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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 FURVELA TENT-TRAP MK 1.1 FOR THE COLLECTION OF OUTDOOR BITING MOSQUITOES Review round: 2 Reviewer: 1

Basic reporting: This manuscript “The Furvela tent-trap Mk 1.1 for the collection of outdoor biting mosquitoes” by Charlwood et al. describes the refinement of the Furvela trap for the collection of anthropophilic mosquitoes. To analyze the new configuration of the Furvela trap for endophagic biters, they compared twice-a-week usage of the tent to daily window-trap collections in Northern Tanzania, and for exophagic biters to outdoor light traps in the PAMVERC trial area in Mozambique. Overall, I believe the trap design to be a good one, especially due to the flexibility of the general design, cost, and due to the ubiquitous nature of CDC light traps. I do think the manuscript suffers from not including more from the large PAMVERC trial data as this has hundreds or thousands of trap nights, and from ambiguity in the methodology. I fully agree with the premise that human landing catch is becoming unacceptable for use, and its use needs to be phased out. With this, I do feel that a more direct comparison between the Furvela design and HLC should be done. I do not believe this is necessary for publication, but I do think it is necessary to a widespread adoption of the technique. I do recommend the paper for publication, though think a major revision of the text should be undertaken to improve clarity for the reader. I also feel the format and quality of the figures needs to be improved prior to publication. Basic Reporting: The Furvela trap, while previously described, needs to have a basic description in the text to orient the reader to the design. This is especially pertinent as I can’t locate the (what I presume) is the original description of the Furvela trap, as this reference (Charlwood JD: A new efficient and cheap trap for the collection of outdoor-biting mosquitoes. Acta Trop 2005, 95:S1–S506) seems to be to the entire supplementary info covering conference proceedings from the 4th MIM Pan-African Malaria Conference. The supplementary video and SOP do help this, but still I feel the paper would benefit from something brief in the text before the “improvements” part of “The Furvela tent-trap Mk 1.1” section. Figures as included have the below issues: • They may not be of sufficient resolution. • Figure 1 Legend: I would highly recommend listing in the legend what each picture is showing. Defaulting to the text makes description unnecessarily taxing. • Figure 2 Legend: “Tanzania” is misspelt. • Figure 2: I don't see an arrow. • Figure 2: Why is only the blue line labeled? • Figure 2: The gaps in sampling would be better presented with a broken axis as you then lose information from the time periods you have sampling (i.e. reduce the “white space” in the figure). • Figures 3 and 4: I'd highly recommend the use of bar graphs over pie charts. It would convey the data in a smaller and more informative package. Additionally, this allows for easier comparison between groups. Stacked barplots may make comparisons between before/after easier. See below figure (in attached pdf) for example (I will also attach the R code to generate this figure). This presentation also allows you to more quickly look between species without having to go back to the figure legend to see what is what. (see attached PDF for figure) • Figures 3-4: Make sure that colors are consistent between graphs, i.e. Furvela is always blue. • Figure 4 Legend: A numbered reference is cited, but the references aren’t numbered. • Table 2 uses “DRR” which is not defined. The text describes “IRR.” Other comments: • Lines 206-207, Where is this time data? If in the supplemental I only see times "1, 2, 3". Was the night divided for collection period? This needs to be said explicitly how the night was split if so. • Overall, the supplemental data could be cleaned up significantly. i.e. in the indoor_outdoor_furvela document, there are extraneous datasheets that aren’t included in the paper, sheets are labelled poorly (“Sheet 1”, “Sheet 2”, “Collections”, etc..), and some of them have no/poor labeling (no dates, shorthand for species). Fix sheet labels, remove datasets not utilized, and remove figures not used. • The supplementary file names need to be explicit and the same as described in text, i.e. include “Supplementary File 1 – xxxxxxxx.xlsx”. Experimental design: The design is significantly muddled by the cobbling together of various studies using the Furvela trap, of which it is poorly described which tents are used (models, etc.), which “improvements” are included, and sampling locations/times/methods compared, etc. I think the authors would benefit from a descriptive table of what sampling was performed. The methodology needs to be expanded, i.e. number of trap nights, be explicit about what data is re-used from studies published elsewhere, design of the study (i.e. are collectors being rotated through tents, etc.?). Validity of the findings: A central premise regarding the furvela trap’s uniqueness is that an “entry” behavior is not required for collection. However, as mosquitoes are caught while attempting to enter the hole in the tent (while following the odor plume)---I feel this distinction is subtle from other “entrance” traps. The design is like that of the “infoscitex tent” design we described in Krajacich et al. as mosquitoes collected to odor plumes pushed out of mesh windows are instead collected by suction ports above the mesh (albeit in a simpler design with the Furvela trap). Mosquitoes have been known to be very adept flyers, entering even small holes in a bed net. While the fan I can imagine makes entry difficult, as there is a large gap in the zipper- how often have collectors found mosquitoes in the tent? How often have you caught full or partially bloodfed mosquitoes in the furvela trap (indicating they were possibly caught while exiting after feeding), and does this proportion differ from that caught in the CDC Light traps? Additional comments: Line 147: Need a comma after “light-traps” Typo in line 151, “In Kagera Province, northern Tanzania.” should not be a separate sentence. Line 214: Remove comma after "In studies" Lines 224-226: Is it true that only An. funestus and Culex spp. have changed indoor/outdoor ratios? It seems only Mansonia africanus is the same before/after spraying. The two mentioned are the only ones who went from dominant indoor to dominant outdoor, but most changed in proportion. The discussion should address whether any of the vectors (especially those such as Coquelletida fuscopenata that are less-commonly described) have been reported to be more commonly exophagic in other studies (from indoor/outdoor light traps or ideally HLC). Line 249, need comma between "outdoors" and "suitable" Line 254: "Window-traps may be a substitute for light-traps." What is this based on? They may be for indoor collections, but obviously not for outdoor collection. Lines 279-280: Has this light repellence been seen with CDC light-traps with/without the bulb (but having a CO2 source) in this area? This finding is interesting as I am unaware of a documented repellence effect due to light for anthropophilic vectors, though I am aware of many cases of lights attracting vectors (i.e. sandflies) and other non-target dipteran (making sorting more difficult).

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 FURVELA TENT-TRAP MK 1.1 FOR THE COLLECTION OF OUTDOOR BITING MOSQUITOES Review round: 2 Reviewer: 2

Basic reporting: The manuscript "The Furvela tent-trap Mk 1.1 for the collection of outdoor biting mosquitoes (#16725)" describes a modified version of the Furvela tent-trap and compare it to other trapping methods. The English language requires significantly improvement, e.g. have to be checked by a native speaker. I have indicated several sentences which stay unclear for me due to language and grammar issues. Although the general structure of the manuscript conforms to PeerJ standards, the structure of the introduction is not very easy to follow. Please start with the general background (malaria, number of cases, distribution, ...), then explain the Motivation of your study (indoor collections to not necessarily reflect the outdoor situation, ...) followed by the State-of-the-art (pros and cons of methods used in the past (HLC) and current methods (light traps, tent traps)). Then highlight the lack of knowledge (there are already tent traps available, explain why we need your improved system (entry behavior, ...)) and finally give a description of the explicit study aims (in short: What exactly are you doing and why). In addition, several information are not well referenced. The figures and tables are generally okay. However, I recommend using bar plots instead of pie charts, which are generally expected to be more comparable between different groups. Some information are missing in the labels of the figure and tables, e.g. explain all abbreviations in table 2 in the label (DDR, TT, CI, adjusted, ...). Finally, although the raw data are supplied, the data are not very useful. Every table has a different, mostly unstructured format and use different abbreviations, which are not explained. Experimental design: The research is in the scope of PeerJ, but the explicit knowledge gap and research question is not well described. As highlighted for the introduction, my critique of missing structure also applies to the methods section. You are comparing the Furvela tent-trap in different settings, but due to the unstructured presentation, it does not become clear what you did in which site. Please give different subsections, e.g. "In the first experiment, we did ..." & "The second experiment was conducted....", etc. In addition, from the description in the methods, it does not completely becomes clear, that the monitoring was conducted after mosquito control interventions. Furthermore, important information on the mosquito sampling (e.g. window traps) and statistical methods (e.g. why negative binomial) are missing. Validity of the findings: The findings are valid and supported by sufficient data, but are not presented very well. As in the introduction, there is a lack of references for some of the statements and therefore sometimes look like speculation. The research question was not clearly indicated and therefore there is no link between the introduction and the conclusions. Additional comments: INTRODUCTION #before line 39: please provide general information on the impact of malaria on public health in Africa (number of cases, distribution, etc.) #lines 40-42: "This success ..." --> unclear sentence, please rephrase. #lines 42-44: "Outdoor transmission [...]" --> unclear sentence, please explicitly explain why both, indoor AND outdoor monitoring, is needed, i.e., because indoor monitoring does not necessarily reflect the outdoor situation #lines 44-50: "Indeed, ..." --> I do not understand why you include this citation. Delete? #line 52: "untested," --> unnecessary comma #line 55: "require" --> do you mean "lead to"? #lines 56-58: "Although ..." --> Please be more specific: malaria vectors = Anopheles spp. or specific Anopheles species (Anopheles gambiae complex). In addition, the vectors for malaria can also transmit other pathogens. #lines 59-61: "Outdoor ..." --> Please give references for the statement that human landing collections is the best method. #lines 61-62: "HLC ..." --> Please give a reference. #lines 62-63: "Such ..." --> Please give a reference. #lines 63-67: "The objective ..." --> Unclear. As far as I understood, here you want to explain why alternative methods for HLC are needed. #line 70: What do you mean by "risk-free"? #line 72: What do you mean by "at least as far as malaria vectors go"? #line 74: At the first report of a species in the manuscript, please give the full name of the species, e.g. "Anopheles gambiae s.l." instead of "An. gambiae s.l." #line 75: replace dot by comma: "CDC light-traps." --> "CDC light-traps," #lines 93-94: "The ‘Furvela’ tent-trap, ..." --> Why does it not require an entry behavior? Is there a reference of the first description of this trap? #lines 94-95: "The trap remains ..." --> reference? #lines 110-112: "We supply ..." --> Was the aim of the study to show that the Furvela trap catach mosquitoes? I thought this was clear before. What exactly are the aims of your study? METHODS #before line 116: please give some more information as introduction of the method section, e.g. "in order to improve the Furvela tent-trap Mk 1.1 ..." or information why the improvements were necessary #line 116: please give a reference for the first description of the Furvela tent-trap Mk 1.1 #line 147: include comma after "In addition to CDC light-traps" #lines 147- : I completely missing details on the exit window-traps (e.g. construction, references, etc.). #line 154: "The best ..." --> What trap do you mean? Window-traps or Furvela traps? #lines 155-156: "As mentioned ..." --> As mentioned before? You have to explain, which risk a linked to HLC and give a reference for the statement. #line 158: What is "PAMVERC trial"? Explain. #lines 165-167: Why did you use negative binomial regression, e.g. Why did you not use ANOVA or Poisson regression or .... Please explain how you observed overdisperion in the data, which is one important information to select negative binomial regression as appropriate method. Which explanatory variables were used? Was this statistical method also used to analyze the collected data described in the lines 178-183. RESULTS #line 203: comma after "Between the 23/10/2014 and 14/3/2015" #lines 205-206: "r = 0.93, P = 0.0003" --> What kind of statistical method was applied here? Spearman? There is no explanation here or in the method section. #lines 206-208: "A larger proportion ..." --> As far as I understood from the method section, you only aimed to compare the different kind of traps? Why did you also analyze the temporal pattern? Explain in the description of the study aims in the introduction and explain in the methods how you analyzed the temporal pattern. # lines 208-212: "Immediately following the application ..." --> Please explain this mosquito control measurements in the method section and not in the results section. # lines 214-216: "In studies, from Tanzania, ..." --> Please move to the discussion section. # lines 216-217: "The other species ..." --> Which other species? Describe. # lines 217-219: "Comparable results were ..." --> What do you mean by "comparable results"? Describe. # lines 233-234: "The incident rate ratio ..." --> I do not understand what you are doing here. Give more information on the IRR and the variables TT and L. # lines 234-236: "Variables including collector, ..." --> The modelling approach must be explained in the method section. # lines 234-236: "Variables including collector, ..." --> What do you mean by the variable "collector". Might be better included as a random effect? # line 239: "Anopheles" in italics DISCUSSION #general: Please also discuss potential problems with the tent traps. For example, please discuss the problems of the sampling standardization, which might be highly dependent on the collector in the tent. # lines 247-248: "Because of the risks ..." --> Give a reference. # lines 258-249: "In order to determine ..." --> You do not determine the transmission, but the density of potential vectors. Please rephrase. # lines 249-252: "The ratios ..." --> In general, the comparison does not give any information about the suitability of the Furvela tent-trap to trap outdoor biting mosquitoes, e.g. because you do not have information about the size of the real outdoor biting mosquito population, but indicate a similar performance like the light-trap. Please rephrase. line 254: "Window-traps may be a substitute for light-traps." --> What do you mean by "substitute"? I do not understand. #line 258: "sp." --> not in italics. In addition, please use "spp." as there are several species present in Cameroon. #lines 267-268: What do you mean by "chaotic"? Spatial-temporal variability? Please explain and rephrase. #line 269: "indoor light-trap" instead of "light-trap" #line 269: "For example" instead of "Thus" # line 277: "With the current ..." --> You mean the elimination of mosquitoes in order to eliminate malaria. Rephrase. # lines 285-288: "Ease of transportation ..." --> Why are vector maps needed? Explain. # lines 288-289: "Such information ..." --> How do the tent traps help to estimate the flight range of mosquitoes? Completely unclear for me. Explain. # lines 289-290: "They may ..." --> How to tent traps help in the regarded to determine the intervention area? Explain. # lines 307-308: "Presently this ..." --> Why is there a restriction to indoor insects? Explain. # line 308: "The use ..." --> I do not understand the sentence. Rephrase.

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 FURVELA TENT-TRAP MK 1.1 FOR THE COLLECTION OF OUTDOOR BITING MOSQUITOES Review round: 3 Reviewer: 1

Basic reporting: I feel the manuscript has been greatly improved since the initial version, both in terms of clarity and the overall formatting. I feel that the changes that have been incorporated are appropriate and sufficient. Below I list some minor corrections/suggestions. Lines 36-39: This sentence is a bit awkward/run-on and would be better split into two. Also merge into below paragraph. Line 239: Report week numbers or change figure to show dates Line 242: Report density drop Line 278: Remove “really” Line 319-321: I’d mention explicitly that light being a repellent has not been shown previously. Overall comment: The discussion is somewhat disjointed and doesn’t flow easily from paragraph to paragraph. I’d suggest some improvement of transition sentences between paragraphs, and the paragraph order. I think it’s publishable in this format, but could be improved. Experimental design: no additional comment Validity of the findings: no additional 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: THE FURVELA TENT-TRAP MK 1.1 FOR THE COLLECTION OF OUTDOOR BITING MOSQUITOES Review round: 3 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: The authors significantly improved the manuscript and can be accepted. However, to my opinion, the supplementary tables still need significant improvement. Especially please do not use unexplained abbrevations (e.g. "coll" = collection method, "afr" = "Anopheles funestus", AG part = ???, ODDS = ???). Please tidy these tables up, there are a lot of blank cells or unused columns.

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: NORTH ATLANTIC OSCILLATION DRIVES THE ANNUAL OCCURRENCE OF AN ISOLATED, PERIPHERAL POPULATION OF THE BROWN SEAWEED FUCUS GUIRYI IN THE WESTERN MEDITERRANEAN SEA Review round: 1 Reviewer: 1

Basic reporting: I outline specific issues to be fixed below: -This manuscript uses clear language throughout, although it would benefit from an additional readthrough by a native English speaker. -The last paragraph of the introduction should be removed -- it is more suitable for inclusion in the abstract, or perhaps the discussion. -In lines 160-161, the authors state that the 'AO is characterised by a merdional dipole in sea level atmospheric pressure between polar regions and mid-latitude'. This is the NAO, not the AO. The latter half of the sentence '...and could be interpreted as the surface signature of modulations in the strength of the polar vortex aloft' is verbatim from the abstract of Thompson & Wallace 1998 and should be rephrased. -Throughout this manuscript, the authors write out and explain equations that are standard in the field. (A particular example of this is the approx. 1 page devoted to explaining chlorophyll fluorescence on lines 271-289.) Replacing this text with one-two sentences and a citation for chlorophyll fluorescence would suffice. Similarly, equation 1 and the text following (lines 127-131), equation 2 and the lines following (137-142), etc. could be removed without losing any clarity in the manuscript. -In the figures (3, 5, 6) please use different shaped symbols in addition to different colors to distinguish among your results. This is necessary for any color-blind readers, as well as printing in black&white. -In figure 4, please clarify what the grey and orange boxes represent. And, please make the shading/hatching different between the two colors (see color blind readers, above). Experimental design: The method of sampling for Fucus guiryi presence/absence needs to be described in the Methods: how was it checked? Quadrats and transects? Visual searching over a defined period of time? Validity of the findings: The most significant issues with this manuscript are the assumptions made about the basic biology of Fucus guiryi, which then impacts the interpretation of the authors' findings in the abstract, conclusions, etc. The authors assume that the presence of F. guiryi in the summertime is solely due to the transport of thalli and embryos from neighboring populations to the west. However, it is always present at their key study site in the winter. (It is not stated how the winter populations appear.) No mention is made of the reproduction of F. guiryi and the possibility of microscopic stages/remnant holdfasts of macroscopic stages remaining even when juvenile or adult thalli are not present (and thus it appears to the naked eye that F. guiryi is gone). The authors could have tested specific issue for this by putting out settlement plates at their sites during different years -- any F. guiryi that appeared during a summer season would have to be due to the settlement of new individuals. Also, the fact that F. guiryi was present _every_ winter of the survey indicates the low likelihood that this population is solely dependent on recruitment from a western source population. In a broader context, it is possible that the appearance of summer F. guiryi is correlated with the NAO...not because the NAO brings F. guiryi to this site, but instead because it creates favorable conditions for existing F. guiryi to grow into macroscopic sizes. The authors should consult Schiel and Foster 2006 doi:10.1146/annurev.ecolsys.37.091305.110251 for a review of the population biology of fucoids. The authors state that Tarifa is the 'seeding' population. As this was not conclusively demonstrated/experimentally tested, they should state instead that it is a 'likely seeding' population. (see comments in #1 above) The authors do not examine or discuss the impact of herbivores on blade shape, which can significantly impact their discussion/results of developmental instability. Some discussion of the impacts of herbivory on blade shape is needed. 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: NORTH ATLANTIC OSCILLATION DRIVES THE ANNUAL OCCURRENCE OF AN ISOLATED, PERIPHERAL POPULATION OF THE BROWN SEAWEED FUCUS GUIRYI IN THE WESTERN MEDITERRANEAN SEA Review round: 1 Reviewer: 2

Basic reporting: MAIN: This paper reports a long (26 years, 1990-2015) time series of records of occurrence of a marine alga at an isolated warm range limit. This is a fantastic opportunity as such a data set is difficult to obtain and has allowed the authors to make a more detailed analysis of causes of variability in presence / absence than what is usually possible. The analyses conducted allowed a correlation of the presence of the species with the overall mean value of the NAO (an index reflected in mean weather conditions) from the spring months. Spring is the main reproductive / recruitment season for the species, This is an important topic that deserves publication. I recommend that it should be published quickly. Main suggestion 1. The paper could be even further improved if the authors could try to use a better proxy for the local conditions of this very localized study area rather than just the NAO. Main suggestion 2. The introduction could be more clear in the formulation of hypotheses/questions behind the other measurements conducted. The measurements conducted in July 2011 (radiation, temperature, photosynthetic parameters) are a single point in time relative to the time series and it is hard to related the two things and to understand the aim of these point measurements relative to the goal of the paper. I recommend a better explanation of what question are you trying to answer with these measurements. Also, a comparison conducted in the field, under distinct environments can be due to population differences or environmental differences. So, again, please explain better what is the question. Main suggestion 3. I recommend a better literature review of other such cases of variability of edge populations of Iberian algae. 3a. First, please compare your results with the very relevant paper on variability in Fucus guiryi at its southern limit by Carla Lourenço et al (2016). It reports the dependence of this species on specific local climatic effects namely the upwelling conditions in isolated spots. It also reports the southern limit of Fucus guiryi. Lourenço CR, Zardi GI, McQuaid CD, Serrao EA, Pearson GA, Jacinto R, Nicastro KR (2016) Upwelling areas as climate change refugia for the distribution and genetic diversity of a marine macroalga. Journal of Biogeography 10.1111/jbi.12744 3b. Likewise, there are other papers for the Iberian Peninsula that have studied long-term variations of species at their range edges, not just for the warm edges in the south but also for the warm edges in northern Spain, where surface seawater warms towards East. Other suggestions: 4. The index of developmental instability is interesting but can be highly affected by grazing rather than climate stress. Fucus guiryi are extremely consumed by grazers, namely Sarpa salpa fishes that get to it at very high tides. Did this not affect your measurements? Was this population located so high on the shore that fish could never get to it thus not affecting thallus development? This is a tradeoff with the physical stress factors that increase further up on the shore. 5. The chlorophyll fluorescence parameters are widely used in the literature and do not need to be described in detail, with equations, in this paper that simply uses them. 6. Disappearance and recovery in the same site, so isolated from the remaining populations, suggests that the population might have kept microscopic latent stages (embryos of Fucus can remain undeveloped but alive if conditions are limiting) during unfavourable years, that recovered the population afterwards. This hypothesis has not been addressed in the paper. See for example our discussion of such issues for an iberian kelp species (despite the differences between these in being subtidal and having gametophytes): Assis J, Coelho N, Alberto F, Valero M, Raimondi P, Reed D, Serrao EA (2013) High and distinct range edge genetic diversity despite local bottlenecks. PLOS ONE 8(7): e68646 7. Is fig. 4 really necessary? It can be simply reported in the text. Experimental design: no more comments (see above) Validity of the findings: no more comments (see above) Additional comments: no more comments (see 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: NORTH ATLANTIC OSCILLATION DRIVES THE ANNUAL OCCURRENCE OF AN ISOLATED, PERIPHERAL POPULATION OF THE BROWN SEAWEED FUCUS GUIRYI IN THE WESTERN MEDITERRANEAN SEA Review round: 1 Reviewer: 3

Basic reporting: I found the manuscript well structured, the analyses adequate and sound, the conclusions of an environmental control for species occurrence supported by the data. Text in general was also well written and ideas very easy to follow. Experimental design: No comment Validity of the findings: I only have one concern (see also a few minor comments below) relating to the authors interpretation of the nature of the marginal Calaburras population. The authors argue throughout the text that F. guiryi is present every year at least during the winter and that some unfavourable years the population does not survive to midsummer. The sole explanation proposed for the recurrent recruitment during the winter is the putative transport of drifting thalli and unattached embryos from neighbouring Gibraltar populations (some 80km away) by the Atlantic Jet (lines 59-61; 396-401; 424-426). Genetic data shows that Fucus (and canopy-forming brown seaweeds in general) are very poor dispersers at km scales (e.g Neiva et al. 2012). There is a much more parsimonious explanation for the recurring appearance of F. guiry at the very same site – the presence of cryptic, hardy microscopic stages that survive unfavourable months before developing again during milder months or after catastrophic events (Carney & Edwards 2006; Creed et al 1996). Local(not immigrated) few-cell zygotes (something like a seed bank) or remnants of thalli (as in many cryptic perennial Cystoseiracea that lose most biomass during the unfavourable winter months) are good candidates, and both avoid the need for a constant supply of propagules from a 80 km distance source. This hypothesis should be at least discussed as an alternative to the immigration scenario. Additional comments: Minor comments: line 48: F. guiryi occurs as far south as Dahkla in the Western Sahara (Lourenço et al. 2016, also discusses the importance of upwelling and lower SST in this African range) line 49: “sometimes a population develops at Punta Calaburras”. If I understand correctly this is not very clear in the sense that the population is present throughout the years at least during some months and not missing some years line 52: “(sub F. spiralis and F. spiralis var. platycarpus)”. I don’t understand what these refer to – subspecies? To avoid confusion over the focal species it should be removed or clarified. Same in line 62. line 67: “university of AFM”- in full if not defined earlier line 73: “presence at Punta Calaburras – add something like “during/until mid-summer” to avoid confusion. Same in line 394 – add “mid-summer” before “occurrence” line 166: it could be explicit the institutional source of the data (NOAA?) for AO and NAO, as for SST, instead of just the link line 423: “persistence until midsummer” would be clearer than “success of occurrence in midsummer”, since the species is there already during the winters. Same in 445 – “proliferates until midsummer” may be more accurate. line 449: In calaburras the tidal range is narrower but also overall lower. Stating something like “the narrower, [and overall lower] tidal range” reinforces the differences discussed immediately after Figure 1: correct “Westarn” Alboran in legend Figure 2 and 6: clarify UTC in legend Figure 3: correct “ornage” in legend References Neiva et al. 2012 Drifting fronds and drifting alleles: range dynamics, local dispersal and habitat isolation shape the population structure of the estuarine seaweed Fucus ceranoides. Carney & Edwards 2006 Cryptic processes in the sea: a review of delayed development in the microscopic stages of marine macroalgae Creed et al. 1996 The development of size structure in a young Fucus serratus population Lourenço et al. 2016 Upwelling areas as climate change refugia for the distribution and genetic diversity of a marine macroalga

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: NORTH ATLANTIC OSCILLATION DRIVES THE ANNUAL OCCURRENCE OF AN ISOLATED, PERIPHERAL POPULATION OF THE BROWN SEAWEED FUCUS GUIRYI IN THE WESTERN MEDITERRANEAN SEA Review round: 2 Reviewer: 1

Basic reporting: This revised manuscript is much clearer to read than the original version -- thank you! However, there are still grammatical English errors throughout the manuscript that need to be fixed. I highlight some, but not all, in this review. (e.g. the third line of the Abstract reads 'The presence of the alga at Punta Calaburras is supposed could be due to the growth of....' ) The authors should remove the last part of the last sentence in the Abstract 'an aspect practically unknown before'. Lines 56-57 should be rephrased - unclear. Line 67: remove 'uninterrupted' Line 70: remove 'must' -- this has not been experimentally confirmed. Remove the two sentences that start on lines 136 and 138 - they are not needed in this manuscript. Line 143: fix the ... Lines 172-174: remove these first two sentences, as they do not belong in the Methods section. Line 184: change 'image' to 'images' Line 232: change 'cultured' to 'placed' Line 363: replace 'last' with 'recent' Experimental design: The authors have not yet clarified how the sites were surveyed for the presence/absence of the algae. Was the entire site visually surveyed? Were quadrats/transects used? The presence of small macroscopic stages may require careful surveying techniques, and this is not clear from the text written. The authors need to describe how the thalli were selected for the developmental instability analysis. Was this random? Haphazard? Both Random and Haphazard selections of individuals have specific ecological criteria. The authors need to demonstrate how bias was avoided when thalli were selected. Similar comment for line 230 in the fluoresence Methods section. The authors should change dry weight to dry mass, as they were recording mass, not weight. Also, the equation in line 243 is wrong. The denominator should be fresh mass, not dry mass. Please note that this equation is no longer Equation 5. Similarly, Equation 7 is not equation 7 anymore. Validity of the findings: Line 278-279: rephrase this sentence. A trend of what through time? Binary logistic regressions (line 280-294) are not linear. I am not sure where the equation came from in line 285, but it does not represent the model output that is shown in Figure 3. This section needs to be rewritten to reflect this. The authors state that there was not a difference in developmental instability between the two sites, but they had a p=0.04 value for difference by location. This is significant (as the authors do not state that they used a different p value as a cutoff for significance), but the authors treat it as if it were not. They also do not state which site had the higher developmental instability values. Figure 2 should also show the tidal range for Pt. Calaburras. Additional comments: The first paragraph of the Discussion is repetitive from the Introduction. It should be shortened substantially to focus on how the new results from this work fit with current ecological theory on peripheral populations. Additionally, the second part of this paragraph (~371-401) should be significantly shortened for clarity. The authors should simply discuss that the population most likely re-emerges from existing microscopic stages already present at Punta Calaburras, and that dispersal from the Strait of Gibraltar is possible, albeit unlikely. Remove 434-437 - these sentences are not relevant for this manuscript. Change the Discussion text regarding the developmental instability 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: NORTH ATLANTIC OSCILLATION DRIVES THE ANNUAL OCCURRENCE OF AN ISOLATED, PERIPHERAL POPULATION OF THE BROWN SEAWEED FUCUS GUIRYI IN THE WESTERN MEDITERRANEAN SEA Review round: 2 Reviewer: 2

Basic reporting: The authors have addressed the reviewers concerns adequately. Experimental design: The authors have addressed the reviewers concerns adequately. Validity of the findings: The authors have addressed the reviewers concerns adequately. 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: NORTH ATLANTIC OSCILLATION DRIVES THE ANNUAL OCCURRENCE OF AN ISOLATED, PERIPHERAL POPULATION OF THE BROWN SEAWEED FUCUS GUIRYI IN THE WESTERN MEDITERRANEAN SEA Review round: 2 Reviewer: 3

Basic reporting: This is the 1st revision of the manuscript by Melero-Jiménez et al. investigating the climatic and oceanographic correlates of mid-summer persistence of a range-edge population of the seaweed F. guiryi and its comparison (developmental and physiological traits) with a less marginal population. Globally I found the revision adequate as most comments raised were addressed. I only have a few additional minor comments/suggestions that the authors can address without the need for an extra round of revision. Some ambiguity remains in some sentences concerning the seasonal nature of F. guiryi at Calaburras – it should be clear to the reader that macroscopic F. guiryi is always detected in the winter and either these individuals persist until mid-summer or they disappear, depending on the years. Specifically, that midsummer occurrence is related to survival of winter alga and not recruitment (this happens all years during winter). The authors can consider some suggestions (see minor comments below) that hopefully improve clarity. Experimental design: R1 mentioned in the first review that it was not clear how presence/absence had been assessed (transects, quadrats, searching haphazardly, other?). This information may be relevant for the readers and should be made available. Validity of the findings: My personal view (based on available literature) is that migration from Gibraltar is much less plausible than local recruitment. In any case, long-distance (non-local) dispersal by kelp and fucoid seaweeds is much more often mediated by drifting, reproductive thalli (that release their fertilized zygotes in situ after being washed ashore) than by direct travel of spores or zygotes, that in the case of Fucus are actually negatively buoyant (i.e. they sink). This alternative vector of long-distance dispersal (drifting reproductively thalli) from Gibraltar may be worth mentioning. Additional comments: Minor comments/suggestions line 24 – rephrase and correct “is supposed could be due to the growth of resilent, microscopic stages as well as influence of the permanent Atlantic jet” line 43 – the first sequence is too long; it can be break in two sentences or at least use a coma. It may also be more correct to add “being more abundant (towards warmer range edges) in areas characterized by upwelling” line 47 – the “known” southern limit would be more correct line 51, 61- the presence of F. guiryi (named as F. spiralis and F. spiralis var. platycarpus; see Zardi et al., 2011). F. guiryi (named as F. spiralis; see Zardi et al., 2011 was found in 1987… It may be clearer as “previously named” or “previously/originally identified as” F. spiralis etc… lines 56 – upwelling causes lower SST and allows persistence in otherwise too hot areas, but this effect does not necessarily have to do with modern climatic change. It may be more correct to rephrase as “in agreement with the hypothesis of upwelling providing thermal refugia/relief for F. guiryi”. line 67 – “field teaching at the university of AFM”. For clarity it could be added “at the university of the corresponding/senior author AFM”, otherwise readers (like me) may think it is the university abbreviation. line 70 – Seasonality appears to be exclusive of Punta Calaburras. This could be reinforced as “in contrast to the nearby perennial populations in the Strait of Gibraltar in the Strait of Gibraltar AND the remaining range. line 87 - replace “population” by “populations” line 189 – correct “can influences” line 372-374: this phrase is a bit confusing, please rephrase line 375: replace “occurrence of thalli” by “re-appearance of thalli” line 387: “Another, alternative hypothesis is that recurrent re-appearance of F. guiryi at Calaburra is achieved by…” “unfavourable summer months”… 392: local microscopic stages (recruits, holdfast remnants) at PC ca work as a seed bank 393: With our data, we cannot discern whether the recurrent re-appearance of the alga during the winter is due to migration or local recruitment, or both. Taking into… 401: correct F. guiyi 404: “involved in the annual occurrence of the alga” by “involved in the persistence of macroscopic thalli during the summer” line 421: replace “occurrence” by “survival” line 461: replace “modulate the occurrence of the annual survival of” by “modulate the mid-summer survival of macroscopic thalli of” line 470: Dra Ester Serrão

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: INTRASPECIFIC VARIATION IN THE DIET OF THE MEXICAN GARTER SNAKE THAMNOPHIS EQUES Review round: 1 Reviewer: 1

Basic reporting: This is a beautiful data set that covers many years. Well done. The statistical relevance needs to be included in the legends of each figure. I would suggest attempting to combine the final figure of log snake length and prey mass into one chart. the use of different symbols or colors would be appropriate, however, this may prove to be too messy and unreadable. Experimental design: I applaud this manuscript as there are not enough natural history style manuscripts being written. My sole comment in this section is why were the hemipenes or cloaca not everted to determine sex? Simply eyeballing the tail breadth will invariably lead to misidentifying the sex of some animals. Validity of the findings: I would be careful on commenting on the commonness of some of your findings. You have stated your conclusions well and described the diet of these populations clearly. I have added some questions in the text, such as what is the percentage of snakes that had food? I also encourage you to be wary of digestion level of food. I believe that this has led to many circles on the zero line of figure 3. I would consider adding this information to the legend or text, or both. Additional comments: This is a robust data set that you have done a nice job with. I have identified some concerns in the text and in the notes. One thing to add is to include a citation for your delineations of snake size classifications. Have those size classifications been used before, are you classifying them as newborn, etc.. upon personal observation, natural history notes, etc...

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: INTRASPECIFIC VARIATION IN THE DIET OF THE MEXICAN GARTER SNAKE THAMNOPHIS EQUES Review round: 1 Reviewer: 2

Basic reporting: The authors have presented a valuable contribution on the diet of T. eques, a relatively little studied gartersnake. They attempt to describe diet differences between sexes and among size classes, which is an important aspect of the species ecology. This MS needs to have major editing for grammar and language. There are portions that are difficult to follow because of grammar issues. Authors need to follow consistent format for the species name. As is they switch between scientific and common name. Authors need to follow proper and consistent format for presenting stats. I made edits, comments, and suggestions to the PDF. These address questions that need to be clarified and statements that need more justification. Experimental design: The overall design is simple and appropriate. I made questions and comments on the statistical approaches that I would like to see addressed. In the introduction that authors state they pooling older data (Lines 62-64) in their analyses, but it is unclear if this was actually done. As the methods and results currently read the authors only deal with their data. I would like to see them conduct tests with their data, that of the other authors, and the pooled data. Alternatively, instead of doing this independently, they can do a multivariate test using the data source as a variable to determine if diet varied among the data sources. Either way this issue needs to be addressed and clarified. The authors need to clarify the sex body size analyses because it is unclear to me if this analysis was done with only adults or included all snakes (juveniles and adults). Including all snakes may bias their results to no sexual size dimorphism. Validity of the findings: As currently presented the results are novel by contributing to our knowledge of the snakes diet between age classes, but I am not completely convinced about the sex differences. See above comment and those to the MS. I made additional comments to the MS regarding the methods. Many of the questions and comments made to the discussion may be addressed if other sections are more clearly explained. I found it difficult to connect methods, results, and discussion because of the lack of detail in the methods. Sometimes it is hard to follow the logic and methods the authors use, making further interpretations difficult. Additional comments: This MS has a lot of potential and the authors have presented valuable data. But in its current state I feel there is more work to do, especially in regards to more clearly explaining the methods and results. For ontogenetic diet and prey availability discussion I suggest the authors look into the rich body of literature on other Thamnophis species.

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: INTRASPECIFIC VARIATION IN THE DIET OF THE MEXICAN GARTER SNAKE THAMNOPHIS EQUES Review round: 2 Reviewer: 1

Basic reporting: There has been considerable improvement in many areas of the MS. However, it reads more disjunct with this improvements. Certain sections suffer from poor grammar while other sections flow very well. I found it difficult to jump from common name to scientific name throughout the MS. Please identify the scientific and stick with it. Line 71, the citations listed are not complete. i would suggest adding the phrase 'amongst others'. In addition, Line 73 the author identifies that recent studies do not investigate sexual diff in diet. i find this to be4 inaccurate and deserves a more comprehensive literature search to address this. The final paragraph of the introduction is entirely out of place. Experimental design: The question is appropriate and approached correctly. Line 82: snakes were collected haphazardly at 3 different locales on the Mexican plateau. the reader does not need to know about flipping rocks, etc.. Line 92: 16 years on 1.5 +- 0.9 occasions each, what is being referred to here? Line 101 13% of these findings, again, what is being referred to here? Validity of the findings: The additional analysis performed, the MCA contributes to the robustness of the data. It demonstrates the data can be analyzed in multiple ways to produce the same results. I find the MCA analysis much more informative than the cluster analysis and would recommend using that in supplemental materials. The analysis specifically describes slugs and axolotls on the figures, however, table 1 simply classifies these as others. It would be best to stay congruent with these data. Line 233-237 grammar is not present.Line 221 citations are needed because this has been found in numerous other snake species. Additional comments: I found your improvements and grammar and additional analysis evident. However, the MS suffers from being very disjunct at this point, to the reader. I feel like some sections are beautifully written and others are grammatically lacking. I would suggest a couple of more citations (as noted above). I think the new analysis adds to the robustness of the data. I prefer it greatly to the cluster. Please include examples of the "other" section of table 1 as you address slugs and axolotl specifically in Fig 1. overall good improvements.

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: INTRASPECIFIC VARIATION IN THE DIET OF THE MEXICAN GARTER SNAKE THAMNOPHIS EQUES Review round: 2 Reviewer: 2

Basic reporting: I am pleased with the authors revisions, especially with how the addressed the statistical issues. The authors have done a great job on this manuscript. I made a couple of very minor edits which are noted on the attached PDF. Experimental design: Addressed concerns. Validity of the findings: Addressed concerns. Additional comments: Good job!

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: WHOLE GENOME SEQUENCING OF RHODOTORULA MUCILAGINOSA ISOLATED FROM THE CHEWING STICK (DISTEMONANTHUS BENTHAMIANUS): INSIGHTS INTO RHODOTORULA PHYLOGENY, MITOGENOME DYNAMICS AND CAROTENOID BIOSYNTHESIS Review round: 1 Reviewer: 1

Basic reporting: The text is well written and the article is consistently written in professional english. A few long sentences may be reframed to simplify the text for readers. Raw data is shared as required and results are well supported with evidences Experimental design: The methods used are sufficient for the article and the mitochondrial genome elucidation through 'genome skimming' is very innovative. However, I think the readers would like a more detailed account on 'recovery of the complete mitochondrial genome of R. mucilaginosa using the genome skimming approach' as there are few reports on this this and also this is the main attraction of this review. Thee should be more references on similar genome skimming methods used or on other methods for mitochondrial genome recovery. Otherwise, there are several reports on description of the genome and transcriptome of Rhodotorula. I didnot follow why the authors have focussed on Distemonanthus benthamianus in detail. I think the details should be minimized as the site of isolation has no effect on the description of genome. If there is some significance of this plant in this context then it should be clarified in the article Validity of the findings: Data is robust and although there is nothing much to be 'newly' reported, the mitochondrial genome recovery does give this article an edge above the other articles/reports published on genome of Rhodotorula. Additional comments: The results and discussion section and conclusion section can be made more informative by (1) comparison to other similar reports (2) more description on genome skimming (3) comparing the presence of carotenoid pathway gene(s) in yeast Rhodotorula and plants or bacteria, since the article is more focused on carotenoids production and its importance as a valuable anti-oxidant (4) I believe readers would also want to know details on "homing endonuclease from the LAGLIDADG and GIY-YIG families" and a section can be added on this

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: WHOLE GENOME SEQUENCING OF RHODOTORULA MUCILAGINOSA ISOLATED FROM THE CHEWING STICK (DISTEMONANTHUS BENTHAMIANUS): INSIGHTS INTO RHODOTORULA PHYLOGENY, MITOGENOME DYNAMICS AND CAROTENOID BIOSYNTHESIS Review round: 1 Reviewer: 2

Basic reporting: The manuscript describes the isolation of a putative endophyte from the plant Distemonanthus benthamianus, its identified as a strain of Rhodotorula mucilaginosa, genome sequencing and analysis of the genome. Primary observations from the genome were the annotation of the mitochondrial genome and identifying the candidate genes for synthesis of carotenoid molecules. The manuscript is a solid set of data. The following points are aimed to improve the text. A general point is that the preexisting literature has been someone sparsely covered in places, and that then limits some of the conclusions that can be drawn. For example: (1) Line 58 species within the genus and “validating the monophyly of the genus Rhodotorula” and line 307. Here consideration of the two Wang et al. 2015 papers in Stud Mycol. 81: 27-53 and 149-89 would be wise, since these were the study to clean up Rhodotorula, as until then the genus was polyphyletic. The conclusion on line 307 is too strong without all the species in the genus being included. (2) Lines 94-97: a key point is that the genes have been mutated and shown to be involved in synthesis of pigment. This was first done in the closely related genus Sporobolomyces (Abbott et al. 2013 Appl Microbiol Biotechnol (2013) 97: 283-295), and then the two genes in Rhodosporidium (=Rhodotorula) toruloides (Koh et al. 2014 BMC Microbiol 14:50; Sun et al. 2017 Biotechnol Lett 39: 1001-1007). (3) Strain RIT389 should be deposited in one or more culture collections, e.g. CBS. Text edits: Line 83: write as “resources for R. mucilaginosa are surprisingly scare in public databases”. Lines 83-84: JGI has also sequenced R. mucilaginosa strain ATCC58901. Given the discussion on the genus, it may be worth pointing out that there are genome projects for other Rhodotorula species. Line 91: “in at least three different fungal species” is too vague. Lines 107-108: Replace “A relatively recent study showed that” with “The”. Lines 192-193: how the identification was may should be clearer, i.e. PCR of ITS and sequencing. Lines 197-198: there appear to be two unrelated points in this sentence, so better split into two. Line 213: add “levels” for “carotenoid levels when”. Line 226: “i.e.” in place of “e.g.”. Lines 233, 235 and 239: spelling of “toruloides”. Lines 237 and 241: using “Rhodotorula sp.” would be better. Line 274: “Identification of a genomic region”. Lines 280 and 281: better with the addition of two commas, as synthase, and carotenoid, . Line 304: “databases” plural. References: check for formatting, e.g. italics on species names, remove capital letters in article titles. Experimental design: Lines 187-192: (a) This section needs more background on the isolation of the microbe from the chewing stick. (b) While the description may have been what happened, it does not present the authors in a strong light. SEM is over-kill to show something is not a bacterium. Validity of the findings: See comment 1 in section 1 about the claims on monophyly of the genus Rhodotorula. 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: SNAPSHOT RECORDINGS PROVIDE A FIRST DESCRIPTION OF THE ACOUSTIC SIGNATURES OF DEEPER HABITATS ADJACENT TO CORAL REEFS OF MOOREA Review round: 1 Reviewer: 1

Basic reporting: The article is clearly written and provides appropriate references. The figures make sense, although I would like to have supplemental material with a few acoustic recordings so that we could listen to the different soundscapes. Experimental design: I find 2 major flaws with the design which limit the conclusions one can draw from the study: 1) Temporal window: I am concerned that recordings were only made during daytime hours. I think this is a big oversight. You mention that there is high variability during nighttime and early morning hours – but this is exactly when you may see larger differences between habitats. For example, if a species of soniferous fish lives only in deeper water, but they only sign at night, then you would not pick up this difference between depths/habitats by missing their prime singing window. 2) Temporal replication: I understand that perhaps logistical or financial constraints were at play here, and the investigators may not have had access to longer-term recorders or the ability to dive in deeper waters. But 10-minute recordings during subsequent mornings is not very high replication. I think the authors should emphasize the “snapshot” nature of these recordings and put this caveat in their results and possibly in the title. That said, there is still value in this work, but it doesn't knock your socks off in terms of novelty or a grand contribution to the field. There are also a few points of clarification needed: -exact water depth of recordings (in a table) -why no inclusion of higher frequency sounds -how the spectral averaging was done -did you look/listen to recordings and get any explanation for some of these differences/sound sources? One other point about experimental design/analysis: Did you try using any of the newer eco-acoustic indices like acoustic complexity, acoustic dissimilarity, etc? I am guessing that you may have tried, but found no significant patterns or perhaps even misleading results. You would not be alone – several others have tried and have learned that these indices are not “ready” for the marine environment yet. There was a lively discussion about this at a recent ASA meeting, and a recent paper by Staaterman et al (2017, MEPS) demonstrates an example in which the indices don’t work as expected. I know many authors are reluctant to put null results or confusing results into a manuscript, but I would urge you to include these analyses anyway. It would bring further emphasis to the fact that these indices require further ground-truthing for the marine environment. Validity of the findings: Some of the interpretation of the differences between habitat types and distances from shore need to be refined. There needs to be a discussion about the propagation of low-frequency sounds in shallow water. Otherwise, the conclusions are well-stated and reasonable. Additional comments: Given the fact that the authors probably cannot go back and redo these recordings, or perhaps were limited with their instrumentation, it's not reasonable to ask them to increase replication. This is a small study but still a valid contribution to the field. For this reason I am putting "minor revisions" rather than "major revisions". But I would like to see them try running the newer eco-acoustic indices (with existing data), and to clarify about some of their methods for spectral averaging etc. And I encourage them to think about higher spatio-temporal resolution in future work, if time and money allows. small comments here: Introduction – line 48: you start two sentences in a row with “as an example” Methods: - line 93- is Tiahura a natural canal or man-made? Why are there no corals there? Line 110: I am concerned that recordings were only made during daytime hours. I think this is a big oversight. You mention that there is high variability during nighttime and early morning hours – but this is exactly when you may see larger differences between habitats. For example, if a species of soniferous fish lives only in deeper water, but they only sign at night, then you would not pick up this difference between depths/habitats by missing their prime singing window. Please give more specifics about the water depths where recordings were made. I see that you have the range of water depth for each habitat type, but this range is pretty big (~20m). Methods: so the boat was drifting, not anchored? To me this is another potential source of error. I have made recordings of the hull of a boat and the slapping/lapping of waves is indeed a problem. Were you not able to deploy passive recorders for longer periods?? How did you deal with these lapping noises? Why did you not include analyzes of higher frequencies (snapping shrimp band)? I understand perhaps more interest in looking at fish vocalizations, but it would not be hard to also include snapping shrimp here. I understand that perhaps logistical or financial constraints were at play here, and the investigators may not have had access to longer-term recorders or the ability to dive in deeper waters. But 10-minute recordings during subsequent mornings is not very high replication. I think the authors should deemphasize the “snapshot” nature of these recordings and put this caveat in their results and possibly in the title. Lines 143-152: please clarify language here. It is not clear how you got the 12 samples. Also, please explain the “averaged for each sampled frequency” – did you not have power spectral density in 1-Hz bands? From the way your axes on the plots are labeled, it appears that you did not have 1-Hz bins and some averaging across frequencies took place. Please explain what bandwidths were used. Does the recorder automatically spit out SPLs and not the raw waveforms? This part needs clarification. Line 163: I find the phrase “distant habitat” misleading here. You mean distance from shore, I assume, but you may also mean distance from other habitats. I would instead say “deeper habitats”. If you don’t mean distance from shore, this would imply that your hydrophones were not truly “in” the environment you claim to be sampling. Lines 222-234: I think some discussion is required about the propagation of low-frequency sounds in very shallow water. Since transmission of these sounds is restricted by water depth, you would actually expect to have lower sound levels in these lower frequencies for the shallowest sites. The fact that you got the opposite demonstrates that there is a greater contribution from geophony or biophony. Line 235-237: I don’t see how your results lead you to this conclusion. Higher sound levels at different habitats along transect 1 could mean more sound-sources at the other (non-reef) habitats, which you state later in the paragraph. I would remove this sentence or rephrase it. I would also mention later in this paragraph the fact that deep water enables transmission of lower-frequency sounds, so in addition to the fact that there may be additional sound sources in the reef slope/sandy area (fish), it may also have to do with the physics of the environment. Did you try using any of the newer eco-acoustic indices like acoustic complexity, acoustic dissimilarity, etc? I am guessing that you may have tried, but found no significant patterns or perhaps even misleading results. You would not be alone – several others have tried and have learned that these indices are not “ready” for the marine environment yet. There was a lively discussion about this at a recent ASA meeting, and a recent paper by Staaterman et al (2017, MEPS) demonstrates an example in which the indices don’t work as expected. I know many authors are reluctant to put null results or confusing results into a manuscript, but I would urge you to include these analyses anyway. It would bring further emphasis to the fact that these indices require further ground-truthing for the marine environment. A note for future work: I have been to Moorea and noticed the big cruise ships that come through. I would be curious to know how the propagation of the ship noise differs depending on geographic location, again due to the physical constraints of shallow vs. deep water. I would suggest you put out some passive recorders at different depths/transects along the reef slope and see what happens when a ship comes through. Would also be interesting to put some inside those two bays/cracks in the island. Figure 5: could be displayed better – consider using color and putting all on one plot, or adding another panel with average for each habitat type. Would be nice to have supplemental data with recordings from each habitat type.

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: SNAPSHOT RECORDINGS PROVIDE A FIRST DESCRIPTION OF THE ACOUSTIC SIGNATURES OF DEEPER HABITATS ADJACENT TO CORAL REEFS OF MOOREA Review round: 1 Reviewer: 2

Basic reporting: Intro and background are sufficient to outline the problem. In general, the manuscript would benefit from review from a native English speaker for improved clarity and word use. A summary table of the raw data was supplied (although the raw data itself, i.e., wave files) was not. I am not sure if this conforms to PeerJ’s standards. Figures 3-4 are non-normative and I am not sure what they add in terms of interpretation. Experimental design: I have three major problems with this study. Data collection during the day is obviously totally insufficient because this is not, as the authors note, the time when most soniferous organisms on reefs are active. Furthermore, it seems from the methods as if the transects were conducted sequentially and within transects the stations were visited south to north. Then this was replicated in the same way two more times. If that is true then there is a major bias there in terms of the times at which each station and transect were sampled. Finally, the authors attempt to compare the recordings they made to habitat type but bring no evidence of the fauna resident in each habitat type. For these three reasons, I cannot recommend publication of this research. Validity of the findings: For the reasons stated above, I do not interpret these findings as valid. Additional comments: Lines 82-83: “highly specialized habitat requirements” – such as? Lines 82-85: the logical link between “highly specialized habitat requirements” and variations in low-frequency SPL is not clear. Suggest rephrasing. Line 94: “noisy” – first of all this is somewhat subjective, but 90 dB is not, in my opinion, “noisy”, or, more appropriately, high amplitude Lines 98-105: could the authors please bring evidence to support their claims about the species richness of each of these habitat types. Did they conduct visual surveys? Or is this conjecture? Lines 131-133: The close to shore station was always visited first in each transect and replicate? If sound levels change throughout the day then this would introduce a major bias into these results. Did the authors not put recorders at each station for the entire day or ideally over several diel periods? If not, there is no way to know how sound levels were changing during the sampling period. Results section: I found this entire section very hard to understand. Lines 231-234: True, it is very important to consider the physical environment and its influence on ambient sound. Because the authors do not have visual survey data to link to fauna at each reef, it seems as if purely physical differences among the stations would be the most parsimonious explanation of the differences found. Lines 258-262: I agree with the statement here that the short sampling period is insufficient to properly characterize these habitats. A large body of literature now exists that points to the importance of diel sampling because of how dramatically sound levels can change even over short temporal scales.

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: SNAPSHOT RECORDINGS PROVIDE A FIRST DESCRIPTION OF THE ACOUSTIC SIGNATURES OF DEEPER HABITATS ADJACENT TO CORAL REEFS OF MOOREA Review round: 1 Reviewer: 3

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: The Ms provides a first insight into the acoustic features of different habitats adjacent to the coral reef surrounding the north coast of Moorea (French Polynesia). In particular, it focuses on the description of soundscape signatures (sound energy along frequencies) along three transects of four recorded points each (barrier reef; sandy plain; reef slope; drop-off). The Ms is well organized and the results produce interesting advance to our knowledge of the field. These baseline data are strongly needed to produce a benchmark that could be used as a reference for future biomonitoring studies. Here I provide a list of recommendations to improve the MS. The authors have provided a good temporal and spatial diversification of samples by investigating three points for every habitat, and replicating in three different days. Anyway, the total amount of recorded minutes in each of them is quite limited. Soundscape holds a great deal of variation over time of the day and over days. It could substantially change either to the time of the day in which recordings were taken (for instance many fishes vocalize in specific temporal windows) or may also vary from one day to the next (see Pieretti et al. 2016). In order to capture a reliable example of the soundscape of the area, longer recordings would be the optimum solution. The authors’ dataset is limited to 12minutes (divided in 4mins in three replicates) for every recording station. That makes 36 minutes for every habitat type. Moreover, authors do not fully specify the recording time of the day (line 110: Recordings were conducted between 09:00 and 16:00). I still consider the collected data a useful and needed starting reference for future studies, however in the discussion I suggest to explain this limitation (adding details at line 261) or to the reasons that have led authors to consider it as less important (maybe in these tropical environments soundscape fluctuations are weak). I recommend that a native English-speaking colleague review the Ms. In fact, examples of some errors and typos along the paper are at line 93 (depths), 110 (recordings), 181 (did not differed) or the sentence at line 101-103 should be rephrased. The statistics should be better presented and clearly explained. At lines 153-160, the current paragraph makes its understanding challenging. I suggest specifying the number of cases (N) - embedded in the text or in the caption of figures – so that the reader can easily understand which data have been considered for each analysis. Others: Keywords are missing. Line 143: FFT 1024 on the 48kHz? (line 130) Figures seem to have a higher resolution in frequencies. Line 143-144: I would explain why this step was taken Line 173: I would refer to figures before Line 179: from Lines 224-226: it was not stated earlier Figure 1: rescale the distance among transects; the distance among 2 and 3 seems to double the distance of transect 1 and 2 Figure 5: Caption: refer to the statistics used Dr. Nadia Pieretti Dipartimento Scienze Pure e Applicate - DiSPeA Università degli Studi di Urbino "Carlo Bo" Campus Scientifico Enrico Mattei Località Crocicchia 61029 Urbino Italy

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: COLOUR CHANGE OF TWIG-MIMICKING PEPPERED MOTH LARVAE IS A CONTINUOUS REACTION NORM THAT INCREASES CAMOUFLAGE AGAINST AVIAN PREDATORS Review round: 1 Reviewer: 1

Basic reporting: The paper is well written and covers the relevant literature. Experimental design: The experimental design is suitable, though as mentioned below, the authors should justify the switch from spectroscopy to objective imaging (without animal-vision) for the heterogeneity experiment. The authors use the term “chroma” interchangeably with hue without justifying why. In any case, this should be changed or clarified, and will not affect the overall interpretation. Validity of the findings: The findings are valid, though there are some areas that require clarification, or could be improved: A tweak in the “greenness” calculation used might improve the findings and reduce statistical dispersion issues (see general comments). I think the authors should be much more careful when asserting that the larvae were not adopting an intermediate colour in the heterogeneous treatments as they are unable to prove that the background is heterogeneous from the larval perspective instead of plausible mechanistic or behavioural alternatives. Statistics: using ordinal statistics might offer a more clean way to analyse the result of different treatment levels (e.g. cumulative link models using the ‘ordinal’ package in R allows for mixed models), though I think the author’s approach is ok. As mentioned above though, the ‘greenness’ measure used creates a large skew in the dataset, which means the green treatment will have a disproportionally more powerful influence on the fitted models than the other treatments. In practice the author’s findings are very clear in that there are intermediate levels of greenness and luminance on the different treatments. Additional comments: Why was photography used for the heterogeneous treatments, while spectrometry was used for all other treatments? This needs justifying. The images were not converted to bluetit cone-catch quanta images, so will be different to the spectrometer-based measures and the authors should make this more clear to the reader. The use of imaging means the authors could also look at within-individual heterogeneity. e.g. rather than measure average larval colour, they could see whether larvae on heterogeneous backgrounds also have greater variation. To do this the authors could divide the G channel by the sum of RGB channels, then measure the standard deviation in pixel levels. Higher standard deviations would indicate higher within-individual variation in green and brown patches. This extra analysis would add some useful extra information, though is not essential. Habitat specialisation in the heterogeneous environments provides a plausible explanation for the finding that larvae tended to specialise on one background colour over another. e.g. the larvae wouldn’t be expected to switch to an intermediate colour if they could always choose to sit on a single background type. Given the authors were unable to track individual movements I think they should present this as the most likely strategy. If the dowelling were striped (so that the larva’s bodies are always crossing many colour patches) the intermediate strategy might be more believable, however even then it could be due to a simple mechanistic constraint that only allows the larvae to sense very small patches of their substrate colour near their eyes/sensing organs. L152-163 The definitions of colour and luminance used here are mentioned before talking about the visual system being used, and how luminance is calculated (i.e. bluetit double cone catch quanta under a given illuminant). Chroma would normally be the distance from the centre of a triangular (trichromatic) or tetrahedral (tetrachromatic) colour space, and hue would be the angle(s), but the authors seem to use chroma to refer to both hue and chroma (which is often called saturation). I think the authors should change their definitions to more standard ones, or justify why they are combining hue with chroma. To make matters more complicated chroma is later defined as ‘greenness’ in L176. L157-158 What exactly is being being measured in these statistics? Green and brown dowels would probably differ much more in hue more than chroma. L210 This calculation of a ‘greenness’ opponent channel has been used by others before, though using MW/(MW+LW) would generate numbers that are more biologically relevant (symmetrically bounded between zero and one, instead of asymmetric bounding between zero and infinity). The calculation of greenness from the photographs later on does this correctly. This might, for example, result in a better fit in figures 5 and S2, which shows a disproportionate effect by the most green treatment. L216-221 How were the images linearised (the authors should cite the paper and methods used to do the objective image analysis)? Figure 3 I think the y-axis should be labelled “larvae greenness” if this is correct. Also, the dowel measurements should be labelled and perhaps segregated more clearly from the larvae measurements. L240-241 and 254-256 The use of an asymmetric greenness measure (as mentioned above) means that these discrepancy values cannot be compared and should probably be replaced with something more meaningful. i.e. the greenness measure is certainly not a perceptually uniform colour-space in which to look at distances. Distances in tetrahedral colour space are also somewhat problematic as these too are not perceptually uniform. Calculating JNDs would be the easiest way to quote colour differences (and would probably be useful as it is biologically meaningful).

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: COLOUR CHANGE OF TWIG-MIMICKING PEPPERED MOTH LARVAE IS A CONTINUOUS REACTION NORM THAT INCREASES CAMOUFLAGE AGAINST AVIAN PREDATORS Review round: 1 Reviewer: 2

Basic reporting: . Experimental design: . Validity of the findings: . Additional comments: Colour change of twig-mimicking peppered moth caterpillars REVIEW GENERAL I am happy with this very interesting MS that has been thought out conceptually and tested carefully. Excellent literature review in Intro but seems rather duplicated in Discussion – you need only say it once, probably most of it in Discussion. I would like a slight change of emphasis in the Introduction to explain why it is so important to explore the issue of slow colour change. It makes an attempt at this but gets lost somewhere in the prose. You may want to cut the Intro length while doing this – it is really very long at present – aim for 3 paras. For example only last 2 sentences of first para really needed! Paras 2 and 3 are really about mechanism – not what is covered in this MS. There is remarkably little said about masquerade in the MS which is odd – after all, these organisms resemble twigs to our eyes anyway. Would we expect greater or less background matching in masquerading than non-masquerading species? Is it a synergistic trait, or a trade-off between shape and colour. Aren’t there comparable data with Kallima butterflies and striped skunks – and probably more that I don’t know of? You might want to discuss these alternatives ways of looking at the colour/shape masquerade relationship in the Intro? It would be good to have more natural history: are these larvae sedentary on one plant – are there different parts of the plant with different colours? What are the challenges faced by individual larvae in terms of colouration? Are there any data on this model species that pertain to larval colour and the industrial pollution story? Figure 1 excellent SPECIFIC 23 larval stages unclear 35 rigorously is a bit strong 168 what is a B&Q? 291 How do you know visual capabilities? 304 ref not in italics 360 any anecdotal evidence for weak selection? Table 1 to SOM Tim Caro

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: COLOUR CHANGE OF TWIG-MIMICKING PEPPERED MOTH LARVAE IS A CONTINUOUS REACTION NORM THAT INCREASES CAMOUFLAGE AGAINST AVIAN PREDATORS Review round: 2 Reviewer: 1

Basic reporting: basic reporting - pass Experimental design: experimental design - pass Validity of the findings: Validity of the findings - pass Additional comments: The authors have been very thorough in addressing all of my concerns and queries, and I am happy to recommend acceptance. They have done a good job of acknowledging the minor limitations of their study, and making these clear to 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: COLOUR CHANGE OF TWIG-MIMICKING PEPPERED MOTH LARVAE IS A CONTINUOUS REACTION NORM THAT INCREASES CAMOUFLAGE AGAINST AVIAN PREDATORS Review round: 2 Reviewer: 2

Basic reporting: . Experimental design: . Validity of the findings: . Additional comments: I have carefully read the revised manuscript entitled: Colour change of twig-mimicking peppered moth larvae is a continuous reaction norm that increases camouflage against avian predators. The authors have made considerable efforts to address the points raised by the editor and 2 reviewers, and I am happy with the outcome. I am not sure if the Conclusion section is needed but otherwise it is ready 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: GENERATION AND CHARACTERIZATION OF CROSS NEUTRALIZING HUMAN MONOCLONAL ANTIBODY AGAINST 4 SEROTYPES OF DENGUE VIRUS WITHOUT ENHANCING ACTIVITY Review round: 1 Reviewer: 1

Basic reporting: The report is clear and contains relevant results. However, language could be improved and revision is needed. Experimental design: The research question is well defined and the results support the conclusion. Some terminology would have to be spelt out. Validity of the findings: Although the study data is robust, the impact and novelty of the results are not immediately clear. While there are many findings on neutralizing antibodies to DENV for therapeutic usage, the authors would need to highlight the utility and novel functions of the antibody in this study. Additional comments: Major (1) The manuscript was well written and the experiments are well defined and support the conclusion of the study. While neutralizing antibodies are critical in disease protection, your introduction needs to be further clarified as to why the epitope characterization is needed, as some part of the study has been done by other investigators. The objective would need more clarity, for example, are you performing the experiments to develop a potent neutralizing antibody with no ADE activity or are you trying to determine the antibody epitopes? (2) Because the antibody used was developed from a previous study, please clarify which parts of the study (if any) contains data from the prior study. (3) It is known that ADE activity hampers neutralizing activity of an antibody. Please explain why both the modified and original antibodies demonstrated similar NT levels, despite the absence of ADE activity in the modified antibody. (4) Would the concentration of antibody needed for virus neutralization/disease protection be feasible? How much antibody would be required to neutralize DENV at different MOI? Minor (1) Try not to use abbreviations in the title, it would be better to spell out ADE. (2) The manuscript needs to be revised for spelling and grammar. Some examples where language could be improved include lines 239, 246, 292, 319. (3) Describe PhD-12 and C7C. Were they the identification codes for phage display?

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: GENERATION AND CHARACTERIZATION OF CROSS NEUTRALIZING HUMAN MONOCLONAL ANTIBODY AGAINST 4 SEROTYPES OF DENGUE VIRUS WITHOUT ENHANCING ACTIVITY Review round: 1 Reviewer: 2

Basic reporting: Paper is poorly written, English is not clear and unprofessional. Many grammatical, incomplete sentences, poorly chosen words, and conceptual mistakes (e.g humeral instead of humoral) of the English language are found through all the paper’s length. For example (and only 3 examples): Lines 108-109 Purified B3B9 HuMAb was used in phage display panning experiment to characterize their binding epitopes Lines 286-288: After comparing this peptide with the amino acid sequences of E proteins of DENV1–4, it was found that all these LXXXG motif was matched to 107LFGKG111… Lines 393-394: Moreover, N297Q mouse MAb can competed enhancing antibody in polyvalent serum in in vitro study (Williams et al., 2013). A proofread procedure by an English native speaker is strictly needed. The title should include the specificity of the MAb characterized to enhance the relevance of the study. For example: “Characterization of aglycosylated human monoclonal antibody targeting the fusion loop of dengue E protein shows strong neutralizing and no enhancing activity“ Introduction: The introduction is lacking important information: 1. The IgG glycosylation patterns and its interaction with the Fc-gamma receptor is needed in order to expose the importance of the Fc modification in the Mab. Literature from Nimmerjahn F and Ravetch is suggested. 2. “Dengue disease, transmitted by mosquito-borne infection,…” is poorly described. Dengue virus is transmitted by a mosquito, dengue disease is the result of this infection. 3. The aim of the study written in the last paragraph is confusing and a stronger statement regarding the title of the manuscript is suggested. Through all the manuscript the main objective of the paper is not clear. Even though there was a lot of work invested and the results are interesting, the paper lacks the proper communication of its message and there is not a clear relevance of its findings. Figures: Figure 1c, 2 are not of high quality, they look blurry. In Figure 2 is the control just PBS instead of the antibody, a proper control is a staining including non-infected cells. In Figure 4. Please show first the ADE in K562 and in B the THP-1 as it is explained in the text. It may seems that A is B and B is A if the methods and results portion is correct. K562 ADE was measured by counting infected cells and ADE in THP-1 with RNA quantification. Which by the way, is not a quite accurate method for measuring Dengue, since there are a lot of RNA products that are not part of an infectious virus particle. Hypothesis and scientific question is not clearly expressed and needs to be more accurate. Experimental design: Even though the paper is interesting, it is a purely an instrumental paper constructing a potential anti-dengue therapeutic antibody, but it is of concern if it is enough for the PeerJ journal. Maybe if the paper would be improved in clearly stating its objective, it can be taken for consideration for publication. A more profound justification of the reason for choosing the Phage display methodology is needed. Concentrations and virus moi are missing. In Figure 1, an antibody without the specificity in the competititve Elisa is needed as a negative control. The research question is not well defined. From the results, the reader can infer the biological relevance of the paper but it is not clearly stated. The exact reference in some cases is missing, for example, on has to infer the original work showing the origin and description of the Mab B3B9. Results are written with scarce findings and conclusions. Some controls are lacking. For example: - demonstrating the absence of glycosylation in the Ab is needed. - showing the non/binding to the Fc gamma receptors by FACS is missing. - The specificity of the MAb should be confirmed by an assay showing the interaction between the MAb and the fusion loop. - Treatment of the original MAb with enzymes that removes the glycosylations would give more robustness to your results. Validity of the findings: The potential impact and novelty in this manuscript is not assessed by that authors. Even though there is a potential newly produced therapeutic antibody, the paper lacks to send its message. The asseverations of having constructed a new therapeutic antibody are ambitious regarding the results that they are showing. No proper controls are shown. The biological relevance of an antibody cannot be limited only to its neutralizing or Fcgamma receptor binding. Therefore, the paper needs to be concluded in a more conservative way. Additional comments: Even though the paper is not well written, the message is not clear and the biological relevance of this new antibody is purely speculative, if the paper is rewritten in a clearly manner, some of the methods and results are more extensively described, if in the discussion they will be able to suggest the potential of this Ab but lowering their asseverations, the paper could 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: GENERATION AND CHARACTERIZATION OF CROSS NEUTRALIZING HUMAN MONOCLONAL ANTIBODY AGAINST 4 SEROTYPES OF DENGUE VIRUS WITHOUT ENHANCING ACTIVITY Review round: 2 Reviewer: 1

Basic reporting: The manuscript was improved, but it is still no publication material. Even though they included necessary information sometimes ill positioned in the manuscript, English is still not at a level for publication. For example, and just one example of many throughout the manuscript, cells do not "contain" a receptor, cells express a receptor. Scientifically the paper is ok, but the written manuscript is poorly explanatory, with many grammatical mistakes, many sentences without any sense. I still need a more thoroughly revision of the literature about Glycosylation of antibodies and how this affects their function. Also, why the phage displayed was used for the detection of the antibody specificity. In the results section, the subtitles have to show a finding, not merely described what they have done to achieve this finding. Negative controls are intended to be part of the figures. In the IFA figure, the negative control of mock infected cells detected by the antibodies are not included. ADE was not originally described by Schmidt, but by Halstead. Please revise. It is Ab dependent enhancement. please revise through all the MS. I still think that the paper is publicable, but has to be improved. Experimental design: I think that some of the experimental design is lacking. For example to include the negative controls in the figures. I do think that they need to show still Fc binding of both antibodies and if they eliminate the glycosilation of the original one, do they obtained the same results as the new one? When something is Similar...it has to be similar to another thing. a lot of results are just described as "similar"...to what? I do not agree in just using a predictive software for glycosilation, or lack of it. But if they would give more emphasis in showing that just this point mutation is sufficient for an antibody to loose its glycosilation, then I can accept it. Regarding their DNA PCR product electrophoresis for IgG subtyping, how can you differentiate between a band of 211 bp for IgG1 and 207 bp for IgG3? Some trivial Methods are described extensively, and most important methods are not. As a virologist, moi description is mandatory for a FRNT test. Validity of the findings: The results are ok. Data is ok, conclusions can be more extensive and emphatic. But their English level do not allowed the reviewer or the reader to understand everything thoroughly. One examplae of many: "To determine NT and ADE activity in Fc receptor bearing K562 cell, B3B9 rIgG was able to neutralize DENV2 and 3, but not DENV1 and 4".There is no sense in this sentence. Additional comments: Please give the manuscript to an English native speaker for revision. In such a state at it is know, publication is not possible.

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: GENERATION AND CHARACTERIZATION OF CROSS NEUTRALIZING HUMAN MONOCLONAL ANTIBODY AGAINST 4 SEROTYPES OF DENGUE VIRUS WITHOUT ENHANCING ACTIVITY Review round: 3 Reviewer: 1

Basic reporting: The authors claim that the manuscript was proofed by a native speaker and by a professional service. The manuscript has improved, but there are still some mistakes and poor choices of words (I marked everything that needs to be changed in the enclosed document) Results are still relevant and can be published. Experimental design: There are some experimental details that need to be stated. Please take a look in the document attached. Validity of the findings: The last sentence is a bit too out far reaching stating that the antibody produced by the authors can be used in therapy. I would say that it can be used in models testing for a possible anti dengue therapy. Additional comments: Manuscript has improved, but needs still some polishing before 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: GENERATION AND CHARACTERIZATION OF CROSS NEUTRALIZING HUMAN MONOCLONAL ANTIBODY AGAINST 4 SEROTYPES OF DENGUE VIRUS WITHOUT ENHANCING ACTIVITY Review round: 4 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: Lines 460-463, the authors should indicate that these are in vitro results and it is a proof of concept. Additionally, the authors should be clear on potential "problem" of a human anti-mouse antibody response. The sentence should be reworded for clarity and good scientific presentation. Lines 464-468, the authors should be clear on the conclusion. Try to include the phrase "with further studies on...." and emphasize on the potential utility of the antibody. In the abstract, please remove the word "fortunately". Additional comments: Generally the manuscript is in good order, the authors would need to do a thorough proof-reading, with a native English speaker. There are some minor grammar mistakes throughout the manuscript, eg line 396 (balancing vs balance); 472 (advisement vs advise).

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: VEGETATION STRUCTURE OF PLANTAIN-BASED AGROSYSTEMS DETERMINES NUMERICAL DOMINANCE IN COMMUNITY OF GROUND-DWELLING ANTS Review round: 1 Reviewer: 1

Basic reporting: This manuscript is generally well written and the data have been subjected to sound statistical analysis. There are some textual flaws and I have annotated these on the manuscript. I am not entirely comfortable with the use of the term 'plantain based agroecosystem'. If cocoa or coffee is present then aren't such farms likely to be cocoa or coffee farms with some plantain interplanted? In other words, is plantain always the main crop in the farms that were studied? What concerns me is the lack of ant identifications to species level, with one exception. I suspect that the Phedole app. are members of the megacephala group. As presented, the reader is unsure whether this study represents a set of native ants or whether the fields are colonised by 'weedy', cosmopolitan ant species. I would have thought that this was essential information. Experimental design: Since the ultimate thrust of this work is to see if farms harbour useful biological control ant agents, this study has missed an important opportunity to include the sampling of arboreal ants. Baits could easily have been placed in the plantain, although I doubt if plants would occur in each of the 25 subplots. This would therefore demand a somewhat different analysis. As this manuscript stands, the are copious implications about arboreal ants and control of tree pests, without actually having looked at the arboreal system. Maybe this has been done in the thesis, but if so surely some data could be brought into this manuscript. Validity of the findings: The findings presented here are valid but not as profound as they could be due to a) lack of species-level identifications and b) lack of data on arboreal ants. Additional comments: lines 129-133. It is not clear how you qualified dominant versus subdominant species. I realise that this is embedded in the text here, but you really need a separate sentence for definition of dominant species and another for subdominant species. Figure 1. This should be in colour as the grey tones are not well differentiated. Line 201. Refers to functional groups, yet nothing is said about this in the Methods nor in the results. Lines 231-232 and elsewhere in the Discussion. You refer to species dominating the area, yet you have previously dismissed most of these species as being dominants. Change terminology to ''predominating in the area' or something else that does not clash with the word dominant. Many other editorial matters are marked up on the 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: VEGETATION STRUCTURE OF PLANTAIN-BASED AGROSYSTEMS DETERMINES NUMERICAL DOMINANCE IN COMMUNITY OF GROUND-DWELLING ANTS Review round: 1 Reviewer: 2

Basic reporting: This is a manuscript about how vegetation densities at high and intermediate strata can change the numerically dominant ground foraging ant at a local scale in plantain polycultures. For the most part, this manuscript uses clear, unambiguous, and professional language. The Figures are relevant and layout is fine. Raw data is included. However, the manuscript could be improved by considering the following: Line 27: instead for “cropped in association” you may want to consider using the terms “intercropping” or “grown in a polyculture” (also applicable for line 78) Lines 47-49: It would be clearer if this were rephrased to “Ecological factors, which are the focus of the present study, can include both ecological interactions (e.g. foraging interference) and habitat-related factors (e.g. nesting sites). In general it would improve readability and make the manuscript more succinct if the study examples were restructured to include the citation at the end. For example Lines 57-60: This sentence would be clearer if rephrased to something like “For example, in South American savannas tall grass cover has been shown to reduce the incidence of the dominant ant species, Solenopsis substituta (Santschi) (Vasconcelos et al. 2008). Line 73: change “proved” to “showed” or “provided evidence” Line 76: change “understorey” to “understory” Line 122: Add a period after “dominance” Line 127: Remove “of” Line 133: change “genus” to “taxa” Line 134: remove “species” Line 135: change “genus” to “taxa” Line 154: change “(Table 1)..” to “(Table 1).” Line 155: change “genus” to “taxa” Line 158: change “species or genus” to “taxa” and remove “as” Line 177: should be “Abera-Kalibata et al. (2007) found that two morphospecies of Pheidole were among the most abundant” Line 183: change “with a recruitments” to “with worker recruitment” Line 219: change “genera” to “genus” Line 239, 240, and 243: change “habits” to “strategies” Experimental design: The manuscript provides original primary research within the aims and scope of the journal, however there are several areas where the methodology needs clarification: Lines 117-120: Only half of the ant taxa you report are identified to morphospecies in your results, the rest are identified to genus. Your methods should be fixed to reflect this, and explain your reasoning (why not identify them all to morphospecies?). Line 124: Your abstract (Line 29), indicates that you deliberately excluded less abundant ant species (which would explain what appears to be surprisingly low diversity in your reported ant taxa). Please clarify if that is the case and include it in the methods if it is. Lines 127-134: Baccaro et al. (2010) used somewhat different dominance parameters than those used in this ms ( >5% bait presence, >25% bait control, and a minimum mean abundance score of 4 compared to your >10% bait presence, >25% bait control, and a minimum mean abundance score of 3.5). Thus with the methods in Baccaro et al. (2010) none of your ant taxa would be “dominant” (Pheidole spp. has a mean abundance of 3.76). However, if you had lowered the bait control parameter instead of the mean abundance score parameter, the Axinidris sp. would have be “dominant”. I think it would improve the manuscript if you provide an explanation for your reasoning in changing these parameters from the original methods and why this measure of dominance is meaningful in general. Line 121: What do you consider “control” of a bait to be for this study? Validity of the findings: Validity of the findings: Many of the findings appear sound, however I have some concerns about the following: (1) Pheidole is a large genus, and it’s possible that you have a large number of rare species masking as a single abundant taxa, I think this is important to address. (2) Ant biocontrol of pests occurs at the level of the plant leaves, rather than on the ground, and ant dominance will often be quite different on plants than on the ground, this is also important to address in your discussion section. (3) “Dominance” has so many meanings, several of which you use throughout your manuscript. I think it would be useful to clarify why you chose to use your two different measures of dominance (rather than just numerical dominance at the local scale and overall abundance at the broadest scale) or conversely why you didn’t consider bait monopolization at the local scale in your vegetation strata analysis. Additional comments: All general comments are 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: VEGETATION STRUCTURE OF PLANTAIN-BASED AGROSYSTEMS DETERMINES NUMERICAL DOMINANCE IN COMMUNITY OF GROUND-DWELLING ANTS Review round: 1 Reviewer: 3

Basic reporting: Gbéblonoudo et al. have accomplished one manuscripts deals on an interesting topic, with a great effort to collect vegetal information in different strata in plantain crops, however I have some concerns about ant datasets, analyses and description of the results. I think that english must to be check. Experimental design: The design is ok but I'm not sure if evaluate dominance of ant species using only genera is the most correct way.i'ts Validity of the findings: Important results can be found but i think that a the ant taxonomy to specie level shoul be revised. Additional comments: Gbéblonoudo et al. have accomplished one manuscripts deals on an interesting topic, with a great effort to collect vegetal information in different strata in plantain crops, however I have some concerns about ant datasets, analyses and description of the results. Line 39: All your Key Words are present in the tittle. I would suggest that you can make better use of this part to give more ideas of your work and results. Par exemple: Cameroon, Formicidae, density, abundance, baits, vegetation strata, Dominant, subdominant, and subordinate ants, Musa etc, etc…… Introduction Line 47: humidity and temperature are environmental factors not physiological. Maybe you can say that these factors affects ant physiology…. please rewrite this idea to be consistent. Line 47: To be more direct this phrase can be re write as ….. “and ecological factors (Philpott and Armbrecht, 2006), that include ecological interactions (e.g., foraging interference) and habitat-related factors (e.g. nesting sites), which are the focus of the present study. And please if you can, include some references for each factor mentioned. Line 56: (hymenoptera: Formicidae) can be deleted Line 60: Idem Line 62: The phrase: “but that dominance by ants in the Dolichoderinae was higher in pastures” have not sense….. maybe you want to say that: ‘…..but dominance of Dolichoderinae ants was higher in pastures”…. Line 79: Please include a reference at the end of the phrase. M&M Line 67: I’m pretty sure that the phrase is ‘ant diet varies….’ instead ‘Ant diets vary…’ Please check the English. Line 116: Here you speak about species and morphospecies but you don’t give information of them. Line 119: delete “the” Line 122: Please you should include one dot before “We” Line 118: Rewrite as follow: according to Fisher and Bolton (2016)….. Line 127 to 129: you say that you follow Baccaro et al. 2010 and Carval et al. 2016, but they use the specie and morphospecies level in theirs analysis. Why do you decide to use genera level? Can you explain your decision of use genera instead of specie level? As far as I know, is important to take in account the variation inside the genera because one genera can have many species with different diets, behavior, etc, for example inside the genera Pheidole, different species can have marked differences in terms of resource exploitation, (one sp. can be more dominant than others) and in my opinion this is an important factor to take in mind. Also, in some place you should state how many baits did you put on the field, or say the exact number of subplots and say that in each one your put a bait, it’s seem simple but this information is really important. Results You must to begin with a general phrase saying the total abundance of species (genera in your case). Also the number maximum the genera par bait or the minumun… etc… this information is also relevant. Line 154: Please delete one of two dots between (Table 1) and Pheidole….. Line 154 and 155: ‘Pheidole spp.were identified as the dominant genus’…. Maybe this could be due to the fact that is the most diverse genus? Line 153 to 158: You should give the data information of how much each one controlled the bait. And know that this in the table 1 too but you can also give data about the number of baits or the number of workers in each bait, etc…. Line 160: “frecuency” instead “frecuencies” please check the English. Line 164: I’m not sure that the word collected is the most appropriate maybe is ‘recorded’… Discussion Line 178: please use a contrast connector between ‘Uganda’ and ‘We’ …. Juste to differentiate two ideas. Line 184: Please use “on baits” instead “at baits” Line 191 to 197: And how you explain those results… less/more resources to nest, or to feed, etc???? Line 213: “…..in our study, negatively related to the density of arboreal habitats”… I think that this is due to the sampling method used, this is expected when you use soil baits, but if you use arboreal baits? Or if you follow your soil baits in the time…. In those cases the results can show that different species can visit the bait…. Its important to make the difference…. Line 232: Axinidris sp. dominate the area (Vasconcelos et al., 2008)… I’m not sure that this work can say something about Axinidris (an African ant), when they mainly works in Amazonian landscapes. Line 235 to 236: See how is important the specie level? You cannot make a generalization in term of diets or trophic level with certain ant genera, as Pheidole. I like so much the figure 2, its very informative but I think that you show explain it better in the text. A general conclusion is missing. Please check also these references, that can be useful: - Achury et al 2012. Psyque. “Effects of the Heterogeneity of the Landscape and the Abundance of……….” - LeBrun et al 2007. Ecology. An experimental study of competition between fire ants and argentine ants in their native range

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: VEGETATION STRUCTURE OF PLANTAIN-BASED AGROSYSTEMS DETERMINES NUMERICAL DOMINANCE IN COMMUNITY OF GROUND-DWELLING ANTS Review round: 2 Reviewer: 1

Basic reporting: The authors have attempted to address the comments of the reviewers. It is now a serviceable piece of work, although a few edits still need to be attended to, including one uncited reference. I have made comments directly on the manuscript, which appear in blue. I am still not overly excited about the study for the reasons I already mentioned, namely the lack of sampling of vegetation and the lumping of species. This latter has, to an extent, been addressed, but the analysis and figures still treats the species within a genus as lumped together. Experimental design: Adequate Validity of the findings: Adequate Additional comments: The authors have attempted to address the comments of the reviewers. It is now a serviceable piece of work, although a few edits still need to be attended to, including one uncited reference. I have made comments directly on the manuscript, which appear in blue. I am still not overly excited about the study for the reasons I already mentioned, namely the lack of sampling of vegetation and the lumping of species. This latter has, to an extent, been addressed, but the analysis and figures still treats the species within a genus as lumped together.

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: OVERLAND MOVEMENT IN AFRICAN CLAWED FROGS (XENOPUS LAEVIS): EMPIRICAL DISPERSAL DATA FROM WITHIN THEIR NATIVE RANGE Review round: 1 Reviewer: 1

Basic reporting: There are no doubt differences between South African English and US English, but I found the following to be noteworthy enough to include: Make a clear distinction between dispersion and migration Pp. 61, 62: no reason to cite a reference twice in the same sentence. p. 82: Poyton should have a date - a full citation p. 93: delete “When it occurs … Line 183: Why add the word “event” to movement? Not necessary, besides, “occurrence” may be a better term Line 218: This truth of this sentence depends on capture-recapture techniques used. If using drift fence/pitfall trap arrays, this statement is false. Line 240: Replace “to” with “from.” Line 272: How about “burrowed animals” or “buried animals?” This phrasing sounds microbial. Line 304: replace “further” with “farther.” Farther is the term relating to dostance. Lines 316-317: How can sites receive movement? Animals move. Line 324: Remove “do” Line 333: Move this to the bottom of the paragraph – the least important conclusion. Line 345: Delete “would like to” Also, much of the North American literature on migration, dispersal, and population dynamics is left out. There is one citation, though, you must incorporate: Smith, M. A., and D. M. Green. 2005. Dispersal and the metapopulation paradigm in amphibian ecology and conservation: Are all amphibian populations metapopulations? Ecography 28:110–128. You should also make a clear distinction between dispersion, migration, and perhaps habitat abandonment or some such other term. Experimental design: Very good. I have no issues. Validity of the findings: High, plus these findings are important. Additional comments: Nice design, important study, clunky word usage and at least one NA citation should be added.

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: OVERLAND MOVEMENT IN AFRICAN CLAWED FROGS (XENOPUS LAEVIS): EMPIRICAL DISPERSAL DATA FROM WITHIN THEIR NATIVE RANGE Review round: 1 Reviewer: 2

Basic reporting: No comment Experimental design: No comment Validity of the findings: I have some major concerns regarding on the statistical analyses used here. Additional comments: This manuscript examines the dispersal of a native population of Xenopus laevis. Most of individuals moved less than 200 m, with a maximal distance reported of more than 2 km. No sex-biased dispersal have been identified in this study. This is an interesting paper, for which the dispersal data of this invasive species might be very useful in models. The manuscript is well-written. I have some major concerns regarding on the introduction and the statistical analyses used here. I recommend the acceptance of this publication but after several corrections. I have detailed my major and minor comments below, and I hope that they will contribute to improving this manuscript. Introduction Page 6, Line 47. I suggest to change “Locomotion” to “Movement”. In my point of view, the term “locomotion” is commonly used related to the way to move (for instance S-shaped movement in salamander species), whereas the movement is linked to both the migration and dispersal processes (with dispersal kernels, dispersal propensity and distance moved). Page 6, Line 49: Dispersal is a multi-causal process, which could be measured using two methods: either by tracking animals in the field (using capture-recapture for example) or, because dispersal is a movement which could induce gene flow, molecular tools are also used to determine the dispersal ability of a given species. Consequently, dispersal is not only estimated by the observed distance moved by individuals. Doing so, I suggest to change “whereas dispersal represents the observed distance dispersed” to “whereas dispersal represents the distance moved”. More details about dispersal are needed in this part. Page 6, Line 50. Of course, performance abilities might be correlated to the mobility capacities in a species. I am surprised to not see the papers from Herrel and Bonneaud here. Herrel and Bonneaud (2012) and Herrel et al. (2014) demonstrated several relationships between locomotion abilities and performances in a sister species Xenopus laevis. Herrel A, Bonneaud C (2012) Trade-offs between burst performance and maximal exertion capacity in a wild amphibian, Xenopus tropicalis. J Exp Biol 215:3106–3111 Herrel A, Vasilopoulou-Kampitsi M, Bonneaud C. (2014) Jumping performance in the highly aquatic frog, Xenopus tropicalis: sex-specific relationships between morphology and performance. PeerJ 2:e661 Page 7, Line 95. You did not define the difference between dispersal (a movement which could induce gene flow) and migration. Maybe you could add few lines about this difference (from line 49 for instance) that should help for the understanding of the paper. Materials & Methods Page 8, Line 113. Do you have more information about the permanent and temperature water-bodies? Temperature of water, width, depth, or if other amphibian species are living within? Page 8, Line 121. How many tracking session? How many days between them? Page 9, Line 128. Did you applicate a protocol to avoid the dissemination of Bd (Batrachochytrium dendrobatidis) after each tracking session? Page 9, Line 135. Did you anesthetize individuals before pit-tagging? You should be, please details the method used. Page 9, Line 145. Please give the p-value of the Shapiro test for testing the normality of data. Page 9, Line 149. Please explain why you perform performance tests additionally to the capture-mark-recapture session. Page 10, Line 166. Why did you chose the longest distance performed in the shortest time in your analyses? You can keep the 3 distances by individuals and run mixed-effect models by adding the individual as random factor in your models. By taking into account the intra-individual variation of the distance performed in controlled conditions, this statistical method would increase the robustness of your data. Page 10, Line 179. You could give the results using the complete dataset for the tests related to the size. Page 10, Line 184. Your analysis tacking into account both the dry and wet season is interesting. But it could be very pertinent to have the humidity conditions recorded at each capture session in order to relate these data with the movement events. Results Page 11, Line 203. Please, give the range of sex ratio in percent. Page 11, Line 213. Remove “as is usual in amphibian dispersal” (which is more appropriate in the Discussion part). Page 12, Line 225. “The maximum dispersal distance observed was a X. laevis female, which travelled 2 420 m” is a repetition of the line 214. Please add details about this long-dispersal event at line 214. Also, give the distance in km or in meters throughout the manuscript. Discussion Discussion could become reduced by at least 30% without losing essential information. Page 14, Line 294. In fact, sex-biased dispersal found in amphibians have been revealed using genetics, which suggest that juveniles (individuals that we cannot tag in most of amphibian species) are responsible for most of dispersal events (inducing gene flow). Line 341. Do you envisage performing a radio-tracking study in this native population?

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: FOLIAR MICROBIOME TRANSPLANTS CONFER DISEASE RESISTANCE IN A CRITICALLY-ENDANGERED PLANT Review round: 1 Reviewer: 1

Basic reporting: This is a good manuscript. Experimental design: No problem. See General Comments. Validity of the findings: No problem. See General Comments. Additional comments: This is a well written manuscript on interesting results. I don’t see any issues with design and I think that the findings are valid. So, I’ll proceed with more general comments to hopefully help the authors find ways to improve the manuscript. I think that the context could be deepened somewhat here and there. A brief background is needed on species of Phyllostegia endemic to Hawaii and other Pacific islands. Is there any knowledge of what continental plant gave rise in the islands to endemic Phyllostegia, and of when that migration event might have occurred? Is that ancestor a host to this same powdery mildew, at least at the taxon level? A little more context and background on the powdery mildew might be helpful too. It is considered to be currently cosmopolitan but is there any reason to think that the mildew was recently introduced? Or does it seem more likely that the mildew has always been there in the islands as diversification in Phyllostegia progressed? I am not aware of any endophyte study reporting that the “ vast majority of sequences from … were identified as the pathogen”. Might the authors draw a little more attention to this finding if it truly is unique? The authors report relatively little endophyte diversity, and might emphasize that more. Another question that should be raised has to do with the presence in the leaf slurries of Ampelomyces quisqualis. The latter is a known mycoparasite of powdery mildew. Was the mildew that developed on plants examined microscopically for the presence of pycnidia of Ampelomyces? Was it examined for the yeast?

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: FOLIAR MICROBIOME TRANSPLANTS CONFER DISEASE RESISTANCE IN A CRITICALLY-ENDANGERED PLANT Review round: 1 Reviewer: 2

Basic reporting: It was difficult to get hooked into the manuscript details. There was no indication of name of the plant species until Line 65 or the causal agent of the disease in Line 70. I suggest these organisms be identified in the title and abstract to keep the reader engaged. Why did the authors choose this pathosystem to study? What is significant about this plant other than it is endangered, many plants species have this status. The continued use of the generic terms 'pathogen' and 'plant' is vague. The presented research focuses on specific plant species, one specific powdery mildew pathogen species, a specific and potentially important yeast biocontrol agent and a few named fungal taxa in the discussion. The research has potential implications for the conservation of highly imperiled plant species. To sustain the reader's interest, it may be best to relate the pertinent information using clear communication of the taxa involved throughout the manuscript. In Line 63, the use of the term 'pathogen mortality' is not clear. Does the pathogen die or does the host plant die as a result of colonization by the pathogen? In Lines 61, 128, 130 and 165 the authors use quotation marks. When one is unsure of the correct term, it may be preferred to choose words that convey the intended meaning to facilitate comprehension by the reader rather than highlight perceived indecisiveness with quotation marks. If these terms are in fact quotations from another source, then a citation will clarify the use of quotation marks. In Lines 97-98, 116, 146, 147-148, 158, 159, 161, 172, 199, 227, 251-252 and 265 the authors use parentheses. This is unconventional and discredits the strength of the statements. Experimental design: Lines 139-140, the authors discuss 'pathogen exposure and pathogen introduction'. Is this in reference to N. galeopsidis? Continuing on in Lines 140-142 the authors mention the air intake system in the greenhouse. Are you claiming to inoculate your experiment through random chance of air dispersal through the greenhouse ventilation system? If so, this unusual inoculation method will be best supported with citations of its use in other peer-reviewed publications. Were negative controls used in this experimental design? If so, it will be important to report the data collected for comparison to the inoculated plants. Validity of the findings: The research presented claims to be preliminarily successful in the use of a microbiome inoculation on the leaf tissue of Phyllostegia hirsuta to protect the plant from disease after out-planting. This is an important finding, if there is evidence the pathogen is present at the out-planting site to support the claim the slurry inoculation technique shows true efficacy. I strongly urge the authors to become familiar with the biology of the powdery mildew organism and its relationship with a host plant. Powdery mildew infections are almost always host specific. Therefore, P. hirsuta is in all likelihood able to defend against successful colonization by Neoerysiphe galeopsidis even though its close relative, P. kaalaensis is deemed to be less resistant to infection by this pathogen. Moreover, powdery mildew conidia burst in free water e.g., sterile water slurry. The OTU counts of N. galeopsidis the authors obtained in the sequencing effort using the P. hirsuta leaf tissue may represent dead cells of N. galeopsidis rather than viable cells. As the manuscript reads now, the analysis of the experimental results does not give a convincing argument for the underlying biology of the N. galeopsidis and its interaction with P. hirsuta. Additional comments: One interesting finding is the potential biocontrol interaction between Pseudozyma aphidis and N. galeopsidis. This yeast has been shown to be an effective biocontrol agent against Podosphaera xanthii on cucumber. Gafni et al. (Frontiers in Plant Science, 2015, doi:10.3389/fpls.2015.00132).

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: FOLIAR MICROBIOME TRANSPLANTS CONFER DISEASE RESISTANCE IN A CRITICALLY-ENDANGERED PLANT Review round: 1 Reviewer: 3

Basic reporting: The manuscript by Zahn and Amend describes the efficacy of using foliar microbiome extracts on the endangered Phyllostegia spp. as biocontrol agents against the foliar pathogen N. galeopsidis in Hawai’i. The manuscript examines two endangered species. P. kaalaensis and P. mollis, that received inoculations of either fungal endophyte spores or filtered leaf extracts prior to inoculation with the pathogen. The foliar fungal community is monitored at three intervals with NGS amplicon sequencing of the ITS rDNA locus. The manuscript is written clearly and the authors provide sufficient background and context to understand the rationale for the study, the methods, and the results. The figures are professional and easy convey the results. The raw data are shared in an appropriate manner. Experimental design: The research question is well defined and meaningful. In general the methods are clearly described in sufficient detail for the reader to replicate the study. I have noted below one area where additional detail on methodology is necessary to replicate the results. Validity of the findings: The experimental methods and data analysis are experimentally robust and the author's conclusions are well stated and supported by the results of the study. Additional comments: I have some general comments and suggestions. Line 22. ‘Throughput’ is misspelled. Line 38. To help clarify the meaning of this sentence change ‘and are often defined negatively’’ to ‘but are often defined negatively’. Line 125. Please provide citations for primers use in the study (ITS1F and TW-13). Line 129. What method was used for blending? Please describe in greater detail.

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: HOW CAN WE RELIABLY IDENTIFY A TAXON BASED ON HUMERAL MORPHOLOGY? COMPARATIVE MORPHOLOGY OF DESMOSTYLIAN HUMERI Review round: 1 Reviewer: 1

Basic reporting: Overall, this is a well-researched article with useful scientific information for the study of desmostylians. The use of English in this article is mostly clear and unambiguous in its presentation. Some sentences were unclear in their sentence structure or word usage. The author may benefit from having a native English speaker review the article again before submitting the final manuscript to ensure clarity to an international readership. The section that needs the most attention is the Conclusion. This section needs to be elaborated more or simply removed entirely. One single sentence does not help to summarize the important details of this important paper for desmostylian research. Perhaps quickly summarize the differences in all desmostylian humeri and how it could help future research. Below are sections of the article that need revision: 1. Line 35: Barboza et al., 2017 mentions evidence of Desmostylia from the early late Hemphillian Oso Member of the Capistrano Formation, which may extend the known range of Desmostylia on the eastern Pacific Coast if the material is not reworked. 2. Line 44: You may want to mention Santos et al., 2017 as the authors provide an updated ontogenetic sequence for Desmostylus as well as features diagnostic of advanced age specimens based on mandibular morphology. Additionally, the authors synonymized Vanderhoofius with Desmostylus. 3. Line 67: The additional paragraph does not seem needed. This sentence might work best being added to the end of the previous paragraph. 4. Line 75: “desmostylian” is misspelled as “desmosdtyian” 5. Line 75: This sentence is a little confusing as the author states they “examined morphology. . . based on direct examinations of specimens or literature review.” This wording is odd with the two uses of examine. Perhaps exchange “examined” with “analyzed” or something similar. 6. I would suggest moving the Institutional Abbreviation section from line 166 to the end of the Introduction or at the beginning of the Methods and Materials section. This might make it easier for the reader to have the abbreviations first instead of having to go find them below. 7. Line 138: Perhaps make it clear for readers not familiar with the outgroup relationships that the Sirenia are within Tethytheria. 8. Line 179: Depending on time for the author, creating figures the compares the stated features between the different taxa might be incredibly useful for the reader, especially those not familiar with them. I don’t believe it is absolutely necessary, but could improve the ease of understanding of this important section of the paper for the reader. 9. Line 181 and 82: Dugongidae is misspelled as Dugonidae 10. Line 181 and further mentions: Hydrodamalis is within the Dugongidae family 11. Line 185: “desmostylian” is misspelled as “desmostylidans” 12. Line 185-185: The wording within the parentheses is overly complicated and confusing to the reader. Perhaps simplifying the wording to just “Equidae smaller than Merychippus” removing the need to double parentheses. 13. Line 186-187: The line “the intertubercler groove of Perissodactyla covered by greater tubercle” may be missing a word. The sentence reads odd to me, but perhaps not. 14. Line 187: “characteristic” is misspelled as “charavteristic” 15. Line 207: “Behemotops is relatively ompared to other desmostylians.” I believe this sentence may be missing a few words. 16. Line 213: “extends to more distally” perhaps remove “to” 17. Line 229: This sentence is confusing to the reader as I am not sure what is trying to be said. “tubercle is not developed nodule-like unlike those in other desmostylians.” 18. Figures 3-7: These figures are incredibly useful and well-done, but may I suggest improvements to make the figures more useful for readers. - On figures with multiple specimens, please label which image is which specimen for quick identification and greater use. - Please provide explanation for the colored coded boxes and, while redundant, the characteristics associated with each number could be listed again for quick reference. - Please make the numbering larger as some are hard to see. - Is it possible to provide the actual specimen photos below the illustrated line drawings? This may help future researchers identify the characteristics on real world fossils as drawings may not provide enough detail that is present on the specimens. 19. Line 294: This single sentence paragraph could incorporated into the beginning of the next paragraph. 20. Line 303 and 304: This is the first mention of these Trichechus species. It may help the reader to spell out the names fully and provide taxonomic authority for the first instance. 21. Line 308: “Berta t. al” is missing the “e” in “et al.” 22. Line 310: “manatees are “walk” on the sea floor and by using their forelimb” this sentence has slightly confusing wording. Perhaps change to “manatees use their forelimb to “walk” on the sea floor” for a little more clarity. 23. Line 321: “based diagnostic features” perhaps missing “on” between “based” and “diagnostic” 24. The Conclusion section needs to be elaborated more or simply removed entirely. One single sentence does not help to summarize the important details of this important paper for desmostylian research. Perhaps quickly summarize the differences in all desmostylian humeri and how it could help future research. 25. Figure 1: Please label which groups are Desmostylia, Perissodactyla, and Tethytheria. And provide more detail on the relationship between the two others to desmostylia. The connected dotted line might be confusing to readers not familiar with desmostylian taxonomy. One solution might be to split the tree into two trees: one showing the relationship to Tethytheria and the other showing relationship to Perissodactyla. Experimental design: Please see previous comment on suggestions for improvement to figures. 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: HOW CAN WE RELIABLY IDENTIFY A TAXON BASED ON HUMERAL MORPHOLOGY? COMPARATIVE MORPHOLOGY OF DESMOSTYLIAN HUMERI Review round: 1 Reviewer: 2

Basic reporting: Aside from minor misspellings and inappropriate uses of plurals/singulars, all is in good order. See General comments to authors for further explanation. Experimental design: The authors have put a sincere effort in justifying the use of each specimen and exploring the impacts of ontogeny on the findings. This is very good for the experimental design of the study, I have no problems with it. Validity of the findings: The observations that differentiate the taxa are clear and supported by the evidence. Additional comments: The authors have submitted a manuscript outlining anatomical features that are diagnostic to the humeri of most species of desmostylians (all of those for which humeri of associated skeletons are known). Desmostylians have historically had much of their postcrania ignored in terms of taxonomic utility, and generally even though they are not all the same, many studies on locomotion tend to argue for different modes while using different taxa. The obvious thing is that those arguments could potentially all be correct, if only the authors recognized that they were all referring to different animals. Matsui and colleagues have done a great service to combat this tendency by writing to specifically identify differences among the diversity of desmostylian taxa. For this reason, I support publishing this paper. The manuscript is not ready for publication yet, but it is only for a collection of minor revisions that are needed that are mostly related to language used. Some are about the scientific content, but others are simply copyediting matters that are not the role I should be playing here. Rather than outline every single one, here is a description of the groupings that they all fall under. It is important for the authors to go through the entire text and look for these; it will make the paper much more effective, clear, and useful: 1) Grammatical – There are some inappropriate uses of plurals and misspellings, such as “Abstracts” (should be abstract, if there is only one) and “mirabilis” (should be mirabilis). The best advice I can give is to ask a colleague to look through it to make those corrections. After seeing a manuscript so many times as an author, it is easy for one’s eyes to overlook those minor errors and fresh eyes can do a good deal of difference. 2) Clarity of terms – Throughout the text, most especially in the Results, terms such as “weakly developed”, “apparent”, “stronger”, and “robust” are used when describing morphology. Though the author may have a sense for what that means to them, these terms don’t really mean anything precise that easily translates to the same meaning to anyone else. This is a common problem in vertebrate paleontology, and something wise to avoid if you want your writing to be useful to anyone else (which is presumably why you are writing this). For example, does “desmostylians do not have an apparent deltoid tuberosity” mean something different than “desmostylians for not have a deltoid tuberosity”? All that “an apparent” does there is invoke a sense of uncertainty in your observation. If you do not observe a deltoid tuberosity, that’s unnecessary. If someone finds one later on, they can update the observation with that added detail, no harm done. Terms like stronger/weaker/robust are meaningless is shape terms. As this is a morphological study, perhaps the better terms to use are larger/smaller/longer/shorter/wider/narrower. And, that needs to be comparative (see below). 3) Descriptions need to either be clear descriptions of shape/measurement (such as “circular”, or “5cm long”), or comparative. You have plenty of good examples of how to do that correctly here, but there are also incidences where the text includes statements of something being stronger or robust without a comparison to something else it is stronger than or more robust than. Aside from changing that to explain that the structure is larger (see point 2 above), making sure it is compared to something else is the only way to make that useful. For example, one could say that Cornwallius has thick enamel on its molars. But thicker than what? A more useful description would state that “Cornwallius has thicker enamel on its molars than do the molars of Anthracobune, but thinner enamel than similar sized molars of Desmostylus”. If you go through the text and look for incidences in which you use a comparative term without comparing it to anything else, you should either revise it or eliminate it. The entire work will be more useful and unambiguous.

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 RESPONSE OF SOIL MICROBIAL COMMUNITIES TO VARIATION IN ANNUAL PRECIPITATION DEPENDS ON SOIL NUTRITIONAL STATUS IN AN OLIGOTROPHIC DESERT Review round: 1 Reviewer: 1

Basic reporting: Some text, particularly in the Introduction, needs editing by a native English speaker. Specific suggestions to improve clarity are given below, but there are other areas of text where editing could make meaning more clear while reducing length of text. In some cases, clarity could be improved by separating ideas into two sentences (e.g., lines 143-147). Literature references are appropriate. Table and figure structures are professional. Raw data is shared. The paper's theoretical constructs and organization could be improved to better use the results to support the conclusions. The strength of conclusions, as stated, should be toned down. Please refer to the attached document for details. Experimental design: The scope is appropriate for the journal. The research question is relevant, but the approach could be better described through re-organization of the text. See the attached document for details. Data collection met high technical standards. Methods are described with sufficient detail. Validity of the findings: The paper has two primary novel aspects. The authors note, in one case, that such results have not yet been reported. Data is statistically sound. Conclusions are a bit overstated. Reorganization of text could improve linkage to research questions. See attached document for details. 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 RESPONSE OF SOIL MICROBIAL COMMUNITIES TO VARIATION IN ANNUAL PRECIPITATION DEPENDS ON SOIL NUTRITIONAL STATUS IN AN OLIGOTROPHIC DESERT Review round: 1 Reviewer: 2

Basic reporting: The authors of this research present interesting findings that are well supported by thorough attention to various measures of microbial biogeochemistry (microbial biomass stoichiometry, enzyme activity, soil nutrient pools) and analysis in a desert ecosystem. The authors demonstrate that desert ecosystems may respond the variability in rainfall in unique ways depending on soil nutrient content (plant cover) (e.g. under wetter conditions an increase in relative production of P-targeting enzymes in RS soils and higher microbial nutrient concentration in G soils) and also in broad overarching ways as well. These responses are observed both in soil microbial mediated boigeochemsitry as well as bacterial community composition. I believe that the content that is presented in this manuscript is of interest in building understandings of microbial community function in these ecosystems with increasing pressures of climate change and precip variability. I do believe that the communication of this study could stand to improve a bit. I think that the discussion is well related to past studies and prevailing theory, but I think that generally these results are over-interpreted. For example, when one of the wet years has significantly greater enzyme activity in one cover than another and conclusions are drawn about mechanism. I think that the scope of this journal may be okay for this type of study, but I do as the author to include a caveat about the generalizability of their study (being limited). If the study had a more limited scope in its thesis, I think it could be better communicated. Title: Unclear what “vulnerability” refers to. Can you think of a title that more reflects the central findings: That microbial responses to variation in annual precipitation depend on soil nutrient status in a desert ecosystem. This is the type of insight that I draw from this study…. And the narrative could stand to improve my having a clear thesis as such. It appears that this is a discussion of “interannual variation”- so I would specify that in title and after when talking about variation. Line 39: Please introduce the acronym earlier in the paragraph with parentheticals before using. Line 80-81: This sentence does not make sense “… are highly enzymatic activity..” ?? Line 83: This sentence does not make sense “In the rainy enzymatic…” ?? Line 132: I would just ask the authors to consider that there could be great functional redundancy with these enzymes (many microbes produce them) and that the change in microbial structure does not necessarily relate to adaptation to different variable precipitation, but just the turnover of microbial communities and the fact that many different microbes can produce the enzymes (unless some sort of correlation is shown between precip->microbial community structure->enzyme activity. Line 181: word choice… perhaps “dynamic” instead of “aspect” Lines 510-514: Can you move this up from the discussion to frame the hypothesis- that basically high nutrient environments are more resilient to variability in precip? Line 220: 2011 was sampled in Feb. Can you make a note that this timepoint was left out of regression analysis- to clarify it is not confounding this results if so. Lines 292-294: This can go in results Lines 286-291: First please write out J. Craig Venter Insitute (JCVI) if this is what is meant. Secondly, please include a citation that details the JCVI pipeline methods for sequence processing/analysis. Experimental design: My other concern is just to ask if the authors could make sure to communicate how they address that other factors other than precipitation change from year to year. For example, is it clear that the soil moisture content actually reflects the differences in annual precipitation? these two things exist on such different scales and it could be soil moisture driving differences rather than interannual variation in precip, per se. In the results- moisture seems to be significantly different between years, and moisture itself could control variation in nutrient dyanamics (and is known to affect enzyme activity) on temporal scales that are disjointed from annual precipitation- and yet the regression models are all focused on annual precipitation (year). pH also changes… The manuscript could better address how variability is annual precipitation is actually synonymous with year and not confounded by other factors and temporal scales. I don’t believe the study adequately tests responses variability itself, but rather seeks to understand the relationships of variable precipitation with soil/microbial mediated processes. There is no control to test variability over the years or a measure of variability itself, but the study rather show correlations between annual precipitation and dissolved nutrients/microbial nutrient ratios etc. over 3 years. Thus, I believe the manuscript needs to be reframed to communication this and not claim to assess the impact of variability itself (which I think in many sections is okay- but should be attended to). There may be some implications regarding increasing variability in precip with climate change that can be generated from this study…but must be framed as such and not as a study of this in itself. Validity of the findings: Overall, I think if the scope of the study were more focused on the differences in response to changes in precip across different nutrient regimes, the manuscript would be better communicated. Please see above in study design for questions of validity in interpretation of the findings Lines 491-509: No statistics are actually demonstrating any connection between enzyme activity and microbial taxa relative abundances, I think this is a bit too handwavey and should be much more limited if included. Additional comments: Overall the authors to a great job of collected and analyzing interesting and compelling data regarding nutrient dynamics, microbial activity, and bacterial community composition in desert soils across two different vegetation covers (soil nutrient status). I think that the manuscript could just be limited in the scope of its findings/interpretation to avoid taking on questions like variability in precip (interannual- which only has 3 years here, etc.) Rather focus on the interesting differences from year to year within the study's two cover types/nutrient levels. Best

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 RESPONSE OF SOIL MICROBIAL COMMUNITIES TO VARIATION IN ANNUAL PRECIPITATION DEPENDS ON SOIL NUTRITIONAL STATUS IN AN OLIGOTROPHIC DESERT Review round: 2 Reviewer: 1

Basic reporting: All of the criteria are met, with the following exceptions: Text added to the manuscript by the authors since the original submission needs editing. I provided suggestions in my review. Figures 1 and 2 were not in the materials I downloaded for review; but they were part of the original submission's supplementary information. Experimental design: All criteria are met Validity of the findings: All criteria are met Additional comments: The authors have adequately addressed the reviewers’ constructive criticisms. Specific comments below are largely related to editing of text to improve clarity, with the exception of line 583 (see details below). This conclusion needs to be put into the context of functional redundancy of ecoenzyme expression by microbial communities with different taxonomic composition, given that the authors included a citation about functional redundancy in the introduction of the manuscript. Specific comments Line 51 (abstract): change to “Soil communities within both sites …” Line 53: “… to face up the wet years” is not a phrase commonly used in English. I would change the sentence to something like the following: However, the community of the site with lower resources (RS) is well adapted to acquire P resources by ecoenzyme upregulation during years with adequate precipitation, suggesting that this community is resilient after drought occurs. Line 56: change to: the soil microbial communities of both sites could be vulnerable to drought through C and P co-limitation and reallocation of resources to physiological acclimatization strategies in order to survive. Line 95: ‘The’ is not needed at the start of the sentence. Line 370: change ‘AP’ to ‘PME’ Line 418: Figures were not included in the manuscript I downloaded, so I assumed Figs. 1-2 were moved from the supplement of the original submission. Line 474: Needs editing. I suggest: These results suggest that the microbial community in the RS soil, with lower resource availability, must reduce growth as a result of: 1) the physiological cost associated with a low reallocation to P-rich ribosomal RNA, as suggested by the growth rate hypothesis (GRH) (Sinsabaugh & Follstad Shah 2012; Sterner & Elser 2002; Zechmeister-Boltenstern et al. 2015) and 2) the required investment of energy towards the acquisition of P in order to produce ecoenzymes (Evans & Wallenstein 2012; Schimel et al. 2007; Wallenstein & Hall 2012). Line 537: ‘resilient’ not ‘resilience’. I would add the phrase ‘during times of adequate moisture’ at the end of the sentence. Line 557: I am not sure this statement is accurate. I would change it to: ‘Our results show how values of TERC:N and TERC:P may shift with respect to variation in annual rainfall and different vegetation cover.’ Line 568: Needs editing. I suggest: Our results for the dry year (2012) showed that the ecoenzymatic activities associated with C and P acquisition were lowest in the RS and G soils. Values for TERC:N and TERC:P were similar between the RS and G soils, suggesting that both sites may be vulnerable to drought. Line 575: Needs editing. I suggest: Similarly, increased ecoenzyme activities associated with P acquisition and elevated TERC:P values when the water is not limiting (2013 and 2014) suggest that the RS soil microbial community is well adapted to acquire P resources via ecoenzyme upregulation post drought. Line 579: Needs editing. I suggest: We suggested that, under the scenario proposed by Global Climate Change models for desert ecosystems that predict reduced annual precipitation and increased rainfall variability, the microbial community from both sites could be vulnerable to drought events, but the RS soil microbial communities can make adjustments in order to obtain nutrients in wet years, suggesting that this community is resilient post drought. Line 583: This result would occur only if functional redundancy is lost through change in community composition. Line 590-597. Needs editing. I suggest: Soil communities of both sites (RS and G) may be vulnerable to drought. However, the community at the site with lower resources (RS) may have evolved adaptations, such as rapid ecoenzymatic upregulation, under chronic P limitation. This adaptation confers greater resilience within the community to respond to precipitation events post drought. Under the Global Climate Change scenarios for desert ecosystems that predict reduced annual precipitation and an increased intensity and frequency of torrential rains and drought events, soil microbial communities within both sites could be vulnerable to drought through the combination of C and P co-limitation and reallocation of energy and nutrient resources to physiological acclimatization strategies in order to survive.

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 RESPONSE OF SOIL MICROBIAL COMMUNITIES TO VARIATION IN ANNUAL PRECIPITATION DEPENDS ON SOIL NUTRITIONAL STATUS IN AN OLIGOTROPHIC DESERT Review round: 2 Reviewer: 2

Basic reporting: The introduction still has a few English language issues which I tried to edit- In addition, I'm not sure the communication of the study is quite at a level where the manuscript will make the intended impact. I have included an attached version in which I try to make some edits to the intro to improve the logic/flow of the intro/narrative. The authors should consider these possible edits. Edits/comments are in track changes. Experimental design: no comment Validity of the findings: no comment Additional comments: The introduction still has a few English language issues which I tried to edit- In addition, I'm not sure the communication of the study is quite at a level where the manuscript will make the intended impact. I have included an attached version in which I try to make some edits to the intro to improve the logic/flow of the intro/narrative. The authors should consider these possible edits-

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 RESPONSE OF SOIL MICROBIAL COMMUNITIES TO VARIATION IN ANNUAL PRECIPITATION DEPENDS ON SOIL NUTRITIONAL STATUS IN AN OLIGOTROPHIC DESERT Review round: 3 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: The authors have made all of the requested revisions. I believe the manuscript may now be accepted 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: THE RESPONSE OF SOIL MICROBIAL COMMUNITIES TO VARIATION IN ANNUAL PRECIPITATION DEPENDS ON SOIL NUTRITIONAL STATUS IN AN OLIGOTROPHIC DESERT Review round: 3 Reviewer: 2

Basic reporting: I have no additional substantive comments- I think that the manuscript could still stand improvement in narrative organization and editing of a few remaining English grammar issues, but to a certain extent this is a matter of preference and relates to the elements of style more than content. As indicated by the editor, further efforts here are not necessary for the publication of this manuscript. I would encourage the authors to just take one last scan for grammar, in particular, as this may improve paper's impact in terms of readership. The authors have attended to my previous substantive concerns in the last round of review. Good job- I think the manuscript encapsulates substantial work and interesting results. Experimental design: no additional comments Validity of the findings: no additional comments 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: MOBILE ACOUSTIC TRANSECTS MISS RARE BAT SPECIES: IMPLICATIONS OF SURVEY METHOD AND SPATIO-TEMPORAL SAMPLING FOR MONITORING BATS Review round: 1 Reviewer: 1

Basic reporting: It would be great to have a more thorough explanation for why you chose to do this methods comparison. You note that Whitby et al. 2014 found different results in their methods comparison. Can you explain in your hypotheses why you believed another method comparison was necessary in the ENP? E.g. was this because of the high species richness in the area? The diversity of vegetation, etc? I really liked your Conclusion section. The rationale for the topic came through very clearly. Can you highlight some of those main questions in your introduction? Experimental design: Page 4- Can you give a clear explanation for the benefits and issues of using each method type? It would help the reader understand the importance of your later findings to have this background. I would suggest creating a table showing: each method type, advantages/disadvantages, and the prominent literature citing recommendations. If there has not been adequate literature on one method this also highlights the importance for this and future methods studies. I think such a table would be a very useful/quick reference of what studies have previously suggested and been done. The reason for testing the 2 methods against each other on Pg. 10 could use a better explanation. You have clearly set up what you aimed to quantify on Pg. 10 (line 203) in your methods. I would like to see this same clarity reflected in your results. Even by using the same subheadings later. Some clarity on methods and site selection are missing. See my comments below. Validity of the findings: I think you have found a useful result for researchers and conservation managers. It is nice to see the quantification of conservation methods. Additional comments: Line 37- add to “document bat population trends…” Line 69- Hayes 2000 – This ref is not in your citations. Please add. Line 75 – Loeb 2015- This seems like it should be Loeb et al. 2015. Please change this throughout Line 132- Be consistent with your use of “Figure 1” and “Fig. 1” Line 137- I think I would add the make/model of the external microphone in parentheses here and then delete the next sentence starting with: “We conducted mobile transects using….” This just seems to be a bit wordy, but I recognize this is a stylistic recommendation. Line 143- “We collected data with the three methods during a 40 night sampling period…” Your methods here are a little bit vague. Can you add the number of hours of recordings per each method type? Line 164- Can you explain more clearly how you selected your sites? For example, you used water sources, but was this randomly determined based on x distance to a water source? In easiy accessible areas? Near known roosts? Just a bit more detail here might better inform managers that want to follow this method in the future. Line 177- “…accounting for accessibility…” does this mean locations were randomly selected within, for example, 10 m of a track? Might help to clarify this again. Line 178 – Can you report the ~height of the painter’s poles? Line 200- I would delete ‘language’ from this sentence since R is a programming language not a software language as such. Line 215- Please clarify what ‘this analysis’ refers to. Line 228- A lot of repetition of the word ‘test’ in line 228 and 229. Can you clean this a bit? Page 11- Can you mirror the subheadings to your methods so that it matches the subheadings in your results? E.g. spatial…. Temporal Page 13- You report the results of species richness based on season and vegetation in Table 1. I realize you are emphasizing the methods comparison, but it would be nice to see the difference in species richness based on these factors highlighted briefly at the end of PP1 on Page 13 and then reference Table 1 . Line 333-35 – Interesting discovery and provides good info. for managers! Line 348- please add “mobile” before ‘transect surveys’ Line 385 – “… monitors population trends…” I don’t think your research monitors population trends specifically at this point. Rephrase to rather emphasize the trends in species/community composition. Line 449- To be more succinct, I would delete: “… some studies report the # of nights required to reach a specified threshold of the estimated species richness.” Line 473- Can you give specifics on what “slightly more?” Line 647- "pespective,,.." – Reference needs fixing Line 657- "US Geological…"Fill in here.

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: MOBILE ACOUSTIC TRANSECTS MISS RARE BAT SPECIES: IMPLICATIONS OF SURVEY METHOD AND SPATIO-TEMPORAL SAMPLING FOR MONITORING BATS Review round: 1 Reviewer: 2

Basic reporting: - adequate - a few mentions of "population trends", e.g. lines 65, 114 could be removed as this was not the intention of the study - some difficulty locating sampling stations within Fig. 1 within darkest shading; perhaps larger map of ENP at expense of smaller image of Florida. More important to the manuscript to denote detail of study area than to know that ENP is in southern Florida. Experimental design: - question of interest in the study is moderately well-described. There are a few shortcomings however. The given metric against which to measure estimates of species richness is derived from range maps. It is not obvious that range maps are completely accurate; furthermore, because two species cannot be uniquely identified acoustically (Eastern and Seminole), the "true" number of species is different than described by the range maps. Finally, the range maps, I presume, do not differentiate richness by habitat type. Consequently, the validity cannot be assessed at the level of habitat type, but rather, at the level of ENP. That creates a mismatch which makes it awkward for evaluating study design at the habitat level (level of spatial and temporal replication). - it was not clear what randomisation scheme was used to place the spatial replicates. Given concern about distance between sampling stations and roads; I would have thought measures might have been taken to place replicate sampling stations perhaps away from roads; perhaps challenging in pineland habitat. - unclear why there was no attempt to have spatial replication in mangrove habitat--manuscript should make some mention of this - as noted in marginalia of the submitted PDF, the study could not readily be replicated because of the manner in which sampling locations were chosen and the inclusion criteria of Supplemental S1. Validity of the findings: - data appear fine; I could replicate most of the analyses presented via the R script provided - my main concern when species richness is the metric, maximal species richness is small (9) and species identification is auditory is the potential for false positive identification (recording presence of a species not detected). With so few species in the taxonomic target, only a single false positive might alter the inference of the study. Supplemental Appendix S1 discusses this, but the manuscript ought to highlight mitigation taken to prevent false positives and perhaps tabulate number of files falling into various eliminated (<5 calls; <75% calls with ID match, ...) categories. - because of the absence of the mangrove habitat type from spatial replicates, this limits the scope of inference of this study, not just to ENP but to pineland and prairies of the park - I made several comments regarding suitability of confidence interval overlap as an inferential tool as well as whether the R code using a multiplier of "2" on the SE produces the desired "84%CI" - there exists a literature suggesting that CI overlap is not an effective means of examining differences. The "84%" level only resolves this matter when the SEs of the two contrasting estimates are equal, which is not the situation here. a closer reading of the supporting literature cited by Gotelli and Colwell (2011), namely Payton et al. (2003, J. of Insect Science 3:34) states "assuming equal standard errors (k = 1) yields γ = 0.166. In other words, if you wish to use confidence intervals to test equality of two parameters when the standard errors are approximately equal, you would want to use approximately 83% or 84% confidence intervals" - Wolfe, R. and Hanley, J. (2002). If we’re so different, why do we keep overlapping? When 1 plus 1 doesn’t make 2. Canadian Medical Association Journal, 166, 65–66. - Spanos, A. (2014). Recurring controversies about P values and confidence intervals revisited. Ecology 95: 645–651. doi:10.1890/13-1291.1 - this is not a large portion of the manuscript, more details included in the marginalia of the PDF, but it should be noted by the authors - Lines 265-270 cause me concern. Is my logic correct? If so, deserves further discussion in the manuscript: - line 266: note these two numbers of files are roughly equal (although there were double the number of spatially replicated nights) - line 268: this step is unclear to me; <1/3 of files containing bat sequences were identified to species; is that the correct interpretation? - line 269: whereas here the 16K to 26K ratio is roughly back into rough parity with the number of detector nights. - What this suggests is that the "conservative protocol" eliminated a larger proportion of single strategic files than spatially replicated files. -That seems as if it might be a threat to the comparison between the ability of two methods to detect bat species. If so, should that be addressed in the manuscript? Additional comments: It is challenging to experiment with design parameters of a monitoring programme, nevertheless, an important component of any monitoring work is to determine whether it is able to achieve its goals. Hence, the goals of the programme need to be explicit and evaluation of the attainment of the goals is nedessary.

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: MOBILE ACOUSTIC TRANSECTS MISS RARE BAT SPECIES: IMPLICATIONS OF SURVEY METHOD AND SPATIO-TEMPORAL SAMPLING FOR MONITORING BATS Review round: 2 Reviewer: 1

Basic reporting: Suggested changes on basic reporting were addressed to my satisfaction or the authors provided logical reasoning for ignoring changes. I like the addition of Table 1, because it seems like a great resource for methods. Is all information attributable to Loeb et al. 2015? Check consistency around using ENP and 'Everglades National Park.' For example it is written out on line 455. Would suggest only writing out on first use. Experimental design: Satisfied with changes addressed to make methods and questions more clear. Validity of the findings: No Comment. Additional comments: I am happy with the changes the authors have made to this manuscript. I think progress has been made to clarify some of the confusing aspects of project design, methods and the congruity with results. I look forward to seeing this paper contribute to the well-quantified studies that use sensory-based conservation/monitoring.

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: HGTSIM: A SIMULATOR FOR HORIZONTAL GENE TRANSFER (HGT) IN MICROBIAL COMMUNITIES Review round: 1 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: Song et al. describe the open-source tool HgtSIM, which was developed to simulate HGT events among microbial community members taking into account user-defined mutation levels. The authors give an illustrative example how HgtSIM works, starting from the insertion of a set of selected genes into a collection of genomes, followed by modification of these genes simulating mutation and adaption. Song et al. then employ the tool GemSIM to simulate metagenomic reads from their simulated dataset, perform assemblies using the assembler IDBA-UD and finally assess the recovery rate of gene transfer events. HgtSI is not the first tool to simulate HGT in microbial genomes (see HGT options of EvolSimulator by Beiko&Charlebois, 2007, https://doi.org/10.1093/bioinformatics/btm024), but seems to be more straight-forward to use. It could be downloaded from Github together with a comprehensive documentation. HgtSIM is written in python 3 and the code is well readable. After testing HgtSIM I felt that it could be slightly more flexible in terms of query genome input format and user-specified options seemed to be rather limited. HgtSIM was successfully used on the provided test dataset. However, gene insertion was only possible on closed genomes (or single contigs), as soon as additional contigs were added (in the same file) the HGT simulation failed. This was somewhat surprising as the tool supposedly was designed to simulate HGTs in metagenomes - many published metagenome assembled genomes are not closed. Thus, it would be great if HgtSIM could also be used on genomes with more than 1 contig. Flanking sequences are often introduced together with transferred genes. In HgtSIM the user has the option to provide upstream and downstream flanking sequences, which is great. However, it seems that these flanking sequences are then equally used for all inserted genes. I would suggest to implement a feature to automatically generate flanking sequences with varying sequence length and composition (within a given range). This would help to make the simulation result more similar to real world data. An important feature of HgtSIM is that acquired genes do undergo evolutionary drift and adaptation to the new cellular context. However, in its current form this process felt slightly limited due to the fixed identity value provided by the user. Ideally, HgtSIM would include an optional "mixed mode" - which would use a range of different identity values randomly to alter the query genes. Line 119: The here described results imply that recent HGT is most difficult to detect. I was wondering to what extend this observation was affected by the assembler (IDBA-UD) and to what extend a better performing assembler would change the result. An alternative assembler could be SPAdes (or metaSPAdes), it is popular and widely-used and presumably outperforms IDBA-UD considering contig and scaffold lengths (see Vollmers et al., 2017 https://doi.org/10.1371/journal.pone.0169662). Lastly, the authors evaluated the impacts of read length and insert size on the recovery rate but they did not elaborate on the potential influence of gene length. I would assume that successful detection negatively correlates with gene length. If there is such correlation, then a length distribution plot of inserted genes together with their recovery rate would be a helpful reference for the user.

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: HGTSIM: A SIMULATOR FOR HORIZONTAL GENE TRANSFER (HGT) IN MICROBIAL COMMUNITIES Review round: 1 Reviewer: 2

Basic reporting: The manuscript presents a software for the simulation of genomes, which contain simulated horizontally transferred genes. The authors provide an example application to an artificial microbial community. I agree this is an interesting aspect in metagenomics, which besides strain diversity and limited coverage introduces an additional challenge to metagenome assemblies. The manuscript is a brief “Applications Note” rather a full research article, as required for PeerJ. It lacks a central research question and suitable, comprehensive experiments to address it. For example, the manuscript does not introduce the challenges of metagenomic assemblies in detail, and does not address the problem of HGT event prediction, or donor/acceptor inference, for which HGT simulations might also be useful. The results on an artificial set of genomes, using one assembler, demonstrate the general utility of HgtSIM and give some general hints for metagenomics assembly. For those who work on shutgun metagenomes, the results are quite expected. However, they don’t resemble any realistic microbial community and thus have no meaning for metagenomic assembly practice. Experimental design: I have downloaded and tested the software. It is written in Python3 and has only few dependencies, which are easy to fulfil. I did not completely understand the implementation strategy, as parts of the script seem unnecessary (e.g., the genetic code is hardcoded although the script imports Biopython, which already provides more sophisticated genetic code classes and methods). Anyway, I could successfully run a simple test, based on the provided example files. Validity of the findings: Does not apply. Additional comments: To become a PeerJ Research Article, the manuscript would require fundamental expansion. A suitable alternative might be a submission to OUP Bioinformatics, as “Applications Note”.

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: HGTSIM: A SIMULATOR FOR HORIZONTAL GENE TRANSFER (HGT) IN MICROBIAL COMMUNITIES Review round: 1 Reviewer: 3

Basic reporting: The authors present a simulator for horizontal gene transfer in microbial communities. The article is written in unambiguous and professional English and only a few typos need to be corrected. I would suggest to add more background into the introduction and also include a couple of references about more recent tools for detecting HGT, e.g. https://doi.org/10.1371/journal.pcbi.1004408 https://doi.org/10.1371/journal.pcbi.1004095 https://doi.org/10.1093/bioinformatics/btw423 Figure 1B is hard to read. It is impossible to distinguish some of the labels. Please refine. In Table 2 it would be useful to add the size of the genomes, as this has an effect on the simulated coverage (see experimental design), which again might have an effect on the results presented in chapters 3.2 and 3.3. Table 3 is missing a caption which explains the numbers in the table. I guess these are recovery rates (in %) of the HGT genes. Also, the 0% and 5% need to be explained in the caption or table. The authors do not share the raw data, but at least provide accessions for the genomes used for the simulation. Please provide all details about which parameters were used with the different tools (read simulator and IDBA_UD), so that results are reproducible. Experimental design: In general, it remains unclear what the goal of the simulation should be. What should be benchmarked with the simulated data? Is it mainly about assembly performance? If so, the authors have to make sure that they are not measuring a different effect when evaluating the assembly (see below). Or is the goal to benchmark tools for detection of HGT? If so, it would be nice to see how one if these tools performs. I understand that a thorough benchmarking of HGT detection tools might not be within the scope of this manuscript, but if there are benchmarks published already, the “best tool” could be applied on the simulated data set. Lines 54-62: Taking a number of gene sequences as input and inserting them into a number of target genomes seems to be a relative straightforward task. Having an automated way to do so is very useful for simulation and benchmarking. Therefore, the described pipeline is a step into the right direction, if such studies are performed often and in high-throughput. It remains unclear, though, how realistic the chosen model of mutating the horizontally transferred genes is. The authors barely explain the underlying principles of their model. One can immediately argue, that a distinction between one-base (silent or non-silent), two-base and three base mutations is too simplistic. There are examples where two-base or even three-base mutations are silent mutations (e.g. UCU and AGC both code for Serine). There are more sophisticated models available, could they be integrated in the simulation pipeline (e.g. https://doi.org/10.1080/10635150701546231)? Line 71: Even for the simple model, how would one choose C1:C2:C3:C4? What is the default for the pipeline and why was it chosen this way? Line 80: Does the recipient genome have to be a finished genome (i.e. no gaps allowed)? Many genomes are permanent draft genomes which cannot be used for a large scale simulation if the pipeline only works for finished genomes. Line 84: When cutting the genome for inserting the HT genes, this will disrupt genes in the recipient genome? Is this realistic assumption or are insertions into CDSes much less likely? If so, this should be included in the model Line 100: “The category ratios of “0:0:0:1” and “1:0:1:1” resulted in level of amino acid sequence changes that were similar to the user-defined level of nucleotide mutations” This sentence is unclear. What exactly is the user-defined level of nucleotide mutations? And how does this relate to these ratios? In general, details about running the pipeline and assembly (parameter setting) are missing, which makes reproducibility difficult. Validity of the findings: The authors present a small benchmark data set consisting of 20 genomes, where 100 genes from Alphaproteobacteria get randomly transferred to 10 genomes of Betaproteobacteria. I am not convinced that the effects of missing genes in the subsequent assemblies are actually always the degree of mutations. If we assume that the 20 genomes have a length of 4Mbp on average, the sequencing depth for the simulated data set is only about 12.5x (10 mio x 100 bp / 20 * 4 Mbp = 12.5). This is already a pretty low coverage for assemblies based on long kmers and could explain that for kmer ranges >60 most of the genes are lost. What are the total assembly sizes for these assemblies? I would guess they are rather small. Additionally, a couple of genomes are >5Mbp long, which reduces the coverage for these genomes even further. Line 120: therefore, the mink parameter has a general effect on the assembly of low coverage genomes and not on HGT detection. Only using show kmers (20) in the kmer range pick up the low coverage genomes and therefore allow HGT detection Line 131: there are no results shown for longer reads of different input sizes I would suggest to prepare two different data sets, one without HGT and one including HGT. Comparing these two datasets would elucidate if the missing genes are actually an effect of HGT or just an effect of too low sequence coverage. Additional comments: The manuscript seems to be very preliminary and needs a more carefully designed experimental setup. It is not clear, if the findings are an effect of HGT or missing sequencing depth. Also, it would be nice to see an HGT detection tool applied on the data set.

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: HGTSIM: A SIMULATOR FOR HORIZONTAL GENE TRANSFER (HGT) IN MICROBIAL COMMUNITIES Review round: 2 Reviewer: 1

Basic reporting: no comment Experimental design: no 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: HGTSIM: A SIMULATOR FOR HORIZONTAL GENE TRANSFER (HGT) IN MICROBIAL COMMUNITIES Review round: 2 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: The revised version has been substantially improved. The authors have carefully addressed the reviewer's comments. As there is not any substantial issue remaining from my side, I thank the authors and recommend acceptance of 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: HGTSIM: A SIMULATOR FOR HORIZONTAL GENE TRANSFER (HGT) IN MICROBIAL COMMUNITIES Review round: 2 Reviewer: 3

Basic reporting: The authors switched to metagenome assemblers for the simulation study. This improved the quality of the simulation study tremendously. However, the findings are not reproducible, if details of the simulation are not described. This is the case for many details in the study and need to be addressed (see details below). Please correct the following typos: line 29: ...tool offers... line 31: ...events... line 49: ...error... line 124: ...paired-end... line 144: ...optimal... line 163: ...read length... Figure 4A, y-axis: Assemblies Experimental design: lines 122-124: which mutation model were used for the simulation? Were flanking regions added to the transfered genes? If so, what sequences, how long? lines 124-126: does the script introduce sequencing errors? Please add the resulting sequencing coverage to the main text. The methods are still not described in detail. The presented results are not reproducible. Please provide the all input files for the scripts such that the study can be reproduced. The GitHub repository has all the necessary scripts, but e.g. the abundance.txt file or the input_genomes_m0 files are missing. How exactly were recovered genes identified? Alignment against the reference? Which tool was used? What were the alignment parameters? And what the criteria for calling a HGT gene recovered? I found all details in the python scripts on GitHub, but they should be included in the manuscript (Methods section). Also, please include the version numbers of the assemblers. Validity of the findings: line 135: what happens to the HGT genes? Are they not assembled at all? Do you recover only one copy of the gene? lines 146-152: First, it is not surprising that the recovery rate is not increasing linearly with sequencing depth. It is well known, that the assembly in terms of recovered genome sequence behaves in such a way (Lander and Waterman 1988). Very surprising is the fact, that higher sequencing coverage leads to a lower recovery rates. I was convinced that there is a bug in the simulation and analysis pipeline, but the results seem to be correct. I evaluated the assemblies using metaQUAST and from these results, the higher coverage data sets seem to produce better assemblies (less contigs, higher genome recovery, higher N50, etc). I would suggest to add these basic statistics to the manuscript, just to show that there are no flaws in the simulation process. Taking a closer look why the HGT genes get lost in the high coverage data sets, I was able to re-run the analysis using the Assembler_recovered_transfers_iden100.py script from GitHub and the data provided. It was very surprising for me to see that the flanking regions get lost in the assemblies with higher coverage. Here is an excerpt from the BLASTN results generated by the script for the metaspade_m0_k21-125_l100i250_3_million.fasta assembly AMAC_01196 NODE_75_length_51023_cov_1.46669 100.00 1203 AMAC_02518 NODE_2577_length_11107_cov_1.63078 100.00 1269 AMAC_02914 NODE_5740_length_2456_cov_2.52716 100.00 1908 AMAC_03303 NODE_3040_length_9082_cov_1.74892 100.00 1452 AMAU_01212 NODE_938_length_22333_cov_1.55155 100.00 1530 AMAU_01255 NODE_1152_length_20072_cov_1.53219 100.00 1497 AMAU_02414 NODE_3750_length_6844_cov_1.81192 100.00 1290 AMAU_02488 NODE_4885_length_4137_cov_2.12155 100.00 1518 AMAU_04187 NODE_921_length_22539_cov_1.54172 100.00 1176 AMS_00102 NODE_26_length_64149_cov_1.46827 100.00 1395 AMS_01465 NODE_491_length_29719_cov_1.50854 100.00 1413 AMS_01716 NODE_1033_length_21171_cov_1.56804 100.00 1176 AMS_01785 NODE_3328_length_8142_cov_1.76752 100.00 1194 AMS_02653 NODE_869_length_23152_cov_1.47987 100.00 1062 AMS_02811 NODE_139_length_44053_cov_1.48303 100.00 1410 For the higher coverage data set (assembly metaspade_m0_k21-125_l100i250_20_million.fasta) the BLASTN results look like this: AMAC_00685 NODE_684_length_1608_cov_19.649 100.00 1608 AMAC_01196 NODE_766_length_1203_cov_20.8583 100.00 1203 AMAC_02518 NODE_752_length_1269_cov_17.6364 100.00 1269 AMAC_02914 NODE_660_length_1908_cov_18.9573 100.00 1908 AMAC_03303 NODE_711_length_1452_cov_17.7535 100.00 1452 AMAU_01212 NODE_697_length_1530_cov_19.0856 100.00 1530 AMAU_01255 NODE_702_length_1497_cov_18.6773 100.00 1497 AMAU_02414 NODE_744_length_1291_cov_18.9364 100.00 1290 AMAU_02488 NODE_700_length_1521_cov_19.2229 100.00 1518 AMAU_04187 NODE_792_length_1102_cov_28.4966 100.00 1101 AMS_00102 NODE_726_length_1395_cov_17.3326 100.00 1395 AMS_01465 NODE_718_length_1414_cov_20.2716 100.00 1413 AMS_01716 NODE_777_length_1178_cov_19.8879 100.00 1176 AMS_01785 NODE_771_length_1194_cov_19.8293 100.00 1194 AMS_02653 NODE_800_length_1062_cov_18.9562 100.00 1062 AMS_02811 NODE_720_length_1410_cov_17.9443 100.00 1410 Basically, all genes are assembled, but only on contigs which have exactly the size of the HGT gene. Additional comments: The presented results are very surprising and the authors should elaborate on the findings and try to interpret these in more detail. The manuscript would improve significantly from digging a little deeper into the results and trying to explain what has been observed. A way to do this might be to load the metaSPAdes results into a tools like e.g. Bandage (https://rrwick.github.io/Bandage/) and visualize the HGT genes in the assembly graph. I tried to re-run metaSPAdes, but I failed as some files were missing in the GitHub repository, which made the presented results not reproducible (see 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: INDICATORS FOR THE USE OF ROBOTIC LABS IN BASIC BIOMEDICAL RESEARCH: A LITERATURE ANALYSIS Review round: 1 Reviewer: 1

Basic reporting: In general, the manuscript is clearly written. There are some typos and small grammatical issues, which should not be difficult to address. I noted the following: - "methodbut" -> "method but" - "an robotic" -> "a robotic" - "methodswithin" -> "methods within" - "methodsin" -> "methods in" - "methodscan" -> "methods can" - "methodsthat" -> "methods that" - "Finally, While" -> "while" - "This large overlap could have to do with what the skewed distribution of recognized ..": "what" seems ungrammatical. Some sentences are unclear or confusing, and I suggest the authors clarify them. These are as follows: - The authors state that "this ability to have a much more detailed computational view is critical for reproducibility as narrative descriptions of methods are known to be inadequate for this task". I would think the robotic labs would have to start with the narrative description of experiments for replication. How does one go from this narrative description to computational view? - The authors state that "Of the potential 1035 methods only 151 were recognized". Is 1035 the number of relevant MeSH terms? Earlier in the manuscript, they state there are 1036 relevant MeSH terms. - " With the exception of cell culture, the other methods in Table 1 are also comprised of highly automatable tasks." I thought being included in Table 1 meant the method could be fully automated. What's the specific difficulty with cell cultures? Explain. " 26 of these methods are in fact supported by one of these cloud labs, exposing some leakiness in our procedure." The example given is Real-Time PCR. This seems to be a term in MeSH since 2012. Either give a more accurate example or explain the discrepancy. There are a few figures and tables and their content is adequate. I think Figure 2 can be more effectively presented as a table, and percentages can be added. I think Figure 1 is useful, and something similar for method selection/categorization and for article/method matching would be helpful to guide the reader and tighten up Methods/Results. Currently, these numbers can be hard to follow as narrative text. The authors commendably provide the data and the code for their experiments. Since method identification (a named entity recognition task) is core to the study, I think it would be useful to provide some background for research in this area. Currently, only towards the end of the Discussion section, this particular task is mentioned and very briefly. A more detailed discussion early in the paper would be more informative and situate this study better. Experimental design: The notion of automatically reproducing basic research with robots is an intriguing idea that is being increasingly recognized and an investigation of how much of current research can be automated in this manner is timely and meaningful. The authors do a good job of explaining the research goals and providing the context for them. In general, the methods are described sufficiently and since the data and the code are provided, the methods can be assumed to be replicable. My main issue with the study is that the method identification approach does not seem powerful enough to make claims about the use of robotic methods. The authors admit this to some extent, stating that their results are likely to underestimate the use of such methods; however, I believe they could have done more with existing, off-the-shelf tools. Much information seems to be lost in mapping steps and also in method identification. I think some expert feedback or use of additional resources would be appropriate. For example, 71 of 107 methods identified are identified in MeSH (which is a good starting point), the rest (36) seems like a significant proportion to just ignore.Why not try to use other vocabularies/ontologies for these 36 or just generate term lists, since the annotator basically performs simple string matching? For example, OBI (ontology of biomedical investigations) comes to mind as a good candidate. Since the paper is mainly interested in assessing the coverage (and not in only automatic method identification), such interventions would be justified.Some further discussion of these 36 methods and how 71 methods identified in MeSH were mapped to 59 concepts would also be helpful (am I right to assume some methods map to the same MeSH concept?). It is also not clear to me whether the authors tried using MeSH identifiers used for indexing articles in PubMed. I would think this would be the easiest way of answering their core question. Checking a few of the articles they considered, I can see that indexing terms for these articles include some method names. Since these indexing terms are assigned by human indexers, it can be considered a gold standard and would potentially alleviate the coverage issues noted for the annotation method the authors used. Do the authors find that these methods are not consistently or fully indexed? If the authors have not considered this, I would strongly suggest that they do. If they did, some explanation is needed regarding why they chose not to report it. There are existing, broad-coverage biomedical named entity recognition methods such as MetaMap (Aronson and Lang, 2010). Did the authors consider using them, instead of coming up with their own lexicon-based method? They state (in the Discussion section) that state-of-the-art in process/task detection is low (0.44 F1), but they do not provide the performance for their own method, making it difficult to assess whether the analysis can be strengthened by such off-the-shelf NER tools. Since MetaMap can be easily tailored to run against MeSH specifically, I believe it can strengthen their approach and findings without too much additional effort. Validity of the findings: Due to the issues I discussed above, I believe the findings are suggestive rather than conclusive. The authors highlight the difficulties in using automated methods for their task in the Discussion section. I think some of suggestions (using MeSH indexing terms, MetaMap, etc.) can be considered to address some of these challenges (in particular, points 1 and 2 on lines 189-191). However, I believe this paper is still a step in the right direction for understanding the feasibility of such approaches to reproducibility. As a side note, I think it would still be useful (with the limitations of the approach in mind) to know how many articles (of 1011) were found to contain only robotic methods. This is never clearly stated. 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: INDICATORS FOR THE USE OF ROBOTIC LABS IN BASIC BIOMEDICAL RESEARCH: A LITERATURE ANALYSIS Review round: 1 Reviewer: 2

Basic reporting: The basic reporting standards have been met. Experimental design: I see no issues with the experimental design. Validity of the findings: Overall: This paper nicely describes the problem of scientific reproducibility and how this could be better addressed by automating basic lab methods using robotics, where applicable. The authors demonstrate that in the corpus of papers analyzed, the majority of methods used in the experiments were assays that could be performed by robotic labs. The conclusions are supported by the analysis. One thing that I think is missing from the discussion is how this work can impact the future of biomedical research. More specifically, now that we know that the majority of research could be performed by robotics, what are some potential ways we could implement this? A discussion of the potential issues would be nice to include, such as the issue of the expense of running robotic machines, ie not every lab could afford to purchase an automated robotic PCR machine, for example. However, this could be potentially addressed with increased collaborations with labs that already own these tools, and have optimized the protocols. This paper could also be used to promote funding agencies to support automated protocols using robotics, to further ensure scientific reproducibility. Additional comments: Minor edits: Abstract - first line Should it be Robotic labs (lowercase labs) Lines 11-12 I suggest writing 'could be applicable' in "In this article, we investigate whether these labs are applicable in current experimental practice." Introduction- Lines 35-36 In this sentence: "These are labs in which the entire 36 experimental process is performed by robots and available remotely in the cloud (Bates et al., 2016)", what is available remotely in the cloud? The data? Lines 54-56 State the questions as 1a and 1b, otherwise it looks like you are asking two questions. Discussion Lines 159-161 "Our annotation procedure does not attempt to generalize: the MeSH ID a method is labeled with is exactly what is returned in a search for the method without traversing the hierarchy of the method tree." Please reread this sentence, there seem to be some typos here. Line 166 You are asking only one question, in two parts, so I suggest rephrasing to "In terms of the second part of the question..." Line 174 Missing a space in methodswithin Line 175 Missing a space in methodscan Line 191: Missing right parenthesis Line 203 Missing a space in methodsthat Line 205 "While" should be lowercase -- Did you find any missing synonyms in MeSH? If so, did you make requests to MeSH for these new synonyms? -- This link does not resolve, it needs to be reformatted: http://dx.doi.org/http://dx.doi.org/10.17632/gy7bfzcgyd.1 -- Shared datasets: A more extensive description of the datasets in Mendeley would be useful. Currently, the description of data lists the 4 primary datasets, but a detailed description of all the shared files would be helpful. More detailed metadata in each file would be helpful, including a definition of all the acronyms and abbreviations used.

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: INDICATORS FOR THE USE OF ROBOTIC LABS IN BASIC BIOMEDICAL RESEARCH: A LITERATURE ANALYSIS Review round: 1 Reviewer: 3

Basic reporting: In their paper entitled “Indicators for the use of Robotic Labs in Biomedical Research: A Literature Analysis,” Groth and Cox argue that the larger issue of reproducibility across life sciences research could be addressed by the use of “cloud labs.” These new laboratories are built to fully automate certain biological research protocols. According to the analysis by Groth and Cox, these newly arrived cloud laboratories can automate over 60% of the protocols in a subset of papers within the Elsevier collection. The authors do not clearly claim that researchers should be using these cloud labs, but they suggest that using them for certain protocols will decrease the number of errors within the field and increase the reliability of scientific research. While an intriguing concept, the paper as is does not warrant publication. At this point it merely points out that certain laboratory methods can be automation, which is obvious to the reader. To warrant publication, the authors must distinguish methods that can be automated AND effect the scientific conclusion of the paper from methods that merely can be placed on a robot. This would be a worthwhile publication. Experimental design: Point 1 – Definitions of terms The authors must and clarify define various words used throughout the paper. These include: 1. "automated" 2. A "task" vs. "pipeline. Where automation is used and helpful, and when something is "automated" 3. A "robot" vs. "robotic lab". Robots doing automation are plentiful and prevalent, while cloud labs are not. It's not clear if robots constitute "automation" the same way robotic labs do. 4. Automation of "technique" vs. "analysis.” For example, 'microscopy' appears as an robotic method, but it is unlikely that all instances of microscopy automated both the image capture and image analysis. Validity of the findings: Point 2 – Methods that could be automated do not seem to be the ones causing the reproducibly issues. Before publication, the authors must break down methods that can and can not be automated, compared to whether these methods are likely to impact the results of the specific paper. The authors list methods that could be automated by a cloud lab without any examination or discussion of whether automating these methods would have any impact on the final scientific result. Within these, many methods either provide a binary response, and are quite robust to variation and interpretation, or are merely preparatory and the preparation methods are robust to variation. For example, PCR is a robust technique that is usually used in a preparative manner, to obtain enough genetic material for a given experiment. It is difficult to see how outsourcing this method to a CRO would in any way affect the outcome of the primary and important experiment. The same thing could be said for many of the other methods the authors define as automated. A stronger paper would refine further and remove these methods from the list of those that could be automated. Then, a better discussion forms around the methods that can and can not be automated. Point 3 – How is a fully automated CRO any different than a “cloud lab?” Before publication, the authors must draw a distinction between the existing, fully automated CROs and a “cloud lab.” Otherwise, the rest of the paper does not come from a position of strength. The authors make a very good point that automation and use of CROs can increase the quality of lab research, but they take a naïve approach to the topic. They are correct that fully automated CROs, or fully automated internal facilities, are the standard for industrial science, where reproducibility is paramount. Where they fail to convince a reader is how these new “Cloud labs” are any different than existing CROs. For example, Transcriptic is a fully automated laboratory that allows researchers to conduct protocols of their choosing. In industry, however, it is widely accepted that a CRO is a fully automated laboratory that allows researchers to conduct protocols of their choosing. Point 4 -- Analysis lacks depth. It appears the analysis is a simple word count with little post processing. There is inadequate meaningful justification of this simple technique, or explanation of how the analysis goes further. For example, why was a technique selected that lacked recall? Why were only exact matches used? Why not use a hierarchical ontology for categorizing methods. Additionally, there appear to be substantial error rates in the characterization of technique usage. Finally, there is insufficient characterization of the papers which did not list an automated method (38% of papers), or discussion of this metric. It is loosely suggested, from the introduction, that these papers are likely to publish a new technique, but this number seems too high. Point 5 – There is an inadequate characterization of corpus There is some statement about types of papers in the introduction (main question, part I), but types are never meaningfully characterized initially or analyzed sufficiently. Before publication, the authors should better describe the corpus used for analysis. More metrics, such as the number of papers which have a methods section, would illuminate possible skews in the analysis. The analysis would also benefit from a deeper survey of the types of papers included in the corpus, e.g. MCB, study, clinical, new technique, etc. Point 6 – Various copy errors There are various copy errors throughout, but they are not listed here, as the paper must change significantly to warrant publication. Point 7 - Usefulness of Figure 1 Figure 1 shows several steps which really just reduces down to a single screen of selecting papers which appear in Elsevier. 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: INDICATORS FOR THE USE OF ROBOTIC LABS IN BASIC BIOMEDICAL RESEARCH: A LITERATURE ANALYSIS Review round: 2 Reviewer: 1

Basic reporting: The paper is generally well-written. A few typos I pointed out in the previous review still appear in the manuscript, although the authors say that they have been fixed. It might be a PDF conversion error, perhaps. The typos in the current revision are as follows: - "-The list of papers and their DOIs is available" -> are available -Typos involving"method" ("methodbut", "methodswithin", etc.) - "It is an elegant yet straightforward protocol lends itself"-> "that lends itself" - "due to it's vague usage" -> "its vague usage" I also think it would be good to be consistent about the usage of the term robotic lab vs. robot lab. Experimental design: I think the manuscript has been improved with the additional concept annotator and a more inclusive search strategy. One remaining issue for me: I am not convinced that using simple string matching for the 34 (=107-74) unmatched methods would cause potential bias, as the authors argue in their response. These have already been identified as robotic methods by some external entity, as I understand, not by the authors. Therefore, I believe their use is justified, as it mainly addresses the shortcomings of MESH and would strengthen their findings. It would also be good to give a sense of how accurate the concept annotators are on a benchmark corpus, if possible, in the lack of a ground truth for the task. 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: INDICATORS FOR THE USE OF ROBOTIC LABS IN BASIC BIOMEDICAL RESEARCH: A LITERATURE ANALYSIS Review round: 2 Reviewer: 2

Basic reporting: There are several instances where two words are missing a space, for example: Page 6/Line 168 and 170 methodsdetected should be methods detected Page 6/Line 171 and 172 methodsper should be methods per Otherwise, everything looks good, the paper is well written and referenced, and it meets all the standards for PeerJ. Experimental design: No comment. Validity of the findings: No comment. Additional comments: The only other minor issue is this URL still does not resolve for me: http://dx.doi.org/http://dx.doi.org/10.17632/gy7bfzcgyd.1 This URL does work: http://dx.doi.org/10.17632/gy7bfzcgyd.1

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: INDICATORS FOR THE USE OF ROBOTIC LABS IN BASIC BIOMEDICAL RESEARCH: A LITERATURE ANALYSIS Review round: 2 Reviewer: 3

Basic reporting: See final noted. Experimental design: See final notes. Validity of the findings: See final notes. Additional comments: The paper is much stronger and clearer now, but the analysis still leaves something to be desired. If you look back on my initial concerns, they focused on whether automation of the tasks that the authors suggest, such as PCR, would have any major impact on the outcome of the research. My opinion is that they would not. The authors do not address this claim in their responses. That is the major issue I still have with the manuscript. I am not firmly against publishing the work, but do believe the tone of the paper needs to change. After all, this is really a presentation of the workflows that are used in the literature vs the availability of these workflows at CROs or automated labs. It is a survey. Conjecturing that automation of these workflows will increase reliability in the scientific realm is not believable to me, and may not be for the reader either. An honest and thorough address of the topics in my original review would flush a lot of this out. Unfortunately, this did not occur.

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: VISUAL AND ACOUSTIC COMPONENTS OF COURTSHIP IN THE BIRD-OF-PARADISE GENUS ASTRAPIA (AVES: PARADISAEIDAE) Review round: 1 Reviewer: 1

Basic reporting: This paper involves description of the little known male display behavior r of the Bird of Paradise genus Astrapia from videos take by the authors. The detail of the description varies with species, the description of the Huon Astrapia has the greatest detail. The descriptions tend to be detailed and in some cases very limited for some species The authors cite the available literature. There are no hypotheses being tested as this is pure description and some speculation as to the behavior Experimental design: This paper provides only descriptions of videos of male courtships and in one case a copulation with a female. The methods are adequate to to allow descriptions where relevant videos exist. Validity of the findings: The findings appear valid. The first video of each behavior type and the behaviors described match. No statistics are presented due to small sample sizes. Additional comments: The manner in which the male behaviors are described are very mechanical. While there is no doubt that males are following as sequence I would have found it beneficial to see these displays as interactive, and inclusive of the female's behavior. In some instance her behavior is noted, but more detail would be of interest and important in interpreting the actions on video. Even saying that she is off screen would be useful in the early stages. It would also be useful to have a short summary of the results of the behaviors being described at the beginning of the sequence. Courtships leading to copulations and those the do not are often somewhat different as are those in which young males may be involved.

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: VISUAL AND ACOUSTIC COMPONENTS OF COURTSHIP IN THE BIRD-OF-PARADISE GENUS ASTRAPIA (AVES: PARADISAEIDAE) Review round: 1 Reviewer: 2

Basic reporting: This article meets the standards set out for basic reporting. However, I do think the introduction is exaggerated with respect to the importance and implications of this study. Experimental design: This paper meets the standards set out for experimental design. However, there is no research question posed in this study. This work is simply a description of behaviors noted in videos present in a public data base. This paper does not present the results of a 'study' or 'experiment' or 'investigation' nor is there a 'research question'. It utilizes videos available in a public data base, and it describes behaviors that have not been described before. Validity of the findings: It would appear that this study meets the standards for validity of the findings. The videos in the library at Cornell University are 'valid' and thus descriptions of behaviors observed in those videos are also therefore valid. But, again, this paper does not present the results of a study or research project. Additional comments: I appreciate the value of the videos collected by one or more of the authors and it is very important that behaviors previously unknown are described. This 'ethogram' part of the current paper is fine. These behaviors and vocalizations are important to describe. But, that's all this paper is. It is a partial, incomplete, poorly documented ethogram of courtship behaviors based on video recordings. That's okay, but I beg to differ with the authors that these descriptions represent the courtship phenotype of this genus. I generally don't like that term courtship phenotype, but even if it is a good term the genus Astrapia does not have one courtship phenotype as suggested by the title and introduction. Each species would have its own courtship phenotype, and each species would exhibit considerable variation in that phenotype. Specific comments: 1. The authors couch the introduction as it's important to study birds of paradise in order to understand how sexual selection promotes diversity. The notion that sexual selection promotes speciation is a hypothesis, not a well-accepted fact. It probably does, but it is certainly not widely accepted to do so. And, the data in this study have nothing to do with sexual selection. The data in this study are just descriptions of behaviors used in courtship. I think the introduction should be rewritten to emphasize how little is known about various species of birds of paradise and the importance of cataloging their behaviors so that comparative studies can be done. I think the authors are trying too hard to make this study seem relevant to sexual selection or behavioral ecology in general. This study has no relevance to sexual selection or behavioral ecology. It is relevant to the biology of birds of paradise and understanding the diversity of behaviors in that group. That's okay, but I think the authors should not try to overstate the importance of their data. 2. The authors should describe what is meant by 'core bird of paradise' or 'core bird of paradise genera'. 3. The authors conclude that there are considerably more behavioral components to the display of Astrapia species than previously known. Well, considering that there is not a single paper describing any courtship behavior in any species of Astrapia, isn't that obvious? What is the point of even saying this? If zero behaviors were previously described, and now there are 7 behaviors described, does that mean that there are many more behaviors than previously known? I'm not purposefully trying to be flippant, but my point highlights the point above that the authors are trying to make this partial ethogram into something it isn't. These behaviors are important to describe, but I encourage the authors to accept the extreme limitations of their data set and not to venture outside of that data set. 4. I would have appreciated a comparison of the display behaviors across the species. Perhaps there aren't enough videos of all species? But, I think the authors should try to compare the various display elements across the species that show them. Or, compare these behaviors to those seen in other genera, like Parotid that the senior author has studied extensively.

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: VISUAL AND ACOUSTIC COMPONENTS OF COURTSHIP IN THE BIRD-OF-PARADISE GENUS ASTRAPIA (AVES: PARADISAEIDAE) Review round: 1 Reviewer: 3

Basic reporting: All good. Experimental design: No experiments were conducted. Validity of the findings: The authors have been very responsible in restricting the interpretation of their data to generalizations supported by video observations. I would actually prefer more analysis and speculation in conclusions. Additional comments: The authors present some extraordinary observations of the courtship display behaviors of Astrapia birds of paradise. These birds are incredibly poorly known, and found in some of the most remote habitats on earth. Practically ever page of the manuscript elicited at least one involuntary "Holy shit!" from this reviewer. The effort required to obtain these data are amazing, and the novelty is beyond doubt. The real contributions of this manuscript is to further broaden our understanding of the diversity of the displays of birds, and birds of paradise in particular. I think that the manuscript could be improved with a little bit of a change in narration style or perspective. The authors have presented this manuscript as an example of video "data mining". Although I applaud the use of video vouchers to document the behavior, it is not as if the authors are recommending this as a research method. Unlike genbank or other data bases, one can't legitimately say, "Gee, let's mine this video collection for amazing and unexpected data on bird behavior!" Incongruously, the author's don't reveal that Scholes and Laman have spent nearly a decade scouring the forests of New Guinea painstakingly collecting these kinds of video data! I think attempting to present behavior as a more modern, data-mining pursuit actually distorts and harms our understanding of the results. For example, we are told that certain perches are "practice" perches, but we don't know anything about why that judgment was made. Because the results are not presented in terms of how many days were spent where and in what season of the year, etc., we don't know whether the lack of continuity in behavior was because the birds never returned to that perch again, or the researchers had to move off. That sort of thing happens a lot. The authors assume things from knowledge they have, but are not part of the 'data-mining' procedure, and this affects all of the results. Do the authors really imagine that members of the public could "mine" this "publicly accessible" video collection, and do anything like this job of accurate job of analysis? Absolutely not! The difference between these alternatives, of course, is what is missing from the ms. Perhaps brief description of the field effort etc. that went into each batch of recordings, and some further details in supplemental files could address this problem accurately. But it is also one of intellectual stance that comes across throughout the ms. Lastly, I think that the authors could do more to compare, or at least discuss, the elements of the behaviors that are shared or convergent with other birds of paradise. For example, the wing-shuffling mechanical sounds are shared with some species of Epimachus, no? What other birds of paradise perform these relatively rare inverted displays? In the realm of convergence, the Velvet Asity (Philepitta castanea) also does a sort of somersault display around the perch that could be mentioned. Is there anything about the display of A. mayeri that is specifically associated with its extraordinarily long, and derived white tail? Although these comments would be speculative, they would be very constructive. So the analysis at the end seems shorter than needed. Miscellaneous comments (line numbers): 59- No "the" before Astrapia 94-98- Include number of males here. 160-161- What exactly is a pivot? Lots of side to side motions would not be called pivots, so this is unclear. 173- The phrase "a display that is called…" sound very Olympian, like the names of the displays are discovered. But the authors just made them up! Please use "that we call…" 200-01- a side to side rocking motion that makes the tail bob up and down? How is that? Can you help a little more? 247- "Interactive observation (female)" - Sounds like the name of an modern, avant garde painting. It's not even helpful because an interaction requires two individuals, and the sex of only one is identified. How about the straight up- "Male-female interactions" ?? Same goes for "Upright nape-peck (pre-copulatory)". (I can't wait to see that in a gallery in Soho!) Why not Pre-copulatory upright nape-peck ? 254- turn, not tern 279- I am sure that it is challenging to visualize, but I really recommend a sonogram of the shirk-shirk wing sound. Although it may look poorly, acoustical details can probably be extracted from it. I think that needs to be in, or its absence needs to be explained. 316, 318- Should be fig. 5, not 4. 602-3- I cannot visualize the bird and its parts in Fig 2C at all. What parts are wings or tails? Can the authors help with this- perhaps a little inset line drawing? Otherwise, this image is a blackhole.

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 IDENTIFICATION AND CHARACTERIZATION OF GRAS TRANSCRIPTION FACTORS IN TOMATO (SOLANUM LYCOPERSICUM) Review round: 1 Reviewer: 1

Basic reporting: 'no comment' Experimental design: 'no comment' Validity of the findings: 'no comment' Additional comments: 1. In the part of Materials and Methods, your introduction needs more detail. I suggest that you illustrate the detailed methods of sampling materials at different stages in order to explain the results at lines 328- 331. 2. In the part of Materials and Methods, please indicate the criteria of excluding acids. 3. The English language should be improved to ensure that your international audience can clearly understand your text. I suggest that you have a native English speaking colleague review your manuscript. Some examples where the language could be improved include lines 291, 296-298, 319,323, 353-358. And some other problems were revised in manuscript. 4. I commend the authors for their extensive data set, compiled analytical works. In addition, the results of manuscript is useful to information for the functional research in the future for tomato. But it is clearly written unambiguous language. If there is a weakness, its English language is not professional in the manuscript (as I have noted above) which should be improved upon before Acceptance.

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 IDENTIFICATION AND CHARACTERIZATION OF GRAS TRANSCRIPTION FACTORS IN TOMATO (SOLANUM LYCOPERSICUM) Review round: 1 Reviewer: 2

Basic reporting: The English language should be improved to ensure that your message is transported clearly. I suggest that you have a native English speaking colleague review your manuscript. Some examples for the beginning of the Ms: rational root patterning - radial (42)The transcription factors are essential in functional genomics - reword (47)The GRAS transcription factor is - you are talking about a gene family (65) The functional localization of the motif - reword (68) In addition, the N-terminal region of GRAS proteins in the DELLA subfamily contains the other two conserved motifs ......., indicating that the N-terminal domains of GRAS proteins are variable - finding motives does not necessarily make them variable (83) Hitherto, the RNA-seq is developed, following the function of GRAS genes, which are deeply excavated - can RNA-seq lead to function? (90) the inhibition of DELLA proteins was released immediately by the 26S proteasome - the degradation by the DELLA protein through the proteasome released the inhibition Make sure all spaces are at the correct position and check for italic on the plant names. (49) Arabidopsis(Arabidopsisthaliana) first Arabidopsis not italic. I think Brassicaceae is more commonly used in contrast to Cruciferae. Recheck the gene names: e.g. 265 Solyc090830.2.2 Some newer literature references on GRAS proteins seem to be missing. Especially the discussion on the evolvement from methyltransferases could be important for the analysis of conserved motives. Fig. 1 (and others) - please explain colour coding and add some more information to the legend. Fig. 2 - might be too small writing for printing As this is an interesting field and genome wide analysis could tell us much about understanding evolution and the role of these factors in different developmental stages this could be more clarified in the discussion. Experimental design: GRAS sequences were extracted from Solanum lycopersicum but the expression data were from S. pimpinellifolium? Why were those uesed and is there a difference between the GRAS genes in both species? (205) in some GRAS proteins were less than that in the typical GRAS domain, such as Solyc04g011630.1.1 or Solyc06g076290.1.1genes.... Did the authors check if this is just an annotation problem or if these genes might be pseudo genes? Validity of the findings: (220) Strikingly, some clades do not contain the members of GRAS proteins from Arabidopsis or rice; for instance, Gv6 and BolA groups. This might be attributed to the loss of the corresponding member following the separation of the last common ancestor. - is there any prove that it is a loss of this branch not a gain? (320) We also determined that some genes present a time-specific expression, such as, in the stage of fruit ripening; several genes are expressed in the later stage. - This is an important finding and should be elaborated upon. Fig. 5, where this data is derived from, is not very easy to follow - perhaps some symbols of grouping to different stages whould make this easier to read and interprete. Are the genes listed according to their branch (that could be added for clarification) or was there any clustering performed? As you state "As a whole, we found that the same group of GRAS genes shared a similar expression pattern..." this is not compehensible from the figure at first glance. Confimation of these data via qPCR might be recommendable. In 2015 Huang et al. have put together a genome-wide analysis of GRAS proteins in tomato and added also expression data. The authors should make it more obvious in how this study differs from the previous study. 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: A COMPARISON OF CHLOROPLAST GENOME SEQUENCES IN ACONITUM (RANUNCULACEAE): A TRADITIONAL HERBAL MEDICINAL GENUS Review round: 1 Reviewer: 1

Basic reporting: The manuscript is generally well written and, apart from some minor grammatical errors and ambiguous sentences as indicated below, the language used is clear to understand. The introduction section has effectively been written, introducing all the aspects of the study and referencing previous studies on genus Aconitum. However, there is need for more details in order to support the need for further molecular systematics research. The two most recent studies by Hong et al. (2017) and Kong et al. (2017) on phylogenetic reclassification (reconstruction) appear to have clearly outlined phylogenetic relationships within the genus Aconitum by using multiple nuclear and chloroplast markers. The authors in the current study acknowledge the effectiveness of universal markers of cp genome to resolve phylogenetic relationships among species (line 64-68). However, they claim that there is need for further research using data obtained from whole genome (Phylogenomics). To support their claim the authors have stated in line 94-95 and 99-100, that there are some species whose positions are yet to be resolved. The authors should list examples of a few taxa whose phylogenetic positions are unclear and outline how the current data will resolve. The cited literature is relevant and recent. The reporting structure is in line with the standards of the journal. Figures are well presented and of good quality. Information on raw data deposition and availability has been given by the authors. Experimental design: The design used here seems sound, replicable and in line with the research concerns highlighted in the introduction section. The research is within the scope of the journal. Validity of the findings: The results presented here are reliable based on the experimental design. The results and discussion section is sound and the readers can be able to draw conclusions based on the results discussed here. Additional comments: The manuscript is well presented. However, to ensure that international readers understand the intended meaning in some statements, I suggest you further review the manuscript. Below, and as highlighted in the attached pdf, I have listed some of the points that may need your attention. Please compare the suggested statements to the original ones. Line 32 The length of the cp genome sequences was 156,109 bp for A. angustius, 155,625 bp for A. finetianum and 157,215 bp for A. sinomontanum. Line 33 Each of the three species possesses 126 genes including 84 protein coding genes (PCGs). Please use ‘’structural variations’’ instead of ‘’structure variations’’ to refer to changes in genome organization. Line 40 In total, 50 simple sequence repeats (SSRs) were detected in A. finetianum and A. angustius, while 57 SSRs were discovered in A. sinomontanum. 43 A higher percentage of… 45 ..will be of benefit for further/ will benefit further…. 54 The cp genome in angiosperms is a circular DNA molecule with typically quadripartite structure 74 …possesses heterologous chromosomes and is suggested to be a hybrid of A. finetianum and A. sinomontanum. 80 In the past decades, the genus Aconitum was known as a taxonomically and phylogenetically challenging taxon. 86 …ITS, which suggested Aconitum to be a monophyletic clade and a sister group of Delphinium 103 We further compared 114 Fresh leaves were collected from A. angustius, A. finetianum and A. sinomontanum growing in a greenhouse at South China Botanical Garden, Chinese Academy of Sciences. 124 Genomic DNA was fragmented randomly and then the required length DNA fragments were retained by electrophoresis The minimum numbers (thresholds) of the SSRs were set to be 10, 5, 4, 3, and 3 for mono-, di-, tri-, tetra-, and penta-nucleotides SSRs respectively. 134 comments You sequenced and annotated three species, here you report four species. Please confirm if this is correct. OGDRAW should be in parenthesis 194 …. by manually identifying the overlapping regions. 195 In order to further determine the draft genome, quality and coverage of each base position by reads remapping were double checked and corrected accordingly. 200 The chloroplast genomes displayed a typical quadripartite structure, including a pair of IRs (25,927-26,225 bp) separated by LSC (86,664-88,074 bp) and SSC (16,914-17,107 bp) regions (Fig. 1 and Table 1). 202-205 I suggest you use 1 decimal place instead of 2. The GC content of the three species was 38.0%, demonstrating congruence to that reported in A. barbatum var. hispidum and A. barbatum var. puberulum (38.0%) of subg. Lycoctonum as well as in the species of subg. Aconitum (38. 0% or 38.1%) (Table 1). 206 Please avoid listing the names of the species again, when it is clear that you’re referring to them. When duplicated genes in IRs regions were counted only once, each of the three cp genomes encoded 126 predicted functional genes, including 84 PCGs, 38 tRNA genes and four rRNA genes. 212 In the three studied species, and similar to most other plants species, the maturase K (matK) gene is located within trnK intron. 213 In the IR regions, the four rRNA genes and two tRNA genes (trnI and trnA) are clustered as 16S-trnI-trnA-23S-4.5S-5S. This is also reported in the cp genomes of A. barbatum var. hispidumand and A. barbatum var. puberulumas as well as in many other plant species 223-225 However, structural variations were still present in the LSC/IR/SSC boundaries (Fig. 2). The genes rps19-rp12-trnH and ycf1-ndhF were located in the junction regions of LSC/IR and SSC/IR respectively. 225-230 The rps19 gene crosses the LSC/IRa junction in A. sinomontanum, A. barbatum var. puberulum and A.barbatum var. hispidum of subg. Lycoctonum, as well as in A. jaluense, A. volubile, A.carmichaelii, A. kusnezoffii and A. monanthum of subg. Aconitum. As a result, the gene has apparently lost its protein-coding ability due being partially duplicated in the IRb region, thus producing pseudolized Ψrps19 gene. 253 The average nucleotide variability (Pi) value was estimated to be 0.00549, ranging from 0 to 0.03856, based on comparative analysis of sequence divergence of complete cp genome sequences of Aconitum species. 271 MISA identified 50 SSRs, with minimum 10 bp repeats in length, in both A. finetianum and A. angustius, while 57 SSRs were detected in A. sinomontanum. 280 Remarkably the SSRs had a high A/T content with only seven SSRs, including (ATCT)3, (TTCT)3, (CTTT)3, (TAAAG)3, (TTTC)3, (ATAC)3 and (CATT)3, containing one C or G nucleotide. 294 The total aligned length with parsimony informative loci was 178,392 bp with 4,342 for the complete cp genome sequences, and 106,535 bp with 3,164 for PCGs, respectively. 300 Based on the phylogenetic tree, the tetraploid A. angustius was always closely related to A. finetianum, which is also supported by previous research Figure 1. One of the ycf15 duplicates, on the IR, is colour coded as ‘other genes’ while in table two the gene is listed under ‘’genes for photosynthesis’’. Please check alongside the partially duplicated gene of ycf1.Probably this is because the duplicates have been annotated on the same strand of the DNA molecule. Figure 2. Please check the use of ‘inverted’ in the title. On the same, kindly consider using ‘’IR regions’’ or just ‘IRs’ while referring to the two IRs rather than using ‘IRs regions’.

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: A COMPARISON OF CHLOROPLAST GENOME SEQUENCES IN ACONITUM (RANUNCULACEAE): A TRADITIONAL HERBAL MEDICINAL GENUS Review round: 1 Reviewer: 2

Basic reporting: Please have a native English speaker revise the writing. Examples of mistakes include lines 26-27, 70, 80-81, 86, 107-108, 305. There are many others, these are some of the worst. Experimental design: No herbarium specimen vouchers are cited. Many journals now require this for publication. Vouchers are essential for reproducibility, especially in taxa where identification is challenging. Considering that chromosome number is the best way to distinguish two of the species, ploidy confirmation is also desirable. Line 189 describes results from de novo assembly, while line 129 describes a reference mapping method. Which is correct? The phylogenetic analysis describes 3 methods used, but only 2 trees are shown (Fig. 4A & B). The nodal support values are not identified. Lines 295-297: The 2 trees shown are not “mostly concordant” since 4 nodes differ, and 3 nodes in 4B do not have high support values. Figure 1 is not necessary, since the difference between the plastomes is shown in Figure 2. All other aspects of gene organization do not differ from published Ranunculaceae. Table 2 is not necessary, since this is the typical angiosperm gene content and structure. Validity of the findings: Most of the plastomes used in the comparative analyses are unpublished sequences downloaded from GenBank, with no attribution other than citing accession numbers. What evidence do the authors have that these unpublished plastomes are properly identified, assembled and annotated? The comparative analyses fail to incorporate the Aconitum reclinatum plastome, recently published by the same authors. This is unjustified. My recommendation is to remove the phylogenetic analysis, since it does not provide much new information beyond the results already published in the A. reclinatum paper, and limit the gene content/structure analysis to species with reliable published plastomes. 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: A COMPARISON OF CHLOROPLAST GENOME SEQUENCES IN ACONITUM (RANUNCULACEAE): A TRADITIONAL HERBAL MEDICINAL GENUS Review round: 2 Reviewer: 1

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: A COMPARISON OF CHLOROPLAST GENOME SEQUENCES IN ACONITUM (RANUNCULACEAE): A TRADITIONAL HERBAL MEDICINAL GENUS Review round: 2 Reviewer: 2

Basic reporting: I had recommended removing the phylogenetic analysis – but the authors kept it in the manuscript. If it is retained, I insist that the authors fully acknowledge the source of all chloroplast genomes, including those that are unpublished. I am in favor of open data, but this also requires full attribution. Two citations were added for published plastomes (Lim et al., 2015; Choi et al., 2016) but these are not included in the literature cited. The unpublished Aconitum plastomes from Korea need to include a citation of the authors listed in the Genbank records: “Kim, G.-B., Lim, C.-E. & Mun, J.-H. unpublished. Complete chloroplast genomes of Aconitum species from Korea. Obtained from Genbank on Date” Voucher numbers have been added (from Genbank records) for the source specimens of the plastome sequences, but these are incomplete – we also need to know the herbarium where the voucher is stored. This is missing from Genbank and will need to be obtained from the submitters of those sequences. The phylogenetic distribution of the pseudogenes should be described (now that the phylogenetic analysis is retained). Interestingly, only pseudo-infA corresponds to a clade (i.e. single pseudogenization). In the case of pseudo-ycf15 the 3 species A. monanthum, austrokoreense & A. chiisanense are not monophyletic. This suggests the tree is incorrectly resolved – or less likely, the gene was lost once and then restored to a functional copy. These possibilities need to be discussed. Likewise, rpl16 was either pseudogenized twice, or restored to function once in subg. Lycoctonum. “pseudolized” is not a word. Replace with pseudogenized. 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: A COMPARISON OF CHLOROPLAST GENOME SEQUENCES IN ACONITUM (RANUNCULACEAE): A TRADITIONAL HERBAL MEDICINAL GENUS Review round: 3 Reviewer: 1

Basic reporting: The added text on the phylogeny of pseudogenes (lines 360-370) requires substantial English improvement. Experimental design: no comment Validity of the findings: no comment Additional comments: The authors have adequately addressed my concerns.

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: MALE SEX PHEROMONE COMPONENTS IN HELICONIUS BUTTERFLIES RELEASED BY THE ANDROCONIA AFFECT FEMALE CHOICE Review round: 1 Reviewer: 1

Basic reporting: This manuscript is well written with a solid foundation of literature to motivate the research conducted within; figures are legible and appropriately explained in captions. I have no major concerns with the analyses or writing, but please go through the references for consistency in formatting, especially italicizing genus and species names. Experimental design: The authors do a thorough job of investigating male pheromone production in three distinct ways, strengthening the conclusions to be drawn from their findings. Reporting of protocols is explicit and clear from data collection to software used in statistical analyses. Validity of the findings: The authors present strong evidence for the existence of pheromones in mature males that enhance mating success. Results from the structural, chemical, and behavioral experiments work together to show that chemicals produced (or at least stored) in the hindwings of mature males contribute to reproductive success. I have only a few comments about the reporting of these results and their implications: First, in L 333-337, the authors note that courting rates differed between control and treated males in one of the four species, yet they report percentages for successful matings pooling all tested species. What do the percentages of mating success look like when excluding the species in which control males courted more frequently than experimentally treated males? Next, experimental addition of pheromones is the other side of the coin to experimental removal/blocking of such compounds. In L 422-424, the authors give the example of H. cydno males unsuccessfully courting heterospecific females. Assuming that the pheromone composition of H. cydno is made up of a handful of similar compounds to those studied here, how difficult would it be to synthesize and experimentally apply such compounds to these males to do a complimentary follow up experiment? Finally, throughout this manuscript, the male butterflies are said to have both forewing and hindwing androconia. This is a reasonable assessment based on morphology alone, as Figure 1 shows sexual dimorphism in both wing pairs. However, the chemical analyses of different parts of the wings suggest that it is largely if not entirely the hindwing region that produces/stores pheromones. Furthermore, the behavioral studies only blocked regions of the hindwing manipulated males to demonstrate differential mating success. It seems that the dorsal hindwing is the key area for pheromone signals in these species. In light of these results, is it less appropriate to continue calling the forewing regions androconia? Perhaps a sentence or two could be added about if/how results presented here affect the classification of the regions on the forewing? Additional comments: This manuscript and the research within are both of high quality and definitely suitable for publication. As you can see, my corrections and suggestions are all minor. At most, a couple of points could be clarified with another line or two of explanation in the text, but no additional analyses will be required. I have mentioned some suggestions above with specific line references, but please see the attached file for the full list of line-by-line 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: MALE SEX PHEROMONE COMPONENTS IN HELICONIUS BUTTERFLIES RELEASED BY THE ANDROCONIA AFFECT FEMALE CHOICE Review round: 1 Reviewer: 2

Basic reporting: In general, the authors use clear, unambiguous, and professional language throughout. Their introduction and background are sufficient, the literature is well referenced and relevant, and the article structure conforms to discipline norm. The figures are relevant and of high quality, though S3 is highly relevant to the main text of the article, and should be included in the main text. The raw data was also supplied. Experimental design: The authors posed a study searching for the presence of androchonia in four populations of Heliconius butterflies, and tested whether the presence of the chemicals produced by these androchonia influence male mating success. This is a highly relevant and timely research question for speciation and mate preference work, as Heliconius butterflies are a model system for speciation research. Male preference for female wing color is heavily studied in this system, but, to date, very little is known about either female preference or the use of chemical signaling in Heliconius mating decisions. Therefore, this study has the potential to substantially broaden the scope of speciation research in one of the most well used study systems for reproductive isolation and population genetics research. The characterization of the chemical compounds associated with the proposed androchonia is outside of my technical area of expertise, therefore I suggest someone else review those methods. However, they appeared clear to me, and detailed enough that I believe they could be easily replicated. I do, however, have a few comments and concerns about the experimental design of the behavioral assays. 1) Given that the authors only recorded female behavior when males were courting, the authors should describe (or cite a source describing) male courtship in H. melpomene, H. erato, and H. timareta. Male courtship is a very important part of this study, and it is currently undefined. Please include. 2) I am concerned by the large number of failed choice assays in two of the populations (H. melpomene rosina and H. erato demophoon). The authors appear to have used observational data for a portion of the females who did not mate with either the control or the experimental male (and thus have not behaviorally exhibited a clear preference) to assess whether females behave differently around preferred or non-preferred males. However, as these females did not mate with either male, it is unknown which male they prefer, therefore their data may confound the results. 3) I am also slightly concerned about the small sample sizes used for the behavioral assays, given that behavioral data tends to include a large amount of individual variance. The sample sizes are fine for the binary mating outcome analyses, but may be too small to detect biologically relevant female behaviors when searching for female behavior specifically focused towards attractive or less attractive males. The authors do mention these low sample sizes in the results section, but they then over interpret these findings in the discussion. Validity of the findings: The authors did a good job presenting their work in the context of published research on the role of chemical signaling in butterfly mate choice. Their histology and chemical characterization results are clear, and their mate choice study, though with small sample size, has clear results. I am, however, concerned by the selection of which butterflies were used for further analysis of female behavior (which I describe in more detail in the general comments below), and the authors classification of these behaviors as rejection behaviors. The authors describe all female behavior as rejection behavior, however they do not have supporting evidence that these behaviors are, in fact, rejection behaviors in Heliconius melpomene, Heliconius erato, or Heliconius timareta. The authors should take an unbiased approach when describing the female behaviors, and simply state that they observed and recorded four different female behaviors: flutter, wings open, abdomen lifted, and flying away. Unless there is another study in H. melpomene, H. erato, and H. timareta that I am not aware of which clearly shows that fluttering, wing opening, abdomen lifting, and flight happen at a higher frequency when males are courting females than when females are not being courted by males? Fluttering and wing opening are fairly common butterfly behaviors, thus the authors should justify their description of these behaviors as rejection behaviors, or use more neutral language. Additional comments: The authors integrated an interesting set of chemical ecology and animal behavior studies to assess whether chemical signals on the wings of male Heliconius butterflies influence male mating success. In general, this is a well written paper describing a timely set of experiments. However, I have a couple of major concerns regarding the behavioral experiment, as well as concerns about data interpretation, which I detail below. 1) For both H. melpomene rosina and H. erato demophoon, most of the females did not mate during the two-day choice assay (as illustrated in Table 1). This suggests to me that there may have been something wrong with the set-up. Are the authors sure that the females were old enough to mate? Are the authors sure that the males were old enough to mate? While it is clear that the females that did mate preferred control to androchonia blocked males, I am a bit concerned by the shear number of failed mate choice trials. Particularly given the authors interpretation of female behavior during the observed portion of the choice trials. Given that most H.m. rosina females did not mate, I find it unsurprising that the authors did not observe a difference in H. m. rosina female behavior around control or androchonia blocked males (as illustrated in Figure S3). In fact, the authors’ behavioral results, coupled with their finding that over half of the H. m. rosina females failed to mate with either the control or the androchonia blocked males suggests that either these females were not reproductive, or that the females did not find either male attractive. Either way, one would expect these females (who did not mate with either male) to treat both male forms equally. I suggest that the authors remove the behavioral data for the females that did not mate, and only compare the behavior of the females with known preferences (ie mating outcomes). Otherwise the authors are using their assumptions about female preference to define behaviors as rejection behaviors, instead of using actual preference data to assess whether females treat males they prefer differently from males they do not prefer. 2) The authors state that the absence of these chemical compounds in young males and older females suggests that the compounds are unlikely to have been sequestered from plants, but that logic seems a bit flawed to me (Lines 370-373). The compounds could be sequestered from plants in males but not females, and only be released in older males. In order to state that the compounds are unlikely to be obtained from direct sequestration of compounds from larval host plants, the authors need to show that males reared on different host plants produce the same chemical compounds. This does not mean that I do not agree with the authors’ argument that these chemical compounds are important for mate acquisition. I think the authors have adequately shown that these chemical signals increase the likelihood of female acceptance. However, I do not think the authors have sufficient support for their statement in line 372 that there could be genetic control of the production of these compounds. Nor does there have to be genetic control of the production of these compounds for them to play a role in reproductive isolation- if the chemicals were sequestered from host plants, host plant differentiation could be associated with male chemical differentiation and consequent reproductive isolation. Lines 394-401: I am confused as to why the authors expect females from different species and populations to exhibit the same behaviors when interacting with males. The authors found that females did exhibit population specific behavioral differences in response to preferred versus non-preferred males- why not discuss this further? This is a particularly interesting finding, given the small sample size (which the authors do discuss in their results). As I mentioned earlier, since the authors have not yet shown that these behaviors are, in fact, rejection behaviors (and their results here suggest otherwise), the authors should use a more neutral term to describe the documented female behaviors. Minor Comments: Line 193- the authors state that shortening extraction time did not influence results, but do not show the data or cite a paper to support this statement. This should be a relatively straightforward figure or citation. Please include. Line 425-426: The authors state that male androconial compounds differ between H. timareta and H. melpomene, but this data appears to be unpublished. Is it currently in review? Will it be published soon? Can the authors support this statement with published 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: MALE SEX PHEROMONE COMPONENTS IN HELICONIUS BUTTERFLIES RELEASED BY THE ANDROCONIA AFFECT FEMALE CHOICE Review round: 1 Reviewer: 3

Basic reporting: XX Experimental design: XX Validity of the findings: XX Additional comments: XX

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: MALE SEX PHEROMONE COMPONENTS IN HELICONIUS BUTTERFLIES RELEASED BY THE ANDROCONIA AFFECT FEMALE CHOICE Review round: 2 Reviewer: 1

Basic reporting: The authors have addressed my concerns in their revision. Experimental design: no comment. Validity of the findings: The authors have satisfactorily addressed my previous concerns. Additional comments: I like this study. It is integrative and timely. I appreciate the revisions the authors made to the text. Particularly their inclusion of separate analyses of the female behavior in successful and unsuccessful mating trials, the detailed descriptions of the behaviors of the butterflies, and their moving the figure of female behavior from supplemental to the main text. The authors have satisfactorily addressed my previous concerns.

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: MALE SEX PHEROMONE COMPONENTS IN HELICONIUS BUTTERFLIES RELEASED BY THE ANDROCONIA AFFECT FEMALE CHOICE Review round: 2 Reviewer: 2

Basic reporting: I have only seen the revised version with responses to the previous reviewers. In my eyes this paper complies to all criteria, it is well written, well illustrated and both introduction and discussion are sound. Experimental design: The authors have convincingly responded to the concerns raised by the previous reviewers and I have no additional comments concerning the experimental design. Validity of the findings: The authors have perfectly responded to the concerns raised by the previous reviewers, and reformulated parts where a slight over-interpretation might have occurred. No additional comments from my side. Additional comments: This paper accumulates an important amount of work on male sex pheromones and their role in mating in different Heliconius species/races. It is evident that certain behavioural data are difficult to obtain in rare species/races. But this does not at all reduce the value of the presented 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: HCV CORE ANTIGEN IS AN ALTERNATIVE MARKER TO HCV RNA FOR EVALUATING ACTIVE HCV INFECTION: IMPLICATIONS FOR IMPROVED DIAGNOSTIC OPTION IN AN ERA OF AFFORDABLE DAAS Review round: 1 Reviewer: 1

Basic reporting: English language usage is quite OK, although a review could enrich the manuscript. Experimental design: In this manuscript, Wasitthankasem et al propose the use of the core antigen of the hepatitis C virus (HCV Ag) as an alternative marker to HCV RNA when screening patients for HCV viremia. Considering that: “Development of highly sensitive of HCV Ag quantification assays correlates well with and is comparable to HCV RNA(Bouvier-Alias et al., 2002; Ottiger et al., 2013; Florea et al., 2014). Therefore, HCV Ag can be used as a reliable marker for the diagnosis of active HCV infection and the evaluation of response to antiviral therapy (Mederacke et al., 2009; Rockstroh et al., 2017; Alonso et al., 2017).”, what is the originality, or even the main objective of the present work? I understand that authors are presenting specific data from a given human population, but they should keep clear what exactly is the novelty of their approach. Validity of the findings: The data is OK and the experimental work seems to be well conducted, but while reading the manuscript I got a question: Since authors say that “The results of this study suggest that anti-HCV Ab tests had a fair correlation with HCV RNA and are appropriate for primary screening…” are they proposing HCV Ag testing ONLY as a “primary screening” that should later be confirmed through HCV RNA testing? In this case, the discussion about the costs/time needed to HCV RNA testing makes no sense… The proportion of patients that needed diagnosis confirmation through HCV RNA testing seemed still significant to me. Could the authors discuss that? Additional comments: Some points can be addressed by the authors: There are no reasons to include the following phrase “The remaining samples were HCV RNA-negative.” at the Abstract since authors claim that, amongst patients, 222 tested positive for HCV RNA. Actually, at this point, it is not clear if patients negative for HCV RNA are simultaneously negative to HCV Ag and/or to anti-HCV (which seems not be the case since the presented numbers could not totally overlap). Authors mention that disease progression after HCV infection depends on different factors, but they do not present any follow up of the patients. It would be interesting to have some follow up data in order to actually evaluate the efficacy of the proposed diagnostics. Authors claim that “All but two of the HCV RNA-positive subjects were reactive to HCV Ag (≥3 fmol/L).”. What happened to these two patients? Were they subjected to the same therapeutics as the positive patients? Again considering these two patients, at the Discussion it is mentioned that both were HCV genotype 3, but the next paragraph starts saying that “This study demonstrated that HCV Ag showed excellent correlation with viremia irrespective (sic) of the HCV genotype…” In my opinion, these two assertions are conflicting. Please comment on that. Are the authors planning to evaluate the results of direct acting agents (DAAs) therapy as a potential outcome in the present cohort (controlling for HCV genotypes, for example)?

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: HCV CORE ANTIGEN IS AN ALTERNATIVE MARKER TO HCV RNA FOR EVALUATING ACTIVE HCV INFECTION: IMPLICATIONS FOR IMPROVED DIAGNOSTIC OPTION IN AN ERA OF AFFORDABLE DAAS Review round: 1 Reviewer: 2

Basic reporting: Wastthanasem R et al report a study performed in 298 HCV-seropositive individuals to test the usefulness of HCV Ag as a marker to access active HCV infection in an HCV endemic region. Their findings suggest that HCV Ag is a good surrogate marker for HCV RNA. English language and written is clear. My only suggestions in regards to this work are that the authors include some newest references to improve the discussion and how their results relate to these publications. For example, a) Hepatitis C virus core antigen assay: an alternative method for hepatitis C diagnosis. Wang L, Lv H, Zhang G. Ann Clin Biochem. 2017 Mar;54(2):279-285. doi: 10.1177/0004563216661218. b) Can Hepatitis C Virus Antigen Testing Replace Ribonucleic Acid Polymerase Chain Reaction Analysis for Detecting Hepatitis C Virus? A Systematic Review. Khan H, Hill A, Main J, Brown A, Cooke G.Open Forum Infect Dis. 2017 May 26;4(2):ofw252. doi: 10.1093/ofid/ofw252. Also, what is the author's opinion on how this marker could potentially work in patients receiving antiviral treatment in their population setting? (The Role of Hepatitis C Virus Core Antigen Testing in the Era of Direct Acting Antiviral Therapies: What We Can Learn from the Protease Inhibitors. Linh Thuy Nguyen, Emma Gray, Aisling O'Leary, Michael Carr, Cillian F. De Gascun, and Irish Hepatitis C Outcomes Research Network). ¶ Experimental design: No comment Validity of the findings: No comment Additional comments: I find this work useful since it provides more insight on the effectiveness of an alternative method for HCV diagnostics in regions or countries that may not afford full-scale HCV RNA testing.

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: HCV CORE ANTIGEN IS AN ALTERNATIVE MARKER TO HCV RNA FOR EVALUATING ACTIVE HCV INFECTION: IMPLICATIONS FOR IMPROVED DIAGNOSTIC OPTION IN AN ERA OF AFFORDABLE DAAS Review round: 1 Reviewer: 3

Basic reporting: The drafting of the introduction is redundant (second paragraph , line 48-56) with respect to the use of serology as a standard marker for diagnosis and the need for the use of confirmatory techniques, as well as the disadvantages thereof, which is mentioned again in paragraph 4 (lines 71-76). Due to a suggestion in the following section, I consider that the literature reference in line 87 and 95 is needless in that context, so I suggest replacing its justification in relation to the origin of the samples, which come from endemic regions of the infection, demonstrated in this literature reference (Wasitthankasem et al. 2017) . References in line 372 and 441 do not have a DOI number. Experimental design: In materials and methods are not described the evaluation of the combination of Ac and Ag tests to predict active infection. In table 3 , the results of HCV Ag and AntiHCV plus HCV Ag are exactly the same. The content of the statement of line 101 is a description of a result, not of the methodology, should appear in results only. Validity of the findings: The title of manuscript refers the implications for improve treatment option but there are only one paragraph in the manuscript that mention therapeutic agents and does not make specific reference to how it impacts the use of the determination of the antigen in the treatment of the patient. Neither in the objetive, experimental design or results does contemplate the dynamics of the marker during the treatment; in the discussion are not discussed the implications that the use of the antigen could have like marker of monitoring in the patients. There are others articles with the same results that this manuscript and the authors do not let us known which is the importance of repeat this analysis. Roberto Alonso et al. HCV Core antigen assay as an alternative to HCV RNA quantification: a correlation study for assessment of HCV viremia. Enferm Infecc Microbiol Clin. 2017 H.A. Soliman et al. Significance of the hepatitis C virus core antigen testing as an alternative marker for hepatitis diagnosis in Egyptian patients. European Review for Medical and Pharmacological Sciences. 2015 Magali Bouvier-Alias et al. Clinical utility of total HCV core antigen quantification : a new indirect marker of HCV Replication. Hepatology. 2002 Van Helden J et al. Hepatitis C diagnostics: clinical evaluation of the HCV core antigen determination. Z Gastroenterol. 2014. Chevaliez S et al. Clinical utility of hepatitis C virus core antigen quantification in patients with chronic hepatitis C. J Clin Virol. 2014. Hadziyannis E, et al . Is HCV core antigen a reliable marker of viral load? A n evaluation of HCV core antigen automated immunoassay. Ann Gastroenterol. 2013 And the others cited in the manuscript. The demographic data presented is not discussed and/or referred to as relevant to the purpose of the article. That is, the degree of fibrosis, age, gender or level of enzymes directly affect the determination of the HCV core antigen or what is the purpose of presenting them or having collected them in relation to the objective of the work? For example, in line 167, the associations between HCV RNA and elevated liver enzymes levels and advance liver stiffness are widely accepted clinical results, relevant for the HCV treatment and clinical management but irrelevant for the diagnosis of active HCV infection. For the same reason I higly recommend removing Figure 1, because it does not present relevant data that are not addressed in figure 2. The objetive of this work is correlates HCV RNA and HCV Ag, so the study universe is samples but not patients. Hence only is necesary known the total numer of Ac HCV positive samples that was used for the study. In Discussion (lines 274-277), the justification described for the antigenic test refers to cost; should be more sustained with respect to an approximate cost of each of the tests or a percentage of total savings in the test algorithm used for a patient with active HCV. Because the "Optional Screening Strategy" is not based entirely on the results obtained in this manuscript, it is suggested to give credit to the authors on which it bases its proposal. I sugest a statment like this "...These results together with previously reported evidence allow us to suggest a strategy...". On the other hand, the strategy does not contemplate the genotyping of the virus. 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: HCV CORE ANTIGEN IS AN ALTERNATIVE MARKER TO HCV RNA FOR EVALUATING ACTIVE HCV INFECTION: IMPLICATIONS FOR IMPROVED DIAGNOSTIC OPTION IN AN ERA OF AFFORDABLE DAAS Review round: 2 Reviewer: 1

Basic reporting: Authors adequately answered the questions raised by this reviewer. Experimental design: The authors clearly stated the novelty of their approach – meaning, a cost and effectiveness comparison of the different methods of HCV infection detection, in an environment characterized by a high prevalence of HCV infection, in a low-income country. Validity of the findings: No comment Additional comments: Authors adequately answered the questions raised by this reviewer. The authors clearly stated the novelty of their approach – meaning, a cost and effectiveness comparison of the different methods of HCV infection detection, in an environment characterized by a high prevalence of HCV infection, in a low-income country. Also, the Discussion was restructured and the information specifying which patients would be tested by which detection method was added. Specifically, authors included that individuals who possessed anti-HCV antibody would be screened for HCV Ag. Only HCV Ag-negative samples should then be confirmed with HCV RNA-testing. Although no follow-up data were presented in order to actually evaluate the efficacy of the proposed diagnostics, I understand that this was beyond the scope of this study. Also considering treatment, but now concerning the two patients that tested negative to the HCV Ag, the authors just explained that they were referred to the Thailand National Health Security Office as otherwise positive based on the RNA results (gold standard). In summary, and as previously mentioned, I believe that the authors adequately answered the questions raised by this reviewer and that the modifications enriched 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: HCV CORE ANTIGEN IS AN ALTERNATIVE MARKER TO HCV RNA FOR EVALUATING ACTIVE HCV INFECTION: IMPLICATIONS FOR IMPROVED DIAGNOSTIC OPTION IN AN ERA OF AFFORDABLE DAAS Review round: 2 Reviewer: 2

Basic reporting: The authors have improved the manuscript. The English language is clear, references have been updated, and the structure is adequate. Experimental design: The authors have clearly explained their methodology and work algorithm, which in turn is part of their proposal to screen for HCV using the viral antigen. Validity of the findings: These points were covered by the authors and corrected for clarity as suggested by the reviewers. Additional comments: As mentioned in the first review, this work is important because it offers an alternative diagnostic method for HCV screening, thus securing treatment of those who are unaware of their condition in regions of high endemicity.

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: HCV CORE ANTIGEN IS AN ALTERNATIVE MARKER TO HCV RNA FOR EVALUATING ACTIVE HCV INFECTION: IMPLICATIONS FOR IMPROVED DIAGNOSTIC OPTION IN AN ERA OF AFFORDABLE DAAS Review round: 2 Reviewer: 3

Basic reporting: No comment Experimental design: No comment Validity of the findings: The changes in title, introduction and conclusion are adequate and let known which is the importance of this study. I only sugest that in order to unify the terms, use the term : "low- to middle-income countries (LMIC)" in line 66 (introduction) 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: TRANSCRIPTOME DYNAMICS ALONG AXOLOTL REGENERATIVE DEVELOPMENT ARE CONSISTENT WITH AN EXTENSIVE REDUCTION IN GENE EXPRESSION HETEROGENEITY IN DEDIFFERENTIATED CELLS Review round: 1 Reviewer: 1

Basic reporting: The manuscript needs work on sentence structure. Specifically, the overuse of prepositional phrases makes the sentences very difficult to follow. I suggest that the writer limits the use of these phrases and the manuscript will be much more reader friendly. Experimental design: The analysis seems sound, but the interpretations for what may be occurring within the blastema are overreaching and sometimes based upon speculation. Little is known about the cell heterogeneity across the time points studied and almost nothing is known about the chromatin structure in these cell type. The study is interesting and of value, but toning back the interpretation seems appropriate. The following suggestions point to examples where changes could be implemented. Validity of the findings: -I am not disputing the possibility that gene expression heterogeneity decreases during dedifferentiation, but it is unclear if the gene expression measures utilized in this study can support this hypothesis. This argument is based upon several points: 1) Our understanding of cell dedifferentiation during limb regeneration is minimal, which is an assumption required for this analysis to be applicable. As far as I am aware, only one cell type, the fibroblast, is assumed to dedifferentiate during axolotl limb regeneration and the definition of this dedifferentiation is unclear. Could fibroblasts just be “activated” fibroblasts similar to myofibroblasts in mammals, yet have more capabilities for patterning. Is the level of dedifferentiated limb fibroblasts known? As far as I know it is not. The fibroblast population in the limb is a small proportion of the cells that make up the sample used for transcriptomic analysis. Could the conclusions made be a representation of gene expression not actually occurring in the dedifferentiated fibroblasts? 2) The sharp decrease in gene expression heterogeneity after injury does not necessarily represent dedifferentiation. For example, amputation leads to a retraction of muscle fibers and their decrease in expression of many muscle-specific genes, which is likely highly heterogeneous in its extent and temporal profile. This phenomenon would lead to high variability early on and decreased variability after the event occurs. “Somehow surprisingly, not only cvGEBIs were significantly different for the expected timepoints, but they started being significantly negative as early as 1.5 days after amputation (Figure 1, and Table 1). If a reduction in gene expression heterogeneity truly reflected the presence of a significant number of dedifferentiating cells, these results would suggest that cell dedifferentiation leading to the formation of the blastema can be noticed transcriptomically considerably earlier than it can be defined morphologically.” -This statement assumes that dedifferentiation is the only transcriptional profile being collected, which is not the case. During this time, the inflammatory response, wound closure, and histolysis are all beginning to reside, which should also generate a decrease in transcriptional heterogeneity once they subside. I don’t think it is appropriate to say that the signature is due to dedifferentiation. 4) The interpretations made by studying cell heterogeneity could be innapropriate because the readout is the average signals generated from a mass of heterogeneous tissue including muscle cells, vascular cells, Schwann cells, epidermal cells, skeletal cells, and fibroblasts. Assume that the early blastema contains many different cell types, but may be different from sample to sample. Over time, the majority of cells in the blastema are of a single cell type, likely the fibroblast. This scenario would give the same gene expression heterogeneity profile seen here, but the interpretation would be considerably different than the one presented in the manuscript. Without single cell data, it is difficult to prove that the loss of gene expression heterogeneity demonstrated here is due to cell dedifferentiation. -Can a sentence be included in the GO analysis stating that the genes represented in the mitotic re-entry category are significantly different than the chromatin organization category. Chromatin organization in terms of GO is often associated with the cell cycle instead of the euchromatic or heterochromatic nature of the chromatin. -It is argued in the manuscript that chromatin architecture changes globally during regeneration. I am unaware of a study that demonstrates this to be the case. The author argues this point, but it is speculation until a study demonstrates this to be the case. -Can the author provide evidence that 10 biological replicates is sufficient for testing heterogeneity of gene expression? They present data that lower biological replication is likely insufficient, but can an argument be made that 10 replicates is enough? -The authors should make clear how the analysis presented here varies from the original Voss et al., 2015 manuscript. Additional comments: The manuscript by Diaz-Castillo presents some interesting points on the general feature that dedifferentiation is associated with a loss of gene expression heterogeneity. The manuscript uses a deep transcriptional dataset of axolotl limb regeneration to study gene expression heterogeneity throughout the regenerative process. Although the analysis seems sound, the interpretations for what may be occurring within the blastema are overreaching and sometimes based upon speculation. Little is known about the cell heterogeneity across the time points studied and almost nothing is known about the chromatin structure in these cell type. The study is interesting and of value, but toning back the interpretation seems appropriate. The following suggestions point to examples where changes could be implemented. Most points are covered in the above sections.

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: TRANSCRIPTOME DYNAMICS ALONG AXOLOTL REGENERATIVE DEVELOPMENT ARE CONSISTENT WITH AN EXTENSIVE REDUCTION IN GENE EXPRESSION HETEROGENEITY IN DEDIFFERENTIATED CELLS Review round: 1 Reviewer: 2

Basic reporting: NA Experimental design: This manuscript performs a set of investigations and provides empirical evidence for the hypothesis that dedifferentiated cells will lead to reduced gene expression heterogeneity. Motivation and problem are clearly described. Methods are described with proper details, I would suggest to add more biological explanation for each metric used in the study. Validity of the findings: An existing data set is used in the study. The effect of the number of replicates is discussed, highlighting the importance of enough replicates. Several different metrics (including proper statistical test) are used to analyse the data and derive final conclusions. Additional comments: The manuscript presents empirical results for the hypothesis of reduced gene expression heterogeneity in dedifferentiated cells. Overall, the paper is well organized and presented. Conclusions are well derived from the analysis. My only suggestion is to add more biological meaning for the metrics used in 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: TRANSCRIPTOME DYNAMICS ALONG AXOLOTL REGENERATIVE DEVELOPMENT ARE CONSISTENT WITH AN EXTENSIVE REDUCTION IN GENE EXPRESSION HETEROGENEITY IN DEDIFFERENTIATED CELLS Review round: 2 Reviewer: 1

Basic reporting: The author has made significant changes to the manuscript to make it easier to follow. Experimental design: The experimental design was based upon a previously published study, which was strong. Validity of the findings: My general concern in my original review was the interpretation of the findings. Many of the issues cannot be resolved without years of research in the field to determine the actual meaning of dedifferentiation and the cellular heterogeneity of the regenerating limb blastema. The Author has made significant changes to provide support for their idea of dedifferentiation and I accept the arguments made by the author. Additional comments: The author has strengthened the manuscript through the changes made throughout the manuscript and their rebuttal to the reviewers is valid. I am comfortable supporting acceptance of 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: TRANSCRIPTOME DYNAMICS ALONG AXOLOTL REGENERATIVE DEVELOPMENT ARE CONSISTENT WITH AN EXTENSIVE REDUCTION IN GENE EXPRESSION HETEROGENEITY IN DEDIFFERENTIATED CELLS Review round: 2 Reviewer: 2

Basic reporting: The sentences are really difficult to understand. A native speaker is suggested to revise the manuscript. cvGEBIs is the major concept for this paper. But it's not well described in the method. It would be nice to provide a formula or diagram to readers. Experimental design: The idea of studying changes of expression heterogeneity is very interesting. However, the logic is not complete to get the conclusion. Validity of the findings: The logic connection between extensive chromatin relaxation and generalized decrease in gene expression heterogeneity along with cell dedifferentiation is not clear. How extensive chromatin relaxation causes generalized decrease in gene expression heterogeneity is not explained in the background. The normalization method strongly affect the detection of generalized increase in transcription. The author should do more research about it. Other wise it's really difficult to convince people that generalized increase in transcription doesn't contribute to the decreasing of expression heterogeneity. The fact that genes have sharp decrease in gene expression heterogeneity between 1 and 1.5 days post-amputation are enriched in cell cycle and chromatin organization terms doesn’t suggest these two functional terms are the mechanism of decreasing gene expression heterogeneity. Instead the author should test if genes that are deferentially expressed post-amputation are enriched in these two functional terms. 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: INCREASING EVIDENCE THAT BATS ACTIVELY FORAGE AT WIND TURBINES Review round: 1 Reviewer: 1

Basic reporting: No comment Experimental design: One concern about the experimental design is that methods for species identification for acoustic calls needs to be clarified on if all species were identified using auto-ID functions of the software or if there were comparisons with call libraries during manual identification of call files. Another concern about the experimental design relates to the data collected from insect trapping being compared to genetic data of fecal and stomach contents. It appears to me that insects from trapping were not identified to the species level using genetic analyses and therefore, those data can't be compared directly to species identification of the genetic material at the same taxonomic level. See specific comments on the manuscript. Validity of the findings: It needs to be clarified how fecal samples were collected with details of where guano was being obtained. Were these guano samples in small piles or were there fecal pellets stuck to the turbine/door as if the guano was had fallen from a bat in flight. Isopods discovered in the fecal samples, as found in the supplemental tables, raises the question on the validity of identified insects consumed by bats. This is particularly so given that these arthropods are usually on the ground or under substrate and generally photophobic and unlikley to be attracted to the turbines themselves. In light of this, there are possible explanations for its presence in your samples and the need for caveats. See specific comments on the manuscript. Additional comments: This is a great paper that uses a variety of methods to detect the presence of foraging behavior by bats at wind energy facilities! However, I feel that there are some missed opportunities when you don't present more on the acoustic data for the other bat species that show a greater amount of feeding activity than hoary bats and red bats. I do like the fact that there is a comparison of insects captured at the sight vs. the stomach and fecal samples of bats. However, the resolution in species identification from the genetic analyses is difficult to compare to the trapped insects when the same methods aren't employed. I think some of the conclusions related to your findings are fair to speculate but may not be the entire picture of what is happening in this system. I did play devil's advocate by pointing out other options or reasons for explanation that could also be possible. These are addressed directly 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: INCREASING EVIDENCE THAT BATS ACTIVELY FORAGE AT WIND TURBINES Review round: 1 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: The statement in the discussion “Our study provides strong support for the hypothesis that bats are using wind turbines as a foraging resource” can be interpreted in two ways: 1) bats forage around turbines; 2) turbines affect bat behavior and/or resources such that the area is preferred or used more than other areas without turbines. The correspondence between insect composition in light traps and those in bat stomachs and pellets is very suggestive that bats forage in and around wind turbines. It is also certainly possible that turbines can attract insects which in turn attract bats. Alternatively, the correspondence may simply reflect a situation where bats are foraging on available insects (both near and far from turbines) and the turbines do not affect this relationship in any meaningful way. That is, while the study does show that interpretation 1 is valid, it is harder for this reviewer to feel that a strong case has been made for interpretation 2. Foraging behavior at turbines has been observed both visually and acoustically in other studies, and Foo et al. contribute additional evidence that this activity is also prevalent in their study area. (The statement that eastern red bats were likely roosting on the turbine structures after foraging nearby is a particularly noteworthy observation.) However, the study was not designed to falsify the hypothesis that turbines serve as a focal resource (although they may well be). To accomplish this, it would be necessary to sample both bat activity and insect prevalence at sites distant from turbines, and perhaps also assess fecal pellets obtained from bats captured at these locations with those collected at turbines. From this a comparison of the proportion of bat calls with approach and terminal phase components recorded at locations distant could be made with those sampled close to turbines. If proportions were similar, it would indicate that turbines are not necessarily attracting bats and they simply encounter these features as they are foraging across a landscape; that is, foraging activity is similar. A study that applied this method to acoustic samples is Jameson and Willis (2014) who observed “a smaller proportion of feeding buzzes at [meteorological] towers compared to woodlots during all study periods despite the dramatic increase in overall activity at towers during autumn” and concluded that bat presence was likely associated with visual attraction to tall structures for purposes of social interactions. I applaud authors for a solid study showing that there is, as stated in the manuscript title “increasing evidence that bats actively forage at wind turbines”. However, the authors may wish to consider tempering statements such as “abundant foraging opportunities continue to attract migrating bats to wind turbines” with a caveat that “attraction”, that is, relatively greater foraging use of turbine areas was not experimentally determined nor specifically the focus of the study. Additional comments: It would be useful to see the night-to-night correspondence in stomach samples relative to insect trapping. High correspondence would indicate that utilization matched availability (at least for certain orders), and provide strong evidence of prey capture at or near the wind site. Low correspondence would indicate prey capture at a distance from a site, or perhaps a low within-night match of utilization-availability. These results could be added to the supplementary materials.

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: INCREASING EVIDENCE THAT BATS ACTIVELY FORAGE AT WIND TURBINES Review round: 2 Reviewer: 1

Basic reporting: N/A Experimental design: N/A Validity of the findings: N/A 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: INCREASING EVIDENCE THAT BATS ACTIVELY FORAGE AT WIND TURBINES Review round: 2 Reviewer: 2

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: The primary concern this reviewer had with the initial ms was that the authors were making too strong a claim that bats are attracted to turbines for foraging. In the revised ms and response the authors acknowledge this and have made changes to the text that temper this claim while still indicating that it remains a phenomenon that needs further investigation. The authors did demonstrate that foraging does occur at turbines, and correctly conclude that foraging opportunities may be one reason that bats are active at those locations. 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: NEW SPECIES OF THE ENDEMIC NEOTROPICAL CADDISFLY GENUS CONTULMA FROM THE ANDES OF ECUADOR (TRICHOPTERA: ANOMALOPSYCHIDAE) Review round: 1 Reviewer: 1

Basic reporting: The English used throughout the manuscript is sound and there are only a few minor typing mistakes. The literature is up to date and all important references are cited in the text. I suggest the addition of a few authorship references along the text. The manuscript has a coherent structure with no misplacement of text in wrong sections. The authors correctly present background information for the reader in the Introduction section and state their hypothesis. The results are new and relevant to science and in accordance with the issues stated in the introduction. The figures are well drawn and present clear information of the necessary issues. Experimental design: The research presented is within the aims and scope of PeerJ. The methods used by the authors are those mostly used by the entomologists and suit correctly the aim of the manuscript. The methods are described in sufficient detail and references correctly cited. The research question helps filling a biodiversity gap extant in caddisfly studies, especially in the still poorly known Andean area. Validity of the findings: The species presented by the authors are undoubtedly new to science and are presented in a clear way with sufficient illustrations to support their asserts. The additional distributional data the authors provide are meaningful and help us to better understand the biodiversity and distribution of organisms. The authors propose a species synonym, which seems to be correctly stated based on the knowledge and material available. The argumentation they provide for the synonym is logic and the evidences provided are sufficient to lead to this conclusion. Additional comments: The authors present three new species of the high altitude caddisfly genus Contulma. Based on the descriptions and illustrations provided, the species are definitely new and represent a significant contribution to caddisfly knowledge. They also present new records and an identification key of males of Contulma, which is a relevant contribution to science, especially taxonomy and ecology of aquatic insects. The manuscript is well written and contains only some minor issues that should be addressed, such as citation of figures and rearrangement of a few sentences. All those issues are marked in an annotated 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: NEW SPECIES OF THE ENDEMIC NEOTROPICAL CADDISFLY GENUS CONTULMA FROM THE ANDES OF ECUADOR (TRICHOPTERA: ANOMALOPSYCHIDAE) Review round: 1 Reviewer: 2

Basic reporting: This paper is well-written. The figures are very good, and the literature matches with the call-outs in the text. A good contribution to our knowledge of this genus. Some minor corrections are suggested in the PDF. Experimental design: This paper falls within the Aims and Scope of the journal and meets all other stated objectives. Validity of the findings: no comment Additional comments: Please see yellow highlighted areas for minor suggested improvements. A table with all of the known species, their authorities, and country distribution would improve the overall value of this paper. This manuscript was also reviewed by Tatiana I. Arefina-Armitage.

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: NEW SPECIES OF THE ENDEMIC NEOTROPICAL CADDISFLY GENUS CONTULMA FROM THE ANDES OF ECUADOR (TRICHOPTERA: ANOMALOPSYCHIDAE) Review round: 1 Reviewer: 3

Basic reporting: The paper is clear and well written. The authors present an excellent overview of the current state of Trichoptera taxonomy in the Neotropics and ample background information on the distribution, habitat, and previous work of the genus. The illustrations are superb and expertly done. Relevant collection, specimen, and database information are provided. Experimental design: This original taxonomic research is within the scope of PeerJ and fills an important knowledge gap for Neotropical taxonomy. The work meets the highest technical and ethical standards in taxonomy. The methods are described in great detail and in such as way to facilitate replication by another investigator. Validity of the findings: The superb illustrations, detailed diagnoses and descriptions, and identification key will prove to be valuable resources for future workers of this group. The authors present ample evidence for the designation of these new species. The authors explain their rationale for the synonymization of C. duffi with C. penai. Additional comments: Thank you for the opportunity to review this manuscript. This original taxonomic research is within the scope of the journal and appropriate for publication in PeerJ. The importance of describing rare, highly endemic new species, such as these new Contulma from the Andean region of Ecuador, cannot be overstated. This work is an excellent contribution to our overall knowledge of Trichoptera taxonomy and distribution, and provides a rare glimpse into the remarkable diversity of these little known organisms. Climate change, land-use changes, and other threats to high altitude glacial streams and inter-Andean valleys all underscore the critical need to document this diversity. The paper is clear and well written. The authors present an excellent overview of the current state of Trichoptera taxonomy in the Neotropics and ample background information on the distribution, habitat, and previous work of the genus. Methods are clearly described, and include valuable information regarding databases and adherence to ICZN standards. The illustrations are superb and expertly done. The diagnoses and descriptions are detailed and clear. The synonymization of C. duffi with C. penai is well explained and justified. The inclusion of references to previously published illustrations, will make the identification key an especially valuable resource for future workers. I was unable to test the key using actual specimens, but based on previously published illustrations, it worked perfectly. I offer a few very minor changes below as suggestions for improvement. I have also listed some very minor edits (mostly typographical errors). All of these are meant as suggestions to improve the paper and should be left to the authors as to whether or not they are needed. 1.) Please check for consistency in formatting for in-text citations. For example, on line 33, Holzenthal 1988 is written without a comma, however on line 48 Holzenthal and Río-Touma, 2012, the comma is included. I leave it to the PeerJ editors to confirm the proper final formatting. 2.) Lines 45-49: The authors explain that these species are rarely collected using standard light trap techniques and that most specimens are hand netted during the day or collected by Malaise traps, especially at high altitude. They go on to state that infrequency of collection does not equate to rarity in nature. Although not explicitly stated, the authors seem to imply that this may be a reflection of minimal collection efforts or perhaps low temperatures. Although admittedly speculative, perhaps a sentence to this effect would help to clarify. 3.) Line 97: Author & year not listed for first mention of C. paluguillensis. 4.) Lines 99-100: First mention of species listed do not cite authors & years. Are these also described by Holzenthal and Flint 1995? 5.) Line 194: Author & year not listed for first mention of C. lanceolate. 6.) While not critical to the paper, readers might appreciate a short recap of what is known regarding the phylogeny and species relationships of the genus. The authors cite Holzenthal and Flint’s (1995) comprehensive revision of Contulma, yet they make no mention of species groups, although they are somewhat alluded to in some of the diagnoses (e.g., lines 99-101, 152-153, Footnote 2 of key). I recognize a phylogenetic analysis is certainly outside the scope of this paper, but a broader discussion of morphology, species relationships, and how these new species might be placed, would add valuable context. 7.) Line 203: There is an extra “s” in the word process. 8.) Footnote 1 of Key: Robertson is incorrectly spelled. 9.) Line 249: posteromesal is incorrectly spelled. 10.) Line 384: setose protuberances (needs space). 11.) It is unclear why each of the figure legends begins with “Catalog of the Neotropical Trichoptera (Caddisflies)”. Typographical error? In summary, I recommend this manuscript for publication with very minor revisions. It is an excellent 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: COMPARATIVE ANALYSIS OF THE MICRORNA TRANSCRIPTOME BETWEEN YAK AND CATTLE PROVIDES INSIGHT INTO HIGH-ALTITUDE ADAPTATION Review round: 1 Reviewer: 1

Basic reporting: In general, the manuscript is well written. However, the following issues should be addressed. The growing environment of the experiment animals should be provided, especially the altitude. The altitude could potentially affect the overall result. In the section of differential expression of microRNA, more detail on how to normalize read counts should be provided. The method of FDR calculation is also needed. In Result section, Line 120, it is hard to interpret (88.86±4.87%). Lline 131, it is not traditional way to use correlation coefficient to measure experimental confidence, what is the method to calculate the correlation coefficient: Pearson or Spearman’s? Line 166~169, how the Pearson’s correlation of family, cluster and background are calculated is not clear, between yak and cattle or between tissues? If it is between yak and cattle, how are tissues treated in the calculation. More details are needed to interpret the result. Figure1. ‘coserved’ should be ‘conserved’ Figure 2. The color scale for the heatmap is too wide. Figure 3. Legend does not match the figure. There are 5 panels in the figure, while there are only A to C in legend. Figure 4. p-value should use –log10 instead of –log2. Fold change in p value does not make sense and is hard to interpret. Table S1: Format the spreadsheet to show all numbers. Table S2: Round or format the numbers to an easily reading format. Table S5: Does ‘genomeID’ represent chromosome? All the supplementary tables need to be reformatted to a readable and scientifically meaningful format. All supplementary figures should have figure legends. 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: COMPARATIVE ANALYSIS OF THE MICRORNA TRANSCRIPTOME BETWEEN YAK AND CATTLE PROVIDES INSIGHT INTO HIGH-ALTITUDE ADAPTATION Review round: 1 Reviewer: 2

Basic reporting: This manuscript presents the findings from miRNA profiling study of Yak and Cattle. It is well written with a professional article structure. The findings sound interesting to relevant research community. Experimental design: No comments Validity of the findings: Some minor comments on the data analysis In the method section of data analysis for small RNA sequencing, the author wrote "Given the reliability of analysis results, reads with a copy number larger than three were retained for subsequently analysis. Comparable amount of miRNAs were identified for using bovine genome or yak genome (data not shown), we selected bovine genome as the reference genome in this study." More details need to be added here. For example, does the "copy number" mean "read count"? need to be greater than three in all samples or at least one sample? Regarding miRNAs identified with bovine and yak genome, the number of miRNAs might be similar, how about their expression level? If the expression level is not similar, the results from yak genome will have to be used. In the section for identifying miRNA targets, how are the ortholgous human miRNAs identified for bovin miRNAs, by comparing sequence similarity or simply by matching the names? 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: COMPARATIVE ANALYSIS OF THE MICRORNA TRANSCRIPTOME BETWEEN YAK AND CATTLE PROVIDES INSIGHT INTO HIGH-ALTITUDE ADAPTATION Review round: 1 Reviewer: 3

Basic reporting: Revision by a native English speaking colleague or editing services is required. The introduction would benefit from some discussion of the evolutionary relationship between yak and cattle. Related to this point, the introduction should provide some justification for using cattle as the comparison species in this study. The legend for Figure 4 requires additional details for panel D. Figure 4 panel D contains text that has been resized such that the aspect ratio is incorrect. The legend for Figure 4 includes the statement "miRNAs in red indicated upregulated in heart, miRNAs in black indicated downregulated in yak". This color scheme appears inadequate as the two possibilities are not mutually exclusive. The data from this study is available through NCBI GEO under the accession number GSE87833. The GEO record for this study contains errors that should be corrected. For example, the methods section contains information from a different study: "Retinal mRNA profiles of 21-day-old wild-type (WT) and neural retina leucine zipper knockout (Nrl−/−) mice". The GEO summary also includes the word "epigenetic": "we illustrated the epigenetic differences in the microRNA transcriptomes level for heart and lung between yak and cattle". It isn't clear to me why the term "epigenetic" is used in this context--the authors have not demonstrated that these expression differences arise from epigenetic mechanisms as opposed to DNA sequence differences. Experimental design: The sample size is small but appears suitable for interspecies comparisons. However, more information on the animals is needed. Specifically, were they maintained at high or low elevations? The section "Data analysis of small RNA sequencing" contains some confusing and inconsistent statements. First, the statement "Given the reliability of analysis results, reads with a copy number larger than three were retained for subsequently analysis" is unclear. What is the "analysis results" referring to? Also, what does it mean for a read to have "a copy number larger than three"? Does this refer instead to a miRNA with support from more than three reads? Second, the statement "Comparable amount of miRNAs were identified for using bovine genome or yak genome (data not shown), we selected bovine genome as the reference genome in this study." The section up to this point makes no mention of using a reference genome--instead reads are mapped to bovine and other mammalian pre-miRNAs. Thus the meaning of this statement is not clear. The section "Data analysis of small RNA sequencing" refers to a database as "NCBI". A more specific name for this database is needed. Validity of the findings: In general the results are interesting but do not, in my mind, convincingly indicate that the miRNA expression differences between cattle and yak relate to high altitude adaptation. A more convincing study would have incorporated mRNA expression, such that the differences in miRNA expression could be linked to target gene expression differences. In general the language used to describe the conclusions needs to be adjusted to better convey the indirect nature of the findings. For example, the conclusion "revealed extensive roles of miRNAs in high altitude adaptation" could be "suggest extensive roles of miRNAs in high altitude adaptation." The paper does not include a discussion section. A discussion section should be added and should include a discussion of the limitations of this work, including the limitations of miRNA target prediction. The discussions of the implications of the miRNA expression differences do not seem to note which miRNAs are expressed more highly in yak and which are expressed more highly in cattle. Figure 4 does not clearly convey this information. This information should be included this in the discussions and in figure 4. For the miRNA differential expression results to support the hypothesis that miRNA expression is involved in high altitude adaptation it is important that the direction of the expression differences be discussed. 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: COMPARATIVE ANALYSIS OF THE MICRORNA TRANSCRIPTOME BETWEEN YAK AND CATTLE PROVIDES INSIGHT INTO HIGH-ALTITUDE ADAPTATION Review round: 2 Reviewer: 1

Basic reporting: The issues raised in last review have been taken care of. No further comment Experimental design: The experimental design meets the journal standard. No further 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: COMPARATIVE ANALYSIS OF THE MICRORNA TRANSCRIPTOME BETWEEN YAK AND CATTLE PROVIDES INSIGHT INTO HIGH-ALTITUDE ADAPTATION Review round: 2 Reviewer: 2

Basic reporting: Basic reporting --------------- 1. The authors indicate that an editing service has been used. However, the writing does not seem to have improved in quality and thus still requires extensive editing and is not suitable for publication in its current form. 2. The figures still contain text shown at incorrect aspect ratios. 3. The legend for Figure 4 still requires information describing panel D. 4. "reads mapping to bovine or other mammalian miRNAs were defined as conserved miRNAs": based on this statement, reads mapping only to bovine would be classified as "conserved", which doesn't make sense. 5. "reads with a read count larger than three" is unclear. I assume the authors mean "miRNAs supported by three or more reads". 6. line 122: "Given the reliability of analysis results"--In the revised version it is still not clear to me what this statement refers to. 7. line 124" "Comparable amount of miRNAs" should be "Comparable numbers of miRNA species" 8. lines 130 and 131: the normalization methods and FDR methods should be stated rather than just referring, for example, to a "default" method. 9. the discussion includes results that are not presented in the results section, for example "we identified 784 and 807 miRNAs using yak and cattle genome, respectively". The discussion should not present new results. Experimental design: Experimental design: -------------------- 10. Based on the miRBase information on microRNA nomenclature, orthology should not be inferred by name alone: http://www.mirbase.org/help/nomenclature.shtml "The numbering of miRNA genes is simply sequential. For instance, at the time of writing the last published miRNA was mouse mir-352. The next novel published miRNA will get the number 353. However, if you submit an Xenopus miRNA that is identical to human mir-121 for example, we will suggest you also name your sequence mir-121." "Please note that miRNA names are able to convey only limited information, and are entirely unsuitable to encode information about complex sequence relationships. You should not therefore rely on the name to tell you all you need to know about the sequence. Sensible database approaches should instead use dedicated fields and annotation to describe such relationships, such as the "family" data provided here." Based on the author's response to reviewer 2 and on the methods of the paper, the authors have not used a suitable, sequence-based approach to confirm that they have identified the appropriate human ortholog miRNAs for inferring mRNA targets. This raises questions about the validity of all the downstream functional analysis. The authors need to apply a more rigorous ortholog detection procedure, and they need to clearly describe this procedure in the methods. Validity of the findings: I have concerns about the validity of the findings given the issue with the miRNA ortholog identification described under Experimental design. 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 ASSOCIATION STUDY OF PIGMENTARY TRAITS (SKIN AND IRIS COLOR) IN INDIVIDUALS OF EAST ASIAN ANCESTRY Review round: 1 Reviewer: 1

Basic reporting: This is a important piece of work and well investigated. I have only minor points that should be adressed. Unfortunately due to the relatively small sample size no, so far unknown variants, showed significant association with eye and skin colour. I would, however encourage the authors to investiage ZNF804B in more details and in larger datasets if possible.The level of the English throughout the manuscript is very high and, therefore the manuscript is easy to read and well structured. The figures and tables in the manus are overall of high scientific quality and support the content of the manus nicely, however the graphic overall quality of the figures is low and eg. the abscissa in Figure 1 and 2 is inadequate. The use of literature is satisfying, but should be supplemented with more literature. Lines 48-52: Andersen et al. 2013 (Forensic Sci Int Genet. 2013 Sep;7(5):508-15. doi: 10.1016/j.fsigen.2013.05.003). Experimental design: I like the overall study design with a study population and follow up population to replicate the skin pigmentation findings. The statistical analyses are wisely choosen and performed in a very nice way. I see some challenges with the measurements of skin pigmentation and the use of imputatation. The authors have obtained buttock pigmentation measurements for the study population, but not for all of the follow up populations. It is well known that buttock levels resemble the constitutive pigmentation levels, whereas arm levels may be influenced by environmental factors (UVR) (eg. Johansen et al. 2013, PLoS One. 2016 Mar 3;11(3):e0150381. doi: 10.1371/journal.pone.0150381). I find it problematic to compare these two areas of measurements without any further discussion. This should be adressed in the discussion section. Also a better discription upon the sample collection for the follow-up samples is needed. Much could be learned from the very nice description of the study population "Measurement of pigmentary traits". Eg. how many replicates were taken and did the authors use the median of the replicates if any? I would also appreciate a discussion about the use of imputation. Validity of the findings: The outsome of the study is important for research within the field of pigmentation genetics. The East Asians is a neclegted population groups. It is not surprising that no variants show significant association with skin colour as the variation within the investigated group is minor. A the authors also describes, the number of samples needs to be increased in order to find variants with minor effects. 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 ASSOCIATION STUDY OF PIGMENTARY TRAITS (SKIN AND IRIS COLOR) IN INDIVIDUALS OF EAST ASIAN ANCESTRY Review round: 1 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: This manuscript reported a GWAS on pigmentation in Asian populations. Such study is much needed. The strength of this study included instrument-based pigmentation phenotype measurement. The reviewer only has two suggestive comments. 1. The authors did not report meta analysis of the discovery set and replication set. Any reason for it? 2. It would be helpful to list the results of all the pigmentation loci previously identified in white populations. It is important to compare the beta estimates between the two racial groups. Of course this study has a much smaller sample size so that P value is not comparable.

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: MECHANISMS OF OAT (AVENA SATIVA L.) ACCLIMATION TO PHOSPHATE DEFICIENCY Review round: 1 Reviewer: 1

Basic reporting: The manuscript reports the mechanisms of oat acclimation to phosphate deficiency. The major findings include the capacity of oats to use organic phosphate (phytate) as a source of phosphorus and the role of different pools of acid phosphatases in acquiring phosphorus from soil. The paper is prepared professionally, minor language editing is recommended (e.g. "another enzymes" in the abstract, line 57, etc.). The statement that acclimation of cereals to phosphate deficiency remains undiscovered sounds a bit too strong. There are many publications in this field, and the authors cite the most important papers in their manuscript. The manuscript contains valuable sets of data that can be useful for agronomists and plant physiologists involved in the problem of phosphate deficiency in cereals and in oat in particular. Experimental design: The experimental design is acceptable. Four cultivars have been used. The parameters studied mostly refer to the general classical physiology. The biochemical characterization of isoforms of acid phosphatases adds an important information to this study. The methods are described with sufficient detail. This research fills some existing knowledge gaps in phosphorus acquisition in cereal plants. Validity of the findings: The major findings in this study are valuable. They include the capacity of oats to use organic phosphate (phytate) as a source of phosphorus and the role of different pools of acid phosphatases in acquiring phosphorus from soil. Additional comments: In general, this manuscript contains important sets of data on phosphorus acquisition by oat plants. The study has a value for practical applications. It is useful for the agronomists that face the problems of phosphate deficiency in their field practice.