perhaps a graphic to explain the protocol would also be beneficial.

Line 166, how many sections were on each slide? One slide or one section is quite low for analysis. I suggest analysing a minimum of 3 sections per animal.

Line 171-174, “Cells were planted into 6-well plates (Coring, MA, USA) at 37 OC, 5% carbon dioxide (CO2). After the cells were adhere to plates, a few of non-adherent cells were absorbed and added to the fresh medium. For another 3 days culturing…” This section did not make sense initially, however after looking at the references you provided I believe you wanted to write that “non-adherent cells were removed, and fresh medium was added to the adhered cells and allowed to incubate for 3 days”. Please clarify and fix if incorrect.

Line 143: please detail the type of monitoring system used for blood glucose.

Line 182, for the transwell plate, what pore size and pore material was used?
Validity of the findings: Prolonged inflammation is a key problem in wound healing for diabetic patients. Given the promising effects of Rosiglitazone it would be beneficial to determine if key inflammatory markers involved in wound healing such as CCL2, CXCL8, TGF-β… are reduced in your Rosiglitazone treated mice at both time points
Additional comments: Is it possible to get brighter, higher quality images?

Overall a good paper, needs some refining and clarification to get the correct point across. I recommend completing the suggested experiments to strengthen 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: ROSIGLITAZONE ACCELERATES WOUND HEALING BY IMPROVING ENDOTHELIAL PRECURSOR CELL FUNCTION AND ANGIOGENESIS IN DB/DB MICE
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1.	Clear and unambiguous, professional English used throughout.
English has been reviewed and corrected
2.	Literature references, sufficient field background/context provided.
Sufficient introduction and background has been included, and appropriately referenced.
3.	Professional article structure, figures, tables. Raw data shared.
The structure of the article is adequate. Figures are relevant to the content of the article, and of sufficient resolution. Figures are now appropriately described and labeled.
4.	All appropriate raw data have been made available

5.	Self-contained with relevant results to hypotheses.
The submission is self-contained, represents an appropriate ‘unit of publication’, and includes all results relevant to the hypothesis.
Experimental design: EXPERIMENTAL DESIGN
1.	The Original primary research is within Scope of the journal.
2.	Research question are well deﬁned, relevant & meaningful.
3.	It is stated how the research ﬁlls an identiﬁed knowledge gap.
4.	It is a Rigorous investigation performed to a high technical & ethical standard.
5.	Methods now are described with suﬃcient detail & information to replicate.
Validity of the findings: Methods, results, discussion and conclusions have been revised, complemented and now are clear.
Additional comments: The following is the review for the manuscript entitled “Rosiglitazone accelerates wound healing by improving endothelial precursor cell function in db/db mice”
in this new version all the concerns raised by this reviewer have been answered.
Although the experiment with the antagonist of VEGF,  was not done in cells obtained from db/db mice, it demonstrates the participation of VEGF on wound healing in this model. It would be interesting to demonstrate whether a similar effect to the one observed in cells treated with glucose (acute state) is also observed in cells from db/db mice (chronic state) treated with Rosiglitazone.


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: ROSIGLITAZONE ACCELERATES WOUND HEALING BY IMPROVING ENDOTHELIAL PRECURSOR CELL FUNCTION AND ANGIOGENESIS IN DB/DB MICE
Review round: 2
Reviewer: 2

Output:
Basic reporting: In my opinion, after making corrections, the manuscript contains the necessary information to correctly understand and replicate the study by another authors.
Experimental design: After correction this paper well described all technical details.
Validity of the findings: The paper presented for review raises very important issues related to the possible use of RSG on wound healing and the related mechanisms involved in db/db diabetic mice.
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: ROSIGLITAZONE ACCELERATES WOUND HEALING BY IMPROVING ENDOTHELIAL PRECURSOR CELL FUNCTION AND ANGIOGENESIS IN DB/DB MICE
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have sufficiently corrected the manuscript based on the reviewers 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: FUNDAMENTAL NICHE UNFILLING AND POTENTIAL INVASION RISK OF THE SLIDER TURTLE TRACHEMYS SCRIPTA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript from the authors presents a study about climatic niche differences between native and non-native populations of the slider turtle Trachemys scripta.

I found the manuscript very well written and clear and the methods used. The references were sufficient and well cited. The figures need to be substantially improved due to their poor quality.

The authors consider that the fundamental niche can be defined from physiological limits (e.g., maximum and minimum critical temperatures) However, this is not an accurate definition of the fundamental niche (sensu Hutchinson 1957). Although this has been a pervasive confusion in the literature for years it should be clear that the fundamental niche definition was erected from a demographic basis (see Hutchinson 1957 and Holt 1997). In other words, the fundamental niche is the environmental conditions that influence birth and death rates on a population allowing its survival without immigration. I would like to see a more deep explanation from the authors about this issue and why they decide to use physiological proxies operating at the individual level to operationalize the concept of fundamental niches.

References

Hutchinson, G.E. (1957) Concluding Remarks. Cold Spring Harbor Symposia on Quantitative Biology, 22, 415-427

Holt, R.D (2009). Bringing the Hutchinsonian niche into the 21st century: Ecological and evolutionary perspectives. PNAS 106: 19659–19665.
Experimental design: The authors compiled a lot of physiological information from this species and they use this data to generate thermal tolerance curves and predict potential distribution across the world.

The authors did not include a proper comparison of analog or non-analog climate conditions between native and non-native areas. This comparison likely is crucial to establish whether native and non-native areas are climatically different and can be used to establish whether invasive T. scripta populations are occupying non-analog climates in other areas. Although there is a full set of methods to test this (see for example Elith et al. 2010; Mesgaran et al. 2014, etc.), it is strange the lack of this in this work.

References

Elith et al. (2010). The art of modelling range-shifting species. Methods in Ecology and Evolution 1: 330-342.

Mesgaran et al. (2014). Here be dragons: a tool for quantifying novelty due to covariate range and correlation change when projecting species distribution models. Diversity and Distributions 20: 1147-1159
Validity of the findings: The authors presented two kinds of results about niche comparisons from native and non-native ranges. By one hand, null model tests revealed that there are no differences between native and non-native realized niches (lines 289-293). Randomization tests conducted by the authors suggest that even minor niche differences between native and non-native areas cannot be different from a random point selection through the entire background area. By another hand, their PCA matrix comparisons revealed a difference in climate structure from native and non-native areas (line 294-302). However, these PCA comparisons did not included a randomization test, which is crucial in this kind of exercises. Furthermore, thermal tolerance curves from physiological data suggest that native and non-native areas (see fig. 3) are included within the full curve from temperature (i.e., the "fundamental niche" as authors defined previously).

It is hard to see that native and non-native niches are different as authors concluded based on the data at hand and the statistical analyses. The data and tests used here suggest to me that T. scripta is occupying a large portion of its "fundamental niche" in non-native areas likely due to the elimination of dispersal barriers and/or elimination of potential competitors/predators.
Additional comments: The manuscript from the authors presents a study about climatic niche differences between native and non-native populations of the slider turtle Trachemys scripta. I found this study very interesting because compile a lot of physiological information which is used to generate thermal tolerance curves and predict potential distributions in geographical space. However, the results suggest that there are no differences in climatic niches in native and non-native areas and a proper comparison of analog and non-analog climates across areas is recommended.


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: FUNDAMENTAL NICHE UNFILLING AND POTENTIAL INVASION RISK OF THE SLIDER TURTLE TRACHEMYS SCRIPTA
Review round: 1
Reviewer: 2

Output:
Basic reporting: I have two comments in this section. One is related to figure 3. The part (a) of the figure is smaller than the other ones, please correct it.

My second comment is related to English. I would suggest the authors ask a Native speaker for proof-reading of the entire manuscript, especially after the changes.
Experimental design: I found the methods employed appropriate, in general, but I have the following comments.

Lines 138-159. All these sentences are a good summary of the entire approach used here and it is not bad. However, I think the authors should consider moving this part to the first part of the methods. The introduction is already big and not easy to read because of the diversity of information and the structure in which it was written.

Line 173. 5 km is around 2.5 min. Given the resolution of the raster layers for global analyses, this means that not even duplicates where removed. A larger distance must be used for global analyses to avoid duplicate records. I consider that increasing the distance may not change the findings and conclusion of this work. However, if the authors decide not to improve this part, an explanation of what potential effects can be expected from this caveat should be mentioned in the discussion.

Line 180. In line 275 the authors also talk about BIO10 and BIO11, I consider that those variables should be mentioned here as well.

Lines 192, 199. I disagree in that a distance layer defines a characteristic of the fundamental niche. A population of this species is going to be OK as long as the water body in which it exists has appropriate conditions and individuals do not have to move. Population size can be relatively stable once it reaches its carrying capacity. Whether or not there is another water body close or far won't affect such population. Please argument better why the authors included this variable.

Line 206. How the probability value was calculated? Why weighting is necessary? Conditions are available whether or not the species is using them and depends on other factors especially related to history and mobility? Why not defining accessible areas instead of doing these weighting? Why all turtles and not only the ones that are closely related to the one of interest? Please clarify in the text, the readers will find these explanations very useful.

Line 250. If the distance-derived variable is included, the assumption that the response of the species to this variable is similar to responses to temperature and precipitation is big. Describe why this assumption is not problematic.
Validity of the findings: The findings are interesting and from my point of view, valid. Please see some comments for the results and discussion sections below.

Line 290. I assume this result (the niche has been, at least, partially retained (D=0.287) in the invaded range) is concerning the native environmental conditions. Please be specific.

Line 294. This sentence is part of methods, please move it to that section.

Line 325. Please explain better this part or reword the sentence. How is it possible to invade an area and do not occupy it?

Line 331. How are the authors evaluating niche dynamics? What do the authors mean by that? Dynamics of what niche (fundamental, potential, realized)?

Line 352. Based on figure 3, it looks like BIO01 and BIO11 are not as good variables as the authors suggest. Occupied environmental conditions characterized by BIO1 and BIO11 do not match with the fundamental niche as good as BIO10 does. Please discuss these ideas in the text. What the authors can suggest about variable selection processes in ENM, for instance?

Line 387. I am not sure what the authors are trying to say here. Species fundamental ecological niches are defined by the limits of their tolerance to variables. How come a species can occupy distinct niches within its tolerance ranges? What niches are the authors referring to? Please clarify

Line 399. Do the authors have any evidence of the species going to invade these areas?

Line 412. As from I understood, the authors did not consider what invasive records correspond to established populations or individuals that are only surviving. Please discuss what implications this fact could have in the results found in this work.

Line 432. This is correct but it would be nice to know if the authors have ideas of other potential solutions. What the authors can say about combining records from native and invaded areas (established populations) to create better ENMs for invasive species? Do the authors think it can be an option for species where fundamental niches are unknown?
Additional comments: The main idea behind the manuscript entitled “Fundamental niche unfilling and potential invasion risk of the slider turtle Trachemys scripta” is interesting and worth to explore. I consider this is an acceptable contribution; however changes are necessary to improve it. My general comments are listed below.

- The manuscript is long and considering the analyses performed and the information presented it can be improved by avoiding some information that may not be that relevant. For instance, introduction and discussion are difficult to follow because of its length.

- The authors say they developed a conceptual model and I disagree with that. What the authors did was to use distinct tools and analyses to investigate a specific question. Therefore, they only used an approach that can be modified in many different ways in other studies. What the authors did in this study do not allow to compare if this approach is better than others than can be followed to perform comparable analyses; therefore I consider the authors should refer to their set of analyses as an approach and not a conceptual model.

- The use of terminology in the manuscript is not completely appropriate. Although the authors made an effort in including a Glossary, they are not consistent with terms like niche and niche shift. The distinct types of niches are not used properly in all their mentions. I would suggest the use of three types of niches: fundamental, existent, and realized (see the Book of Peterson et al. 2011).

Especial comments:
- Line 72. "... however, an important limitation when they are used for range predictions is the assumption made that the climatic niche between the native and the non-native (invaded or exotic) range is conserved across space and time". This is not at all a limitation, on the contrary, this is the base to make these methods applicable to such questions. This paper is another proof of the usefulness of this assumption and the fact that the limitation is not the assumption but the way in which ecological niches are catheterized.

- Line 121. Determine is not an appropriate verb here, perhaps identify works better for what you are doing.

- The paragraph in lines 107-123 may be better located after the paragraph that is following it in the current version of 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: FUNDAMENTAL NICHE UNFILLING AND POTENTIAL INVASION RISK OF THE SLIDER TURTLE TRACHEMYS SCRIPTA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors present a revised version of this manuscript with clarification about definitions and proper comparisons of analog and non-analog conditions. The issue of the fundamental niche definition was revised based on a review of classical papers from Hutchinson. Although I disagree with the Soberón & Arroyo-Peña (2017) about the fundamental niche definition, which it should be not related to physiological thermal limits, I think the authors provided a relatively decent explanation about how this data can be used as a proxy. Anyway, they discussed and explained this in the main text.

The authors also corrected the confusions about niche differences in native and invaded ranges and conducted a proper comparison in the environmental space. The comparison of the structure of the covariance matrix in ecological space is a novel approach here to evaluate whether analog and non-analog conditions exist across different geographical settings. I think this part resolves the problem identified in the first version.
Experimental design: The experimental design based on the new analyses included is ok.
Validity of the findings: I think the authors rewording the text to avoid confusions between geography (e.g., native and invaded range) and ecological space (e.g., analog vs. non-analog climates). I'm ok with these changes.
Additional comments: I think the authors present a very interesting study with new statistical approaches to compare realized niches across geography in invasion ecology.


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: REAL-TIME REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION DEVELOPMENT FOR RAPID DETECTION OF TOMATO BROWN RUGOSE FRUIT VIRUS AND COMPARISON WITH OTHER TECHNIQUES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript was written in good English; but it can be improved.
The literature references were good enough to the topic
The manuscript was structured well and the authors expressed themselves in.
Experimental design: The experiments were carried ad hoc and the methods were sufficient
Validity of the findings: The findings are novel and useful. More needed to explain how these can be used as a routine tests. The recommendations need to be improved based on that
Additional comments: The manuscript is original and informative.