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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.

Title: THE ZEBRAFISH AS A MODEL SYSTEM FOR ANALYZING MAMMALIAN AND NATIVE Α-CRYSTALLIN PROMOTER FUNCTION Review round: 2 Reviewer: 3

Basic reporting: No problems here. Clear and well written manuscript. Experimental design: 1. The authors have done a better job addressing the significance of the research to the field. 2. The authors attempt have resolved the issue with qPCR. Validity of the findings: Data are robust and controlled. Additional comments: A genuine and sincere effort was made to improve the manuscript.

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.

Title: THE RELATIONSHIP BETWEEN EGG SIZE AND HELPER NUMBER IN COOPERATIVE BREEDERS: A META-ANALYSIS ACROSS SPECIES Review round: 1 Reviewer: 1

Basic reporting: Strong. See below for some points to consider. Experimental design: Strong. See below for some points to consider. Validity of the findings: Revisions are needed to bring the conclusions in line with the results. Additional comments: This is an interesting paper that presents a novel meta-analysis of whether egg size correlates with helper number in cooperative breeders. This relationship would be predicted if, for example, mothers plastically adjusted their egg size according to helper numbers. This is an issue of broad current interest in the cooperative breeding and maternal investment literature. While the study struggles with sample size limitations it does provide a novel contribution to knowledge in this area. Revisions could be usefully made, however, to better align the overall conclusions with the findings, increase transparency regarding the use of repeated measures of species, clarify a few points, and provide some more inclusive coverage of the relevant literature. I cover these points below. KEY POINTS 1. Greater caution is needed with interpretation, as the meta-analytical findings do not currently provide direct evidence of maternal plasticity or helper effects. Evidence that egg size varies with helper number does not necessarily reveal (i) that mothers actually show plasticity in egg size (as the observed variation in egg size could arise from among- rather than within-female variation in egg size, and while some studies considered this many did not), or (ii) that this variation, whether plastic or not, is causally attributable to variation in helper number (as there could well be confounds of helper number; e.g. territory quality). The manuscript would therefore benefit from… - Acknowledgement of both points in the discussion - Attention to the potential alternative explanations for the findings that recognition of these points highlights - Adjustments to the phrasing of arguments and conclusions throughout the manuscript regarding plasticity and effects of helper number (including the abstract and title, see point 2). 2. The title needs to be revised to more acurately reflect the findings. The title “Cooperatively breeding mothers reduce their investment in eggs in larger groups: a meta-analysis across species” currently seems misleading for two reasons: (i) the statistical evidence for such a correlation across all species falls shy of the significance threshold once repeated measures of species are controlled (ii) the meta-analysis is not based exclusively on within-female relationships between helper number and egg size (and so the meta-analytical result could be a product principally of among-female variation in egg size rather than within-female plasticity; see point 1). A more general title based on the question rather than the answer would resolve this easily. E.g. “Investigating egg size variation with helper number in cooperative breeders: a meta-analysis across species” 3. Please clarify the classification of species. In Supp Table 1, it is not currently clear why, when using the data for Malurus elegans from Lejeune et al 2016, the species has been classified as showing compensatory care for the within-mother effect sizes but additive care for the among-mother effect sizes. Surely the species either shows compensatory or additive care? Indeed, why use among-female effect sizes at all where within-female effect sizes are available, if the goal is to study plasticity? Please clarify / resolve. 4. Please adjust the figures to increase transparency regarding the repeated measures of species. In the funnel plots for Figures 2 and 3 it would be great if the authors could place a number next to each data point indicating which species it comes from (there aren’t many so this should work well), and then have the numbered list of species in the figure legend. This will allow the reader to consider the extent to which any apparent pattern is influenced by the inclusion of repeated measures of species. SPECIFIC POINTS INTRODUCTION 5. It would be good to see the introduction better address two relevant areas of the literature: (i) The introduction currently skates over the rather intensive research effort to date on load-lightening of post-natal maternal investment in cooperative breeders. Given the parallels with the questions asked here (re the impact on pre-natal maternal investment) it would be good to more fully explain the advances in this area to date (in particular the relevant insights from the comparative work in Hatchwell 1999 – currently only cited in passing). (ii) It also seems odd to cite virtually none of the empirical literature addressing maternal plasticity in egg size in cooperative breeders, when this is the focus of the manuscript. 6. Lines 78-81 – please clarify the argument here 7. Lines 102-105 - It seems odd to justify the study’s focus on pre-natal rather than post-natal maternal investment on this basis when … - Its not clear why maternal investment in egg size “cannot be directly influenced by helpers”, as helpers do have the potential to impact maternal condition at laying (e.g. via effects of group size on vigilance/foraging trade-offs etc.), and - Presumably the main reason for focussing a meta-analysis on pre-natal investment in the egg is novelty (much work has already been done on post-natal maternal load-lightening)? 8. Lines 105-109 and in the paper’s Summary Please clarify the definition of ‘additive’ and ‘compensatory’ care. As set up by Hatchwell 1999, doesn’t ‘additive care’ refer to a scenario where the mother does not compensate for the contributions of helpers (and ‘compensatory care’ refers to a scenario where she does)? The wording in this manuscript suggests that the distinction between additive and compensatory care here is made on the basis of whether the helpers (rather than the mother) compensate for each others’ contributions. This is subtly different, and presumably has implications for both the logic and the classification of species. Please clarify. 9. Lines 196-197 – please remove ‘broad taxonomic spread of species (fish to birds) in our sample’ from this argument or rephrase, as this doesn't really help to justify not controlling for phylogeny (there is only 1 fish). METHODS 10. Lines 119-122 – The definition of ‘cooperative breeder’ needs a clearer definition of ‘helper’. The inclusion of dunnocks (presumably polyandrous?) in the analysis, for example, suggests that the authors are comfortable with including species in which the helpers are actually breeders. What then is a ‘helper’? To increase transparency and clarify the logic it would also be good to explicitly acknowledge here and (briefly) justify the inclusion of cooperatively polyandrous species. 11. Conducting a statistical test for a difference between species with additive and compensatory care strategies seems to me to be pushing the data too far - there are data for only 3 species with compensatory care (perhaps 2 depending on the answer to point 3 above), so such a comparison at present seems pretty meaningless and potentially misleading. Wouldn’t it be more appropriate to simply plot the current figures and discuss the patterns by inspection? It would still be good to follow this up with an analysis of the overall data set restricted to species with additive care only (as there are at least 8+ species of this kind), which yields an interesting result, but then also conducting this analysis on the (just 2 or 3) compensatory species alone also seems to be pushing the analysis too far. RESULTS 12. To enhance transparency regarding the data structure throughout, whenever quoting sample sizes it would be good to break down the sample size in to the number of effect sizes and the number of species. So rather than reporting “n = 20” report “n = 20 effect sizes from 10 species”. DISCUSSION 13. Line 290 – rephrase – this isn’t ‘demonstrated’. 14. lines 291-293 – it seems a little late to acknowledge this critical point. To maximise transparency it would be good to include this point in the 1st paragraph of the discussion where the findings are summarised. 15. The final paragraph of the discussion goes a little hyperbolic – please bring the claims in line with the findings (greater caution needed throughout). e.g. Rephrase “The prevalence of LL indicates adaptive plasticity”. A prevalence of LL would indicate plasticity but such a prevalence has not been demonstrated here (as the analyses haven’t focussed on within-female relationships / experimental work) so a more cautious phrasing is needed. e.g. “our findings demonstrate the pervasive influence of the social environment on life-history” – that would be nice, but they don’t - the effects, if they do reflect plastic responses, could presumably be driven instead by correlates of the social environment (see point 1). e.g. “Our results demonstrate the importance of plasticity in maternal reproductive investment in cooperative breeders” – same issue.

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.

Title: THE RELATIONSHIP BETWEEN EGG SIZE AND HELPER NUMBER IN COOPERATIVE BREEDERS: A META-ANALYSIS ACROSS SPECIES Review round: 1 Reviewer: 2

Basic reporting: This is an interesting study on an important topic that is gaining increased interest. I have a lot of comments, mainly editorial, and so easy to deal with, but important nonetheless. The key issues are: (1) Many references from studies of non-cooperative breeders are used in inappropriate places to support points that are particular to cooperative breeders; (2) In many cases were references to cooperative breeding studies are made, they pertain to provisioning and not egg investment, when in this case the point is not necessarily transferable from nestling to egg investment; and (3) As a consequence of each, key information and or references are missing on the theory of egg investment in cooperative breeders (see Savage et al. 2013a, b and 2015) (these references are provided, but key theory presented within is generally missing (e.g. Carranza et al. 2008; Russell & Lummaa 2009). I have provided as much help as I can to address these issues. You might not agree with all, and that is fine of course, but the overarching point is that more theory on egg investment in cooperative breeders needs to be introduced to put your study in context and acknowledge the growing background in this area, and I personally think you need to be quite careful with many of the references you use as they are not necessary transferrable to the point being made, and so are either confusing or mis-leading. Line 65: Other possible references include Metcalfe and Monaghan 2001 and Lummaa and Clutton-Brock 2002, both in TREE. I mention these because they really deal with either other aspects of fitness (the former) or long-term, intergenerational effects (the latter). As such, I feel that both add to your reference list, while at the moment, I feel that the references are really saying the same thing. Line 66-67: I think you are making an empirical point, but mix this up with references of a theoretical nature (e.g. Maynard Smith 1977). To me, what is really needed here is some clear experimental evidence of a cost of not just reproduction, but increased effort. Line 71: Few is subjective, so I would suggest you use Fewer instead. Line 73-74: The word excellent is subjective (maybe switch to apt or appropriate), and the subject of the sentence needs to be made clear. At the moment, I think you are trying to carry the subject from the previous paragraph, but it is not so clear. Line 75: Delete in a single location as this is not true. Meerkat pups following the groups are not in a single location, and nor are the offspring in the numerous plural cooperative species in which helpers provide care to offspring from multiple nests. Line 78: The Hatchwell 1999 reference is fine for this, as it is a review, but the komdeur 1994 reference is a bit random. First it is subsumed within Hatchwell. Second, it is not the first evidence. Third, it is not the best evidence. Either you want to provide the first suggestion and/or the best, latest experimental evidence (preferably both) followed by a review, but not a random paper. Lines 78-81: Several lines are missing here, as you go from something super-general to suddenly in the next sentence talking about eggs in birds. In line 78, all we know is that you are talking about cooperative breeders, maybe in birds, and something to do with investment. But in line 79 you are on eggs in birds. You need several intervening sentences. First, you need to say that theory suggests that mothers should change investment according to workforce (e.g. Crick 1992; Carranza et al. 2008; Johnstone 2011; Savage et al. 2013, 2015; McAuliffe et al. 2015). Second, you need to say that experimental tests have been largely conducted during provisioning (e.g. Hatchwell & Russell 1996; Wright et al. 2008; Russell et al. 2008). Finally, you need to raise the question about whether or not such effects should be also manifest at the egg stage (Savage et al. 2015 deals with this directly). Line 84: Not sure you need to call this load-lightening or maternal load-lightening, this is kind of the same thing. Koenig et al. 2008 termed the specific hypothesis at hand as the concealed helper hypothesis. Line 85 and Lines 88-89: Not sure either Crick or Heinsohn dealt with egg investment, the more appropriate references here are Russell and Lummaa 2009 and Savage et al. 2015. Line 87: Lessells, has 2 l’s Line 91: Again, do any of the references deal with egg investment, again the more appropriate references as outlined above. Personally, I think there is a very important distinction between reducing investment at egg versus chick stages, and the references used throughout should acknowledge this difference. Line 94: You need to be careful here. The Burley hypothesis is distinct from the Sheldon one, and both are distinct from the Russell and Lummaa one. The Burley reference is actually more akin to load-lightening (in a weird way because she allows her mate to load-lighten when high quality), than to Sheldon’s definition of differential allocation, and Russell and Lummaa 2009 expand on the Sheldon definition to make it appropriate to cooperative breeders. Line 96-97: Maybe not here, but you should acknowledge in the discussion that it is conceivable that reduced egg investment with increasing group size is non-adaptive, and results from increased foraging competition. There are some nice studies that can rule this out, e.g. Canestrari et al. 2011. Line 101: Personally, I would set this up as a direct test between the concealed helper hypothesis/load-lightening and differential allocation, rather than a simple description of how mothers react to increasing helper numbers. Russell and Lummaa 2009 as well as Savage et al. 2015 clearly set out some predictions. The former suggest that DA might be predicted in plural cooperative breeders, or other cooperative breeding social structures that generates competition among reproductive females within groups. Savage et al. suggested that DA versus LL is expected as a function of the costs off egg laying, with LL occurring when the costs of egg production are high, and DA when it is low. In other papers, Savage et al. 2013 (BES) also suggested that it will vary as a function of helper relatedness. Further, Carranza et al. 2008 also set out circumstances in which DA can arise. In saying all this, I am not suggesting that you can provide a full test of these hypotheses, but it is important to at least provide the background theory, and the point of your study. I think it would be fine to say that the primary is to shed light on these hypotheses by testing whether helpers generally cause LL or DA effects. Line 103: I would not say that egg size cannot be affected by helper number. You can have evolved responses, and in meerkats, helpers influence maternal condition, through allowing her to reduce investment in pup feeding, which in turn positively influences investment in neonates (see Russell et al. 2003 BE). In some bird species, helpers feed females before egg-laying, which might also impact their condition. I think would be okay to say that they presumably have reduced direct effects in egg compared with offspring, but whether or not direct effects are not yet known. Lines 107-109: I don’t understand this, nor the reference to Langmore et al. 2016. Lines 118-119: This is not strictly true, usually only the female incubates the eggs, I suppose you mean provisioning, but need to be more specific. Lines 122-126: There are plenty of non-eusocial insects where the above constraints are lifted. The main problem with insects for your purposes is that there are almost no studies of egg size across helper numbers. Russell & Lummaa 2009 could not find any non-eusocial cooperatively breeding insects in which maternal investment in egg size as a function of helper number was assessed, but it would be worthwhile checking to see if there are any since this publication, which would help bolster your sample size. Lines 154-158: Sorry about this, I was in the middle of a high load of teaching. I can confirm to you and to the other reviewers and editor, that categorisation of helper effects according to the presence versus absence of helpers is valid in wrens as the number of groups with greater than one helper were very limited. Nevertheless, categorisation will inevitable inflate your effect size estimate. I will provide you with the necessary details, although there will be no qualitative change to your results. I am not sure Figure 1 is necessary, perhaps it could go online? Line 268: I think it would be nice here to re-iterate that this results provides general support for the concealed helper hypothesis. Line 271: It might be nice here to re-iterate that this evidence complements the hypothesis that additive care by helpers should lead to LL in females in terms of provisioning (Hatchwell 1999). Line 280: You need to be a wee bit careful here, as Russell et al. 2007 found the evidence and came up with the idea, but Koenig et al. which did not find any supporting evidence, coined the hypothesis. At the moment, you use the two citations to different ends, but this is not clear, making it suggest that the Koenig et al study is also supportive. Line 286: At the moment, you use the term In contrast, to contrast with LL, but the subject of the previous sentence is about support not LL per se. It therefore sounds, as written, that you are going to provide contrary evidence for LL, but in fact go onto provide contrary evidence for DA. I think I would suggest starting a new paragraph, and making the subject of the sentence clear. I also think it would be appropriate to discuss the suggestion by Russell and Lummaa that DA should be more likely when reproductive females compete within groups, since their fitness will be impacted by the relative success of their offspring. It is at least noteworthy, that the majority of the species that you studied were singular breeders, meaning females do not compete reproductively amongst each other within groups. Line 311: I think that this Savage et al. 2015 reference should be cited and discussed in line with your results, but this is the wrong citation here, and should be Carranza et al. 2008. Experimental design: No comments Validity of the findings: No comment Additional comments: I have added everything above under basic comments

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.

Title: THE RELATIONSHIP BETWEEN EGG SIZE AND HELPER NUMBER IN COOPERATIVE BREEDERS: A META-ANALYSIS ACROSS SPECIES Review round: 2 Reviewer: 1

Basic reporting: see below Experimental design: see below Validity of the findings: see below Additional comments: I am very sorry to say that i am disappointed by this latest version of the MS, and again have spent at least a full day refereeing it. In particular, I still have quite a few significant issues with clarity and citation use, which to me are very linked in this MS. I said this last time, but perhaps I was not clear. For example, there are many examples of using the wrong citations or using a mixture of citations that confuse rather than clarify. When this occurs, it is unclear whether the point being made is wrong and the citation correct, or vice versa. Either way, citations should help clarify a point, not confuse it. A key issue is the use of citations based on theory of post-natal care to support a point made about pre-natal investment (e.g Crick 1992, Hatchwell 1999 or even Brown 1987 or Stacey & Koenig 1990). There are many reasons why one should not do this. Most notably, mothers might reduce investment in offspring provisioning because chicks are less hungry due to the contributions of other group members and consequential effects on offspring begging. By contrast, given that mother must by definition lay the eggs before the contributions of any others, there are obvious reasons why load-lightening at this stage might not be expected, despite showing post-natal LL. This is why LL at the egg stage was not thought of prior to 2007. It is incorrect, presumptuous and mis-leading to use Crick 1992 or Hatchwell 1999, to support the load-lightening hypothesis at the egg stage. Using theory provided to understand post-natal LL as a means to understanding pre-natal maternal strategies (e.g. by citing Hatchwell 1999) leads one inevitably to conclude that it is the post-natal responses to variation in helper number that drive pre-natal maternal investment strategies. By contrast, theory on maternal investment pre-natally make no such assumptions (see Russell et al. 2007, Taborsky et al. 2007, Russell & Lummaa 2009, Savage et al. 2013, 2015). Instead, in the vast majority of the hypotheses or theory provided in the above papers, the point concerning LL are more simple: an increase in helper numbers leads to an expectation of more food (or protection) for offspring, which allows mothers to save resources at the egg stage without losing fitness. The authors are right that if all co-carers fully compensated for each other’s presence, this assumption falls away. But there is next to no evidence that this is the case. Instead, any compensation is partial, and so increases in helper number (or helper presence versus absence) generate additive care. Further, more detailed theoretical attention, particularly by Savage et al., shows that post-natal care responses, are themselves linked with pre-maternal investment. This means that it is much safer to use helper presence/number as a predictor of maternal pre-natal investment (as is typical) rather than helper behaviour (this study). Thus, I am happy with the first analysis on helper effects on maternal investment, but less happy with the suggestion that helper behaviour post-natally drives this pre-natal investment (since it could be the other way around). My worry about the second analysis is exacerbated by the lack of clear definition of compensatory versus additive care. For example, dunnocks appear to be used as an example of compensatory care (Discussion), but increasing group sizes are associated with additive care in this species, because compensations are incomplete. I would like to see a table inset in this MS, which outlines the species used as compensatory or additive with a clear justification for each one. I think that what we will find is that there is a continuum, from rarely fully compensatory to highly, or even rarely, super-additive care. Finally, on this point, I worry that because of the rarity of fully compensatory systems (perhaps absence) the question of whether additive care post-natally is collinear with the first question? Below I outline my numerous specific comments Line 72-73. As currently written this statement is untrue. Not only are there ca. 100 studies in cooperative breeders looking at parental responses to variation in the social environment, but variation in the social environment includes variation in co-carer contributions and so includes bi-parental care systems. There are over 100 more studies in this care system. I am not sure this is fewer than the number of studies looking at variation in response to other traits, and even if it is, the point is moot. Lines 74-75. As written, this includes bi-parental care. I think it is fine to include bi-parental care at this point, but to get to cooperative breeders you need to say how they differ from bi-parental care systems, and the answer here is that they have variation in the number of carers. This allows maternal variation in investment to be linked to variation in the social environment, without direct manipulation of that environment (ie in cooperative breeders the social environment varies among mothers, which is not so obviously the case in bi-parental care systems). Lines 75-79. The point that needs to come across here is not so much what cooperative breeders are, but that there is substantial variation in helper number within and among species, allowing tests of maternal responses. This is one of the key points made by Russell and Lummaa 2009, which formed the basis for their predictions about how this variation might be expected to affect maternal investment at the egg stage. Lines 80-84. You need to be careful here. As the other Reviewer pointed out last time, you cannot use words like plasticity, unless you show within-female variation, and the only way to show this is to do an experiment or to use long-term data which allows sufficient contrasts. This is a classic case of where your message might be correct, but most of your citations are inappropriate, or whether you citations are appropriate but that the point being made need not be restricted to plasticity. Given the sentence before, I am assuming that the statement is correct, but most of the citations are not, including Hatchwell et al. 2004, Heinsohn 2004, Koenig et al. 2011, as none of them use experiments to specifically detail within-parent responses. Cockburn et al. 2008 provides comparisons of long-term data to show adaptive female, but not male responses in superb fairy wrens. One of the Macdonald et al. bell minor papers also uses nice removal and chick begging experiments to test for plasticity in bell minors, and one of these should also be cited. Lines 84-89. This is a good example where multiple points are made, but all the references are added to the end – making it impossible to know which substantiates which point. To make matters worse here, the points are incorrect, and all of the references are inappropriate. First, you are talking about responses here, and so by definition the consequences of experimental manipulation or very carefully detailed statistical analyses based on longitudinal data. There are only a handful of such studies, listed above (none cited in this section), but I am not sure they are sufficiently numerous to talk about a typical response. So, instead. I would suggest you say…One such response is….. Second, if you are somehow trying to talk more generally, you cannot use the word response, it would not follow your previous sentence and again you cannot really talk about a typical response (see Hatchwell 1999). In this case, the Reyer and Manica references are irrelevant, but at least Hatchwell and Kingma are good. Either way, you need to be more careful in your use of words, and more precise in both what you say and how you cite the work. Line 82: Manica and Mirani reference is a poor choice, there are many confounding factors in that paper and compensation cannot be inferred. Compensation can only be shown by experiment and these have been done rarely, e.g. Hatchwell & Russell 1996, Wright 1998, Macdonald et al. in a series of bell minor papers; Russell et al. 2008 Line 92: Neither Brown 1987 nor Koenig and Stacey have anything to do with eggs and should be deleted. Canestrari et al. should also be removed unless you want to cite every single paper that is in your meta-analysis. The only paper that outlined the idea and made any theoretical predictions is Tabosrky et al. 2007 (for protection) or Russell et al. 2007 (provisioning) and Russell & Lummaa 2009 (inclusive of both and other helper effects); and then the formal models of Savage et al. Lies 89 & 96: You have Savage et al. 2015, then Savage, Russell and Johnstone 2015, these formats need to be reversed (full authors first and then et al. 2nd)? Line 101. Crick 1992 makes no mention of eggs. I said this in my last review: one cannot extend theory on responses to offspring provisioning with responses on eggs. They are many reasons why load-lightening at the egg stage is very different, including the fact that no other group member in real time can respond. The only theoretical treatment of helper effects on egg investment are Taborsky et al. 2007, Russell et al. 2007, Russell & Lummaa 2009, and the series of Savage et al. papers; particularly his 2013 and 2015 papers. No other paper is relevant that I know, and certainly not those cited. Line 103-104: Remove Canestrari et al. since this is entirely empirical and does not extend any of the points made previously. And Santos 2016 is a thesis, which should be removed completely. Line 111. Savage et al. 2015 should probably cited here which squarely considers how mothers should invest with increasing helper numbers. Line 116. Remove the word “exists” Lines 117-119. Not sure what is trying to be said here. If I understand correctly, does a meta-analyses really indicate whether detailed studies are required to understand selection and the biology of individual species? Lines 119-122. I am not sure of your logic here. I am not sure you need to justify why you look at pre-hatching rather than post-hatching investment. They are totally different questions. I think the important point is more because it is a stull relatively under-studied area of research, with demonstrable implications for understanding selection on cooperative systems (e.g. Russell and Taborsky 2007 refs). Lines 125-128. Sorry, I have no idea of the logic here. I suggest that the authors check out Russell & Lummaa 2009 and Savage et al. 2015, for logic in when we expect LL of DH at the egg stage. Again the authors cite a paper that has nothing to do with egg investment, and so confuse the point being made. The problem with understanding post-natal investment is that maternal, paternal and helper contributions are dynamic and potentially negotiated as chicks age (McNamara et al. 1999). Post-natally - one cannot say categorically that mothers are more likely to LL when helper effects are additive, because the reverse might be true, helper effects might be additive because mothers load-lighten. The interesting thing about the egg investment stage, is that mothers need to invest before partners or helpers are able to respond. Indeed, the response of co-carers, when it occurs, is itself a part-function of maternal investment at the egg stage (Savage et al. papers). Thus, it is circular, mis-leading and potentially wrong, to use co-carer investment strategies at the chick stage to predict maternal investment strategies at the egg stage. Predictions about LL versus DA at the egg stage need to be articulated from papers that deal specifically with these points at the egg stage. Line 130. Need to say at the egg stage Line 131. Following my comments above, you need to be careful to define what you mean by additive care. You mean at the chick stage, and you mean of all co-carers other than the mother? Or do you mean overall, irrespective of LL by the mother at the chick stage. You state in the methods that you simply used what the authors had reported, but 1-2 lines summary of this is required in the Intro and a table in the methods. For example, in chestnut-crowned babblers, mothers reduce their provisioning contribution to almost zero in large groups. But, because the contributions of male carers are unaffected by the number of co-carers, and because the number of co-carers can be numerous, there is a positive association between carer number and overall provisioning rate of the brood. I am guessing that you would call this additive care, and I would too, but serves to illustrate the point that not all individuals in the group need to show additive care for care to be additive. Indeed, you could also have a situation where all group members LL, but care is still additive, because LL is only partial. Finally, it is unclear whether you are saying that because care is additive, we expect maternal LL at the egg stage (which is what I think you are saying), but I would say that opposite is also possible, additive care at the nestling stage arises because of maternal LL at the egg stage. You do not need to make all these points of course, but I hope you can see that some clarity is required, and you need to be clear that any detected association between LL at the egg stage and additive care at the nestling stage could arises as a cause or effect. Lines 142-146. I fundamentally disagree with this comment, and have predicted elsewhere (Russell & Lummaa 2009) that maternal effects in social insects should be much stronger than in vertebrates. All you need to say here is that the data is lacking. Lines 146-149. This is also incorrect. Broods often receive more incubation and more food in communal birds because the number of nest attendants is far great than the usual 2. The problem here is that you cannot identify which egg came from which mother, and so it is difficult to know what causes any changes in egg size in communal nesters. Line 209, were they not also inclusive of different data, which I would have thought would have been more important. For example, Langmore et al., published 9 years later, used far more years of data than Russell et al.. Line 273. Helper effects can be additive even if they are compensatory. Please can you clarify if these species show additive care, albeit perhaps to a lower level than others species. Lines 288-289. Need to specify that less means reduced egg size not clutch size Lines 293-294. Is this association not just an epiphenomenon of the fact that there is an association between helper number and maternal egg investment across species, and almost all of the species included showed additive care? Lines 294-298. I don’t understand what is being said here. Dunnocks show additive care, females with 2 males receive more food for their young than those with only 1 male. This additive are arises from incomplete compensation, and females increase clutch size as a consequence (Davies and Hatchwell 1992). As a consequence, I do not follow the logic of your point – females increase egg investment and decrease provisioning when breeding polyandroulsy; so is the decrease in provisioning because of the extra male or because of their increased egg investment, or a bit of both? Lines 311-315. Please at least acknowledge that causality is unknown. We do not know whether maternal LL at the egg stage generates additive effects at the chick stage, or not. I do not necessarily disagree, but we do not know the direction of causality at this time. Line 317. Did Woxwold and Magrath not suggest that the relationship between group size and breeding success might be weak because not all group member contribute? If so, this is a very different argument to the one you present here, which was really the foundation of Russell et al. 2007, and then furthered by Koenig et al. when he coined the term concealed helper effects. Lines 340-345. I do not disagree with any of this, but it is at least noteworthy that this analysis does not control for maternal egg investment, and so any role of maternal egg investment (particularly clutch size) in generating differential nestling starvation (Liebl et al. 2016 Anim Behav).

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.

Title: THE RELATIONSHIP BETWEEN EGG SIZE AND HELPER NUMBER IN COOPERATIVE BREEDERS: A META-ANALYSIS ACROSS SPECIES Review round: 3 Reviewer: 1

Basic reporting: Great Experimental design: Great Validity of the findings: Great Additional comments: Very well done for dealing with all of my previous comments, I do think the MS is now far clearer and more informative than before.

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.

Title: THE IMPACT OF SELF-INCOMPATIBILITY SYSTEMS ON THE PREVENTION OF BIPARENTAL INBREEDING Review round: 1 Reviewer: 1

Basic reporting: On the whole, the paper is well written, and the background literature is well reviewed. The figures are clear and well presented. That said, there are times where the literature is incorrectly explained, and the relationship between the major hypothesis and the results is not particularly solid (see below). Another major shortcoming of the manuscript is that the computer code is not provided. It should be uploaded with the next submission, and must be stored permanently on dryad conditional on acceptance. However, all of these concerns can be addressed relatively easily. Experimental design: I believe the research is well executed and suitable for this journal. As said in '1. Basic reporting' it is not repeatable without computer code, but I get the sense of their approach. The investigation is rigorous and honest. Validity of the findings: I believe that the work as stated was well executed. A concern I had is that results were described as significant or insignificant without the use of p-values. I would suggest a different approach (if computationally possible) - that the authors increase their sample size such that differences are apparent without the need to invoke null hypothesis significance testing. If that degree of replication is not feasible, than the authors should provide p-values with their statistical statements. My most pressing concern is the relationship between the motivating question and the results. I describe this below. Additional comments: This is an interesting paper. That said I have a significant concern about the framing of this paper with regard to the major motivating hypothesis. 1. The authors claim to investigate if differences in the extent of biparental inbreeding associated with alternative incompatibility systems can favor chemical based self-rejection, and claim to reject this hypothesis. However, the simulation does not address this problem. Rather, the authors investigate differences in the genetic load and the extent of biparental inbreeding as a function of alternative self-incompatibility systems, and never directly test their proposed hypothesis. I therefore would like to see the authors ask directly if different modes of self-incompatibility change the extent of autozygosity, allozygosity, and inbreeding depression (the question they asked). The question of selection for this form of self-rejection should be relegated to speculation in the discussion. 2. In fact it is not clear what such a test of this hypothesis would be. The authors do not make clear wether they imagine such incompatibility systems replacing one another in natural populations, or if they expect higher level species selection to act. This later hypothesis is often invoked to explain the distribution of plant mating systems (e.g. Goldberg et al 2010). These levels of selection should be clarified in the introduction (lines 34-38). 3. I am somewhat worried about the sensitivity of results to a few somewhat idiosyncratic decisions concerning the nature of the genetic load that was simulated (modest selection for a small number of loci with high mutation rates) and the expression of this load (individuals with high inbreeding depression take up space in the lattice). I would be curious to see results with more, rarer deleterious recessives with more extreme fitness effects (as we generally assume). I would also be interested to see how early-acting inbreeding depression (such that unfit inbreds did not take up space) would impact the results. 4. There are a few (relatively minor) misstatements in the introduction. As the authors' know being that 95% of plants are hermaphrodites does not mean "that a single plant is capable of self-fertilizing and reproducing without a mating partner," but rather that plants have male and female parts. The authors should clarify this for their audience. I also think that Fisher's automatic advantage could be explained better. The advantage is that selfers can fertilize themselves and someone else not that they pass a higher fraction of their genes to their offspring (if all pollen that self-fertilized an individual fertilized a different individual instead, fitness would be identical). Another minor point is that heteromorphic incompatibility systems often have genetic based rejections, however, I don't think this would impact the modelling or the results.

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.

Title: THE IMPACT OF SELF-INCOMPATIBILITY SYSTEMS ON THE PREVENTION OF BIPARENTAL INBREEDING Review round: 1 Reviewer: 2

Basic reporting: This manuscript is nicely written, with professional language and clear figures. Ideas and relevant literature frame the context well (except for the specific concerns noted below). There are no data to be archived, but the code is available. Experimental design: This is a primary research manuscript with a clear motivating question. Investigation of the question is rigorous (except for the specific concerns noted below). The code provided and settings described are enough to replicate the research. Validity of the findings: The findings are summarized appropriately. Conclusions are mostly stated clearly, though some improvements are noted below. My concerns about the overall biological interpretation are noted below. Additional comments: This manuscript is focused on the general question of how different systems of self-incompatibility affect biparental inbreeding. This is an interesting question to ask for both evolutionary and ecological reasons. The authors have done an excellent job of clearly and concisely presenting the results from many simulations. Before this work is published, however, it is essential to clear up some issues about the interpretation of the SI systems and conclusions. (1) My one big concern is about "heteromorphic SI." The model implements a system called PSI, in which selfing is prohibited but there are no additional mating incompatibilities. This system is useful as a control, in that it does nothing to reduce biparental inbreeding. However, it bears no resemblance to real heteromorphic SI systems, in which intra-morph matings (not only selfs) are prohibited by an S-locus that is linked to one or more morphological loci. The authors only admit that PSI is an entirely artificial system near the end (lines 375-378), and the entire introductory framing, as well as much of the discussion, is cast as heteromorphic versus homomorphic SI. This is extremely confusing and misleading. Please either completely remove the discussion of heteromorphic SI, or simulate new results using a genetic system that reflects how heteromorphic SI works in nature. (2) My other main concern is about the central conclusion: that homomorphic SI systems do not do much to prevent biparental inbreeding. For one thing, some of the statements about angiosperm evolution are far too sweeping to be based on this one small simulation study (e.g., lines 27-28, 322-323). Additionally, I suspect this finding from the model may in part be an artifact of the large number of S-alleles (Table 1). I think these are at least twice as large as what's known from natural systems, which would greatly reduce the potential to avoid biparental inbreeding. It would actually be quite interesting to see how the inbreeding avoidance scales with the number of S-alleles, at least a comparison between "few" (say, 10) and "many" (say, 40). Alternatively, the rate of generating new S-alleles could be lowered. Either way, please provide some references to support that the number of S-alleles in the model is biologically realistic. (3) My remaining comments are much smaller, ordered by appearance rather than importance. throughout: "biparental" is more usual than "bi-parental" throughout: "Mating system" typically refers to the selfing rate rather than the type of SI. But admittedly the literature is frustratingly inconsistent with this kind of terminology. throughout: SI does not always act in the stigma. For most or all GSI, it acts in the style. 30: Renner (2014) says that 5-6% of species are dioecious, but there are lots of other sexual systems (monoecy, gynodioecy, etc.) besides hermaphroditic. 30-31: Just because an individual is hermaphroditic doesn't mean it is capable of uniparental reproduction (e.g., SI). 35: The logic of "consequently" is not clear. 52-54: Using "haplotype" here means it isn't correct for sporophytic SI. 67-68: Can an empirical citation be provided? 81: Purging hasn't been explained. 91: It's not clear what "continuous" means here. 115: "if they share *only* the same recessive" 129-143: I think of inbreeding as defined at the level of which *individuals* mate with which, so it sounds a bit odd to say that inbreeding is reduced at particular parts of the genome. Maybe homozygosity would be clearer? 148: The statements here are true (but see a comment below), but "for this reason" is not their logical connection. 158: For isolation by distance, a toroid doesn't seem appropriate. Better to allow dispersal off the edges? 209-211: Why is autozygosity defined by grandparents rather than by parents? If the latter, it seems like selfing would automatically be separated from biparental inbreeding. I'm wondering if the current "proportion autozygous" results (Fig 3) might be dominanted by selfing, obscuring differences between the SI systems. 220-227: This would make more sense if there were some indication of what quantities will be compared. Results: It would help if each subsection began with an indication of what is being checked or what questions are being answered, rather than just jumping into descriptive results. Lines 259-262 do a good job of this. The Discussion leads off with a small matter that is not the main point and sounds like an artifact. It would be more effective to start with big questions and perspective. 313-314: I don't follow this sentence. Several generations from when? 341, 346, 348, and 148-149: The claim is that there are many S-alleles in a population *because* that's what's necessary for enough compatible matings. This reads like population-level group selection in the sense that real populations or simulation runs without enough S-alleles preferentially went extinct. Is this the intent? Natural SI populations with few S-alleles do exist, e.g., after a bottleneck. 373-375: This seems like an extremely aggressive claim with no foundation in logic or data. 386-389: SI can also weaken in older flowers simply because proteins denature, rather than because it's a beneficial strategy. Table 1: The s^2 column doesn't seem necessary, or it could at least be placed right next to the sigma column. Figure 1: This is a nice depiction. One small adjustment is that the pollen's inner red circle looks orange (on my screen, at least).

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.

Title: FORELIMB MUSCLE AND JOINT ACTIONS IN ARCHOSAURIA: INSIGHTS FROM CROCODYLUS JOHNSTONI (PSEUDOSUCHIA) AND MUSSAURUS PATAGONICUS (SAUROPODOMORPHA) Review round: 1 Reviewer: 1

Basic reporting: This is a well-written and well-executed paper examining muscle moment arms and joint ranges of motion in the forelimb muscles of an extant ‘sprawling’ archosaur and an extinct ‘parasagittal’ archosaur. Basic reporting The English is generally clear and unambiguous throughout. There is some confusion of UK vs. US English throughout; e.g. meters (US), but modelling and centre (UK). I have identified a few typos in the text and picked them on the attached PDF. The paper is set in an appropriate context, the literature is appropriately referenced, and the structure is appropriate. The figures containing moment arm graphs are far too small. I was unable to read the text in the figures, and while they might be reproduced slightly larger in final print, they are still way too small. I suggest breaking the moment arm figures up and making the text at least 10 points at final size. The manuscript states in one place that models are available upon request, but in another that they are included with the study. My preference would be to see the models included with the study, but that is the prerogative of the authors. You state at the beginning that extension is negative, but throughout there is confusion about flexion and extension and which is negative. Maybe I missed something but I can’t see any figure captions for the supplementary information figures. Experimental design: The research question is clearly framed, well defined, relevant and meaningful. It has been performed to a high standard and the methods have been described in sufficient detail. Validity of the findings: The data appears robust, is clearly described, and is appropriately interpreted. Conclusions drawn are linked to the original research questions and supported by the results. I have some comments regarding your interpretations about quadrupedality. 1) You state that not being able to flex the humerus anterior of vertical would preclude quadrupedalism. I don’t think that’s the case. In obligate quadrupedal ornithischians, I don’t think the humerus could have been protracted past vertical because of the location of the humeral head on the caudal surface of the humerus and the morphology of the glenoid. Because the elbow is strongly flexed, however, this doesn’t prevent quadrupedalism. I’ve said this somewhere in print and can probably dig it out for you. But these animals were clearly quadrupedal. It must have meant they had a very short stride, admittedly, but I don’t think it precludes quadrupedality; 2) A pronated manus isn’t necessarily necessary for quadrupedality. Trackways of iguanodontids show quadrupedal locomotion with an apparently supinated manus; 3) In sauropodomorphs, quadrupedality seems to be considered to only have occurred once. I’m not sure we necessarily have to assume this. It’s possible that there was mosaic evolution of the forelimb in sauropodomorph evolution and that facultative quadrupedality could have been present in some forms and not in others. Thus contradictory findings might be expected on the sauropodomorph ‘stem’ when examining different basal sauropodomorphs. Additional comments: There are many minor comments on the attached pdf.

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.

Title: FORELIMB MUSCLE AND JOINT ACTIONS IN ARCHOSAURIA: INSIGHTS FROM CROCODYLUS JOHNSTONI (PSEUDOSUCHIA) AND MUSSAURUS PATAGONICUS (SAUROPODOMORPHA) Review round: 1 Reviewer: 2

Basic reporting: 1) Clear, unambiguous, professional English language used throughout. Excellent. Just some minor typographical errors were noted with sticky notes. 2) Intro & background to show context. Literature well referenced & relevant. The introduction and background are good, but the section on pronation appears to have overlooked the most recently published tests of the references that they rely on most heavily in their results and discussion. I provided detailed comments, explanations, and references to ask for some clarifications in terminology and their interpretations of pronative ability in their study specimens. 3) The structure of the submitted article should conform to one of the templates. No comments. 4) Figures should be relevant to the content of the article. The figures are good, and I trust that the published version will indicate which side is Crocodylus, and which is Mussaurus. 5) The submission should be 'self-contained,' should represent an appropriate 'unit of publication,' and should include all results relevant to the hypothesis. No comments 6) All appropriate raw data has been made available in accordance with our Data Sharing policy. No comments Experimental design: 1) Original primary research. - No comments. 2) Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap. - In regards to their stated knowledge gap for dinosaurian pronation, I inserted comments as needed to help identify what some of the newest studies have tested in relation to their research questions. Hopefully these comments can help identify how their pronation study fits in with the latest research. 3) The investigation must have been conducted rigorously and to a high technical standard. - Excellent. 4) Methods described with sufficient detail & information to replicate. - Excellent. 5) The research must have been conducted in conformity with the prevailing ethical standards in the field. - No comments Validity of the findings: 1) The data should be robust, statistically sound, and controlled. - No comments 2) The data on which the conclusions are based must be provided or made available in an acceptable discipline-specific repository. - No comments 3) The conclusions should be appropriately stated, should be connected to the original question investigated, and should be limited to those supported by the results. - Excellent, but with some inserted concerns as to their support for their pronation results. 4) Speculation is welcomed, but should be identified as such. - No comments 5) Decisions are not made based on any subjective determination of impact, degree of advance, novelty, being of interest to only a niche audience, etc. Replication experiments are encouraged (provided the rationale for the replication, and how it adds value to the literature, is clearly described); however, we do not allow the ‘pointless’ repetition of well known, widely accepted results. - No comments. 6) Negative / inconclusive results are acceptable. - No comments Additional comments: Very nice piece of writing - This paper is carefully written, with thoughtful consideration to different points of view and terminologies so common to myological/arthrological studies. The layout is very organized and flows nicely; the figures are easy to interpret. I am impressed with the support for your interpretations of how to categorize movements based off of your moment arms; this is the type of quantitative support needed to help standardize some of the terminology for sprawling versus parasagittal clades. I inserted a number of sticky notes and highlighted text throughout the pdf markup. Most of them are minor suggestions, typographical error notations, and questions. My only concern was with the pronation aspect of the study. My latest research is testing some of your primary references on forearm pronation in dinosaurs, and I noticed that your arguments could benefit by incorporating them. I tried to outline how your interpretations of forearm pronation could be affected by this research.

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.

Title: FORELIMB MUSCLE AND JOINT ACTIONS IN ARCHOSAURIA: INSIGHTS FROM CROCODYLUS JOHNSTONI (PSEUDOSUCHIA) AND MUSSAURUS PATAGONICUS (SAUROPODOMORPHA) Review round: 2 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: I believe that the changes in discussion and interpretation are more than satisfactory in helping identify how your study fits in with the latest pronation research.

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.

Title: ASSOCIATIONS OF IGF2 AND DRD2 POLYMORPHISMS WITH LAYING TRAITS IN MUSCOVY DUCK Review round: 1 Reviewer: 1

Basic reporting: English writing still need to be improved, because there are some issues on the wording, spelling, and sentence transition and connection. For example, you should pay attention on the consistent italic style for gene names, in your manuscript, some names are italicized but some are not, please correct them. Line 74, "Identified" change to "identified"; Line 75, changed "guide" to "reference"; Line 79, " please write full name of "dH2O". Line 80, "3 min pre-denaturation at 94ºC" change to "pre-denaturation at 94ºC for 3 min" Line 96, delete " mRNA" and "present", change "quantity" to "expression". Line 106, change "sequined" to "sequenced". Line 107, delete "using" Line 110, change "using" to "in". Line 116, change "egg traits' to "egg production traits". Line 119, add "Difference with" before "P value". Line 124-128, gene sequence of Muscovy duck is identical to "that of" Anas and Anser. Line 137, add "also" after "other tissues". Line 169, add "However" before "we only locate..." Line 173, change "IGF2 high expression" to "high expression of IGF2" Line 189, " However" change to "In addition" Line 202, change "with" to "on". In addition, there are some statements that lack important information for proper understanding. For example, line 32-33, IGF2 in the dominant follicles support function of which hormone? Line 70-71, cDNA was used as template to amplify what region of IGF2 and DRD2 genes? Line 110, how many females are genotyped through sequencing? Line 146, 2 SNPs of IGF2 and 11 SNPs of DRD2 were selected for further association study based on what? Line 173, IGF2 high expression in the ovary many be related to whose development? Line 181, IGF2 might regulate cell proliferation during follicular development in what animal? Line 212-213, are the two SNPs with high linkage? You never mentioned in the results part. Literature references should also be selected carefully and properly based on the purpose of your study. In this study, your aim is to investigate association of IGF2 and DRD2 with egg production in Muscovy ducks. In this case, you should focus more on the literature about reproduction of Muscovy ducks and research of the two gene in the reproduction system of avian species, and suggest how your research is important to fill the current knowledge gaps. Why do you choose some literature on their relationship with human and fish diseases (line 35-38, line 195-199, line 204-206)? In addition, are you sure you understand the reference paper correctly? In line 197, it is DRD2 agonist used as a reagent for the treatment of the disease rather than DRD2 itself used as a agonist for the disease. I totally doubt whether your corresponding advisor has read it or not. Experimental design: This study is a primary research within aims and scope of the PeerJ journal. However, the authors did not make enough justification for their experimental design in the introduction part. For example, why do they study Muscovy ducks instead of other birds? why do they choose IGF2 and DRD2 to study? how did they find these important genes? why do they choose E300D and E59W instead of egg production at other time points as important traits in their study? In addition, the method is also not described sufficiently. For example, how are the primers designed for sequence cloning, qPCR and SNP detection? Although the authors listed all the primers in Table 1, they did not describe how are the primers used in each step.You should cite the primer names in the methods section and also put a note under Table 1 to explain the meaning of primer names. In addition, I suggest you to put table 1 to supplementary materials and add a figure to explain the structure of the two genes and the location of each primer in the gene sequence. In Figure 1, you made a phylogenetic tree of muscovy duck DRD2. But you never mentioned in your method section how the tree was generated and how the sequence alignment was performed. Why do you only made phylogenetic tree for DRD2 but not for IGF2? In Figure 2 and 3, how do the gene expressions differ among different tissues? Please label letters above each bar to denote the significant differences. In Figure 4, you should include the whole heatmap in the figure rather than the significant block you are interested in. In Table 2, I suggest you to add frequency of each mutation as another column. Finally, in the animal experiment, you did not give enough information on how the animal were obtained from the company. How many ducks were offer? How were they were bred before the study? What feed were they provided? Validity of the findings: The authors did not discussed enough to assess the impact and novelty of their study. Although the data is statistically sound and some significant SNPs were identified to be associated with egg production traits, they failed to make logical connection with current knowledge in the field, and make further biological speculations on how the SNPs may affect function of the genes and egg production traits. I think they still need work hard to improve their introduction and discussion part. Simply listing findings of previous studies in human, mammal and fish but lack of necessary rationale and linkage does not make a sound discussion at all. Additional comments: This article described two genes and their polymorphisms, specifically high expressions in ovary, and significant SNPs related to the reproduction traits in Muscovy ducks. The study is important and interesting. However, you still need to do more work to improve English writing make it smooth to read, add more information in the methods part for sufficient description, and read more and think more to make a logical discussion to speculate possible reasons and convince the importance of your discovery. Based on the reading, I doubt whether the corresponding author has proofread and revised it or not.

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.

Title: ASSOCIATIONS OF IGF2 AND DRD2 POLYMORPHISMS WITH LAYING TRAITS IN MUSCOVY DUCK Review round: 1 Reviewer: 2

Basic reporting: This paper is well organized, but sustains grammatical errors. The introduction is informative, but does not speak to the importance to this study, nor the relevance of this study to industry. Inclusion of this information would make this a more successful manuscript. The images used in Figures 4 and 5 are of poor resolution. Using a higher quality version of this image would be advantageous. According to the instructions for authors: multiple references listed within parenthesis should be listed chronologically; in the reference section, there should be a period between the first initial of the last author’s name and the year; the full title should be used in each of the references (see Lines 242, 250, 260, 265, among others); references with 3 authors should all be spelled out, while 4 and more use et al. and the year (these references on these lines are not cited this way in the manuscript: 255, 298, 303, 312). The following are a sample of grammatical errors which should be addressed: Line 32 – Especially is not the correct word to use in this sentence. It should be removed. Line 58 – First egg age acronym not consistent as compared with the rest of the manuscript. Line 117 – statement should state “effect of each genotype”; genotype should be singular Line 120 – a space should appear before and after the less than or equal sign. Line 149 – should read “C-1704G SNP of the IGF2 gene” Lines 154 & 159 &160– “Highly significantly” should be replaced with “indicate highly significant associations” Line 157 – Should read “highly associated with E59W” Line 172 – “Was” should be replaced with “is” Line 172 – “but predominantly” should be replaced with “with the highest expression found” Line 181 – add a comma before “while” Line 183 – should read “highly productive” Line 183 – change “were” to “are” Line 184 – change “ones” to “chickens” Line 266 – remove extra space Line 352 – Should read “or are highly significant” Experimental design: Answering the following questions would provide clarity to the manuscript: Why were egg collection data taken specifically at 300 days and 59 weeks? Where the ducks all raised in the same run, or were portions raised at different time points/seasons/locations? What age were the ducks euthanized? Where the same ducks used to gather production data and tissue data for expression analysis? Why were some SNPs chosen over others for association studies? Validity of the findings: Be specific when defining the tissues expression of IGF2 occurs in in the Tissue Expression of IGF2 and DRD2 Genes section. The explanation of Table 3 in the Results section should be re-assessed to make it more clear. The superscripts differentiating AA, AG, and GG of A-1864G for the E59W trait should be listed as a, ab, and b, respectively. The p-values reported in the text, and those in the tables are different. Clarifying the reasoning behind this would make this paragraph much more understandable. The presence or absence of associations in T+3024C and A+3183C for DRD2 should be addressed in the Results section. After reading this manuscript in its entirety, it remains unclear the purpose of this study, and implications of its findings relevant to the duck industry. This information would make this paper much stronger. Additionally, the conclusion does not explore reasonings behind association of certain SNPs to specific production parameters measured, more than a just a repeat of basic findings from the Results section. This manuscript would greatly benefit from a re-written conclusion that goes further into interpreting results from this study. Concerns should be raised about the validity of the findings in the expression analysis, since age of tissue collection is not explained. The information does not appear to help add to the findings of the manuscript. At which point in the duck’s production timeline tissues are collected could play a major role in the expression levels. This information would be more useful if gathered at the same time points the production traits were collected (AFE, E300D, E59W). Having both SNP association data and expression data would present a more informative discussion as to the results in this manuscript. Additional comments: Information regarding functions of IGF2 are reported in mammals, but would be more compelling if linked to poultry, or a statement should reflect that the author could not find any such evidence in poultry to clarify. Table 4 superscripts are not consistent in capitalization. Listing the table number within the first sentence of the explanation of the results would improve the flow of the findings. In two cases (Zhang on lines 38 and 42; Ferrero on lines 198 and 200) one of each of these author’s references were not correctly cited in the text, therefore, there is no way to distinguish which reference is being referred to.

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.

Title: ASSOCIATIONS OF IGF2 AND DRD2 POLYMORPHISMS WITH LAYING TRAITS IN MUSCOVY DUCK Review round: 2 Reviewer: 1

Basic reporting: English writing can still be improved. In introduction section: Line 2: change "prized" to "priced". Line 5: change "pay" to "paid" Line 8: E300D and E59W are both egg production traits at certain time age, how can they be peak time and end time for laying. Please revise this sentence. Line 9: add "Therefore," before "we" In sample collection part of Material and Methods section: Line 1: change "800" to "Eight hundred" Line 2: add "were" between "which" and "in" change "distillation-distillation" to "double distilled" In Polymorphisms of IGF2 and DRD2 Genes part: change "S" to "s" in "sequencing" In Association of IGF2 and DRD2 with egg production traits part: Line 2: Change "Linkage disequilibrium" to "linkage disequilibrium analysis" Line 8: change "were" to "had", delete "indicate" Line 13: change "were" to "was" In discussion section: Line 1: delete space in "poultry", change "it" to "which" Line 2: change "i" to "I" in "improving" Line 5: change "I" to "We" Line 14: delete "found", change "suggest" to "suggests" Line 22: add "that" after "than", change "low productivity" to "lowly productive" Line 34: It is "DRD2 agonist" rather than "DRD2" that "the production and secretion of vascular endothelial growth factor protein in granulosa cells" Experimental design: The experiment design is good and enough to answer the research question. Methods is also described with sufficient details and information. Validity of the findings: Data in this study is robust and statistically sound. Reasonable conclusion and speculations are also made based on the results. Additional comments: Generally, the manuscript have been greatly improved after your revision. But English writing still needs to be improved. I believe it is a good study that would be published soon.

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.

Title: ASSOCIATIONS OF IGF2 AND DRD2 POLYMORPHISMS WITH LAYING TRAITS IN MUSCOVY DUCK Review round: 2 Reviewer: 2

Basic reporting: Content added by the authors in the Introduction, Methods, and Discussion sections contain substantial grammatical and syntax errors in the revised manuscript. The information included did not aid in clarifying the relevance of this study. This manuscript contains potentially important information for the duck industry, but the authors have not adequately conveyed this message in these sections. Additionally, without appropriate citations to back up their claims which are used to substantiate the significance of this study, overall the argument is less compelling. Table 3 remains hard to understand. The current title is not sufficient in allowing the reader to understand everything going on, and does not make clear how to differentiate the meaning between lower case and capitol letter associations in the LEM. Despite mention of the correct formatting for references in the initial review comments, two-authored references are not cited correctly in the text of the manuscript. Finally, sentences ending in the citation of multiple references are not listed chronologically. Experimental design: A brief statement as to the rationale of eggs at day 300 and week 59 is warranted, even if widely known in the duck production community. PeerJ readers are likely to not know of the specifics of duck egg laying cycles. The sampling methods indicate in the manuscript that all ducks were run together, while in the rebuttal, the author states that ducks sampled for production and tissue analysis were from a “different batch”. The rationale behind selecting 2 out of the 5 SNP identified in IGF2, and 11 out of 28 SNP identified in DRD2 needs to be explained in the text. Validity of the findings: The impact of this study was not sufficiently addressed in the revision. The discussion includes statement in reference to non-avian species, but fails to acknowledge whether any information is known within birds, or if positive associations of the data are being backed up by the authors for different taxa. The authors did a good job of noting that only positive associations were identified, but that further investigation is warranted in regards to causation due to the SNPs Additional comments: No additional comments

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.

Title: AN UPDATE ON ANTICANCER DRUG DEVELOPMENT AND DELIVERY TARGETING CARBONIC ANHYDRASE IX Review round: 1 Reviewer: 1

Basic reporting: The title of the review article is not reflecting at all the content of the work, since only sulfonamides are trated, and more precisely the few sulfonamides described by Matulis group. Although the authors mentioned the five types of small molecule CA inhiibtors targeting thismisoform, then only a very limited number of compounds is considered. Experimental design: There are no experiments presented. The various methods of inhibitor assay are not very meaningful in a revie article on such a specialized area Validity of the findings: Two structures of CA IX inhiibtors are presented which are of little interest since such compounds did not ptogress even to preclinical work. The part on antibodies is very badly written and antibody-drug conjugates not even mentioned Additional comments: Overall this is a badly written ms which has a mixture of diverse topics whoch do not for, a solid ms, being too heterogeneous. For example it is difficult to understand what the zebra fish model did for the discovery of CA IX inhibitors.

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.

Title: AN UPDATE ON ANTICANCER DRUG DEVELOPMENT AND DELIVERY TARGETING CARBONIC ANHYDRASE IX Review round: 1 Reviewer: 2

Basic reporting: The language is clear, but the selection of the articles for this review is absolutely authors-oriented! Experimental design: not applicable Validity of the findings: Table 1: I could not understand or quantitate the differences between "minor effect on pH" and "significant effect on pH". is CA IX expressed in Xenopus transmembrane (as in humans) or cytosolic? This could be detrimental for the significance of these data. And maybe this part, dealing with only two CA inhibitors could be deleted because it is not validated for this target in the literature. Additional comments: No additional comments

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.

Title: AN UPDATE ON ANTICANCER DRUG DEVELOPMENT AND DELIVERY TARGETING CARBONIC ANHYDRASE IX Review round: 1 Reviewer: 3

Basic reporting: See comments to the authors Experimental design: See comments to the authors Validity of the findings: See comments to the authors Additional comments: This review was written by an expert team who has developed new molecules to target carbonic anhydrase IX and has contributed significantly to the field. However, I think it is highly focussed on their own work rather than a general review, for example, only focussing on one subclass of inhibitors, the aromatic sulfonamides, so it is more of a report of their work only, where they, for example, comment “for the first time we emphasised the use of mammalian models”. However, they have already published this in 2016 and 2017, so anyone looking at the literature would find this. They also make several claims, for example, “the first review to combine information about animal models and other methods used in the field”, whereas there are many reviews on carbonic anhydrase IX as a target. In fact, it would be useful to include other recent reviews in the references. Essentially, it is partly a technical description of work they have done in terms of the sorts of assays that have been around or published already. They also cover the zebra fish work already published and is well recognised as a useful phenotypic screen. CA-targeted strategies focused mainly on preclinical work and the clinical studies are poorly reported. For example, the carbonic anhydrase IX inhibitor in clinical study was indisulam, yet it has been discontinued from clinical study a couple of years ago now. Similarly, the Willex antibody was actually effective at predefined set points of expression of CA9, yet has not been commercially developed. Finally, SLC0111 has gone through phase I but there is no information on the pharmacodynamics used, toxicity, or the dosing or any other details. So for someone who is interested in the clinical development, there is out of date information, not enough discussion as to why apparently successful compounds are counted as failures or update on the most recent compounds or how they are proven or not to have an effect on carbonic anhydrase IX. Overall, therefore, I felt it is mainly concerned with their own publications in the last two years and summarising those data without an adequate discussion of the clinical problems and why with over 1000 compounds reported by Supura, onlya single compound has progressed to phase II studies in over 15 years since it was recognised an important therapeutic target. A much more detailed discussion of these problems would be of more interest.

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.

Title: AN UPDATE ON ANTICANCER DRUG DEVELOPMENT AND DELIVERY TARGETING CARBONIC ANHYDRASE IX Review round: 1 Reviewer: 4

Basic reporting: Kazokaité et al. have submitted a review article summarazing the current status of development of anti-tumor agents targeting carbonic anhydrase IX (CA IX). CA IX acts in control of tumor pH, modulates cell adhesion and invasion and contributes to tumor progression. Therefore, CA IX is ranked among promising anti-tumor drug targets with many new developed CA IX-targeting molecules every year. For that reason the topic of submitted review is actual and beneficial. Professional English is used throughout the review, but in some parts is the text more complex than necessary. The number of references is fully sufficient for this type of a review, some of the references that I would recommend to add will be mentioned later There are no figures or charts present in publication, I would suggest adding at least one or two charts to improve readability. The review contains two helpful tables, but Table 2 has incorrect text wrapping in several columns. Experimental design: 'no comment' Validity of the findings: 'no comment' Additional comments: 1. Line 55: „The unregulated pH is a common feature of many aggressive tumors to adapt to hypoxia.“ I lack the clarification of the relationship between hypoxia and the problem with pH regulation. Explanation of the extracellular acidification of tumor microenvironment is given below, but there is no link to hypoxia. 2. In text, the terms „antitumor“ „anti-tumor“ „anticancer“ „anti-cancer“ are used extensively. It is necessary to unify the writing of this term, with or without a dash. 3. I suggest that the text from lines 157-166 is not thematic in the chapter. The division of inhibitors according to CA inhibition mechanisms is not important in describing the SFA method. 4. Lines 240-242: I wonder what resulted from discovery of a compound that is so tight bound to CA IX that the binding can not be measured by the SPR method? Is such compound a good candidate for CA IX-targeting agent? And is the SPR method appropriate for searching for suitable ligands? 5. Lines 291 and 297: It is necessary to add the number of carbonic anhydrase IX to headings. There is only „CA-targeted“ but the meaning is „CA IX-targeted“. 6. Line 305: I think that in the phrase "who are at risk of relapse" it would be appropriate to add „who are at high risk of relapse“, because unfortunately there is a risk of relapse for all patients. 7. Capture 4.1: In this part you do not mention the monoclonal antibody VII/20, which also has the therapeutic potential. I would recommend adding data from the publications Zatovicova et al. 2003 and Zatovicova et al. 2010. 8. Line 340: I would recommend changing the word order in the sentence to: „To demonstrate the anti-tumor effect of CA IX inhibition in vivo,...“ 9. Line 359: There is no specification which „compounds may be suitable...“ 10. Line 430: The use of the term "hypoxic cancers" does not seem to me to be appropriate. In my opinion, it would be better to use, for example, "hypoxic tumor areas". 11. Line 442: You note that antibodies binding does not affect the catalytic activity of CA IX. Could it be possible that it is affected by antibodies with the ability to internalize CA IX protein?

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.

Title: AN UPDATE ON ANTICANCER DRUG DEVELOPMENT AND DELIVERY TARGETING CARBONIC ANHYDRASE IX Review round: 2 Reviewer: 1

Basic reporting: the manuscript is now suitable for publication Experimental design: not applicable Validity of the findings: suitable Additional comments: none

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.

Title: ANTIBODIES TO BORDETELLA PERTUSSIS ANTIGENS IN MATERNAL AND CORD BLOOD PAIRS: A THAI COHORT STUDY Review round: 1 Reviewer: 1

Basic reporting: The article meets all basic reporting criteria. Experimental design: The work is original and the study design is appropriate and clearly explained. The methods are well described and the analyses and presentation of results are adequate. Validity of the findings: The investigators establish the poor level of protection against pertussis in both mothers and infants in a Thai population, setting the stage for additional work in support of maternal immunization with Tdap. Interestingly, this work in a location where whole cell pertussis vaccine is given to infants provides an opportunity to understand the role of adolescent and maternal vaccination with acellular pertussis vaccine followed by whole cell vaccine in infants. The discussion and conclusions of this study are well stated and supported by the data presented. Additional comments: Well written manuscript, with relevant and up to date references, clear description of methods, analyses and results. The figures are illustrative of the key findings of the study.

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.

Title: ANTIBODIES TO BORDETELLA PERTUSSIS ANTIGENS IN MATERNAL AND CORD BLOOD PAIRS: A THAI COHORT STUDY Review round: 1 Reviewer: 2

Basic reporting: Figure 1, showing the distribution of declared pertussis cases according to age from 2011 up to 2016 is not very informative, as only for 2015 (n=51) and 2016 (n=72) the figures can be split in reasonable numbers. I would suggest to put the data of Figure 1 in a Table and also indicate how many of the cases were laboratory defined and by what method. Experimental design: No comment Validity of the findings: One minor comment on the conclusions: -Line 149 states: There were no statistically significant differences in the proportion of recently infected rates in women born before or after the EPI program: statistics on 1 vs 2 cases? This can not be used for a statistical analysis Additional comments: A general comment: Line 227: Our results explained the susceptibility for pertussis among newborn infants in Thailand and supported the requirement for a pertussis booster vaccine during pregnancy. It is clear that pertussis booster vaccination during pregnancy will increase the antibodies in newborns, but it is also clear there are no actual correlates of protection for pertussis. Although antibodies to PT and Prn certainly play a role in protection, there is also evidence that other factors such as local IgA levels and cell-mediated immune responses are important.

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.

Title: ANTIBODIES TO BORDETELLA PERTUSSIS ANTIGENS IN MATERNAL AND CORD BLOOD PAIRS: A THAI COHORT STUDY Review round: 2 Reviewer: 1

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

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.

Title: MORPHOLOGICAL AND MOLECULAR DATA CONFIRM THE TRANSFER OF HOMOSTYLOUS SPECIES IN THE TYPICALLY DISTYLOUS GENUS GALIANTHE (RUBIACEAE), AND THE DESCRIPTION OF THE NEW SPECIES GALIANTHE VASQUEZII FROM PERU AND COLOMBIA Review round: 1 Reviewer: 1

Basic reporting: The writing is usually clear (but the discussion and part of the results should be rewritten) and there is strong evidences (molecular and morphological) supporting the taxonomical work. The literature is cited purposefully. However, the text sometimes overwhelmed the reader with useless details (see e.g. M&M section), while the figures have been rather sloppily prepared (see comments below). Some subheadings should be modified to reflect their content and the result section should be rearranged (see details comments below). Furthermore, the text can be simplified and shortened in several places (see some suggestions below). Part of the results should be rewritten and I will suggest removing the chapter discussion that is unnecessary (as this MS is a taxonomical treatment). Experimental design: This MS deals with the taxonomy of three plant species, there is no research questions or experimental design to evaluate. Validity of the findings: no comment Additional comments: Review of MS for PEERJ – Manuscript ID 18193 – Florentin et al., Morphological and molecular data confirm homostyly in the mainly distylous genus Galianther (Rubiaceae). The 28th of June 2017 This manuscript deals with taxonomic changes among the “Spermacoce clade” of the tribe Spermacoceae (Rubiaceae). In particular the authors transfer two species Diodia spicata and D. palustris to the genus Galianthe and describe a new species Galianthe vasquezii R.M.Salas & E.L.Cabral from Peru. The work is mainly based on results found in previous study (i.e. Dessein 2003 and Groeninckx et al. 2009). It provides four new DNA sequences for the species Diodia palustris and uses herbarium-based morphological observations. The writing is usually clear (but the discussion and part of the results should be rewritten) and there is strong evidences (molecular and morphological) supporting the taxonomical work. The literature is cited purposefully. However, the text sometimes overwhelmed the reader with useless details (see e.g. M&M section), while the figures and part of the text have been rather sloppily prepared (see comments below). As stated in the review guidelines, I will not comment on the impact and novelty of this study. As this is a MS dealing with the taxonomy of three plant species, there is no research questions or experimental design to evaluate. It is yet unclear to me if taxonomical treatments are considered as “Research Article” by PEERJ. If yes, my impression is that this taxonomical note should be considered as worth to be published. However, in my opinion, there are some issues to be addressed before publishing this study. The authors emphasize, in the MS title, the presence of homostylous species within a mainly distylous genus. But do not discuss the evolutionary implications. They rather treat this biologically interesting phenomenon (see Darwin 1877 book on the different forms of flowers on plants of the same species or the Webb & Lolyd 1986 paper in New Zealand Journal of Botany 24: 163-178 and many others) as a character state. Therefore, the title does not reflect the work done. I will suggest two options: The first one: to adapt the title to reflect the work (e.g. Galianthe vasquezii, a new Rubiaceae species from Peru and the transfer of two Diodia species to Galianthe) The second one: to use the current phylogenetical framework (i.e. the one published by Groeninckx et al. 2009 and the authors’ addition of D. palustre) to discuss the evolutionary implication of homostylous species within a distylous clade. In that case the author should be aware that with only 6 species (out-of the 50 of the genus) their discussion would remain highly speculative. Additionally some subheadings should be modified to reflect their content and the result section should be rearranged (see comments below). Furthermore, the text can be simplified and shortened in several places (see comments below). Part of the results should be rewritten and I will suggest removing the chapter discussion that is unnecessary (as this MS is a taxonomical treatment). Suggestions and minor issues: P2, L15 “The remaining species described …” How many species of Dodia were transferred to the different genera. P2, L18-20. The sentence is unclear. Do you mean that current species of Galianthe subgen Ebelia are all distylous? P2, L21-28. I would suggest simplifying the whole paragraph, by acknowledging that you follow the work of Dessein (2003) and as a result move Diodia spicata, along with a recently sequenced species (i.e. D. plaustre) to Galianthe. By doing this you also describe a new species G. vasquezii. (as stated in P2, L30). P3, L11. Remove “Acronyms according to Index Herbarorium”, as this is obvious. P3, L11. Change: “were analysed” by “were observed” or “were studied” P3, L12-13. Are you sure PEERJ allow website citation within the main text? To be checked. P3 L22-29. Is this necessary? It appears as obvious to me (following the last International Code of Nomenclature for algae, fungi and plants, art 29). P4, L26-28. I believe you can spare some words here by saying that you “align your sequences with MAFFT and subsequent manual corrections in Geneious”. P4, L24. change “Data analyses”, by “phylogenetic analyses” P5, L13. Result section should be reorganized. The “molecular study” at P15L18 (but I guess you mean “phylogenetic results”) should come first and the “Taxonomic Treatment” second (Because you based your taxonomic treatment on your phylogenetic analyses). P5, L19. You may want to have the “Description-” subheading here, to be consistent with the following subheading (e.g. Distribution-, Ecology-, Conservation status-, etc.). I cannot recall if a Diagnosis is mandatory for a name to be validly published. You may want to check. P5, L22. Replace “o” by “or” P6, L2. What are P= 31 and E =29. You should state to what P and E refer. P6, L3. “muri nanospinose”, what does this mean? It should be clarified for the readers. P6, L3. “with notorious papillae” do you mean “conspicuous papillae”? P6, L6. “caducous.; seed…” remove the “.” (i.e. “caducous; seed…”) P6, L14. “21 Jul 1944” replace by “21 Jul. 1944” P6, L15. “15 Dic 1933” replace by “15 Dec. 1933” Throughout the text, when a month is abbreviated it should be followed by a point. P6, L23. Change “nov. comb.” by “comb. nov.” P7, L10-29. Same remarks than above for the subheadings, the P= and E = meanings. P7, L28. What does “u” means? P8, L5 to P10, L5. “Additional Specimens Examined” I did not review that part! P10, L19. Add the subheading “description-” P11, L5 what does P and E mean? P11, L17 to P12, L 19. “Additional Specimens Examined” I did not review that part! P15, L18 to P16, L23. “Molecular Study”: I guess you mean “phylogenetic results” needs to be rewritten in a clearer way. I am lost in your results, which sometimes contradict Figure 4. A few none-exhaustive comments: P15, L26. Are the genera unresolved or do you lack of statistical support? P16, L2, remove the “s” or did you mean “statistical”? P16, L3 what is a “low supported (40) to unsupported clade”. Is that statistically supported or not? The answer is “statistically not supported”. I suggest removing the discussion, as this is a taxonomical treatment. Furthermore, It is unclear to me what you are discussing. Figure 2: Instead of presenting 2x6 pictures, you may want to present 4x3 pictures. The caption should be rewritten as follow: Morphological characters distinguishing Galianthe vasquezii (pictures A-F from the Isotype in MO) and G. Palustris (Pictures G-L from A.-A. Cabana 19 in CTES). And then the details of each pictures Figure 4: In the caption, replace the “y” by “and” Figure 5: In the caption explain what are the numbers above the branches… I suggest to collapse the branches statistically unsupported. (i.e. <51)

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.

Title: MORPHOLOGICAL AND MOLECULAR DATA CONFIRM THE TRANSFER OF HOMOSTYLOUS SPECIES IN THE TYPICALLY DISTYLOUS GENUS GALIANTHE (RUBIACEAE), AND THE DESCRIPTION OF THE NEW SPECIES GALIANTHE VASQUEZII FROM PERU AND COLOMBIA Review round: 1 Reviewer: 2

Basic reporting: Authors analyzed the taxonomic position of two species that were before assigned to Diodia genus: Diodia spicata and D. palustris; but currently they have been translated to Galianthe genus. They use morphological and molecular data (ETS and ITS). The uncertainty position occurs due to some morphological differences exist between these two species and some Galianthe subgen. Ebelia species, mainly the homostylous flowers. Also authors describe a new species (Galianthe vazquezii) and provide a dichotomous key for taxa with indehiscent mericarps. They conclude that both analyzed species fall among Galianthe species subgen. Ebelia. The English language of the manuscript is clear and correct. The Intro and background are well supported. Figures are in high quality and are well labeled and describe. However, references along the manuscript don’t seem to follow the PeerJ style (are in cursive font). Some details in the Intro can help to improve the manuscript. 1. In the abstract, and along the manuscript, authors mention the phrase: “generic concept” of Galianthe subgen. Ebelia, however in the introduction they never clarify which is this concept. They just mentioned some traits with taxonomic importance. It could be helpful to extend this concept. 2. In the Intro (Page 2; Line 22-26) authors describe three different taxonomic proposals in order to relocate the taxonomic position of D. spicata and D. palustris. Finally, they decided “to verify the position of…. within Galianthe” genus. It is not clear why they decided to keep the Galianthe hypothesis over the Borreria subgen. Dasycephala hypothesis. Although they mention that species “share most affinities with Galianthe subgen. Ebelia” (Page 2; Line 27), is not clear if the decision is due to is the position currently accepted. They could clarify the point. 3. Authors mention (Page 2; Line 27-28) that studied species have some differences with other species of the subgen. Ebelia, but they don´t mentioned these differences. It could be clarify with the point 1. Experimental design: The method used was well defined and analyses are generally appropriate. The design of the morphological and molecular study was based on a good sampling of species of Spermacoce clade, however, it is noticeable that there is just one species of Galianthe subgen. Ebelia. (G. brasiliensis). Although this was correct due to this especies is the type of subgen. Ebelia, it is recommended the use of more species. Molecular markers in general showed well resolution. The methods and results are described with sufficient detail. Some suggestions: 1. Page 4; Line 9. Authors mention three accession numbers added, but they just report four of them (Page 4; Line 22-23). Check. 2. Why you don´t use Bayesian inference analyses instead of ML bootstrapping to obtain the posterior probability values as a measure of clade support?. 3. Page 6; Line 3: be consistent with decimal separators (dots or commas). 4. Page 6; Line 6: caducous; (without dot) 5. Page 7; Line 27-28: be consistent with decimal separators (dots or commas). 6. Page 11; Line 5: be consistent with decimal separators (dots or commas). 7. Page 14; Line 6: x=15, (without dot) 8. Page 14; Line 12: branch. Brazil. (space) 9. Page 16; Line 2: an extra “s” Carajasia has moderate. 10. Page 16; Line 7-9: write values of clade support by each minor clade mentioned. 11. Page 16; Line 13-14: write values of clade support of Borreria species from the Americas. 12. Page 16; Line 15-18: Mention values of clade support at least for the two subgroups of Spermacoce. 13. Page 17; Line 15: “in” or “is” Figure 5. Could be better if also highlight Borreria and Spermacoce clades. Validity of the findings: The data and the results are robust and improve the knowledge of the Galianthe genus. The conclusions are well stated, linked to the main question and limited to supporting results. Additional comments: I consider that the manuscript could potentially become acceptable for publication in PeerJ. Corrections could be considered as minor corrections but they can help to make it easier for the reader.

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.

Title: MORPHOLOGICAL AND MOLECULAR DATA CONFIRM THE TRANSFER OF HOMOSTYLOUS SPECIES IN THE TYPICALLY DISTYLOUS GENUS GALIANTHE (RUBIACEAE), AND THE DESCRIPTION OF THE NEW SPECIES GALIANTHE VASQUEZII FROM PERU AND COLOMBIA Review round: 2 Reviewer: 1

Basic reporting: This is the second review of this manuscript (previously submitted to PEERJ). This MS deals with taxonomic changes among the “Spermacoce clade” of the tribe Spermacoceae (Rubiaceae). In particular the authors transfer two species Diodia spicata and D. palustris to the genus Galianthe and describe a new species Galianthe vasquezii R.M.Salas & E.L.Cabral from Peru. The writing is clear and the discussion and results section have been improved. I made some comments below. Before being published, the MS need to be carefully checked by a native English speaker (I am not in a position to correctly assess this part – but my feeling is that there is room for improvement). As mentioned in my previous review, the authors emphasize, in the MS title, the presence of homostylous species within a mainly distylous genus. It is a strategy that will attract readers and at the same disappoint some of them because of the absence of discussions regarding the evolutive implications of this finding. Nevertheless, as the authors consider distily/homostly as a morphological character state, it is acceptable. The aims of the MS need some clarifications. First, in an unclear sentence (P2L16-17; see suggestion below), the authors say “in this work we studied” … either the genus Diodia or the species with indehiscent mericarps (this is unclear to me). Then, to exemplify their focus, they list four species (P2L18 - namely: D. bogotensis, D. brasiliensis, D. cymosa, D. hispidula). Later in the introduction (P2L27-30), they discuss more specifically the position of two related species (D. spicata and D. palustris). Then (P3L1-2): “As for Diodia spicata, Dessein (2003) proposed to transfer it to Galianthe based on molecular data, pollen grains (double reticulum), and fruit morphology”. Followed by “the aim of this work is to verify the position of D. palustris and D. spicata within Galianthe based on molecular and morphological data.” The problem is that Dessein (2003) already did it. Thus (as mentioned in my previous review), I believe that the aim of the paper is rather the taxonomical transfer of D. spicata (along with its close relative D. palustris) to Galianthe. Which, by the way, is a honourable, perfectly publishable, aim. Now, what about D. bogotensis, D. brasiliensis, D. cymosa, D. hispidula nothing is mention in the M&M or the Result sections about those four species?!? Additionally, before publishing this MS, I recommend the authors to go through the text again and make clearer that they consider distyly (respectively homostyly) as a morphological character state (see e.g. comments below). In some instance homostyly or distyly is presented e.g. like a syndrome (P1L28) but without explicitly mentioning a syndrome of what? Or as “scarcely manifested” (P18L22) - what does this mean? Experimental design: Because this MS deals with the taxonomy of three plant species, there is no research questions or experimental design to evaluate. Validity of the findings: the data support very well the taxonomical changes made by this paper. Additional comments: Review of MS for PEERJ – Manuscript ID 18193 – Florentin et al., Morphological and molecular data confirm homostyly in the genus Galianthe (Rubiaceae), based on the new species G. vasquezii from Peru and the two new combinations of Dioda species. The 9th of September 2017 This is the second review of this manuscript (that have been previously submitted to PEERJ). This MS deals with taxonomic changes among the “Spermacoce clade” of the tribe Spermacoceae (Rubiaceae). In particular the authors transfer two species Diodia spicata and D. palustris to the genus Galianthe and describe a new species Galianthe vasquezii R.M.Salas & E.L.Cabral from Peru. The writing is clear and the discussion and results section have been improved. I made a few comments below. The MS need to be carefully checked by a native English speaker (as I am not in a position to correctly assess this – but my feeling is that there is room for improvement) I am not commenting on the impact and novelty of this study. Because this MS deals with the taxonomy of three plant species, there is no research questions or experimental design to evaluate. As mentioned in my previous review, the authors emphasize, in the MS title, the presence of homostylous species within a mainly distylous genus. It is a strategy that will attract readers and at the same disappoint some of them because of the absence of discussions regarding the evolutive implications of this finding. Nevertheless, as the authors consider distily/homostly as a morphological character state, it could be acceptable. The aims of the MS are not clearly presented. First, in an unclear sentence (P2L16-17; see suggestion below), the authors say “in this work we studied” … either the genus Diodia or the species with indehiscent mericarps (this is unclear to me). Then, to exemplify their focus, they list four species (P2L18 - namely: D. bogotensis, D. brasiliensis, D. cymosa, D. hispidula). Later in the introduction (P2L27-30), they discuss more specifically the position of two related species (D. spicata and D. palustris). Then (P3L1-2): “As for Diodia spicata, Dessein (2003) proposed to transfer it to Galianthe based on molecular data, pollen grains (double reticulum), and fruit morphology”. Followed by “the aim of this work is to verify the position of D. palustris and D. spicata within Galianthe based on molecular and morphological data.” The problem is that Dessein (2003) already did it. Thus (as mentioned in my previous review), I believe that the aim of the paper is rather the taxonomical transfer of D. spicata (along with its close relative D. palustris) to Galianthe. Which, by the way, is a honourable, perfectly publishable, aim. Now, what about D. bogotensis, D. brasiliensis, D. cymosa, D. hispidula nothing is mention in the M&M or the Result sections about those four species?!? Furthermore, before publishing this MS, I recommend the authors to go through the text again and make clearer that they consider distyly (respectively homostyly) as a morphological character state (see e.g. comments below). In some instance homostyly or distyly is presented e.g. like a syndrome (P1L28) but without explicitly mentioning a syndrome of what? Or as “scarcely manifested” (P18L22) what does this mean? Further comments and suggestions: Title: P1L2: change “Peru and the two new…” with “Peru and two new….” Abstract: P1L18 and L27 are in contradictions - e.g. L27 mention, “bifid stigmas and indehiscent mericaprs” has morphological characters supporting the position of Dioda palustre and D. spicata within Galianthe. But those characters are not “diagnostic features” of the genus as listed on L18-21. P1L28. Is homostyly a syndrome? If yes a syndrome of what? Has, within this paper, homostyly is considered soly as a combination of morphological character (because there is no references to its evolution implications – in particular the transition from outcrossing to selfing. I will suggest modifying this sentence to make it clearer. Introduction: P2L14. “Galianthe was linked to Diodia” how was it linked and by whom? Later you say: “due to certain morphological similarities”. I would avoid this imprecise phrasing and state immediately what “link” those two genera. P2L16-17. It is not clear to me if the authors focus on the genus Diodia or on the indehiscent mericarps? P2L17-18. What about all those species you mentioned? Where are they now? Should they be moved to Galianthe? This is very confusing. I though that you were focusing on only two species. Why then didn’t you add those species to your phylogeny? P2L21. “numerous” please indicate how many are “numerous” P2L25 “several”, please be more precise and indicate how many. P3L1. Please indicate on what bases (which morphological or molecular data) Delprete transferred those species to Spermacoce. P3L2-4. About the aims of the paper: you state, “The aim of this work is to verify the position of D. palustris and D. spicata based on molecular and morphological data”. But this has already been done by Dessein (2003) (at least for D. spicata). Thus what are your aims? I believe (as suggested in my previous review) that your aims are to do the taxonomical transfer of D. spicata along with its close relatives to Galianthe. Key to Galianthe species: P16L1. I would avoid mentionning chromosome numbers in the key. This character is not observable in the field. Furthermore, very few is known about the karyology of this group and it might well be possible that a lot of variation (e.g. aneuploidy, dysploidy, along with polyploids) occurs. Too few material have been studied in this genus to provide reliable results. Discussion: P18L22 what does “scarcely manifested” means. Is it distylous or not? Please explain in more details what you mean. Is that an intermediate state (a transitional state?) from distyly to homostyly (or the inverse)? P18L13. Change: “the transference of” by the “the transfer of” I hope those comments may be useful to the authors.

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.

Title: MORPHOLOGICAL AND MOLECULAR DATA CONFIRM THE TRANSFER OF HOMOSTYLOUS SPECIES IN THE TYPICALLY DISTYLOUS GENUS GALIANTHE (RUBIACEAE), AND THE DESCRIPTION OF THE NEW SPECIES GALIANTHE VASQUEZII FROM PERU AND COLOMBIA Review round: 2 Reviewer: 2

Basic reporting: Authors have included most of suggestions made before. The manuscript has been improved significantly. Experimental design: No comment. Validity of the findings: No comment. Additional comments: No additional comments

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.

Title: MODUS OPERANDI AND AFFECT IN SWEDEN: THE SWEDISH VERSION OF THE REGULATORY MODE QUESTIONNAIRE Review round: 1 Reviewer: 1

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: No comment. Additional comments: The article is well written, clear and respects the professional standards of structure, introduction and background, literature references, ethical issues. It is an article well-founded theoretically, with a clear and precise objective. It was intended to verify the independence of the two regulatory modes and the psychometric properties of RMQ in the Swedish context. These objectives have been achieved in an appropriate manner. No flaws were found in the experimental design, although the student-only sample could be improved. However, for the validation of the scale is a correct sample. The discussion section is very interesting and complete. The study will serve to improve the literature on this topic.The section Limitations and suggestions for future research is very extensive, even in some moments the wording is unclear. I suggest reviewing this section.

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.

Title: MODUS OPERANDI AND AFFECT IN SWEDEN: THE SWEDISH VERSION OF THE REGULATORY MODE QUESTIONNAIRE Review round: 1 Reviewer: 2

Basic reporting: This manuscript investigated two regulatory modes using structural equation modeling. The manuscript is well-organized. However, the statistical analyses need improvement. Experimental design: Structural equation models presented on Page 14 need to be further specified before any analysis. The model specification is usually guided by a combination of theory and empirical results from previous research. Validity of the findings: It would be helpful to present a table of parameter estimates (i.e. standard deviation, test statistics) and equation models for the SEM. Additional comments: Summary This manuscript investigated two regulatory modes using structural equation modeling. The manuscript is well-organized. However, the statistical analyses need improvement. Major comments 1. Preliminary analysis In the abstract, there were 5 participants did not report their gender. However, the percentage of male is much higher than female. That difference may lead to selection bias. Can the authors explain the data? In addition, preliminary descriptive statistical analysis such as collinearity issue and outlier detection need to be conducted carefully. 2. Model Structural equation models presented on Page 14 need to be further specified before any analysis. The model specification is usually guided by a combination of theory and empirical results from previous research. 3. Results It would be helpful to present a table of parameter estimates (i.e. standard deviation, test statistics) and equation models for the SEM.

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.

Title: MODUS OPERANDI AND AFFECT IN SWEDEN: THE SWEDISH VERSION OF THE REGULATORY MODE QUESTIONNAIRE Review round: 1 Reviewer: 3

Basic reporting: No comment. Experimental design: It seems that the analysis combines the university and high school students. I did not get any good sense as to why this was done. It is not clear whether separate analysis should be done for these two groups of students or not. Some clarification is needed. Validity of the findings: No comment. Additional comments: 1. Some sentences are repeated word by word: the sentence ``However, other values (the goodness of fit index and the root mean square error of approximation) indicated that the hypothesized model fits the data well.’’ appears in line 235-236 and again in lines 253-255. 2. Related to the previous point, what are these values (the goodness of fit index and the root mean square error of approximation)? Please report the numbers.

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.

Title: MODUS OPERANDI AND AFFECT IN SWEDEN: THE SWEDISH VERSION OF THE REGULATORY MODE QUESTIONNAIRE Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: The authors have taken into account the suggestions and recommendations that the reviewers have indicated in their reports. The article may be published.

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.

Title: MORPHOLOGICAL CONVERGENCE IN ‘RIVER DOLPHIN’ SKULLS Review round: 1 Reviewer: 1

Basic reporting: Summary: Charlotte E Page and Natalie Cooper present a 2D geometric morphometric analysis of odontocete skulls with the aim of testing for convergence in river dolphins. The dataset contains 24 species with between 1 and 5 individuals of each species. The methods appear sound and thorough e.g. the checks of repeatability and multiple tests of convergence. I have a number of issues with the reporting that I think should be improved prior to publication. Line edits: Title: the sub title “a disparate grouping justified” generates a little confusion because the term “disparate” is often used to describe morphologically dissimilar species. Please consider rephrasing to a clearer title. Line 6: I think the novelty of this study is overstated within the abstract. You state: “This has led many to using the ‘river dolphins’ as an example of convergent evolution. However, these morphological similarities have never been quantified.” McCurry et al. 2017a quantifies morphological similarities between a wide range of crocodilians and odontocetes, including each of the river dolphin species. Please re-write the introduction as to clarify what the knowledge gap is, and how this study fills it. Line 13: “The cause of this morphological convergence remains unclear, but our results support hypotheses of shared feeding mode or diet and thus provide the foundation for future work into convergence within the Odontoceti.” Please state exactly how your results support hypotheses of the cause of convergence? Line 24: Why is the “tree of life” written in Caps? Line 42: “One iconic example of convergent evolution is in the ‘river dolphins’, a group of distantly related cetaceans that secondarily entered river systems from the ocean, evolving riverine lifestyles and similar morphological characteristics (Hamilton et al. 2001).” The term “river dolphin” is more convoluted than this. Pontoporia is not a riverine species, inhabiting both estuaries and oceanic environments. Furthermore, some dolphins outside of the “river dolphin” grouping are found in riverine environments e.g. Sotalia fluviatilis. Please rewrite this section in a way that describes the complexity of habitat similarities/differences. Line 62: “Here we present the first quantitative investigation of the morphological variation present in the skulls of river dolphins, and quantify whether the four river dolphin genera are convergent.” This statement ignores past work, please fit Barroso et al. and McCurry et al. into the introduction and explain what you do differently to these studies. Line 75: What do you mean by “equally closely related to river dolphins”, this needs more clarification. Why did you include the species that you did? Line 76: “we photographed specimens across all Odontoceti” please state the number of species sampled here, otherwise it sounds like you sampled all species within the group. You could even include an odontocete tree showing which species were sampled and the sample size for each species? Line 78: “Sexual dimorphism varies among odontocete genera, being present in some species as differences in size, but with no differences in shape between sexes recorded (Amaral et al. 2009; Higa et al. 2002). We therefore chose males and females from different collection locations where possible to provide a representative sample of each species.” This is confusing, do you mean that some species have sexual dimorphism in size and other have differences in morphology? Or that only differences in size exist? Some odontocetes do show variation in cranial morphology dependant on sex e.g. Mesoplodon (Besharse 1971), monodon etc.. Please rewrite to clarify. Besharse, J. C. (1971). Maturity and sexual dimorphism in the skull, mandible, and teeth of the beaked whale, Mesoplodon densirostris. Journal of Mammalogy, 52(2), 297-315. Line 106: Please state here that it was 2D GMM that was used. Line 109: Which “putatively convergent features”? Please provide more detail. Line 133 Is there a full stop missing after the reference? Line 225: “Mandibular PC1 describes variation in the elongation of the mandible resulting in an elongation of the rostrum, symphysis and alveolar tooth row (Figure 4D)”. The word use of the word “resulting” here inappropriately implies cause/effect. Line 253: The four genera seem to have experienced evolution in the same direction. Consider rephrasing to “evolved similar skull shapes”. Line 296: What are the limitations/benefits of 2D vs 3D GMM? Please consider adding this to this paragraph. Line 299: “Skeletals s” is a typo. Line 313: “Here we have presented the first quantitative investigation into convergence in the river dolphins.” As stated earlier, this is not the first study to use GMM on a sample including river dolphins, or to identify convergence. Figure 2: Why used A and B for both the panels and the curves. Consider changing to lower case letters to identify curves or even C1, C2 etc. Experimental design: Generally, the experimental design and use of methods is sound. However, in the introduction and conclusions the study makes several untrue claims about being the first to quantify variation in river dolphin skull shape (e.g. Line 7, Line 62, Line 313). Several studies have quantified variation in odontocete skull shape, including river dolphins. For instance, Barroso, Cranford, and Berta (2012) identified a river dolphin morphotype in mandible shape. Also, McCurry et al. (2017a) identified a river dolphin morphotype, tested for convergence and discussed the cause of this convergence. Neither of these were cited in your introduction, but appear later in your discussion. There are several differences in your approach and sampling that allow your study to fill a knowledge gap, but being the “first quantitative investigation of the morphological variation present in the skulls of river dolphins” is not one of them. Please rewrite your introduction and conclusions as to better identify a knowledge gap and alter your conclusions in relation to this. Validity of the findings: Overall, I think that the conclusions are scientifically sound and supported with enough evidence. As stated above, and in the line edits, I think that the rational, benefit to the literature and conclusions need to be re-written. As stated in the line edits I think there needs to be more clarification of why the species sampled were sampled. Additional comments: No additional comments

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.

Title: MORPHOLOGICAL CONVERGENCE IN ‘RIVER DOLPHIN’ SKULLS Review round: 1 Reviewer: 2

Basic reporting: The paper is generally clear and easy to follow. There is some detail lacking at times, but generally I see no issues with language or clarity of the text. Figures 3 & 4 are hard to read. The dots are quite small, and don’t contrast from the other elements well enough. Correcting this would make the figures much clearer. In Figure 2, I think it would be more helpful to show the placement of the semi-landmarks, as opposed to the outline only Experimental design: The introduction is centred on the principle that morphological convergence in river dolphins as never been quantitatively assessed, however this has been done by other studies to some extent (Werth 2006; Barroso et al. 2012; Kelley and Motani 2015; McCurry et al. 2017a, b). In particular, the recent study by (McCurry et al. 2017a) was centred not only on the convergence between river dolphins, but also with crocodiles sharing a similar ecology. I suggest a better way to phrase this would be to state that although this has been done, a detailed analyses of the cranial features that converge (or not) in river dolphins is still lacking. This would also be more in line with the guidelines of the journal, of emphasising a gap in current knowledge rather than the novelty of a study per se. Lines 98-99: there is usually more variation in the dorsal side of odontocete skulls than in the ventral, so the authors need to justify why this analyses focus only on the ventral side. I think I know why, but best to be explicit and clear so readers don’t have to guess. References Barroso C, Cranford TW, Berta A (2012) Shape analysis of odontocete mandibles: Functional and evolutionary implications. J Morphol 273:1021–1030. doi: 10.1002/jmor.20040 Kelley NP, Motani R (2015) Trophic convergence drives morphological convergence in marine tetrapods. Biol Lett 11:20140709. doi: 10.1098/rsbl.2014.0709 McCurry MR, Evans AR, Fitzgerald EMG, et al (2017a) The remarkable convergence of skull shape in crocodilians and toothed whales. Proc R Soc B Biol Sci 284:20162348. doi: 10.1098/rspb.2016.2348 McCurry MR, Fitzgerald EMG, Evans AR, et al (2017b) Skull shape reflects prey size niche in toothed whales. Biol J Linn Soc 121:936–946. doi: 10.1093/biolinnean/blx032 Werth AJ (2006) Mandibular and Dental Variation and the Evolution of Suction Feeding in Odontoceti. J Mammal 87:579–588. doi: 10.1644/05-mamm-a-279r1.1 Validity of the findings: I don’t think these results show independent convergence between all individual river dolphin lineages as implied, but rather convergence between Platanista and all other river dolphins. From the phylogenetic vs phenetic tree comparisons, only Platanista shifts its position relative to other river dolphins. These also group together in the molecular tree, so their placement is largely consistent in the phenetic tree. The section where the possibility of plesiomorphy is discarded is not well justified. It’s not that the authors are not right in dismissing it necessarily, just that they don’t provide a convincing argument for it. I suggest the authors present more details to explain their position. Lines 62-63: as per guidelines of PeerJ, statements like this highlighting the novelty of a study should be avoided. Also, please note my comments above as to why I feel this is not strictly true. Additional comments: Overall, I think the manuscript is well written, and focus on an interesting scientific question regarding morphological evolution in cetaceans. The data analysis appears to be done competently, although I would like a bit more consideration of the phylogenetic uncertainty in molecular studies of river dolphins. However, I think the narrative is slightly inconsistent as the paper states in the introduction this has never been done, and then cite in the discussion earlier papers that already suggested it. I think the paper would be much improved if the authors focused on the details of which aspects of the morphology change more, and how these fit with possible mechanisms driving the convergence. For this, I think it would be useful if the author could analyse the dorsal side of the skulls, where most of the variation is likely to be found. Therefore I’m recommending the paper be rejected in its current form, but encourage a resubmission as the authors have the data to allow a more interesting analyses to be presented. In addition, please find below some minor comments on formatting and wording: Lines 118-119: (McCurry et al. 2017a, b) also carried out GMM analyses on mandibles. This must be acknowledged Lines 57-58: The authors probably mean cervical vertebrae, not caudal. Also, references to Figure 1B are not entirely appropriate, as Figure 1B does not show the vertebrae. Lines 75-77: the phrasing here is unclear. It suggests that all species were photographed, but this is clearly not the case from the list in the supplementary material. If the authors meant that the specimens analysed cover all the odontocete families, then they need to specify that the family Kogiidae is also not represented. Lines 81-83: Please specify how individual specimens were determined as being full grown adults. References McCurry MR, Evans AR, Fitzgerald EMG, et al (2017a) The remarkable convergence of skull shape in crocodilians and toothed whales. Proc R Soc B Biol Sci 284:20162348. doi: 10.1098/rspb.2016.2348 McCurry MR, Fitzgerald EMG, Evans AR, et al (2017b) Skull shape reflects prey size niche in toothed whales. Biol J Linn Soc 121:936–946. doi: 10.1093/biolinnean/blx032

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.

Title: DEVELOPMENT OF MEDICAL INFORMATICS IN CHINA OVER THE PAST 30 YEARS FROM A CONFERENCE PERSPECTIVE AND A SINO-AMERICAN COMPARISON Review round: 1 Reviewer: 1

Basic reporting: This paper is well structured and written. Experimental design: The data used in this paper cover 30 years. The results are comprehensive and constructive. Validity of the findings: Review the progress of medical informatics in China over the past 30 years to summarise the gains and losses even give constructive suggestions for future development is an important task for the whole country. Additional comments: To show the development and do a comparison from a conference perspective, especially academic conferences, it would be better to show the impact of the precedings. An impact can be the number of citations or downloads of the publications of a conference.

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.

Title: DEVELOPMENT OF MEDICAL INFORMATICS IN CHINA OVER THE PAST 30 YEARS FROM A CONFERENCE PERSPECTIVE AND A SINO-AMERICAN COMPARISON Review round: 1 Reviewer: 2

Basic reporting: The English language in the manuscript should be improved. Others are fine. Experimental design: No comment Validity of the findings: Data is statistically sound and controlled. Conclusion are well stated. Additional comments: In this manuscript, the authors compared four main Midical Informatics (MI) conferences held in China, and two main MI conferences held in USA from different perspectives. Some conferences are more industry-oriented, and some are academy-oriented. The authors concluded that, the conferences reflected that the Midical Informatics discpline in China is behind of that in USA, either in participants or topics involved. The content of the manuscript is informative, while the English language needs to be improved. The followings are some specific points in the manuscript. 1) Something is wrong in the lines 88-90. The market was "23.7" billion US dollars. After 5 years, it will shrink to "14.5" billion US dollars. In 2015, the market of hospital informatics alone reached 23.7 billion US dollars; by 2020, this market is projected to exceed 14.5 billion US dollars, at an annual compound growth rate of ≥ 24% 2) line 160: "choose" should be in the past tense "chose". Similar for many other verbs 3) lines 271-275: It is better to list "health administrative authorities" as a specific category for the comparison purpose. If it is treated as "other", it is difficult for the readers to identify the difference from the Figures. 4) line 276: what's the meaning of SCMI? 5) The last Figure is Figure 7, while the figure legend is Figure 6(b). Need to be corrected.

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.

Title: DEVELOPMENT OF MEDICAL INFORMATICS IN CHINA OVER THE PAST 30 YEARS FROM A CONFERENCE PERSPECTIVE AND A SINO-AMERICAN COMPARISON Review round: 1 Reviewer: 3

Basic reporting: no comment Experimental design: no comment Validity of the findings: The 2020 estimate of the market volume (p.6) seems to be either miscalculated or mistyped. I suggest to be more specific in Results and Conclusions of the Abstract. Most of the figers and half of the tables don't touch USA MI therefore I suggest to exclude "the Sino-American comparison..." from the paper's title and to compare Chinese conferences in more detail. P.12: The high variability in the paper's quality is intuitively acceptable but not proved. The low acception value may indicate the funding bias as well. Therefore I suggest to decrease the level of confidence when stating the conclusions about this. Additional comments: I liked the paper: the authors managed to address an important issue by simple and transparent methods. The paper is written in excellent English and I've read the Appendix with special interest.

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.

Title: DEVELOPMENT OF MEDICAL INFORMATICS IN CHINA OVER THE PAST 30 YEARS FROM A CONFERENCE PERSPECTIVE AND A SINO-AMERICAN COMPARISON Review round: 2 Reviewer: 1

Basic reporting: no comments Experimental design: no comments Validity of the findings: no comments Additional comments: In this revised version, the authors add a new part about preceding impact. The new finding from impact perspective is consistent with the conclusion of the whole paper. I feel this work meets the criteria as a research publication and suggest to accept this paper.

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.

Title: DEVELOPMENT OF MEDICAL INFORMATICS IN CHINA OVER THE PAST 30 YEARS FROM A CONFERENCE PERSPECTIVE AND A SINO-AMERICAN COMPARISON Review round: 2 Reviewer: 2

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

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.

Title: GENOME-WIDE SURVEY OF SINGLE-NUCLEOTIDE POLYMORPHISMS REVEALS FINE-SCALE POPULATION STRUCTURE AND SIGNS OF SELECTION IN THE THREATENED CARIBBEAN ELKHORN CORAL, ACROPORA PALMATA Review round: 1 Reviewer: 1

Basic reporting: Durante and Baums report on the genetic diversity and population genetic relationship of the coral Acropora palmata from a portion of this species' distribution in the Caribbean. The overall presentation is clear and the tables and figures are of good quality. The authors compare results from a small panel of microsatellite loci with genome-wide SNPs derived from genotype-by-sequencing. The authors suggest these results are important for considerations regarding coral connectivity and management. Overall, I found the description of the methods, thoroughness of the analysis and interpretation of the results to be quite limited. The shortcomings of microsatellite analysis, particularly of small number of loci, are now generally known and likelihood of detecting selection with confidence (as opposed to genetic drift or demographic impacts) is even more limited with a relatively low sample size from each location. The RAD-tag data could be utilized more effectively to address some of the authors hypotheses, particularly about relationships of individuals and populations with respect to geographic hypotheses related to the Mona Passage. Experimental design: The experimental design is adequate by genotyping individual corals from a number of locations. I detail major concerns below as well as indicate positions in the manuscript where additional clarification would be useful. 1. RAD-tag methodology. The authors should provide additional details and support for their approach to RAD-tag. It is unclear from the current text how the libraries were pooled and sequenced (Lines 132-135). In Lines 163-166 it would be helpful for the authors to provide references and support for these filtering steps. It is unclear how and why the authors came to the decision to only use loci present in at least 60% of individuals and a minor allele frequency of 0.05. Similarly, I also found it surprising the authors only used STRUCTURE based approaches when addressing individual data. Why did the authors not use phylogenetic based methods to look at relationships of individuals across the sites? The 5X coverage is alarmingly low (which the authors acknowledge on Line 335) for interpreting their results and I would suggest the authors compare results using a data with lower and greater coverage (based on Figure 1). 2. Mantel tests and environmental data. The authors correlate the FST values with geographic distance and a combination of environmental data. The authors should provide a methodology for how the geographic distance was determined and support for their approach. The environmental data comparison is an interesting comparison but the value of the confidence in these correlations appears suspect. The authors note (Line 280-282) that these environmental data are coarse, which suggests to this reviewer they could be misleading when compared with the geographic distance as a potential explanation for the genetic differences between populations. Validity of the findings: There are a few places in this manuscript where the findings could be described more clearly or in a more balanced way. 1. Outlier analyses and results. The identification of one microsatellite locus as under potential selection was interesting but given the low number of loci investigated it is difficult to interpret the confidence in this determination. The authors used standard approaches for detecting potential SNPs under selection but the results from these are equivocal. The different methods produced largely unique sets of potential SNPs and the authors should provide an explanation for this difference. Detecting regions of the genome under selection is difficult and I think these results would suggest the possibility that they are false positives. When combined with the data that these loci do not strongly match to any transcribed parts of the genome I would suggest these data and the corresponding Discussion section were removed from the manuscript. 2. Genetic diversity comparisons. The authors invest a significant portion of the Discussion on comparisons of genetic diversity between animal species. These comparisons should be prefaced with a number of caveats about where these numbers were derived (i.e., lab strain, population-averages, etc.). More importantly, in Lines 372-375, the authors correctly state that their estimate from this species could simply be due to the small sample size. This would be a more parsimonious explanation. I think putting in this explanation (a technical one based on methodology) at the end of a more complex biological explanation is not a balanced discussion. Additional comments: Small comments and suggestions Table 3. Fis should have capitalized "is" Figure 2 and 3. I would suggest the authors have a STRUCTURE analysis of with and without SNPs potentially under selection, similar to the microsatellites. Also, the authors should include a STUCTURE analysis of the microsatellite data when comparing only the same sites and individuals as used for SNP analysis. Figure 4. Please check the figure legend for this figure. I assume panel (b) is also microsatellite data but not clear from the legend. Also, please label y-axis on panel (d). Supplemental Table 3. It is unclear why some of the sequences in this table have "Ns". I am concerned that these positions were called as SNPs although they are apparently not identified base positions.

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.

Title: GENOME-WIDE SURVEY OF SINGLE-NUCLEOTIDE POLYMORPHISMS REVEALS FINE-SCALE POPULATION STRUCTURE AND SIGNS OF SELECTION IN THE THREATENED CARIBBEAN ELKHORN CORAL, ACROPORA PALMATA Review round: 1 Reviewer: 2

Basic reporting: The two data files in Supplementary Info that involve microsatellite calls "...STRUCTURE.txt" are unreadable, formatting problem. In general the paper is well-written but there are a few ways in which it could be clarified. 1. the abstract mentions "an extended set of 12 microsatellite markers" but in Introduction authors refer to "...structure derived from ten and eleven microsatellite loci." First of all, address the mismatch between the 11 loci that were examined and the abstract. Second, to put this ambiguity at the end of the introduction was confusing and unnecessary. My understanding from the rest of the paper is that all of these samples were genotyped at 11 loci - and any dataset may have some missing data, after all - and then in the present analysis one of those loci was determined to be an outlier, so the analysis was repeated using only the 10 non-outlier loci. This is also quite typical, so it will make more sense if you say you used 11 loci, and then re-analyzed after the outlier analysis. 2. another confusing way this was written - Methods/Library Preparation you discuss concentrations without complete units, e.g. ng should be ng/µl, and then "Samples were pooled into four libraries..." please make it clearer that all libraries (individual coral colonies) are uniquely barcoded, to distinguish this from pooled-RAD-seq in which Fst can be determined but not individual-level statistics. This was confusing to me at several points in the manuscript. otherwise the Intro/background writing is good and supports this paper well. There are places in the Results, e.g. lines 280-285, that are probably more appropriate for the Discussion section of the paper. Table 1 explain the distinction between A (SNPs) and B (microsatellites) in the legend. Also explain the missing latitude/longitude data? Experimental design: Experimental design and question are straightforward. A large dataset of individuals with 10 microsatellites are analyzed in the context of a RAD-seq study on a subset of these individuals from 4 primary geographic regions. This analysis allows the determination of any outlier (perhaps locally adapted) diversity, and re-assessing hypotheses of spatial variation in Acropora palmata. For some readers who are less familiar with this region/system, a map may be helpful to distinguish between the competing hypotheses. Genotyping, sequencing, and bioinformatic approaches are typical and appropriate. Validity of the findings: The overall result - concordance of SNP and microsatellite data once outlier loci are removed and considered separately - is a useful contribution to understanding spatial distribution of diversity in Acropora palmata and other species in the region. Clarifying some of the reporting/writing as mentioned earlier will make these results make better sense to the reader; again I think a map contrasting the hypotheses and the results in this paper may be of value. Make clear where the Supplementary Figure fits into the general story - that is the IBD analysis when all 11 loci are used, not referred to in manuscript. All the IBD tests are relatively weak with only 6 data points (pairwise contrasts) but with the Structure analyses this is a working hypothesis for the pattern of diversity until more data come along. The argument in lines 280-285 should move to Discussion, but I also don't agree that the spatial scales are so distinct - if the environmental data have spatial resolution of 0.25-1° latitude, that is about 28-111km, not so disparate from the spatial separation of reefs within the 4 regions? So please clarify that argument perhaps with more information on those spatial separation distributions. I would also consider Meirman 2012 when evaluating the approach to IBD, the consideration of outlier loci under an island model, and so on for interpreting these results at this scale. The second paragraph under "Genetic diversity indices in A. palmata" is confusing. You are mixing diverse summary statistics, and it isn't clear until late in the paragraph why the specific examples are being chosen - there are many 1000s of estimates of nucleotide diversity in metazoans, and even synthetic analysis across multiple cnidarian species. The contrast with the Romiguier paper starts to clear up what your goal was with those paragraphs, ostensibly to tie observed diversity with known life history to suggest that diversity is quite low in this particular species, which may be an effect of recent devastating losses in A. palmata among other things. I would clarify these paragraphs tremendously, recognize that this type of comparison is common especially in the plant literature (e.g. older work by Hamrick and Godt), but that of course every species has idiosyncratic issues driving their diversity. So just make it more apparent up front what this paragraph/section is attempting to communicate. Additional comments: Overall a nice contribution that will be a useful resource for Caribbean biogeography and coral diversity projects in the future once it is improved and clarified.

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.

Title: GENOME-WIDE SURVEY OF SINGLE-NUCLEOTIDE POLYMORPHISMS REVEALS FINE-SCALE POPULATION STRUCTURE AND SIGNS OF SELECTION IN THE THREATENED CARIBBEAN ELKHORN CORAL, ACROPORA PALMATA Review round: 1 Reviewer: 3

Basic reporting: The article is mostly written in clear professional english. There are some parts where grammar needs to be corrected to improve clarity: - Line 22. Suggest substituting the word 'place' for 'infer'. - Lines 37-39. Should be split into two separate sentences. - Line 52. 'permitting agencies'? should it be 'managers' instead? - Lines 62-63. Not clear meaning - Lines 91-94. Statements are redundant - Line 163. 'More' than what? - Line 172. Replace 'within' with 'in' - Lines 251-253. This sentence is awkward, suggest splitting in two to make more clear. - Lines 330 - 334. Sentences are contradictory, not sure what the overall message here is. The literature references are appropriate, however, given the emphasis on the authors place on the novel use of RAD markers in corals, these results should be placed in a broader context of studies that have used this technic in other animals, particularly invertebrates and non-scleractinian anthozoans. The results are relevant to test the population structuring hypothesis presented (Mona Passage as a location of major genetic discontinuity) The article is wells structured. Figures and tables are appropriate, however, Figure 1 should be supplementary as it does not contain critical information to deliver the main message of the paper. Specific comments: - Tables 1 and 4. Define in legend what A) and B) indicate -Figure 3. Is not clear what each pane represents. There are references to (a-c), (d-f) and (D-E). Please make sure it is clear how they are grouped Experimental design: The article constitutes an original contribution to our understanding of the mechanisms that structure the genetic diversity of species in the ocean. It also provides a nice example where a previous result is reevaluated and updated through the use of newer methods that increase resolution and accuracy. As such, this manuscript nicely fills a knowledge gap in marine biology with conservation implications. The research was conducted to an acceptable technical (see comments below) and high ethical standard. The methods are described in detail, however it would be useful to see the actual commands run as supplementary material to ensure its reproducibility. Specific comments: - Are the samples utilized for RAD-seq the same as the ones used for microsatellites? If so, details of individual samples should be presented as supplementary information. - Additional methods to select K should be explored (see Janes et al 2017 Molecular Ecology). How were the tested values of K selected? (they differ between RAD-seq and microsatellite analyses). - Was there a check for clones performed? The authors mention that samples belonged to different genets, but there is no mention of how this was determined. It would be good to show these results. - The authors present point estimates of population statistic values, such as Fis. With hundreds/thousands of markers these statistics are more informative when shown and discussed as distributions - The logic behind considering Read 1 and Read 2 of the paired end as replicates is not clear. How are they replicates? This needs to be better justified/explained. - Lines 327-329. Numbers of fragments are discussed. Please provide the actual numbers (expected vs. observed). Postulate an explanation for discrepancy (see Herrera et al 2015 GBE). - Genetic diversity is presented in values of pi and also in terms of SNPs/bp. Please present in consistent units. - Paragraph lines 350-367. What is the main message of this paragraph? It ends without a tying end or conclusion. - Line 371. What is the predicted nucleotide diversity of A. palmata? Please specify. Also see Arnold et al 2013 Molecular Ecology for a discussion of genetic diversity estimation from RAD-seq data. Validity of the findings: The data is seemingly robust and the statistical analyses performed are appropriate. However, a greater exploration of the parameters used in the populations analysis in STACKS should be performed. The authors selected conservative parameters regarding the number of individuals per population and the number of populations for a particular locus to be included in the analyses. These parameters have great influence in the number of SNPs that are included in the analyses. The authors correctly identified the relatively low number of SNPs as a weakness in some of their analyses. Performing the population structure and selection analyses in SNPs datasets produced with a variety of parameters is necessary to understand the robustness of the conclusions. Furthermore, the authors should explore additional methods to evaluate population structuring such as PCA, which does not necessarily bins populations into discrete K categories. Additional comments: No additional comments

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.

Title: GENOME-WIDE SURVEY OF SINGLE-NUCLEOTIDE POLYMORPHISMS REVEALS FINE-SCALE POPULATION STRUCTURE AND SIGNS OF SELECTION IN THE THREATENED CARIBBEAN ELKHORN CORAL, ACROPORA PALMATA Review round: 2 Reviewer: 1

Basic reporting: The authors have done a fine job in their revision of this manuscript. The revision clarified the ambiguous methods and results in the originally submitted manuscript. The addition of the supplemental figures for analyses exploring impact of cut-offs for inclusion of loci is useful. I have no further comments for this section. Experimental design: The experimental design is good and the authors have clarified questions I had with the analysis of the data. I have no further comments for this section. Validity of the findings: The authors have done what they can do in their revision to address my concerns with the RAD data. Frankly, I am not entirely convinced that the sequence-based data are robust because of the low coverage. I understand that sometimes projects do not go as planned. The authors now include some statements in the manuscript to explain these shortcomings. I have no further comments for this section. Additional comments: The authors have done a good job with this revision, overall. The section in the Discussion that summarizes the genetic diversity is various animals (beginning on Line 390) still seems unnecessary because these are different types of comparisons (e.g. whole genome compared, small regions of the genome, transcriptomes), thus difficult to compare with any degree of confidence. The authors acknowledge their estimates are potentially not representative for this species (Lines 426-429). I would suggest again that the section "Genes under positive selection" adds little to the manuscript and the manuscript would present better if this were removed. There are few hits to a potential protein and the function would be even further removed.

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.

Title: GENOME-WIDE SURVEY OF SINGLE-NUCLEOTIDE POLYMORPHISMS REVEALS FINE-SCALE POPULATION STRUCTURE AND SIGNS OF SELECTION IN THE THREATENED CARIBBEAN ELKHORN CORAL, ACROPORA PALMATA Review round: 2 Reviewer: 2

Basic reporting: The authors have satisfactorily addressed the review points. Experimental design: The authors have satisfactorily addressed the review points. Validity of the findings: The authors have satisfactorily addressed the review points. Additional comments: The authors have satisfactorily addressed the review points.

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.

Title: THE ABILITY OF LAYING PULLETS TO NEGOTIATE TWO RAMP DESIGNS AS MEASURED BY BIRD PREFERENCE AND BEHAVIOUR Review round: 1 Reviewer: 1

Basic reporting: This article is very well-written and clear to read, the level of English is satisfactory. The introduction is well-referenced and provides a clear background for the need for the study. The raw data file is supplied and is clear to read. Figures are relevant and clear although I found the direction of the x-axis labels difficult to read in Figure 4, I think they should be flipped around if possible. I think Figure 1 is extremely valuable and good to see it included. Minor comments: Comments on the abstract are first impressions, some information becomes clear when you read the entire manuscript but these were things that were identified when reading the summary. Lines 29-30: Definition of attempts? Line 33: Is there any other way to navigate a LR than jumping it? Lines 35-36: The LR is a clear potential perching apparatus, therefore it would be expected that birds may rest on it more than the GR. Line 73: ‘ramps’ not ‘rams’ Line 83: comma after ramp Experimental design: This experiment is original research that is needed for understanding how birds use ramps that are increasingly used in alternative housing systems yet there is currently little scientific literature on the best ramp designs. The controlled individual testing allows for detailed observations of individual bird responses. The experiment was well-designed with a high standard of habituation/training regimes and controls in place but I had a few queries throughout the methods section for additional clarification (see comments below). However, my biggest query is why the effects of rearing were not included in the current manuscript (written they are to be included in a separate manuscript). As the current manuscript is not that long, it seems like the effects of rearing would be a great addition to this paper rather than being included separately. Particularly as the birds were tested during the rearing age as 12-14 week pullets. Comments: Line 97: More information on why this particular design of the ladder ramp was chosen. Line 104: I assume birds were divided evenly between rooms/groups. Could birds see/hear each other? It is not clear how the rooms/groups are divided? Mesh? Solid walls? Line 111: comma after ‘room’ Lines 111-112: Clarify here that all birds were used in the current study despite being reared differently. Line 131: Company details for the camera system? Line 149: Clarify if any food deprivation prior to testing and how hunger/lack of hunger was potentially controlled for as a motivation factor. Line 150: Age of birds during habituation stated here would be useful. Lines 150-152: Did this occur within the home pen? Lines: 152-154: Were all birds given the same amount of habituation or did habituation depend on individual bird responses. I imagine some birds were quicker to habituate than others? Lines 154-157: It is not entirely clear to me the difference between test room and test pen. Where precisely were the birds placed for each of these habituation periods? Line 160: Delete the word ‘because’ Lines 164-165: Was this pencil-tapping also done during habituation or just started during training? Lines 165-166: Was training always in the same direction first? Lines 173-174: I am not clear on what is the difference between the 2 groups. From my understanding, birds were reared in two rooms, with 2 groups per room. Therefore, are you only testing birds from 2 groups within 1 room? Or are you referring to 2 rooms (2 groups per room) here? Line 180: change to ‘testing order per ramp type’ Lines 231-232: Were these the only behaviours used as indication of hesitation? If so, I think the sentence should read ‘including head orientations, crouches…..etc’. Right now it reads as there were also several other behaviours included as indicative of hesitation and I think all should be listed here as this is a novel approach for behavioural observations. Lines 242-243: Further clarification as why were these not counted? Were these interpreted failed attempts or birds just using the ramps for resting? Based on your observations, if a bird did not complete a transition within 1 min, it was likely the bird was not using the ramp for transition purposes but was just perching on it (if you can make that assumption from the observations). Thus is this why the 5-min scan samples were included to look at ramp use for resting/perching? Line 251: ‘included’ not ‘analysed’ Lines 255-256: Would be useful for the reader to state how many birds these exceptions applied to. Particularly for the 2 or 3 min test if this was only a small number of birds. Lines 269-270: Further details on how interactions were assessed using t-tests. Lines 271-272: I don’t understand what two variables are being combined. Further clarification for this sentence. Line 273: ‘some of the data’ Validity of the findings: I think the data are thoroughly analysed with statistical analyses presented where possible. The group tests just provide visual data due to the low sample size but the visual presentations are still valuable. The discussion is well written and conclusions drawn from the findings are valid. I have a few comments on the results and discussion as detailed below. Lines 303-306: Perhaps I missed somewhere earlier any statement about measuring/analysing attempts. Line 307: But sometimes 3 mins? For how many birds? Any effect of ramp type on whether 3 mins was required? Lines 317-320: Can you comment on pauses with the LR. It almost seems by design, there are pause breaks inserted as there are rungs compared to the continuous nature of the GR. Would you therefore expect birds to pause on the LR compared to the GR, regardless of whether one is more difficult than the other or not? Line 357: in individual bird behaviour Line 359: Lambe et al. Lines 419-425: I think here it would be more valuable to include some discussion of using the LR as a perch and whether this will have impact on bird movement in the system compared to a GR that is typically not used as a perch. Since the LR appears to have a dual purpose, in a commercial system this may impact bird movement in different areas of the house if it is consistently blocked by birds using it for perching rather than transitioning. Discussing diurnal patterns of perching seems a bit out of place here, but perhaps could be included in relation to what is discussed above and any interplay between perching patterns and transition patterns within a housing system. The LR is potentially confounded by its dual purpose and this may then be affected by available perch space within the system, perhaps this could be discussed. Lines 426-429: For clarification, could birds in the group testing room go from the slats to the litter without using a ramp? Or did the ramp placement eliminate this possibility (I think by your figure the two ramps had no gaps inbetween)? If birds could bypass the ramp completely, did you look at the numbers of birds that did this? Whether the perching on the LR meant more birds were jumping down to the litter and not using a ramp at all? Would be valuable to discuss whether just ladder ramps in a commercial setting that could be consistently blocked may mean more birds execute jumps that could injure them. Lines 441-443: another reason why the effects of rearing might be valuable to include in this current manuscript rather than as a separate manuscript. Lines 448-454: I think the confound of the LR being used a perching space needs to be mentioned here as this is likely to have impact in a commercial system as to what the birds use the ladder for. Additional comments: No additional comments

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.

Title: THE ABILITY OF LAYING PULLETS TO NEGOTIATE TWO RAMP DESIGNS AS MEASURED BY BIRD PREFERENCE AND BEHAVIOUR Review round: 1 Reviewer: 2

Basic reporting: Line 33: Collisions with the food reward bowl were “greater.” Specify more frequent instead of greater, as greater collisions implies more significant impact, the forces of which were not measured in this study. Line 54: define “pop-hole” and/or, if possible, provide a citation. Line 58: define “end of lay” for the non-specialist in poultry rearing. How long does this interval last, or how old are the birds at the end of lay? Line 73: typo “ram” should be “ramps” Line 96: spell out MDF Line 131: Spell out CCTV. Add sampling rate (frame rate) and video pixel resolution. Line 180: “Systematically balanced” give slightly more detail. Presumably this means non-random, that every other bird was treated to a different order, or was it a different order for a given group of birds (tested individually within a group). This is relevant to interpreting the confidence intervals for individual performance criteria. Lines 261-266: It seems that only Z statistics are reported. If that is the case, it would be better to state that non-parametric tests were used because assumptions of parametric tests could not be met, then delete the specific reference to paired t-tests and repeated measures ANOVAs. Line 266 (and figures): 95% CI’s are reported in the figures. Why not use standard deviations instead? Otherwise, clarify how you calculated the 95% CI’s. It would seem that an N of 4 groups would lead to unnecessarily large CI’s. Line 286: Add percentages here to the numbers, as percenages are more broadly relevant than the exact numbers Line 323: I suggest starting with sentence two and putting (“Fig. 4”) at the end of the sentence instead of using the present opening sentence (to eliminate redundancy). Line 343: As previous, start with second sentence and then direct the reader by putting (“Fig. 5”) as the end of the sentence (delete current topic sentence). Line 351: Here it is not clear what you mean by “fully accounted for” Expand slightly on detail. Discussion, General: Two issues would be useful to explore, even if briefly, in the discussion (for example, perhaps near lines 391-394, or 426-427). You do not describe whether the birds ever fell or appeared to lose locomotor control, and you do not mention whether they ever used their wings to assist during climbing or to outright fly. For the research into keel-bone damage, it is worth mentioning if they birds never fell due to loss of control. Likewise, even in general terms, it would be helpful to understand whether wing flapping and flight increases or decreases in relation to ramp design. Lines 437-438: this is somewhat confusing because you earlier wrote that you could not analyze the data according to a repeated-measures ANOVA. Clarify with additional detail what you mean by “accounted for”. Figure 1 (legend). It seems that the A figure is a ladder and the B is a grid, but these are reversed in the legend. Experimental design: Nothing to add. Validity of the findings: Nothing to add. Additional comments: Your research is interesting and worthwhile for improving our understanding of ways to design housing systems for layer hens that will lead to reduced frequency of keel bone damage. Your experimental design and analyses are well conceived and rigorous, and I have no major criticisms. I offer minor comments to help improve clarity in selected instances (basicreporting section).

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.

Title: THE ABILITY OF LAYING PULLETS TO NEGOTIATE TWO RAMP DESIGNS AS MEASURED BY BIRD PREFERENCE AND BEHAVIOUR Review round: 2 Reviewer: 1

Basic reporting: Article is well written and the authors have made all suggested changes. Experimental design: The methods section has been clarified as suggested by both reviewers and is now much clearer to read. Validity of the findings: The discussion is much improved and reads very well. Additional comments: No additional comments

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.

Title: UNCOVERING THE RELATIONSHIP AND MECHANISMS OF TARTARY BUCKWHEAT (FAGOPYRUM TATARICUM) AND TYPE II DIABETES, HYPERTENSION, AND HYPERLIPIDEMIA USING A NETWORK PHARMACOLOGY APPROACH Review round: 1 Reviewer: 1

Basic reporting: Nice and highly relevant paper. It is a report on important results of the investigation, based on a comprehensive systemic approach integrating drug target prediction, network analysis, and molecular docking simulation, developed to reveal the relationships and mechanisms between the putative targets in Tartary buckwheat and Type II diabetes, hypertension, and hyperlipidemia. Authors are reporting that some pathways discovered in the study, such as the insulin resistance pathway, AGE-RAGE (Advanced glycation end products) signaling pathway, and insulin signaling pathway, have a direct relationship with the three studied diseases. They revealed the relationship of Tartary buckwheat and the three diseases to provide more information on the clinical applications of Tartary buckwheat and to further research on Tartary buckwheat properties and quality. Mainly clear professional English is used. Paper with nice novel results. Title of the manuscript is suitable, abstract is enough informative. Literature references are sufficient. Conclusions are of broad interest and based on the results. Abstract, Introduction, Results and Discussion are clearly presented. Professional structure of manuscript is well performed. Figures are clear, but the reviewer has a specific suggestion to Fig. 1. Results are self-contained with the results relevant to hypotheses. Experimental design: Research questions are well defined and within the aims and the scope of the journal. Materials are accordingly defined. Methods are suitable, properly described and used, in a way that is possible to replicate experiments. The investigation is performed to high technical standards. It is no ethical problem involved. Validity of the findings: Data are obtained in a sound way, they are statistically evaluated in a proper way. Conclusions are very well stated and based on the results. Discussion and conclusions are sound and relevant. Additional comments: Specific suggestions: Line 1 and elsewhere in the manuscript: Instead of »tartary buckwheat« correct to »Tartary buckwheat«, where applicable. In difference to »common buckwheat« (with lower-case first letter), »Tartary buckwheat« should be in English always written by upper-case first letter; this way of writing was internationally accepted, among others in the discussion on International Buckwheat Research Association (IBRA) Assembly in 2013, as word »Tartary« comes from the name of the Tartar people. Line 51, delete: amount of Line 67, instead of: shell, better: husk Figure 1, Nice and illustrative figure, however »PT« letters should be better placed outside the »T2D« area, to be more clear, including to be clear on black/white prints.

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.

Title: UNCOVERING THE RELATIONSHIP AND MECHANISMS OF TARTARY BUCKWHEAT (FAGOPYRUM TATARICUM) AND TYPE II DIABETES, HYPERTENSION, AND HYPERLIPIDEMIA USING A NETWORK PHARMACOLOGY APPROACH Review round: 1 Reviewer: 2

Basic reporting: The manuscript reported new and relevant results on systemic approach integrating drug target prediction with the network analysis, and molecular docking simulation. Results are important for understanding relationships and mechanisms between the putative targets in Tartary buckwheat and Type II diabetes, hypertension, and hyperlipidemia. Professional English of the manuscript should be improved Experimental design: Experimental design is well performed and within the aims and the scope of the journal. Materials and methods are well defined and used. It is thus possible to replicate experiments. The investigation is performed according to high technical standards. There seems that no ethical issues are involved. Validity of the findings: Data are obtained in a suitable way,and correctly evaluated. Conclusions are properly based on the experimental results. Additional comments: Reviewer suggests improvement of English style. In the title and manuscript Tartary buckwheat should be written with the capital first letter. Text in lines 44-47 is not clear enough, needs rewriting. Line 54: correct »which have« Line 54, punctuations are not correct. Line 132. punctuations are not correct. Line 263, the reference should be inserted. Conclusions are not clear enough, they should more clearly pointed out the purpose, results and focus of the paper.

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.

Title: THE REANALYSIS OF BIOGEOGRAPHY OF THE ASIAN TREE FROG, RHACOPHORUS (ANURA: RHACOPHORIDAE): GEOGRAPHIC SHIFTS AND CLIMATIC CHANGE INFLUENCED THE DISPERSAL PROCESS AND DIVERSIFICATION Review round: 1 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: Orlov, Poyarkov, Vassilieva, Ananjeva, Nguyen, Sang, and Geissler, 2012, Russ. J. Herpetol., 19: 23-64 noted extensive confusion with Rhacophorus robertingeri in the literature and mentioned R. chuyangsinesis considered as a junior synonym of R. calcaneus. Thus, the authors need to correct data. Additional comments: Because computer programs change over time and the default parameters are not necessarily rigid, it would help to specify what the default parameters are. This also helps the reader to know that you judged particular parameters and associated assumptions to be the best ones for your data.

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.

Title: THE REANALYSIS OF BIOGEOGRAPHY OF THE ASIAN TREE FROG, RHACOPHORUS (ANURA: RHACOPHORIDAE): GEOGRAPHIC SHIFTS AND CLIMATIC CHANGE INFLUENCED THE DISPERSAL PROCESS AND DIVERSIFICATION Review round: 1 Reviewer: 2

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: Excellent manuscript by the authors. Since the manuscript talks about geological phenomenon, I felt Two important papers from Indian sub-continent were mission from the manuscript. I would be good if the following papers are cited in the revised manuscript. 1. Taxonomic revisions of Rhacophorus genus in Indian sub-continent was given in Biju et al 2013. This paper did not find a place in the manuscript. 2. Similarly, Vijayakumar et al 2016 provided insights on glaciation, gradient and geography of Western Ghats, that perhaps shaped diversification in Raorchestes genus. This can be cited in the manuscript.

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.

Title: THE REANALYSIS OF BIOGEOGRAPHY OF THE ASIAN TREE FROG, RHACOPHORUS (ANURA: RHACOPHORIDAE): GEOGRAPHIC SHIFTS AND CLIMATIC CHANGE INFLUENCED THE DISPERSAL PROCESS AND DIVERSIFICATION Review round: 1 Reviewer: 3

Basic reporting: Format of text is not consistent throughout the manuscript. For example, "et al." are italicized in some case, but not, in normal, in other case. Correct such basic formatting throughout the manuscript before submission. Experimental design: The results of the present study and the former one (Li et al., 2013) were very different. Especially, the estimated ancestral area is quite different. The present authors must clarify any difference in samples, analysis, and interpretation on the results between them. Validity of the findings: As shown on Fig.S1, 1) a lot of nodes lacked a significant supports. Further, 2) the topology is not consistent between Fig. S1 and Fig. 2. Need to improve or explain on these two major concerns. Additional comments: No additional comments

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.

Title: THE REANALYSIS OF BIOGEOGRAPHY OF THE ASIAN TREE FROG, RHACOPHORUS (ANURA: RHACOPHORIDAE): GEOGRAPHIC SHIFTS AND CLIMATIC CHANGE INFLUENCED THE DISPERSAL PROCESS AND DIVERSIFICATION Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: Excellent work by the authors. Congrats!

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.

Title: THE REANALYSIS OF BIOGEOGRAPHY OF THE ASIAN TREE FROG, RHACOPHORUS (ANURA: RHACOPHORIDAE): GEOGRAPHIC SHIFTS AND CLIMATIC CHANGE INFLUENCED THE DISPERSAL PROCESS AND DIVERSIFICATION Review round: 2 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: Thanks for addressing the comments.

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.

Title: THE REANALYSIS OF BIOGEOGRAPHY OF THE ASIAN TREE FROG, RHACOPHORUS (ANURA: RHACOPHORIDAE): GEOGRAPHIC SHIFTS AND CLIMATIC CHANGE INFLUENCED THE DISPERSAL PROCESS AND DIVERSIFICATION Review round: 2 Reviewer: 3

Basic reporting: I confirmed the improved MS. Experimental design: No problem. Validity of the findings: OK. Additional comments: I am looking forward for seeing this publication.

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.

Title: PBXPLORE: A TOOL TO ANALYZE LOCAL PROTEIN STRUCTURE AND DEFORMABILITY WITH PROTEIN BLOCKS Review round: 1 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: Some conclusion and discussion need more clarification (see below in general comments). Additional comments: The manuscript entitled ‘PBxplore: a tool to analyze local protein structure and deformability with Protein Blocks’ by Barnoud et al presents the implementation and development of a python tool based on Protein Blocks definition to characterize local structures in a protein. The protein blocks (PBs) used in this work have been previously published and have been shown to be useful in many applications such as peptide design, local protein conformation predictions etc. The suit of tools presented by the authors can be used either for a single structure or for a trajectory. The work presented is technically sound, however a few points could be clarified when comparing the results of PBxplore and RMSF calculations for instance. Even though more details about the RMSF calculation are present in the cited paper (Jallu et al 2012), the authors could specify that it was calculated on Calpha atoms. Since RMSF values are obtained when aligning each frame of the simulation on a selection of atoms, what would be the values if the authors align their trajectory on the loop 33 to 35? It should be clarified that indeed RMSF values if performed on all the structure including the loops for instance will give a more global view of flexibility, but if calculated with respect to a subset of atoms of interest it could give a more local picture. It is not very clear what the authors imply when stating the residues 32 and 36-37 around the loop of interest are acting as hinges. From the Neq plot it seems that indeed residue 32 has an elevated value, but it is not as striking for residues 36 and 37. Also, the sentence on page 6 ‘These results question the relationship between MD simulations and allostery and the role of long range effects on protein structure’ is a bit of an over-statement in this context, as long range effects have not been explored thoroughly in this work. It is rather the difficulty to extract from MD simulations meaningful information on structural changes which play a role in allosteric signaling. Notwithstanding the above, it is interesting to see that regions displaying elevated global RMSF values can easily be pinpointed in a straightforward fashion using PBxplore for further analysis regarding local vs global flexibility. Therefore this tool should be a valuable tool for analysis in computational structural biology.

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.

Title: PBXPLORE: A TOOL TO ANALYZE LOCAL PROTEIN STRUCTURE AND DEFORMABILITY WITH PROTEIN BLOCKS Review round: 1 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: It will be good to develop a statistical measure to identify the residues which are not deformable and flexible, yet their RMSFs are high (example of beta-3 integrin). It will help PB analysis on the top of RMSF. On the other hand it will also help to highlight regions in an analysis, if any, for which the RMSFs are low yet deformabilty and flexibilty are high (as around residue 20). It will also help users if a web based server could be setup for PBxplore (should not be difficult). Small suggestion: Name of the tool should be "PBxplorer" and not "PBxplore".

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.

Title: DETECTION OF BACTERIAL CONTAMINANTS AND HYBRID SEQUENCES IN THE GENOME OF THE KELP SACCHARINA JAPONICA USING TAXOBLAST Review round: 1 Reviewer: 1

Basic reporting: Unclear, ambiguous writing and need some language editing Scientific writing need significant effort. e.g. no references, poor hypothesis and synthesis Experimental design: No sufficient evidence/detail to highlight that this tool is anywhere near the state of the art in this category Validity of the findings: Zero Novelty Data is not robust, e.g. used only one genome, no benchmarking Additional comments: Thanks for your efforts. According to me this paper/tool is not publication ready in its current form and needs a significant effort (especially in benchmarking and scientific writing) Major Where is the benchmarking? You should compare the tool with the existing mainstream tools and provide some comp. Stats. Why using only one genome? Why not Bacterial Genomes? Since authors have mentioned that this tool can be used for “other genomes” let me give some bacterial genome scenarios. (a) If I have multiple genomes from diff. strains of single species, do you think this tool can distinguish the intra-species contamination? Here the strain means whole genome based ANI >99%? I don't think its possible. (b) How can we use this tool to identify the contamination in genomes assembled across shotgun microbiome denovo assemblies? where we have very few ref. Genomes? You have used word “matched” all across the text, mentioning the blast hits. Honestly the word “matched” is very unclear to me, what is the cutoff that makes you decide that its a match? Overall, its not a well written paper and need some significant effort. Line 79 “but if such searches are carried out with the entire scaffolds, they may be biased by highly conserved regions, which frequently have very little discriminatory power (transposons, virus insertions etc.)” This statement need ref. Line 81 “ Alternatively, protein-based searches may be performed with all predicted proteins of a genome against a reference database (frequently NCBI nr or uniref90).” where is the ref.? Who created these two categories? Line 83 “Based on these results,” what results? Please re-frame Line 84 “The advantage of this approach” what approach? Please write the text clearly. Line 141 “also detected via a 16S sequence in a different scaffold, see above” According to me the above line makes no sense, what is “via a 16S sequence” do you mean a blast hit? If yes, what is the aignment parameters? e.g. length, e-value? Line 142 “large regions that were homologous” what is the defination of large here? Line 147 “This clearly supports the hypothesis of diverse phylogenetic origins of the scaffolds classified as bacterial.” The above statement needs ref. Line 150 “the 90% threshold” what is that threshold Line 151 “These scaffolds may comprise assembly artifacts the have merged contaminating sequences with sequences of the target species as well as recent horizontal gene transfers.” The above sentence is very confusing, poorly written and has no evidence mentioned in this paper. Line 152 “It is not the aim of this publication to re- analyze the S. japonica genome, but for the purpose of illustration we have selected the two scaffolds with the highest numbers of blast hits from this category, and manually examined them.” If its not the aim why to do this illustration at the first place? Line 155 “The first, scaffold159, is approximately 500 kbp long and has 557 blast hits for different 500 bp- segments, 26% of which (towards the beginning of the sequence) are with bacteria.” above senetence needs formatting and reframing Line 157 “GC profile server” I think its GC-Profile server

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.

Title: DETECTION OF BACTERIAL CONTAMINANTS AND HYBRID SEQUENCES IN THE GENOME OF THE KELP SACCHARINA JAPONICA USING TAXOBLAST Review round: 1 Reviewer: 2

Basic reporting: The manuscript and the provided software-tool Taxoblast addresses the need of molecular biology scientists to analyse scaffolds from genome assemblies for possible contamination or lateral gene transfer. It describes the tools functioning and features and explains the utility based on an example assembly from the brown alga Saccharina japonica. Advantages of the method in relation to screening reads before assembly are shown. Overall the writing is good instructure, language and well understandable in expression. The provided tool is described as helpful for biologists that do not have capacity to perform similar analyses as effectively as with the tool (abstact: "usable also by researchers with little background in informatics"). This is an important added value of bioinformatics tools in general, thus not worth mentioning for publishing of this specific tool. Experimental design: Validating the tool by an example data set was done carefully and has led to new insight, useful for improving the publication of the chosen genome data. Validity of the findings: The findings are well documented, and the summary given in statistics data is complete. Additional comments: I recommend the paper for publication after minor revision indicated above.

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.

Title: DETECTION OF BACTERIAL CONTAMINANTS AND HYBRID SEQUENCES IN THE GENOME OF THE KELP SACCHARINA JAPONICA USING TAXOBLAST Review round: 1 Reviewer: 3

Basic reporting: No comment (see below). Experimental design: No comment (see below). Validity of the findings: No comment (see below) Additional comments: The authors describe in the manuscript a pipeline named “Taxoblast”, which uses a BLAST-based approach and predefined taxa to identify contaminants, as well as hybrid sequences, in assemblies of genomes. The pipeline is publically available. As a case study, the authors examined the genome assembly of the kelp Saccharina japonica and identified some bacterial contaminants as well as bacteria–kelp hybrid contigs. The manuscript is well-written and easy to follow. The described approach is simple, and it is laudable that the pipeline is freely available. However, the manuscript reads rather like a case study. It remains uncertain whether the kelp genome harbours particular problems, or whether similar problems occur in other datasets. For example, the authors state that internal tests found very little differences between blastn and blastx searches. However, this may not apply for other genomes, in which the possible contaminants are less well-known, or less diverged from the host (for example eukaryote parasites). I expect that in these cases, a blastx-based approach has clear advantages. It would also be useful to compare the present approach with some other programs or platforms, e.g. KRAKEN, which are commonly used for identification of contaminants. Other issues: l. 40: I would not call a paper published in 2011 as “recent”. Moreover, it would be useful if the authors cite a study showing that contaminants are an even more eminent issue in NGS-derived genomes. l. 50 (and other places throughout the manuscript): Something went wrong with the in-text citations, for example here: “(e.g. [3,4])” . l. 98-99: I think the authors mean: "blastx searches against the National Center for Biotechnology Information (NCBI) _protein_ nr database and blastn searches against the NCBI _nucleotide_ nr database". nr = non-redundant.

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.

Title: DETECTION OF BACTERIAL CONTAMINANTS AND HYBRID SEQUENCES IN THE GENOME OF THE KELP SACCHARINA JAPONICA USING TAXOBLAST Review round: 2 Reviewer: 1

Basic reporting: no comment Experimental design: See below. Validity of the findings: no comment Additional comments: The authors have submitted a revised version of their manuscript that describes the pipeline named “Taxoblast”, a BLAST-based approach to identify contaminants and hybrid sequences in contigs. The authors have added a brief benchmark estimate and have corrected minor errors. The manuscript has significantly improved, and I acknowledge the rationale behind the study. I would have preferred a broader approach that compares, e.g., the results obtained with different methods and with different data. However, the paper is sound as it is.

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.

Title: GINSENOSIDE RK1 BIOACTIVITY: A SYSTEMATIC REVIEW Review round: 1 Reviewer: 1

Basic reporting: . Experimental design: . Validity of the findings: . Additional comments: This manuscript handles Ginsenoside Rk1 bioactivity: A systematic review. This is an interesting and well-written manuscript. However, the manuscript contains some weakness that should be revised before it is considered for the journal publication: Line 66: Genus Panax can be classified by origins. Chinese ginseng is Panax notoginseng and Korean ginseng is Panax ginseng “traditional Chinese medicines” should be changed or rephrased to “traditional medicines”. Comparison with other ginsenosides will be needed.

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.

Title: GINSENOSIDE RK1 BIOACTIVITY: A SYSTEMATIC REVIEW Review round: 1 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: Authors curated 21 original research papers showing biological and pharmacological effects of ginsenoside Rk1 and summarized findings of each paper in the manuscript. Due to the style that seems to be common in medical fields, authors carefully enlist endless effects of Rk1 on endless lists of disease without providing any conceptual framework on how Rk1 works. This could be due to original papers that hardly provide any mechanism. Ideally, authors should have tried to make a sense out of all those different effects by extrapolating signaling mechanisms behind all those diseases. Nevertheless, the manuscript is resourceful and should help readers who want to know reported effects of Rk1 on various disease.

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.

Title: GINSENOSIDE RK1 BIOACTIVITY: A SYSTEMATIC REVIEW Review round: 1 Reviewer: 3

Basic reporting: 1) The manuscript needs to be checked and re-edited for grammatical and punctuation errors. I would suggest that the authors revise the abstract, as lines 51-58 seem repetitive in nature. 2) The manuscript summarizes all relevant papers and provides a good overview of the literature but this study has only been performed till Aug 2015 and not till the present day (in this case May/June 2017). Thus, it is recommended to revise this manuscript to include all the literature that has been reported till present date. 3) How many different types of ginsenosides are there in total? It would be better to include a brief description along with a figure in the introduction, which talks about all the different ginsenosides, and how they are isolated. This will help the readers understand better, how Ginsenoside Rk1 is an entirely different class of compound and exhibits unique bioactivity when compared with its sister analogues. 4) The results section on the anti-cancer activity including sub-titles liver cancer, melanoma and gastric cancer is very descriptive, repetitive and needs to be re-edited. The authors have already provided similar information in table 2. I recommend thoroughly revising this particular part of the manuscript to reflect critical findings. Avoid using the word “they” excessively within the anti-cancer section. The other half of the bioactivity section (anti-platelet, anti-inflammatory and so on) is well-written. 5) The references are adequately cited within the manuscript 6) Figures and table provide enough relevant information and conforms to the publisher guidelines. Experimental design: 1) The research presented within the manuscript is original and within the scope and aims of the journal. 2) The systemic review or meta-analysis is carried out in a detailed and systematic manner. 3) The GRADE method followed by the authors is a good example of methods that need to be employed to avoid bias in reporting findings. The grading system has also been adequately described by the authors in Supplemental Table 2. Validity of the findings: The authors tend to be quite positive about the findings published in other articles, without critically assessing related risks and disadvantages. This may in some cases sketch an unrealistic view of Ginsenoside Rk1 bioactivity. For example, the authors describe Rk1 as anti-cancer drug, however, not a whole lot of work has been conducted with this particular compound and majority of the work published has been primarily carried out in vitro. How would the drug behave in vivo when it is administered to tumor bearing mice or cancer patients ? Is the compound stable ? What are the solubility and bioavailability parameters that need to be taken into account to develop them into anti-cancer therapeutics? Such statements would greatly contribute to the novelty and originality of the manuscript, and I strongly recommend revising this in the conclusions/discussions section. Additional comments: Line 46: Introduce space before we Line 55: Shouldn’t the word be “on” liver cancer, melanoma and gastric cancer? Line 71: Re-edit the sentence Line 128: Change to includes or included rather than including Line 268: “in the” is repeated twice Line 272: Remove space between I and t Line 301: Use “by” instead of “through” Line 391: “Confirmed the anti-cancer effects” is a strong statement to use considerably due to all the in-vitro studied conducted. Please revise this statement.

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.

Title: GENE AND GENOME-CENTRIC ANALYSES OF KOALA AND WOMBAT FECAL MICROBIOMES POINT TO METABOLIC SPECIALIZATION FOR EUCALYPTUS DIGESTION Review round: 1 Reviewer: 1

Basic reporting: No comment Experimental design: The majority of this project is excellent. This is the first in-depth culture-independent metagenome-based analysis of koala and wombat. A weakness relates to the use of fecal pellets as the sole source of microbiome analysis and then relating the microbiota to koala and wombat digestion. Eventhough there was an early publication in the koala, this only had a very low number of animals. The digestive microbiota communities in the gut and caecum and likely to be different to those in the fecal pellet. Can the authors perhaps include the comparisons of the metagenomes of a single animal. Validity of the findings: No comment Additional comments: No additional comments

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.

Title: GENE AND GENOME-CENTRIC ANALYSES OF KOALA AND WOMBAT FECAL MICROBIOMES POINT TO METABOLIC SPECIALIZATION FOR EUCALYPTUS DIGESTION Review round: 1 Reviewer: 2

Basic reporting: The basic reporting in this paper is fine. Overall it is clearly written. My most substantial comment is that sometimes information appears in the wrong section and is sometimes redundant with what has already been stated. For example: Lines 80-90 present a summary of the results. This does not seem appropriate for the introduction. I have flagged several of these instances but the authors are encouraged to conduct a thorough review. The figures for the most part are highly useful. Some required additional explanation. E.g., Fig 2: indicate the significance of the different number of circles in panel A. Experimental design: The design and analysis of the results are well defined. The knowledge gap being addressed is articulated. The only criticism of the methods is that it would have been better if there were more control over the origin of the samples (ie more samples from the same zoo), but given the difficulty in keeping these species, the design is reasonable, and did not appear to impact the outcome. Validity of the findings: Overall the findings seem valid, robust and statistically sound. The notion that there are members of the koala microbiome that function to degrade PSMs is likely true, and the authors for the most part acknowledge that this is a hypothesis yet to be experimentally tested. Additional comments: In this manuscript, the authors present a comparative study on the gut metagenomes of two iconic mammals from Australia, the koala and the wombat. This is the first investigation into these metagenomes. They found that while there are many similarities in these metagenomes, the koalas is larger with greater representation of metabolism of plant secondary metabolites. The authors propose some key players in the microbiomes of these animals related to PSM metabolism, lignocellulose degradation and urea recycling. S24-7 is mentioned in the abstract as being one of the unique members of the koala microbial community. The role of family is not further discussed in the discussion, yet it has been implicated in the degradation of PSMs, particularly oxalate, which likely occurs in Eucalyptus. Some references of potential interest: Ormerod et al 2016 4:36 Microbiome and Millar et al AEM 82:2669; Along these lines, the putative function of the two taxa common in the wombat were not mentioned in the discussion. The discussion could be improved through the inclusion of additional recent literature. recent experimental Such as Kohl et al Ecology Letters 2014; Welte, 2016 AEM 04054; Haiser et al Science 2013 341:295, Spanogianopoulos Nature Reviews Microbiology 2016 14:273 Figure 1 is beautiful. Does it include all genera in the Diprotodonts or is it an abbreviated tree? Suggest removing “toxic” from Eucalypt. The concept of high levels of plant secondary metabolites can be explained in the text. “’putatively” detoxifying bacteria Lines 80-90 present a summary of the results. This does not seem appropriate for the introduction Lines 275-283 mostly contain information that should be in the methods. Fig 2: indicate the significance of the different number of circles in panel A. The term CDS seems to be defined long after its first use Fig 3: add an explanation of the use of colored dots. I figured it out eventually but it would be useful as an aid to the reader. Lines 348-359: I could not identify the results in this paragraph. Seems like it belongs in the methods. Interpretation that may be better suited to the discussion appears in the results, check lines 375-400 for examples. The paper would be significantly shorter if the methods were not reproduced in the results 425: I think it would be more accurate to say microbe populations from koalas instead of koala populations; same comment line 471 443-455: most of this is methods 461-463: discussion 504-507 methods 524-527: discussion 619: restate the phylum’s name of Synergistaceae here 694-5: citation for phylosymbiosis? See Bordenstein’s work

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.

Title: ANTIMICROBIAL CONSUMPTION ON AUSTRIAN DAIRY FARMS: AN OBSERVATIONAL STUDY OF UDDER DISEASE TREATMENTS BASED ON VETERINARY MEDICATION RECORDS Review round: 1 Reviewer: 1

Basic reporting: In general my opinion is that the manuscript is written in an extensive way. There is a lot information that is actually not really needed for the reader. I believe that the more interesting information that is presented in this study would/could be more convincing when it is presented as short communication. In its current version it contains too much side information that distracts the reader from the essential and good/nice results of the study. For instance, the introduction and discussion are rather long. The results focus quite extensively on the number of treating farms, while I believe the essential information is included in the nDDDvet results. Furthermore, the statistical comparison between acute and chronic treatments is nice, but in my opinion not necessary information. L12-13: 89.4% and 10.8% does not sum up to 100% Some sentences are formulated in a strange/difficult way, e.g. L27 “the highest mean number”. I suggest that the manuscript is checked by a native speaker. E.g. L262-263. I believe you mean “6334 single antimicrobial treatments of dairy cows were observed…” L43 these products are actually “antibiotic-free”, since there is a withdrawal period. Consider rephrasing L93 “population correction unit” instead of “corrected” L183 per prescribing “veterinarian” or “practice”? The results are presented per veterinary practice. L194 the multiplication sign “x” should not be in italic L230 drop “the calculation of” L268 drop “As would be” L285-286 this information is not necessary in the text, below the table is sufficient. L305-309 please rephrase, the test and its properties were already mentioned in the M&M section, so no need to repeat them here. L310-314 Please rephrase, this is very technical and not clear what you mean. The sentence is 5 lines long and the message is unclear to me. L327 there were no 6334 datasets. There were 6634 observations (= single antimicrobial treatments) in the dataset… L350 “other” do you have an example of this? L376 drop “that” Please check the references. E.g. L523 Preventive Veterinary Medicine with capitals. Experimental design: The study design is well described. However the authors should discuss the disadvantages of their design more thoroughly (see validity of the findings). Validity of the findings: The discussion should stress more the external validity of these results. Although it is already present in the discussion, I would start the discussion with the novelty of the results (as actually present) and the drawbacks of the study design (currently mentioned at the end of the discussion): - Convenience sampling - No dry cow therapy - Only 30.6% of AM treatments in dairy cattle used - L216-217. Although this will probably only be a minority of the treatments, instead of not including these treatments, you could consider to estimate the AMC of oxytetracycline sprays, in analogy with Postma et al., 2015: “…1 mL will be sprayed per second and the fact that 3 s of spraying is the average duration mentioned in the SPCs for topical spray product…” How do all these drawbacks influence the comparison with other studies? Additional comments: No additional comments

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.

Title: ANTIMICROBIAL CONSUMPTION ON AUSTRIAN DAIRY FARMS: AN OBSERVATIONAL STUDY OF UDDER DISEASE TREATMENTS BASED ON VETERINARY MEDICATION RECORDS Review round: 1 Reviewer: 2

Basic reporting: Basic reporting and the use of English are good. The authors explained the context of antimicrobial consumption in Austria well, and used relevant literature. There are a few things that have to be addressed. The are many acronyms in the article that are not explained, for example TGD (line 62 - it is explained in line 131), OIE (line 79), LKV (line 132),RDV (line 152), ESVAC (line 338), EDC, EFSA (line 423). Line 207; "...., rather than per kilogram." (I assume you are referring to live weight?) Lines 340-342. Are you referring to Germany (Merle et al), Austria (Obritzhauser et al), the EU, Europe or globally? Please state. Lines 346-348 that refer to the New Zealand study: it is not only the calculation methods that distort the comparison, but in Austria dairy and beef production systems have been combined, while the NZ findings refer to dairy farms only. This makes a big difference and should be stated in the paper, or the NZ comparison should be deleted. There seems to be an issue with one reference. Line 548. As it is, the authors have a mix of first and second joint report, and I believe the reference should be changed. In APA 6th format it is: ECDC (European Centre for Disease Prevention Control), EFSA (European Food Safety Authority), & EMA (European Medicines Agency). (2015). ECDC/EFSA/EMA first joint report on the integrated analysis of the consumption of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from humans and food-producing animals. EFSA Journal, 13(1), 4006-4120. doi: 10.2903/j.efsa.2015.4006 In HARVARD is should be: ECDC (EUROPEAN CENTRE FOR DISEASE PREVENTION CONTROL), EFSA (EUROPEAN FOOD SAFETY AUTHORITY) & EMA (EUROPEAN MEDICINES AGENCY) 2015. ECDC/EFSA/EMA first joint report on the integrated analysis of the consumption of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from humans and food-producing animals. EFSA Journal, 13, 4006-4120. Experimental design: Experimental design is sound and the method is described well. The limitations of the research have been well described. Using the standardised "Defined Daily Doses" for veterinary medicine of the EMA and use of AMC of active substances is a useful approach. I believe the paper is within the scope and aims of the journal. Validity of the findings: The findings are a good benchmark for Austria and a good comparison and contrast with other EU states. Additional comments: I have gone through what the previous referees have said and I believe you have responded well to their comments.

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.

Title: HRV-DERIVED DATA SIMILARITY AND DISTRIBUTION INDEX BASED ON ENSEMBLE NEURAL NETWORK FOR MEASURING DEPTH OF ANAESTHESIA Review round: 1 Reviewer: 1

Basic reporting: This article introduces a heart rate variability based method to measure anesthetic depth. In the introduction section, authors stated that EEG based indices are not suitable for deph of anesthesia (DoA) evaluation. They also stated that HRV would be more suitable to monitor DoA. To my own knowledge EEG based indices like BIS or GE entropy are considered as gold standards and are widelly used worldwide for DoA evaluation. I think authors should provide a more exhaustive literature references list to affirm that EEG is not suitable for DoA monitoring. In the discussion, authors cited Jeanne et al 2009. This paper is related to nociception ; not to DoA. Experimental design: The research question is well defined, relevant and meaningful. Though the research question is well defined, the material and method section need to be described with more details. Regarding the anesthetic procedure ; - How anesthesia was performed ? Did all the patients received gas (sevoflurane, desflurane,...) ? How hypnosis was maintained (propofol ? gas ?) ? How anesthetic agents were adapted ? - Were EEG indices like BIS or entropy measured during anesthesia? - Can authors list all the physiological/anesthesia parameters monitored during the procedure ? Validity of the findings: The statistical analysis is not clear and should be completly rewrite ; - How do authors justify the sample size ? - Statistical analysis used for validation (ROC curve) are not clearly explain. Additional comments: The statistical analysis needs to be explain more clearly. I do not understand why authors didn't compared their method to the commonly use EEG based indices.

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.

Title: HRV-DERIVED DATA SIMILARITY AND DISTRIBUTION INDEX BASED ON ENSEMBLE NEURAL NETWORK FOR MEASURING DEPTH OF ANAESTHESIA Review round: 1 Reviewer: 2

Basic reporting: The English language needs to be substantially improved in order for the paper to be accessible to a broad, international audience. This is perhaps the biggest issue with the paper. The literature review and structure of the paper is appropriate. There are several details regarding the methods that are, however, opaque. This includes (i) the criteria by which the experts scored the level of consciousness (i.e., the EACL); (ii) the construction of the ensemble neural network; (iii) the criteria for artifact removal in the ECG time series. Experimental design: The experiment design is sound, and it is clear that substantial effort was put into execution. In particular, the effort to deploy five different anesthesiologists to obtain expert labels is commendable. Also, the cross validation for the machine learning steps looks to be well designed. Overall, the study seems rigorous. However, there are methodological details that should be provided in the text in order to make the paper results more reproducible. This includes being more systematic about the criteria used by the anesthesiologists to score the data; as well as providing better clarity on the design of the ensemble neural network (especially for readers who do not have a background in neural networks for classification applications). Validity of the findings: The major contributions of the study are in the use of artificial neural networks to enable classification of depth of anesthesia from the so-called SDI, a measure of non-stationarity in the ECG time-series. Here, non-stationarity is assessed by obtaining an empirical distribution over sliding windows of the raw data, minus manually removed artifacts. I would encourage the authors to concisely state these contributions in the introduction of the paper, so they are clear up front to the paper. As mentioned, I believe the results are overall interesting and sound. My major concern is whether the correlation coefficient (between the SDI/ANN-derived classifier and the EACL) is the best way to measure performance. From an application standpoint, what seems most important is determining when a patient is or isn't conscious, and thus I wonder why the authors didn't also do a more direct analysis on predicting these states in a more binary fashion (i.e., rather than attempting to obtain correlate against a continuous index). As well, in understanding the power of the method it would be very important to compare performance to other DoA measures, though I accept the authors' argument that this is best reserved for future work. There are also key questions about how the performance varies with different anesthetic regimes. The authors provide the different types of drugs used, however do not disassociate the classifier performance in these terms. For example, to the poorly performing cases all correspond to one particular drug regime? Additional comments: This is a well-designed study that highlights the potential utility of ANNs to classify ECG-derived biomarkers for DoA classification. However, since the main contribution of the paper is the use of the ANN, I think a more complete study may use other covariates in order to highlight why the SDI is the best one for this application; as well as how performance varies with different drug regimes. The paper must be carefully copyedited in order for it to be accessible to a broad, international scientific audience.

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.

Title: HRV-DERIVED DATA SIMILARITY AND DISTRIBUTION INDEX BASED ON ENSEMBLE NEURAL NETWORK FOR MEASURING DEPTH OF ANAESTHESIA Review round: 2 Reviewer: 1

Basic reporting: English editing is OK. Literature references is more exhaustive. Experimental design: Method is more clear. Authors added details about the anesthesia procedure and the statistical analysis. Validity of the findings: Authors added comparison with the EEG based indexes as requested. Additional comments: The reference to Jeanne et al. has not been deleted. This reference is about HRV and analgesia ; not HRV and DoA.

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.

Title: HRV-DERIVED DATA SIMILARITY AND DISTRIBUTION INDEX BASED ON ENSEMBLE NEURAL NETWORK FOR MEASURING DEPTH OF ANAESTHESIA Review round: 2 Reviewer: 2

Basic reporting: The writing has been improved since the original submission. There are some minor typos in the Figure captions that should be addressed (e.g., Figure 13 caption is truncated). Experimental design: The experiment design is rigorous. Validity of the findings: The findings appear valid and well-constrained. I appreciate the authors' contextualization of their results. The new figure 13 is helpful in understanding the merits of the proposed approach in contrast to BIS. While the gains are modest according to certain metrics, it is clear that the proposed approach is accomplishing the intended goal of predicting DoA. Additional comments: I recommend revisiting the sentence on p4, line 82 motivating neural networks. We do not fully understand the mechanisms of ANNs and it is an overtstatement to say they operate similarly to manner to biological networks.

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.

Title: ADULT ZEBRA FINCHES REHEARSE HIGHLY VARIABLE SONG PATTERNS DURING SLEEP Review round: 1 Reviewer: 1

Basic reporting: excellent throughout Experimental design: query point re method is elaborated in general comments it is an original and innovative design and research question Validity of the findings: see below-- relates to method Additional comments: The paper ‘Silent songs: What birds rehearse during sleep’ is both innovative and remarkable (in technique) and results and opens a window to mechanisms of control of error-coding in song learning practice in zebra finches. The paper is also beautifully written, clear in outline and outcomes and belongs to one of the best papers I have read in a while. There are two important queries that I have for this paper-or really one major point. It concerns the syringeal muscles, or rather a missing explanation/ description in the method detailing which were used and why, ultimately affecting the results and conclusion. 1. To make the point, one can perhaps begin with a supplementary figure (Fig.S1), in principle the same as in the Larsen/Goller paper of 2002, but not as well marked in the muscle groups. In this paper under review the caption might confuse readers. Songbirds/oscines have at least 4 pairs of syringeal muscles—so the description saying that there are ‘at least six’ does not work well here. Why not give the exact number of pairs of syringeal muscles in the zebra finch and present this as a definite statement? In the Figure, four muscles are labelled (the largest, it says) leaving the reader wondering where the other muscles might be. Moreover, as only three of these of these 4 identified muscles per side have been used for recording, it is not clear which ones. This is confusing for the reader. It would make it easier if a reader were not to be referred to a Table in order to figure out which of the four muscles shown in the figure were, in fact, used for recordings. My suggestion 1) Provide and identify either all syringeal and tracheobronchial muscles of the zebra finch in this image if there are indeed more (as the caption suggests)—or the caption explains that the image showing only the largest has in fact x number more.. 2) Drop the ‘at least’ in the caption and stay with the number of muscles in the finch so the reader gets to know how many muscles zebra finches actually have. Since muscle numbers vary between song birds and between oscines and sub-oscines (sub-oscines have three pairs) it is important to be told exactly how many there are in the study species. 3) It would be helpful if, in the figure itself, information was provided and identified of those muscles of which recordings were made (especially since the number in the figure is not the same as in the text) Under methods, lines 95-97, it is stated in each bird, recordings were obtained from either 2 different muscles on the same side, or from left and right muscles (Fig. S1; Table S1). Again, this is vague- so did it vary between finches whether recordings were made using q, 2 or 3 muscles and when using both sides, were the same muscles used on either side used or different ones? It is best to spell out these methodological details. If I remember the important Larsen/Goller paper of 2002 correctly, the various syringeal muscles have different functions. According to an older paper by Goller and Suthers 1996, the largest syringeal muscle, m. syringealis ventralis has no gating function but in its EMG activity was positively correlated with frequency modulation and with fundamental frequency so it would make sense to me if recordings were taken from this muscle but what about the other muscles? Can the authors please provide a rationale for the muscles chosen and what the authors hypothesised specific muscles would be able to show, (what can adductors or abductors show?). It would also be very useful to know whether the recordings were taken equally from left and right side (laterality is a consideration). It has been shown in a number of songbirds that song is not just produced bilaterally but certain frequencies (often at the higher end) may be produced exclusively by the left side (several publications especially by Suthers, but also by Suthers,Wild and Kaplan).. I am concerned that, if the hypothesis of why certain muscles were chosen and what they are assumed to show is not presented and discussed convincingly, readers may not believe the results. The errors may be a result of the manner in which the recordings were sampled. Equally the authors may convincingly show why any syringeal or even tracheobronchial muscle will do for recording but, either way, any such explanation and detail, in my view, would strengthen the paper considerably. I have no doubt that this is easy to fix, at least for these authors! 2. a smaller point but important Lines 63,64—not all songbirds produce song that is highly stereotyped—but this sentence reads as if stereotyped song is universally applicable to all songbirds and, of course, that would be incorrect. The sentence simply needs and identifyer e.g., such “in zebra finches” or “in many songbirds, including zebra finches” --it’s stated clearly in lines 158-159 where it needs no addition or change. I have no further comments to make other than to express the hope that this paper can address the method query satisfactorily. In my view, it is overall a very strong and innovative paper that should be published as soon as possible. It will be of wide interest to those working in neuroethology, psychology and in avian cognition.

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.

Title: ADULT ZEBRA FINCHES REHEARSE HIGHLY VARIABLE SONG PATTERNS DURING SLEEP Review round: 1 Reviewer: 2

Basic reporting: In this paper Goller and colleagues exploit the muscle activity (EMGs) that coincides with neuronal activity of song control regions of songbirds during sleep in order to evaluate the potential meaning of this neuronal activity. The latter is thought being important for memory consolidation, and the activity of song control areas is one of the prime examples of rehearsals during sleep. The authors computed EMGs that represent the various motor units (so called syllables) in the song of adult zebra finches. Then, they compared the envelop of the EMGs with the activities that they recorded during the night in the same muscles and animals. With this procedure they showed that most muscle activity during sleep does not correlate with the pattern found in singing birds. In particular, rarely the animals produced EMGs during the night that would represent the entire sequence of motor units (called the motif). From this, the authors conclude that it is unlikely that the birds replay the song during the sleep and conclude that the nightly neuronal activity of song control regions is not involved in song memory consolidation. Experimental design: The methods are sound. Validity of the findings: The methods are sound and the results are convincing. However, the discussion is somewhat disappointing and the data representation (figures and legends are somewhat careless) needs more work. Additional comments: In general the methods are sound and the results are ok. However, the discussion is somewhat disappointing and the data representation (figures and legends are somewhat careless) needs more work. Further, the authors should replace/delete “silent songs” in/from the title. This wording is misleading since certain birds indeed produce silent songs. Abstract: I suggest to delete the last sentence. Further, the data are not supporting the introductory sentence of the abstract. Thus, the authors might want to adjust the abstract. Discussion: Lines 281-296: This entire paragraph should be re-written. The authors make first the statement that the nightly activity does not resemble the song motif EMG and as such does not help to consolidate the song motor memory. But then they state that the nightly neuronal activity might produce noise that helps to avoid the decay of the motor memory. What is the difference between consolidation and avoiding decay? Actually, this reads as if the authors have no clue about the meaning of the nightly activity. So, either they should really explain what it might mean (not just using unspecified wording) or make the statement that the function of the nightly activity is unclear. Here, the authors should critically refer to previous evidence that nightly replays are indeed replays and important for consolidation. Figure legends: Line 475: Include SLA. Line 485: The authors should state that this example is a rare observation. Line 489: explain what are the counts based on, time? Replays? Line 503: What do you mean with timing of syllable like patterns during SLA (red)? I guess inter-syllable intervals. Please rephrase the sentence. Lines 510-514: Numbering A-D seems wrong. Please use the same number of motifs/SLA in both cases and state that the same colors were used so that the overall color difference would be meaningful. Further, state how many replayed “syllables” were indeed significantly similar to the song syllable of these 5 animals. Figures: Fig. 1B: Include correlation values of the various syllables Fig. 3C: Include vSL, vSR. Fig. 3D: Break down “2 or more syllables” and “2 or more syllables +unknown” in order to support your point that entire motifs were rarely produced. Fig. 3E: Occurrences: Counts, percentage? Include 80, 40 in the upper panel. Fig.4B-C: Please adjust the number of included data sets (See above). Fig. 4C: include vSL, VSR. Fig. 4D: Include syllables 1-5 and animals 1-5 into the figure. Minor points: Line 164: Move explanation of SLA to line 148. Lines 174/175: Include statistic for this statement. Line 183: Move this sentence to the discussion or delete. Line 191: What do you mean with “visually categorized 457 SLA events into one to seven categories of activity?” Aren’t you using some statistical method for this? Line 204: Is “3 ms longer” a statistically relevant difference. If so, include STAT or delete. Lines 252-256: This statement seems out of place at this position? Line 258: do you mean: during singing?, please specify. Line 266-268: Please include the evidence for night-time replay in other systems?

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.

Title: 5-FLUOROURACIL AND IRINOTECAN (SN-38) HAVE LIMITED IMPACT ON COLON MICROBIAL FUNCTIONALITY AND COMPOSITION IN VITRO Review round: 1 Reviewer: 1

Basic reporting: Although the use of language is generally quite good, I would recommend asking a native English speaker to review the manuscript as some sentences are a little difficult to fully comprehend. Examples of this can be found on lines 114, and 150-152. Also, if the donors described on line 155 were the same used for the 5-FU experiment, it would be clearer if the text read ‘five of the six healthy human donors.’ and delete the remainder of the sentence. Care should be taken with the use of abbreviations: abbreviated names are shown on lines 28-29 without previously being written, the abbreviation for irinotecan is written on line 35 but not always used later (e.g. lines 39, 41, 42), on line 58 it should read 'nuclear factor-kappa B (NF-κB)'. Lastly, could the authors clarify whether changes described on lines 73-74 are proportional or actual numbers, consider changing line 363 to ‘after the simulated chemotherapy treatment’, and I couldn't find any reference to Figure S7 in the text. I would like to commend the authors on Figure 1, which gives a very clear view on the experimental set-up. Experimental design: In order to aid experimental replication, could the authors define 'mucus solution' (line 92), add suppliers/catalogue numbers to media components (lines 121-122), detail how the SCFAs were validated e.g. use of standards, or reference library (line 161), quantify the 'maximum speed' (lines 176, 180), and briefly describe or reference the phenol-chloroform method (line 177). The research presented here is the logical next step to build on the previous research in the literature, and I strongly agree with the authors potential follow-up work described on lines 380-386. Validity of the findings: No comment Additional comments: No additional comments

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.

Title: 5-FLUOROURACIL AND IRINOTECAN (SN-38) HAVE LIMITED IMPACT ON COLON MICROBIAL FUNCTIONALITY AND COMPOSITION IN VITRO Review round: 1 Reviewer: 2

Basic reporting: The manuscript is written in an easy-to-follow English, with only a few typos and unclear statements (see section 4, general comments for specific things to change/consider). Relevant literature cited. Tables and figures generally of acceptable quality, though some improvement is needed (see section 3 for specific comments). Experimental design: The manuscript reports primary research within the scope of the journal. Materials and methods are generally sufficiently described, though with some shortcomings (see below). Specific comments to Materials and Methods section: Line 92. The mucus solution is not defined (composition?) Line 118 and elsewhere: Samples seem to be stored at -20C before further analysis. Storage beyond a few days at -20C is known to influence GM profiles as determined by high throughput amplicon sequencing. How long was the samples stored at -20C? Were some samples stored longer than others at -20C and do the authors have any data to support, that storage at this temperature did not influence the results? Line 125-128: This information does not belong in Materials and Methods section (but rather in the results section) Line 164-165: Lactic acid not detected? Sometimes present in rather high amounts. Please comment. Line 176: FastPrep running at 1600 rpm? Does not make sense. Perhaps "oscilations pr. minute" is what is meant? Line 176 and elsewhere: What is maximum speed? Line 177: Provide more details or reference for phenol-chloroform extraction. Line 189-206: Generally adequate details are given, but it is not clear how e.g. how the amount of 20 ng "amplicon" was determined (line 197). Line 257-258 and 300-304 not described in Math&Meth. Leave information out? Validity of the findings: Data generally robust, though there is some room for improvement (see below). One big concern is that it does not seem to be investigated how stable 5-FU and SN-38 is in the fermentors. One could speculate, that the main finding of the study (no direct effect of 5-FU and SN-38 on GM) could simply be a consequence of 5-FU and SN-38 being rapidly broken down. Please provide evidence (data or references) that this is not the case. Other, smaller comments: In the text examples of specific taxa differing significantly with treatment within the same donor are stated, but p-values are not stated in e.g. fig. 3 and 6. Further, was FDR or Bonferroni-correction carried out to avoid false positives? Figure legends are not always as informative as they could be. P-values (and where relevant R-values) are rarely stated. It is not stated how GM-characterisation was carried out in the relevant figure legends - include information such as "as determined by 16S rRNA gene amplicon sequencing" etc. Figure 4 is very messy and difficult to follow. Try to make it easier to understand. Perhaps by further dividing the figures into separate figures for mucus and lumen? Also state p and R-values. Table 1: How was the confounding factors determined? By Permanova? Consider doing e.g. a canonical correspondance analysis (rCCA) also/instead. Suppl. Fig. 4 indicate a strong run-effect. Was this taken into account (e..g by randomising samples to different runs) when doing the analysis (DGGE)? Was PCR1 for amplicon-sequencing done in one run or is there also a potential run-effect/systematic effect influencing data interpretation here? Additional comments: Interesting study addressing a very relevant research question. Specific comments, not already covered above: Line 19: Add "dysbiosis" after "gut microbiota" Line 31: Consider "softening" the conclusion to "our findings strongly indicate that..." Line 72: What is meant by lower abundance? Absolute abundance? Relative abundance of certain taxa? Line 251-255: Are these findings significant? Could there be an run-effect as discussed above? All over the manuscript: "Illumina sequencing" is a very vague and unspecific term. Be more specific and call it e.g. "high-throughput 16S rRNA gene amplicon sequencing" Several places: Phyla-names are not in italics

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.

Title: 5-FLUOROURACIL AND IRINOTECAN (SN-38) HAVE LIMITED IMPACT ON COLON MICROBIAL FUNCTIONALITY AND COMPOSITION IN VITRO Review round: 2 Reviewer: 1

Basic reporting: I am happy with the changes made to the manuscript, particularly regarding the improvement of the language, and enhanced clarity regarding methods. Experimental design: no comment Validity of the findings: no comment Additional comments: No additional comments

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.

Title: 5-FLUOROURACIL AND IRINOTECAN (SN-38) HAVE LIMITED IMPACT ON COLON MICROBIAL FUNCTIONALITY AND COMPOSITION IN VITRO Review round: 2 Reviewer: 2

Basic reporting: All OK in the revised version of the manuscript. One small remark: In line 76 "microbiota" should be exchanged with e.g. "taxa". Experimental design: All OK in the revised version of the manuscript. Validity of the findings: All OK in the revised version of the manuscript. Additional comments: The revised version is much improved and acceptable for publication in PeerJ

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.

Title: LOWLAND EXTIRPATION OF ANURAN POPULATIONS ON A TROPICAL MOUNTAIN Review round: 1 Reviewer: 1

Basic reporting: I found this manuscript to be well-written, with the discussion flowing particularly well. The introduction, however, is a bit too brief and needs another paragraph that focuses on anurans in the tropics and what we know about their spatial distributions and how those have changed from historic records (and highlight potential reasons why). As is currently written, the transition from second to third paragraph is too abrupt. Experimental design: The methods could use a bit more detail, as noted in my line-by-line comments. In regards to the analysis, it is unclear why the authors modeled detection probability as a function of the linear and quadratic effects of elevation. Unless sufficient justification can be provided, I think this needs to be eliminated and the analysis re-ran for each species. Displaying AIC tables with AIC values, model weights, and log likelihoods would also be helpful to the reader. Validity of the findings: The scope of the authors current sampling is far from all encompassing. Making specific conclusions on species extirpation and range shifts from spatially and temporally limited data is slightly concerning, and I would suggest that the authors identify this issue. Additional comments: Introduction: The shift between the second and third paragraphs is very abrupt. There is no lead-in about anuran issues in the tropics. Another paragraph should be added before the last paragraph that focuses on what we know about how anuran distributions in the tropics and how they have been changing and, briefly, some hypothesized reasons for this change. Methods: L189-192: How much of an attempt was there to match up current sites with historic sites? How were current sites selected? Looking at Figure 1, there are 4 or so northern historical sites that don’t have current counterparts, which is slightly concerning. In addition, why were these months selected to do surveys? L246: Did you look at model fit and examine assumptions for these models? This needs to be addressed in the methods and evidence of model fit should be provided. L256: Should be “closed” rather than “close”. L258: What is this average and SD referring to? Please make clearer. L264: There needs to be some reasoning as to why the authors thought detection probability would vary with elevation. Is there any reason why other factors on detection probability were not considered (e.g. date, precipitation, temperature, etc.)? L267-268: Why would there be a quadratic effect of elevation on detection probability? I cannot think of a reasonable explanation for this and the authors have given no justification. L278-279: Same comment as above regarding model fit and assumptions. Results: L312: Table 1 does not show any AIC values. It is unusual not to include this somewhere in the manuscript if you are using model selection. Including AIC values and model weights would allow the reader to see what the top models were and how much better they fit than the null model. Also allows the reader to see if there are any issues with pretending variables. L325: What about E. coqui occupancy probability? Did it increase from historic levels? Discussion: L343-344: I do not feel entirely confident that the range of sampling was thorough enough to state that certain species suffered extirpation or experienced range declines. At lower elevations, only about 6 sites were surveyed, and the range of the acoustic recorders was only 50 m. Have you really covered enough ground to make these definitive conclusions? L373-383: This is good supporting information and does make me feel more confident in your results. Figure 1: I would suggest changing the colors on this figure. I do not see the dark gray and am having trouble distinguishing between the two orange colors on the legend. Figure 4: Based on how you did your sampling, I am not fully convinced that species are extirpated from certain elevations. I also think this figure is somewhat redundant given what you’ve already presented. Table 1: The cumulative weights for many of these species are not very high. I would be curious to see model sets for each species with AIC values, weights and log likelihoods included. This could be included as a supplementary table but I do think it is important for the reader to get a better picture of what is going on. Typically if you are using AIC, you do include an AIC table with the above-mentioned items (and it doesn’t necessarily have to be a full model set, perhaps only the top models).

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.

Title: LOWLAND EXTIRPATION OF ANURAN POPULATIONS ON A TROPICAL MOUNTAIN Review round: 1 Reviewer: 2

Basic reporting: ok Experimental design: Needs more detail about the comparison between historical and current data to determine range shifts. Validity of the findings: Historical data should encompass the same timeframe as the current data. Additional comments: This manuscript examines changes in anuran distributions in the mountains of Puerto Rico by comparing historical data from museum specimens and previous studies with current data gathered with audio recorders. I have a few concerns about the paper: 1) Current data is temporally biased to the months of March-May, while historical data includes the full year. The authors could resolve this problem by limiting their historical dataset to include the same months as the current data. 2) The method to estimate range shifts seems novel, how does it differ from other published methods? Minor edits Line 41: Remove comma after ‘diseases’. Add comma after ‘species’. Line 46: Replace ‘contrasting’ with ‘comparing’. Line 49: Please provide more info about historical data, what are the data? Occurrence points? Transect counts? Line 57: Instead of saying three lines of evidence, authors can say that Land use change is not responsible for these changes because LM has been a protected reserve for the past 80 yrs. However, previous studies indicate that 1) climate change has increased temperature/dry periods, and 2) Bd is present in X # of species. Line 98: fix typo ‘chytridiomycosis’ Line 99: fix typo ‘characteristic’ Line 105: remove comma after ‘regions’ Line 110: start new sentence with “The flora”. Before going into the goals of the paper, I feel like the authors should add a paragraph about the general patterns of amphibian declines in the Caribbean and Puerto Rico. Line 153: Please provide list of species. Line 157: replace ‘vocal’ with ‘active’ Line 159: add ‘of’ after ‘one’. Should read ‘one of the most studied…’ Line 161: all are endemic to Puerto Rico/ Puerto Rican bank. Line 166: I would suggest to limit the historical data to those records collected during the same time as the current data (March-May). Line 256: add ‘d’ to word ‘close’ Line 278-285: This seems like a novel way to estimate range shifts. How does it compare to other methods presented in literature? Line 324: What do you mean with ‘the level’? please clarify. Line 342-353: I think this summary paragraph is missing previous studies documenting the decline of some of these species (e.g. Burrowes et al. 2004). In addition, it seems that the authors are solely attributing range shifts to climate change-temperature, but then as the discussion progresses Bd and dry periods are also invoked as potential causes. Line 355: Historical data is also problematic because includes all records throughout the year, vs current data only focuses March-May.

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.

Title: LOWLAND EXTIRPATION OF ANURAN POPULATIONS ON A TROPICAL MOUNTAIN Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: L70: Should anura be a key word as well? L124: Should be an “and” between Central Africa and Australia. L184: “Comprehensive” rather than “comprehensible” L 191: remove “ in addition we” L192: What does GBIF stand for? L202: “transect” rather than “transects” L316-219: Is site the same thing as a unique sampling locality? So E. coqui was found in 27/51 sites? It would be easier for the reader if you put the percentage of sites rather than raw number (i.e. 53% of sites). L340: Should be “an increase in elevation” rather than “the increase of elevation” L343: I would recommend consistently using either occurrence or occupancy rather than both terms. L360-363: There could be a little more discussion on detection probability given the emphasis on modelling detection as a function of elevation, quad effect of elevation. I wonder—did elevation indeed influence detection probability for most species? L459-460: I think you could expand slightly on how climate change acts directly on the pathogen to trigger outbreaks. What is it about climate change that causes chytrid to be more of a problem? L445-463: You could combine these two paragraphs by moving the sentence of “While Bd may pose a threat..” to the last sentence of the entire paragraph. L464-475: This paragraph can be eliminated. It doesn’t add much to the discussion and it seems tangential to the main issues at hand. You also never brought up the hurricane threat in the introduction, so there doesn’t seem to be much stock in this as an actual contributory threat. The two papers cited here by the same author stating that amphibians “may have been extirpated” by hurricange hugo is not very convincing. Table 1: The cumulative weights for the current models do not seem to always match what is reported in Table S7. Particularly for E. brittoni and L. albilabris. This is concerning and all should be checked. Table S6: It should be noted in caption that this is for historic dataset. Also, the formatting of this table should be consistent with S7 (i.e. this table doesn’t denote occupancy, it has superscript, it includes loglikelihood whereas Table 7 does the opposite.) Table S7: Include in the caption what Psi and P stand for. The authors should include deviance values in AIC table too, as they help determine how much your model is explaining. #Par is usually referred to as K. It should also be noted in the cpation that this is for the current dataset.

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.

Title: VIRUS LIKE PARTICLES AS A PLATFORM FOR CANCER VACCINE DEVELOPMENT Review round: 1 Reviewer: 1

Basic reporting: The summary highlights the importance and qualities of VLP's, however, I suggest improving the wording on lines 23-28 and creating a single paragraph, to avoid repetition of "VLP's" in each sentence. In line 88, I suggest that more information about the VLPs should be included in the text, for example: classification, synthesis method, architecture (wrapped or not wrapped) or chimeras. This will give a greater impact to the review. The revision would benefit from the inclusion of a figure where the production of VLPs is schematized, for example as shown by Antonina Naskalaska et al. (Polish Journal of Microbiology 2015, Vol. 64, No 1, 3-13), Dan Yan et al. (Appl Microbiol Biotechnol (2015) 99: 10415-10432) by mention some authors. Experimental design: Does not apply Validity of the findings: Does not apply Additional comments: - Has the field been reviewed recently? If so, is there a good reason for this review (different point of view, accessible to a different audience, etc.)? I consider the review to be of great interest since it exposes the use of VLPs as a prophylactic platform for the prevention and treatment of various types of cancer of importance in the world. The last review that I could find about VLP's as a vaccination strategy is 2014, so I consider that this work brings an update in this field so nove. - Does the Introduction adequately introduce the subject and make it clear who the audience is/what the motivation is? The summary highlights the importance and qualities of VLP's, however, I suggest improving the wording on lines 23-28 and creating a single paragraph, to avoid repetition of "VLP's" in each sentence. I made this recommendation because I consider that from the first moment that the VLPs are mentioned it is clear that the information that will be read in the text will be referring to those particles so it is not necessary to repeat VLP's at the beginning of the sentences in lines 23- 28. In line 88, I suggest that more information about the VLPs should be included in the text, for example: classification, synthesis method, architecture (wrapped or not wrapped) or chimeras. This will give a greater impact to the review. In line 88, where the author begins to describe the VLP's, I suggested that the author provide more information on the origin of the VLPs because in the introduction little is said about these particles and their classification, although the revision is not focused on obtaining VLPs, I consider that to provide general information about the various types of VLP's will provide more information to readers The revision would benefit from the inclusion of a figure where the production of VLPs is schematized, for example as shown by Antonina Naskalaska et al. (Polish Journal of Microbiology 2015, Vol. 64, No 1, 3-13), Dan Yan et al. (Appl Microbiol Biotechnol (2015) 99: 10415-10432) by mention some authors. I cited these authors as an example because in their publications they showed schemes that explain very clearly how the VLPs act. I believe that these works can provide the author with a different and clearer perspective on how to outline the role of VLPs as a prophylactic or vaccine platform against cancer. But if the editor thinks the scheme is appropriate, please ignore my recommendation. - Is the Survey Methodology consistent with a comprehensive, unbiased coverage of the subject. If not, what is missing? The research platforms used to obtain the information allow to review several areas of research carried out with these VLPs, which is why I consider it to be an adequate methodology for this manuscript. - Are sources adequately cited? Quoted or paraphrased as appropriate. All references are properly quoted. - Is the review organized logically into coherent paragraphs/subsections? Yes

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.

Title: VIRUS LIKE PARTICLES AS A PLATFORM FOR CANCER VACCINE DEVELOPMENT Review round: 1 Reviewer: 2

Basic reporting: The authors performed an excelente review of several paper about the potential use of VLPs as cancer therapeutic molecules. The review is well redacted and profound. Minor changes. Authors mention that VLPs are promising molecules to fight cancer, however, they must include a paragraph with the potential drawback of this approach. For example, several reports have showed that preexisting human immunity against several VLPs reduced the potential use of this particles. Authors should declare why only few of them has been successfully enter in clinical trials and less are have been licensed. Experimental design: no comments Validity of the findings: no comments Additional comments: no comments

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.

Title: VIRUS LIKE PARTICLES AS A PLATFORM FOR CANCER VACCINE DEVELOPMENT Review round: 1 Reviewer: 3

Basic reporting: The review is well written and follows the guidelines of the journal. However, the manuscript has some deficiencies since, according to its title “VLPs as platforms...”, it should address the issue from two points of view: VLPs against cancer-causing viral infections (preventive) and VLPs developed against cancer (therapeutics). Nevertheless, most of the content is only focused on VLPs as preventive vaccines against cancer-associated viral infections such as those related to HBV, HCV, HPV and HHV-4 (EBV). It is also important to consider that these oncogenic viruses are associated to malignant processes, thus VLPs vaccines are developed to prevent viral infection that in turn has an impact on the incidence of cancer. VLPs as therapeutic vaccines are only addressed in the section 2.3, about pancreatic, prostate, breast, skin and lung cancer. In the case of HCC, the information in that section is mixed. On the other hand, authors do not discuss all the strategies that are being used for development of VLPs as a carrier of antigens, but only show in Figure 1, one of two main strategies for generation of VLPs (genetic fusion). It would be important to contrast at least with a second strategy; authors might consider Eriksson et al., 2011 to extend information. Another important point that the authors do not address is the viral origin of the VLPs, since origin is important from the point of view of the immune response that could be presented Therefore, authors should consider dividing the review into two topics: preventive VLPs and therapeutic VLPs or should focus the review only on therapeutic VLPs. Experimental design: No comment Validity of the findings: No comment Additional comments: Minor revision • In line 119 the word ("Table 2") would be better if it said ("see Table 2") • In line 207, the drug is Gardasil not Gadrasil. • The paragraph on lines 431-433 is confusing or repeated Major revision • In lines 105-107, compared to that, do VLPs represent “a much lower risk of autoimmunity”? Which vaccines have autoimmunity problems or do they exhibit auto-antigens? • In line 150 quotes the work of Pleckaityte et al. 2015, and it is mentioned that the VLP developed by this group has therapeutic potential, but in the work show in vitro results of viral neutralization. Can the vaccine cure hepatocellular cancer or only prevent HBV infection? Or control and eliminate chronic HBV infection in people but without hepatocellular cancer? • The paragraph of line 197 to 200, is a conclusion of the work of Ding et al. 2009? Or it is a contribution of the work of Su et al. 1998, if it is the latter, then that work should be better described. • The paragraph of lines 250-251, refers to the VLPs developed by Martin Caballero et al. 2012 and Jemon et al. 2013, these proved to be therapeutic, then, according to the paragraph would be prophylactic or therapeutic? • All section 3.7.2 EBV should go after section 3.2 HPV. • The finding of mRNA in the EBV VLP in the work of Russ et al. 2011 could be more discussed (Line 436). • Section 4.0 (Summary), needs more discussion about the different ways to obtain preventive and therapeutic chimeric VLPs. The works of Martin Caballero et al. 2012 and Jemon et al. 2013, can be used to extend the discussion. On the other hand, the conclusion is very general, because some VLPs have been promising, but many others have not. In the figure it is not clear if what follows after the mouse are more viruses or are dendritic cells?

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.

Title: VIRUS LIKE PARTICLES AS A PLATFORM FOR CANCER VACCINE DEVELOPMENT Review round: 1 Reviewer: 4

Basic reporting: This is a very interesting, original, and well-written review by Ong et al., regarding virus like particles as platforms for cancer vaccine development. The authors presented almost all existing literature in an innovative and entertaining form. However, I would like to see some more recent literature which describes extracellular vesicles and their close resemblance with VLPs. I would like to see authors critically reflect on how VLPs are different than exosomes/microvesicles and that role can these extracellular vesicles play in vaccine development platform ? Please see articles such as 1) Extracellular vesicles and viruses: Are they close relatives? Nolte-'t Hoen E, Cremer T, Gallo RC, Margolis LB. Proc Natl Acad Sci U S A. 2016 Aug 16;113(33):9155-61. doi: 10.1073/pnas.1605146113. Epub 2016 Jul 18. 2) Microvesicles and Vesicles of Multivesicular Bodies Versus “Virus-Like” Particles. Albert J. Dalton. JNCI: Journal of the National Cancer Institute, Volume 54, Issue 5, 1 May 1975, Pages 1137–1148, https://doi.org/10.1093/jnci/54.5.1137 3) Extracellular Vesicles and Their Convergence with Viral Pathways. Thomas Wurdinger,1,2 NaTosha N. Gatson,3 Leonora Balaj,1 Balveen Kaur,3 Xandra O. Breakefield,1 and D. Michiel Pegtel4. Advances in Virology. Volume 2012 (2012), Article ID 767694, http://dx.doi.org/10.1155/2012/767694 Experimental design: Original content within the scope of the journal Validity of the findings: Conclusions are well-stated, and data has been presented in a very entertaining manner, Additional comments: No additional comments

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.

Title: VIRUS LIKE PARTICLES AS A PLATFORM FOR CANCER VACCINE DEVELOPMENT Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: The modifications made along with the information that was added and the figures positively increases the academic contribution of this article

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.

Title: VIRUS LIKE PARTICLES AS A PLATFORM FOR CANCER VACCINE DEVELOPMENT Review round: 2 Reviewer: 2

Basic reporting: Without comments Experimental design: Without comments Validity of the findings: Without comments Additional comments: Without comments

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.

Title: VIRUS LIKE PARTICLES AS A PLATFORM FOR CANCER VACCINE DEVELOPMENT Review round: 2 Reviewer: 3

Basic reporting: In Figure 2, I think it would be best if the self-assembly was first and then the release and budding to continue with the VLPs Experimental design: no comment Validity of the findings: no comment Additional comments: No additional comments

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.

Title: GENOME-WIDE IN SILICO IDENTIFICATION OF MEMBRANE-BOUND TRANSCRIPTION FACTORS IN PLANT SPECIES Review round: 1 Reviewer: 1

Basic reporting: 1. I suggest having an English language editing service or an English-speaking colleague proofread your paper to ensure proper language usage and to improve logical flow. 2. There is sufficient background about the importance of MTFs in rapid response to stress. However, to aid the reader in interpreting the results and understanding their significance, more background is needed on the proteolytic processing of MTFs (and how this is related to the location and number of transmembrane domains), the alternative splicing of MTFs and the methods used to identify MTFs. 3. The Materials and Methods section is too brief. Most of the methods (including those presented in the Materials and Methods) are described in the Results and Discussion section, and these are not explained in sufficient detail. 4. The Titles/labels do not always accurately reflect what is shown in the figure or table, and in some cases more information is needed to interpret the figure. -The x-axis in Fig 1d should be labeled “Arabidopsis gene families that contain MTFs”. -In Fig 2b, I suggest providing the percentage of MTFs that have each topology. -A more accurate title for Figure 3 would be “Alternative splicing of membrane-bound transcription factors”. -In Fig. 3b, it is more accurate to say that alternatively spliced forms of AT5G10510 lack a TM domain. In addition, the colored lines/blocks should be explained. -In Fig. 3c, The description “ALS-dependent activation” is not appropriate because you have not shown that these TFs are activated by alternative splicing. -Table 1: TOPCONS-single is not included. -Table 2 title: It is not correct to refer to these transcription factors as the “MTF family” because they belong to several different TF families. -Table 3. The title does not reflect what is in the table, which is the number of MTFs in each TF family. 5. The TM predictions from all seven programs are not provided, and it is not clear what predictions are shown in Supplemental Table S2. Are these the combined TM predictions? Experimental design: 1. The need to identify MTFs in other species is stated (Lines 68-71), but I suggest more clearly articulating the gap in knowledge and your specific goals. For example, did you develop a new approach to identify MTFs rather than using an existing approach because there was a need for a more robust MTF identification pipeline? 2. There needs to be more discussion about the discrepancies between your results and those of Kim et al. (2010) (lines 133-134). What were the differences in your approaches that may have led to this discrepancy? You cite the fact that you identified all previously characterized MTFs as evidence that you have obtained high-confidence MTF predictions. Was this not the case for Kim et al. (2010)? If it is difficult to conclude which method is more reliable (line 134), would it be better to use both approaches when trying to identify all possible MTFs? 3. The methods need to be more fully explained. -More detail about the TOPCONS-single algorithm (lines 117-118) and how it was implemented is needed (there is no mention of this program in the Materials and Methods). Based on Hennerdal and Elofsson (2011), only four prediction programs seem to be used by the best performing version of TOPCONS-single (http://single.topcons.net/). If this is the case, the workflow (Fig. 1) is incorrect. -How the other prediction programs (lines 86-89) were implemented also needs to be described, including the criteria for calling a transmembrane domain. -The reason for making TMHMM 2.0 predictions separately should be clearly stated (lines 119-120). -Table 2 should be cited when mentioning the plant species included in the analysis (line 82). 4. The rationale for using the seven prediction programs (lines 97-101) is not fully explained. Why did you choose to use a different approach than Kim et al. (2010)? Validity of the findings: 1. The conclusion that the location of transmembrane motifs in the N- or C- terminal regions of most MTFs supports the conservation of a proteolytic mechanism for MTF activation (lines 26-28, 74-76, 192) needs to be clarified. I interpret conserved in this context to indicate evolutionary conservation, but given that the MTFs belong to different gene families, this does not seem to be correct. It is more accurate to say that the location of TMs indicates that MTFs are processed by the same proteolytic mechanism. However, why the location of the TM domains indicates this needs to be explained. 2. The conclusion that half of the MTFs in Arabidopsis and cotton could be regulated by alternative splicing (Lines 29-31, 77-79, 207) also needs clarification. It is not clear whether your conclusion that MTFs are regulated by alternative splicing is based on the presence of an alternatively spliced transcript or on the fact that these transcripts have different protein domains. In Table S3 you list the alternatively spliced transcripts and note whether they contain a TM domain, but you don’t discuss how often alternative splicing has the potential to regulate the MTF. When you say that ALS was involved in the activation of MTF families (Lines 208-209), is this based on the fact that alternative splicing removes/adds a TM domain? 3. The conclusion that MTF activation involves at least two independent mechanisms (lines 218-221) was reached by other studies. The activation of MTFs was not directly assessed in this study, and this section should be revised to specifically summarize the conclusions of your study. Additional comments: 1. In the abstract lines (31-33) and in the conclusion (lines 215-216) you say that you have done the first systematic analysis of MTFs in plant genomes. This wording is not correct because systematic analyses have been done in Arabidopsis and rice (Kim et al 2010), which you appropriately cite in the Introduction (Lines 65-68). 2. The explanation for the discrepancies in TM prediction between algorithms is vague (lines 110-112). It would be helpful if the advantages and disadvantages of each method were explained. 3. I suggest not listing the plant names (Lines 146-150) because they are already listed in Table 2. Similarly, all of the gene families with MTFs (lines 162-169) don’t need to be listed in the text because they are already shown in Table 3.

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.

Title: GENOME-WIDE IN SILICO IDENTIFICATION OF MEMBRANE-BOUND TRANSCRIPTION FACTORS IN PLANT SPECIES Review round: 1 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: This manuscript describes the identification of membrane-bound transcription factors (MTFs) using in silico computational analysis. 1089 MTFs belonging to 52 gene family were identified from 14 plant species, most of which contain one or two transmembrane region. The authors also found that approximately half of the MTFs can be regulated by alternative splicing. The manuscript provides an important resource for readers who might be interested in MTFs in plants. However, there are several major issues that need to be addressed. 1. A previous study (Kim et al 2010) has identified MTFs from Arabidopsis and rice, and the results were quite different from the current study. 50% of the previous identified MTFs were not recovered by the current study (Fig 1 e). What could be the reason for the difference, and which method is more reliable? How many of the MTFs with known functions were identified by the current study but not by the previous study? Further analysis and discussion on this topic should be provided. 2. It is recommended to include experimental verification for some identified MTFs, for example, the 21 MTFs identified only in the current study. Arabidopsis TF resources are widely available and the experiments should be easy to carry out. 3. Do the authors have an estimation of the false discovery rate among the 1089 identified MTFs? 4. A phylogenetic analysis should be conducted to see how many of the MTFs are conserved between different plant species? Do the NAC MTFs from different species belong to the same sub-group among all NACs? How about bZIP, SBP, bHLH, and other MTFs? 5. The analysis of MTFs regulated by alternative splicing (Fig 3) depends on the accuracy of gene model annotation. While it could be assumed that the Arabidopsis gene models are quite reliable, the author should discuss how reliable are those for cotton and other plant species. 6. Table 2, Picea abies is not a Bryophyta. 7. The English language of the manuscript should be improved, such as those in lines 51 to 53.

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.

Title: GENOME-WIDE IN SILICO IDENTIFICATION OF MEMBRANE-BOUND TRANSCRIPTION FACTORS IN PLANT SPECIES Review round: 2 Reviewer: 1

Basic reporting: 1. The English has been improved, but there are still grammatical errors and awkward phrasing. For example: Pg 2 Lines 49-51. By “attached” I think you mean “located adjacent to” Pg 2 Line 59 “been” should be deleted. Pg 3. Line 75 “that” should be changed to “those” Pg. 3 Line 80 “database” should be deleted. Pg 4 Line 90 “conserve” should be “conserved” Pg. 6 Line 153 “researches” should be “studies” Pg. 6 Line 160 should be “workflow for MTF identification established in this study can identify MTFs” Pg. 7 Line 176. “families of MTFs” here should be “families containing MTFs” because not all members of these families are MTFs. Similarly, in lines 186-187, it is better to say “the six families with the highest number of MTFs” On pg. 9 line 233, “nuclear transcription factors” is not correct. Do you mean “transcription factor domain” or “nuclear localization signal” or both? 2. In the Materials and Methods the in silico analysis of alternative splicing is still not included. On pg. 4 Line 93- the table containing the list of species should be cited. On pg. 4 Lines 106-107- what is the “the strict standard”? 3. For Supplemental Table S2 a legend that describes the contents of this table is needed. Experimental design: 1. The parameters/criteria used for membrane protein prediction are not described. If the default parameters were used, this should be stated. 2. My original confusion about how the analysis was done stems from the fact that 4 out of the 7 prediction programs in Table 1 are used by TOPCONS-single. From Hennerdal et al. 2011: “The best-performing version of TOPCONS-single, using four individual methods (Table 1), is available as an easy-to-use web-based prediction server at http://single.topcons.net/. It uses the globular protein filter of SCAMPI to weed out non-membrane proteins and then proceeds to run the rest of the predictors–HMMTOP, MEMSAT-1.0 and S-TMHMM—on the remaining set.” Thus the use of TOPCONS-single seems redundant. Why were the predictions from the 6 programs not simply combined or TOPCONS-single used directly? Note that there is a more recent reference for TOPCONS-single (Tsirigos et al. Nucleic Acids Research, 2015 6:21383). 3. On page 5 Lines 138-141, you claim that TMHMM 2.0 is the most accurate software available. However, the reference you cite (Flugge and Kunze, 2003) does not make this claim. Is the wrong reference cited? 4. I still think the rationale for using these programs needs more explanation. The points you make in your response (that you wanted a method that uses resources that are freely available to the scientific community and are applicable to more plant species) are convincing and should be mentioned in the paper. Also, you don’t explain why you used multiple algorithms, which I think was to identify as many membrane proteins as possible. Finally, you argue that these prediction programs have been frequently used and have led to good results. But the references you cite (Che et al. 2010 and Li et al. 2011) do not mention these prediction programs. References directly supporting this statement should be cited. Validity of the findings: 1. The discrepancies between your results and those of Kim et al. are still not discussed in detail. You don’t explain how the approach by Kim et al differed from yours and whether they also identified all previously known MTFs. You also don’t directly address why you found less than half of the MTFs found by Kim et al. in Arabidopsis. Do you think there are more false positive predictions in their dataset? That your datasets are complementary? What implications does this have for the discovery of MTFs in other species? Your statement that the databases from other species are “highly reliable” (pg. 6 line 169) is not justified because you have not demonstrated that they are reliable. This does not mean that your analysis is not valid, but you should be clear about the limitations. 2. Pg 8 lines 210 and 212- The explanation for why the location of transmembrane domains indicates conservation of the proteolytic mechanism needs further clarification. For example, I think your point is that TM domains need to be located at the N- or C-terminal ends of the protein to allow a functional TF to be released by proteolytic cleavage, which occurs next to the TM domain. Are there references to back up this observation (eg. all characterized MTFs have this type of protein structure)? Does this mean that the putative MTFs with TMs located in other regions of the protein are not likely to be functional? Additional comments: No additional comments

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.

Title: GENOME-WIDE IN SILICO IDENTIFICATION OF MEMBRANE-BOUND TRANSCRIPTION FACTORS IN PLANT SPECIES Review round: 2 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: The authors have addressed previous concerns to certain degree, though some of the points might not be resolved due to technical difficulty, such as the issue of FDR rate. As it currently stands, the manuscript is acceptable now for publication.

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.

Title: FITNESS IMPLICATIONS OF SEX-SPECIFIC CATCH-UP GROWTH IN NEPHILA SENEGALENSIS, A SPIDER WITH EXTREME REVERSED SSD Review round: 1 Reviewer: 1

Basic reporting: This is a well-written paper and the results are interesting. Experimental design: This is basically a well-designed laboratory experiment. Some methods need to be described with more detail (see comments for the author).. Validity of the findings: I am concerned whether the results obtained under the artificial laboratory conditions can be used to infer selection of life-history traits in nature (see comments for the author). Additional comments: This is a well-written paper on a basically well-designed laboratory experiment and the results are interesting. However, I am concerned whether the results obtained under the artificial laboratory conditions can be used to infer selection of life-history traits in nature. The cost of delayed maturation on fitness, in which female spiders that mature slower in the field are more likely to be eaten by predators before they reproduce, cannot be assessed in a laboratory experiment. Although this cost is briefly mentioned in the Discussion, I am not convinced that it is unimportant in nature. Although large spiders may be too big to be eaten by invertebrate predators, they may be more attractive to larger vertebrate predators. Other factors like physical disturbance by storms may also add to the cost of delayed maturation. A more comprehensive view is that delayed reproduction at large size may be advantageous in a low-predation, low-disturbance environment but non-adaptive in a high-predation, high-disturbance environment. The nutritional value of the flies fed to the spiders needs to be addressed. Drosophila may not contain enough nutrients for spiders to grow optimally and reach maturity. Although the methods are not clear, it seems that spiders in the low-food conditions were fed only or mostly Drosophila, whereas those in the high-food conditions may have received more of a mixture of Drosophila and Calliophora. Could the increased nutritional value of a mixed diet influenced the results? In particular, if males in the Low-High treatment were fed only or mostly Drosophila (because they were too small to capture Calliophora), this might have impeded their catch-up growth potential, whereas females in the Low-High treatment probably received a mixed diet during the second half of the experiment. The first section of the methods states that the spiders were housed in small plastic cups. This may have been alright for small juveniles, but the cups would have been too small for larger individuals. Hopefully, at some point the larger spiders were transferred to larger containers, but this is not stated.

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.

Title: FITNESS IMPLICATIONS OF SEX-SPECIFIC CATCH-UP GROWTH IN NEPHILA SENEGALENSIS, A SPIDER WITH EXTREME REVERSED SSD Review round: 1 Reviewer: 2

Basic reporting: The ms is well written and all relevant raw data are available. The discussion is too spider-centered. There is a large body of literature on sex-specific growth in insects which is not sufficiently cited (e.g. papers by Stillwell & Davidowitz). Detailed comments: L246: These headings are awkward. With regards to the analysis, why did you not model the interactive effects of family and treatment here? Also, why is the sex effect not explicitly tested? See comment below. L304-305: Again, why is not sex included as a predictor, especially in the interaction? L313-314: But High-Low and Low-Low might still differ with regards to fecundity. L317-318: Why are size or mass not included as covariates? L377: Please inform us of the phenology of N. senegalensis. L417: ...PROBABLY result from an… L418: …is VERY LIKELY facilitated… L458: Detecting variation in growth responses due to additive genetic variation or maternal effects in a population does not equate to a "range of developmental strategies". L514: You do not formally test for sex-specific growth responses or for any interactive effects of sex. Experimental design: The study can address the research question. The rearing effort is laudable. It is very painstaking work to rear that many orb-weavers. There are design limitations; i.e. growth was not measured for each instar, which was probably prohibitive logistically. Hence our understanding of sex-specific growth remains pretty basic. Validity of the findings: In this study, the sex-specific effect of food limitation on growth and size at maturity was measured. That is, the results provide information on sex-specific growth mechanisms, with implications for possible sex-specific selection processes. Hence, any discussion of patterns of natural and sexual selection must be speculative and should be treated as such. However, the discussion reads as if the study provides direct insight into selection processes, which it cannot. Thus, I recommend toning down the discussion throughout. The statistical approach of multiple pairwise comparisons falls short of providing a comprehensive analysis. It is quite peculiar that the study aims to identify sex-specific growth responses to food limitation during early and late stages of development, but no model is built with sex as a predictor or part of an interaction with treatment. SSD is of course very pronounced in adults in this species and we don’t need stats to tell us that, but sex-specific growth responses should be tested explicitly. Additional comments: No additional comments

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.

Title: FITNESS IMPLICATIONS OF SEX-SPECIFIC CATCH-UP GROWTH IN NEPHILA SENEGALENSIS, A SPIDER WITH EXTREME REVERSED SSD Review round: 2 Reviewer: 1

Basic reporting: All my - and the other reviewer's - comments on the first version of the ms have been adequately and nicely addressed. I have have no further comments. Experimental design: All my - and the other reviewer's - comments on the first version of the ms have been adequately and nicely addressed. I have have no further comments. Validity of the findings: All my - and the other reviewer's - comments on the first version of the ms have been adequately and nicely addressed. I have have no further comments. Additional comments: The paper is now in great shape and I recommend publication. Best wishes, Matthias Foellmer

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.

Title: MITOCHONDRIAL GENOMES OF THREE TETRIGOIDEA SPECIES AND PHYLOGENY OF TETRIGOIDEA Review round: 1 Reviewer: 1

Basic reporting: Clear, unambiguous, professional English language used throughout but I noticed a couple of things worth revising: Lines 128-129 “The positive relativity were found between A+T content and AT-skew, and also between G+C content and GC-skew.” Replace “mt genomes” with mitogenomes (e.g. in line 132). The graphic resolution of the main figures should be improved in the final submission for publication. Experimental design: No comment. Validity of the findings: No comment. Additional comments: This is a well-structured and well written work reporting the near-complete mitogenomes of three Tetrigoidea species. The paper is adequate in its scope, and fits well in the standards of PeerJ.

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.

Title: MITOCHONDRIAL GENOMES OF THREE TETRIGOIDEA SPECIES AND PHYLOGENY OF TETRIGOIDEA Review round: 1 Reviewer: 2

Basic reporting: Although the article is written in clear English, the authors should check again carefully for typos. Striking examples: pdf line 37: "Batraehididae, Cladonltidae, Diseotettigidae, ..." Fig. 3: "Tridactuloidea" instead of "Tridactyloidea" The names of the species studied should be given in full at first mentioning with the genus not abbreviated. Experimental design: pdf lines 30-38: The authors compare two systematic divisions of Tetrigidae (OSF versus Zheng) that - apart from the level subfamily versus family - is nearly identic except that two taxa are missing in the latter system. Is this due to different arrangement of species or only due to the two (sub-)families missing in China? pdf lines 56-61: The exact purpose of the study, apart from adding new data, is not clear from the introduction. The authors state that they "further study of the phylogenetic relationships among Tetrigoidea". How will they reach this aim? Are the species selected representatives of different (sub-)families or do they represent (sub-)families, of which genome data are not yet available from genbank? The aim of the study should given more precisely and in accordance with what is presented in results and discussion. Validity of the findings: no comment Additional comments: No additional comments

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.

Title: MITOCHONDRIAL GENOMES OF THREE TETRIGOIDEA SPECIES AND PHYLOGENY OF TETRIGOIDEA Review round: 1 Reviewer: 3

Basic reporting: The authors present the description of three mitogenomes of Orthoptera of the family Tetrigidae, basically in a data release manuscript. The manuscript is well written and easy to understand. The structure is adequate, as the results are rather descriptive and leave comparably little room for discussion. Experimental design: The methods are suitably applied, largely well described, and the results are described in high detail. Validity of the findings: Overall, the findings appear valid. My main point of criticism is the following: Molecular phylogenetic studies of Caelifera are notorious for the difficulties presented by mitochondrial haplotype sharing (see references pp. 6/7 in Hawlitschek et al. 2016, http://onlinelibrary.wiley.com/doi/10.1111/1755-0998.12638/full). While most previous studies focus on Acrididae, not on Tetrigidae (or related families in Tetrigoidea), the authors should take into account that hybridization (which has been postulated also across genera) or other phenomena causing misleading results may also influence their Tetrigoidea results. The authors should introduce these problems and carefully discuss their implication for their results. Additional comments: Other than that, I only have minor comments, a list of which is given below. Kind regards, Oliver Hawlitschek l. 16: Replace "vaule" with "value". l. 30 / 31: Add citations for the first two sentences of the introduction. l. 39: "minor damage to crops": better write "minor importance as agricultural pests". l. 47: "original taxon": does this mean "sister group to all other Tetrigoidea"? l. 53: Replace "widely used molecular components" with "a widely used molecular component". l. 69: "PCR amplification and sequencing by primer walking": Text should be bold. l. 89 / 90: "18 members of Caelifera and F. qinlingensis, […]": But the species of Tetrigoidea studied here are members of Caelifera, too? l. 128: "positive relativity": Do you mean "positive correlation"? l. 132: I do not understand the importance of true bug mitogenomes here. If this is a standard method I do not know it. Please explain in the methods section. l. 172-174: "Some highest […] encoded tRNAs.": These sentences are difficult to understand, please check the wording. l. 224: "original taxon": See my comment to l. 47. l. 230: Replace "Melanopline" with "Melanoplinae". l. 237/238: "topologies of Tetrigoidea based on 16S rRNA": It is not clear where these results come from. Did the authors conduct specific analyses based on 16S only? This is not described in the methods section. Or are they from Eades et al. (2014)? Please clarify. l. 270: Replace "Zhemin zheng" with "Zhemin Zheng". l. 393: Replace "Potua Sabulosa" with "Potua sabulosa". l. 397: Please give the website for the download of Tracer. l. 406: Replace "Molecular evolution" with "molecular evolution". l. 422: Italicize "Tetrix tenuicornis". l. 440: Replace "Comparative & phylogentic" with "comparative & phylogenetic".

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.

Title: MITOCHONDRIAL GENOMES OF THREE TETRIGOIDEA SPECIES AND PHYLOGENY OF TETRIGOIDEA Review round: 1 Reviewer: 4

Basic reporting: no comment Experimental design: no comment Validity of the findings: There is one aspect in the paper which I find speculative at the moment. I explained it in a comment in the attached pdf (line 150). Additional comments: I added some comments and suggestions in the attached pdf.

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.

Title: MITOCHONDRIAL GENOMES OF THREE TETRIGOIDEA SPECIES AND PHYLOGENY OF TETRIGOIDEA Review round: 2 Reviewer: 1

Basic reporting: Following on my previous comments: Section 'Nucleotide composition and skew': The phrase strating with 'The approximate positive correlation were found (...).' need revision. A-skew and C-skew should be replace by A+T-skew and G+C-skew for the sake of consistency. All figures still need to be improved with regards to resolution for publication. For example, I found it difficult to read a printed version of the phylogenetic tree. Experimental design: No comment. Validity of the findings: No comment. Additional comments: No additional comments

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.

Title: MITOCHONDRIAL GENOMES OF THREE TETRIGOIDEA SPECIES AND PHYLOGENY OF TETRIGOIDEA Review round: 2 Reviewer: 2

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

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.

Title: MITOCHONDRIAL GENOMES OF THREE TETRIGOIDEA SPECIES AND PHYLOGENY OF TETRIGOIDEA Review round: 2 Reviewer: 3

Basic reporting: No further comments. Experimental design: No further comments. Validity of the findings: No further comments. Additional comments: The authors have thoroughly revised their paper, improving it considerably. I only have a few minor details, mostly stylistic, that should be taken into account. Kind regards Oliver Hawlitschek 1. l. 33: "and one tribe": Xerophyllini is a tribe within Cladonotinae. Other subfamilies of Tetrigidae have more tribes. Best remove this. 2. l. 53: Change "Cong and Grishin., 2016" to "Cong and Grishin, 2016". 3. l. 59: Change "represent for Scelimeninae, make it possible" to "to represent Scelimeninae, making it possible". 4. l. 60/61: Change "Combined with other 18 Caelifera mitogenomes in the database, the comparative analysis was made" to "A comparative analysis with other 18 Caelifera mitogenomes was conducted". But see my next comment. 5. l. 54-66: Most of this entire paragraph is a summary of the methods section and therefore redundant. I suggest keeping only the first, second, and last sentence and removing the rest of the paragraph. 6. l. 156: Change "the identify of Tetrigoidea" to "the identification of Tetrigoidea". 7. l. 176: Change "NNU were relatively" to "NNU was relatively" and "NNC were lower" to "NNC was lower". 8. l. 178-182: "Some highest … tRNAs": This sentence is still not clear. Please rephrase again. 9. l. 245: Hawlitschek et al. (2016) is not the ideal reference here, as it does not really discuss hybridization. Check p. 2 of this paper for more suitable references (e.g., Gottsberger 2007, Hochkirch & Lemke 2011, Rohde et al. 2015).