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: DOUBLE LIVES: TRANSFER OF FUNGAL ENDOPHYTES FROM LEAVES TO WOODY SUBSTRATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, this paper was well written. Figures were professionally made, the writing was well organized.

Some things that need to be fixed:

Data: I didn't see mention of raw data deposition (sequences to NCBI, tables of data in Dryad/Figshare/etc).

Citations: I was surprised to see only one of Takashi Osono's papers cited or discussed, as many of the articles he and colleagues have written are closely related to the topic of this paper. For example, see

Fukasawa, Y., T. Osono, and H. Takeda. 2009. Effects of attack of saprobic fungi on twig litter decomposition by endophytic fungi. Ecological Research 24:1067-1073

It also seems that the Discussion in particular would be strengthened by engaging more thoroughly with the existing body of work on post-senescence decompositional trajectories of fungal communities in litterfall. One that comes to mind in particular:

Voříšková J, Baldrian P.  2013. Fungal community on decomposing leaf litter undergoes rapid successional changes. ISME J. 7(3):477-86. doi: 10.1038/ismej.2012.116
Experimental design: My primary concerns with the experimental design are twofold. One is that there are no negative controls, which seem to me to be important for demonstrating that the fungi growing on the tongue depressors are not a result of contamination. The second issue is mentioned by the authors: the samples are not independent, since all samples in a given container are more likely to be similar to one another (e.g. Figure 3). Since the results are not independent, there are some elements of the results that I do not think are appropriate to report as they are currently. For example, any estimate of the mean number of taxa per leaf is likely to be spurious, since the most parsimonious explanation fo the data would be that sporulating fungi in a box contaminated other samples in that box, and so the attribution of which taxa came from which leaf fragments is at best unclear.
Validity of the findings: As mentioned above, I did not see where the raw data were deposited. It would also be useful to have the R analysis code provided. This could be either in an archival repository like FigShare/Dryad/Zenodo, or else as a supplement to this manuscript.

Given the issues with the experimental design, I think the authors need to rethink what results they present and consider what information is robust to the issues of cross contamination and the related lack of independent replication between samples.

Given the issues and as written, I don't think all of the findings are necessarily valid, but I could imagine restructuring the paper to focus on the subset of things that can still be learned from the experiment.
Additional comments: Overall, I applaud the authors for engaging (in a series of papers they have now published and some in review) with the understudied topic of the ecological roles of endophytic fungi. They have brought new ideas/thinking to the discussion that I think have a lot of merit (foraging ascomycete hypothesis, viaphytism) for explaining observations of these systems in nature. At their core, these ideas are likely to be really useful ways to think about what these organisms are doing in the hosts/ecosystems they inhabit.

My overall concern with this manuscript, however, is the emphasis on the creation of a new term, 'viaphytism'. I think the concept is useful, and I see how the previous terminology used, 'foraging Ascomycete', is not appropriate, since numerous Basiodios seem to follow the same pattern, but don't see how a new term is necessary given the existing terminology used to describe similar concepts (latent decomposers, latent saprotrophs). I wonder if the argument the authors are making, that what many endophytes are doing in transitioning between substrates of different types (live leaves -> dead woody debris) as opposed to staying put in the same substrate as it senesces (live leaves -> dead leaves) is distinct enough to warrant a new term. Many fungi found endophytically have been shown to have the ability to degrade senesced or necrotic tissue, and many of these are also known to be very cosmopolitan genera and species. In that case, it may be possible for many of these fungi to persist on/in healthy leaves, but prefer or specialize on a particular woody substrate. In other words, if the 'via' part of the term is not essential (in other words, they can be in leaves but don't have to be), then is it worth using as a description of an organism as a whole? And if it's just a temporary state, then why not just say 'saprotroph with an endophytic life stage'?

Specific comments:

Line 71: Would be good to add some citations here -- my impression is that a good number of saprophytic endophytes can at least produce conidia on fallen leaves (and thus are not trapped in a 'dead end'), even if they don't produce sexual fruiting bodies.

Lines 92-93: I think there is a distinction of kind between 'endophyte' and 'saprotroph'. The former refers to location, the second to consumption. The more appropriate comparison would be between endophytic and free-living or between biotroph and saprotroph.

Line 106: But isn't this tautological? Or are you saying in contrast to pathogenicity? Because it seems logical that pathogens of one plant existing as endophytes of another plant would also benefit in much the same way as saprotrophs might.

Line 230: Citation 'Elizabeth Arnold' is improperly formatted.

Line 251: ibid

Line 276: Typo 'viaphyptic'

Line 294: This citation is no longer in review.

Line 303: I don't get the logic here. If they can colonize wood, then aren't they saprotrophs by definition? The experimentally observed ability to grow on dead woody substrate seems to me much more convincing evidence of its ecological mode than how a taxon is described in a particular database.

Figure 1: Small point, but if there is only a single isolate for a give taxon, then it seems it should be sp. instead of spp.


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: DOUBLE LIVES: TRANSFER OF FUNGAL ENDOPHYTES FROM LEAVES TO WOODY SUBSTRATES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No major issues remain.
Experimental design: No major issues remain.
Validity of the findings: No major issues remain.
Additional comments: I applaud the authors for engaging so constructively with the review. I appreciate the thorough response to comments and have been convinced by their argument for including the term 'viaphyte'. The discussion in the response to reviewers helped me to understand some of the subtlety that I missed the first time through. I think this is an exciting paper as much for the ideas it contains as the data it presents, and it is likely to be a valuable contribution to the field.

I did notice a number of outstanding proofreading issues, which I do not list here, including some issues with the formatting of the references (e.g. the last two are out of order and one is repeated). However I assume those will get resolved in the proof stage prior to publication (or I would certainly hope so).


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: COMPARISON OF SERUM EXOSOME ISOLATION METHODS ON CO-PRECIPITATED FREE MICRORNAS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written and clearly and logically set out. It cites a range of relevant literature. The figures are presented logically with detailed legends and the results are all relevant to the aims of the manuscripts. The materials and methods are detailed and easy to follow. Raw data is provided for the experiments presented in the paper.
Experimental design: The field of exosome biology and their microRNA content and role in health and disease is growing rapidly. There are many different approaches to isolating and characterising exosomes and their contents. Although clear guidelines are being constantly updated regarding appropriate QC for exosome work there are few papers published that directly compare different methods for exosome isolation and determine the amount of free microRNA carried through purification. This is important knowledge and will help researchers refine their studies and ensure that conclusions are robust.
The ethics declaration form is in Mandarin with the exception of reference to the mouse strain "Balb-c". An English language version should be provided which has relevant ethics for the studies presented in the manuscript.
Validity of the findings: The results are clearly presented in a logical manner. It is usual to present more than one exosome/ EV marker by western blot. The authors should add in additional western markers as quality controls for their isolated EVs, e.g CD63, or another of the accepted markers. Furthermore, the TEM images, particle size distribution and western images in Figure 1 are not very clear. The authors should adjust the size/ resolution of these images so that they are clearer for the reader.The discussion and conclusions are valid and clear.
Additional comments: This is a useful paper which should be informative to researchers working in the field of exosome/ EV biology and who wish to ensure the methods they use for purification are appropriate and that their results and conclusions are robust.


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: COMPARISON OF SERUM EXOSOME ISOLATION METHODS ON CO-PRECIPITATED FREE MICRORNAS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper by Cheng et al. reports on the comparison of serum exosome isolation methods, including ultracentrifugation and precipitation-based commercial kit, on co-precipitated free microRNAs. The subject of this study is topical and relevant for the expanding field of exosome isolation since new methods currently are commercially available with considerable benefits in terms of exosomal purity, amount and labor consumption. Thus, in this work, the authors have attempted to validate methodologies that will help better providing details of serum exosome isolation methods for further research on exosomal miRNAs.
Overall, the topic is of interest and the work carefully executed. The paper is well written.
Experimental design: Some reservations regarding the conclusion and the methodology:

The miR126 and miR152 copies of the blank control sample in the different figures as well as the control sample are not stable.

Please, specify why has not any housekeeping gene been used as an endogenous control for the quantification of the number of copies of miR126 and miR152.

Thus, I encourage the authors to discuss and clarify this point to prevent misleading potential readers
Validity of the findings: -is the exosome characterization by size using Nanosight a single experiment? Please, specify in the legend or add the other biological replicates

-please, add the concentration of miR-39 in the figure 2, even if it is included in the legend, to be clearer

-figure titles and legends, please add spaces between words and add/remove spaces into the brackets

-please, add the molecular weight-size in the western blots in the supplementary figures

The data are robust and statistically sound
Additional comments: Overall, this is an interesting paper which should help the scientific community better to improve the methods developed for exosome isolation eliminating serum contaminants


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: INVESTIGATING SELF-RECOGNITION IN BONOBOS: MIRROR EXPOSURE REDUCES LOOKING TIME TO SELF BUT NOT UNFAMILIAR CONSPECIFICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I enjoyed reading this paper, which is clearly written, well organized along standard lines, and provides relevant and appropriate citation and discussion of past research.  Given, I assume, that the raw data file provided is not going to be published with the article, it would be helpful if the authors provided lines between the dots in Figure 2, so that the reader can see how individual bonobos responded to the different conditions.  I would have liked the authors to discuss more elaborately the work with young children in western cultures showing a time lag in indicating self-recognition in response to video-self-images; what is on lines 122-127 seems ambiguous.  I found Table S2 particularly helpful. I do not believe that the authors provide evidence to support their contentions about bonobo self-recognition (see below).
Experimental design: The experimental design is interesting and largely appropriate to testing differences in looking time under various conditions, and the few bonobos available easily accounts for the lack of counterbalancing in the design.  However, given that the authors are suggesting that the image of the bonobo itself had become, because of the mirrors, comparable to a familiar bonobo, the design would have benefited from (1) having the bonobos also look at a video of a familiar (same sex) bonobo following the mirror intervention, and (2) collecting data on self-observation of bonobos during the mirror exposure period.  And given that the authors were concerned to examine differences in responses of the bonobo to his or her own image to responses to another known or unknown bonobo, it would have made sense to have the bonobo respond to a bonobo of the same sex in all cases. In looking at the images of the unknown animals, the background is strikingly different from that of the animal itself and its known conspecifics, which introduces a further layer into the interpretation of the bonobos’ responses to the images. It would be helpful to know the dimensions of the visual image on the Apple iPad, to compare with the other two image presentations. Unfortunately, the design itself cannot provide evidence that distinguishes recognition (identification) of self, from recognition of the image of oneself as a familiar bonobo.
Validity of the findings: I do not find compelling the authors’ interpretation that the shorter post-mirror exposure looking times at the self (compared to pre-mirror exposure), but lack of difference (between pre- and post-mirror exposure) in looking times at an unknown bonobo, indicates self-recognition. I suspect that the findings could be explained by the bonobo’s recognizing the familiarity of the bonobo in the “self” image, rather than its recognizing the identity of the bonobo as itself. It seems to me that a useful test in the post-mirror phase would have included a familiar (same-sex) bonobo in the sequence shown to them. If the bonobos’ response to this image had also shown a diminishment in time spent looking, it would suggest that the “self” image was just as familiar as the known bonobo’s image.  In addition, the fact that the unknown bonobo had a different background may have made it more interesting, and thus maintained the looking time of the bonobo. The fact that the bonobos did not move in the Cself and mirror conditions is frustrating, and makes comparison to the other “pre-mirror” conditions difficult to interpret, as the authors note.  I would also have liked to know how much time the bonobos actually spent looking in the mirror when it was available during the 3 months; my own observations of bonobos and mirrors suggests that they don’t spend all that much time looking, but I’m sure there is variability.

Unfortunately, given that the methods cannot answer the question the authors are concerned with, namely bonobo self-recognition, I cannot see how the paper can be rehabilitated to be publishable. This is unfortunate, as it is evident that the authors put a lot of thought, time and effort into the study.
Additional comments: Specific concerns and typos:
29-30: Although true that the bonobos “may” form stable visual mental representations of themselves from interaction with a mirror over an extended period, the data do not necessarily support this interpretation: The data are consistent with the idea that bonobos may find images reflecting their own background that they see every day, and which they observed in the mirror and the NCself image, less interesting than that of an unknown bonobo in a very different background.
65-66: I suggest a more nuanced view of lesser apes’ self-recognition skills, especially as bonobos have never been tested with the mark test, so may show comparable skills (see Heschl & Fuchsbickler, 2009, who is mentioned by the authors as providing a different view, but also see Ujhelyi et al., 2000).
94: My memory is that not all chimpanzees show robustly present self-recognition (see Povinelli et al., 1993, e.g.).
95: I’d write “some individuals do not provide evidence for the capacity when tested”, otherwise it sounds like they are tested numerous times, and most aren’t.
125-127:  The trend is from non-self-recognition to self-recognition here I believe.
130: “fist” should be “first”.
175: “recognize visual representations of themselves” is problematic here, as it suggests self-recognition.  I suggest changing to “find visual representations of themselves familiar”.
190: What are the dimensions of the image presented on the ipad?
210-211:  Given the authors’ concerns with self-recognition, it would seem same-sex images would be important.
216: Delete “upped and”.
246: “dependant” should be “dependent”.
271: “interested” should be “interest”.
285+: By using the same data in two analyses, the authors are increasing the probability of falsely detecting a difference. Given that, not taking this into account, the authors found an interaction “significant” at p = .057, I recommend they correct their alpha level appropriately, or use an alpha of .10 (which seems reasonable, given so few subjects). Effect sizes would be useful for interpretation.
303: Did age have the predicted effect?  No findings for the influence of age are presented. Was age an influence at all? If not, it should be deleted as a predictor variable.
320: I don’t understand this line.
321: End line with “of”.
341: Change “indicate” to “suggest”, as there is no evidence provided that the prolonged mirror exposure is the definitive cause of any difference.
348-349: Again, I agree that the mirror MAY have allowed for a visual self-image to be developed, but I do not see that it MUST have allowed it to.
362: Change “if” to “of”.
375-377: But the authors did not test the response to a known bonobo in the post-mirror condition. In addition, in the pre-mirror condition, 6 of the 8 bonobos were in the same range for Cself and known bonobo conditions, suggesting that the suggested “intermediate” description here is not strikingly accurate.
381: Delete extra “that”.
407-413: The methods of the authors could not get rid of the simple familiarity of the bonobo’s image from the predicted effects of mirror exposure.  Thus, it may be recognition of familiarity that diminished the looking time, rather than recognition of self. The authors note this on line 423, but focus on the self-recognition interpretation in the abstract and conclusion.
420, 426:  The word “suggest” indicates “may” and that something “appears” to be true to the describer, so I recommend a more direct statement following “suggest”: “exposure has allowed,” “bonobos acquire”.
431: The p = .057 is not, traditionally, significant, and again, the decrease may be explained as a result of recognition of familiarity, rather than recognition of self; and the lack of decrease with the unknown bonobo may result from the interesting (and novel) background in the video.
440: Change to “bonobo’s”.
439-441: I disagree that we have gained insight into the bonobo’s “capacity to form a visual representation of self and the ability to later recognize the self-image from non-contingent video footage”; this seems an overstatement, as I don’t believe we have any new information from the research in relation to self-recognition.
445: Similarly, we already knew from earlier work that that bonobos recognize themselves.
446-447: This seems a leap.
453-455: Again, the data are mixed as to how robust self-recognition is.
458: Put comma after “species”.
460-461: Note that Mitchell (2012) suggests a different “package of cognitive capacities” related to self-recognition.
462: Change “De” to “de”.
References are not in any consistent style.
Figures 2 and 3: The legend says that the looking time was square root transformed for modeling, but the y axis states that it represents looking time in seconds, but it seems to be transformed in some way.

References mentioned:

Heschl, A., & Fuchsbickler, C. (2009). Siamangs (Hylobates syndactylus) recognize their mirror image. International Journal of Comparative Psychology, 22, 221-233.
Mitchell, R. W. (2012). Self-recognition in animals. In M. R. Leary & J. P. Tangney (Eds.), Handbook of self and identity (2nd ed., pp. 656-679). New York: Guilford.
Povinelli, D. J., Rulf, A. B., Landau, K. R., & Bierschwale, D. T. (1993). Self-recognition in chimpanzees (Pan troglodytes): Distribution, ontogeny, and patterns of emergence. Journal of Comparative Psychology, 107, 347-372.
Ujhelyi, M., Merker, B., Buk, P., & Geissmann, T. (2000). Observations on the behavior of gibbons (Hylobates leucogenys, H. Gabriellae, and H. lar) in the presence of mirrors. Journal of Comparative Psychology, 114, 253-262.


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: INVESTIGATING SELF-RECOGNITION IN BONOBOS: MIRROR EXPOSURE REDUCES LOOKING TIME TO SELF BUT NOT UNFAMILIAR CONSPECIFICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comments
Experimental design: I find the article very interesting, the experimental design is appropriate. I do not see any issues with using the same order of presentation; otherwise you would have introduced a factor that could have interfere with the results.
Validity of the findings: This article is interested in self recognition in non human primates. The authors have used an original paradigm based on looking time toward video instead of using the more classic and controversial mirror task. The originality of their work lies also in the fact that they tested the same participant at different time; First when they were mirror naïve then when they had been confronted to mirror for a long period.
They tested bonobos showing them contingent or non-contingent videos of themselves, videos of known or unfamiliar individuals. They were mainly interest in watching non-contingent images of themselves and of unfamiliar individuals.
After being able to see themselves in a mirror for a long period, it was found that the bonobos were less interested with their non-contingent own mirror-image compared to the video of unknown individuals.
The authors conclude that bonobos do recognize themselves in contingent videos but need training to do so in non contingent videos.
The data are clear and the authors are drawing fair conclusion.
Additional comments: I have few minor remarks:
In the introduction, there are some evidence of self recognition on moving images from 8 months of age (Legerstee et al., 1998; Child Development). I believe that this article can be cited.
Discussion: I agree with the authors that their subject might have had experience with contingency. Shadow is an other example of contingency that is naturally happening. It may explain why they are not surprised and/or interested by the contingent video. It is however evidence of self recognition and the fact that after training they are not interested anymore in the non contingent one is even showing that they understand the delay!
I think that the subject had already a representation of themselves and that the mirror experience is making this representation more familiar and stronger.
I wonder if there is any work on contingency in non human primates that can be cited here? Else, it would be interesting to discuss the fact that non contingency is an attraction parameter.


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: INVESTIGATING SELF-RECOGNITION IN BONOBOS: MIRROR EXPOSURE REDUCES LOOKING TIME TO SELF BUT NOT UNFAMILIAR CONSPECIFICS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Mostly fine, but see “General comments for the author.”
Experimental design: There are problems in relation to what the authors discovered and what they hoped to find. I’m also somewhat concerned as to how much the bonobos used the mirror when available for an extended time.  See “General comments for the author.”
Validity of the findings: I believe that the findings are, in a limited way, valid, even with my concerns, providing the authors can say why they have such confidence that the bonobos in their study actually looked at and interacted with the mirror when available for an extended period. I also have concerns about the authors’ attempts to tie their data to so many aspects of the MSR literature, when it is really tied to a small part of that literature. See "General comments for the author.”
Additional comments: I enjoyed reading the paper, but find several problems.  I believe these can be easily corrected, but it will require the authors to refocus their attention on what they actually found out, rather than making elaborate connections to the self-awareness literature (much of which I found uninformative and incorrect). The authors are arguing that mirror exposure results in knowledge of what one looks like such that one is no longer interested in looking at images of oneself. This is an interesting idea. But it means that the authors’ focus needs to be on this idea, not on issues about self-recognition per se.

Issues:

37-38: This would imply that blind people could not be self-aware.
45-79: This seems irrelevant to the research. I also found it confusing.  How is the evidence for MSR in gibbons and siamangs “contradictory”?  It seems some of the animals show evidence of MSR, and some don’t.  Perhaps the authors meant “controversial”, as Gallup and Anderson don’t accept the evidence. I don’t see how this uncritical presentation of the literature gets us anywhere, especially because the authors are trying to provide evidence for noncontingent self-recognition, and all these studies are about contingent self-recognition.
81-91:  This is a difficult topic to represent, as the evidence from American kids is most relevant to the study, but the other evidence suggests that American (and Western kids) are likely to be very different from the rest of the world. I don’t know if this literature needs to be discussed, except perhaps to indicate that photo self-recognition occurs in western children after MSR.
93-97: Again, this information would be relevant if the focus were on MSR or other forms of contingent self-recognition, but it is not the focus.
99-115: I think it is important to discuss what one gains from MSR, as this section does.  I would note that Mitchell points out that MSR results in knowing what you look like, which is directly relevant to what the authors are studying. The way it is currently written, it sounds as if every time the animals recognize themselves they must use kinesthetic-visual matching; but the idea is that once they do recognize themselves, they can know “that is what I look like.”
121: I do not see this as an “alternative explanation,” but rather as an explanation for what is happening at 5 months in these infants. No one is suggesting that this is the same thing as self-recognition.
124: Not sure what “tell-tale spontaneous behavior regardless of contingency cues” is, but on 364-365, the authors write “tell-tale behaviours of self-recognition, such as exploration of visually inaccessible body parts and contingency testing”, which seems contradictory to 124.
130-132: This statement seems an overstatement. If I know what I look like, then I should recognize that the image is of me, and I might check to see if the image is an accurate depiction by seeing if I have a sticker on my head. How does this indicate that “the self possesses a temporal continuity”?
144: Delete “a sense of self”, as it adds nothing to the sentence.
161-162: Change to “themselves (i.e., without the help of kinaesthetic feedback), and”. You can’t use kinaesthetic feedback to recognize yourself in noncontingent images (or at least you can’t use kinaesthetic feedback DURING noncontingent imagery).
169-171: I agree.
186: Change “15 cm” to “15”.
207-208: An alternative would have been to simply use same-sex footage. It would have been fine to use the same male and female images for the males and females, respectively. Given that some males had male unknowns and others female unknowns, and same for females, it would be interesting to know which are which, even if you can’t do a statistical analysis.
213-214: Writing “All” here suggests that the 4 video conditions add up to 30 seconds total.
256: “we are confident”—how?  Can you provide even anecdotal evidence that the animals looked in the mirror. I was stunned at how little time bonobos spent looking in the mirror when it was repeatedly provided for them in a study I worked on. So did you observe the animals looking in the mirror a lot?
271: Change to “a random intercept. We added age as a”—this adds “a” at beginning and end.
312 and elsewhere: Do you need to indicate that these numbers are means? Also, you should provide standard deviations.
324-332: I must admit that my understanding of LMM testing is limited. However, on 311-315, the author uses information from Table 2 to say that the bonobos spent more time looking at NC self and unknown than at C self and mirror. The t-tests in Table 2 supporting that these differences. However, here (324-332) the t-tests in Table 4 appear to show NO differences (except for age, about which the authors remain mute).  In addition, my statistical training taught that if you use the same data in multiple analyses, you need to adjust your p-value.  In the second analysis, the authors use the NC self and unknown data from the first analysis.  The p-value is just over .05, but shouldn’t they be comparing it to a lower p-value?  What are the effect sizes here?  If large, that would be important to mention. Please make sure what I’m saying is correct by consulting a statistician before accepting it as accurate.
324, 355, and elsewhere: Change “high” to “higher” in this context. Although the animals had 30 seconds only to look, I’d say that looking for 17 seconds is not exactly “high” interest.
360-361: Exactly!  Could the small size of the images have influenced this as well?
381-385: Why would water not have provided info about their physical features?
419-421: If “‘self’ is a mental construct, accessible only to its owner,” how would we ever have developed the idea?  While self may be a mental construct, it is one that has a community-wide understanding (or several community-wide understandings!), and must be accessible to others to have existed as a concept in the first place.
433+: Much of this belongs in the introduction, as this is the point of the study.
448-452: But the bonobos also know the background in the NC self imagery, but not the background in the unknown. I don’t see why bringing up the difference in looking time between contingent and noncontingent conditions from the first study is relevant.  There might be one explanation for one difference, and another explanation for another difference. Or perhaps I’m missing something?
455: Change to “In hindsight, it would have been desirable”.
475-477: If the evidence to support your conclusions comes from research we already know about, then why do your study? Rephrase.
491-493: Mitchell also suggests that MSR “may just be one part of a package of cognitive capacities, obtained from convergent evolution”, so not clear why “but see” is here.  He presents a different suite of cognitive capacities, but a suite nonetheless, that must have occurred by convergent evolution, given dolphin and elephant MSR.
Figure 2: It would also be helpful to have the lines connecting the data points in this figure.
Figure 3: Thanks for adding the lines in Figure 3. Also, could you provide the lines connecting exposed NC self to non-exposed unknown? It is interesting to see that almost all of the bonobos lessened in their interest in the NC self image after the mirror had been present for an extended period, and that two of them did the same thing for the unknown image. The lines would allow the reader to see if there are connections among each animal’s choices.

I think that without knowing why the authors have such faith that the bonobos they tested used the mirrors over their extended presentation (and I’m willing to accept anecdotal evidence from daily observations), it’s hard to know if the extended mirror exposure had an effect.  Of course it seems likely that it did.  I do find the results interesting (and presume the statistical queries I posed can be easily resolved). The authors need to reframe the introduction to be about how animals would gain knowledge of what they look like from mirror exposure, such that they could be aware that the NC self image is of themselves. In that context (and I fear only in that context), the study is interesting, such that discussion of the mysterious self is really not necessary, nor is discussion of all the animals that have shown MSR and other self-related activities.  The authors are presenting evidence to answer a very particular question, and their data provides little evidence beyond that answer.


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: BIOMASS, ABUNDANCES, AND ABUNDANCE AND GEOGRAPHICAL RANGE SIZE RELATIONSHIP OF BIRDS ALONG A RAINFOREST ELEVATIONAL GRADIENT IN PAPUA NEW GUINEA
Review round: 1
Reviewer: 1

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: This paper makes a valuable contribution to our understanding of bird range sizes and diversity along an important tropical elevational gradient. It does, however, need a great deal of work to get it into publishable shape, and I have placed many comments directly on the manuscript. All comments with no entries at all signify typographical errors, of which there are many. Below are some of the major issues, others are left on the MS.

The introduction takes a long time to get to the main question and should start with it.

Two main points:

1.	An important issue is what ‘mean elevational abundance’ actually is. The statement is “We recorded the mean number of individuals of a species recorded at 15-min interval (for all 16 points together) as “mean elevational abundance”.  How were the 14 visits included? did you sum or divide across the 16 points? In particular why are there no values less than 1.0 in the data? It seems 0s were omitted, but this doesn’t make sense to me. One could also drop both the word mean and elevation from ‘mean elevational abundance’; just say a measure of abundance at a location, which would be clearer.

l.189 the reason total elevational abundance differs from mean elevational abundance took me a very long time to understand, and I am not sure I have got it even now. Please state explicitly what mean elevational abundance is in fig. 4A as everywhere else it is used it refers to individual species. What I think is that fig. 4B is the sum of abundances at an elevation, and fig. 4A is that sum divided by species richness.

2.	The second issue is with the estimates of abundance, which were not corrected for detectability. I agree with the authors that this is difficult to do accurately. Nevertheless, I am sure that point estimates will produce some biases in the results (in the E Himalaya for example Ficedula hypereythra is one of the commonest, but most secretive, of birds). I feel that something can be done with your data to at least evaluate how results might be affected. For example, Sam et al. (J Biogeog) present mist-netting results (and perhaps Freeman’s could be used as well). How does relative bird abundance among those species that are readily mist-netted correlate with bird abundance in the point estimates? What happens if you look at range size and mist net estimates? Is there any other way to evaluate consistency of results (e.g. compare wet vs dry seasons when birds may be more or less conspicuous?). As you recorded the species in concentric circles, you could also do some crude detection corrections and see if these make a difference.

The issue of detectability is the reason Price et al. (2014) decided to adopt the British Trust for Ornithology Breeding Bird surveys, and map territories through thorough surveys on 5ha plots. That of course only surveys a fraction of all breeding birds at a location, but gives good estimates of relative abundance, and captures all common species.  Note that, despite statements in the paper, all nonpasserines were surveyed and reported in the supplemental data (e.g. Gallus gallus), but they were not analyzed in the paper (indeed as the authors note, the censuses of nonpasserines were not analyzed at all in the paper, we only used inferences from overlapping elevational ranges for that group.) The reason in the overlapping range data several orders were omitted is because they were considered to interact only weakly with other species (more weakly than ants!). Lack in his book ‘Island Biology’ made the same argument.

lines 127-> it is unclear what the difference between the mean point and mid-point is. Is the mean point summed elevations, weighted by abundances?

l. 177 Is it sensible to average abundance across sites, unless one includes all zeros, which would then give an estimate of total abundance on the mountain?  Otherwise it seems that the difference between recording a single individual and recording none is very large. I would have thought another reasonable test would be to use simply maximum abundance.

why not test for an association with elevational range as well as geographical range?

why not show a scatter plot of each species mid-point elevation against abundance, and of abundance against geographical range?

Another thing that probably should be done is generating a model of range size vs. abundance + body mass (or if  you think abundance is determined by range size, abundance vs range size + body mass. Large species should a priori have larger ranges I think. Although I would certainly not require this for publication, many reviewers would ask you to use phylogenetic corrections in these analyses. Personally, for this paper I don’t think it is needed.

It would be very helpful if the authors were to give a better breakdown in the data supplement: separating wet and dry seasons for example. I think this is quite important to do, and will make the paper much more valuable to other workers going forward.

Trevor Price


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: BIOMASS, ABUNDANCES, AND ABUNDANCE AND GEOGRAPHICAL RANGE SIZE RELATIONSHIP OF BIRDS ALONG A RAINFOREST ELEVATIONAL GRADIENT IN PAPUA NEW GUINEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper describes abundances of birds along a tropical elevation. It employs different perspectives, basically: (i) different classifications of bird species and (ii) combination of geographical range sizes with abundances. Its form fits international scientific standards.
Experimental design: The paper is rather descriptive but it contains tests of relevant hypotheses. It is well elaborated and the data well statistically analyzed. It based on field data, which collection was undoubtedly difficult and demanding work under harsh tropical conditions.
Validity of the findings: The general finding, that montane birds species live in high abundances, was already done elsewhere. However, such evidence is still very limited and replicates are highly appreciated. This paper tries to decompose the question by classification of birds to different groups. It is a possible way to put it forward, even though I do not see too much news after reading the paper. For example, the finding mentioned even in conclusions i.e. that the patterns are driven by insectivores and frugivores just say that these two groups are the most abundant. I guess it would be better to discuss more the exceptions fro the general pattern. I am relatively sceptical about using the overall biomass information as it looks as driven by species richness. However, the conclusions are correct in my opinion, biomass decreases towards high elevations due to relatively lower productivity in the montane regions. The interesting hump-shaped pattern in total abundances is most likely driven by the hump-shaped pattern of species richness. I think this can be discussed.
Additional comments: I like the paper as it provides evidence about high abundances of montane species in the tropics, which is still rare, it provides a thorough analysis makes efforts to put it into the context of available literature. I would suggest putting more efforts into the reasoning why to use different analysis for avian functional groups and later on discuss the differences more, did it help us somehow to understand the pattern?


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: BIOMASS, ABUNDANCES, AND ABUNDANCE AND GEOGRAPHICAL RANGE SIZE RELATIONSHIP OF BIRDS ALONG A RAINFOREST ELEVATIONAL GRADIENT IN PAPUA NEW GUINEA
Review round: 2
Reviewer: 1

Output:
Basic reporting: see my general comments to the authors. the english still needs work, and there seems to be one major conceptual issue that should be addressed, on the use of mean abundance.
Experimental design: the authors have done a great job at improving the analysis over the first version, but comparing mist net and observational studies and repeating analyses for different seasons.
Validity of the findings: I am sure the findings are valid, but would like to see analyses repeated with max (mean elevational abundance) cf. mean abundance
Additional comments: This is an important paper, presenting much original data and analyses, which will be of great value. I also appreciate the data being presented in a supplement, which still does not happen often enough. The authors have done a nice job and validating their findings through the use of seasonal comparisons and mist net results. The writing could still use some work. I have made some comments on the MS, and on the supplement, both of which I am returning. Major comments, including responses to the rebuttal are below:

I noted one should quote the new Frontiers in Biogeography paper, which the authors could not locate. I meant the one on which Sam is herself a co-author.

One other comment needs addressing. Is it sensible to average abundance across sites, unless one includes all zeros, which would then give an estimate of total abundance on the mountain? Otherwise it seems that the difference between recording a single individual and recording none is very large. I would have thought another reasonable test would be to use simply maximum abundance. – We added the statistical results also for the total abundances, which were actually nearly the same as mean abundances. L 189- 190.

l.133 I still cannot see what mean abundance tells us. It has been used in the main figures (fig4 and 5C,D) as well as several supplemental figures. Supposing 100 birds were recorded in one location and 1 in another, then we would get mean abundance of 50.5. Now suppose that the 1 bird in the second elevation was missed. Then we would get mean abundance of 100.  To take a more concrete example, the fact you observed Eclectus roratus occasionally at 1200m results in its mean abundance being reduced from 5.43 to 3.94. In other words, the mere presence of a species across a broader range reduces its mean abundance. At a range limit, species will always go to low abundance.
Eclectus roratus	7.08	3.78	1

It is of interest that total abundance gives a similar result, but I am not sure what the measure is. Is it the summed abundance of an individual species across all elevations? Elsewhere it is defined as summed abundance across all species at one elevation, which makes sense. But if it is summed abundance of an individual species across all elevations, it seems that this also is not the test you want.  Supposing you found one species at low abundance across all sites, and one at high abundance at one site. They may have the same total abundance, but surely the test you were looking for was local density vs. range size.

As noted in the earlier review I think you should use maximum (mean elevational abundance) for each species, which is what you use in several places already.

Explain what is mean abundance from mist-netting data (Figure S2). If similar to the census data, then I think it should also be changed.

I am struck with the similarity of these results to those of the east Himalayan gradient, compare fig. 1c of Price et al 2014 with fig. 2b of this paper. The differences are small and perhaps explained by the different census methods.  I feel that a reader will be interested in this, rather than a simple dismissal with the statement that the studies are not comparable because not all non-passerines were include. Only 4 non-passerine species, all of low abundance from New Guinea, would not have been included in our censuses (as detailed in a comment in the MS). I guess New Guinea is much less seasonal than 1700m in the east Himalaya, which makes the similarities more impressive.

Trevor Price


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: DISTRIBUTION, EVOLUTION AND EXPRESSION OF GATA-TFS PROVIDE NEW INSIGHTS INTO THEIR FUNCTIONS IN LIGHT RESPONSE AND FRUITING BODY DEVELOPMENT OF TOLYPOCLADIUM GUANGDONGENSE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Summary
The authors present a study on the GATA-type transcription factors in the fungus Tolypocladium guangdongense partly based on transcriptomics data from a previous study (Zhang et al., 2019). They perform basic bioinformatic analyses of the GATA factors (gene structure, conserved motifs, cis-elements and phylogenetic analysis) in order to classify them in accordance to the already well-described GATAs from species such as Neurospora crassa and Aspergillus nidulans. The authors also investigate the potential light regulation of TgGATAs and their involvement in fruiting body development via qPCR-based expression analyses. While the current state of knowledge about the fungal GATAs is nicely summarized and a good overview of the respective literature is given, the small amount of experimental evidence hardly allows for speculations about the functions of the newly identified TgGATAs. Taken together, the study suffers from several major shortcomings discussed in the following sections.

Introduction
The introduction gives a good overview over the functions of GATA transcription factors in fungi but fails to convey the importance of the analysis in the organism of choice, T. guangdongense by simply describing it as an “edible-medicinal fungus” (line122). I suggest that the authors give some information on the classification and phylogenetic position of T. guangdongense in order to elaborate its importance as model organism for their study.

Figure 1
The alignment of zinc finger domains needs some improvement. I recommend that the authors use a monospaced font like Courier in order to avoid certain letters being cropped. I furthermore suggest mentioning the threshold for consensus highlighting in the figure’s description and adding the position of the zinc finger domains. The authors should also consider including the GATA domain classification, e.g. type IV a and type IV b.

Figure 2
The authors only show one GATA ortholog in S. cerevisiae whereas the literature (e.g. Cooper, 2002; Park et al., 2006) mentions at least ten GATA factors in yeast. I assume they want to express that neither of these are functional/structural orthologs to GATA1-7, but this is still highly misleading to readers unfamiliar with the details of fungal GATA factor classification. In my opinion, the authors should find a way to include all GATA factors for the respective found in literature and databases such as FTFD. Adding a sub-group for non-orthologues GATA factors such as the ones found in yeast would help understanding the full complexity of GATA factor classification and subdivision in fungi.
Cooper, T. G. (2002). Transmitting the signal of excess nitrogen in Saccharomyces cerevisiae from the Tor proteins to the GATA factors: connecting the dots. FEMS microbiology reviews, 26(3), 223-238.
Park, J. S., Kim, H. J., Kim, S. O., Kong, S. H., Park, J. J., Kim, S. R., ... & Lee, Y. H. (2006). A comparative genome-wide analysis of GATA transcription factors in fungi. Genomics & Informatics, 4(4), 147-160.

Figure 3
The phylogeny unambiguously shows the GATA factor subdivision and is perfectly valid in that sense. The authors should restrain from drawing “evolutionary” (line 250) conclusions from a NJ phylogeny since it is merely based on a distance matrix rather than an evolutionary model. I suggest the use of maximum likelihood or Bayesian methods, which use models of sequence evolution, if they want to speculate on GATA factor evolution in fungi. If they simply want to subdivide the GATA factors based on sequence similarity NJ is acceptable, but this should be stated in the method section.

Figure 6
The authors should give information on the phylogeny construction and name the already well characterized motifs, e.g. zinc fingers and PAS domain, in the figure’s legend. I recommend also performing domain identification on the so far uncharacterized motifs in table S5 via e.g. Prosite and annotate them as far as possible.

Figures 8 + 10
I suggest separating the plots not by gene but by duration of light treatment (Fig. 8)/developmental stage (Fig. 10) or showing the data in grouped bar chart in order to facilitate comparing the expression of the different genes at the same time point.
In lines 447 – 448 the authors assume that WC1 expression “may peak within the first 30 min” while WC1/TgGATA1 still seems to be downregulated at 30min and expression is increasing from 2h to 4h of light treatment in Fig. 8.

Materials & Methods
I suggest using [µmol photons m-2 s-1] rather than [lux] for light intensity.
The authors should also provide the light conditions for fruiting body development.

qPCR-based expression analyses (Table S7):
The authors use amplicons drastically different in size, ranging from 158 bp for CCG_01872 up to 474 bp for CCG_00651, and still assume constant amplification efficiency for all samples in using Livak’s 2-ΔΔCT. Bustin and Huggett have shown that longer amplicons are detected earlier in SYBR Green qPCR assays. I therefore suggest that the authors use a variation of the Model based analysis of real-time PCR data (Alvarez et al., 2007) described in Grimes et al., 2014.
Bustin, S., & Huggett, J. (2017). qPCR primer design revisited. Biomolecular detection and quantification, 14, 19-28.
Alvarez, M. J., Vila-Ortiz, G. J., Salibe, M. C., Podhajcer, O. L., & Pitossi, F. J. (2007). Model based analysis of real-time PCR data from DNA binding dye protocols. BMC bioinformatics, 8(1), 85.
Grimes, B. T., Sisay, A. K., Carroll, H. D., & Cahoon, A. B. (2014). Deep sequencing of the tobacco mitochondrial transcriptome reveals expressed ORFs and numerous editing sites outside coding regions. BMC genomics, 15(1), 31.

Comments on language, grammar and misc.
Line 107: “Light is an important environmental signal for their sexual and asexual growth, circadian …“
Lines 436 – 438: The authors use the word “process” for what I assume should be “possess”.
Line 480: The authors identified seven GATA orthologs, not seven groups of GATAs.
Lines 468 – 469: Missing citation for involvement of fungal GATAs in sexual and asexual development.

Possible Experiments to further investigate the role of TgGATAs
•	Expression analysis of TgGATAs in response to blue/red/white light
•	Identification and expression analysis of orthologs of known downstream targets of NcWC1/2 in response to different light signals
•	Generation of TgGATA k.o. mutants in order to verify the assumptions about the involvement in fruiting body development
Experimental design: see above
Validity of the findings: see above
Additional comments: see above


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

Input:
Title: DISTRIBUTION, EVOLUTION AND EXPRESSION OF GATA-TFS PROVIDE NEW INSIGHTS INTO THEIR FUNCTIONS IN LIGHT RESPONSE AND FRUITING BODY DEVELOPMENT OF TOLYPOCLADIUM GUANGDONGENSE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript “Distribution, evolution and expression of GATA-TFs provide new insights into their functions in light response and fruiting body development of Tolypocladium guangdongense”by Zhang and co-workers describes the systematic identification and characterization of GATA-TFs in T. guangdongense. The authors use a pool of pre-defined software tools that have become a standard for similar studies over the past years. Finally, they conclude by identifying putative GATA TFs that play a crucial role in light response and fruiting body development in T. guangdongense.
The manuscript is mostly well written with a little tweaking of the English language required. Nevertheless, I am not a native English speaker and it would be a good idea to get advice from a native English speaker.
In general, the introduction is well structured gradually gliding the readers to essential background information with appropriate literature citations and the need for undertaking the present study. The figures are well illustrated with descriptive legends and are of publication quality. Appropriate raw data has been provided.
Experimental design: The authors describe a regular meta-analysis for identifying and characterizing GATA TFs in T. guangdongense which falls well in the scope of the PeerJ journal. The chosen topic of the study seems justified as there are limited studies describing the characterization of GATA-TFs in T. guangdongense. The methodology adopted is widely used for identifying and characterizing TFs and is described adequately for other researchers to replicate the analysis. The results are described in enough detail and interpreted well. The discussion section is well constructed with a clear balance between the interpretation of the results obtained and associated speculations.
Few minor points:
1.	How many numbers of replicates were used for qPCR expression analysis: biological and technical replicates? Also, were the results statistically significant?
2.	The authors state the availability of transcriptome data for different light conditions. It would be nice to compare the GATA expression profiles obtained from RNASeq and qRT-PCR.
3.	Please cite the qRT-PCR primer table in the method section as this section appears first.
Validity of the findings: The present study is preliminary in nature and is identified as such by the author which is appreciable. The exact functional mechanisms of the GATA TFs identified are far from complete which requires concrete functional studies involving knock out/overexpression. I hope the authors will venture into such studies in the near future to come up with meaningful conclusion of their present findings.
Additional comments: I commend the exhaustive analysis performed by the authors and recommend the publication of the manuscript after a minor revision.


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: DISTRIBUTION, EVOLUTION AND EXPRESSION OF GATA-TFS PROVIDE NEW INSIGHTS INTO THEIR FUNCTIONS IN LIGHT RESPONSE AND FRUITING BODY DEVELOPMENT OF TOLYPOCLADIUM GUANGDONGENSE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript "Distribution, evolution, and expression of GATA-TFs provide new insights ...", by Zhang et al, describes a detailed sequence analysis of the seven GATA TFs found in the genome of the edible-medicinal fungus Tolypocladium guangdongense. Putative functions of the genes are attributed according to the similarities to well-investigated orthologs in other fungi, in agreement with their sequence features. The work is very weak: a major body of results in the manuscript resides in sequence analysis and bioinformatic predictions, making many of the conclusions too speculative. The results are combined with expression data on the effect of light or developmental stage on mRNA levels of the seven genes, measured by RT-PCR. In general, the results are insufficient for solid conclusions.
Experimental design: I have several major concerns:

1) About the mycelia age used to illuminate the samples: 20 days seems a too long incubation time for a fungus. Why do the authors use old mycelia to be illuminated? Experiments with other fungi usually used in research, including Neurospora, are carried out with much younger mycelia. I assume that the mycelia used in the illumination experiments are in a very late stationary phase. Why was this time chosen? Have experiments to investigate the effect of mycelial age on the response An experiment is missing to look for a more appropriate incubation time.

2) It is not clear the reason to use the vacuolar protein sorting (VPS) as a single reference control gene in the RT-PCR studies. Is it really a constitutively expressed gene under the conditions investigated? It is strange that the genes for such different GATA factors, involved in very different metabolic aspects, have similar regulatory features. Changes in mRNA levels for the investigated GATA genes are not very strong (induction level after illumination, for example, is much higher in N. crassa for the wc-1 gene). E.g, the transient reduction of mRNA levels after illumination that affects similarly to different GATA genes might be explained by a minor increase in the VPS mRNA levels in the same conditions. It is necessary to use at least a second reference gene. Usually, house-keeping genes, like those for actin or beta-tubulin, are normally used in RT-PCR quantifications. Which is the basis to use VPS?

3) Experimental conditions are so poorly explained. Please, note that descriptions should allow other researchers to repeat the experiments under the same conditions. A separate section for strains (the strain is not indicated!) and culture conditions should be indicated. How were inoculations done? The samples for the developmental conditions, how were they taken? Are they supposed to be in liquid culture? Do the authors take samples from specific structures or the total mycelial samples? With the provided information it is not possible to reproduce the experiments.

4) I have doubts about the co-expression analysis. I do not doubt on the usefulness of mathematical or bioinformatics resources, such as the WGCNA, but the results should be consistent with what the reader may visually grasp. In the graphs displayed in Fig. 8, I see more correlation in the expression pattern between GATA2 and GATA3 than between GATA1 and GATA2. However, in Fig. 9 GATA2 and GATA3 are linked by a green line. Is is also strange the red line for GATA5 and GATA6 in Fig. 9, which however show quite different patterns in Fig. 8. I am not very confident of an approach that goes against what your eyes perceive.

5) It is stated in the figure legends of RT-PCR experiments that the error bars indicate standard deviation of three experiments. Do the authors mean technical repetitions or biological repetitions? An extremely low variation is observed in three experiments for some of the genes (see, e.g., GATA 1 and GATA 6 in Fig. 8, or GATA1 and GATA 7 in Fig. 10). In my experience, RT-PCR data are always subject to biological noise, which is added to technical noise, which makes these extremely low variations quite unusual.
Validity of the findings: Bioinformatic findings may be reliable, but without experimental support, they are just preliminary clues awaiting for experimental demonstration. As indicated in section 1, irrespective of the experimental concerns, available data are very insufficient for solid conclusions.
Additional comments: In general, I find this work too preliminary and speculative. I have no doubts about the reliability of the bioinformatic analyses, which are very rigorous and exhaustive, but at the same time, they are to a large extent very conventional and straightforward to achieve, in many cases consisting just on the output of online tools. On the other hand, experimental work is not sufficiently explained and reliability is more doubtful.
The weakness of the results makes the conclusions too speculative. This is especially visible in the paragraph of lines 441-457 in the discussion. The data are too poor to establish possible regulatory connections between different GATA genes. In addition, there are sentences that make no sense. E.g.: " ...transcripts of TgGATA1 increased as light treatment increased from 30 min to 4 h, speculating that the expression level of WC1 may peak within the first 30 min..." Within the first 30 min does not fit with an increase from 30 min to 4 h. I do not understand also the logic of the following sentence "Besides, the expression levels of TgGATA4, TgGATA6, and TgGATA7 were also down-regulated, speculating that they may function as early light-induced genes to regulate the downstream target genes". Downregulation is transient and rather small, but, why does it mean that "they may function as early light-induced genes"?

Some minor comments that may help the authors for future resubmissions:

Line 32. "Seven subgroups of TgGATAs were identified". The same idea is found in the conclusion (line 480). The authors mean seven TgGATA genes, not subgroups.

Lines 71-72. WC-2 is not a photosensitive protein. In the WC photoreceptor complex, the only photosensitive protein is WC-1.

Line 94, People working with S. cerevisiae usually ignore that there are plenty of different yeasts in nature. Better to say S. cerevisiae.

Line 226: chromosomes have no orientation, it is just arbitrary and, in any case, it is irrelevant information unless referred to closely linked genes.

Line 397: "proteins of GATA1 and GATA2 are rarely found in S. cerevisiae due to lowest identities" The authors mean that they are not found. Also the next sentence "It is considered that photoreceptors, like WC-1 and WC-2, may not exist in S. cerevisiae". It is well known that S. cerevisiae lacks light responses and actually lacks WC photoreceptors.

Line 436: "...that TgGATA6 process similar..." should be "...that TgGATA6 possess similar... "


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: DISTRIBUTION, EVOLUTION AND EXPRESSION OF GATA-TFS PROVIDE NEW INSIGHTS INTO THEIR FUNCTIONS IN LIGHT RESPONSE AND FRUITING BODY DEVELOPMENT OF TOLYPOCLADIUM GUANGDONGENSE
Review round: 2
Reviewer: 1

Output:
Basic reporting: I think the English can still be improved here and there but is much better than in the previous version. (Comment to reviewer #3, line 397: "....photoreceptors are not exist in this fungus (Part et al., 2016).
Experimental design: The revised version is much improved and all my comments have been addressed.
Validity of the findings: The findings are still highly speculative and there is not much depth in this paper. However, all the criticisms to the existing paper have been addressed.
Additional comments: Please revise the use of the English language once more.


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

Input:
Title: DISTRIBUTION, EVOLUTION AND EXPRESSION OF GATA-TFS PROVIDE NEW INSIGHTS INTO THEIR FUNCTIONS IN LIGHT RESPONSE AND FRUITING BODY DEVELOPMENT OF TOLYPOCLADIUM GUANGDONGENSE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have adequately addressed all the queries raised by the reviewers.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Although no additional studies have been conducted to improve the functional role of GATA TFs, the manuscript has been revised adequately with an appropriate response to the queries raised and hence could be accepted for publication in PeerJ journal.


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: DISTRIBUTION, EVOLUTION AND EXPRESSION OF GATA-TFS PROVIDE NEW INSIGHTS INTO THEIR FUNCTIONS IN LIGHT RESPONSE AND FRUITING BODY DEVELOPMENT OF TOLYPOCLADIUM GUANGDONGENSE
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have made a considerable effort to improve the manuscript and have generally responded satisfactorily to the reviewers.
In overall, the manuscripts dealt in big detail with the subject of the GATA Proteins in Tolypocladium, especially at the in silico level. Data on expression, although reliable, must be considered only preliminary from the point of view of functional assignations, but they are now technically more sound than in the former submission and are worthy of publication. Data are in general reasonably described, discussed, and referenced, and figures and tables have been carefully done.
I am not a native english speaker, and therefore I am no competent to judge grammar aspects.
About manuscript content, I only suggest some minor changes. I consider the manuscript in its current state to be an interesting contribution to the understanding of GATA family of proteins and I consider that only minor improvements are necessary before publication.
Experimental design: In my opinion, the issue of mycelial age or illumination conditions has not received enough attention in this research. However, there are too many data obtained with the present growth conditions, and so the present work should stay in its current form. However, I recommend the authors to consider for future works to carry out time course experiments to check the effect of age, or try alternative growing conditions. For example, if colonies grow very slowly, many colonies can be inoculated in the same petri dish, or cultures can be grown by spreading spores on agar surface to obtain a mycelial mat.

It is also a weak aspect to use the entire mycelium at the different stages of sexual maturation when this only occurs at well delimited structures in the mycelium. If development occurs at localized points, the data are logically mixed with those of mycelium that does not participate in these phenomena and might be subject of other regulatory scenarios. It is difficult to interpret the data without having information on how abundant are such structures or how much do they represent over the total mycelial mass.
These are possible improvements to take into account in future work, but the data in the present study should be already sufficient for publication.
Validity of the findings: I still have my doubts about the functional extrapolations from expression data, especially when differences are not so drastic. It should be borne in mind that regulatory proteins control the expression of other genes, and often respond to signals with post-transcriptional changes, and do not necessarily have to be importantly regulated at their mRNA level. As an example, the Neurospora Wc-1 gene is regulated by light (actually, self-regulated), but its ortholog in Fusarium is not, despite the fact that both have similar functions.
Thus, interpretations must be especially cautious. Eventually, the verb "indicate" should be rather replaced by "suggest" in some places. See. e.g., in lines 630-632: "TgGATA4 and TgGATA5 were down-regulated at stages of fruiting body development, indicating that they may negatively regulate the sexual development", better to say "TgGATA4 and TgGATA5 were down-regulated at stages of fruiting body development, SUGGESTING that they may negatively regulate the sexual development"
Additional comments: As a major comment to the authors, thanks for the efforts to improve the expression data, which are now more reliable.

Please, consider some improvements in the data:

Data in Table 2: there are some species with additional GATAs. Could they be in some case paralogs of the "official" GATAs?
An observation about the display of data in the table: the symbols chosen are too similar for a clear distinction. It would be visually more clear if, e.g., colored circles are used for the positives. Also, please note that pale colors (typically yellow) are not very visible, especially when printed in black and white.

Bars for dark conditions in Fig. 7 are not real and can be removed; it must be indicated that values are referred to those in the dark, that were taken as 1. Displayed in that way, it leads to the misleading impression that all genes are expressed equally in darkness. Actually, it would be a good idea to provide information on real absolute values in the dark, e-g., from RNA-seq data.
The same can be said for the mycelium values in Fig. 8.

Some improvements in the text:

Lines 156-158: The sentence "To identify the TgGATAs, the online analysis platform, InterProScan (Zdobnov and Apweiler, 2001) was used to select the proteins with conserved GATA-domains by scanning the T. guangdongense protein database" should rather be "To identify the TgGATAs, the online analysis platform, InterProScan (Zdobnov and Apweiler, 2001) was used to screen the T. guangdongense protein database for proteins with conserved GATA-domains"

Lines 392-394: the newly added sentence "Based on the results of reference genes selection, α-tub1 and β-tub1 were selected as reference genes for analysis the relative expression levels of TgGATAs" should rather be "Based on the results on reference genes, α-tub1 and β-tub1 were selected as reference genes for analysis of relative expression levels of TgGATAs"

Line 536: "regulatory connections may be exist between..." should be "regulatory connections may exist between..."

Line 538: "may be a downstream target gene" rather than the ""may be the downstream target gene"·


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: USING BIOINFORMATICS AND METABOLOMICS TO IDENTIFY ALTERED GRANULOSA CELLS IN PATIENTS WITH DIMINISHED OVARIAN RESERVE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors conducted this study aiming to understand the molecular mechanisms behind diminished ovarian reserve with bioinformatic approaches. They utilized published gene expression data, including mRNA and miRNA dataset, and further analyzed and integrated them with several bioinformatic tools. They found inflammatory and steroidogenic signalings could responsible for diminished ovarian reserve. And they also supplemented this observation with their own metabolomic experiment which showed decreased steroids in the granulosa cells from patients with diminished ovarian reserve. The manuscript is well-written and logical sound. Several concerns will be listed below.
Experimental design: 1. Most of the analysis in this manuscript highly depends on the E-MTAB-391 dataset the authors retrieved from a previous publication (Skiadas CC, et al.). However, there are multiple studies compared the gene expression of granulosa cells between normal and diminished ovarian reserve individuals (Greenseid K, et al., Chin K-V, et al., Pashaiasl M, et al.). These studies should be cited in the manuscript and the authors should justify the reason they only select a specific dataset as their input. For example, couple of the studies observed the changes in expression of IGF1 and IGF2. However, they are not on the DEG list of this manuscript. Why is that? And then why is the current dataset more optimal for the authors' purpose?

2. To identify differential expressed genes, the authors use log2 (FC) >0.58 as their cutoff. Several studies used log2 (FC) >1 as filter and it is not necessary the standard way to do it. But the authors has the responsibility to explain how this number (0.58) was chosen. If the filter was log2 (FC) >1, there would be no genes related to steroidogenesis in the list and the steroidogenesis is one of the major players being discussed in this manuscript.

3. For the differential expressed miRNA, the authors directly used the list from the publication of Chen D., et al. Again, it is not the only study toward this question. For example, Woo I., et al. did deep sequencing on the granulosa cells from normal and diminished ovarian reserve individuals. The differential expressed miRNA in their study are not the same list as in the study of Chen D., et al. So the authors should justify why they chose one over the other one (and please cite the publication) or they should consider to use both of them as input.

4. The authors then utilized the differential expressed genes and miRNAs to study the miRNA-mRNA regulatory network. The intention here is good. However, the authors did not compare the patient selection and sample collection methods in details. The authors should make a table to compare different cohorts of patients in their manuscript which should include their own cohort for the study of metabolomics. And the authors also need to explain how the differences between different selection methods could influence the results of their own if there is any. For example, the FSH level of these three cohorts are different.
Validity of the findings: 1. In general, I appreciate the authors presented their data pretty well as well as discussed their findings in details in the discussion. At the same time, I do think the figures can be upgraded a little. The major concern here is that most of them will not be readable (figure2-6). There are a lot of labels and the fonts are way too small. In Figure 3, as far as I can see, there is only one color. And it is very hard to tell if the darkness of the green are different from each other. Please try to present the figures in a way the readers can read and digest.

2. I believe the metobolomic study in the end of paper can be interesting and beneficial to  the filed. But the authors should provide the whole list (now there is only metabolites related to steroidogenesis) of their discovery as a supplementary file.

3. Please also provide the list of differential expressed miRNA used in this publication.
Additional comments: I believe the study can provide valuable information to the field. But as a manuscript heavily replies on bioinformatics, the selection of input is very critical. The authors need to be very careful on the way they select dataset and provide strong rationale on how they select the data.


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: USING BIOINFORMATICS AND METABOLOMICS TO IDENTIFY ALTERED GRANULOSA CELLS IN PATIENTS WITH DIMINISHED OVARIAN RESERVE
Review round: 1
Reviewer: 2

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

The manuscript by He et al was designed to understand the molecular mechanism of diminished ovarian reserve. They provide some preliminary data on the effects of steroidogenesis and inflammation at diminished ovarian reserve. Although observations made here are of interest, this study is rather too descriptive, and lack of mechanistic insights. Also, the writing is not very competent, with many awkward sentences.

Additional minor comments:

1: The authors need to proof read the entire manuscript carefully, and to make correct statements.

2: In results part, these titles are not interpret fitly.


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: USING BIOINFORMATICS AND METABOLOMICS TO IDENTIFY ALTERED GRANULOSA CELLS IN PATIENTS WITH DIMINISHED OVARIAN RESERVE
Review round: 2
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: I appreciate the responses from the authors to each comments. The current manuscript is better scientifically and logically sound. However, the writing is somehow downgraded in this version, particularly for the newly added context. It is not only that the structure of the sentences is not reader-friendly but the contents are not professionally written. For example, the authors added a paragraph to describe the importance of miRNA in reproductive system. The most compelling evidence is certainly the Dicer-KO experiments, which were done by multiple groups. The description toward importance of miRNA in this current version is very superficial. And similar issues can be found across the manuscript. It is highly recommended that the authors should make the efforts to polish the manuscript and make sure the manuscript can communicate with the scientific community with clear and scientific language. .


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

Input:
Title: USING BIOINFORMATICS AND METABOLOMICS TO IDENTIFY ALTERED GRANULOSA CELLS IN PATIENTS WITH DIMINISHED OVARIAN RESERVE
Review round: 2
Reviewer: 2

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


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

Input:
Title: THE OLDEST RECORD OF THE STELLER SEA LION EUMETOPIAS JUBATUS (SCHREBER, 1776) FROM THE EARLY PLEISTOCENE OF THE NORTH PACIFIC
Review round: 1
Reviewer: 1

Output:
Basic reporting: Main comments:

This new paper by Tsuzuku and Kohno redescribes a fossil mandible originally reported by Kaseno in the 1950s, and discussed a number of times since. Originally misidentified as the extinct pinniped Allodesmus, subsequent authors reassigned the specimen to the extant genus Eumetopias. This study redescribes and statistically evaluates this specimen, and the authors conclude that it is referable to Eumetopias jubatus. The paper is well-written and concise, and the statistical analyses are competently executed. I have a few points that need to be addressed before I can recommend acceptance.
1)	This manuscript avoids mentioning the extinct sea lion Proterozetes. Proterozetes is coeval with this specimen, and recently reported from the Pleistocene of Japan in a conference presentation. Could this specimen belong to Proterozetes? Comparisons are needed to A) demonstrate that this specimen is not Proterozetes rather than Eumetopias and B) why Proterozetes was left out of the statistical analyses. On that note, what about Eumetopias ojiyaensis?
2)	This specimen has double rooted teeth. Are single rooted teeth present in any extant specimens of E. jubatus? If not, this feature would actually fall outside the range of variation for the extant species and therefore would not be assignable to it. Pending the outcome of comparisons with Proterozetes, I think a strong case can be made for identification as Eumetopias. However, I have reservations over a species-level identification as E. jubatus. At best, I think such a dental difference could mean this may correspond to a chronospecies of Eumetopias ancestral to E. jubatus.
3)	A more minor point, but some reference to other specimens of Eumetopias (e.g. Eumetopias ojiyaensis) would be useful.
Owing to these issues, I suggest major revision – and have every reason to believe these issues can be satisfactorily dealt with. Aside from these, I have several minor comments below:
Title: jubatus is misspelled.
Abstract: “Among then” should be “them”
Abstract: the age cited for the Omma Formation is incomplete, and should include the maximum age as well. Why not just state the precise age rather than rounding – e.g. 1.36-0.83 Ma?
39: “One such important fossil specimen has been recorded…”
53: recent should not be capitalized
60: “One such important…”
66: Awkward phrasing; when using the word yields, something yields something else, rather than something being yielded by another. In other words, it is “yielded by” rather than “yielded from”, if that makes sense. I’m no grammarian, so I don’t really know how to explain this very well! Regardless, I suggest changing this (here, and elsewhere) to “It was derived” or “It originated from” or “It was collected from”.
76: “turned out to be early Pleistocene”
140: Be specific – cold temperate? Is there a paleotemperature estimate?
174: “In form” can be deleted.
196: “The crown is tall and conical…”
197: delete ‘also’
244: I think the term ‘deeper’ is more appropriate here than ‘higher’
248: use the word ‘lacks’ instead of ‘doesn’t have’

Kind regards, Robert W. Boessenecker
Experimental design: I think that the statistical analyses are executed well and the results are informative, however the study does suffer form the exclusion of the similarly sized (and closely related) sea lion Proterozetes.
Validity of the findings: See above.
Additional comments: No additional comments


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

Input:
Title: THE OLDEST RECORD OF THE STELLER SEA LION EUMETOPIAS JUBATUS (SCHREBER, 1776) FROM THE EARLY PLEISTOCENE OF THE NORTH PACIFIC
Review round: 1
Reviewer: 2

Output:
Basic reporting: See general comments.
Experimental design: See general comments.
Validity of the findings: See general comments.
Additional comments: This is an interesting manuscript and will make quite an important addition to our current knowledge of crown Otariidae. I commend the authors on the informative figures and descriptive text. I have carefully read the manuscript and have made some minor comments and stylistic suggestions as specified below, and there are two main issues which I would like to bring attention to. I hope the authors consider these recommendations and suggestions.

Major comments
Given the age and morphology, it would be advisable to do a thorough comparison with the mandibles of Proterozetes ulysses described by Poust and Boessenecker (2017) as this is relevant to the identification of GKZ-N 00001.
Being the oldest record of Eumetopias jubatus it could be considered as a calibration point that could help improve divergence estimates in crown Otariidae. This should be added to the discussion part of the manuscript. See for example, Parham et al. (2012), Syst. Biol. 61(2):346–359, and other related papers on this matter.

Minor comments.
Title has a typo: Eumetopias jubtus should be changed to Eumetopias jubatus
Line 36: “then,” should be changed to “them”
Line 58: I suggest changing “... that is now only around Hokkaido…” to “... which is now only around Hokkaido…”
Lines 59-60: I suggest changing “... relatively wide, because their mandibular and tooth fossils have…” to “... relatively broad, as dental and mandibular remains have…”
Lines 66-67: I suggest changing “It was yielded from…” to “The specimen came from…”
Line 69: I suggest changing “... that was an extinct…” to “... which is an extinct…”
Lines 82-83: I suggest changing “Therefore, exact identification and classification of GKZ-N 00001 is meaningful.” to “Therefore, highlighting the meaningfulness of proper identification of GKZ-N 00001.”
Line 108: Typo, reference “Veletz-Juarbe, 2017” should be “Velez-Juarbe, 2017”
Lines 119-123; Emended Diagnosis: I suggest the emended diagnosis should be more descriptive. As in, is there a proportion that can help illustrate the large size of the canines? For example, what is the diameter of the canines relative to the width of the rostrum or relative to the postcanine toothrow length? Is there a numerical range that can illustrate the large angle between the horizontal and ascending rami?
Line 137: I suggest changing “... Diplodonta are collected…” to “... Diplodonta have been collected…”
Line 140: “cold water” is there a known range of degrees that can help illustrate this better?
Line 163: I suggest changing “... and is slot shape.” to “... and is slit-like.”
Line 167: I suggest changing “... and rounded in form. It is 11 mm in diameter.” to “... and rounded in outline, with a diameter of 11 mm.”
Line 170: I suggest changing “... and is rounded in form. Its diameter is 6 mm.” to “... with a rounded outline and 6 mm in diameter.”
Line 171: I suggest combining the two sentences as in line 170.
Line 172: Same as above.
Line 174: Same as above.
Line 179: I suggest changing “... foramen is large. Its diameter is 7 mm.” to “ … foramen is large, with a diameter of 7 mm.”
Line 190: is the accessory cusp of p2 located mesially, distally or more central?
Line 191: I suggest changing “... bilobed in form.” to “... bilobed in outline.”
Line 248: Typo, change “laceorate” to “lanceolate”
Line 265: The first part of this sentence needs to be more clear, as it is, it can be misinterpreted as if otariid dentition would change from double to single rooted condition during ontogeny. It should be clarified that it has been shown to occur within a genus (i.e. Callorhinus) and more broadly, across other pinniped groups, although not necessarily in the same order as in otariids (see Velez-Juarbe, 2018, J. Vertebr. Paleontol. 38(4):e1481080).
Line 268: I suggest changing “... to be a primitive conditions in…” to “... to be a plesiomorphic condition in…”
Line 270: I suggest changing “... primitive…” to “... plesiomorphic…”
Line 276: I suggest changing “... jubatus has been known from…” to “... jubatus was from the upper…”
Figure 1: In figure 1B the digastric prominence is labeled as positioned on the posterior surface of the symphysis. This needs to be changed. In pinnipeds, the digastric muscle is “single-bellied” with its origin at the paroccipital processes, while its insertion is along the posteroventral edge of the mandibular ramus (see Howell, 1929). Also, see for example, the label of the digastric insertion in fig. 3 of Boessenecker (2011).


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: HIGH MOON BRIGHTNESS AND LOW AMBIENT TEMPERATURES AFFECT SLOTH PREDATION BY HARPY EAGLES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Although the manuscript is well written and clear, there are some sections that should be revised. A major concern is that the introduction and discussion are not written in the same way. While the introduction focuses on the prey, the discussion focuses on the predator. I suggest authors review these sections. Furthermore, a variable that seems to be important in the predator-prey system, such as the brightness of the moon, is only mentioned very slightly in the introduction.
On the other hand, I suggest that the methods section in the abstract have to be revised, you should explain better the lines 60-61.
After reviewing the data and the script, I suggest the authors send Table 1 to the supplementary material and replace it with a table showing all the final models and their variables. Table 2 and Figure 3 show information regarding the same issue. I suggest authors try to synthesize the information into one.
Experimental design: Despite the fact that the research is original and delves into a very interesting and not studied topic such as harpy eagle-
 sloth predator-prey systems I think the working hypotheses are somewhat confusing. In the introduction, different approaches to the proposed hypothesis are evaluated. The objectives of the study are not clear and the last paragraph is somewhat confusing. I suggest reviewing this section. I have some comments to help improve the manuscript:

1. The material and methods and results section don´t have the same framework. The authors explain several subsection within the material and methods section, which are not reflected in the results. Also the subsections within the methods are somewhat messy. I suggest checking the HARPY EAGLE PREY PROFILE subsection, as it mixes tagging GPS information and previous experiments. I suggest including more information about the type of gps device, method of placement, type of programming to get data .

Minor comments:
ln133: falconary is not a properly term here

ln 137: What is the age of the eagles? Born this year? please specify

ln142-146: I suggest include in the results section. I don't understand why it compares to a water bird. I suggest deleting the quote (Daunt F, WANLESS S, HARRIS MP, Money L, Monagham P, MONAGHAN P. 2007. Older and wiser: improvements in breeding success are linked to better foraging performance in European shags. Functional Ecology 21: 561–567 ) and look for a more suitable one, for example other raptors in reintroduction programs.
Validity of the findings: I thank you for providing the raw data and the script. After a review I suggest the authors include the year variable as a random factor in the models to check if there is an annual variation.
The authors' conclusions are consistent with the results, although as they indicate, it would take a few more studies to verify their final hypothesis.
Additional comments: The manuscript addresses some important ecological issue related with the harpy eagle-
sloth predator-prey system. While the study topic is interest and contribute to the knowledge about an endangered raptor I found the manuscript to be somewhat confusing and lacking some details.
I hope the suggested changes help improve this 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: HIGH MOON BRIGHTNESS AND LOW AMBIENT TEMPERATURES AFFECT SLOTH PREDATION BY HARPY EAGLES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The data, analysis, writing, review of literature and images are very good. But part of the analysis and some explanations of some biological definitions must be restructured.
Experimental design: It is a very good design of experiments, however it would be important to take the species Bradypus variegatus and Choloepus hofmanni separately because they show great differences in their behavior.
Validity of the findings: Supporting some conclusions in anecdotal facts can be very dangerous. It is well known that the species of the genus Choloepus are nocturnal, but assume that there is significant nocturnal activity of Bradypus due to an article by an individual defecating is not good, in my experience in radio-tracking in Colombia and Costa Rica nocturnal activity in Bradypus variegatus is almost non-existent.

"we show that as 240 temperatures increase, predation rates decline". An explanation for this pattern is that Bradypus variegatus will spend less time basking in the sun and therefore exposed when ambient temperatures are higher. In articles cited in the manuscript it is explained how the temperature of Bradypus vaariegatus varies as the environmental temperature does (Pauli et al 2016), for its activation and activation of their microbiota this species has a basking behavior, which can vary in duration depending on the body temperature reached by the species, in some mountainous localities where the environmental temperature is lower this behavior may have a longer duration (Urbani & Bosque 2007).

Finally, the explanation of lunar phobia have to improve, several articles exhibit that in dark nigths the activity of nocturnal mammals increase because the amount of enviromental ligth dcrease, thus, the predation risk.
Additional comments: The current manuscript has an incredible data set with which many analyzes of the predation by the arpya eagle and its relationship with its prey could be made; however, there are major problems in the analysis and structure of the document.

First, the differences in the natural history of the sloths species of the genus Bradypus and the denus Choloepus are unknown or despised, For example the differnces at the level of daily activity and metabolic needs.

On the other hand, there are some flaws in the structuring of the introduction where important topics for the development of the manscript are omitted such as lunar phobia and the general biology of the arpya.

Third, an analysis taking the sloths of the genus Bradypus separately could clarify some of the  effects of the variables on the predation of sloths.

Finally, there is a management of the term specialization that may be confused, one of the evidences of specialized animals in a prey is that its geographical distribution is less than the distribution of the resources in which it specializes, contrary to what was reported by the authors in this 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: HIGH MOON BRIGHTNESS AND LOW AMBIENT TEMPERATURES AFFECT SLOTH PREDATION BY HARPY EAGLES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The new version of the manuscript is much clearer and better structured. The authors have included the changes suggested by both reviewers and this can be seen in the present manuscript. However, I think the introduction could still be improved. I suggest that the authors review the paragraph that talks about the Harpy Eagle (108-123). They should talk about the eagle and its status first and then the predator prey- system.

In the previous review, I suggest the authors send Table 1 to the supplementary material and replace it with a table showing all the final models and their variables. They  response that accept this suggestion; the new table has been added as suggested.However, the same table appears in the pdf file as in supplementary material and not the one I suggested. The autor should review thats Table.

However there is a misstmach between text and tables. In the text, the authors say “ Statistical results are summarised in Table 2”, but Table 2 is related with “Prey composition in the diet of harpy eagles”. They should review that issue.
Experimental design: Now the objectives and the Hypotheses are clear. The authors improve the methods section. They included all the suggestion of the previous review.
Validity of the findings: No comment
Additional comments: The manuscript has improved since the previous revision. However, the authors can improve the manuscript. I have suggested some small changes that I hope the authors appreciate.