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

Input:
Title: GENOME-WIDE SEQUENCE IDENTIFICATION AND EXPRESSION ANALYSIS OF ARF FAMILY IN SUGAR BEET (BETA VULGARIS L.) UNDER SALINITY STRESSES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised manuscript has improved overall and satisfied the required standards for publication in its current form.
Experimental design: All corrections are satisfactory in the revised manuscript.
Validity of the findings: The change made by the authors in the revised manuscript is satisfactory.
Additional comments: The changes made by the authors in the revised manuscript has improved and strengthened their research work to the publication standard in this journal and would add more knowledge to the understanding of plant auxin response factors. I recommend it to be accepted for publication.


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

Input:
Title: GENOME-WIDE SEQUENCE IDENTIFICATION AND EXPRESSION ANALYSIS OF ARF FAMILY IN SUGAR BEET (BETA VULGARIS L.) UNDER SALINITY STRESSES
Review round: 5
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I'm satisfied with the revision now.


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

Input:
Title: GENOME-WIDE SEQUENCE IDENTIFICATION AND EXPRESSION ANALYSIS OF ARF FAMILY IN SUGAR BEET (BETA VULGARIS L.) UNDER SALINITY STRESSES
Review round: 5
Reviewer: 2

Output:
Basic reporting: The inclusion of GO terms and sequences in the supplemental files has further improved the  accessibility of the results and sequence data used. It has addressed editor's concerns and satisfied the required standards for publication.
Experimental design: Satisfactory in the revised manuscript.
Validity of the findings: Satisfactory in the revised manuscript.
Additional comments: The inclusion of GO terms and sequences in the supplemental files has further improved the  accessibility of the results and sequence data. It has addressed editor's concerns and satisfied the required standards for publication. The revised manuscript has further improved and strengthened their research work to the publication standard in this journal and would add more knowledge to the understanding of plant auxin response factors. I recommend it to be accepted for publication.


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

Input:
Title: A HIGH-RESOLUTION GROWTH SERIES OF TYRANNOSAURUS REX OBTAINED FROM MULTIPLE LINES OF EVIDENCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is a long-awaited addition to the knowledge of Tyrannosaurus ontogeny from one of the foremost authorities in the field. In total, the 1,604 hypothetical growth characters from 41 specimens represent the largest and most comprehensive ontogenetic data set presented for a dinosaurian species. Generally, this paper was written with precise and clear language throughout; though I have highlighted a few areas below where changes can be made for consistency, accuracy, and brevity.

A. Commas are mostly used to separate multiple internal references, though line 820 and 939 has semicolons separating citations instead.

B. OMNH should be the Sam Noble Oklahoma Museum of Natural History.

C. Line 217, “After 81 hours” seems like unnecessary detail. The timing of the analysis will depend on the machine used.

D. It Is unclear which definitions for growth categories (juveniles, subadults, adults, etc.) you are using in the methods (e.g., “subadult specimens” from Line 154). You define these well in the results, but the best mention of them previously to the methods is on lines 76-77 in the Introduction when discussion Erickson et al. (2004). Which begs the question, which definition of growth categories are you using early in this paper?

E. Line 604 (and others), you mention halfway points in life. What are you considering the maximum lifespan to be?

F. Line 1374 seems unnecessarily enthusiastic with the exclamation point.

G. Figures 24, 25, 26 would be best represented as a table; figure 29 is largely redundant with Figure 1 and could easily be combined; figures 3 and 4 should also be combined; Figure 7 is difficult to read as presented.

The references are thorough and contain all of the most important literature for the discipline. Errors, largely in the form of missing citations, were found throughout the reference section and are outlined below.

A. Lines 137-139, there is no consistency in the order of citations.
B. Line 187, Swofford, 2003 is cited as Swofford 2002 in the references section.
C. Lines 305, 351, Wurneburg et al., 2019 is cited as Werneberg in the references section.
D. Line 820, Leslie et al., 2018 is not in references section.
E. Line 1121, Molnar, 2013 is not in the references section.
F. Line 1176, Moss, 1957 is not in the references section.
G. Line 1196, Hurlburt et al. 2013 is not in the references section.
H. Lines 1302, 1305, Osborn, 1905 is not in the references section.
I. Lines 1316, 1320, Osborn, 1906 is not in the reference section.
J. Line 1348, Wilkinson and Rhodes, 1997 is not in the reference section.
K. Line 1357, Lincoln et al., 1982 is not in the reference section.
L. Line 1447, Pira et al., 2010 is not in the reference section
M. Line 1448, Dodson, 1975 and Hall and Portier, 1977 are not in the reference section.
.
Experimental design: The scope of the paper fits well with other articles published in PeerJ, including a paper I authored in 2014 using this very method. The research question, goals, and assumptions are all well-defined early in the paper. Overall, I had only a few issues with experimental design.

A. I'd like to see more about how you defined the nascent from the adult state in the methods. If the smallest specimens are used to create the outgroup (artificial embryo), it would be proper to describe how these are unequivocally immature by all vertebrate growth proxies (small size, few LAGS, unfused sutures, etc). This will also make your decision to remove juvenile and subadult specimens from some of the Spearmen Rank analysis (such as those comparing size to growth stage) more understandable because the least mature juveniles are being defined as such on the onset of the analysis.

B. Line 154, you discuss reducing your data set due to uninformative characters. I believe greater explanation is needed to warrant pruning data. If there is only a single character state found in all specimens, removal is justified to reduce tree length. However, the other three criteria need more explanation. “Codings lacked pattern” seemingly implies that you were not completely objective when running the analysis and that you had a predicted topology. The same goes for removing potential autapomorphies (autontomorphies?) from subadult specimens. Last, removing characters only scored in 3 or fewer specimens is intuitive but also seems to lack justification.

C. Explain how you decided which specimens to keep from redundantly coded individuals.

D. Line 168, The comment on Holliday et al. (2019) is most relevant in the context of how it affects your coding. I suggest you add a line on which characters these observations change.

E. An a posteriori analysis to determine the most mature specimen is appropriate and your method is with merit. However, on the final tree, FMNH PR2081 has nine autontomorphies, while CM 9380 has nine reversals and two autontomorphies. This begs the question, why can’t the individual with the most adult character states be assumed the most mature?
Validity of the findings: A. Line 237-239, states that the ontogram would divide into branches if sexual dimorphism is present. This is what would theoretically occur if the dimorphic characters outnumbered the synontomorphies. This phenomenon, however, has not been demonstrated in a published analysis and should thus be treated with caution and skepticism. Instead, I suggest investigating reoccurring character reversals found in the adult specimens.

B.  In the section on Synontomorphy Trends, the location of peaks will change based on skeletal completeness and the type of optimization used in the analysis. At least two of the peaks (at GS 5 and GS 18) occur before polytomies with relatively incomplete specimens. I suggest you map skeletal completeness based on the percent of scored characters for each specimen in your growth categories and compare those as a discussion of a potential source of bias in the ontogenetic record.

C. I strongly don't believe that the Spearman Rank Correlation can be used to identify geographic or stratigraphic bias in this particular sample. Geographically you may be able to code location for every specimen (line 837) but you’ve lumped them together by state, an artificial invention of modern politics and geography. Instead, I would use latitude to make a more precise delineation of spatial differences. As for the stratigraphic position, the data set is just too small to have any confidence in this result. Even if two species were present in the data, the growth series would likely overlap too significantly to differentiate. As with sexual dimorphism, this is another testable hypothesis that needs to be explored further in future studies.

D. Line 874, are Carpenter (1990)’s characters of ischial orientation included in the analysis? I was not able to find them in the supplementary file based upon the description in the text, but believe they should be included to best capture any potential dimorphism in the sample.
Additional comments: Overall, I found this to be an enjoyable and important paper to review. The data set itself is comprehensive and will undoubtedly prove valuable for future tyrannosaur researchers. Given time constraints and my ignorance regarding Tyrannosaurus anatomy, I have taken your description of specimens at face value and have instead focused on your methods, assumptions, and interpretations. In all cases, this paper is worthy of future publication with minor revisions and clarifications.


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 HIGH-RESOLUTION GROWTH SERIES OF TYRANNOSAURUS REX OBTAINED FROM MULTIPLE LINES OF EVIDENCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Firstly, this is a monumental piece of work requiring a tremendous amount of observations, which is sure to generate substantial future lines of research.

The writing and argumentation is clear and the data employed are well-documented in the body and supplementary materials.

A particular aspect that is appealing is the set of predictions laid out for future testing: I look forward to results of these tests. Additionally, the author is very clear in addressing one by one the various alternative explanations (geographic, stratigraphic, sexually dimorphic, etc.) for variations among the adult specimens.

The figures are very informative; not only the documentation of the essential patterns discovered herein, but also the mapping of the results of other studies onto the newly completed growth patterns. This helps to put other research into a new context.
Experimental design: One issue that should be addressed at least in passing is the possibility of multiple ontogenetic trajectories within one taxon in the same population. This pattern has been documented in dinosauromorphs in papers by Griffin & Nesbitt. To be fair, they conclude that this pattern may be primitive to dinosauromorphs but transformed in derived dinosaurs which seem to have a more rigidly fixed ontogenetic pathway, but they did not have the immense database presented here to confirm that. Thus this might be a good opportunity to at least briefly address this concept, given the substantial data set now available:

Griffin, C.T. & S.J. Nesbitt. 2016. The femoral ontogeny and long bone histology of the Middle Triassic (?late Anisian) dinosauriform Asilisaurus kongwe and implications for the growth of early dinosaurs. Journal of Vertebrate Paleontology 36: e1111224. Doi: 10.1080/02724634.2016.1111224

Griffin, C.T. & S.J. Nesbitt. 2016. Anomalously high variation in postnatal development is ancestral for dinosaurs but lost in birds. PNAS 113: 14757-14762. Doi: 10.1073/pnas.1613813113
Validity of the findings: Lines 149-150	The author is clear on his coding philosophy, allowing for repeatability. However, since parsimony-based phylogenetic analysis software can often incorporate polymorphic observations for individual OTU, it might be informative in this or a future study to run the analysis with both observed states present. We cannot know a priori if it would affect the results, but it might (for instance) reveal particular growth phases of transition found in multiple individuals, and thus more completely characterize the ontogeny of Tyrannosaurus rex. (I would not regard this as a requirement for the present study, however.)

Lines 168-174	It is worth noting that Holliday et al. (2019) did not solely rely on bone surface texture in arguing that the frontoparietal fossa was occupied by vascular tissue. They also discuss the unlikelihood of it being occupied by the adductor musculature in dinosaurs, as it would require a right-angle muscle reorientation with no apparent sesamoid. They might be inaccurate in this presumption, it is true. The present study does not sufficiently explore all the lines of evidence that Holliday et al. used to come to their conclusion. Therefore, it might be more appropriate to say here that based on the present author’s observation of the bone texture of this region, he will accept the traditional view of this fossa rather than the new interpretation. (This was a longwinded way of saying that the present author didn’t actually reject Holliday et al.’s interpretation as he didn’t examine all the relevant lines of evidence but that he is preceding for the purpose of this study with the traditional assessment.)

Line 217	Just for clarification, the author should indicate that the 81 hours was the completion of the analytical run (rather than the author quitting the process prematurely).

Line 336ff	For the discussion of growth categories, the author states mass ranges which are given to the kilogram. This is following the results of previous works cited within. But this propagates a problematic issue of these studies: a false impression of precision in mass estimations. These studies often give results far beyond the number of significant digits of the particular measurements actually observable on the fossils.
Additional comments: Again, this is a spectacular piece of research, and I look forward to seeing its publication.


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

Input:
Title: A HIGH-RESOLUTION GROWTH SERIES OF TYRANNOSAURUS REX OBTAINED FROM MULTIPLE LINES OF EVIDENCE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall, this work is a major, self-contained synthesis of nearly all available data on T. rex biology, in the context of the most comprehensive cladistic ontogenetic analysis of a fossil taxon to date. The integrative approach provides a clear, replicable framework for distinguishing sources of anatomical variation among tyrannosaurids. The study design and interpretations of ontogenetic data are broadly sound. My most significant comments concern the specifics of how the cladistic methodology was employed. I look forward to the publication of this manuscript following a response to the comments and revisions I outline below.

The manuscript is generally very clearly written. See “Specific Comments” and “Typos” below for instances in which further clarification is needed and spelling mistakes should be corrected. The structure conforms to both PeerJ standards and discipline norms.

The introduction provides a good background on both recent research on tyrannosaurids and the cladistic ontogeny method. Given the high degree of variability present among the mature individuals present in the sample, the author should discuss Ontogenetic Sequence Analysis (Colbert and Rowe 2008), why it was not applied to this study, and what it could reveal about variation in T. rex ontogenetic trajectories if applied. Other instances where further citations/discussion of background literature are needed are listed under “Specific Comments” below.

The figures are generally effective, though I provide some suggestions for improving their clarity under “Figures” below.
Experimental design: The integrative approach to analyzing the “three axes of ontogeny” together is an important step forward in analyses of this kind. It fills a knowledge gap in identifying whether or not size and age correlate with other morphological measures of maturity in T. rex. I have no concerns about the originality or ethics of the work.

Replication of the study could be enhanced by providing a supplementary list of commands/steps performed in the cladistic analysis.

The cladistic results need further interrogation before a tree topology can be settled on for purposes of discussion. Support for clades should be identified by bootstrap, jackknife and Bremer decay analyses. There should be a discussion of which specimens have the most labile placement, and consequently lead to the reduced resolution of the strict consensus tree. The influence of the duplicated characters already identified by the author on the results should be examined. If the author is intent on presenting a majority rule consensus tree, problems with this consensus method (Sumrall et al. 2001) and their influence on the results should be discussed. Confidence in interpretations of relative maturity obtained from a majority rule consensus tree should be tempered appropriately. In any case, the author should emphasize that only those growth stages (“clades”) retained on the strict consensus tree are clearly supported. All other groupings are more ambiguous. See “Specific Changes” below for more detailed comments on these points.

I do not anticipate that these additional experiments will greatly alter the overall conclusions of the study, but they should be performed to fully explore the data and indicate degree of support for each “clade”. I am recommending major revisions largely because the topology of the cladogram is the foundation upon which the results and discussion sections rest, and further work needs to be done to test the robustness of support for any topology presented. If some clades are found to be weakly supported, this may have downstream effects that would necessitate further revisions to the rest of the manuscript. “Major revisions” should not be taken to indicate anything fundamentally flawed about the overall intellectual approach or the interpretation of experimental results. The results and discussion sections are generally excellent and, assuming no major changes in tree topology, should be largely acceptable.
Validity of the findings: The extensive, detailed descriptions provide a comprehensive framework for interpreting anatomical variation in tyrannosaurids.

The discussion of individual variation is thorough, and the thoughtful interpretations provide much-needed clarity to discussions of intraspecific variation in dinosaurs.

Likewise, the discussion of T. rex biomechanics, geographic localities, and stratigraphic positions in ontogenetic context are welcome and greatly add to the already considerable value of this study as an integrative work on tyrannosaurid biology.

The conclusions are appropriately limited to the results presented.
Additional comments: I am commenting as a researcher experienced with methods used to study the ontogeny of fossil vertebrates and not as someone with specialized knowledge of tyrannosaurid anatomy, beyond that reasonably expected from a dinosaur worker who focuses on the anatomy of basal theropods and basal ornithischians.

This paper needs a justification of why Ontogenetic Sequence Analysis (OSA) (Colbert and Rowe 2008) was not used, particularly given the high degree of variability in T. rex ontogenetic characters (analogous to homoplasy in a phylogenetic analysis) that produces a poorly-resolved strict consensus tree. OSA can be used to examine the variation in ontogenetic sequences that a population can undergo (Colbert and Rowe 2008, Griffin and Nesbitt 2016). The author should engage with the arguments of Griffin and Nesbitt (2016—see supplementary information quoted at length below) about the similarities/differences between cladistic ontogeny and OSA, and reasons to use one method or the other. As Griffin and Nesbitt (2016, supplementary information) state:

"OSA is similar to cladistic ontogeny (8, 9) in that it is a parsimony-based method of determining the sequence of skeletal character change through ontogeny. However, whereas OSA uses information from all trees returned in a parsimony analysis of the ontogenetic character data (see Methods), and thus reconstructs all developmental pathways consistent with the data, cladistic ontogeny utilizes a consensus tree, minimizing the ability to observe any variation in developmental sequences in a population. Cladistic ontogeny is therefore a faster method of analyzing morphological changes during growth, and unambiguous ontogenetic character transformations shared by all sequences within a population are easily discernable using that analysis. However, any variation in the population will appear mainly as a lack of resolution in ontogenetic characters, and not an interpretable signal in its own right, limiting ability to understand and quantify variation. Furthermore, it becomes increasingly more difficult to derive a meaningful signal from cladistic ontogeny with increasing amount of sequence polymorphism, so that a dataset with the level of variation of Coelophysis bauri results in a consensus tree consisting of a single large polytomy. Thus, each method has strengths and weaknesses, but except in instances where variation is virtually absent or explicitly intended to be ignored, we advocate using OSA to reconstruct morphological changes during growth. One of our major conclusions of this contribution is that using OSA is necessary in early dinosaurs when reconstructing growth."

I think a dialogue in the literature between the practitioners of these complementary approaches would serve the field well.

The author consistently mentions that T. rex bite force increased through ontogeny to produce the high values seen in adults. However, there are no citations to either the paper that established this quantitatively for the juvenile T. rex BMRP 2002.4.1 (Bates and Falkingham 2012), nor to papers establishing high bite force in adult T. rex (Erickson et al. 1996, Gignac and Erickson 2017), nor any comparisons to the literature on ontogenetic changes in crocodylian bite force (Bates and Falkingham 2012; Erickson et al. 2003; Erickson et al. 2014; Gignac and Erickson 2015, 2016; Gignac and O’Brien 2016). Such comparisons between crocodylian bite force ontogeny and that of T. rex should be discussed in the sections on bite force in the current manuscript.

If the author has not done so already, he should check to make sure that the 2015 corrigendum to Erickson et al. (2004) does not affect any of his interpretations. As there were no major corrections made to the 2004 study, I doubt there will be any effect on the current work, but I bring it to the author’s attention just in case.

Specific Comments:
Line 168: This paragraph on the texture surrounding the dorsotemporal fossa seems somewhat out of place here. I suspect it is included here because the interpretation of this anatomical region is important to character definition and construction, but the author should add a statement clarifying this.

Line 176: A more explicit rationale and justification of the method by which the quantitative characters (e.g., size) were divided into discrete states is needed here. It’s also possible in TNT phylogenetic analysis software to analyze the measurements as continuous characters, without first breaking them into discrete states (Goloboff et al. 2006). This approach could be worth trying to see if it alters the results.

Line 180:  This sentence could use some clarification. I think the author meant “relative size is used only when a specimen is represented by a single bone or a partial skull or skeleton.” Note the position of the word “only” in this sentence compared to the original.

Line 187: How many random addition sequences and rounds of branch swapping were performed? This should be stated. The author should consider including a list of the specific PAUP* commands/procedures utilized as supplementary information to ensure ease of replication by future researchers.

Line 206: The use of terminology paralleling phylogenetic analysis (e.g., autontomorphies, synontomorphy) is very effective. As a whole, I hope that this paper will further advance attempts to standardize cladistic ontogeny terminology.

Line 229: Increased resolution is not an appropriate reason to favor presenting a majority rule consensus tree over another type. As Sumrall et al. (2001) state:

"A systematist should acknowledge all most parsimonious trees, whether one is preferred over the others or not; because we can never know if a given tree is the ‘‘real’’ phylogeny, each of the most parsimonious trees must be considered as a viable candidate until explicit criteria for selecting among them (or even for selecting among less optimal trees) are presented. Numerical minority of a node in a set of most parsimonious trees is a spurious rationale for rejection."

In the context of this study, the other 50% of trees obtained by the heuristic search, but not summarized by the 50% majority rule consensus tree, are still equally parsimonious to those presented. A tree that lacks a clade present in 99% of the other trees is still an equally parsimonious “solution” (as measured by tree length) to the “problem” of optimizing a given set of characters for a given set of specimens. In this study, there are equally parsimonious trees that, to give two examples, do not place FMNH PR2081 as more mature than UMNH 11000, or show CMNH 7541 as less mature than BMRP 2002.4.1 (as shown in Figure 1, neither of these groupings was recovered in 100% of the most parsimonious trees). Locally labile terminal specimens in an analysis (i.e., those that bounce between two relatively close placements) can introduce bias into majority rule consensus results (Sumrall et al. 2001). An Adams consensus tree may be useful to identify the specimens with the most labile placement. If the author wishes to use a majority rule consensus tree, the concerns of Sumrall et al. (2001) should be discussed, and confidence in the results qualified appropriately.

As Nixon and Carpenter (1996) state: “If the goal of using consensus trees is to summarize the agreement in grouping among a set of cladograms, then only the “strict” consensus tree fulfills that goal. The other methods may all result in groups that are not supported by the data at hand, or supported only ambiguously.” Alternatively, the author could present a reduced strict consensus tree following “safe” pruning of unstable specimens (Wilkinson 2003). It is necessary to investigate how the choice of consensus method influences the tree topology, because all subsequent parts of the manuscript currently rely uncritically on the topology of the majority rule consensus tree.

Likewise, the frequency at which a clade appears among a set of most parsimonious trees is not really a true measure of support for that clade. The author should perform bootstrap, jackknife, and Bremer decay analyses on this dataset and report the resulting values at the nodes as more informative measures of support for those clades.

Line 456: Is there any precedent among studies of extant taxa for using a functional/ecological definition of somatic maturity? Perhaps the author is getting at this with comparisons to metamorphic extant taxa, but this should be more explicitly stated, especially in terms of how it differs from other definitions of somatic maturity used for extant taxa.

Line 788: The author should restate what he’s using as the “growth rank” and what is meant by “maturity” in this context. Is “maturity” the placement of the specimen on the ontogram?

Line 1085: Which recent work is the author referring to that shows that the T. rex skull was akinetic? I think a citation is needed here for clarity.

Line 1207: It would be helpful to the reader to briefly restate the distinction between a phylogenetic synontomorphy and a nonphylogenetic change here.

Line 1278: What happens when the oversplit characters are either reduced down to single characters or removed? The author should investigate how much influence these characters are exerting on the topology of the resulting ontogram.

Line 1329: I think some discussion is warranted about whether or not the reversals of ontogenetic character states seen in some adult T. rex may have been actual reversals over the lifetime of an individual, or whether those individuals simply retained juvenile character states into adulthood, never passing through a transition for those characters in the first place. In other words, how do we distinguish a reversal in a character state optimization on an ontogram from a reversal over the course of an individuals’ lifetime (such as growth followed by later resorption of the horns of some marginocephalians—e.g., Horner and Goodwin, 2009). I realize that it may not be possible to test this, but to me, it’s important to think conceptually about whether these T. rex individuals arrived at state 0 in adulthood by either a transformation of 0->1->0 or simply retained state 0 from an earlier ontogenetic stage, having never achieved the “1” state. A sentence indicating this distinction would provide a useful conceptual framework for future authors to think about reversals.

Line 1437: The author should explicitly indicate that Proterosuchus is an outgroup to Archosauria (i.e., outside the T. rex + Crocodylia clade).  The subsequent discussion of changes in skull shape among archosaurs should take into account additional ontogenetic shape change data gleaned by Bhullar et al. (2012, and associated supp. info.). This study provides additional data on crocodylians, other non-dinosaur archosaurs, and dinosaurs themselves that could help infer plesiomorphic cranial changes.

Line 1539: Point 4: How might this prediction be tested? It is clear enough what evidence is needed to test the other three predictions, but I think the author should explicitly state what a hypothetical specimen either supporting or disproving prediction 4 would look like.

Line 1559: The author might mention that this is certainly not unique to T. rex among non-avian dinosaurs; indeed, it’s probably a plesiomorphic trait shared with crocodylians. I recommended citing Erickson et al. (2007), Lee and Werning (2008), etc.

Line 1562: Point 14. This point is more of a prediction/hypothesis than an observation from the current study. This should be explicitly stated.

Figures
Figure 1. Growth stages should be color-coded on these trees. See comments above about problems with presenting a majority rule consensus tree. At the least, this tree should have Bootstrap/jackknife and Bremer Decay values listed at the nodes.

Figure 3. Font size could be increased on these graphs to enhance readability.

Figures 7 – 9. Given the number of different colors used, the color palette should be checked to see if it can accommodate color-blind people, if the author hasn’t done so already (https://www.color-blindness.com/coblis-color-blindness-simulator/). Additionally, each anatomical domain should be symbolized by differently shaped points along each line (e.g axial skeleton w/ triangles, scapula with squares, etc.)

Figures 11 and 12. Further clarification should be added to the figure captions about what exactly is meant by dentary tooth rank and maxillary tooth rank.

Figure 18. Color coding of specimen numbers should be used in combination with the existing superscript scheme to help distinguish gracile vs. robust specimens. For example, all specimens marked “G” could also be colored blue, and all those marked “R” could also be colored red.

Figure 19. If a color scheme for gracile vs. robust specimens is adopted for Figure 18, incorporate the same color scheme into this figure.

Figure 29. The taxon sample in this figure could be expanded by incorporating data from additional studies of ontogenetic shape change in archosaur skulls (e.g. Bhullar et al. 2012).

Tables:
Given that PeerJ does not have page length restrictions, I advocate keeping these tables as part of the paper and not relegating them to supplementary information. However, I think they would be best placed as appendices at the end of the paper so as not to needlessly interrupt the flow of the main part of the manuscript.

Typos
I did not exhaustively check the manuscript for spelling, but noted typos as I found them.

Line 783: the word “count” should be added after “tooth” in the sentence beginning “Among adults, the hypothesis of dentary tooth as a proxy…”

Line 1084: “compares” should be changed to “compared”.

Lines 1192 and 1193: Both instances of “laterophenoidoprootic” should be changed to “laterosphenoidoprootic” (note the “s”).

Line 1215: I think the following sentence is missing a word: “However, only six of ___ unambiguously optimized synapomorphies are congruent…”

Line 1672: “lestest” in the reference should be “latest”

Line 1728: “Crcocodylia” in the reference should be “Crocodylia”

References cited in this review
Bates, K. T., & Falkingham, P. L. (2012). Estimating maximum bite performance in Tyrannosaurus rex using multi-body dynamics. Biology Letters, 8(4), 660-664.
Bhullar, B. A. S., Marugán-Lobón, J., Racimo, F., Bever, G. S., Rowe, T. B., Norell, M. A., & Abzhanov, A. (2012). Birds have paedomorphic dinosaur skulls. Nature, 487(7406), 223.
Colbert, M. W., & Rowe, T. (2008). Ontogenetic sequence analysis: using parsimony to characterize developmental sequences and sequence polymorphism. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, 310(5), 398-416.
Erickson, G. M., Van Kirk, S. D., Su, J., Levenston, M. E., Caler, W. E., & Carter, D. R. (1996). Bite-force estimation for Tyrannosaurus rex from tooth-marked bones. Nature, 382(6593), 706.
Erickson, G. M., Lappin, A. K., & Vliet, K. A. (2003). The ontogeny of bite-force performance in American alligator (Alligator mississippiensis). Journal of Zoology, 260(3), 317-327.
Erickson, G. M., Makovicky, P. J., Currie, P. J., Norell, M. A., Yerby, S. A., & Brochu, C. A. (2004). Gigantism and comparative life-history parameters of tyrannosaurid dinosaurs. Nature, 430(7001), 772.
Erickson, G. M., Curry Rogers, K., Varricchio, D. J., Norell, M. A., & Xu, X. (2007). Growth patterns in brooding dinosaurs reveals the timing of sexual maturity in non-avian dinosaurs and genesis of the avian condition. Biology Letters, 3(5), 558-561.
Erickson, G. M., Gignac, P. M., Lappin, A. K., Vliet, K. A., Brueggen, J. D., & Webb, G. J. W. (2014). A comparative analysis of ontogenetic bite‐force scaling among Crocodylia. Journal of Zoology, 292(1), 48-55.
Gignac, P. M., & Erickson, G. M. (2015). Ontogenetic changes in dental form and tooth pressures facilitate developmental niche shifts in American alligators. Journal of Zoology, 295(2), 132-142.
Gignac, P. M., & Erickson, G. M. (2016). Ontogenetic bite‐force modeling of Alligator mississippiensis: implications for dietary transitions in a large‐bodied vertebrate and the evolution of crocodylian feeding. Journal of Zoology, 299(4), 229-238.
Gignac, P. M., & Erickson, G. M. (2017). The biomechanics behind extreme osteophagy in Tyrannosaurus rex. Scientific reports, 7:2012, 1-10.
Gignac, P., & O’Brien, H. (2016). Suchian feeding success at the interface of ontogeny and macroevolution. Integrative and comparative biology, 56(3), 449-458.
Griffin, C. T., & Nesbitt, S. J. (2016). Anomalously high variation in postnatal development is ancestral for dinosaurs but lost in birds. Proceedings of the National Academy of Sciences, 113(51), 14757-14762.
Goloboff, P. A., Mattoni, C. I., & Quinteros, A. S. (2006). Continuous characters analyzed as such. Cladistics, 22(6), 589-601.
Horner, J. R., & Goodwin, M. B. (2009). Extreme cranial ontogeny in the Upper Cretaceous dinosaur Pachycephalosaurus. PLoS One, 4(10), e7626.
Lee, A. H., & Werning, S. (2008). Sexual maturity in growing dinosaurs does not fit reptilian growth models. Proceedings of the National Academy of Sciences, 105(2), 582-587.
Nixon, K. C., & Carpenter, J. M. (1996). On consensus, collapsibility, and clade concordance. Cladistics, 12(4), 305-321.
Sumrall, C. D., Brochu, C. A., & Merck, J. W. (2001). Global lability, regional resolution, and majority-rule consensus bias. Paleobiology, 27(2), 254-261.
Wilkinson, M. (2003). Missing entries and multiple trees: instability, relationships, and support in parsimony analysis. Journal of Vertebrate Paleontology, 23(2), 311-323.


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 HIGH-RESOLUTION GROWTH SERIES OF TYRANNOSAURUS REX OBTAINED FROM MULTIPLE LINES OF EVIDENCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The errors and inconsistencies in the first review have been corrected.
Experimental design: The experimental design, especially those of the a posteriori tests, has been substantially improved. My original criticisms have all been satisfied with modifications to the tests or thorough explanation.
Validity of the findings: I see no argument with the validity of the findings. The author addressed my concerns over testing for sexual dimorphism and geographic variation sufficiently.
Additional comments: You gave the manuscript substantial revision beyond what was requested. I appreciate your exploration of different ways to identify the most mature specimen and sexual dimorphism in the ontogram.  Undoubtedly this is an important contribution to our knowledge of tyrannosaurs and I recommend it for publication in its revised form.


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 HIGH-RESOLUTION GROWTH SERIES OF TYRANNOSAURUS REX OBTAINED FROM MULTIPLE LINES OF EVIDENCE
Review round: 2
Reviewer: 2

Output:
Basic reporting: This revision of an already exceptional manuscript has only improved it. The clarity of the arguments have been strengthened, and the new version actually contains even more important descriptive and analytical information than the first.

The concerns of myself and the other previous reviewers have been appropriately addressed, either by incorporation into the present manuscript or by clear explanation as to why the suggestions cannot be implemented at the present time.
Experimental design: The modifications to the previous incarnation of the manuscript (for instance, the tree generation process) are clearly described and repeatable. The assumptions and recognition of the limitations of the procedures are made clear.
Validity of the findings: The author has presented strong evidence for his conclusions.
Additional comments: These points are trivial enough that they do not reach the level of me indicating "minor revisions"; nevertheless, they might mildly improve the manuscript.

I did find a minor typographical error on line 1133: "immatuirty"

The discussion of the dentary groove (starting on line 1150) should including the response paper of which the manuscript author was a contributor: doi: 10.1016/j.cretres.2016.02.007

---
I apologize for being slower to work my way through this version of the manuscript than other authors, but I wanted to be secure that the previous reviewers' comments had all been addressed and that the new material was fairly evaluated.


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 HIGH-RESOLUTION GROWTH SERIES OF TYRANNOSAURUS REX OBTAINED FROM MULTIPLE LINES OF EVIDENCE
Review round: 2
Reviewer: 3

Output:
Basic reporting: All of my comments are in "General Comments" below.
Experimental design: All of my comments are in "General Comments" below.
Validity of the findings: All of my comments are in "General Comments" below.
Additional comments: Dr. Carr has done a very thorough job of addressing my concerns. His responses to my comments and those of the other reviewers universally improved my confidence in the results, deepened the discussion, and clarified the figures. In particular, I applaud the reanalysis of the data matrix in TNT and the use of a single most parsimonious ontogram that avoids the problems associated with using consensus ontograms. I am also very satisfied with his discussion of Ontogenetic Sequence Analysis (OSA). I have no further important comments or changes to recommend. This manuscript stands as a landmark work for ontogenetic studies in vertebrate paleontology, and I urge its acceptance for publication.


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

Input:
Title: BEE DIVERSITY IN SECONDARY FORESTS AND COFFEE PLANTATIONS IN A TRANSITION BETWEEN FOOTHILLS AND HIGHLANDS IN THE GUATEMALAN PACIFIC COAST
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well-written. The study is well-justified in terms of knowledge gaps and the application of the results. The introduction and discussion draw from the literature to an appropriate degree. Figures and tables seem clear.
Experimental design: There seems to be a potential weakness in the design in terms of statistical analysis. There are three farms, each with three subplots of different land type in each farm. It is not clear how far apart these farms are or how distant the subplots within a farm would be. As such, there are only three replicates per treatment (i.e farm), so it makes it challenging for stats. There was quite a bit of sampling effort per farm, but it's repeated measures so the non-independence of the samples should be accounted for in the analyses. It's not clear to me if, how or when that happened. There is some reference to linear mixed-effect models, but I don't see any indication of the model structure (i.e. fixed vs random effects). ANOVAs are used throughout, but I wonder if that is the best approach. I would expect the error structure of bee collection data to be more appropriately measured using a Poisson or negative binomial distribution. In any case, the authors should indicate that their data satisfy the assumptions of whichever model they choose to use. It's my understanding that the Tukey HSD test is post-hoc so you should report the primary test (e.g. ANOVA) and then use Tukey HSD to determine differences. At times Tukey HSD is reported alone.

One of the authors is a well-known taxonomist with expertise in the region. However, there remain some issues with the treatment of the taxonomic component of the study. 1) Morphospecies are listed in the Appendix without even a family level identification. 2) Taxon authorities are not listed next to species that are identified. 3) The authors cite only two papers (Ayala 1999, Michener 2000) for the taxonomy, however the taxon concepts for the species do not and cannot come from these studies alone. I realize that taxonomists often know species by sight, but that doesn't mean the taxon concepts don't rely on the previous work of others. These should be cited in the same way you would cite an R package (p.s. you should cite your R packages, too!)
Validity of the findings: I would like to see a more careful analysis of the data. I don't think all the conclusions are necessarily supported by robust statistics.
Additional comments: This seems like a good effort to me, but the limited number of sample sites need to be dealt with carefully.


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: BEE DIVERSITY IN SECONDARY FORESTS AND COFFEE PLANTATIONS IN A TRANSITION BETWEEN FOOTHILLS AND HIGHLANDS IN THE GUATEMALAN PACIFIC COAST
Review round: 1
Reviewer: 2

Output:
Basic reporting: The overall manuscript is of high quality and self-contained.

The manuscript is very well-written in an English language that is clear and unambiguous. I did, however, notice a few minor linguistic mistakes, which I indicated in the attached PDF document.

The introduction is well-written and demonstrates how this research contributes to existing knowledge gaps. However, to my opinion, some background on the pollination of coffee is missing. This should not go into too much detail, as the actual pollination of coffee flowers is not studied in this research, yet some background would be useful in order to motivate why it is important to study bee diversity in Arabica coffee farms. Since the importance of pollinators and their diversity is different for Coffea arabica compared to Coffea canephora, I would also advice not to wait until the Materials and methods section to mention that this study concerns Coffea arabica.
The provided background information is supported by sufficient and relevant literature references.

The manuscript is structured in a professional way.

Figures and tables are of high quality, relevant and appropriately described. A few minor remarks:
-	Legend of Figure 4: the description of graphs b and c should be switched.
-	Legend of Figure 6: I think ‘(B) Bee richness’ in the legend should be replaced by ‘(S) Bee richness’?
-	Table 2: It is not explained in the legend why certain estimate-values are presented in bold.

Raw data have been made available in appendix.
Experimental design: This study presents original primary research that fits within the aims and scope of the journal.

The research questions are well defined, relevant and meaningful.

The methods are described in a detailed way. I do, however, have some minor remarks:

-	A map showing the location of the three studied coffee farms could be a nice addition as it would provide information about the distance between these studied farms, their size, and the area and position of the surrounding secondary forests and other land-use types (e.g., villages or primary forest). This is some valuable information that I think is lacking in the manuscript.

-	Perhaps some short motivation could be presented as to why the bees were grouped into honeybees, native bees, and stingless bees. What is the main ecological difference between these three groups of bees?

-	The reference to the different plot types (coffee, early, late) throughout the text and tables/figures is not always consistent. Sometimes this is referred to as ‘forest type’, ‘plot type’, ‘vegetation type’, or ‘landscape type’. To avoid confusion, I would recommend to stick to one term only.

Statistical analysis methods are carefully selected and the choices made are motivated.
Validity of the findings: No comment.
Additional comments: Dear authors, to my opinion, you performed an interesting and relevant study in a well-organized and high-quality way, and you wrote this manuscript in the same way. It was a pleasure to read this well-written 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: EXOGENOUS HORMONES INFLUENCE BRASSICA NAPUS LEAF CUTICULAR WAX DEPOSITION AND CUTICLE FUNCTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: An interesting and well written article, although the English could use a little polishing. I enjoyed reading the manuscript.

The introduction gives appropriate background on what is known concerning the role of hormones and environment on wax biosynthesis, and outlines the hypothesis being tested.
•	The pathways of wax biosynthesis are not described. This would make it difficult for someone not familiar with wax biosynthesis to interpret the data, and to understand the precursor/product relationships between the various wax components. A simple figure in the text or as a Supplementary figure should be provided. This should include the gene targets for the qRT-PCR.
Experimental design: Materials, methods and experimental design.

The experimental design and technical aspects of the manuscript are well described and appropriate. It is good to see that the authors used GC rather than GC-MS for the quantitate analysis and set the plants up in a randomized design. The authors were careful to use a leaf that was still expanding, with active wax biosynthesis, for the work. Classical permeability assays were used, water loss, chlorophyll leaching and toluidine blue staining.
•	The spot application of toluidine blue, rather than leaf or leaf segment immersion makes this assay rather subjective. It is not easy to see any difference in permeability based on the leaf pictures in figure 5. This is a common problem with this test, but the authors also provide data on water loss and Chlorophyll leaching. Are there any better pictures that could be used?
•	In this work, the hormones were applied to the soil. It would be helpful if the authors could provide some evidence that is an acceptable application method and that plants can take up MeJA, SA and ACC when applied in this manner.
•	For the qRT-PCR section, better identification of the target genes is needed. This is important because some targets appear to be members of gene families and because B. napus is a tetraploid. As an example, BnCER6 would correspond to BnaA07g24600D and BnaC06g25830D. Using EnsemblPlants would be one way to obtain this information, although the sequenced genotype is not exactly the same as the ones used in the paper. Alternatively, the authors could reference the target by its accession number.
•	From the primer sequences provided in Table S1 it is clear that the primers may not be appropriate for the study. Primer targets were checked using both the NCBI BLAST algorithm (https://blast.ncbi.nlm.nih.gov) and BLAST at EnsemblePlants (https://plants.ensembl.org).  The BnCER1 primer will amplify a single gene product, when two would be expected. A similar situation exists for the BnCER4 primer pair. It is highly recommended that the authors redesign the qRT-PCR primers to be gene specific, or to target both (or more) of the targets in this tetraploid species. Alternatively, evidence should be provided that the qRT-PCR is indeed targetting all of the relevant genes. If this cannot be provided, then the qRT-PCR should then be repeated to gain more accurate results. Use of ACTIN-7 as a control could also be reconsidered (see Yang et al 2014, Gene 538:113-122)
Validity of the findings: •	The first question to ask is whether the soil based application of hormones was effective. The authors have addressed this by looking at expression of PR1, PDF1.2 and ERF2. This should be moved to the beginning of the result section rather than the end.
•	It would be helpful if the authors could comment on the typical variation in wax amount seen in cultivars of B. napus. Are the wax loads of ZS9 and YY19 within the limits of typical variation, or are they unusual? Tassone at al 2016 (Industrial Crops and Products 79:77-83) would be useful to address this question.
The data presented in the manuscript indicates that there are some differences in wax load and composition in plants subjected to the hormone treatment compared to their controls. The two cultivars clearly respond differently.
•	Differences in the expression levels of genes associated with wax biosynthesis were also reported, although this may need to be reconsidered (see above). It is difficult to interpret the data without confidence in the qRT-PCR results.
•	The paper does not report the alkyl-esters (wax-esters) present in the cuticular wax. These can account for a significant proportion of the wax and play a role in permeability (Tassone et al 2016).
The authors studied cuticle permeability using a number of methods. This is good, but raises questions.
Please comment on:
•	Water loss over 150 minutes is greater for control plants of ZS9 (11.5%) than control plants of YY19 (8%) (figure 3), whereas chlorophyll leaching over 150 minutes is slightly greater for YY19 than ZS9.
•	The lines differ significantly in leaf area for the control plants (table 3) suggesting different growth characteristics.

Conclusion
•	The authors comment that all three hormones have fairly similar effects on the cuticle. This is a little misleading as the individual hormones appear have different effects on wax composition (Figure 2B) and gene expression (Figure 6).
•	The authors conclude that changes in wax profile caused changes in cuticle permeability (line 378). This is not supported by the data. Correlating cuticle permeability to wax chemical composition is speculative without considering cuticle thickness and structure. This should be assessed using electron microscopy.
•	The evidence does supports the hypothesis that hormone treatment may influence wax composition, but in contrasting ways for the 2 varieties. This is an intriguing observation.
Additional comments: The authors describe selecting two lines with different wax coverage for study (line 106). These lines also seem to differ in leaf area (table 3) additional phenotypes should be reported if known. Ideally, near isogenic lines differing in wax load would be better for comparison.

Based on figures 1 and 2, hormone treatment of YY19 does not change wax load, but increases secondary alcohol levels with a corresponding decrease in total alkanes (primarily the C31 alkane). The lack of increase of the C29-ketone is worthy of comment considering MAH1 is thought to be responsible for the synthesis of both the secondary alcohol and ketone (Greer et al 2007)


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: EXOGENOUS HORMONES INFLUENCE BRASSICA NAPUS LEAF CUTICULAR WAX DEPOSITION AND CUTICLE FUNCTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript "Exogenous hormones influenced the cuticular wax depositions on Brassica napus leaf and the cuticle function" by Yuan et al. describes the characterization of two cultivars of Brassica napus and how they respond to different phytohormones in terms of leaf wax accumulation, leaf wax composition, epidermal permeability, and cuticle-related gene expression.

I appreciate that the language was clear and easy to understand. Overall, the English was quite good. A very minor review of the English should be sufficient to make it even more comprehensible.

The article is well structured with a logical flow. The majority of the figures are well designed and easy to understand. In figure 5 I found the close up images of toluidine blue staining (bottom rows of A and B) difficult to discern, the images were very dark and it was difficult to make out any of the detail (especially in printed form). I also noticed some extraneous lines in Figure 1. Apart from that, the figures were well prepared.

One detail that I think should be included in the figure legends is the statistics used for each analysis. These are described in the materials and methods but I find it useful to include details about whether a 1-way or 2 way ANOVA was used including the post-hoc analysis.
Experimental design: I do believe that this manuscript fits within the aims and scope of this journal.

I do, however, have several concerns.

1. The gap of knowledge that this research fills in is not clearly stated.
2. Some methods were not clearly defined
2.a. Lines 125 and 126: It was not clear what the phytohormones were dissolved in. MeJA is difficult to dissolve in water. Was MeJA perhaps dissolved in ethanol? Were the control plants watered with nutrients containing an equivalent amount of solvent (e.g. methanol)? (were these "vehicle controls" or "mock treatments"?
2.b. For the experiment on the effect of SA, MeJA, and ACC on cuticle permeability it is stated in the text (line 269) "....when compared with water sprayed leaves..." this seems to imply that for this experiment the phytohoromones were applied via spraying. However, there is no mention of a spray phytohormone application in the materials and methods, only an irrigation-based treatment. Can the authors please clarify?
3. I noticed in the raw data under the "water loss" tab that for the ZS9 cultivar replicate 3 of the CK (control) and replicate 7 of the MeJA treatment are the averages of the other 6 replicates. Can the authors please explain this?
Validity of the findings: I do feel that this research does present some interesting results. However, I feel like the presented data and conclusions aren't entirely in agreement and these discrepancies weren't well discussed in the paper.

One of the major assertions of the paper is that the data presented in the manuscript are in agreement with data from other species. Line 310-311: "our results on alkane contribution to cuticle barrier function match previous results from other species...." The general trend from other species is that there is a negative relationship between alkane content and epidermal/cuticular permeability. That is to say that plants with elevated alkane contents have less permeable epidermis/cuticles and vice versa. The data in this manuscript show that ACC treatment, of both cultivars, results in decreased amounts of alkanes yet the ACC-treated ZS9 cultivar demonstrates lower water loss and chlorophyll leaching rates than control plants (and plants from all other treatments). The authors conclude that their data are supportive of the model that alkane content is negatively correlated with cuticle permeability...the results do not support this conclusion.

I think that these results present a unique discovery but the discovery hasn't really been discussed or framed properly.

It is curious that the cultivars have different responses to phytohormones in terms of epidermal permeability and associated phenotypes. Incorporation of more discussion on this topic (i.e. why do the cultivars differ in these responses) would most certainly strengthen the manuscript.
Additional comments: Overall, I find that this manuscript presents some interesting results. However, I feel that addressing some of my concerns would certainly improve the quality 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: EXOGENOUS HORMONES INFLUENCE BRASSICA NAPUS LEAF CUTICULAR WAX DEPOSITION AND CUTICLE FUNCTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: Data are over-interpreted.
Additional comments: The manuscript by Yuan et al., presents a large set of analytical, physiological and molecular data to describe the effects of the plant “stress” hormones salicylic acid (SA), methyl jasmonate and ACC, an precursor of ethylen, on cuticular waxes and cuticle barrier properties. Using sophisticated GC-MS analysis the authors detected diverse changes in the monomeric composition of leaf waxes upon hormone treatments in two Brassica napus cultivars (ZS9 and YY19). The studies were complemented with physiological assays to evaluate potential changes in leaf cuticle permeability and molecular studies monitoring the expression of wax biosynthetic enzymes.

I enjoyed seeing the comprehensive analytical data set on the leaf wax composition. The data are appealingly presented in adequate graphs. I liked that appropriate statistical tests were applied but I wish standard deviations (biological variation) instead of standard errors would be shown in the future. I also have some concerns regarding the interpretation of the data and the conclusions drawn.

The data of the permeability assays presented in figure 3 and 4 look to me like two-phase-kinetics. According to Zeisler-Diehl et al. 2017 (https://doi.org/10.1093/jxb/erx282) the final water loss or Chlorophyll leakage values are less indicative, but the slope of the changes in the linear phase best describe the permeability. Using the slope in the linear phase as a measure of permeability a different picture arises with some hormone treatments increasing water permeability (e.g. MeJA) and other hormones slightly decreasing water permeability. These data should be re-evaluated.
Consequently some parts of the discussion may need to be edited. Furthermore based on the current data the general statement (line 301) that the changes in permeability (Line 295, decrease in ZS9) are accompanied by shifts between more and less polar monomers is not justified since the alkanes in ZS9 do not change.
As the effects of the hormone treatments on wax monomers are very different in the two cultivars a direct linkage between hormone treatment and changes in wax monomers is not established. The authors should consider investigating a dose depended response of the different hormones to support their interpretation.
Although exciting, the discussion of the expression data (Line 342ff) is a bit biased. The explanation of the discrepancy between high CER1, CER3 expression and a reduction in alkanes by the specificity of these enzymes for C29 compounds seems over-interpreted. First the authors used “primers designed to the conserved region of the gene family” and thus don’t know which specific CER1/CER1-like etc. they amplified in the expression studies. Secondly, in YY19 the nonacosane only slightly changes (decreases and increases) by the different hormone treatments (Table 2) but all three hormones drastically increase the expression level of CER1. In ZS9 MeJA strongly reduces the expression of CER1 but nonacosane remains unaffected.
In my opinion the conclusions (Line 370ff) do not represent the data. “all three hormones had fairly similar effects” is totally different from the data presented when looking at waxes, permeability and expression. This should be rephrased much more carefully.
Although the authors discussed potential effects of the hormones on leaf growth and development they should also consider hormone induced effects on the extractability or “metabolism” of compounds (crosslinking, ester formation/release, etc.) in their discussion.

Minor:
Line 29 write “ethylene” in abstract.
Line 77 permeability of plant cuticle rather than “surface waxes”
Line 129 “elicitor” is not correct in this context.
Line 168 what was the room humidity / how was it kept constant?
Line 310 rephrase. The contribution of alkanes was not specifically investigated.
Figure 6: Clarify “control”. Control conditions or control gene ACTIN7?


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: EXOGENOUS HORMONES INFLUENCE BRASSICA NAPUS LEAF CUTICULAR WAX DEPOSITION AND CUTICLE FUNCTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No further comment
Experimental design: Generally good.
Validity of the findings: See general comments
Additional comments: The authors have thoroughly assessed and responded to the comments from the first review.

Addition of the pathway figure and the brief description of wax biosynthesis will certainly be helpful.

Point 4. Changing the contrast on the Toluidine blue figure has not really resolved the issue. I find the data unconvincing. Leaves have spots of different intensities and the spots shown in the magnified images could easily have been chosen to fit a hypothesis. This is really a problem of the technique and I have no criticism of the authors. I would suggest removal of this figure. Water loss and chlorophyll leaching assays appear more convincing,

Point 7. Adding accession numbers has improved the manuscript as it enables better evaluation of the target genes. Confidence in the choice of primers and target genes really requires an improved assembly of the B. napus genome sequence, which would be beyond the scope of this paper.

Minor improvement of the English is required.


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: EXOGENOUS HORMONES INFLUENCE BRASSICA NAPUS LEAF CUTICULAR WAX DEPOSITION AND CUTICLE FUNCTION
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The resubmitted manuscript by Yuan et al., shows advantageous improvements. The reorganisation (e.g. expression first) make sense. Similarly the removal of TBO staining. Also most of my comments and of other reviewers have been addressed in a satisfactory way. This includes all minor editing, adding details and rephrasing and more carefully presenting statements.
However I am not completely satisfied by the response to comment 6) – permeability. In the response the authors talk about a water loss and chlorophyll-leaching RATE. This is exactly what I requested. A RATE is a value (e.g. % water loss) per time unit. Such calculated rates (e.g. % water loss per min or % chlorophyll leaked per hour) are not presented or listed in the manuscript. Figure 3A shows the accumulated % water loss over a selected time period, not water loss per time unit. E.g. After about 120 min the % water loss per 15 min might be very similar for all four treatments.
In addition the permeabilities shown for ZS9 are clearly two phases (e.g. positive Y-axis intercept of trend lines in Fig 3) and are not one “linear phase” as the authors comment. Obliviously the water losses of ZS9 are different in the first 30 min compared to the rest of the time. To make my point a bit more obvious to the authors I made a graph using their data (provided in file peerj-44364-total-data-hormone-canola_wax.xlsx, data sheet “water loss”) but starting at the 30 min time point/ using the 30 min time point as reference. From 30 min on the curve looks somehow linear. As a result ACC is almost identical to control whereas SA and MeJa cause higher water losses. Until here I only focused on Fig 3A but similar conditions and changes may apply for other figures.
However, I do not insist on my point of view and I might be totally misguided. Nevertheless the authors are free take this example to reconsider some views.

I might be also mistaken in reading the data provided in file peerj-44364-total-data-hormone-canola_wax.xlsx (data sheet “water loss”). Nevertheless I got the impression that either data or labels in that table are not consistent with data presented in the Figure 3A, or data sets are mixed up. Reading the numbers from the figure (e.g. using a ruler) and comparing them to the data in the excel sheet it looked to me that:  What is “Control” in the data table (mean 10.4) is “ACC” in Fig 3A,  “SA” in table is “Control” in figure, “ACC” in table (mean 8.4) is “MeJa” in figure and “MeJa” in table is SA” in figure. If this really happened, than data between varieties and treatments may better fit at the end.
However, I may have read that table in an incorrect way and everything is fine. If so I greatly apologize for the confusion.


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: CONTRIBUTIONS OF LATIN AMERICAN RESEARCHERS IN THE UNDERSTANDING OF THE NOVEL CORONAVIRUS OUTBREAK: A LITERATURE REVIEW
Review round: 2
Reviewer: 1

Output:
Basic reporting: This a very interesting and timely report describing the research in a region, now affected by the COVID-19 significantly.
Experimental design: Good design.
Validity of the findings: Descriptive, valid findings.
Additional comments: Good initiative. My only recommendation is to update as much as possible, as this topic change daily.


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: CONTRIBUTIONS OF LATIN AMERICAN RESEARCHERS IN THE UNDERSTANDING OF THE NOVEL CORONAVIRUS OUTBREAK: A LITERATURE REVIEW
Review round: 2
Reviewer: 2

Output:
Basic reporting: This paper employs clear, unambiguous and professional scientific writing throughout the manuscript. The English is generally excellent. The entire paper was clearly laid out and easy to follow. One thing the manuscript's paper asks is "What are the contributions of Latin American researchers in understanding the nCoV outbreak?" It would be worth considering adding this somewhere in the early part of the manuscript in a style befitting the excellent standard of English employed in the paper.

Throughout the manuscript, there is a necessary use of abbreviations, acronyms, author names necessitated by the language of the pandemic as well as the style sheet used by PeerJ—and yet the text does not feel weighted down. Figures and tables are cited within the text, and readers are referred to the back of the paper or appendices where they can be viewed. Overall, the paper reaches a high professional standard in structure. Only a few quibbles around the use of language and expression, which I will leave to later in this section.

A literature review of this type is of considerable interest to scientists within the countries examined, and by other countries. A review and characterization of the medical literature of COVID-19 is indicative of clinical, scientific and research activities in a region affected by the pandemic. While not a systematic exploration, this paper is an important type of bibliographic analysis. Another strength of the manuscript is how it teases out national perspectives where there is interdisciplinarity on research teams, and when cross comparisons with those countries affected by the pandemic in similar / different ways. This will be of value to research funding bodies, policy makers and researchers in other fields.
Experimental design: The study design is stated clearly in the title of the manuscript, and elucidated in the study methods. At times, I was looking for a bit more clarity which I will describe below.

A literature review whose methods are explicitly stated can be very effective in characterizing a corpus of literature. While not a systematic review, as long as a search is well-articulated and performed, and a system is described as to how the authors found and evaluated papers, this type of review can be useful. To orient readers from the outset as to your aims and purpose in the manuscript, I would recommend adding a sentence or two such as, "The aim of this paper is to do X" and "our method is a literature review". Do it as early as sentence #2. You describe your aims at lines 23-29, but it feels as though it would make sense earlier, "right off the top", and then think about deleting the sentence comprised by lines 20-22 (or move elsewhere).

With respect to the "Introduction", this section is sufficiently detailed and provides good background to the emergence of the Novel Coronavirus worldwide. Nearer the section's end, at lines 61-65, the purpose of the review is stated finally. The English could read a little more clearly; for example, say "literature survey" or "survey of the literature") not just survey) and say "submitted by these types of international research groups".

Search strategy 2.1 is appropriately reported but could be clearer and made more transparent. An overall statement should be made about why you selected those search engines, websites and biomedical databases. Was it because they were "openly available" as in PubMed? Easy and simple to use? Also, indicate the interfaces used if relevant. The keywords reported in your search strings as reported (you call them a "search equation" which is a phrase I have never heard before) are OK but some major omissions are evident.

For example, re: SARS-CoV-2 and COVID-19, why didn't you use the keyword phrase "novel coronavirus" OR "novel coronavirus disease*"? With respect to your geographic / Latin American countries filter, it is minimal and not sufficiently sensitive to capture all papers. As a result, some statement around the lack of consulting a search librarian expert, or doing a rather simplistic search in your paper, should be made. It is not clear to me whether a better search would have resulted in the retrieval of a more representative set of papers or not, but it's worth saying that it might be a limitation.

Sections 2.2--23 clearly state what the authors did in terms of data analysis but it was not clear how you selected the various section headings "Bibliometric analysis” and the following “Phylogenetic and molecular understanding” and “medical contributions” sections. Does this come out of a system of classifying papers? Or themes that emerged? It would be useful to have a sentence about how you came to those categories.
Validity of the findings: The validity of the findings in a literature review is contingent on a close reading of the papers, and a process chosen to characterize the papers. Again, it's not a systematic or even a rapid review. However, part of a literature review or survey of papers is to describe the papers retrieved in sufficient detail to know what they discuss. I feel that this is the leanest part of the paper. You cover other aspects well which are directly related to the aims of the paper, such as "Countries of authors" and affiliations (submitting lab at Table 2) which are sufficently described and illustrated.

Figure 2 is welcome. It would be interesting to see a range of topics "at a glance" as you have elucidated in the text of the paper but expressed as a table or even a word cloud.

The limitations of the search should be added to the conclusion.
Additional comments: This is a good paper, congratulations. I enjoyed reading it and hope you can provide periodic updates.


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: CONTRIBUTIONS OF LATIN AMERICAN RESEARCHERS IN THE UNDERSTANDING OF THE NOVEL CORONAVIRUS OUTBREAK: A LITERATURE REVIEW
Review round: 2
Reviewer: 3

Output:
Basic reporting: This review considers the contribution of Latin American research concerning Covid-19, which sets it apart from other reviews on this disease. Why is it important to consider the regional contributions to new science on this subject and does this contribute to understanding the unique aspect of every society when dealing with this pandemic? This should be covered in your introduction and conclusion sections.
The English language in this manuscript should be reviewed. There are many instances where the English language could be polished and I have indicated some of them below.
In general, when describing results reported by other studies, the past tense should be used.
On your abstract: “we searched in different databases, such as Scopus…” could be written as “we searched different databases, such as Scopus…”. “Finding that its contribution was 2.7…” would be clearer if written as “finding that the contribution of this continent was 2.7…”. Also, it is not clear from this sentence what this 2.7% represents. Finally, “visibility to the region contributions” could use an apostrophe (region’s). This is also true for line 61.
Line 21: on the first time the word “national” is mentioned, it could be deleted. Or maybe this affiliation could be better explained: are these 1797 affiliations or 1797 mentions to Latin American countries in the affiliations section?
On the introduction, the number 170000 could be written as 170,000. The same applies to 100000 and 5500.
Line 39: “as shown in table 1”
Line 43: “Coronaviridae, has a zoonotic origin, and can remain on some surfaces for considerable periods.”
Lines 45-47: this sentence needs rephrasing.
Line 81: “due to” should be replaced by “because”.
Line 101: “manually excluded eight additional publications due to affiliations from New Mexico…”
Line 124: “in consulted databases” can be deleted.
Line 127: delete “the.”
Line 130: The expression “science gap” is not very clear. Is it a gap in science funding, technology, facilities?
Line 144: “second, that if…”
Line 150: “a higher ability to infect humans when compared to other coronaviruses”
Line 157: “receptor, providing SARS-Cov-2 with a higher infectiousness”
Line 169: delete “While”
Lines 182-185: Please rephrase these sentences with active voice (table 2 summarizes…).
Line 202: “the geo-positioning of some cases and the heterogeneity of the outbreak progress among countries have been reported”
Line 212: “Meta-analyses and the search for potential treatments”
Line 221: “Oxygen support in critical patients…”
Line 229: “Concerning respiratory viruses”
Line 252: “use” should be deleted
Line 262: delete “regarding”
Line 303: “take into account”
Line 205: “the latter being a treatment that not all medical centres”
Line 309: “Finally, mental health care”
Experimental design: Line 83: Were there any articles with Latin American affiliation and no Latin American authors? Do you believe these articles should be completely excluded? Although they do not contain Latin American researchers, if there are any, you could mention them in your analysis since they were at least partially conducted in Latin American institutes.
Line 111: to avoid confusion, you could explain what the national affiliations correspond to. If one paper has authors affiliated to two different institutes of the same country, does this count as one national affiliation or two?
Lines 166-168: this information should be moved to the next paragraph.
Lines 226-228: this sentence could be rephrased (high levels of NLR and low levels of LCR). Was this confirmed or is this an initial hypothesis?
Lines 232-234: this study should be cited.
Validity of the findings: Reviews should be very cautious when reporting results from other studies; wording should be careful so as to not twist other studies’ results or imply different meanings.
I understand that your review considers Latin American contributions to understanding the epidemic, but the Latin American contribution should be compared with information published worldwide especially when there is a lack of published information or contradictory studies.
Line 39: This sentence is not very clear. Did you mean that the amount of testing per million inhabitants is low because of indigenous populations, vulnerable groups, and Venezuelan refugees? I have checked your reference (Torres & Sacoto, 2020) and the article stated that this happened due to limited test availability, and case monitoring was challenging because of the lack of cellphone use among these populations. Regarding Oliveira et al. (2020), I have not found any information on this subject, please check this reference citation.
Lines 141-142: was the highest of what? Among the analyzed viruses? Which ones?
Line 224: how old is this group exactly? What do you mean with most of the data came from China? Is there a need for more studies? Please make this clear.
Line 287: which are these positive effects of hydroxychloroquine and chloroquine on diabetic patients? Is this regarding blood glucose levels or COVID-19? According to your reference, these medications affect glucose levels. This should be made clear.
Line 320: It is important to state in which country these questionnaires were applied, since cultural and social aspects could also be involved in this matter, and please clearly state that their results represent that population and could not necessarily be the same for all countries.
Line 331: It is important to clarify if this lack of information about the use of face masks is observed only in studies published by Latin American authors or if this is a global issue.
Line 367: I believe the effects of angiotensin receptor blockers on Covid-19 infection have not yet been clearly established (see Reynolds et al., 2020, doi: 10.1056/NEJMoa2008975), therefore this topic could either relate the Latin American articles with other papers or clarify that this is a hypothesis.
Additional comments: No additional comments


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

Input:
Title: A NEW AGGRESSIVE XENOGRAFT MODEL OF HUMAN COLON CANCER USING CANCER-ASSOCIATED FIBROBLASTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Methods section should be written in preterit (line 143,144). αSMO in line 287, did the authors mean αSMA? In line 156 replace the comma with a point.
References 6 and 24 are the same (Rashidi et al., Anticancer Res 2000; 20:715-22). In terms of CAF, one of the most comprehensive reviews is missing (Kalluri, Nat Rev Cancer 2016; 16:582-598).
Figure 1: In the figure legend αSMA is missing, for green only VIM and CK19 are indicated. In the legend the scale bars are indicated with 30 µm but under the bars within the pictures is written 20 µm. An explanation about the insert in 1C is missing.
Figure 2: The label and numbering of the Y-axis are partially overlaid. Depending on final figure size the axis numbering might be too small. Moreover, statistical analysis of standard deviation would gain more relevance than the indicated SEM.
Figure 4: misspelling in figure legend: HT29 + FN should be HT29 + NF; also in Table 2 head of column
Figure 3, 4, 5, S1, S2, S3: No or too small scale bars are provided. The specification of magnification is not sufficient, for example after enlarging an image section the cells appear larger but the magnification stays the same.
Experimental design: The authors confirmed in the online form that for animal handling the name of the approval organization and approval number appear in the Methods section, but it does not.
αSMA is missing in methods section of immunofluorescence.
In Table 1: The antibodies were used for immunofluorescence and not for immunohistochemistry as stated. What is meant with dilution 11:00:00 for Vimentin antibody?
The authors should provide details how they performed growth kinetics, i.e. were the counted cell replated or were samples for each time point plated initially? Might the decrease in cell number for CAF be a result of overgrowth/contact inhibition?
Validity of the findings: In the results section (line 193) the authors state that cells had positive immunofluorescence to vimentin and Col11A1, but vimentin staining is not shown in Fig S1. How was the staining performed? It does not look like immunofluorescence as described in the methods section.
Did the authors performed negative controls esp. for CK19 staining to exclude unspecific staining? If there are differences in SMA and Col11A1 expression between normal fibroblasts and CAFs as stated in the discussion (line 310) the authors should provide resp. data.
Additional comments: The authors show that CAFs support tumor growth of HT29 colon cancer cells more than normal fibroblast. They show that tumor cell injection in pancreas head results in tumor growth and metastasis with the finding that the larger the tumor the more metastases were observed. These are interesting findings, but in the discussion section relevant literature regarding these findings is missing.
That CAFs support tumor and xenograft growth has been widely described (Olumi et al., Cancer Res 1999;59:5002–5011; Orimo et al., Cell 2005;121:335–348; Kalluri et al., Cancer 2006;6:392-401; Erez et al., Cancer Cell 2010;17:135-147; Hammer et al., Neoplasia 2017;19:496-508; and others).
Also the injection in pancreas for tumor growth also of small injected cell numbers was already published (Partecke et al. Eur Surg Res 2011;47:98–107; Sato et al. Int. J. Mol. Sci. 2017;18:1678).
It would be interesting using a tumor cell line that shows less metastasis than HT29, as this cell line regularly develops metastases even if subcutaneously injected with correlation of tumor burden and metastases load (Jojovic, Cancer Letters 2000;152:151-156; own observation), or to compare different cell lines.
Furthermore it would be very interesting to detect human stromal cells in tumors using human specific antibody to look whether there are differences in the amount of integrated human stromal cells in tumor stroma in dependence of injected cells (NF vs. CAF).


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 NEW AGGRESSIVE XENOGRAFT MODEL OF HUMAN COLON CANCER USING CANCER-ASSOCIATED FIBROBLASTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The overall text is written in proper English, albeit there are some mislabeling/spelling errors that need to be corrected.

Spelling error in M&M: “Oncomatrix” should be “Oncomatryx”
Experimental design: Author should describe the methods used for assessing growth of fibroblasts.
-Figure 3: No indication in the legend of what the circled region in panels (a), (c) and (d) represents.
-Figure 4: Mislabeling/spelling error in the title should be “NF” instead of “FN”.
-M&M: Authors mentioned using the proColXIa1 to stain the tissues. However, there was not mentioned of the staining pattern in the mouse tumour to indicate the presence/absence of transplanted human CAFs/NFs.
Validity of the findings: Overall it is an interesting model but some of the concerns regarding source of CAF, nature of transplantation needs to be addressed.
Additional comments: In this study, the primary aim was to describe a new animal model to study metastatic colon cancer, which may eventually be used for evaluation of new therapies. Unlike conventional PDX, the authors demonstrated that the co-transplantation of cancer-associated fibroblasts (CAFs) with human colon cancer line (HT-29) lead to an increase in hepatic nodules, tumour burden, lung metastases and severity score. This is in comparison to mice that had tumour alone or tumour with normal fibroblasts (NFs). In addition, they evaluated the difference between their patient derived CAFs and NFs; showing that CAFs have a higher growth and NFs. Overall, the authors discuss on a new area of cancer research, which is the study of how the tumour microenvironment affects cancer and metastasis development. It is in agreement with the authors that the use of conventional PDX strategies to date may be limited due to the lack of stromal and immune factors. Here the authors attempted to address this by showcasing their efforts to develop a metastatic mouse model with the addition of CAFs.
Authors should provide comments on following points:

1. Authors derived CAFs from the peritumoural area instead of the core tumour. Given the heterogenity of CAFs and hypoxic tumor core one wonders if CAFs from tumor core and peritumoural area are functionally similar.

2. The authors transplanted their tumour (+/- fibroblasts) in the pancreas. How this method is similar or diffrent to other well-known established methods such as orthotropic models, experimental liver metastasis (intra-splenic) or experimental lung metastasis (intravenous). Does this method recaptures the natural tumour development?

3. How did the authors decide on the ratio of fibroblasts to tumour cells? Is this from literature/ understanding of the ratio of stromal to tumour in colon cancer?


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 NEW AGGRESSIVE XENOGRAFT MODEL OF HUMAN COLON CANCER USING CANCER-ASSOCIATED FIBROBLASTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Pass
Experimental design: Pass
Validity of the findings: Pass
Additional comments: Authors have address all the questions thoughtfully. I recommend the acceptance of 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: ISOLATION, CHARACTERIZATION, AND CRYOPRESERVATION OF COLLARED PECCARY SKIN-DERIVED FIBROBLAST CELL LINES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The work of Borges et al. is a continuation of the previous works of the same group on collared peccary. They reported the isolation, characterization, and cryopreservation of fibroblasts from the collared peccary.
Experimental design: There is incomplete information and data presentation that should be revised in the current version of the manuscript.
Validity of the findings: The presented data requires more clarification.
Additional comments: There is incomplete information and data presentation that should be revised in the current version of the manuscript.

L143: Provide information about the trypsinization process (Concentration of trypsin and duration for cell separation) that was used for fibroblast isolation from other skin-derived cells, and also that was used for subsequent passaging.

L150 & L184: Provide figures for the cultured cells in bright-field or normal light microscope. Fibroblasts are mostly spindle morphology, however, in the provided figure of vimentin the cells look irregular shape. Also, provide if you have ever observed epithelial cells, keratinocytes and other cells with the primary cultured explants.

L162: I suggest separating this sub-heading into 2 parts: (1) The morphological characterization of fibroblast, (2) Vimentin immunofluorescence.

Figure 1: I think there are some missed parts of Fig. 1. Describe the abbreviations EG, SUC, and DMSO in the figure caption. What are A, B, C, D, and E? What is meant by scale bar?!

Figure 2: Revise the caption; this is a “merged” image, not “double-stained”.

Figure 3: Why there is a drop in cell viability from 30 to less than 10 X 10^4 in both cells? Is trypsin used appropriately? In L287: latency does not mean a drop in the cell viability if the cells were perfectly dissociated. Moreover, Population Doubling Time is very long, it is more than 8 days! It is very long when compared to your previous report, which was around 2-3 days. https://doi.org/10.1007/s11626-018-0270-6

Table 1: Delete the data of “total number or sum” for avoiding misunderstanding.

L48, and throughout the manuscript: you provide the actual P-value, therefore, replace “>” with “=”.

L50: Indicate the P-value.

For paragraph 84-90: support this statement with recent trials for isolation of skin-derived fibroblasts from other mammals and the usage of trypsin for discrimination between different cells, and cite these two references: (doi: 10.7717/peerj.4302 & doi: 10.3390/ani9060378). You can use the results of these two references for the comparative skin cell culture that you have described in L309-329.

L292: Provide images for results of ROS fluorescence and mitochondrial membrane potential.

The title: please correct “skin-derived fibroblast cell lines”.


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: ISOLATION, CHARACTERIZATION, AND CRYOPRESERVATION OF COLLARED PECCARY SKIN-DERIVED FIBROBLAST CELL LINES
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the manuscript, the authors reported that they have established fibroblast somatic cell lines from skin biopsies of collared peccaries. They reported that the cryopreservation process does not affect the viability, metabolic activity, and proliferative activity, except ROS and ΔΨm. The established somatic cells are prerequisites for somatic cell nuclear transfer experiments and other biological experiments. In my view, established somatic cell lines can be used for animal cloning experiments in the future.
Experimental design: This study aimed to establish somatic cell lines of collared peccaries. The multiple experiments were made to prove that methods used to establish somatic cells are efficient, and all culture conditions supported the efficient culture and maintenance of established cells.
Validity of the findings: the results are well stated, and all findings supported the original research question.
Additional comments: In the title, cell lines instead of cell line, since, five cell lines established.


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: ISOLATION, CHARACTERIZATION, AND CRYOPRESERVATION OF COLLARED PECCARY SKIN-DERIVED FIBROBLAST CELL LINES
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.	Manuscript was poorly written. The writing of the manuscript should be reviewed.
2.	The introduction does not justify the relevance of the objectives set at work.
•	The authors present a work based on the isolation of fibroblasts from adult specimens of Collared Peccary (pecari tajacu) to generate a biobank for conservation purposes.
The International Union for Conservation of Nature (IUCN) considers stable the pecari tajacu population and only establishes as objectives to investigate (https://www.iucnredlist.org/species/41777/10562361#conservation-actions):
•	Population size, distribution & trends.
•	Harvest, use & livelihoods
•	Population trends.
•	The authors justify the objectives of the work by being aimed at the conservation of other species phylogenetically (White-lipped Peccary-tayassu peccary and Chacoan Peccary-catagonus wagneri). In the discussion of the work, they justify that the isolation of fibroblasts will allow their use in cloning by SCNT. But the use of SCNT for biological conservation requires that the donor cell belongs to the threatened species.
Moreover, until now SCNT has not had the potential to be considered as a true option for biological conservation efforts.
c) For the establishment of a somatic cell bank of an endangered species, the isolation of pluripotent or multipotent cells such as mesenchymal stem cells (Liu et al 2013. Stem cell and development) is of greater relevance.

3. The figure legend 1 describes containing 5 figures: A, B, C, D and E. On the other hand, the image corresponding to figure 1 does not correspond to the figure legend and consists of only 1 figure.
Experimental design: 1.	The sample size is statistically insufficient and does not present a correct sex ratio nor show adequate age distribution. The authors have only used 5 specimens of the species, four females and one male and all adults.
2.	The different experiments do not show n or the number of replicates performed. Cell viability by trypan blue, metabolic activity, ROS and population doubling time analyses do not show n and number of replicates.
Validity of the findings: The experiments have not been done with sufficient scientific rigor, nor are they explained with sufficient clarity.
1.	How many times did the authors clean the tissue samples and what solution did they use? How did the authors clean the tissue samples? Centrifugation? Speed?
2.	What is the subculture ratio?
3.	The authors do not mention the surface of the culture plates used.
4.	What are penicillin, streptomycin amphotericin, and Trypsin concentration?
5.	What are the cellular passage and thawing protocols used?
6.	For cell viability, how many cells were determined from a field?
7.	How many replicates were done for each technique?
8.	For detection of primary cultures contamination, the authors do not use any specific technique for bacterial, fungi (Doyle et al 1990) mycoplasma (Masover and Becker 1998; Freshney's method 2000) or virus detection, using positive controls.
9.	Authors do not perform a karyotype analysis of the cell cultures.
10.	Figure 3: cell proliferation after the 8th day was reduced. The reason is the cells began to enter the plateau phase because of contact inhibition?
Additional comments: No additional comments