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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: GENE-BASED ASSOCIATION STUDY FOR LIPID TRAITS IN DIVERSE COHORTS IMPLICATES BACE1 AND SIDT2 REGULATION IN TRIGLYCERIDE LEVELS Review round: 2 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: The authors have addressed the issues, and I have no further 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: COST-BENEFIT ANALYSIS FOR INVASIVE SPECIES CONTROL: THE CASE OF GREATER CANADA GOOSE BRANTA CANADENSIS IN FLANDERS (NORTHERN BELGIUM) Review round: 1 Reviewer: 1

Basic reporting: Following the guidance of PeerJ I have checked figs and tables, the raw data and language. The raw data and code can be opened, and are well described in English. Language is clear, intelligible and professional. At a few places in the text minor details were found that can easily be corrected in a final stage. The references to literature provide sufficient context to Invasive Alien Species management, environmental economics and the model species. The structure of the article is professional. There are no hypotheses or research questions, but there is a clear aim and relevant results in relation to that. Experimental design: The study reports on original primary research within Aims and Scope of the journal. A research question or an hypothesis is not provided. However, in my view, there is no need for them given the aim of the paper to demonstrate comparison of scenario’s using a bio economic model. The application of such a model is relevant & meaningful and fills an identified gap. The study uses techniques that suit the purpose. There are no ethical issues that could be an objection to publish the study. The methods are described with sufficient detail & information to replicate the study, provided the reader has a good mathematical background. Below, I will identify how the authors may facilitate readers that are less proficient in mathematics. Validity of the findings: I appreciate the study as a worthwhile exercise. The data that are used refer to goose census data, harvest data and costs of water sanitation and crop damage. The authors rightly point out that data availability is limited. Still, they are able to put it to good use with their model. The data have not been gathered for this particular purpose but they are used in a logical way and the analyses are statistically sound. The parameters that feed into the models can be regarded as good approximations and the conclusions will certainly be robust for changes in parameter values. The conclusions are well stated, linked to the original aim and limited to supporting results. Additional comments: By applying this model, it becomes clear under what assumptions the enhanced scenario of population management can be economically justified in comparison to business as usual. This is a general advantage of using (quantitative) models and it also works well in this case. The principal point that application of a Cost Benefit Analysis provides a useful framework to support decision making is well-elaborated. Maybe it would be good if the authors mention explicitly why an alternative scenario of ‘enhanced scaring of geese’ is not considered. The value of a Decision Support System is greater when all relevant alternatives are considered. In theory, goose damage may be prevented by scaring geese. Probably, the authors have reason to believe that scaring is not effective in practice, rendering the ‘enhanced scaring of geese’ scenario ‘not relevant’? The sensitivity analysis is restricted to exploring variation in a few relevant parameters (observed capture rate, discount rate and unit costs for eutrophication). The authors have made a selection for parameters they consider relevant. It thus remains unclear whether variation in other parameters may affect the conclusions and what the order is in parameter-sensitivity. For example: The parameters of the logistic growth model have been derived from limited census data. Thus, the value for carrying capacity (K) will be biased if the census results for the last years in the time series are biased. It would be good if the authors could explain that the results are robust for variation in the value for r and K and the unit price of hay. In words the authors have identified what the main assumptions are and to what extent they can be considered justified. One of these is the assumption that costs of maintaining a constant removal/capture rate are constant over a range of population densities. For this species, under these conditions, I think it is fair to make this assumption. Still I would suggest stating explicitly in the text whether the model conclusions will be sensitive for a change in model formulation with regard to these costs. Am I right to believe that this will not be the case, because costs of management are small relative to damage costs? Personally I would have appreciated more insight in the (calculation of the) population development under the two scenario’s and the observed field data from 2010-2015. From the text (lines 447 and 328) I read that “the predictions from this model were close to the observed results of a subsequent eradication “ and “at a lower than observed capture rate Bc could not be eradicated within the time horizon “. Is it possible to plot the observed data after 2009 in fig 3 with different symbols? Please consider adding more information on the predicted population development under the two scenarios (either as a graph or in words). From the information provided (line 276 “...the population level before moult capture in year t equals the remaining post-moult capture population in year t-1 projected one year ahead on the logistic growth curve”), the principle of the calculation of predicted population trajectories is clear. It does, however, require some mathematics to actually implement that into the formula for the growth curve provided (equation 1; in that formula the reference is the start population). The reader will be facilitated if the authors indicate that formula 1 can be used to calculate the population level before moult capture in year t, by replacing p0 by the remaining post-moult capture population in year t-1 and by replacing t by 1. Maybe it is also helpful to provide the script for the full bio-economic model. The authors point out that the benefits of management may be offset by increase in abundance of other species of geese (native or non-native). Would it be good to provide a little more background information on the status and numbers of relevant species in this respect in the introduction? What proportion of current damage is caused by Canada geese and what by other species? Given my proficiency in English, I am not in a position to comment upon language and grammar. But to me the text is entirely clear. Below, I present the scant editorial comments that I think are useful to make. In line 174 remove the word ‘and’ (double word) Line 244-246. It appears as if information is repeated? Please rephrase Line 447-449 I do not entirely understand the meaning of the sentence “The predictions from…..” Could you please rephrase this sentence? Table 1 line 3: a bracket points in the wrong direction

You are one of the reviewers, your task is to write a review for the article. You will 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: COST-BENEFIT ANALYSIS FOR INVASIVE SPECIES CONTROL: THE CASE OF GREATER CANADA GOOSE BRANTA CANADENSIS IN FLANDERS (NORTHERN BELGIUM) Review round: 1 Reviewer: 2

Basic reporting: see below - general comments Experimental design: See below - general comments Validity of the findings: See below - general comments Additional comments: Summary This study presents a cost benefit analysis (CBA) of the management and control of an IAS in Flanders. The BAU scenario is compared with a scenario that includes coordinated moult captures in addition to the BAU actions. The results show that the enhanced scenario had higher benefits (estimated as avoided costs from damage to grasslands and avoided damage from eutrophication) than additional costs. Some sensitivity analyses were performed with respect to estimate of N-damage, capture rate and discount rate. General comments The question addressed in this study is an important one. Given a limited availability of funds and resources, biodiversity management options should be carefully selected and motivated. Cost benefit analysis provides a useful tool for making such motivated policy choices. While the case study is an interesting one, the current version of the text raises several issues. Firstly, the structure of the text could be greatly improved. Most of the motivation regarding the assumptions that were made when performing the CBA were only mentioned in the discussion section, while they should be mentioned in the method section. Examples of these include the selection of damage types, the selection of parameters for the sensitivity analysis and the option not to include rebound effects. In addition, it is only in the section ‘sensitivity analysis’ that the study period is mentioned, while this should again be included in the method section. Secondly, the text (especially the introduction) seems to use the concepts ‘efficiency’, ‘effective’ ‘economically optimal’ and ‘cost-effectiveness’ as synonyms while they are clearly not. Thirdly, the CBA is not correctly described and implemented. - In the introduction (lines 116-135) the authors mention that a CBA is used to search for the scenario with the ‘lowest total costs’, to select the scenario that ‘maximizes avoided costs’ or the scenario with ‘the highest net PV (=total discounted avoided loss minus total discounted management costs)’. These three statements are contradictory and only one is correct (the last one). - In addition, when implementing a CBA it is important to clearly state the standpoint from which costs and benefits are assessed. The current study seems to perform a social CBA from the viewpoint of society in order to minimize the total net social costs associated with Bc management in Flanders. However, the text is not always consistent with this viewpoint: e.g. line 158 mentions ‘… do not represent a direct cost for the regional government or local authorities’ which seems to imply that the CBA is excluding costs from private citizens. - In a CBA costs and benefits are valued based on opportunity costs and not based on market prices, since the latter can be distorted by taxes, subsidies, imperfect information and imperfect competition. Thus market prices are unlikely to reflect the true social value of resource. Yet in the current study, the damage to grasslands is based on market prices for hay (line 222) without correction for sales taxes and agricultural subsidies. In addition, the damage from N and P is estimated based on avoided costs (line 193-195) which is a convenient method but has serious disadvantages since it is typically leading to an underestimation. - The geographic scope of the CBA should be mentioned explicitly and evaluated. Why look at Flanders and not the whole of Belgium? Are these geese population so local that they do not cross borders? - I cannot support the conclusion that ‘limiting the scope or complexity of a CBA is advisable without rendering the analysis unrealistic (lines 519-520)’. The essence of a CBA is to include all costs and benefits to all affected parties. Even though not all these impacts can be valued in monetary values, they should still at least be mentioned and not dropped for convenience reasons. Moreover, the current study is not exactly a good example in this sense since both the costs and the benefits of management actions are underestimated (as the authors acknowledge themselves in the discussion section). When both costs and benefits are underestimated it is not clear how the total result (PV values) are related to the true total impact. Specific comments - Introduction. Line 133. Several other interesting applications of CEA or CBA to invasive alien species management could be mentioned and used in this study (see, e.g., Waigner et al. 2010 or Schou & Jensen, 2017) - Line 141: please define ‘Anseriformes’ when you use the term for the first time. - Line 172: How do you know these are ‘the main economic impacts’ if the other impacts are not valued? - Line 193: The estimated range for the P-value is very high (80-800 euro/kg). Why is this range so large? Why did you decide to use only the lower estimate in the CBA? - Line 211: How are these compensation payments determined? What is their typical level? - Line 237-248: It is not clear how this ‘number of captures needed to reduce the Bc population’ is calculated. Especially the second step is confusing. - Line 275: How realistic is the assumption that the gees population does not migrate? - Line 411: ‘were’ instead of ‘ware’ - Line 429: by applying - Line 514: by applying - The datasets are not clear to use as an outsider: o What is included in the agricultural data? How are these data determined? Sources? How are they used? o Why are the Canada goose data not in chronological order? References Wainger, L. A., King, D. M., Mack, R. N., Price, E. W., & Maslin, T. (2010). Can the concept of ecosystem services be practically applied to improve natural resource management decisions?. Ecological Economics, 69(5), 978-987. Schou, J. S., & Jensen, F. (2017). Management of invasive species: Should we prevent introduction or mitigate damages? (No. 2017/06). University of Copenhagen, Department of Food and Resource Economics.

You are one of the reviewers, your task is to write a review for the article. You will 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: DISTRIBUTION OF BACTERIAL COMMUNITIES ALONG THE SPATIAL AND ENVIRONMENTAL GRADIENTS FROM BOHAI SEA TO NORTHERN YELLOW SEA Review round: 1 Reviewer: 1

Basic reporting: This manuscript describes a field study designed to examine microbial biogeography in surface water and benthic boundary layer water in the Bohai Sea of coastal China. I am pleased that this contribution focuses on the microbial diversity in marine and brackish environments of China and broader east Asia as this region has traditionally received less attention in the marine microbial ecology/oceanography literature. I commend the authors for their hard work collecting this dataset and for also sampling multiple biogeochemical parameters in addition to collecting samples for sequencing. I also commend the authors on the well curated datasets/tables/figures submitted with this manuscript. The writing of the manuscript is generally understandable, although there are many places (highlighted below) with awkward English phrasing and syntax coupled with rather simple grammar mistakes. The authors might benefit from having a native English speaker help them clarify certain sentences, remove unnecessary words, and ease overall comprehension. I have noted several examples below where the English writing could be improved to aid in reader comprehension. Figure 2: “explained” is misspelled Physiochemical is misspelled throughout text Abstract: Line 21: change “matters” to “material” Line 24: replace “spatial” with “the” Line 25: replace “communities” with “taxa” Line 25: remove “the” before “spatial” Line 26: “coastal gradients” Gradients of what? Please specify Line 28: “sunlit surface layer” Unclear if this refers to the “shallow coastal water (depth < 20m)” or is a unique depth strata in addition to the bottom layer. Please clarify. Line 33-34: “Geographic distance enhanced bacterial community dissimilarity and limited bacteria distribution in both surface and bottom water.” Unclear to me what is meant by this sentence... Are you saying that communities became more taxonomically dissimilar as geographic distance between samples increased? And that this trend held in both surface and bottom water? Please clarify Introduction: Lines 60-63: “Hence, spatial effects may superficially mask environmental effects when physicochemical factors are spatially autocorrelated, while in fact persistent environmental heterogeneity is possible to overwhelm geographic separation in generating and maintaining microbial diversity at intermediate spatial scales.” This sentence is unclear to me and should probably be split into two sentences. Are you saying that community composition is a function of both dispersion (so called spatial effects) as well as selection due to variable habitat conditions? And that this balance is effected by the scale at which you are defining your study area? As I understand you essentially have a two-factor study design where you must account for the effects of spatial separation/dispersion between similar habitats and combined niche adaptation+spatial separation differences between dissimilar habitats (i.e bethic boundary layer versus surface water). Again, I think this sentence should be clarified. Line 63-66: “As Wang et al. (2015)…” sentence probably not needed Line 71: “In the surface water” Please specify. For example, “in the upper 20 meters of the water column” Line 85: In contrast instead of “by contrast” Line 86: “shallow coastal water” Again please be specific about depths Line 91: located instead of “locating” Results: Line 197: Strange wording... I suggest instead "Bacterial diversity and abundance" Lines 198-199: “Thereinto, fourteen phyla including the main classes of phylum Proteobacteria were abundant …” This sentence has unusual wording… Try instead “We focused our analyses on fourteen taxonomic groups that were greater than 1% relative abundance in at least one sample. These groups included the Phyla … and the classes Alphaproteobacteria, Gammaproteobacteria, Betaproteobacteria, and Deltaproteobacteria.” Line 205: confusing syntax “This indicated the abundant taxa of P1 and B8 distributed in surface and bottom water with great similarity.” Line 209: replace “taxa of them were found to be the discriminant taxa in these distinct clusters” significantly discriminant clusters Line 223: remove “remarkable negative/positive” Line 226: “III were also strongly” Do you mean Cluster II here? Line 227: remove “Despite of these discriminant taxa” Line 227: “Despite of these discriminant taxa, there were still 50 abundant taxa, whose relative abundance together reached over 50 % in each cluster, dispersed indistinguishably in three clusters” Unclear wording... I'm not actually sure what you mean. Do you mean "50 taxa accounting for over 50% of total OTU abundance were evenly distributed amongst all three clusters."? Line 229: “Moreover, Cluster I-III shared 70 % of total OTUs, and the shared OTUs between surface and bottom bacterial communities accounted for over 89 %” Again unclear wording/syntax/grammar… I'm not entirely sure what you are saying here, but I think it would be clearer if you wrote "Samples from clusters I-III shared 70% of all detected OTUs. Furthermore, 89% of all OTUs were shared between surface and bottom water samples." Line 247: remove “Driving forces of” Line 248: Please be explicit about the input data for your PCoA analysis. Line 248: Replace “exhibited clear clusters” with suggest separation Line 270: remove “striking” Line 271: remove “close” Line 271: “correlationships” should be correlations Line 276: remove “intriguingly” Line 276: “geographic distance” Unclear - geographic distance of what? Do you mean pairwise physical distances between two samples? Please specify Line 248: Replace “exhibited clear clusters” with suggest separation Line 270: remove “striking” Line 271: remove “close” Line 271: “correlationships” should be correlations Line 276: remove “intriguingly” Discussion: Line 283: remove “Generally, environmental variables presented remarkable spatial gradients.” Line 283: “Salinity and turbidity were related to longitude, shaping horizontal gradients from the Bohai Sea to northern Yellow Sea; collection depth led to vertical dissimilarity of temperature, salinity, pH, DO, Chl a, nitrate, phosphate and silicate between surface and bottom water (Dataset S1).” Again, rather unclear wording and phrasing... I suggest something like "We observed horizontal salinity and turbidity gradients ranging from the mouth of the Yellow River to the northern Yellow Sea while gradients in temperature, salinity, pH, dissolved oxygen, Chl A, nitrate, phosphate and silicate were reflected in sample collection depths." Line 288: Replace “non-random distribution pattern of bacterioplankton” with patterns of bacterioplankton distributions. Line 295: replace “actually” with likely Line 295: “to their physiological requests for adequate light and oxygen, as well as the other beneficial conditions in surface water.” I don't think you have enough evidence to support this statement as it is written. I would say something like "Bacterial taxa discriminating Cluster I were likely adapted to surface water conditions as exemplified by the differential abundance of cyanobacterial OTUs and known aerobic heterotroph OTUs in surface samples. Line 309: Replace “the microbial hypothesis that some microorganisms are biogeographically restricted.” with their close proximity to mouth of the Yellow River. Line 369: “This could explain why nitrite was more relative with the bacteria diversity than nitrate in our results.” This statement needs to be revised for proper English grammar. I cannot decipher its meaning as it currently stands. Experimental design: I think the research question, motivations, and description of the methods is sufficient for publication in PeerJ. Validity of the findings: The results appear to be derived from sound sampling, processing, and sequencing methodologies, and for the most part (with some exceptions noted below) the statistical analyses appear to be robust. The conclusions are not overstated, appear to be adequately supported, and are largely consistent with the oceanographic features of the region (again I have some additional recommendations below). Minor Comments: Figure 1: It would be useful to have a figure inset displaying where the Bohai sea is located with respect to the rest of coastal China. This would give extra context for readers who are not familiar with the region. Instead of using terms like “surface waters” I would recommend being more specific and choosing something like “the upper 5 meters of the water column,” “the upper 20 meters” and so on. When mentioning N/P ratios please be more specific if you are talking about total nitrogen or inorganic nitrogen. DO should be replaced with “dissolved oxygen” throughout the text. Results: When discussing correlations between variables in this section please write the correlation and the P value. Line 248: Please be explicit about the input data for your PCoA analysis. Line 276: “geographic distance” Unclear - geographic distance of what? Do you mean pairwise physical distances between two samples? Please specify Discussion: Line 381: “Additionally, adaption to different pressure might also explain the bacterial community variation in bottom water.” I suspect that pressure differences are not driving the patterns in your study. Pressure is a relevant parameter at great depths (over 1 km) but I don’t really think it would be important at depths of < 100 meters such as in your study. Line 387: remove “Unlike geographic distance, depth and salinity,” Major Comments: Please explain your rationale for assigning sample clusters in this this study. More specifically, how did you classify clusters in Figure 2? Why is B8b classified as Cluster II and not Cluster III? I see strong evidence in Figure 1 for two well-supported clusters but is not obvious to me that Clusters II and III should be separated. Figure 3: How do you define “most significant taxa of each cluster” are these from the results of your LEfSe analysis? Why did you not include all the discriminant taxa in Fig S1? This should be briefly explained in the figure legend. Please be precise and clear when discussing sample collection depth and overall water depth, otherwise this terminology can be confusing. I would recommend trying to distill your tables down to the most relevant correlations for your story. It will help your reader grasp the main relationships presented in your study. For example, “latitude” has no significant correlations in Table 1 at all. I also recommend omitting the P value column altogether and simply only highlight a correlation if it exceeds 0.5 and has a P value < 0.05. Then mention this in the table legend. For me P values are rarely interesting in and of themselves, but that is my own preference. The structure of Table 1 and Table 2 is somewhat confusing to me. What I think you are intending to convey is that the correlation for “Longitude, ect” is due to the alpha diversity index calculated for all OTUs at that sampling station while the correlations underneath “Longitude, ect” denoted by “surface” and “bottom” refers to correlations for alpha diversity indices calculated only for the surface and bottom depths respectively. Is this correct? If so, you should explain this more clearly in the table legends. Additionally, it is unclear why you need a separate correlation analysis for Wat-Depth and Col-Depth… Indeed, the “bottom” correlation for the two categories are identical and there is no comparison for surface, which I am assuming is because all your surface samples have the same collection depth… But (assuming my above understanding is correct) then why does it make sense to have an integrated surface+bottom Col-Depth comparison here? Again, I think this needs some clarification… Please see my comment above. Table 2: Please clarify what your distance matrix inputs are for your mantel tests. I am assuming you are performing the mantel test on UniFrac distances versus a distance matrix of pairwise sample distances in each category (Geo-Distance, Wat-Depth, ect) while controlling for the effects of each category in your partial mantel tests. This information would be useful in a table legend. Lines 388-392 (Conclusion): “meanwhile the N/P ratios in most of samples were much higher than the Redfield's ratio 16 (Redfield 1958), suggesting a severe phosphate insufficiency for biological communities relative to inorganic nitrogen.” You discussed the N/P ratio little in the results, yet conclude with a statement referencing it which does not seem logical to me. N/P should either be mentioned more in the discussion prior to this sentence. Here I suspect the deviation of the measured N/P ratio from Redfield ratios is due to terrestrial inputs – probably from local agriculture. I find it unlikely that these communities would be experiencing “severe” P limitation, but rather they just have a large excess of N relative to P due to riverine inputs resulting from human land use. This distorts the N/P ratio from Redfield, but doesn’t really imply anything about limitation. I think you would need to do a number of additional experiments to claim “P limitation.” Lines 164-165: (Methods) “Pearson’s and Spearman’s correlations between variables were calculated by R program.” When conducting many statistical tests (in this case correlation) one should correct P values for multiple testing artefacts (ie false positives simply due to the increasing number of comparisons). This can be done using the conservative Bonferroni Correction, but I would recommend using the Benjamini-Hochberg multiple test correction which controls the false discovery rate. Did you use multiple test correction? If so you should include this in your methods. 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: DISTRIBUTION OF BACTERIAL COMMUNITIES ALONG THE SPATIAL AND ENVIRONMENTAL GRADIENTS FROM BOHAI SEA TO NORTHERN YELLOW SEA Review round: 1 Reviewer: 2

Basic reporting: Overall, the article is clear and well written. However, there are enough small grammatical errors that I think the authors would benefit from having it edited by a native English speaker in order to avoid any ambiguity. The figures are well constructed and add to the overall understanding of the text. I do think, however, that a figure perhaps in the form of a few section plots of important environmental parameters across the sampling region would greatly benefit the understanding of the text and give the reader a better sense for the study area. The supplemental data and figures are accessible and clear to understand. I find Figure S4 highly valuable, however, it is slightly confusing because the color scheme used to identify each of the three regions are the same colors used repeatedly in the Venn diagrams to represent different stations. Consider using a different color scheme to identify each region. Experimental design: The study goals are clearly stated in the introduction as well as the motivation for undertaking the study specifically in this region. The methods are well described and within the current standards for the field. I think seeking to understand the factors that determine the composition of bacterioplankton in the marine environment as well as factors that may limit their dispersal are important factors to consider. One of the main questions posed by the authors is whether geographic distance/depth contribute more than environmental factors to the structure of bacterial communities. However, truly separating the effects of these factors is difficult as many of them co-vary together. And the authors acknowledge in their results that their statistical analysis indicates they are at least partially dependent on each other, making their question difficult to clearly answer. I think the authors could benefit by basing their analysis on the physical structure of the water column across their study region and using a water mass analysis rather than simply distance/depth to determine what may be more important in structuring bacterial community composition and potentially limiting dispersal. Because the study region contains three distinct bodies of water (the Yellow River, the Bohai Sea, and the Yellow Sea) I think this would be feasible and allow the authors to make clearer conclusions. Validity of the findings: The authors perform a rigorous statistical analysis of their data to help support their findings and very clearly address their first posed question. As stated above, I think their analysis for the second question posed could be strengthened by basing their analysis on a physical structuring of the water column and investigating the influence of various water masses and mixing processes rather than geographic distance and depth which co-vary with many environmental factors. 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: DISTRIBUTION OF BACTERIAL COMMUNITIES ALONG THE SPATIAL AND ENVIRONMENTAL GRADIENTS FROM BOHAI SEA TO NORTHERN YELLOW SEA Review round: 2 Reviewer: 1

Basic reporting: The writing has been properly clarified in response to most of my comments. The authors have done well at improving the English writing and generally increasing the clarity and depth of explanation for many sections. Experimental design: Thank you for including multiple test corrections in your statistical analysis. 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: INTRA-COLONIAL DIVERSITY IN THE SCLERACTINIAN CORAL, ACROPORA MILLEPORA: IDENTIFYING THE NUTRITIONAL GRADIENTS UNDERLYING PHYSIOLOGICAL INTEGRATION AND COMPARTMENTALISED FUNCTIONING Review round: 1 Reviewer: 1

Basic reporting: See below Experimental design: See below Validity of the findings: See below Additional comments: In this study, the authors examine nutritional gradients within coral colonies finding distinct heterogeneity in symbiont cell number, protein, lipid and energy content across the colony. The rationale for carrying out the study is strong. The authors state that this knowledge could contribute to coral nursery restoration practices where fragmenting colonies is key to propagation and increasing survival of fragments increases yield. Overall the work is strong. The experimental design and methods are well done and described. The results are backed up with appropriate statistical rigor. I believe the study is a good contribution to coral physiology and restoration science. I do feel, however, that the manuscript could be much stronger than it is currently with some work on the writing. To me, the message of this work is fairly straightforward and could be told in a much, much shorter manuscript. Succinct, but important, results call for a relatively short paper. I put this work into that category. The Introduction and especially the Discussion are overly long. The authors can tell a more succinct and better story. In the discussion, in every section, the authors recap what they have already said in the results. Get rid of these! You don’t need to say things twice. If you are struggling with this, then combine the results and discussion together. The discussion desperately needs subheadings. The discussion of the lipids is way too detailed and strays from the central theme of the paper. I believe that much of this could be omitted. Another problem that makes the lipid section of the results remote to the average reader (me) is the lack of any background on lipid chemistry. The authors need to think about how to bring the uninitiated up to speed in short order before they dive into their data. I would suggest a simple table describing the lipids discussed, whether they are structural or storage etc. This could go in supplemental materials. Without this background it is very hard to follow this section. Minor concerns/suggestions 1. Figure 1 is very important to understand how the sampling was done. But the authors could add a section somewhere – intro, or results and figure legend? – that describes in words how A. millepora grows – as multiple finger-like branches arrayed roughly in 2 dimensions. This will help the reader understand how the branched were sampled. 2. Put N numbers in the figures 3. Discuss the parts of figure 2 in order. If you keep the text the same, then you need to rearrange the figure. 4. What does proximate analysis mean? I am not familiar with this term 5. Stony corals are not soft corals – they are distantly related. There are multiple citations in the manuscript where soft coral findings are lumped with stony coral findings. These either need to be called out as different or removed. For example, whereas there is ample evidence that symbionts are transported around soft coral colonies, there are no data in hard corals to suggest this. Very different biology between the two. Compare apples to apples.

You are one of the reviewers, your task is to write a review for the article. You will 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: INTRA-COLONIAL DIVERSITY IN THE SCLERACTINIAN CORAL, ACROPORA MILLEPORA: IDENTIFYING THE NUTRITIONAL GRADIENTS UNDERLYING PHYSIOLOGICAL INTEGRATION AND COMPARTMENTALISED FUNCTIONING Review round: 1 Reviewer: 2

Basic reporting: The manuscript’s main idea and general focus are good. It has been a long-standing goal in coral biology to understand the relationships between polyps within a colony. Specifically, It has been a major focus to understand if and when there are nutritional allocations within the colony that allow for subfunctionalization of different parts of the colony. The authors do a good job summarizing the motivation of their experiments in the introduction. However, some of the statements asserted as fact in the introduction are not supported by the references provided. For example, the reference provided in line 75 refers to a mass-spec experiment trying to determine the proteins that accumulate in the skeleton. Unless I am mistaken, this paper does not speak to whether or not “protein fuels tissue growth, organogenesis, and calcification.” The same can be said about line 272 and 273. The authors should take more care in writing the introduction and discussion not do overstate conclusions or observations made by other authors. Generally, the data presented here suggest some significant differences in the nutrition allocation across the coral. However, many of the differences are marginally significant. Given the variance in the data (all error bars in figures are SE) and the degree of multiple testing within the paper, it is hard to interpret the biological significance of the findings. Experimental design: The experimental design attempted to be replicative both technically and biologically. The authors conducted well motivated experiments. However, they saw high variance in most of their measurement precluding robust statistical significance. Major comments: The authors aimed to determine if there is differential nutritional compartmentalization along across coral colonies of two different size classes. The authors deploy a sophisticated set of analytical approaches to address this question. However, it is not clear based on the paper how many of the stated conclusions are supported by significant statistical tests (Examples of this are discussed below). It is important for the reader to know, which results are significant in both the original ANOVA and post-hoc tests. Please report the both of these P-values for each statement in the text to help clarify this for the reader. For example, the end of Line 205. I would like to see raw data points plotted over the SE plots for Figure 2 and 3. In general, it is not clear how meaningful discussing the trends in the data are when the underlying differences are not significant (especially when contradicted by other corals in a different size class). For example: Line 196-198: "Within the size classes, there were no significant differences in total lipid (P>0.05) (mg g sample-1) (Fig. 2a), although both edge positions contained lower lipid compared to the next, immediate position." Line 198- 200: "A similar trend was apparent in zooxanthellae densities for the larger colonies (cells cm2 -1) (Fig. 2e), whereby the density at the edge was again lower than the next immediate position." Line 206-208: "The caloric enthalpies showed that, for both size classes, the edge and next immediate position contained the highest energy contents, while the S-centre and L-centre and L-33 positions were low (Fig. 2f)." There are many examples of this within the text, and they should be removed or modified to avoid confusing the reader into thinking these trends are significant. Validity of the findings: As stated above, more effort should be made to ensure the reader can easily determine, which statements and claims came from significant statistical analysis. These limitations of the study should be addressed directly in the discussion. Minor comments: Line 67: 'reasonable to hypothesise that this' - spelling Line 129: Elaborate on air-spraying method. Line 135-137: Redundant with next paragraph Line 142: Not clear how to crush something in a mortar and pestle under a french press. Can you please elaborate? Line 153: Is four digits the appropriate number of significant figures for this measurement? Line 212: "The proportion of storage lipid in the larger colonies followed a similar.." It is not clear what this proportion is similar to based on the text. Figure 1: It would be helpful to reorder the figure panels to match the order in which they are 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: INTRA-COLONIAL DIVERSITY IN THE SCLERACTINIAN CORAL, ACROPORA MILLEPORA: IDENTIFYING THE NUTRITIONAL GRADIENTS UNDERLYING PHYSIOLOGICAL INTEGRATION AND COMPARTMENTALISED FUNCTIONING Review round: 2 Reviewer: 1

Basic reporting: All looks good. Experimental design: Looks good. Validity of the findings: Looks good. Additional comments: The authors have addressed my concerns/suggestions. I would still ask that they simply define 'proximate' in the manuscript - they don't have to remove it, but just define it for the uninitiated.

You are one of the reviewers, your task is to write a review for the article. You will 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: INTRA-COLONIAL DIVERSITY IN THE SCLERACTINIAN CORAL, ACROPORA MILLEPORA: IDENTIFYING THE NUTRITIONAL GRADIENTS UNDERLYING PHYSIOLOGICAL INTEGRATION AND COMPARTMENTALISED FUNCTIONING Review round: 2 Reviewer: 2

Basic reporting: No Comment Experimental design: No Comment Validity of the findings: The authors did a fine job indicating significances in the updated manuscript. However, I am still worried about discussion "trends" in data as "biologically meaningful." In many cases, there is no support that the means are actually different (i.e. not statistically significant). I suggest removing these discussions from the paper. because if these experiments were done again, these trends could go in the opposite direction. Illustrating this, many of these trends, are not supported across the different coral size classes. 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: ESTABLISHMENT OF AN IMMORTALIZED MOUSE DERMAL PAPILLA CELL STRAIN WITH OPTIMIZED CULTURE STRATEGY Review round: 1 Reviewer: 1

Basic reporting: Relevant prior literature on human immortalized human dermal papilla cell line should be referenced. For example, Shin et al., Establishment and characterization of an immortalized human dermal papilla cell. BMB Rep. 2011 Aug;44(8):512-6. Also, Won et al., The establishment and characterization of immortalized human dermal papilla cells and their hair growth promoting effects. J Dermatol Sci. 2010 Dec;60(3):196-8. 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: ESTABLISHMENT OF AN IMMORTALIZED MOUSE DERMAL PAPILLA CELL STRAIN WITH OPTIMIZED CULTURE STRATEGY Review round: 1 Reviewer: 2

Basic reporting: The authors have clearly referred to relevant literature. However, sometimes the prose is confusing. For example in the introduction the phrase 'when the culture system was changed to 2D' is misleading. This suggests that intact DP which are being placed in 2D are in a culture system as well, which is not the case. The idea that intact DP are in a 3D culture system is also suggested within the paper, which again is incorrect. Cells within intact DP are in a 3D environment, but this is not a culture environment. The formatting and structure of sentences should be checked to ensure they are not misleading. There are also numerous typos in the manuscript-eg Western Bloting rather than Blotting. Experimental design: The aim of the paper is to 'optimise isolation and culture' methods of DP and 'establish an immortalised line'. The reason for establishing an immortalised line is not clear-suggestions as to why an immortalised line would be beneficial over a primary line would help here. One of the reasons for performing this work is that DP cells are difficult to isolate, however the method of isolation in the paper appears the same as that described within the literature. Please explain how the isolation method is changed to make DP isolation easier? n numbers should be included-i.e. how many times were the immunostaining protocols performed on cells. How many times was the immortalisation reversing protocol attempted. Did it work on multiple iDP lines? The control media used is dMEM with 10% FBS, however this is media commonly used for human DP cells. Mouse DP cells (which are being assessed here) are usually grown in dMEM with 10% FBS, with additional growth factors such as FGF2. Why did the authors not have murine DP media as the control medium? Temperatures used for PCR should be indicated. Product size should also be indicated in Table 1. Validity of the findings: One of the main results of the paper is that the authors optimised a media to culture DP cells. Optimisation suggests that several media were attempted, but these are not shown. How many other medias were attempted, and what enabled the conclusion that the selected one was the best. In addition, please explain how the optimised media is different from that which is usually used. Another finding in the paper is that iDP6 is the optimal induced line as it has similarity to primary DP cells. However, the profile of primary cells is not shown. To make this argument more convincing, the PCRs and staining (Figures 4/5) should include DP cells which are not immortalised. What do the authors mean by 'agglutination'? Please add a sentence to explain. 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: ESTABLISHMENT OF AN IMMORTALIZED MOUSE DERMAL PAPILLA CELL STRAIN WITH OPTIMIZED CULTURE STRATEGY Review round: 2 Reviewer: 1

Basic reporting: N/A Experimental design: Methods are much better, with n numbers and more detail on the experiments performed. Validity of the findings: N/A Additional comments: It seems from the expanded methods, and the response to reviewers that the main addition to the media, was sodium pyruvate and non-essential amino acids, and FGF2 had little effect by itself. This should be reflected in the discussion, especially page 6, lines 12-16. page 6, line 28-add the following reference after 'But exactly how many kinds of cells are in DP remain to be discovered' Epithelial-Mesenchymal Micro-niches Govern Stem Cell Lineage Choices. Yang H, Adam RC, Ge Y, Hua ZL, Fuchs E. Cell. 2017 Apr 20;169(3):483-496.e13. doi: 10.1016/j.cell.2017.03.038. I wouldn't call primary DP cells 'wild-type'. This makes it seem like the others were from a transgenic/mutant mouse. Just call them primary. Update text and the figure legends for Figure 4/5 accordingly.

You are one of the reviewers, your task is to write a review for the article. You will 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: REMOTE COLLECTION OF MICROORGANISMS AT TWO DEPTHS IN A FRESHWATER LAKE USING AN UNMANNED SURFACE VEHICLE (USV) Review round: 1 Reviewer: 1

Basic reporting: The report is well written. Objectives, methods, discussion and conclusions are clear. Some minor editing is needed as described below. Experimental design: Experiments utilized a custom designed sampling system to successfully collect water for microbial analyses at two different depths. Need some minor additional information to include approximated depth of the lake at the sample sites. Add this to Table 1 reference line 88. end of line 133....How was the system sterilized between sample set collections line 144....250ml.....spell out as it starts a sentence line 162.....how many colonies......10 as the table indicates?....but several samples recovered less than 10cfu.......specify here and correct the table that states 10cfu sampled for these few samples you should refer to the Pseudomona cfu's as presumptive Pseudomonas....as you ice nucleation experiments show that many were not.....this is common - colilert will indicate positive with certain species of Vibrios and Pseudomonads Validity of the findings: Looks OK with addressing above comments. Check references of genus/species italics and standardize as one or a few use caps for many title words while the most use all lowercase 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: REMOTE COLLECTION OF MICROORGANISMS AT TWO DEPTHS IN A FRESHWATER LAKE USING AN UNMANNED SURFACE VEHICLE (USV) Review round: 1 Reviewer: 2

Basic reporting: Language used throughout the manuscript is clear and well written. Only a few comments on this topic. There is a sentence in line 221 that may be correct, but sounds like it might has been a mistake during typing: “to collect samples and multiple depths and locations”, probably meant to be “to collect samples at multiple depths and locations”. Also lines 255 – 256, repetition of terms (location, due) should be avoided. In line 279, repetition of the preposition “in” should be corrected. Revision of sentence from line 297 to 304 is recommended. Bibliography cited along the manuscript is extensive and recent, showing the authors are updated in their research field. There are however, corrections that have to be made to this section. Several papers cited in the References section are not mentioned in the text. Authors should include those citations where needed in the text or remove them from the References section. There is also one reference: Pietsch et al. (2007) (Line 44) which is not in the Reference list and it seems to be an error for what it should be Pietsch et al. (2017), which is widely cited along the manuscript. Although PeerJ Journal do not have a strict policy regarding the References section, they do have some guidelines. Authors should revise the entire section to comply to those guidelines, e.g. provide the full list of authors. Article structure follows PeerJ Journal format. This paper contains 4 figures, which are helpful to the reader for the understanding, not only of the scientific results, but also for the development of the technology used to obtain them. They are presented in good resolution formats and well described. Table 1 and the supplemental table with all raw data are also well presented and clearly labeled. Work presented in the manuscript is well organized and follows the objectives derived from the initial hypothesis stated by the authors in the introduction. Experimental design: Research described in this manuscript fits within the Aims and Scope of PeerJ Journal. As mentioned before, it states a clear hypothesis with its specific objectives in the introduction that are fulfilled along the article. At first sight, it may seem a simple research work, with only 4 different samplings using culture-based methods, in a world where papers with big amounts of data are imposing. But the technology developed by the authors has a great potential in all kinds of research approaches and sometimes small research initiatives are needed in order to optimize that technology and/or methodology that could later be applied to more complex objectives. It is also known among the scientific community that environmental studies, although increasingly more frequent, haven’t provided the information needed to completely understand them. The use of USVs in environmental studies allows not only to reach possibilities beyond the human range, but also to those that could represent a risk to them. Description of methodology allows replication of the experiments. Figure 3 and the ones included in the Supplemental files, plus the explanation in the Material and Methods section are elaborate, providing the capability of reproduction. Data in lines 186 – 192 from the Results section seem to repeat information already described in Material and Methods. Only new information related to the results should be included in this subheading. Validity of the findings: Results obtained in the work presented add knowledge on the aquatic microbial community of a freshwater lake and the presence of ice nucleation active bacteria is a very important feature for the water cycle, as describe by the authors. As considered by the authors in the discussion, collecting samples in different seasons as well as in different locations of the lake, would contribute to increase the understanding of how microbial communities change throughout the lake and are influenced by different environmental factors. Regarding this last topic, authors state in lines 90 - 91 of Material and Methods that “Though water temperature is an environmental variable, it was assume to be constant across the distances and times within and among samples collections in this study”, although no references or hypothesis that would back up this consideration are given. But later in the discussion section, lines 236 – 237, they acknowledge that “Other environmental factors, such as seasonal temperature variation and pH, are additional factors that could have an impact on bacterial distributions (Lindströem et al., 2005)”. Future efforts to complement this work should consider all environmental variables and authors may contemplate the possibility of including in the USV platform different sensors that would allow collecting that kind of information on site during the sampling process. No conclusions are made in this manuscript, but speculations based on previous publications and the hypothesis of the authors. This is considered to be a wise decision regarding the small amount of samples collected and the statistical diversity of the results obtained. 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: REMOTE COLLECTION OF MICROORGANISMS AT TWO DEPTHS IN A FRESHWATER LAKE USING AN UNMANNED SURFACE VEHICLE (USV) 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: REMOTE COLLECTION OF MICROORGANISMS AT TWO DEPTHS IN A FRESHWATER LAKE USING AN UNMANNED SURFACE VEHICLE (USV) Review round: 2 Reviewer: 2

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: Authors have addressed reviewers and editor's comments. Changes made and text added have improved comprehension of the manuscript. They have also adjusted bibliography format to the journal guidelines, and corrected those citations that were missing in either the main text of reference section.

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

Title: IDENTIFICATION BY SHAPE-BASED VIRTUAL SCREENING AND EVALUATION OF NEW TYROSINASE INHIBITORS Review round: 1 Reviewer: 1

Basic reporting: The overall data in manuscript is interesting and informative. Experimental design: Good experimental approach was used in manuscript Validity of the findings: Authors tried to verify their results using various approaches. However, further techniques are required to validate their results. Additional comments: Comments The present manuscript screen the possible inhibitors of mushroom tyrosinase. The manuscript is well written and the findings are interesting. Therefore, article can be consider for publication after revision. • The manuscript title “Identification of new tyrosinase inhibitors with shape-based virtual screening” is just emphasize on in-silico based study, whereas authors also performed in-vitro experiments. Therefore, manuscript title must be rephrased. Moreover, add few studies such as (Eur. J. Med. Chem. 141: 273-281; DOI: 10.1007/s12539-016-0171-x; Comput Biol Chem. 2017 Jun; 68:131-142; Chem Biodivers. 2017 Sep;14(9); Drug Des Devel Ther, 2017, 11:2029-2046; Bioorg Chem, 2017, 74:187-196) in introduction part. • Authors used Neorauflavane as a template, is it possible to use Kojic acid and Arbutine as a template for screening as you used in in-vitro experiment as a positive control. • Author did not discuss about size of protein structure, domain and binding pocket. I would suggest to make one graphical image of all inhibitors or your potent inhibitor against tyrosinase to show binding pocket and ligand conformation. • Authors did not discuss how many conformations he selected in docking experiments and on what basis he selected best pose of among all either just energy value or binding interaction pattern. In docking figure authors did not mentioned the binding distances of interactions. Moreover, authors claims “One hydroxyl established a hydrogen bond with the side chain of Asn260, while the other hydroxyl interacted with the copper ion” in docking results. However, it’s not clear from graphics either it is hydrogen bond or some other type of interaction. In discovery studio mostly the hydrogen bonds are represented by green dotted lines. Moreover, authors did not mentioned docking energy value for 5186-0429 in the results part. • Moreover, I would suggest (if possible) to run MD simulation using any software to check the stability of target protein. • For in-vitro analysis Table 1, Authors only mentioned IC50 value for 5186-0429, however, it’s better to calculate IC50 values for all other compounds in μM. It would help in comparison amongst all screened compounds with control. • In MM section, authors mentioned “Vmax and Km values (for Michaelis-Menten kinetics) were obtained with Graph Pad Prism 5.0 from the nonlinear regression of substrate-velocity curves”. However we could not found Vmax and Km values in manuscript file. Moreover, also provide Ki value (Ki is the EI dissociation constant). • Authors used two substrate (L-Tyro and L-Dopa) for inhibition purpose as mentioned in MM section, while in kinetic study only L-Dopa was used to generate Lineweaver-Burk plots. Therefore, I would suggest to use both substrates and make separate graphs for both substrate. • Furthermore, I would suggest (if possible) to use Zebrafish model for melanin quantification along with toxicity assay for your most active compound.

You are one of the reviewers, your task is to write a review for the article. You will 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: IDENTIFICATION BY SHAPE-BASED VIRTUAL SCREENING AND EVALUATION OF NEW TYROSINASE INHIBITORS Review round: 1 Reviewer: 2

Basic reporting: No comment Experimental design: *In which solvent the L-DOPA, Tyrosine and test samples were prepared? "0.9 mL of sodium phosphate buffer solution (0.05 M, pH 6.8)" This volume of PBS was fixed for all samples? If this is true, we will have difference in the concentrations after the addition of the test samples. Validity of the findings: Some of the data are without error bars. For example: Fig. 3A-B and Fig. 4. How about the reproducibility of these experiments? At least two independent experiments, preferably three with error bars, are required. In graph 3C determine the values of R-saquared and P value and Calculate the values of Ki, The major point is: the discussion of the results needs to be reviewed, relating the theoretical data with in vitro Additional comments: The design of the study appears appropriate and most of the data are satisfactory. However, there are some points that need to be improved, as summarized 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: THE OLDEST ARCHAEOPTERYX (THEROPODA: AVIALIAE): A NEW SPECIMEN FROM THE KIMMERIDGIAN/TITHONIAN BOUNDARY OF SCHAMHAUPTEN, BAVARIA Review round: 1 Reviewer: 1

Basic reporting: The description of the new specimen is thorough, both in text form and as documented photographically. I was somewhat concerned about the legality of and potential access in posterity for the specimen. However, the authors seem to have found an effective and appropriate means of making the specimen permanently available to scientific study. Experimental design: The paper is essentially all descriptive, not analytical. Thus there is no significant part of this particular study which involves experimental design. The relevant specimen data, however, all all provided. Thus, future workers would be able to independently evaluate the morphological observations of the author, and could potentially study the original locality in search of additional specimens or new stratigraphic/sedimentological data. Validity of the findings: The morphological observations presented are sound, and measurements of the skeletal elements are provided. Additional comments: Additional comments and corrections: p. 9, line 62: Origin of Species should be in italics, not quotation marks. p. 9, lines 69: “1970ies” should be “1970s”.

You are one of the reviewers, your task is to write a review for the article. You will 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 OLDEST ARCHAEOPTERYX (THEROPODA: AVIALIAE): A NEW SPECIMEN FROM THE KIMMERIDGIAN/TITHONIAN BOUNDARY OF SCHAMHAUPTEN, BAVARIA Review round: 1 Reviewer: 2

Basic reporting: Clear and unambiguous, professional English used throughout. Sufficient field background/context provided. Additional references could further enhance the paper but not essential (see reviewer PDF). Professional article structure, figs, tables. Some minor suggestions to the figures. Suggestions for two non-essential additional supplementary tables (see reviewer PDF). Self-contained with relevant results to hypotheses. Experimental design: Original primary research within Aims and Scope of the journal Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap. Rigorous investigation performed to a high technical & ethical standard. A paragraph of the UV imaging methodology should be added as their are a range of UV imaging techniques currently available. The methodology is otherwise sufficient in detail and information to replicate the results. Validity of the findings: Valid, well-supported and impactful findings. Data are robust. Conclusions are clear and speculation is stated as such. Additional comments: This is an impressive amount of work, well done! I am actually supportive of publication as is (with a few typo and grammar corrections and a short paragraph on your UV methodology), but it would be great if you can take my minor corrections on board as I think they will improve the quality and value of the paper. Importantly, I think the manuscript should not be shortened. In fact my suggested corrections would involve a very slight lengthening of the paper.

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

Title: THE OLDEST ARCHAEOPTERYX (THEROPODA: AVIALIAE): A NEW SPECIMEN FROM THE KIMMERIDGIAN/TITHONIAN BOUNDARY OF SCHAMHAUPTEN, BAVARIA Review round: 2 Reviewer: 1

Basic reporting: Meets PeerJ's standards. The authors addressed the comments from my first review. - Clear and unambiguous, professional English used throughout. - Literature references, sufficient field background/context provided. - Professional article structure, figs, tables. Raw data shared. - Self-contained with relevant results to hypotheses. Experimental design: Meets PeerJ's standards. The authors addressed the comments from my first review. - Original primary research within aims and scope of the journal. - Research question well defined, relevant & meaningful. It is clear how the research fills identified knowledge gap. - Rigorous investigation performed to a high technical & ethical standard. - Methods described with sufficient detail & information to replicate. Validity of the findings: Meets PeerJ's standards. The authors addressed the comments from my first review. - Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful details for replication provided (rationale and benefit to literature clearly stated) - Data is robust. - Conclusions are well stated, linked to original research question & limited to supporting results. - Speculation identified as such. Additional comments: Oliver, Christian and Helmut, Thank you for taking my comments on board. Here are some final items to address. I do not need to see the manuscript again and fully support its publication. - line 139: 'artificial' not needed - lines 165 and 166: replace '.' with ',' in '50.000' and '8.000' so it is clear you mean fifty-thousand microwatts and eight-thousand microwatts. - typos in the marked changes on lines 1166, 1327, 1381, 1419 and 1564. - line 1921: the potential cartilaginous pad shown in the LSF image in Fig. 4b of Wang, Pittman et al. 2017 is visible as a orange-coloured feature under white light. You could perhaps change the text to support the fact that soft tissues can preserve differently in the same specimen e.g. the orange-coloured pad vs. the white-coloured areas that reveal patagia. This might mean that some 'absent' soft tissues could be possible to reveal in the future using a wider suite of imaging techniques. I look forward to seeing the published paper. Best regards, Michael Pittman

You are one of the reviewers, your task is to write a review for the article. You will 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: LINKING PANGENOMES AND METAGENOMES: THE PROCHLOROCOCCUS METAPANGENOME Review round: 1 Reviewer: 1

Basic reporting: The Authors show how pangenomes and metagenomes can be linked and provide proof-of-concept of how this metapangenomics provides unique insights. The English should be improved to ensure text is clearly understood. For example: 1/ Line 27 to 32. In the abstract, the authors give two statements, “Rapidly growing number of ... …of populations across microbial genomes.” The first statements is a general statement that is followed by a second statement that is supposed to provide more clarity of which aspects of the general statement is the key focus of this manuscript. However, the current phrasing makes comprehension difficult. 2/ Line 65 to 68. Also rephrase these statements to make comprehension easy. 3/ Line 274. Rephrase these statements to make comprehension easy. 4/ Line 411 to 415. Rephrase these statements to make comprehension easy. 5/ The entire document needs to be proofread. Most of the references used are from the Nature Journal but some references are old and newer published manuscripts with impacting findings have not been included. For example: 1/ Line 69: include after reference (“Lorenz & Eck 2005; Thies, Stephan, et al. "Metagenomic discovery of novel enzymes and biosurfactants in a slaughterhouse biofilm microbial community." Scientific reports 6 (2016): 27035.) 2/ Line 70: include after reference (“Tringe et al., 2005; Al-Amoudi, Soha, et al. "Metagenomics as a preliminary screen for antimicrobial bioprospecting." Gene 594.2 (2016): 248-258) 3/ line 71-72: include after reference (“Tyson et al., 2004; Haroon, Mohamed F., et al. "A catalogue of 136 microbial draft genomes from Red Sea metagenomes." Scientific data 3 (2016): 160050; Delmont et al., 2017) Overall, I commend the authors for the thorough data analyses and on conciseness of style of writing. If there is a weakness it merely is with respect to making comprehension easier (as I have noted above). Experimental design: Research question well defined and meaningful. Validity of the findings: Conclusion are well stated, linked to original research question & limited to supporting results. 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: LINKING PANGENOMES AND METAGENOMES: THE PROCHLOROCOCCUS METAPANGENOME Review round: 1 Reviewer: 2

Basic reporting: I am putting my entire review in this section, as nearly all, if not all, of my comments are related to basic reporting. Overall: This is a nice contribution by Delmont and Eren describing the utility of a new software pipeline in the existing Anvi’o tool, along with a few new insights into Prochlorococcus ecology. The pipeline links genes from isolate genome sequences to their abundances in the environment via metagenomic read mapping, and it can identify specific genes (or protein clusters) that exist in isolate genomes but may be very uncommon in the environment. I have few, if any, scientific criticisms, but I found a lot of the text confusing, mostly due to undefined terminology and some long, confusing sentences. I think that this is a relatively straightforward study that would benefit from some streamlining of the text. Specific comments are below. Abstract: -The abstract is somehow both well written and deeply confusing. Please simplify the language. I am left not really understanding what the main question(s), methods, and results are. I know intuitively what both metagenomics and pangenomics (or at least pangenomes) are, but it would be helpful for the authors to explain these terms in the context of how they were considered for this study. Is the sentence starting with “While pangenomics offers …” meant to define both terms? If so, please restructure it along the lines of “The pangenome of a population (or genus?) consists of both core (shared) and accessory genes and genomic features …” or however you want to define it, and please similarly define metagenomics and its use in this study. If metagenomics is being used for abundance estimates (abundance estimates of what – SNPs, populations, and/or genes within pangenomes?), then consider calling it something more direct (metagenomic abundance estimates?). To me, the term metagenomics primarily evokes community predicted functional profiling and/or population genome assembly and metabolic reconstruction. Even though I have personally also made abundance estimates from metagenomes in much the same way as the authors, that would not be what first comes to mind. [edit: that is almost exactly how this is described by the authors in ln 69-74, so this needs to be much more clear in the abstract] -Along those lines, I do not think of metagenomics and pangenomics to be inherently different. I would assume that metagenomics could be used both to define the pangenome (i.e., to find core and accessory genes in metagenomic assemblies) and to determine the abundances of subpopulations and/or specific genes or regions of the pangenome (through read mapping to metagenomic assemblies, SAGs, isolate genomes, or any combination of the above). Which, if any, of these possibilities apply to this study is not clear. -Metapangenomics is not defined, and I do not find it to be a helpful term. Consider removing it from the manuscript, including the title. Based on my reading of the abstract alone, it looks like metapangenomics is meant to describe an abundance-informed pangenome, and if so, why not call it something like that, with some useful meaning in the term itself? Main Text: -ln 32: complementary -ln 38-42: What does this sentence mean? Consider splitting it into two sentences. How does a metapangenome correlate with something? What is it correlating with? I am not sure that “traits” is an appropriate word – are the authors describing core and accessory genes here? What are “sub-clade demarcations”? How would these results differ from phylogenetic analyses (wouldn’t phylogenetics by definition separate sub-clades?)? Do the authors mean some specific phylogenetic analyses that are typically performed at a coarser resolution? If so, please provide more context on the phylogenetic analyses. Main text: -ln 54: “have been” should be “has been” -Throughout: consider changing “shared” to “core” in the context of core genes across pangenomes, as this is more common in the literature and therefore more intuitive. If the authors mean shared among some but not all populations, then that should be explicitly mentioned, but I assume that they mean core genes shared among all populations. -ln 74-76: What does this mean? Wouldn’t metagenomic assembly + read mapping do that too? It might help if you explain the particular utility of isolate sequences here. -ln 77-78 (or earlier): Please define functional traits in the context of a microbial genome or pangenome. I think that you also mean isolate genomes here, so please change the end to “… mapping of closely related isolate genomes.” -ln 80-83: This sentence is a bit long and confusing and can probably be broken down. The authors can rework it for clarification as they see fit, but here are some examples of what I find confusing: What are “well-established practices in pangenomics”? Please give a few examples. What are “emerging opportunities from metagenomic data”? Is this just using metagenomic read mapping for abundance data? If so, just say that. What is a genome-centric framework (I assume that this involves the use of closed, isolate genomes), and how does that differ from what you would get from a combination of metagenomic assembly and binning to identify populations + read mapping across a number of metagenomes to get abundance estimates? What are “pangenomic traits” and how do you define which ones are “key”? Are “key” traits just those that are linked to “niche partitioning” and “population fitness”, and if so, how do you determine that? -ln 85: please state exactly what you mean by integrating pangenomic and metagenomic data. Again, what is “pangenomic data” and what is “metagenomic data”? Are there better terms for these types of data in this context, for example, “… integrating population pangenomes from multiple isolate genome sequences with their abundance profiles across environmental samples from metagenomic read mapping?” That might not even be correct, but the point is that I do not understand. -I think that the focus of both the abstract and introduction and maybe even the title should be on the need for and development of this software pipeline, as that seems to be the key novel result of the study, tested on Prochlorococcus as an example, right? [[later edit: wait, but the tool is Anvi’o, which is fabulous but not new; please use the introduction to very clearly walk the reader through what is known vs. what is new in this study, both in terms of the visualization software pipeline and the Prochlorococcus biology]]. It seems dangerous to imply that metagenomics has never been used to identify the ecological niches of specific subpopulations (for example, the Banfield lab has worked in that general area, at least in AMD systems; how would isolate pangenomes add further information in that context?) and much safer to say that your software and visualization pipeline can help to identify and show these differences more clearly. -ln 93: How many genomes? That number seems important if all of these genomes are going into your downstream analyses. -ln 95: Were these 16S rRNA gene amplicon surveys or otherwise not metagenomic studies? That seems like a worthwhile point of clarification to help make the case for the current study that links isolate genomes to metagenomic data. -ln 96: dynamics plural -ln 97-98: Correlations between the “genomic traits” of isolates … are these just groups of genes that correlate with environmental variables? Were there correlations to variables other than HL and LL? If so, maybe call these “other environmental variables.” -ln 98-104: This is a long sentence, and I got lost halfway through. Do “these two groups” refer to the core and accessory genes? How many metagenomes? What does “independently” mean here, and what is “their differential occurrence”? Does “in Prochlorococcus populations” mean in the same 12 as at the beginning of the sentence? If so, change to “in the 12 Prochlorocuccus populations,” otherwise define these populations. I don’t understand the last part of the sentence at all. Maybe summarize these three studies in three separate sentences and explain which part(s) of each are being included in the current analyses, and then explain clearly how the current study will expand on what is already known from these previous studies. -ln 104-105: This seems like an important distinction. To this point, the general implication is that this is the first time that anybody has thought of exploring niche partitioning in pangenomes or metagenomes, yet here the authors say that the difference is that previous studies have not had resolution at the level of protein clusters. Again, please dispense with the fancy sentences and terms and use the introduction to tell the reader what has been done in the past that is relevant, both in terms of biology and visualization/software, and then explicitly state what knowledge gap will be filled by this study. For example, it would be useful to explicitly say why monitoring protein clusters is useful. -ln 106: Again, what are “pangenomic traits”? Maybe I am just stuck on “traits” as an ecological term and the authors just mean similarities and differences across populations? -ln 106: How do these 31 Prochlorococcus isolates relate to the 12 (or more?) populations described from previous studies above? -ln 108: Please give an exact number for billions -ln 110: Define ecological niche; is this just HL vs. LL here? -ln 109-115: These are results that do not belong in the Introduction. Consider either reworking to frame these as hypotheses (or similar) that will be explored in this study, or remove this. -ln 113 and 117: These are the first mentions of SAGs. This seems to be of abstract-level importance in how you are defining your pangenomes (i.e., a combination of SAGs and isolates). Or am I not understanding how your pangenomes were defined? After reading more of the results, I do not see much in the way of SAGs there, so how did you decide when to use isolates and when to use SAGs in your analyses? SAGs are presumably less complete genomes, so if a particular gene is not detected in a SAG, it does not necessarily mean that it is actually absent. The authors know this, I am sure, but if this is part of the rationale for using only isolates for some of the analyses, it should be mentioned. -I think that Anvi’o can also be called out specifically in the Abstract and/or Introduction. It is not clear to me what aspects of the Anvi’o workflow are new in this study, though the Introduction suggests that this is a novel pipeline. Based on the text to this point, I was expecting the presentation of a novel workflow, and this needs to be made more clear. I think that a paragraph in the Intro with Anvi’o background would be appropriate – how has Anvi’o been used in the past, and what specifically is the new application here? It seems like more than just plugging new data into the software, so maybe a flowchart figure would help? There is a section of the methods dedicated to this, which is good, but I wonder if at least some of that should be moved to the main text, given that the pipeline is one of the key outputs of the study and not just an ancillary method. -ln 134: Has phylogenomics not been done on these 31 genomes before? -ln 176-199: Is this the new part? If so, you could start with something along the lines of “The Anvi’o pangenomic workflow developed for this study consists of …” -ln 179: What is a “genome of interest”? Is this just every genome that will be considered for a given analysis, i.e., 31 Prochlorococcus isolates for this study? [I see later that this is the case, so please rephrase to make this more clear] -ln 234 and 264: What about the SAGs? -ln 234-243: Has this been done already for Prochlorococcus in any of the TARA Oceans publications? I would guess so, but maybe not all 31 isolates were included. It would be worth clarifying what parts of this analysis are new vs. what just needed to be done again here to feed into the Anvi’o pipeline. -ln 244-256: These specific clades have not been described anywhere. I realize that a description of each could get tedious, but is there something general that you could say along the lines of, e.g., “All LL lineages come from low-light niches and include subclades I-IV defined by x, y, z” Otherwise, the description of these clades is not particularly useful. The figures just say that these are “literature-defined” lineages, which is fine, but the authors could briefly elaborate on these clade distinctions in the text. -ln 277-285: Cool! -ln 298-299: ECGs and EDGs -- do we really need more acronyms? I saw these again a couple of pages later and had to dig back to this section to remind myself of what they are. [and again when I came back to the manuscript after a break] When these acronyms appear again a couple pages later (ln 356), the next sentence has four different acronyms occurring eight times … -ln 313: Okay, metapangenomics is finally defined! I still don’t fully understand the utility of this term. Maybe it is just me, but I do not find the introduction of new terms and acronyms in nearly every new manuscript in this field to be helpful. -ln 313-320: What is the result here? The result cannot just be the figure; there has to be some interpretation or guidance for what the reader should be seeing. -ln 354-356: This seems like an important contribution, and it is buried near the end of the Results. -ln 366-367: Change to plural -ln 370-371: Please rephrase this sentence for clarity. What is “they”? -ln 420: Is this really only a little effort?! -ln 464-466: Okay, the authors have confirmed the obvious application that I mentioned above, which is that this can also be applied to metagenome-assembled genomes. Why were those not considered here? I don’t think that this is a hole-in-the-paper offense, but I am puzzled, as it seems like a relatively easy addition that would boost the size of the available pangenome significantly. Figures: These are nice. If the authors insist on keeping the ECG and EDG acronyms, please define them in each figure legend. Experimental design: See above (Basic reporting section) for a few specific comments related to better defining the research question and knowledge gap(s) filled by this study in the Introduction. 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: LINKING PANGENOMES AND METAGENOMES: THE PROCHLOROCOCCUS METAPANGENOME Review round: 2 Reviewer: 1

Basic reporting: The manuscript has been extensively reviewed and English use is clear and unambiguous. Experimental design: In the current form, the manuscript results is relevant to the hypothesis and it is now clear how the work fits into the broader field of knowledge. Validity of the findings: The findings are appropriately stated, and connected to the original question investigated. 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: LINKING PANGENOMES AND METAGENOMES: THE PROCHLOROCOCCUS METAPANGENOME Review round: 2 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: I thank the authors for addressing my concerns and making the manuscript much more clear. I have no further 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: MOLECULAR AND SEROLOGICAL DYNAMICS OF CHLAMYDIA PECORUM INFECTION IN A LONGITUDINAL STUDY OF PRIME LAMB PRODUCTION Review round: 1 Reviewer: 1

Basic reporting: Chlamydia pecorum is an important, and globally significant livestock pathogen causing pathology and production losses. The authors in this study applied molecular and serological tools to investigate the age-dependent distributions of C. pecorum in a lamb production. The manuscript is well written, and conclusions are well supported by the data. This is an important and well-designed study, and significantly improves our understanding on the C. pecorum dynamics. Experimental design: Well-designed study. Validity of the findings: The findings are novel. Comments: For table-3, the data are shown as mean,max and SD. Normally, we show the data as mean with SD, or mean with range (max, min). The authors may consider to modify the data. Additional comments: 1. Beyond qPCR and CFT used in this study, we have also culture and ELISA approaches for diagnosis of Chlamydia. The authors may address briefly the rationale to use only PCR and CFT in this study. 2. The authors should address clearly the specificity of CFT against C. pecorum alone; In the lamb production, it is not unusual to see the presence of other Chlamydia species; 3. Please reduce the length of the Statistical Analysis if possible; 4. The readers will benefit if the results for statistical analysis can be provide in the figures or legends of Figures 1 and 2;

You are one of the reviewers, your task is to write a review for the article. You will 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: MOLECULAR AND SEROLOGICAL DYNAMICS OF CHLAMYDIA PECORUM INFECTION IN A LONGITUDINAL STUDY OF PRIME LAMB PRODUCTION Review round: 1 Reviewer: 2

Basic reporting: Well written, but a little too many self citations in the introduction. Would be nice to see corrected. Experimental design: no comment Validity of the findings: no comment Additional comments: Nice, clear, well written manuscript. No doubt about the validity and importance of the findings.

You are one of the reviewers, your task is to write a review for the article. You will 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: MOLECULAR AND SEROLOGICAL DYNAMICS OF CHLAMYDIA PECORUM INFECTION IN A LONGITUDINAL STUDY OF PRIME LAMB PRODUCTION Review round: 1 Reviewer: 3

Basic reporting: The authors have presented a longitudinal study comparing detection of Chlamydia pecorum in lambs from 2 to 10 months of age via qPCR and CFT and discussed the relative merit of both techniques in terms of monitoring the presence of Chlamydia pecorum over time and as tools to guide the management of disease within a flock. Longitudinal studies are hugely beneficial to the study of diseases especially those caused by intracellular pathogens. The study design has been well thought out and executed. The structure of the article is satisfactory and the literature cited is appropriate and sufficient and supports a strong knowledge of the topic. The abstract, introduction and material and methods are well written. However, material and methods needs more detail (see below). The results, including tables 1 and 2, need a major revision. I think the authors have attempted to extrapolate more from their data than is available. It is clear that the authors have established interesting patterns within in and between their tests but I think they have overstated the interpretation of these results with respect to the progression of disease Tables 1 and 2 are extremely confusing and should be reconsidered and reformatted. The authors have presented to much data in each table and should consider revising to make it easier for the reader. For example: Table 1. A total number of animals qPCR+ at each time point, regardless of repeat positives from different samples, would aid in making sense of the comparative columns. Also, the 68 at the bottom of the second last column is a number not a percentage and equates to the 96% positive by both tests stated in the text. The totals at the bottom of columns 2-4 and the # footnote make no sense. It might be worth having two tables, the first with the expanded explanation of the qPCR results and then these summarised to number of animals and compared to CFT in a second table. Table 2. As with Table 1, this table is very confusing and should be revised as described in Table 1. The combination of numbers and percentages, time points and new detection permutations is not easy to follow. Once again, consider two tables as described for Table 1. I am also concerned about the terminology used to describe the inferences from the qPCR results. Infectious load should be bacterial load as there are no references to how infectious load has been determined in the paper. A qPCR positive result is an indication of shedding but how does this correlate to the progression of disease without other information. The authors have also indicated the presence of “Supershedders” in the flock and animals that were qPCR positive but CFT negative. Could these animals have chlamydia simply passing through theire system following ingestion of faecal material from the “Supershedders” The “Supershedders” were eluded to a source of infection but not passive transmission. I would encourage the authors to revise and simplify their results and the interpretation of results in accordance with what is achievable with qPCR and the progression of disease in the absence of other information, most notably pathology. At the very least some indication on the quantitative nature of the qPCR results needs to be correlated to disease if they choose not to revise. Suggest revising the use of the term detections throughout Suggest standardising infection load" "qPCR load" and "qPCR copy load" throughout Line 52; Suggest Point-of-Care (POC) first and then POC throughout Line 100: Has qPCR been correlated any better Line 191 Delete, qPCR and CFT detection, with Line 193: Revise sentence "meaning these lambs tested C. pecorum DNA qPCR positive and also seroconverted and developed a detectable antibody response measured by CFT" Lines 197-200: "Interestingly, 3.9% (3/76) of lambs were qPCR positive and CFT negative throughout the study period and 2/3 of these lambs had recurrent C. pecorum infection as measured by repeated qPCR positivity" Surely this raises questions about the correlation between immune response, infection and shedding? Or the accuracy of the CFT test. Line 273: unapparent Line 474: Delete second use of word detections Line 477: qPCR+ not PCR+ Experimental design: I think this is a new and valuable contribution to this field of research and would be valued by readers when revised. The aim is well defined and the results, when revised, will make a valuable contribution to field of research and progress to contributing to the gap in knowledge. In describing how qPCR was performed the authors refer to Marsh et al. 2011. There are several sets of primers in this reference and the set used here should be either shown in full or referred to by name with respect to the original reference. No mention is made of the qPCR reagents and should be included. A description of how the bacterial load was determined quantitatively and what this means should also be included. Generally speaking it would appear that the study has been undertaken to an acceptable standard and that all ethical requirements have been met. Validity of the findings: Overall the study is quite straight forward. The data when revised will be a sound foundation to build on with future research in this area, particularly the application of qPCR and CFT, for monitoring the presence of chlamydia in research trials and as management tools on farm. this should be emphasized. The discussion has been adequately written but will also improve with the revision of the results and their interpretation. As will the conclusion. Whilst a revision of the results and toning down of speculation of these with regard to the progression of disease is warranted. The results do support differences in the dynamics of qPCR and serological dynamics of sheep exposed to Chlamydia pecorum. The study should be published and will contribute to the knowledge of Chlamydia pecorum in prime lamb production Additional comments: You have a great story, don't over complicate it. Be consistent in the use of terminology and be mindful of the use of terminology with respect to presence/absence of pathogens and how far this can be used to speculate on the progression of disease. Disease studies usually integrate pathology and histopathology to confirm findings.

You are one of the reviewers, your task is to write a review for the article. You will 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: CHARACTERIZATION OF SYMPATRIC PLATANTHERA BIFOLIA AND PLATANTHERA CHLORANTHA (ORCHIDACEAE) POPULATIONS WITH INTERMEDIATE PLANTS Review round: 1 Reviewer: 1

Basic reporting: Authors need to improve the flow and the clarity of the text in introduction and discussion parts. Literature is almost sufficient; information of the reproductive strategies of Platanthera in American (where there are more diverse) would be added. Line 68. P. bifolia is also largely present in calcareous grasslands Experimental design: Line 104: In the end of introduction, the main research question is curiously presented “In order to determine whether intermediates are indeed hybrids, ...”. What are the alternative hypotheses? This part needs to be better explained. Why intermediate individuals are investigated in only two populations while mixed populations between these two species are frequently observed (as said line 71). Why scents are investigated in only one of these two mixed populations? And why scents are not investigated in two control population? Why autogamy is investigated in only one population and with so few number of individuals (see table 1)? Why morphology is investigate on the base of only four traits? Why authors did not justify the selection of these morphological traits? What is the interpretative value of the number of days of co-flowering between only two populations of two species and during only one year? All these points strongly reduce the explanative power of this study. This situation renders the results very poorly convincing. All interpretations at species level are then based to a comparison between individuals from only two (and sometimes one) populations! Validity of the findings: See before. Method used explained that results are poorly convincing. In the figure 2A, how authors explain that sympatric and allopatric populations of P. bifolia are significantly different? In the table 4, authors have to add both the retention index for each compounds, and the occurrence of each compounds of each species (and repeated the number of sampled individuals per species). The last information about occurrence is a key indicator to determine the evolutionary stability of each compound, especially when the number of compounds is very low. The sentence Lines 435-436 “The results of this analysis show a significant similarity of chemical patterns in floral scent composition with P. bifolia of all intermediate morphotypes sampled” is indeed in contrast with the opposite values of relative proportions for 3,7-Dimethyl-1,3,6-octatriene and 1,2-Hexanediol-2-benzoate indicated in table 4. How authors explain this contrast? Several parts of discussion are too speculative, like the involvement of mimicry system (549) or genetic drift (L 575). For example, how mimicry can occur if morphology and scent are different? Additional comments: The resuls presented here mainly concern one population (for scents and pollination experiment) and only two for morphology and AFLP analyses. The absence of pollinator observations is a pity. Thus the generalisation of these results is quite low. Several points may be improved by additionnal experiments in order to reach the scientific value of PeerJ.

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

Title: CHARACTERIZATION OF SYMPATRIC PLATANTHERA BIFOLIA AND PLATANTHERA CHLORANTHA (ORCHIDACEAE) POPULATIONS WITH INTERMEDIATE PLANTS Review round: 1 Reviewer: 2

Basic reporting: This is a generally well written manuscript on the genetic, chemical and morphological characterization of plants of the terrestrial orchid species Platanthera bifolia and P. chlorantha with intermediate morphologies occurring in Belgium to investigate the possibility that hybrids are evolving in mixed populations in response to local pollinator-selected selection. Experimental design: My main concerns are with the fact that it is not clear whether the genotypic frequency classes were assumed or experimentally verified and that the maternal effect is not mentioned and discussed to explain the results obtained. Validity of the findings: Adding more information about the assignment of the genotypic frequence classes and the maternal effect is recommended. Additional comments: Abstract line 25: The sentence ‘The results of the genetic analyses showed the same genetic patterns of morphologically intermediate individuals with the P. bifolia group’ is inclear so I recommend rephrasing it into ‘Morphologically intermediate plants had similar genetic patterns as the P. bifolia group’. Introduction line 68: What is meant with a ‘fresh’ meadow? Please rephrase. Materials and Methods line 120: ‘… for their attachment to the base of the pollinator’s head’. Please rephrase into ’… to various organs on the pollinator’s head’ to make the text more compatible with the information provided two paragraphs further down on attachement of pollinia to either the proboscis or eyes. Materials and Methods lines 156-158: Was this done noninvasively? Materials and Methods lines 263-264: The authors state that they ‘used six genotypic frequency classes to classify the analyzed individuals: pure parental species, F1, F2, backcross to each parental species. It is unclear how the authors assigned these classes. Were F1, F2 and backcrosses made by the authors themselves? If not, the authors should make more explicit here that they assumed the individuals to belong to these classes but that this was not verified experimentally. Results line 335: Please remove ‘the’ in ‘… traits explained the 89%...’ Results line 420: Please change ‘the honey bee colony’ into ‘a honey bee colony’. Discussion line 439: In the sentence ‘… individuals with intermediate column…’ is not clear what exactly is intermediate here so please add a specification such as ‘shape’ or ‘size’ here. Discussion line 464 and further: I agree that the hypothesis that P. chlorantha evolved from P. bifolia is plausible. The authors could provide a bit more information about the timeframe in which this happened, though, for instance by referring to Inda et al. (2012) Annals of Botany, in which a molecular clock analyses is presented for a.o. the divergence of P. chlorantha and P. bifolia. According to these analyses, this divergence started only quite recently (i.e. in the Pleistocene), which could explain the fact that lineage sorting is still ongoing. Discussion line 483: What is meant with the word ‘expanded’? Elongation? Widening? The authors should make this more clear. Discussion in general: What I missed is the possible influence of the so-called maternal effect in plants (Roach and Wulff, 1987 Annual Reviews), where the mother contributes most to the phenotype of the offspring. It might very well be that the plants with intermediate phenotypes are genetically, chemically and morphologically most similar to P. bifolia because this species received most of the pollinia from B. chlorantha! The authors should mention this and the possibility to verify this by carrying out man-made crosses and investigating the resulting phenotypes. Discussion line 571: Please add that this particular study was carried out ‘in Austria’ as most readers will not know this and assume that Durka et al. (2017) also studied plants in Belgium. Likewise, please add ‘in Belgium’ to the sentence ‘… our observed populations of intermediate plants…’ Acknowledgements line 606: please provide information on permits obtained for the experimental crosses by hand pollination carried out in the field to determine the level of compatibility between the species investigated.

You are one of the reviewers, your task is to write a review for the article. You will 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: BREEDING AND MULTIPLE WAVES OF PRIMARY MOLT IN COMMON GROUND DOVES OF COASTAL SINALOA Review round: 1 Reviewer: 1

Basic reporting: The paper is fine and relatively clearly written in English but would benefit from a widening of focus to explore a wider view of the scope of the difficulties in scoring molt in a field study were there will always be unknowns related to seasonality or lack of, lenght of breeding/non-breeding period, molting/non-molting periods, multiple broods related to all of the former. Two additional references not included I noted in general comments. Both authors have extremely strong backgrounds and are leaders in this field of research The paper is structure professionally The paper is self contained for the narrow look at stepwise molt given the limitations induced by unknown is this type of field work. Experimental design: Methods could use some clarification and checking of numbers of cited in text based on supplemetal data tables, see General Comments. Validity of the findings: Data are intriguing and the paper provides unique data that will become a much cited reference for future research. I spent several hours checking and proposing what if scenarios and I'm sure others will do the same. With this in mind, the authors should consider a more open framework to outline alternatives rather than forcing data into Stafflemauser and arrested molt for breeding scenario. Consider patterns from arrests of molt from seasonality, for breeding, combinations of the two, age structure of populations, and arrests with or without re-initiation because feathers have a latency to molt. And perhaps most interesting, why juveniles are replacing primaries so soon after growing them. But I'd like to stress that this is my opinion and should be construed as necessary. Additional comments: First task is to proof and spell check supplemental data table and verify numbers. For example, the first sentence of Results indicate there were 50 juveniles when there are 44 in the supplemental table. Methods indicate 175 birds when there are 182 in supplemental table. See comments. These are minor inconsistencies perhaps reflecting birds included or excluded for various reasons but clarification would be helpful. One colorful addition is the conditional formatting option in Excel that color codes molt values - I left this in on the Supplemental Table sent to the Associate Editor (I couldn't attach the Excel sheet as a pdf). When completed, move Table 6 to Table 1 adding ages to provide an introductory summary of data. Data in the manuscript are crucial to further understanding patterns of molt. However, data were subjected to narrow interpretation and need a more careful and thorough analysis and summaries without preconceived notions regarding Stafflemauser. There seems to be sufficient info in Stresemanns’s, Pyle, BNA, especially Rivera-Milan, F. F. 1996. (Nest density and success of columbids in Puerto Rico. Condor no. 98:100-113) to assume molt is slow sequential descendant with arrests possible, but it is useful to verify this as the ms does. Ground-dove data seem similar to woodpigeons (cited in ms) except for unknown impacts of the length of seasons on ground-doves. A relevant paper to include is Ashmole's (1968) study of Fairy Terns stepwise molt, near year-around breeding, and arrests for breeding. There may be an inconsistency in consideration of Stafflemauser. The discussion of omissive molt and the multiple replacement of proximal primaries (or Ps) once or twice while setting up Stafflemauser are said to be maladaptive in the paper. The last sentence of abstract suggests the latter is the “single greatest flaw”. If so, then is Stafflemauaser simply an artifact of incomplete P molt? Alternatively, if Stafflemuaser is an adaptation, an incomplete P molt is a prerequisite to setting up waves. Thus the retention of outer Ps for a year or two are putative costs that immatures incur in order to benefit from Stafflemauser as adults. As for multiple replacement of inner Ps, I believe this refers to yearly replacement which birds would do typically so not seeing how it is a flaw or cost. Once waves are set up, benefits are speculative depending on how many Ps are replaced as adults. With complete replacement, benefit might be more efficient replacement with 3 waves than in a single wave of molt. With incomplete replacement, the outer Ps can be replace every other year. The discussion of omissive molt and multiple replacement of proximal Ps need some minor rewriting to reflect this overview. However in revising consider that if data are filtered to only show the 13 adults that are molting P10, the interpretation of Stafflemauser is suspect. A prediction from Stafflemauser is that waves are synchronized more or less. If so, if P10 was molting and appears to be a nodal feather (proximal neighbor was old (0=old), then proximal and medial waves would also be re-initiating with a similar number of Ps renewed. However only two wings, UWBM 82497 and CWS 9167 appear as predicted with similar numbers of new (=1) Ps in proximal and distal waves suggesting a lack of synchrony on re-initiation. In others the proximal wave has progressed many more Ps than would be expected by an assumed roughly synchronized re-initiation of waves. Perhaps this results from proximal waves abutting more distal leaving a continuous sequence of similar wear class Ps as discussed in the ms? Alternative, P10 is molted asynchronously from proximal Ps or as Broody for woodpigeons. P1 appears to be similar. Of 9 growing P1, 5 appear to initiate at P2 so it appears P1 molted independently or perhaps with secondaries? So for ground-doves, is the molt observed a result of a slow (150 days in ms, 200 days in Rivera-Milan 1996) simple sequential descendant pattern that takes place yearly with some arrest & re-initiation or an adaptation/modification worthy of being considered Stafflemauser similar to Fairy Terns (Ashmole 1968). Data indicated that only 30% of ground-doves have multiple waves suggesting the pattern might be an artifact. This might be a better way to frame hypothesis testing rather than the idea that there are distinct series in primaries (similar to secondaries). Add that since 70% of adult have a single wave, multiple waves may be SY and TY (second or third year) birds ironing out the wrinkles to set up a single slow descendant wave as adults. For clarity, redo the direction summary using pairs of growing primaries for P2-9, excluding P1 & P10 as focal feathers because they lack prox and distal neighbor Ps, respectively. This will affirm (or reject) that molt is directional. Then summarize waves more simply than Table 3 & 4. The hypothesis to disproof appears to be that are set nodes, e.g. P1, P4, P8 and therefore node and terminus of waves would be predictable. The difficulty here is that this is only true if all Ps are replaced in which case the Stafflemauser and the hypothetical set-node Stafflemauser would be indistinguishable in adults. With incomplete replacement, assuming re-initiation, there would be no predictability of either accept perhaps a node at P1. Most the set-node patterns in primaries start in middle or proximal Ps and go both ways. Owls, not mentioned, likely have the record for slow molt. Regardless of direction, terminal and nodal Ps are predictable only if all Ps were replaced. Incomplete molt results in unpredictability. On constraints from breeding and crop milk for feeding young. Milk is fed to young nestlings but then I believe adults shift to regurgitated seeds as young grow so not sure that an active crop signals an energetic constraint from feeding young and manifest as a molt arrest. If it takes 150-200 days for adults to molt all Ps, and there are multiple broods, seems more likely the extremely slow molt is an adaptation to prolonged window when breeding & molt is possible. And assuming molt is extremely slow, there is likely a latency in some wings to completion of one P and dropping of the (distal?) neighbor, which might appear to be an arrested molt (a check for molting secondaries might provide a clue here) (also mentioned by Boody for woodpigeons). One could turn the discussion around and suggest that so many were molting (~80%) that they molt continually while breeding and most successful adults do both without arrest. Leaving multiple waves an artifact of incomplete molt or a rudimentary Stafflemauser. A latency to molt hypothesis might be presented as a contrast to Stafflemauser in highly seasonal species. Once renewed a feather will not be physiologically ready to molt until it is X months old thus stopping or merging waves and introducing the idea for what was observable in P1s and P10s. An interesting offshoot is why are juveniles molting primaries so early? If the poor feather quality, then the juvenal feathers should be able to be identified and if no obvious different wear categories, then Ps probably aren't juvenal. E.g., 5 "juveniles" had class 0 (old) primaries in the wing distal to J primaries. These would have to be scored as "J" or "J0" if the birds were juveniles. Perhaps they SYs (second year) that have gone through one non-molting period. A complication, briefly mentioned is that if a pair had multiple broods over a breeding window, the first fledged would likely complete molt while there would be a progressively fewer completing molt in subsequent broods with the last fledged only molt few Ps. Following a non-molting period, if it exists, the SY birds would likely be classed as adults unless juvenal plumage hints were retained. This would create a scoring nightmare with birds in all stages of complete, incomplete, and some erratic molt patterns with the underlying pattern of sequential descendant molt. To further non-temperate molt studies introduce molt latency as a controlling factor and provide a simple figure showing what P wear categories might look like with staggering introduction of juveniles that undergo a period of arrest. Depending on the age structure of population of ground-doves and similar species, SY birds might be most common. Also suggest as Ashmole did that researchers might punch primaries if they had an opportunity to recapture the birds. Figure 1. Number of feathers between the outermost growing primaries in the 15 adults that had two active waves of primary replacement. (in supplemental table there are 19 adults with 2 active waves, 17 scored and 17 shown in figure) Figure 2 legend. "sailed population" an autocorrect for "Sinaloa population"? Table 1 Totals 43 41 95% should be 44 total sampled and 42 replacing primaries Table 2 check counts of growing, correct except P1 looks like it is 14 rather then 13 Table 3 delete simultaneously as it isn’t possible for a single feather Table 4. Summary table for 100 adult Common Ground Doves that were growing primaries. But there are 112 in supplemental table, some were not included? Previously noted UWBM 90871 incorrectly designated no molt but P1 is growing so make P1 growing 9 rather than 8, check others growing, most are correct but there may other discrepancies depending on how many are included Table 6 132 adults in table 138 in supplemental data, 28 not molting (one –UWBM 90871 - molting at P1 designated not molting in the supplemental table) Throughout change Ground Doves or Ground-Doves Some images of wings showing various wear classes of primaries would be useful for future reference A few comments on the Supplemental data table and second worksheet summarizing growing P1 and P10 were sent to the Associate Editor because formatting was lost while saving as a pdf. I can forward these to the authors on request.

You are one of the reviewers, your task is to write a review for the article. You will 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: BREEDING AND MULTIPLE WAVES OF PRIMARY MOLT IN COMMON GROUND DOVES OF COASTAL SINALOA Review round: 1 Reviewer: 2

Basic reporting: The manuscript is written clearly and overall well referenced, and the introduction provides good background on the subject. The structure conforms the journal’s standard. Raw data were provided but need more descriptions of symbols they used (for example, I was not sure what “r” means in the primary molt score columns). Also, I felt that some of the conclusions were not well-supported by the data and/or analyses (figures and tables) they provided. Experimental design: The research was within the scope of the journal and performed to a high technical/ethical standard, and research questions were well defined in the Introduction. As the authors stated, the molt sequences and cycles of tropical species are poorly studied, and this study fills a knowledge gap and describes interesting molt patterns that have never been previously examined. Methods were described in sufficient detail and in a replicable manner. Validity of the findings: Figures and tables are well described in the captions and text but lack associated statistics to support the authors’ conclusions. The conclusions are linked to the original research questions but not rigorously supported by their data and analyses, and the authors need to present alternative hypotheses. Please see the comments in the “General Comments” section for details. Additional comments: L. 102–103: It was not clear why juveniles “should” initiate primary molt at the same loci as adults if they replace the primaries in multiple series. Could they not have different nodal points? L. 134–137: Need more explanation for the relationship between aggression and retainment of bursa. L. 162–164: If the sample collection was not random, the data would still not be “unbiased.” Obviously, the samples were not collected randomly throughout breeding and molting seasons. L. 212–217: The fact that only 17%–31% of adults replacing primaries in two or more waves appears to indicate that this is not their dominant molt strategy. Is it possible that only younger individuals (especially second-year birds) that have not completed the mot of distal primaries in the previous year molt in two waves? L. 214: It is stated here that two groups of primaries were separated by one to several older primaries, and Fig. 1 shows that the number of feathers between two molt waves are less than 6 for more than 1/3 of individuals. Because of the sample size, I am not sure if the distribution shown in Fig. 1 is significantly different from the normal distribution. This seems contradictory to the later statement that the molt of P1 does not usually start until the distal wave reaches to about P6, which is the mechanism to reassure the fresh inner primaries are not replaced redundantly. L. 221–228: I didn’t follow the logic here. If P1 is growing and P2 is new, should the direction of molt be ambiguous as P1 is the first primary? Same for P10. L. 222: The phrase “P1 being too short to be compared for wear and fading with its adjacent inner primaries” does not make sense as P1 doesn’t have adjacent inner primaries. L. 254–258: The overlap of breeding and primary molt appears to occur within a small window based on the supplemental raw data provided (mid July to early August). Most of birds do not molt during the most intense period of breeding season. I understand that the molt may be arrested during that time, but based on the data author presented, I am not convinced that the arrests during the breeding season “cause” the multiple waves of the primary molt. L. 256: The authors do not show statistically that the arrested primary replacement was “strongly” associated with having an active crop gland. L.271: Regarding the sentence starting “Two birds directly support…,” I feel the data did not support their conclusion exclusively as data on other birds showed that molt on P1 started before the distal wave reached P6 (Supplemental data). Since there are many birds that do not molt primaries in two waves, it seems that these examples instead support that the molt arrests during the intense breeding period do not “cause” the two waves of molt. L. 282: Need to show the statistics if the authors are claiming “significantly positive.” L. 282–289: These two flat lines are clearly the artifacts of sampling in July and September intensely. I agree that their molt is little synchronized, and it would be difficult to estimate molt duration reliably. However, the figure seems to indicate that most birds were in the latter half stages of molt by September. Does this indicate that their molt cycles designed to complete their primary molt before winter for most birds? L. 324: Need statistics if you are stating that molt arrests are “strongly associated” with active crop glands. L. 369–371: The “surprisingly” greater frequency of growing feathers in the outer than in the inner primaries in adults (Table 4) corresponds well with the greater frequency of growing feathers in the inner primaries in juveniles (Table 2). This made me think that some of the “adults” replacing primaries in two waves are second year birds that failed to replace the outer primaries in the hatch year. Figure 2: It would be helpful if you could mark the individuals that were molting primaries in two or more waves.

You are one of the reviewers, your task is to write a review for the article. You will 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: DO PATIENTS WITH CHRONIC UNILATERAL OROFACIAL PAIN DUE TO A TEMPOROMANDIBULAR DISORDER SHOW INCREASED ATTENDING TO SOMATOSENSORY INPUT AT THE PAINFUL SIDE OF THE JAW? Review round: 1 Reviewer: 1

Basic reporting: This paper does an excellent job referring to past literature both in terms of background and referencing relevant past work. Raw data is shared - one small suggestion would be to also provide a key that identifies what each abbreviation refers to (for example, add an extra tab to the spreadsheet entitled Variable Key that has one column that lists each variable name and one column that provides an explanation of what the variable name refers to). Overall, professional article structure. I would recommend an extra read of the paper from a person with English as first language. Overall, the paper is very well written and clear, but there are some sentences where the grammar used is a bit awkward and could be improved, which would assist in readability. For example, lines 51-52: "Also observed are abnormalities in tactile processing". This could be combined with the first sentence to read: "Temporomandibular disorder (TMD) is typically characterized by chronic pain in the temporomandibular joint that is often accompanied by abnormalities in the processing of tactile input to this area." And lines 60-61: "However, these studies did not include unilateral TMD patients, but also bilateral TMD patients who were "asked about the most painful side"." This could be re-worded to: "However, these studies did not exclusively include patients with unilateral TMD; rather, patients with bilateral TMD were included, using the most painful side (self-reported) as the TMD-affected side in analyses." Also lines 103-104: "The majority of the sample (70%) did not yet receive treatment at the moment of testing". This could be re-worded to "The majority of the sample had not received treatment for their TMD pain at the time of testing". Also line 343 - "To pain further insight...." I am not sure what this is meant to read. Experimental design: Overall, well done. Hypotheses: Currently, hypotheses in both directions are given. That is, according to the 'spatial neglect hypothesis', patients will have slower processing of tactile stimuli at the painful vs non-painful joint, where as based on the 'hypervigilance hypothesis' the opposite is expected. I think it needs to be clearly written that the overarching hypothesis of the paper is that there will be a difference in spatial processing of tactile input in people with unilateral TMD, but based on the literature, the direction of this spatial bias (prioritising painful vs non-painful tactile input) is unclear. At the moment, it is not clear if the authors have gone with the last hypothesis presented at their main hypothesis. Also, it is unclear to the reader why there is no hypothesis specific to comparisons between the TMD patients and the healthy control group (we find out later that this hasn't been compared). Analyses: I do not understand why a statistical comparison cannot be made between TMD and healthy controls. The current argument is that because people with TMD had pain on different sides (left vs right-sided jaw pain), then the meaning of the PSS is different than in the control group. However, if healthy controls have no difference in spatial processing bias between stimulation on the left and the right, and if TMD patients have no difference in spatial processing bias based on whether they had left-sided TMD pain or right-sided TMD pain, then a between group comparison should be fine to make. At the least, you could take half of the healthy control sample and switch the test stimuli to the left joint and the reference stimuli to the right joint such that the data is matched to test and reference stimuli of the left-sided TMD patients (n=10 left sided pain and n=10 right sided pain). Or perhaps compare the right-sided TMD patients to the whole sample of the healthy controls (and vice versa for the left, switching the healthy control test and reference stimuli). I would suggest including some sort of comparison of PSS between groups in the analysis. Validity of the findings: Abstract: I don't think that the first sentence of your abstract's discussion aligns with your results. The results found no difference in spatial processing in patients with unilateral TMD (although I do agree this was close - 0.07 - and may have been underpowered). However, the way it currently reads is that there was a difference in tactile processing even in people without fear-avoidance beliefs. I would argue that this should read: "The results suggest that patients with unilateral TMD show a tactile processing bias toward the painful side of the jaw, but only when high levels of fear-avoidance beliefs are present." Then you could specifically state that this supports the hypervigilence hypothesis. Similarly, the conclusion of the paper needs to be updated to reflect this. Additional comments: 1. Introduction: Consider adding in the 'why do we care' aspect of spatial processing bias, e.g., understanding the presence and nature of any spatial processing bias may help us determine new treatment for these patients. 2. Methods, Line 115: "attaining the requested performance criteria during the task". At the moment, the reader is unaware of what this refers to. Please reference the appropriate section, e.g., (see TOJ data handling). 3. Cronbach Alpha results for self-report questionnaires: This is commendable that this was evaluated in the study sample. I might suggest providing an overall statement at the start of this section saying that internal consistency was acceptable for all questionnaires and then refer to a supplementary table that has the Cronbach alpha values for all measures. At the moment, this section is quite text/number heavy. 4. Methods, lines 210: Perceived intensity of tactile stimuli during testing - if it differed between left and right side of the jaw, was the intensity used updated in real-time during testing? I don't think this is a problem if not, but important to know. 5. Results, TOJ, Line 282: Should this read mean difference? 6. Perceived tactile intensity between painful and non-painful side, lines 295 - 297: Given that tactile stimuli was matched between sides at baseline, is this not just a validity check (i.e., to ensure that any differences in spatial processing were not due to differences in perceived intensity)? 7. Discussion, line 403: contextual factors - please provide an example of what you mean by 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: DO PATIENTS WITH CHRONIC UNILATERAL OROFACIAL PAIN DUE TO A TEMPOROMANDIBULAR DISORDER SHOW INCREASED ATTENDING TO SOMATOSENSORY INPUT AT THE PAINFUL SIDE OF THE JAW? Review round: 1 Reviewer: 2

Basic reporting: I think this is very high quality. My only suggestion is that the authors think whether there might be better papers to cite in some instances than their own. This is particularly relevant for the methods and discussion. Experimental design: I think the study is within the scope of PeerJ. I think the knowledge gap is defined but should focus on the potential benefit of knowing this stuff in TMD, which is a highly problematic condition. I think the experimental design has solid fundamentals, but it is not well suited to test the hypotheses the paper suggests it is testing. I actually think that the study is testing the hypothesis that TMD pain patients will have a spatial bias to the affected side. The other hypothesis they mention has not been proposed for TMD and in fact has been discussed as a potentially characteristic to just CRPS and perhaps unilateral low back pain. It seems odd and the rationale not well presented, to pitch these two hypotheses, which relate to different situations, against each other. I think any experiment nowadays should build on a protocol and analysis plan that is ideally peer reviewed - if the analysis here was as planned, it would not have got through peer review. These things should also be locked prior to data collection and analysis so the reader can be assured that the study is undertaken in line with the principles of transparency and replicability. That this process was not followed represents a limitation of this work, which should be mentioned in the discussion. The wider design is in my view highly problematic because there are about 15 analyses and no mention of the risk this presents to findings. Moreover, there is not a clear presentation of what results would be required to support or refute the hypothesis about vigilance. That is, the authors use several measures and several sub factors but don't stipulate a priori what would be required to refute. This leave it highly vulnerable to false results and as it stands, i can't interpret the results in light of this substantial threat to validity. Methods are very well described and could easily be replicated. Validity of the findings: The authors do not accept the negative results. Rather they suggest that despite the statistical analyses clearly not supporting the hypotheses, that a positive result existed. I find this very surprising. This means that the discussion, which focusses to a large extent on the positive result, is inappropriate because the result was negative. In my view, the discussion needs to be completely rewritten to reflect the results, not the predicted results. The authors report that 65% of the patient group did what they predicted they would, but they don't state how many of the control group did. The positive finding on one sub factor of one outcome for one hypothesis can not in my view be given any weight on the grounds mentioned above. There is a large literature on spatial biases and their implications and possible interactions with other systems, yet this literature is not really addressed even though the current findings are very relevant to it. That unilateral TMD does not have a spatial bias is important and interesting (i suspect that they may have a smaller one, but your study wasn to powered to detect that). Additional comments: I have attached an annotated PDF with a range of specific comments that i hope you find helpful.

You are one of the reviewers, your task is to write a review for the article. You will 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: DO PATIENTS WITH CHRONIC UNILATERAL OROFACIAL PAIN DUE TO A TEMPOROMANDIBULAR DISORDER SHOW INCREASED ATTENDING TO SOMATOSENSORY INPUT AT THE PAINFUL SIDE OF THE JAW? Review round: 1 Reviewer: 3

Basic reporting: The paper is very well-written. It has a clear structure and is easy to follow. I particularly liked the clear explanation of how the data may help us differentiate between two rival hypotheses. The dataset should have a key to explain what each of the variables are and how they are coded. For example, hand dominance and pain location are both coded as 1 and 2 but I don't know which side is 1 and which side is 2. Experimental design: The paper meets all of the criteria for this section and I have no suggestions for improvement. Validity of the findings: For clarity I would prefer exact p values to be reported rather than "ns" (e.g. lines 118 and 119). The authors say they used the TOJ analysis guidelines proposed by Spence, Shore and Klein (2001), but they appear to be slightly different. Spence et al said "Participants were excluded for one of three reasons: if any of the eight correlations were less than 0.4, if two or more of the calculated PSSs were greater than +/-250, or if the z score for a particular stimulus pairing at the +250 ms and -250 ms SOAs were, when averaged, less than 1.29 (i.e. less than 90% correct)" (p. 805-806). Could the authors add an explanation for the differences and/or add a note to say whether the results would be any different if using the original exclusion criteria. I thank the authors for including details of their power analysis. It is a shame that there wasn't sufficient power for the key effect, which is reported as d=0.42 (i.e. medium-sized), to be significant. Future studies should be powered to detect the smallest effect size that would be considered interesting or relevant, rather than that found in previous studies, possibly even with 90% power. At several points in the results I wondered whether the control was really necessary since the key hypotheses being tested didn't require one and the PSS was not compared between groups. Perhaps the authors could make the need for the control group clearer in the manuscript. I think the authors could say more about their analyses of the perceived intensity of the tactile stimuli in the introduction and discussion. The intensity on both sides and in both groups was tailored to be a 3 on a 1-5 scale (lines 132-141) so was the analysis comparing sides (lines 295-298) and groups (lines 293-295) intended as a manipulation check or a hypothesis test? What are the implications of the difference between groups? Does this mean that the TMJ group must have experienced increasing pain over time? On line 340 I think it would be better to say "did not reach significance" rather than "just failed to reached significance". Also I think the authors could be more nuanced on line 338 by saying something to the effect of "which had it been significant would suggest enhanced...". On line 354 the authors note that the correlation should not be overstated given the small sample size, but equal important is that is was an exploratory analysis rather than a hypothesis test, so I think this should be noted here too. On line 364 I think it is incorrect to say that the result contradict previous findings, since there were significant. It may be more correct to say that the results failed to support previous findings. Additional comments: I enjoyed reading the paper and I think it should be published. Although I have written quite a bit in the 'Validity of findings' section, these concerns should be fairly easy to address. I appreciate that the authors have stated whether most analyses were hypothesis tests or exploratory (which many authors do not do) but I hope that pre-registeration of analysis plans will become more common in this field. A few minor points: Line 82: "Rivalry hypotheses" should be "Rival hypotheses". Line 127: "procedure is highly similar as" should be "procedure was highly similar to". Line 168: is "...your relation..." correct? It would make more sense without the "your". Line 226: Should this be "(virtual) interval", as with the PSS description on line 227? Line 375: "and is this feature" should read "and this feature is". Line 377: "More specific" should be "More specifically". Line 380 "Anyway" sounds quite informal and "Nevertheless" may be better. Figure 1 would benefit from a note in the description to say what the scale of the y axis was (-200 to +200?).

You are one of the reviewers, your task is to write a review for the article. You will 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: DO PATIENTS WITH CHRONIC UNILATERAL OROFACIAL PAIN DUE TO A TEMPOROMANDIBULAR DISORDER SHOW INCREASED ATTENDING TO SOMATOSENSORY INPUT AT THE PAINFUL SIDE OF THE JAW? Review round: 2 Reviewer: 1

Basic reporting: I thank the authors for their changes. The grammatical changes make the manuscript much easier to read. Experimental design: Thank you for clarifying the analyses and for including analyses with the healthy control sample. Validity of the findings: The only thing that I would recommend would be to avoid stating that you found a non-significant trend (abstract and conclusion). Otherwise, using the same logic, we could argue that the correlation between PSS and the somatic focus subscale of the TSK (p=0.03) is actually supporting a trend towards non-significance (given that it is quite close to p = 0.05). I would just state that you had non-significant results. You could then clarify that based on your findings, you could not rule out inadequate power as a reason for this (given the higher variability in PSS than anticipated). Additional comments: Excellent work on the revisions!

You are one of the reviewers, your task is to write a review for the article. You will 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: DO PATIENTS WITH CHRONIC UNILATERAL OROFACIAL PAIN DUE TO A TEMPOROMANDIBULAR DISORDER SHOW INCREASED ATTENDING TO SOMATOSENSORY INPUT AT THE PAINFUL SIDE OF THE JAW? Review round: 2 Reviewer: 2

Basic reporting: Still very happy with this. Experimental design: I think this has been greatly improved, particularly with regard to clearly labelling primary and exploratory analyses. Validity of the findings: The new version is much more faithful to the results in my view. I appreciate the timing of data collection may have preceded the real push for publishing protocols etc, but i do not think that the rarity of the practice is very good justification for not doing it. I am pleased the authors mention this in limitations. I am also still submitting papers that were started before we adopted this policy and I too see it as a limitation of those papers. Additional comments: I think the response to all reviewers has been excellent. I think the paper is very interesting and moves the field forward.

You are one of the reviewers, your task is to write a review for the article. You will 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: DO PATIENTS WITH CHRONIC UNILATERAL OROFACIAL PAIN DUE TO A TEMPOROMANDIBULAR DISORDER SHOW INCREASED ATTENDING TO SOMATOSENSORY INPUT AT THE PAINFUL SIDE OF THE JAW? Review round: 2 Reviewer: 3

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: I think the authors have done a good job in responding to my and the other reviewers' previous comments. There are a few typos throughout (e.g. in line 40 "my" should be "by" and in line 387 "we the spatial bias" does not make sense) but otherwise I think the paper is suitable 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: GENOMIC SIGNAL PROCESSING FOR DNA SEQUENCE CLUSTERING Review round: 1 Reviewer: 1

Basic reporting: Overall, the paper is well-written and easy to understand. There are a few minor spelling and grammatical errors present in the manuscript that the authors should correct. Here are some examples:  1 Line 13 should read "...popular method that has been used to..."  2 The sentence on lines 33-35 should read "However, the K-means algorithm relies on the frequent computation of similarity metrics.."  3 The reference on line 159 is incorrectly formatted.  4 The algorithm's name, STARS, is misspelled as SATRS on page 5 of the installation guide and page 1 of the user manual.  5 The title is shown as "Genomic signal processing for DNA sequence clustering" and "Genetic signal processing for DNA sequence clustering." I suggest using the former, as it reflects the wording in the main body of the text. 6 The word "Matlab" on line 322 should be stylized as MATLAB I also would like to make the following minor suggestions (starting from most important to least important): 1 The figures have very small font, making them very hard to read. I suggest that the authors increase the font across all figures, especially for Figures 3-6, which have a lot of unused white space. 2 According to the user manual in the link provided by the authors, the name of the algorithm is STARS. However, this name does not appear anywhere in the manuscript. The authors should include the algorithm's name at least once in the manuscript. 3 The first two lines of the abstract (11-12) and the introduction (23-24) are exactly the same word-for-word. For some readers, this may come off as repetitive and distracting. I suggest that the authors change the wording of either the openings of the abstract or introduction. 4 I suggest removing the word "novel" in the sentence on lines 58-60. 5 Lines 303-309 are a little confusing to me. I am not exactly sure what the authors mean by phylogenetic trees requiring an external class. Either the authors should clarify this statement or remove the paragraph altogether. I have one major comment for this section: The papers "A new method to cluster DNA sequences using Fourier power spectrum" (Hoang et al., 2015) and "A novel clustering method via nucleotide-based Fourier power spectrum analysis" (Zhao et al., 2011) also cluster DNA sequences by first computing power spectra and then converting spectra to constant length vectors. I suggest that the authors cite these works and describe what sets their method apart from these previous methods. If possible, the authors should include a comparison in terms of running times and accuracy against these methods. Experimental design: The authors describe their method with sufficient detail for others to replicate. Briefly, they convert DNA sequences to power spectra, and then group DNA sequences together using the K-means clustering algorithm applied on the frequency components of the power spectra. In order to make the power spectra of varying length DNA sequences comparable, they apply zero padding, as suggested by Borrayo et al., 2014. They applied their method to a set of 141 COXI sequences from several different organisms. I have the following major comments for the author that I believe will help improve the manuscript: 1 The authors claim in lines 50-57 that sequence alignment algorithms like ClustalW and UCLUST are limited in the number of sequences they can cluster due to their heavy demand of computational resources. However, the authors do not provide a comparison of their algorithm with any of these algorithms, nor do they mention the running time of their algorithm. I would like to see a comparison against other algorithms like ClustalW and UCLUST that includes running time, memory usage, and/or clustering quality to justify their claims. 2 As stated in lines 322-324, the authors have made their program as a standalone package. I commend the authors for making their algorithm readily available for users. However, I was unable to actually test their algorithm since it could only run on Windows. Most bioinformatics researchers use either Linux or Mac OS X, and hence replicating the authors' results would be difficult for such readers. The authors should provide the code open source (preferably on Github) so that users can run the code on their own copies of MATLAB or Octave. 3 As depicted in Figure 2, hierarchical organization of the selected organisms plays a very important role in the manuscript's analysis. The hierarchical clustering algorithm is an alternative to the k-means clustering algorithm, and seems like a more appropriate fit for phylogenetic studies. I suggest that the authors repeat their experiments using hierarchical clustering and report the results in the manuscript. 4 The authors should explicitly state how many frequency components each COXI DNA sequence is converted to (i.e. the value of "n" in line 103). Validity of the findings: The authors applied their method on COXI sequences from various species and find that the resulting clusters reflect known biology. They also provide a thorough discussion of their results.  1 I would like to see the authors repeat their experiments for sequence sets other than COXI. The Hoang et al. 2015 paper, for example, applies their clustering algorithm on 5 different datasets. Perhaps the authors can include the analysis on other sequence sets as a supplementary.  2 Figures 3-5 show clusterings for the COXI set for various values of k. The values of k are chosen to reflect kingdoms, phyla, and classes. However, the clusters do not exactly correspond to these taxonomic ranks. The authors should compute centroids corresponding to the "true" kingdoms, phyla, and classes and compare the "true" clusters with the discovered clusters. A comparison of the inertia, or the within-cluster sum of squares criterion, between the "true" and discovered clusters should suffice. 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: GENOMIC SIGNAL PROCESSING FOR DNA SEQUENCE CLUSTERING Review round: 1 Reviewer: 2

Basic reporting: In this manuscript, the authors present a method for clustering sequences in which the K-means algorithm is applied to a numerical representation (the Voss representation) of DNA sequences to which spectral analysis (a discrete Fourier transform) is applied. The authors also present a visualization method to report the resulting clustering. Data sharing ------------- - Link to the method is mentioned (lines 322-324) but not provided. Thus, I could not test the method. - Datasets not provided - The authors mention that they will provide "... executable and the dataset" (line 323). The executable is not enough. They need to provide the source code as well. Without the source code, the paper should not be published. Professional English --------------------- The article reads well. Only comment: Avoid repetition of whole sentences. The first two sentences at the beginning of the Abstract are identical word-by-word to those at the beginning of the Introduction. Self contained -------------- In order to fully understand the methods, it would be relevant to have a full definition of the "power spectral density (PSD) {\hat S^\alpha}}" (lines 86-87), as well as the "frequency power spectra d(Sa,Sb)" (lines 89-90) Experimental design: The authors apply their method to one gene: Cytochrome c oxidase I (COXI), using different numbers of clusters. It is unclear why this particular gene was selected, and why only one was analyzed. The authors make a point of this method being fast. I would like to see a experiment in which they show how much faster they are, compared to, for instance, the other two methods they mention as slow: CD-HIT and UCLUST Validity of the findings: The description of what is found in the different clusters for COXI is not very informative, as we do not know what the ground truth is. Any clustering algorithm, is going to produce clusters. Assessing the validity of the method presented in this paper, would require some proof of principle. I would like to see the analysis of a gene for which the phylogeny is well established (as different methods agree), and how that compared with the method presented here. Additional comments: Here I report some small changes that could improve the manuscript - Abstract: GPS abbreviation used prior to definition - All Figure need a more descriptive legend, and explanation of terms. - Figure2: name of species are not readable - Figures 3, 4, and 5: labels are not readable.

You are one of the reviewers, your task is to write a review for the article. You will 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: GENOMIC SIGNAL PROCESSING FOR DNA SEQUENCE CLUSTERING Review round: 2 Reviewer: 1

Basic reporting: Grammar and spelling is still a minor issue in this paper. Here are a few examples from the added text: A) "To determine the validity of the results, we computed centroids for true kingdoms and we compare these centroids to these you discovered with our method.” (263-264) B) "UCLUST have a constant time, of 1 second for every experiment…” (282-282) 3) "However, since the K-means method promote the generation of centroids in highly populated regions of the feature space it results are more likely to obtain clusters of organisms that are highly related among them instead of organisms related by possible common ancestors or groups with a small number of less homogeneous organisms" (317-320) 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: GENOMIC SIGNAL PROCESSING FOR DNA SEQUENCE CLUSTERING Review round: 2 Reviewer: 2

Basic reporting: This revision has taken into account most of the questions raised by the reviewers. I do not have any further comments. Experimental design: no comment Validity of the findings: no 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: EU PROTECTED AREA NETWORK DID NOT PREVENT A COUNTRY WIDE POPULATION DECLINE IN A THREATENED GRASSLAND BIRD Review round: 1 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: Dear authors, I really enjoyed reading the manuscript. The study has important implications for the conservation of the Little Bustard, and it can help support the reporting obligations of Portugal in relation to the Bird Directive. It also shows how management policies should be targeted at maintaining not only habitat availability but also habitat quality within protected areas, and highlights the need of going further from a mere designation of protected areas if an effective conservation wants to be achieved. I consider the data robust and the statistical analysis is sound. Therefore, I recommend accepting the manuscript after addressing a few minor issues: Minor changes Line 31. “within the species remaining European stronghold”. Sorry, but it is not clear to me what the authors mean. The authors could indicate that Little Bustard is included in the Annex I of the Bird Directive, which is clearly related to the objectives of SPAs. Lines 32. Please remove “(the European Union network of protected areas)”. Natura 2000 has been already defined in line 30. Line 34-35. These SPAs are designated only for farmland bird conservation? I would say that SPAs are designated under the EU Bird Directive to protect bird species listed in the Annex I. In general SPAs are aimed at protecting also other species that do not have to be necessarily farmland species. Also, I am wondering if these “30 areas without EU protection” can be under national or regional protection. Please clarify it. Line 36. Awkward sentence. Please rephrase, e.g. “the bustard national population declined 49% over the last 10-14 years, especially outside SPA.” Please indicate the population trends inside and outside the SPA system, or the difference between within and outside to show more explicitly the degree of effectiveness of SPAs. Keywords: I would suggest “steppe birds” instead of ‘farmland bird’ which is already mentioned in the abstract Introduction Lines 57. Can be the author a bit more specific? Some examples of these biodiversity values? Do the authors refer to e.g. habitat or climate suitability? Lines 58. I mostly agree, but instead of using Cabeza (2013) as reference (there are many others), I would show one of the few studies that uses population trends as follows: “(but see Pellissier et al., 2013).” Reference: Pellissier, V., Touroult, J., Julliard, R., Siblet, J. P., & Jiguet, F. (2013). Assessing the Natura 2000 network with a common breeding birds survey. Animal Conservation, 16(5), 566-574. Line 85. This reference is very old, I would suggest adding a more recent reference to show the last trends for this species. According to BirdLife International (2016) “The European population is estimated to be declining by 30-49% in three generations (30.9 years)”. Reference: BirdLife International. 2016. Tetrax tetrax. The IUCN Red List of Threatened Species 2016: e.T22691896A90095419. http://dx.doi.org/10.2305/IUCN.UK.2016-3.RLTS.T22691896A90095419.en. Downloaded on 07 November 2017. Line 170. What do the authors mean by “proportionally”? Appendix S1 seems to be incomplete (see last sentence). “Because these IBAs were not subjected to any sort of management, for the …” Please revise it. Discussion Although the discussion and the main conclusions are well stated and linked to the research questions, previous studies have already showed that field management and timing can play a key role in the aptitude of fallows for steppe birds such as Little Bustard during the breeding season, affecting the vegetation structure as well as the amount and type of food resources at microhabitat level. For instance, early herbicide application and shredding were found to be the best treatments for Little Bustard in steppe areas of NE Spain (Robleño et al 2017). Thus, to improve the effectiveness of management actions, conservation guidelines for farmland birds should consider microhabitat preferences, given the importance not only of the amount of habitat provided but also of vegetation structure and food availability. I think that these issues deserve attention and could be mentioned in the discussion to better understand the relevance of habitat quality (and not only availability) for the conservation of Little Bustard in particular, and farmland species in general. In addition, previous research also highlighted the importance of intraspecific interactions in the definition of the habitat selection pattern of females and families (Tarjuelo et al 2013), issues also relevant for the conservation of the species. Ponjoan et al (2012) can help to support the statement in lines 238-239, these authors found that ranging behaviour of males could be partially explained by age, habitat quality and site. References Ponjoan, A., Bota, G., & Mañosa, S. (2012). Ranging behaviour of little bustard males, Tetrax tetrax, in the lekking grounds. Behavioural processes, 91(1), 35-40. Robleño, I., Bota, G., Giralt, D., & Recasens, J. (2017). Fallow management for steppe bird conservation: the impact of cultural practices on vegetation structure and food resources. Biodiversity and Conservation, 26(1), 133-150. Tarjuelo, R., Delgado, M. P., Bota, G., Morales, M. B., Traba, J., Ponjoan, A., ... & Mañosa, S. (2013). Not only habitat but also sex: factors affecting spatial distribution of Little Bustard Tetrax tetrax families. Acta ornithologica, 48(1), 119-128.

You are one of the reviewers, your task is to write a review for the article. You will 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: EU PROTECTED AREA NETWORK DID NOT PREVENT A COUNTRY WIDE POPULATION DECLINE IN A THREATENED GRASSLAND BIRD Review round: 1 Reviewer: 2

Basic reporting: The manuscript seeks to evaluate the efficiency of Natura 2000 areas in truly protecting habitat and encompassed biodiversity. The authors show a remarkable decrease in the population size of the little bustard (Tetrax tetrax) in Southern Portugal, one of the most important areas for the conservation of the species. Tough results are valuable and discouraging, I consider that they only constitute an initial step in the more profound and important evaluation that the authors propose. Fundamentally, I found as main problems that: (1) collected data and used methods do not allow to properly evaluate a trend of the population size of this or any other species (only two periods, 2003-2006 and 2016 were considered), (2) causal factors of changes in the phenomenon under study are not evaluated and poorly discussed (e.g. climatic conditions, hunting pressure, farmland use), except for the area under grasslands. (3) the area under grasslands is analyzed as a dependent variable, and not as a causal factor. I acknowledge that data collection can be too expensive in economic and human effort terms, but alternatives exist to achieve to a more exhaustive analysis (e.g. by reducing the number of areas sampled but increasing temporal sampling, by exploring the land use or cover trends). I hope that my rejection decision does not discourage the authors since I found that they have many options to improve their findings and finally manage in a more efficient way these protected areas. 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: ANTIFIBROTIC EFFECT OF XANTHOHUMOL IN COMBINATION WITH PRAZIQUANTEL IS ASSOCIATED WITH ALTERED REDOX STATUS AND REDUCED IRON ACCUMULATION DURING LIVER FLUKE-ASSOCIATED CHOLANGIOCARCINOGENESIS Review round: 1 Reviewer: 1

Basic reporting: (i) Very unclear figures and its legends Quality of figures seems to be good, but it is unclear what should be focused. I recommend to use arrows and circles to point out important parts so that readers can focus. It is also unclear how many sections the authors analyzed to make their conclusion. If they prepared immune-stained sections from all the treated mice (5 mice in each group?), that should be described. (ii) No legends in supplementary figure. What do weigh 1~8 mean in body weight? Hamster No.5 seems to die 60 days after XON treatment. How did author measure liver weight per body weight of Hamster No. 5 at later stages? (iii) It is very confusing because figure numbers are sometimes missing in Result section. Readers will not understand which figures they need to see in each description. Experimental design: No treatment control (without Ov infection and NDMA) should be performed. If authors have done, the results should be shown or clearly described. Validity of the findings: (i) Quantitative and subsequent statistical analysis should be done to compare the difference between control and treated groups in immune-histochemical analysis. That will enable us to evaluate the effects of drugs more precisely. Especially, it is difficult to judge whether there is any synergistic inhibitory effect of XN and PZ treatment on periductal fibrosis development. Evaluation of fibrosis stages (describe in methods) is not reflected on any figures. (ii) XN and PZ treatment reduced several abnormalities, which are induced by Ov infection and NDMA treatment, such as DNA damage, CD44v8-10 expression, and fibrosis. Main concern is that only one of five hamsters in the ON group developed CCA. Although Ov-mediated ROS responses, inflammation and fibrosis result in CCA genesis, it seems very difficult to evaluate the effects of XN and PZ on CCA genesis under the condition used this study (just “suggest” will be better). In addition, it is not clear whether the provided figures in ON group are from CCA-developed hamster or from non-CCA hamster. (iii) Figure 7 is confusing. For example, XN suppresses periductal fibrosis through reduction of the oxidative stress responses or independent of them? If the former is the case, what does the line from XN to periductal fibrosis mean? 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: ANTIFIBROTIC EFFECT OF XANTHOHUMOL IN COMBINATION WITH PRAZIQUANTEL IS ASSOCIATED WITH ALTERED REDOX STATUS AND REDUCED IRON ACCUMULATION DURING LIVER FLUKE-ASSOCIATED CHOLANGIOCARCINOGENESIS Review round: 1 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: In this manuscript, Jamnongkan et al. described the effects of xanthohumol (XN) in combination with praziquantel (PZ) on various aspects of liver fluke (Ov)-associated cholangiocarcinogenesis, including DNA damage, redox status change and periductal fibrosis induced by Ov and NDMA administration in hamsters. The overall experiments seem technically sound. However, the interpretation of the results is hard to follow (particularly the data on immunohistochemistry) due to the lack of quantitative data on immunohistochemistry as well as the lack of experiments with control hamsters. A number of suggestions are as follows. 1) A major problem along the Results section is the lack of quantitative data on immunohistochemistry. The authors’ interpretation on the effects of XN treatment with or without PZ on histopathological findings, DNA damage, redox status, iron accumulation is largely based on their subjective impression, and no statistical analysis was performed to objectively evaluate the effectiveness of XN on liver fluke (Ov)-associated cholangiocarcinogenesis. They should classify the abnormal findings associated with (Ov)-associated cholangiocarcinogenesis, such as bile duct proliferation, hyperplasia, dysplasia of bile ducts, cholangio-fibrosis in each group during the progression of CCA (Figure 2). The graphs showing the number and/or levels of pathological abnormalities along time course should be included in the Figures. 2) In the same line with item 1, in order to validate their claim that either XN or PZ treatment could effectively reduce the accumulation of 8-oxodG, and that XNOP group could reduce DNA damage more effective than other groups (lines 253-255), the quantitative and the statistical analysis on the effect of XN with or without PZ on DNA damage should be performed. Also, the immunohistochemical data in Figure 4-6 requires quantitative measurements to validate their interpretations such as “no synergistic effect was observed in XNOP (line 265) or effective reduction in iron accumulation and TfR-1 expression for the XNOP as compared with other groups (lines 274-275). 3) In addition, the authors should include the data from untreated hamsters in Figures 1-6. Furthermore, the authors should indicate (by arrows or arrosheads) abnormalities such as the “white granules and small foci (line 229)” and “the obstruction of gall bladder” in the relevant Figures. 4) In the Introduction section, their description and the rationale on PZ treatment was insufficient. 5) I don’t’ understand the rational for measuring the CD44v8 to evaluate the redox status. Authors should describe the mechanisms as to how XN-induced alterations in oxidative stress would alter the expression of CD44v8-10. 6) In the Conclusion section, it is very vague whether their data indicate the effectiveness of sole XN treatment on Ov-associated CCA or their data indicate that the synergistic effect of XN in combination with PZ. These ambiguous conclusions could be derived from the lack of the quantification and rigorous statistical analysis. 7) There are numerous grammatical errors and typos. 8) Several papers are cited without being listed in the References, including Andriopoulos et al. (2007)

You are one of the reviewers, your task is to write a review for the article. You will 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: ANTIFIBROTIC EFFECT OF XANTHOHUMOL IN COMBINATION WITH PRAZIQUANTEL IS ASSOCIATED WITH ALTERED REDOX STATUS AND REDUCED IRON ACCUMULATION DURING LIVER FLUKE-ASSOCIATED CHOLANGIOCARCINOGENESIS Review round: 2 Reviewer: 1

Basic reporting: See general comments Experimental design: See general comments Validity of the findings: See general comments Additional comments: 1. In the first sentence of Results, "The liver per body weight of carcinogenesis in hamster groups either …." does not make sense. "The liver per body weight of hamster groups either …." will be better. 2. Body weight in supplementary figure is still confusing. If one column corresponds to the average of body weight from 5 mice, what does “Time 1-8” at each day (60, 90, 120, 180) mean? 3. Hamster No.5 seems to die 60 days after XON treatment. How did author measure liver weight per body weight of Hamster No. 5 at later stages? Authors’ response: We measured liver per BW in the hamsters that were alive at each time point. This is not the answer! You put “death” into a column of hamster No.5, XON groups in the liver per BW sheet. If the mouse died 60 days after the treatment, you can’t measure anything as in the case of ON groups. However, you put values in 90, 120, 180 days about the hamster. 4. Showing figures are not necessary. However, the authors should, at least, describe the results from untreated groups anywhere 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: ANTIFIBROTIC EFFECT OF XANTHOHUMOL IN COMBINATION WITH PRAZIQUANTEL IS ASSOCIATED WITH ALTERED REDOX STATUS AND REDUCED IRON ACCUMULATION DURING LIVER FLUKE-ASSOCIATED CHOLANGIOCARCINOGENESIS Review round: 2 Reviewer: 2

Basic reporting: In this manuscript, Jamnongkan et al. described the effects of xanthohumol (XN) in combination with praziquantel (PZ) on various aspects of liver fluke (Ov)-associated cholangiocarcinogenesis, including DNA damage, redox status change and periductal fibrosis induced by Ov and NDMA administration in hamsters. The overall experiments seem technically sound and their conclusions were well supported by various experiments with quantification analysis. Experimental design: The manuscript has been significantly improved by the revision, with additional data that strengthen the authors' model. Validity of the findings: All the concerns I raised about the previous version have been adequately. Additional comments: The revised manuscript is much improved and I have no further 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: MORPHOLOGY AND STRUCTURE OF HOMO ERECTUS HUMERI FROM ZHOUKOUDIAN, LOCALITY 1 Review round: 1 Reviewer: 1

Basic reporting: The following numbers refer to line number, followed by my comments. All are minor corrections. 55-58: I don’t think the catalogue of citations is necessary to support this point. Please pick the few (five at most) most relevant citations out of this list. 68-74: Very long sentence. 76: Grammar: Missing word – “broaden [the] current understanding” 78: Grammar: “humeri” not “humerus” 108: “seminal”—please replace with “initial” or some synonymous word. “Seminal” is gendered and archaic language. 121: Grammar: “modern-human like”, correct to “modern human-like” 134-136: A citation to support this statement would be appropriate. 276: Referencing format error: “…mobility)(Shang et al.,…)” ”…mobility; Shang et al.,…” 346: Referencing format error: (Weidenreich, 1941) not italicised 361: Referencing format error: (Weidenreich, 1941) not italicised 396: Missing word—“5% [of] length” 418: Missing terms: “identify AP and ML”  “identify anteroposterior (AP) and mediolateral (ML)”. As a matter of form, these should always be defined before they are abbreviated, despite being common terminology. 499: Syntax: the opening sentence alludes to the %CA of Humerus II being similar to something else, other than KNM-ER 1808. Is something else being compared to Humerus II, or should the sentence read: “The midshaft of Humerus II exhibits a high estimate of %CA, similar to the %CA of the KNM-ER 1808 cross section…”? 674: Language accuracy—Change “inferiorly-rotated and human-like scapula” to “human-like laterally-oriented scapular glenoid” Figs 3 & 4: Labels for the axes required, as well as a legend for the abbreviations in Fig. 3. Tables 1 & 2 should be swapped. The results for Humerus II are discussed before the results for Humerus III, but Table 1 refers to Humerus III. Please make Table 1 the results for Humerus II and Table 2 the results for Humerus III. Figure S4: Grammar—“proximalmost”  “proximal-most”. Please also label the unit of measurement, either in the figure itself or in the caption. Figure S5: Please include a legend for the abbreviations used in the figure. Experimental design: With regard to the methodology and experimental design, I have few criticisms; they are, for the most part, appropriate to the objectives of the study. I do, however, have a few comments on the comparative samples and would like some clarification on other points. The African Homo erectus sensu lato sample quite understandably consists only of KNM-ER 1808 which shows evidence of pathological apposition of bone on the long bones, though significantly without entheseal involvement. Initial assessments by Walker et al. (1982)attributed the pathology to hypervitiminosis A by way of the consumption of bee brood. Hypervitiminosis A, however, affects the entheses. Noting the lack of entheseal involvement, the pathology has more recently been attributed by Rothschild et al. (1995) to treponematosis in the form of yaws, which does not affect muscle attachment sites and causes extensive bone destruction. While Ruff (2008) used Leakey and Walker’s “corrected” cast of KNM-ER 1808 in his analyses and his values are used in the present paper, a justification of the use of this specimen would be appropriate at this juncture. If not a justification, then at least a discussion of the limitations and possible effects the pathology of the specimen may have on the results, especially as they could be profound. 278-280: “we report separate right and left humeral properties of Tianyuan 1 and emphasise same-side comparisons when possible” – why were left humeri not sampled for the recent modern Chinese samples, especially as you emphasise same-side comparisons? 339-356: This section is somewhat convoluted to read, so perhaps some time could be spent clarifying it. Did you create an artificial volume rending of Humerus II by rescaling a volume rendering of Humerus III? These two humeri differ quite notably in cortical thickness it seems, so was some warping/morphing conducted to match the published measurements or the radiograph? If so, this needs to be made more explicit. I understood all the steps independently, but the reasoning for doing it was not clear. Validity of the findings: The results appear to be of interest to the field and the findings have the potential to add much-needed information about temporal and geographic variations in humeral strength and rigidity properties in H. erectus sensu lato. I do have concerns that the pathological status of KNM-ER 1808 may be confounding the results, and the authors will need to address this. In lines 636—665, the authors’ discuss observed humeral robusticity, rigidity, and strength differences between African and East Asia hominin populations. The African H. erectus material, including KNM-ER 1808, has previously been attributed to a different species, Homo ergaster. They do not touch on it in the text, but it would be worth discussing the implications of a separate species attribution for the African material in relation to their findings. Additional comments: Xing et al. have conducted much-needed humeral diaphyseal robusticity and strength comparisons of material attributed to H. erectus sensu lato from Africa and East Asia. The article is well-written, barring the occasional grammatical error. The results, while interesting, must be tempered by acknowledgement of the pathological status of KNM-ER 1808 and the unclear impact this might have on the interpretation of their results. Greater clarity is also required in the methods section when explaining how the Humerus II medullary cavity estimates were arrived at considering the fossil itself was lost and only Weidenreich’s documentation exists. These and other concerns will need to be addressed before publication. This study comprises largely of direct comparison of values for populations without any statistical analysis. This is understandable due to the small sample sizes; however, Humerus II has been associated with a femur from Locality 1 and KNM-ER 1808 has associated femora. It would have been interesting to perform reduced major axis regressions (a la Ruff, 2009) with the African and East Asian samples to add some statistical power to the evaluations of relative strength proportions in the present 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: MORPHOLOGY AND STRUCTURE OF HOMO ERECTUS HUMERI FROM ZHOUKOUDIAN, LOCALITY 1 Review round: 1 Reviewer: 2

Basic reporting: The manuscript if very clearly written, in professional English, and easy to follow and understand. The background and context is adequate, and the tables and figures are professional. Experimental design: The research hypothesis is very clear. Due to the nature of the fossil samples, multiple levels of estimation were necessary in order to complete the analysis. The authors are quite clear about the process involved in these estimation, and provide details on exactly how the estimation was undertaken. Their commitment to total transparency in this is notable. While estimations were necessary, they explain them well, and I feel they are justified within the scope of the analysis. Validity of the findings: Conclusions are clear and robust, and based on the evidence. Additional comments: In general, this is a clear, well-written manuscript about an interesting and important topic. Little has been published explicitly quantifying levels of long bone robusticity prior to Neandertals, and this paper will expand this slowly growing body of literature. I recommend this manuscript for publication with only one small revision: Please mention somewhere how the KNM-ER 1808 cross-sections were corrected for the periosteal pathology on the long bones of this specimen. I realize this data was borrowed from Ruff, 2008, but I had to go look up that article to figure out whether or not this current analysis could possibly be affected by the documented pathology on this specimen. Just a sentence or two in the methods section would spare readers from having to look this up in the original 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: MORPHOLOGY AND STRUCTURE OF HOMO ERECTUS HUMERI FROM ZHOUKOUDIAN, LOCALITY 1 Review round: 1 Reviewer: 3

Basic reporting: In this manuscript, Xing et al provide an assessment of cortical robusticity (using uCT) from the East Asian humeri of H.erectus (HE) to compare with their African counterparts, and to provide the first anatomical description and cortical data on humerus III from Zhoukoudian Locality 1. They conclude that the humeri of East Asian HE are more robust than African HE, but tend to fall within the lower end of the range of standardized robusticity for more recent hominin samples from the same geographical area. The paper is well written, detailed, and technically competent. However, I found it to be overly long and descriptive, and had lost interest by the time I had begun reading the results. The material and conclusions could have been delivered in about 1/2 the space, without compromising the scientific integrity or relevant details. I would suggest severely reducing the length of the manuscript, even if there are no page limits in this journal, it is the readers that should be kept in mind. As an example of places that could be cut, the methods of dating are not germane to the study, only the dates. Readers can move onto the original material on this, on the environmental reconstructions, etc. if they choose to. The anatomical description of Humerus III was also overly detailed for this reviewer - perhaps some of this duplicates Woo and Chia (1954) as well. I would like to see them summarized in graphical format, with labels pointing out the most salient features on the excellent digital photographs in Figure 1. This would also benefit future users of the material for presentations, etc. I think it is also important for the authors to provide a clearer rationale for the study. Specifically, what new information is gained that was not available before in Weidenreich (1941) + Woo and Chia (1954)? It seems we might have arrived at the same conclusion from reading those two papers without the additional benefit of the uCT cortical data. Experimental design: Inasmuch as this is mostly an anatomical description of Humerus III and other East Asian HE humeri, the data reporting and experimental design are sound. I do have two comments, however: 1) I am unclear on why there is a section that reports on standardization by length only (L478-494), and one (more correct) on standardizing by the product of length and estimated body mass? It was not clear why the former was included, since body masses were estimated for the latter anyway? Perhaps I am missing something, but I could not determine the point of the length-only standardization, as all cortical variables were reported in the L*BM standardization as well in the relevant tables. 2) I would like to know why the standardization for length included up to three possible values (even though these varied only by 5.3%), while the mean body mass values were taken as a single value, for the standardization. The BM data, for example on KNM-ER 1808 varies by as much as 40kg. Using Grabowski et al's estimate of 39.5, rather than 60kg mean, would dramatically increase the robusticity of the African HE, putting it much closer to the East Asian hominins. Since much of the results depend on this estimate, it seems more prudent to include a range of values for BM as well as for HL. Validity of the findings: The findings are valid, to the extent that one agrees with the chosen body mass estimates of Tianyuan and KNM-ER 1808. For the former, I have a hard time seeing that this individual would be 85 kg with an inferred humeral length of 327mm. By my admittedly quick calculations, this individual would have had a stature around 170cm (Trotter and Gleser 1952, though there may be better ones out there now). This would give this individual a BMI of 29.4, very close to what would be considered obese by modern standards. This is not to say that such large individuals couldn't exist around 40kya, but I personally find this to be a stretch. While on this topic, can the authors speculate on the more than 25% difference in total cortical area between the left and right humeri of this individual? Seems extreme as a R-L asymmetry. For the latter, see my comments in point 2. I would like to see ranges of body mass estimates here to provide a proper comparison with the other samples. Additional comments: I have attached an annotated manuscript with some minor editorial suggestions in addition to the 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: MORPHOLOGY AND STRUCTURE OF HOMO ERECTUS HUMERI FROM ZHOUKOUDIAN, LOCALITY 1 Review round: 2 Reviewer: 1

Basic reporting: The authors have done an excellent job of addressing the concerns of all three reviewers, and I am satisfied with the response to my own comments. The manuscript reads much better after these edits. Experimental design: No further concerns, the authors have adequately justified the choice of methods, and the incorporation of multiple estimates of body mass for KNM-ER 1808 is appreciated. Validity of the findings: No further concerns Additional comments: Thank you for addressing these comments thoroughly, and for the opportunity to review this 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: COMINIGUT—A SMALL VOLUME IN VITRO COLON MODEL FOR THE SCREENING OF GUT MICROBIAL FERMENTATION PROCESSES Review round: 1 Reviewer: 1

Basic reporting: The paper is well writen and the English is, as far as I can evaluate, appropriate. Appropriate references have been used and the structure is clear. However, the article addresses basically two topics; the CoMiniGut and the in vitro fermentation of selected substrates. It would better to discuss these topics in more detail (in particular the CoMiniGut) separately Experimental design: The manuscript describes the targets and these are in scope with the Journal. The research question is well defined, but not fully achieved as far as the CoMiniGut is concerned. Methods are described in sufficient detail. Validity of the findings: The novelty is not as much as it could be. In the end, the simulations are pH-controlled faecal batch fermentations, which are assessed after 24 h. While this generates interesting preliminary data on what groups in the faecal microbiota are or are not able to grow in the presence of the tested HMOs, this could have been investigated in more depth as the authors actually suggest themselves in the discussion of the model. 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: COMINIGUT—A SMALL VOLUME IN VITRO COLON MODEL FOR THE SCREENING OF GUT MICROBIAL FERMENTATION PROCESSES Review round: 1 Reviewer: 2

Basic reporting: The manuscript of Weise et al develops a low volume batch system for modelling microbial changes in the gut. The systems low volumes lends itself to sparse compounds. This could therefore prove a useful model. I have a few associated questions for the authors. Line 115 - FOS often isolated from vegetable sources, rather than enzyme can extraction. Experimental design: I note that fecal stocks frozen in glycerol; has this been tested to see the loss in viability brought about by freezing? Were fresh samples run side by side? Were timings kept the same for infant and adult models? Literature suggests adult RT is longer than 24hrs. Were samples taken at the different times to echo the differences caused by changing the pH? Did this match what would be expected at the colonic regions? Which time-points chosen - could you have missed peaks in microbial growth? Validity of the findings: The points raised in 2 would impact on the validity of the findings. However, the authors have related the findings to literature Additional comments: No additional comments

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

Title: COMINIGUT—A SMALL VOLUME IN VITRO COLON MODEL FOR THE SCREENING OF GUT MICROBIAL FERMENTATION PROCESSES Review round: 2 Reviewer: 1

Basic reporting: The authors have answered the concerns I raised in a satisfactory way. However, I noted that in the description of the basal colon medium, no reference is made to Saulnier et al. 2008 eventhough the article is included in the references, it is not included in the text; I guess it should be mentioned in lines 186-187. Furthermore, I noted there are actually some words in UK English while the overall paper seems to be meant to be written in US English; e.g. 'harboured' (lines 432-434) Experimental design: No further comments. Validity of the findings: No further comments. Additional comments: No further 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 IMPORTANCE OF LOCAL SETTINGS: WITHIN-YEAR VARIABILITY IN SEAWATER TEMPERATURE AT SOUTH BAY, WESTERN ANTARCTIC PENINSULA Review round: 1 Reviewer: 1

Basic reporting: The paper is well written, clear in the aims, results and in the take home messages. The use of English is correct and the language intelligible. The MS structure conforms to what is expected by PeerJ and the kind of research performed well fall within the Journal aims. The most important ‘issue’ is that the results are based on local findings recorded in an area, i.e. the WAP, which is extensively studied by the scientific community in terms of climate change. Authors report for the first time (and as far as I know this is really the first time that such data are reported) that in shallow areas summer temperature increase already match levels that at the moment are only predicted to occur in the future. The article is logically and correctly based on this. However, I doubt that there are no other research programs in this area monitoring sweater temperature, at least in deeper waters (e.g. based on the use of oceanographic moorings, see for example here: http://www.soos.aq/activities/soos-at-sea/moorings) and these should be cited/commented. I would therefore improve the Introduction by placing their study in a wider international and geographical context, and by better explaining and characterising the uniqueness of this dataset (i.e.: “this is the first of ever record of shallow water temperatures recorded in the WAP showing this this and this” or “despite there are several research stations recording seawater temperature in the shallow in this area (say where these are, if any) this is the unique yearlong record available showing this increase”…or “this is so far the unique available temperature record showing this increase”). These sentences will help the reader in understanding the scale of their finding and, in general, will underline since the introduction the importance of studies at the local scale (not only the big picture is important!) as well as the desperate need of more observation in the shallow which are urgently needed. At the moment I consider the Introduction too brief (although correct) and I would suggest adding these details to complete the background information needed to properly introduce the topic. The title, despite being clear in reporting the topic of the paper, does not contain any indication that the focus is the Antarctic and, since the fact that “treatments might be just controls” has been documented only here, it would be more correct to adding this information in the title as well in order to give a complete overview of the topic since the beginning. Introduction, line 45. It is true that Antarctica has been considered one of the most stable environments on Earth but we are now well aware that major changes are occurring in some areas, such as the WAP, as well as that not all areas are warming up at the same speed. Some, such as the Ross Sea, are not warming up at all and show instead a reversed trend. Again, I would suggest completing this brief outline of the current Antarctic climatology taking into account (and referencing) this variability at the continental scale. This info will complete the findings reported in this paper and will underline even more the need to design site-specific thermal experiments in the future. Introduction, lines 62-63. This sentence is not clear: what do Author mean with “to examine water temperature directly from observations”? Here in situ instrumental records are reported, which are (for sure) not commonly available for most inshore Antarctic environments. On the other hand, in situ measures of temperature are currently and constantly registered by a number of moorings deployed at sea all year-round in off-shore areas (see above link to SOOS). If their intention is to compare the accuracy and reliability of in situ measures vs indirect ones, e.g. those that can be obtained from satellites or remote sensing, this concept has to be better explained. Another point is the general lack of data in form of long-term series…and this is true both for shallow and deep environments. It is reported in Discussion (line 143) that long-term observational data sets in the WAP are limited to a few areas, but Authors do not mention which are these areas and if these data sets are available. It is not therefore possible to properly verify the uniqueness of their dataset. The findings reported in this paper are about of two summers only and the real temperature trend (if any) is still unknown as well as the real interannual variability. This is implicit in the duration of their observation, but I think that other data about interannual variability for the WAP might be available. I would therefore invite Authors to comment about the possible causes of this variability and, again, if available, report about other projects from the same area. All the literature cited is relevant. Figure 1 is clear but I think it would be more useful if also bathymetric contours were included. I have no idea of the water circulation in the area but if any local pattern of is know (and relevant to explain any of the observed variability) This should be commented as well. Figure 2 is not really necessary but, if considered relevant, add scale bar on Fig. 2B and depth information for both figures in the legend. Figure 4 shows a series of ‘outliers’ for the station PY2 at -20 m (red dots) at the beginning of February. I have found no comments on this in the MS. Any hypothesis? It is possible that these data are due to instrumental failures or can be interpreted with a temporary introgression of colder waters? Does the general picture change if removed? Please comment on these records. R scripts, raw data and input files have all been provided by the Authors and the scripts run. There is just a mismatch in the names of files compared to that given in the scripts (files now start with the “peerj-19157-“) but this is not an issue to run the scripts. However, in the final version of the paper this difference should be avoided. Figures do not appear to have been altered in any way. Experimental design: The registered temperatures have been acquired to answer to a different research question (i.e. which are the responses of Antarctic sponge assemblages to climate change) from the one reported in the paper. Despite this, this is the first time to my knowledge that this topic is tackled in such a clear way and the finding that temperatures expected to occur in the next future are already met is of relevance to a variety of studies. For your info only: there is a specific SCAR group (ANTOS), which will provide protocols for monitoring nearshore environments and, hopefully, in the next future there will be a coordinated network of monitoring stations from which year-round temperature data could be obtained. The analyses of temperature trends are all necessary, well done and effective in supporting the findings. The experimental design is very simple and based on the use of HOBO data loggers at three different sites. Despite these loggers are of common use there may be differences in models and sensitivity. Please report manufacturer nominal data about sensitivity and specify the model used in order to allow comparison with other similar data in the future. M&M line 77. Selected sites cannot be separated by 1.5 meters only. Please correct and report max and min distance between these sites. A scale on Fig. 1 would also help in figuring out the distance between these sites. Validity of the findings: The data produced in this contribution are of relevance for the whole scientific community working on the thermal tolerance of Antarctic fauna as they indicate that the design of such experiments has to be carefully reconsidered and adapted to local conditions and temperature trend (of which a good knowledge is now required in advance!). Since the WAP and the Ross Sea, for example, do show opposite temperature trends, with the latter not warming up at all, temperature experiments even on the same species would have to be modified and adapted in order to expose the specimens to temperature that are outside the range of those reached locally. In short, this means that temperature ranges cannot be ‘generalized’ in a ‘one size fits all fashion’ and that the same species might have populations, which have different thermal tolerances. All these facts increase the complexity of thermal experiments, which will have, from now on, to take into account this ‘newly observed’ variability This introduces a higher level of complexity in studies dealing with thermal tolerance and will represent a challenge for researchers. Given this, I think that this paper is really important. I would suggest to mention the paper by Ashton et al., 2017 (Current Biology 27, 2698–2705, http://dx.doi.org/10.1016/j.cub.2017.07.048) where ‘dramatic’ changes at the population and assemblage levels were found on settlement panels constantly heated just at 1 to 2 °C above the surrounding seawater temperature. In their experiment the increased temperature has been maintained for about one year, i.e. animals were exposed to higher temperature for a longer time. These two experiments, i.e. Ashton et al., 2017 and the data here presented, both cast new light on this topic and increase our understanding of the effect of climate changes and the need of carefully design thermal experiments. 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 IMPORTANCE OF LOCAL SETTINGS: WITHIN-YEAR VARIABILITY IN SEAWATER TEMPERATURE AT SOUTH BAY, WESTERN ANTARCTIC PENINSULA Review round: 1 Reviewer: 2

Basic reporting: The manuscript requires more editing. I started to make notes, but pretty quickly gave up as the amount of grammatical issues appeared too large to be handled in a review. Instead, I suggest the authors hire an editor or have a native speaker edited the manuscript. The figures appear well developed, data are shared. Article structure is ok, but the article does not test any specific hypotheses. Experimental design: The study does not test any specific hypotheses. It simply summarizes data from three sites (different location and depth). The site selection does not have any apparent logic (or is related to a hypothesis) and locations are not sufficiently described, i.e., there is no reason to assume that site conditions are comparable, what specific inference scope the sites represent, or what factors are responsible for differences among sites. Statistical tests indicate whether temperature is constant throughout various times of year, but the results of significant tests are not described in the text. Thus, the results only show that temperatures vary with seasons. Validity of the findings: A basic assumption appears to be that quantifying the speed of temperature change within the year will provide information about the ecosystem responses to slower temperature changes due to climate change. The authors provide no convincing evidence that this is the case and it appears to me to be unlikely (note that I am not a marine biologist). Instead, it is more likely that organism have adapted to the annual temperature patterns and consequently critical temperature values will vary throughout the year, making the basic assumption invalid. Similarly, the data provide no information that changes in temperature (warming rates) are increasing, making a discussion about “heat shock” pure speculation (lines 161+). Why is the difference in warming rate before and after October relevant, is the difference even significant? In the same context, the data provide no information which site conditions are indicative of higher and lower environmental variability (lines 175+). Additional comments: The general argument that experimental conditions should reflect natural trends makes sense, but is not new. The data at this stage are not sufficient to support the conclusion of the study. A better description of the sites and longer measurement period may be necessary. Alternatively, the data can provide a baseline for comparison with future measurements.

You are one of the reviewers, your task is to write a review for the article. You will 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 IMPORTANCE OF LOCAL SETTINGS: WITHIN-YEAR VARIABILITY IN SEAWATER TEMPERATURE AT SOUTH BAY, WESTERN ANTARCTIC PENINSULA Review round: 1 Reviewer: 3

Basic reporting: The authors clearly and professionally report their methods and findings in the present manuscript in which they report the temperature variation at three sites around Dourmer Island, Palmer Archipelago, WAP over the course of one year to better understand local variation and inform experimental set point in climate change experiments. The authors present multiple figures describing the temperature dynamics, however I think that some of the figures depicting the raw data measurements might be better suited to be included as supplementary material. It seems like the same data are depicted in multiple graphs and it is unclear to me as written, what the added value is for including each of these figures. Perhaps more detailed description and interpretation would help put these different figures into context. Otherwise, maybe consider including some of these figures as supplementary material. The map (Figure 1) is very helpful in putting the present study site into geographical context. The premise that temperature treatments should be put into a local context of exposure is extremely important. However, I think that the relatively narrow focused area observed somewhat geographically limit how applicable these results will be to other studies outside of this geographic region. Maybe some additional information on percent and duration of sea ice cover might help other researchers better gauge how applicable the results in the present study are their region of interest. They cite Schram et al. 2015, who appears to have data mined the Palmer LTER site for additional sea surface temperatures (range similar to that reported here). Perhaps the inclusion of this type of freely available data could add some weight to the data presented here? Experimental design: The research presented is within the scope of the journal and their research question is well defined, relevant and meaningful – particularly in light that it appears that not all studies take local conditions into account when setting experimental levels for climate change experiments. Validity of the findings: While I agree that it important know something about the local exposure of your experimental organisms, this information is not always available and therefore I would recommend caution when discussing the temperature levels used in previous experiments. I also believe that the authors could perhaps include some additional discussion on why studies that expose organisms to different temperatures are/are not still informative in the light that organisms may briefly experience those temperatures. The authors point out that for their one year of data, organisms were exposed to “temperatures of more than 2.5C for more than 10 days within a year and over 2.0C for more than 25 days, temperatures that have been used experimentally to test responses to acute heat stress” (lines 124-126). I am not convinced that this is sufficient exposure to call these temperatures a control temperature. These elevated temperature days do not appear to have all occurred within the same week, therefore this may still be at the extreme temperature range of what organisms experience. Perhaps some additional justification is needed here? As it stands, I do not agree that the data presented for this one year study is sufficient to make the case that all Antarctic organisms experience these temperatures often enough to call this another control temperature. Additional comments: When listing citations, if not an exhaustive list, perhaps precluding the list with"e.g."

You are one of the reviewers, your task is to write a review for the article. You will 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 FRENCH PRESS: A REPEATABLE AND HIGH-THROUGHPUT APPROACH TO EXERCISING ZEBRAFISH (DANIO RERIO) Review round: 1 Reviewer: 1

Basic reporting: The study proposes in a clear and a well-grounded way a novel, cost-effective and high-throughput method for exercising zebrafish, using a coffee plunger and magnetic stirrer. The introduction has a good scientific background and makes the study’s aim clear, in addition to being well-contextualized regarding the literature. The study was developed and discussed so that it answers the main questions that were proposed. The structure of the text and the format of the figures are in accordance with this journal’s norms and the authors supplied enough data to understand the results analysis. There are, however, some minor shortcomings as follows: Experimental design: a. Bearing in mind that this study aims to standardize a new technique using animals and this kind of study is very sensitive to various interfering factors, it is paramount to add a “Zebrafish maintenance” paragraph in the “Materials and Methods (line 70)” section, detailing: the strain, age and nourishment of the zebrafish; the water control parameters; light/dark cycle. It is also important to add the ethics committee approval for the use of these animals in the experiments. b. Please include the time of the day in which the “Maximal swim performance (line 90)” and “Exercise training (line 101)” were conducted. c. Please add the French Press brand used in the tests (line 72). d. In the “Maximum swim performance” section (line 90), what was the method of weighing, sexing and measurement of the animals? e. In the “Exercise training” section (line 101), how long after the “maximal swim performance” test were the animals taken to the following “pre-intervention maximum swimming speeds” test (hours, days etc.)? f. The authors should provide a better explanation on how the effects of exercise training were assessed (line 149-151), at the end of the 5 days of the swimming regime at a sub-maximal exercise speed, to evaluate the difference in relation to the control group. g. As the study has different stages, the methodology would be clearer if a figure were added with a timeline to better express the events, at the authors’ discretion. Validity of the findings: a. Both the statistical analysis and graphs seem adequate for this type of study. b. In the results section is described “Maximum swimming speed significantly increased in the exercise-trained, but was unchanged in the control group (lines 149-150)”. It would be interesting to include a graph of these data. c. Although the authors wrote a conclusion at the end of the discussion (lines 201-203), it would be appropriate to emphasize the conclusion, by writing a conclusion section in a new paragraph. Additional comments: Taking into consideration that this study proposes an innovative method that could be used in different approaches, I consider this article relevant to be published after improvements are made.

You are one of the reviewers, your task is to write a review for the article. You will 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 FRENCH PRESS: A REPEATABLE AND HIGH-THROUGHPUT APPROACH TO EXERCISING ZEBRAFISH (DANIO RERIO) Review round: 1 Reviewer: 2

Basic reporting: The manuscript describes improvements to existing exercise protocols for zebrafish and proceeds to investigate the repeatability of the measurement approach. The manuscript also reports to investigate improvements in maximum speed following exercise training, mass increases following training, and the effects of sex and length on the results. Most of the results are missing from the manuscript and those that are present are largely reported in supplementary tables. 1) In order to address this the results relevant to each of the questions listed above need to be included in the main text. 2) Data investigating the similarities between different plungers can be moved to the supplementary data Experimental design: In the methods it is reported that 40 fish were measured. 1) Given that multiple questions were asked this should be further explained to give the n number for each experiment. For Example to look at the effects of sex and exercise there was only 10 fish in each group. 2) A single experiment with n=10 was carried out to address the questions raised. It is not clear if this is sufficient power to detect the effects proposed. The authors need to justify the size of the experimental group based on power analysis to indicate the level of change in swimming speed, mass etc, they are able to detect If the number of fish is insufficient the experiments should be repeated with greater numbers to support the findings. 3) The repeatability was assessed by using measurements pre and post-exercise training. It is not possible to use the same data to assess repeatability as the training could induce a difference. Repeatability should only utilise the unexercised fish. 4) For the analysis of water speed and stirrer speed using different French press units, 2 units is not sufficient to say there is no variation between units, and additional measurements are required. Standard practice would suggest a minimum of 3 but given that the authors have access to many more they are encouraged to increase the number significantly. 5) The make and model of the coffee plunger used should be supplied in the methods section. Validity of the findings: The authors introduce a useful advance on the previous exercise approaches for zebrafish. The conclusions regarding the effects of exercise however, are questionable given the low number of fish utilised and the lack of experimental repetition. It will be vital to demonstrate that the experiments were sufficiently powered to address the questions raised to demonstrate the validity of the findings. 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 FRENCH PRESS: A REPEATABLE AND HIGH-THROUGHPUT APPROACH TO EXERCISING ZEBRAFISH (DANIO RERIO) Review round: 2 Reviewer: 1

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: No comment. Additional comments: Thank you for making the proposed changes. I consider that your article is now much more clear and elucidative. Therefore, the article 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: THE FRENCH PRESS: A REPEATABLE AND HIGH-THROUGHPUT APPROACH TO EXERCISING ZEBRAFISH (DANIO RERIO) Review round: 2 Reviewer: 2

Basic reporting: The new figure (Fig2) presenting the change in swim speed with exercise is a very useful addition. However, measurements from individual fish should be presented to allow visualisation of the variation, which is currently hidden by only presenting the estimate from the mixed model? (presumed, not given in figure legend) and standard error bars rather than standard deviation or confidence intervals. Experimental design: The reviewers have made some changes as a response to the previous review but some key concerns remain to be addressed. The clarification of n numbers and variables in the mixed model is an improvement. However, the response to the question of sample size has not addressed the concern. The authors are correct to suggest that post-hoc power analysis are not ideal, but it is still critical to ensure that sufficient animals have been examined. The authors themselves have previously published on the important of prospective power analysis, and if they are not willing to conduct a post-hoc analyses then details of the prospective power that determined that n=20 would be sufficient. The authors also state that the sample size was sufficient to detect an improvement in swimming speed in the experimental group. This does not indicate that sufficient numbers were present in the case of sex, length, or mass where the effect may be less. Furthermore, for the effect of sex on exercise response there were only 10 exercised males and 10 exercised females (n=10). Identifying issues with post-hoc analyses is not sufficient address the concern that the number of animals in this study is probably too low to identify effects which must be addressed. The lack of experimental repetition has also not been addressed and there is no indication in the methods that experimenters were blinded to treatment group. Validity of the findings: Given the lack of justification of the number of animals examined, lack of experimental repetition, and the fact that observers were not blinded to treatment group there are still concerns over the validity of findings. Additional comments: Overall this is an interesting method that is of interest to the field but the examination of the effect of exercise on maximal swimming speed is not conducted with sufficient numbers and experimental repetition to address the questions raised.

You are one of the reviewers, your task is to write a review for the article. You will 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: ALPHA-ACTININ OF THE CHLORARCHINIOPHYTE BIGELOWIELLA NATANS Review round: 1 Reviewer: 1

Basic reporting: In this manuscript, Backman follows up his previous work on microbial alpha-actinin. This paper analyses the first example of an alpha-actinin from a photosynthetic organism that I'm aware of. The aim of the paper is well stated and generally the manuscript is written in a clear and engaging manner. The manuscript is well structured and well presented. In terms of reporting the current data, I have some points for the author. 1.1 It would be helpful to include a table of Pfam and Smart E-values for the domain assignments alongside the Superfamily data already given in the text. The spectrin triple helix in the rod domain is clearly very divergent, as the author indicates. It is not recognised by Pfam or Smart as far as I can tell, and this is a worthwhile point to bring out. 1.2 On the same issue, I think it would be appropriate to put in a sequence alignment of the triple helix in the rod domain with those of Entamoeba histolytica, and S.pombe alpha-actinin, and representative repeats of one (or a few) four-repeat actinins. 1.3 Am I right in thinking that this is the first alpha-actinin that has just one spectrin repeat to be experimentally characterized? If so, I think this should be brought out more clearly in the text. This is particularly important in terms of the mechanism of dimerisation. In other alpha-actinins, dimerisation occurs by interaction of R1 with R2 or R4 (depending on the number of repeats). This alpha-actinin presumably dimerises by homotypic interactions of a pair of R1-equivalents. This should be stated clearly, and used to qualify the abstract (line 29:"all common characteristics of a typical alpha-actinin). 1.4. It would be helpful to include a diagram to indicate where Bigelowiella sits in eukaryotic phylogeny, and how its structure relates to other alpha-actinins. Experimental design: The experiments are designed generally very well and reported in a way that would enable reproduction by another worker. 2.1. It would be helpful to explain a little further how the assignments for the beginning and end of each domain construct were made. I assume these were essentially from the superfamily assignments? The construction of the rod domain is reasonable, but I wondered if the author attempted to make other constructs of the triple helix that would be soluble? It's generally the case that triple helical repeats can be prepared in soluble form providing the phasing of the repeat is correct and there is a minimal number of residues at the end of the construct. In that sense I am not surprised that the rod domain construct was insoluble. Major point 2.2. It is characteristic of alpha-actinins that they dimerise. The author correctly states that a dimeric alpha-actinin should bundle actin filaments, and this is indeed observed. But it does not prove that this alpha-actinin is a dimer. In order to substantiate the author's contention that this alpha-actinin is indeed a dimer (e.g. line 26 - "probably forms anti-parallel dimers"), I think it is important that this should be directly investigated. The gel filtration data in figure 2 indicates that unaggregated alpha-actinin can be obtained from the column. In my view, the author should analyse this for the oligomeric state of the protein. There are numerous biophysical methods that could be used for this, or alternatively chemical cross-linking and gel electrophoresis. In this context, given that the alpha-actinin has a tendency to aggregate, the results shown in figure 6 and figure 7 for bundling actin could result from aggregation of the protein rather than specific dimerisation. The author is correct to point to the tendency of the full length alpha-actinin to aggregate in the conditions used for the bundling assays, therefore the dimerisation is not proven. Validity of the findings: Subject to my concerns in point 2.2, the data seem entirely valid. The protein clearly has functional actin-binding and Ca2+-binidng domains, characteristic of alpha-actinins. The structural modelling of the domains seems good (and for my own interst, I obtained the same results using Phyre). Additional comments: Overall, I think this is a generally very helpful study of a microbial alpha-actinin from a novel class of organism. The author is to be commended on the clarity of the paper and the direct and straightforward experimental design. However, I think the author should directly address my point 2.2 before this 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: ALPHA-ACTININ OF THE CHLORARCHINIOPHYTE BIGELOWIELLA NATANS Review round: 1 Reviewer: 2

Basic reporting: With the exception of a few minor errors the article is well written and referenced and the data are appropriately presented. Experimental design: The research question is well defined and the experimental design is appropriate and well documented. Validity of the findings: The number of times that each experiment was performed needs to be stated in each figure legend. This is important so that the reader can easily assess the reproducibility of the data. Statistical analysis has been performed where appropriate. Additional comments: The manuscript represents a thorough analysis of the a-actinin like protein from Bigelowiella natans. Given the interesting phylogeny of this organism, this analysis has some significance in terms of understanding the evolution of the actinin family of actin cross linking proteins. Overall the experiments are executed carefully and interpreted appropriately. I would recommend publication upon completion of the following minor revisions. Line 76 typographical error: isolatiOn Line124/125 reference needed for sentence ending …. as before. Line 199/200/201 This sentence is repeated in Line 212/213 – it seems a bit redundant to describe this in both places. Line 219 change reliable to reliably. Line 333 change a-actinin to alpha-actinin Legend for figure 2: Use rcf rather than rpm when describing centrifugation conditions Each figure legend containing experimental data should state the number of times that the experiment was performed. For example: “Data shown is representative of 3 independent experiments.” This is important so that the reader can easily assess the reproducibility of the data. Figure 6: It would be useful to present the quantification of the co-sedimentation data. For example, plotting the proportion of actin in the pellet against actinin concentration in a line graph.

You are one of the reviewers, your task is to write a review for the article. You will 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: ALPHA-ACTININ OF THE CHLORARCHINIOPHYTE BIGELOWIELLA NATANS Review round: 2 Reviewer: 1

Basic reporting: OK Experimental design: OK Validity of the findings: OK Additional comments: The issues raised in my review of the manuscript have been adequately addressed in the revised version.

You are one of the reviewers, your task is to write a review for the article. You will 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: SOCIAL NETWORK COMMUNITY STRUCTURE AND THE CONTACT-MEDIATED SHARING OF COMMENSAL E. COLI AMONG CAPTIVE RHESUS MACAQUES (MACACA MULATTA) Review round: 1 Reviewer: 1

Basic reporting: The study investigates patterns of commensal E. coli strain sharing in relation to individual, social and community characteristics in 3 captive-housed group of Rhesus macaques, a social non-human primate. Commensal E. coli genetic diversity in subtypes or strains is extracted from feacal swabs and obtained through multiple cultures and DNA fingerprinting procedure. Social networks are built from observations of behavioural interactions during 6 weeks. The study concludes at a significant effect of group membership and of social community (based on social network clustering analysis) on the % of similarity between E. coli subtypes, and a lack of a significant effect of age, sex, matriline membership and dyadic interaction frequencies. Overall, the study is well-designed and has the potential to be of major significance for animal epidemiology. (There is always a) But the writing is quite verbose; the introduction and discussion are a bit long-winded; and there are many repetitions throughout the text (and quite some typos). This makes for a lengthy read where the reader loses focus rapidly. I also find that the introduction could be punchier by delving right away into epidemiology, transmission, infection, and linked social processes, instead of too much advocating for social network analysis tools and describing what others did or did not do. An on-point straightforward presentation of the state-of-the-art and objectives of the study would be more interesting. This is also the case for the discussion. Specifically, I don’t think that nowadays we can say that “conventional epidemiological assessments of infectious agent acquisition often assume, or treat the probability of contact for every pair of individuals in a population as equal” lines 71-72 so the authors might want to jump right in the nowadays state-of-the-art and rather insist on the level of resolution microbial genetics offer for tackling our questions. Paragraph starting line 98: the authors might also want to mention that E. coli expresses enough genetic diversity to be able to build individual host genetic profile with enough between individual variability (see Craft, M. E. (2015). Infectious disease transmission and contact networks in wildlife and livestock. Philosophical Transactions of the Royal Society of London, Biological Sciences, 370(1669), 1–12. http://doi.org/10.1098/rstb.2014.0107). Furthermore, there are some decisions made by the authors with regards to thinking or methodology that only appear clear in the discussion whereas they would have their space in the introduction or methods already (for instance, lines 480-484 could support the assumption that environmental contamination is low and so any sharing might be attributed to x,y,z instead, either in the intro or method; or lines 544-550 about group III social instability). I am also missing a short paragraph about the significance of the study not only in terms of epidemiology and captive population health management but also on more fundamental questions such as social processes and evolution or host-parasite interaction and evolution. This would give a bit more meat to the article. Finally, in general, the study is well referenced but some important ones are missing, for instance see above Craft 2015 as well as other reviews more accessible than a book chapter like: Godfrey, S. S. (2013). Networks and the ecology of parasite transmission: A framework for wildlife parasitology. International Journal for Parasitology. Parasites and Wildlife, 2, 235–245. http://doi.org/10.1016/j.ijppaw.2013.09.001 Rushmore, J., Bisanzio, D., & Gillespie, T. R. (2017). Making New Connections: Insights from Primate–Parasite Networks. Trends in Parasitology, xx, 1–14. http://doi.org/10.1016/j.pt.2017.01.013 Schmid-Hempel, P. (2017). Parasites and their social hosts. Trends in Parasitology, 33(6), 453–466. http://doi.org/10.1016/j.pt.2017.01.003 Vanderwaal, K. L., & Ezenwa, V. O. (2016). Heterogeneity in pathogen transmission: mechanisms and methodology. Functional Ecology, 30, 1606–1622. http://doi.org/10.1111/1365-2435.12645 Experimental design: The experimental design is very good and it is fantastic to be able to have such high resolution data. The authors give all the material for one to reproduce their results if one fancies. However, I feel that there are some missing bits and pieces in the analysis. Although the authors argue against not including those bits and pieces, I feel they would complete the story. One concerns including proximity or some kind of space sharing/use network as well as purely contact networks. I know that proximity and grooming are very often well-correlated but although the authors state several times that conditions (sunny and dry) are unfavorable for E. coli survival in the environment and that they did not find E.coli is tap water, they also state that E.coli can still survive up to 25 weeks. The fact that the monkeys are “forced” into proximity, a fact that the authors also highlight in the discussion, might indeed “expos[e] individuals more to environmentally deposited feces, [and] also increases the likelihood of social contact among animals that share the same space” lines 588-589. At least, I missed some more explicit discussion of this point. A second one relates to the AMOVA. I understand the logic of conducting hierarchical modelling when one attempts to explain a pattern or phenomenon without assumptions or clear predictions. But here, 1/ there are clear predictions, and 2/ we know that factors influence each other, such that age and sex similarities and kinship influences grooming frequencies for instance which in turn could influence genetic similarity %. I am just wondering whether building a more integrative model with all factors at once would be possible? The separate testing of the effect of group membership, kinship, dyadic frequencies of interactions and individual characteristics leaves a feeling of incompleteness somehow. I am not sure but maybe a more classic generalized linear mixed model (with permutations) or Bayesian regression modelling may be able to sort out the variance linked to each factor at once? Validity of the findings: Pending some methodological precisions and discussions, I think the findings will be robust and conclusions well linked to the questions. As said before, I also find the discussion in need of more straightforwardness. For instance, the authors spend 16 lines summarizing their results, lines 443-459, then another 6 lines shortly after, lines 464-470, repeating parts of those results before entering the actual discussion. And of a broader picture viewing as well. Additional comments: No additional comments

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

Title: SOCIAL NETWORK COMMUNITY STRUCTURE AND THE CONTACT-MEDIATED SHARING OF COMMENSAL E. COLI AMONG CAPTIVE RHESUS MACAQUES (MACACA MULATTA) Review round: 1 Reviewer: 2

Basic reporting: overall great! Although there is a lot of unnecessary pointing (e.g. see below). See general comments to the authors. Experimental design: Excellent. The only issue is the fact that a single isolate was collected from an individual requiring cation in the interpretation of results. Some statistical tests are inappropriate for the kind of data. Again look at general comments to the author. Validity of the findings: All findings are valid. However, caution is required in the interpretation on discordant findings. See general comments to the authors below. Additional comments: The manuscript on social community structure and contact mediated sharing of E. coli in captive maca presents data indicating the value of social interactions as a proxy for inference of the transmission of infectious agents in group living organisms. The manuscript is worth publication in PEERJ after these issues have been addressed. 1. Some statistical tests are inappropriate and needs redoing. Kolmogorov-Smirnov tests are compare the similarity in distributions and are not appropriate for comparing differences in percent similarity in E. coli between and within social clusters. A permutation / randomization test whereby percent E. coli similarity observed within DCG communities are compared with similarity obtained after shuffling individual across DCG and calculating the percent similarity between random pairs to generate the expected sharing assuming there is no influence of DCG on percent E. coli similarity among dyads. 2. The authors make a claim that social processes are important in E. coli sharing BUT do not fully and comprehensively address the issues of non-concordant results between dyadic and group level social processes in the discussion or methods section of the manuscript. Because a single individual animal can have as many as 13 E. coli strains (see refs 1-3 below). Typing a single strain per individual can bring about anomalies in results observed in this paper such as similarity at the group level or some aggregated level such as behavioral DCGs but no signal is detectable at the dyadic level. To obtain a detectable signal of dyadic interactions a single clone in a species where an individual may have several clones co-occurring will require repeated temporal sampling as the authors allude or intensive sampling more than four or more isolates to capture the full diversity within individuals and the latter is more powerful for E coli sharing and the former for tracking transmission. This should come out clearly in the discussion of discrepancy of results 3. To make a solid claim on social processes in E. coli sharing, the authors need to run their data using hierarchical analysis of molecular variance (AMOVA) incorporating group and behavioral DCG social as to partial variance between group level effects which are environmental and those that are social (behavioral DCG) 4. Specific comments. Please see attached copy with comments Methods: Non parametric tests do not solve issues of interdependence but issues non-normality issues in data. You need tests that take into account such dependence. References 1. Ahmed S, Olsen JE, Herrero-Fresno A (2017) The genetic diversity of commensal Escherichia coli strains isolated from non-antimicrobial treated pigs varies according to age group. PLOS ONE 12(5): e0178623.https://doi.org/10.1371/journal.pone.0178623 2. Anderson, M.A., Whitlock, J.E. and Harwood, V.J., (2006). Diversity and distribution of Escherichia coli genotypes and antibiotic resistance phenotypes in feces of humans, cattle, and horses. Applied and Environmental Microbiology, 72(11), pp.6914-6922. 3. Bok, E., Mazurek, J., Pusz, P.A.W.E.Ł., Stosik, M.I.C.H.A.Ł. and Baldy-Chudzik, K., (2013). Age as a factor influencing diversity of commensal E. coli microflora in pigs. Pol J Microbiol, 62(2), pp.165-71.

You are one of the reviewers, your task is to write a review for the article. You will 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: SOCIAL NETWORK COMMUNITY STRUCTURE AND THE CONTACT-MEDIATED SHARING OF COMMENSAL E. COLI AMONG CAPTIVE RHESUS MACAQUES (MACACA MULATTA) Review round: 1 Reviewer: 3

Basic reporting: No comment. Experimental design: See general comments below. Validity of the findings: See comments below. Additional comments: This paper tests social predictors of E. coli strain sharing in three groups of rhesus macaques. It adds to the growing literature on the consequences of social interactions on microbial sharing/transmission, and is distinguished by a relatively large sample size and degree of environmental control. The main finding is that higher level social interactions, captured through “clusters” of socially interacting individuals, appear to better predict E. coli strain sharing than dyadic interactions. This result is likely to suggest new analysis approaches to researchers interested in this topic. I have a number of suggestions that I hope will be of use in any revision process. Major: 1) The finding that DCG clusters are a better predictor of strain sharing than dyadic interactions is interesting, but it is unclear whether it arises as a consequence of biology versus technical artifact. With limited behavioral sampling (6 weeks per social group, mostly collected through scan sampling), dyadic estimates of grooming, huddling, and aggression rates may simply be less accurate than cluster/group-level membership. Can you show (e.g., using subsampling) that the amount of observational data collected produces asymptotically stable estimates for the three types of dyadic interactions you consider, compared to inference of cluster membership in the DCG approach? Are dyadic grooming, huddling, and aggression correlated? (If so, would combining these measures into some sort of sociality index produce a more stable measure of contact?) 2) K-S tests are used to compare E. coli similarity across communities. K-S tests are quite sensitive, since they test for differences in the cdf of two distributions, not differences in means—which I think is what you are most interested in knowing. If a non-parametric test is needed, why not use a Wilcoxon rank-sum test instead? Also, I could not tell if these tests were one-tailed (as suggested by the motivating hypothesis) or two-tailed. 3) Estimates of kinship are based on either matriline membership or a coarse binarization into close versus distant kin. How is a “matriline” defined? (how many generations back does it go?) Why binarize estimates of kinship? In the current scheme, distant cousins are treated as equivalent to full sibs; this approach seems less likely to detect effects of relatedness on strain-sharing, if they exist, than using pedigree or genetic marker-based point estimates. 4) Can you explain what DCG levels “mean” from a biological perspective? Do they tend to capture kin groups or sets of animals with close social bonds (c.f. Silk?) Minor -A single E. coli strain was isolated from each animal. Did you confirm that animals only carry a single E. coli strain? If they do carry multiple strains, can you explain how it might affect your results? -line 27/28, 559, 563: “in the absence of phenomena like social buffering” What phenomenon are you referring to, and what’s its relevance to microbial sharing? -line 132: “largely, these studies have defined…pairs of individuals from whom identical E. coli subtypes were isolated” Tung et al (and also other studies, e.g., Moeller et al in wild chimpanzees) actually use community composition (beta diversity) instead, or in addition to, sharing of individual OTUs. These studies have not analyzed E. coli isolates. -The sample appears to contain adults only. Any ramifications of leaving out infants and juveniles as potential links in the social networks and/or routes of transmission? -line 414: “dyads with an E. coli similarity >70%”—does that mean 70% of bands were shared, or 70% of 0/1 values for bands? What does it mean that this threshold is a “reliable indicator of pulsotype cluster membership and similarity?” -Figure 2: I would find it helpful to see E. coli similarity levels for between group pairs to place the within group pair results in context

You are one of the reviewers, your task is to write a review for the article. You will 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: SOCIAL NETWORK COMMUNITY STRUCTURE AND THE CONTACT-MEDIATED SHARING OF COMMENSAL E. COLI AMONG CAPTIVE RHESUS MACAQUES (MACACA MULATTA) Review round: 2 Reviewer: 1

Basic reporting: No further comment Experimental design: No further comment Validity of the findings: No further comment Additional comments: Great study. No further 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: SOCIAL NETWORK COMMUNITY STRUCTURE AND THE CONTACT-MEDIATED SHARING OF COMMENSAL E. COLI AMONG CAPTIVE RHESUS MACAQUES (MACACA MULATTA) Review round: 2 Reviewer: 2

Basic reporting: clear and unambiguous Experimental design: Research questions are well defined Validity of the findings: Statistical analysis is robust and the authors addressed the comments from previous revisions 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: EXPRESSION STATUS AND CLINICAL SIGNIFICANCE OF LNCRNA APPAT IN THE PROGRESSION OF ATHEROSCLEROSIS Review round: 1 Reviewer: 1

Basic reporting: Meng et al have conducted a study of APPAT lncRNA and its use as a biomarker in circulating blood samples of angina and MI patients. The study seems to have an impressive depth and is overall well-conducted. However it suffers from lack of structure and stringency in reporting and could be much improved. While the English language itself is reasonable, I believe that the manuscript would benefit a lot from a work-through by a key-author, at each section asking if 1) the key points are clearly communicated, and 2) if superfluous information not necessary for key points could be removed. I have tried to give some examples below: 1. One overall structure-suggestion. Shorten materiel and method (5 pages now) It would be much better that each result section match med each figure. 2. How to make the ox-LDL treated VSMCs of rabbit? What is the treatment? How? What is control for the treated cells? 3. ‘Randomly’ selected 2 up and 2 down regulated ? – I believe the word ‘randomly’ can be better described. It is important for the structure of the article. 4. Figure 1B, there are no significant stars. How to calculate the significance? How many times was the experiment repeated? This should clearly be shown, also in caption 5. Short description of results in figure captions. 6. In figure 2, co-expression of APPAT and α-SMA is hard to see. There should be better indication of two kinds of scale bar. 7. How to evaluate the expression level in fig 3?? There is no any description about it. Experimental design: adressed in previous box Validity of the findings: adressed in previous box (but seems valid) 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: EXPRESSION STATUS AND CLINICAL SIGNIFICANCE OF LNCRNA APPAT IN THE PROGRESSION OF ATHEROSCLEROSIS Review round: 1 Reviewer: 2

Basic reporting: The authors have done a fine work in terms of professional English, providing background and describing the need/caveat the address. Some points to address: In the abstract, at lines 32-to-38, it states "To determine expression alteration of APPAT in coronary artery, we performed qPCR on samples of healthy and pathological coronary artery. Same test of APPAT level was performed on circulating blood samples from the normal and patients with angina pectoris or myocardial infarction, followed by calculation of prediction power of APPAT. Additionally, we predicted microRNA target of APPAT and the expression level of the target was primarily measured in human VSMCs cell line, coronary artery, and blood samples." To me this doesn't read well. Something like "...artery. We also assessed circulating APPAT levels in blood samples from healthy individuals, and patients with angina pectoris (AP) or myocardial infarction (MI). Additionally, we predicted microRNA targets of APPAT, and show that the expression level of the targets was primarily measured in human VSMCs cell line, coronary artery, and blood samples. Lastly, we examined the predictive power of APPAT for the risk of AP or MI." would perhaps be better. Experimental design: A power analysis of - discovery experiment - predictive power (ROC and AUC) is lacking. Please add this. Also include an assessment of the validity of the discovery given the many potential non-coding and coding RNAs that exist. In other words, is p = 0.05 a valid threshold? This threshold of discovery should be corrected for multiple testing. Validity of the findings: Figure 1A: I am missing the explanations for the abbreviations in the figure. What does CPC, PFAM, etc stand for? Figure 1B: Perhaps I missed that in the manuscript, how do you explain the relative lower expression of APPAT in human cells as compared to rabbit cells after treatment with oxLDL? Figure 2: Please include non-fluorescent HE, EvG and alpha-SMC stained histological slides of the same artery/plaques. This will aid in orientation with respect to the lumen and the remaining non-plaque area as compared to the IF-FISH images. This will also mean that Figure 3 can be dropped, unless it is the same sample. But together these Figures are not informative at the moment. Replication & Figure 4A: - Please include data on the comparison between the two AUCs for APPAT and troponin. - Is the AUC of APPAT *significantly* better then the one for troponin in predicting MI? In other words: what is the added value? Why not just use troponin? - This is also lacking in lines 274-282: what is the statistical significance of these ROC/AUC analyses? - Please, also include a power analysis of the ROC/AUC analysis. - Furthermore, what is lacking is a replication of these results in a (larger) independent study. This is absolutely necessary and of great value to understand the relevance of APPAT as compared to for instance troponin as well as the validity of APPAT in predicting disease outcome. - This has also implications for the discussion at lines 366-370 regarding the potential value of APPAT as a prognostic marker. I don't believe there can even be a discussion about this, as the validity of APPAT has not been tested in independent studies. This should be addressed in experiments and in the discussion. The above also has implications for the conclusions made in lines 390-392. Things that would aid in support of the conclusions, and that would aid in assessing the validity of APPAT and miRNA-647: - Please add in data on correlations (of APPAT and miRNA-647) in larger datasets with sub-phenotypes of atherosclerosis, for instance coronary artery calcification, and carotid IMT as these are strongly correlated to cardiovascular outcome. - Are APPAT and the miRNA-647 correlated to other cardiovascular risk factors? One would assume there should be correlations with CAC, cIMT and risk factors, if APPAT and miRNA-647 are valid candidates diagnosis and prognosis. - Are APPAT and miRNA-647 also present in carotid plaques? Or is it merely constraint to coronary arteries? - Are APPAT and miRNA-647 also expressed in biosamples (i.e arteries) from GTEx Portal? Figure 5: In figure 1B 'treated' is noted as 'oxLDL', for consistency that should also be the case for figure 5A. Additional comments: In general nicely written, and well structured. What is really necessary though with this type of discovery efforts is to assess the validity of novel targets (verify) and replicate findings in independent and larger samples.

You are one of the reviewers, your task is to write a review for the article. You will 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: EXPRESSION STATUS AND CLINICAL SIGNIFICANCE OF LNCRNA APPAT IN THE PROGRESSION OF ATHEROSCLEROSIS Review round: 1 Reviewer: 3

Basic reporting: The authors propose the lncRNA APPAT as new player in the progression of atherosclerosis. The work is very interesting and potentially useful in future clinical studies. English could be improved in some parts of the manuscript. The authors should share the raw data in public archives such as SRA or GEO before publication. It's possible to submit the data in a private format and then release them after publication. An accession number has to be provided in the manuscript, in the section regarding RNA-seq (methods section). The section regarding miRNA target prediction is not completely clear. How did you select the miRNA candidates? Did you performed an intersection of target prediction algorithms? From the text this part is not clear. I suggest to write a paragraph about that in the methods. Another point to be clarified is the p-value. Did you adjust the p-value according to FDR or other approaches? From the text it seems you used only the p-value in all the analyses. Experimental design: The experimental design is correct and the use of NGS improves the quality of the results compared to the use of RT-PCR. As suggested by the authors at the end of the paper, it is interesting to explore the mechanism of action of APPAT and also the potential interactions with miRNAs. I suggest to focus on extracellular vesicles extracted from serum and plasma for future experiments. In this way, you could be able to understand if APPAT is actively released in the bloodstream or not, and if the content of these vesicles is released inside target cells. Consequently, you could identify the "sponge" effect of APPAT in the target cell. Validity of the findings: The findings support the hypothesis but future experiments are needed in order to validate the role of APPAT as non-invasive biomarker and its function and role in the progression of atherosclerosis. 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: EXPRESSION STATUS AND CLINICAL SIGNIFICANCE OF LNCRNA APPAT IN THE PROGRESSION OF ATHEROSCLEROSIS Review round: 2 Reviewer: 1

Basic reporting: The authors made good efforts to improve the quality of the article. There are no further questions from me. I hope that the authors will give more contributions in the field of atherosclerosis pathogenesis in the near future. Experimental design: - Validity of the findings: - Additional comments: The authors made good efforts to improve the quality of the article. There are no further questions from me. I hope that the authors will give more contributions in the field of atherosclerosis pathogenesis in the near future.

You are one of the reviewers, your task is to write a review for the article. You will 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: FUSTR: A TOOL TO FIND GENE FAMILIES UNDER SELECTION IN TRANSCRIPTOMES Review round: 1 Reviewer: 1

Basic reporting: In figure 2, a clearer description of the bar plot would be helpful. It is not very clear whether the x-axis represents the mean dN/dS of each gene family. If yes, the authors may also consider to add error bars. Experimental design: 1. In the validation section, the authors may consider to include the performance comparison with other existing tools such as VESPA. 2. In the validation section, 2nd paragraph, the authors show that their method has very high sensitivity by identifying all 50 families that were simulated to undergo pervasive positive selection. The authors may consider to add genes that don’t undergo positive selection into their simulated data. It could be helpful to demonstrate specificity of their method. Validity of the findings: No comment. Additional comments: The authors developed a useful tool to detect the gene families and amino acid sites under positive selection based on transcriptomic data. This tool differs from other existing methods by allowing de novo transcriptome assemblies that are not predicted ORFs. The automated tool also simplifies the installation and analysis process. I think this paper will be of interest to the readership of PeerJ.

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

Title: FUSTR: A TOOL TO FIND GENE FAMILIES UNDER SELECTION IN TRANSCRIPTOMES Review round: 1 Reviewer: 2

Basic reporting: Besides the inconsistent "raw data", this report fits PeerJ's other "basic reporting" requirements. It must be stressed that while the dataset used for validation is very problematic (see "Problems with the validation dataset" in the "general comments"), the main issue here is that it is not adequate to test the performance of the pipeline reported here (see "Main issues" in the "general comments") and thus the pipeline should be tested using a different dataset. Experimental design: Methods need to be described in further detail. In particular authors need to provide a more thorough documentation in the git repository (e.g. describe each subroutine and the intermediate files created by the pipeline) and a brief outline of the underlying procedures in the report for publication. Take advantage of the online-only nature of PeerJ and be as descriptive as possible. Remind that the better your documentation is and the easier for the user to understand how the pipeline is handling the data, the more users you'll have. I celebrate that the authors have made available their method, so I was able to reproduce their analysis. For my testing I generate a non-redundant dataset of the sequences in Supplemental table 1 (530, removing 53 that are duplicates) and launched the FUSTr in a Linux machine with 4 processors. I labelled each sequence with its corresponding accession number, a short code for the species, and the "family classification" from the original publication (there were 9 "families" in the original publication). >{gb ID}|{spe}|{family} I then generated one fasta file per species (trying to emulate different transcriptomes for different "samples") and named the files as the short species codes. The 49 files contain between 192 and 1 sequences (there are 18 species for which contributing with only one sequence to the dataset). Trying to reproduce the potentially conflictive redundancy of annotation of the original publication and test the ability of FUSTr to recognize header patterns, I left four duplicated sequences labelled as both "ICKs" and "Huwentoxin-1_family" (i.e. they share the first two fields of the header). The input fasta files thus contain a total of 534 sequences, with four duplicates. The pipeline is encoded in a Snakefile , for which I tracked back the calls and subroutines. The first subroutine is to duplicate the fasta files and stored them in "{sample}.clean" files, then headers are changed to comply with the conventions for the next steps. At this point, headers patterns are extracted from the files by the rule "newHeaders" and stored in <headerPatterns.txt> file. Trinity's Transdecoder.LongOrfs is next called with a predefined "-m 30" option (minimal length for an ORF), which generates as much as six potential ORFs (the longest per frame), and the protein and nucleotide sequences are extracted with TransDecoder.Predict run on the <longest_orfs.pep> file, which predicts a single "best" ORF per input sequence. An important control for validation at this point would be to check if the predicted protein sequences correspond indeed to the known translated sequences (a quick check at UniProt reveals that there are 133 transcripts in the dataset for which there is evidence at protein level). In my test, these steps removed 12 sequences from the analysis for no clear reasons as inspecting the Transdecoder intermediate files reveals that for all of them an ORF containing all of the known coding sequence was detected. The pipeline then assign a specific short ID for each best transcript model and writes two fasta files with all sequences as amino acids (pep) and nucleotides (cds). An all-vs-all blastp is then run on the <all.pep.combined> file with a predefined -evalue 1E-5 and the output piped into SiLiX for clustering with a predefined minimum overlap of 0.9 (-r option). Per "family" fasta files are then generated by the node2families subroutine and large enough families (>14 sequences, it would be good if the authors provide a rationale for this choose) aligned with MAFFT, trimmed with trimAl and transformed into phylip (so codeml can be run). The protein alignments are also use to compute gene trees with FastTree. For a dataset as the one used for validation, all the previous steps are essentially completed in no time (less than a minute in my Linux with four 2.93GHz cores). I didn't have time to systematically check all retained ORFs, but in at least one "family" containing sequences named "lycotoxin" in the original publication (corresponding to IDs labelled as families 10 and 11 in the Supplemental Table 1) the predicted ORFs do not correspond to the known translated sequences. For instance, the "best" ORF of FM863950 is >Gene.187::FM863950_Lycsi_U1-lycotoxin_family::g.187::m.187 Gene.187::FM863950_Lycsi_U1-lycotoxin_family::g.187 ORF type:5prime_partial len:47 (-) FM863950_Lycsi_U1-lycotoxin_family:459-599(-) TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGAATCCTCAGAACTTTATCCAATTTAAAGAATGTTACAAACAAAAAAAATCAAAACAATTTTCTATGTCATTTTTGCTATCGTTTTACGAAATAATTTAA Translated as >Gene.187::FM863950_Lycsi_U1-lycotoxin_family::g.187::m.187 Gene.187::FM863950_Lycsi_U1-lycotoxin_family::g.187 ORF type:5prime_partial len:47 (-) FM863950_Lycsi_U1-lycotoxin_family:459-599(-) FFFFFFFFFFFFFWNPQNFIQFKECYKQKKSKQFSMSFLLSFYEII* But the original sequence is >FM863950.1 Lycosa singoriensis mRNA for toxin-like structure LSTX-A23 precursor (LSTX-A23 gene) GACAGAACTTCGGTTTAGTTCCCCAGGAAATTTTGACTAAGTGACATCTTGAGGTTTCCTCCCAGCCAATCATGATGAAGGTTCTAGTGGTCGTTGCTCTTTTGGTTACTCTTATCAGTTACTCTTCAAGTGAAGGGATTGACGATCTTGAAGCTGACGAACTGTTGTCTTTAATGGCCAACGAGCAAACCAGGAAAGAATGCATTCCCAAACACCACGAATGTACGAGCAATAAGCACGGCTGCTGTAGGGGTAACTTTTTCAAATACAAATGCCAGTGTACAACAGTTGTTACCCAGGACGGAGAACAGACCGAAAGATGCTTCTGTGGAACTCCCCCTCACCACAAGGCGGCCGAATTGGTGGTTGGCTTCGGGAGGAAGATTTTCGGATAAAAGAACAGCTTTATCGGAATATGTGAAGACACGTTATTTGACGTAAATGAACCTCTGTAGAGTTTAAATTATTTCGTAAAACGATAGCAAAAATGACATAGAAAATTGTTTTGATTTTTTTTGTTTGTAACATTCTTTAAATTGGATAAAGTTCTGAGGATTCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Translated as >CAS03548.1 toxin-like structure LSTX-A23 precursor [Lycosa singoriensis] MMKVLVVVALLVTLISYSSSEGIDDLEADELLSLMANEQTRKECIPKHHECTSNKHGCCRGNFFKYKCQCTTVVTQDGEQTERCFCGTPPHHKAAELVVGFGRKIFG Thus, the identification of the coding regions by Transdecoder on this dataset is not optimal (notice that the predicted ORF contains the polyA region of the original sequence). This means that the gene tree and the selection tests were not performed with the coding sequences, and the results are therefore bogus. The authors must debug this problem and document their findings (e.g. is there a way to optimize the selection of the best ORF?). Moving on, the last and most computationally expensive step performs codeml analysis (using a biopython-inspired wrapper) under 7 different models of dN/dS distributions and testing for the most likely model of selection (achieved by comparing the log likelihoods). In my test, 9 families were automatically processed in just under 20 hours. If I understand correctly the summary file (a three-column file not described in the report, nor in the git, with four model comparisons per family), in my test three families are under "positive selection" (i.e. p<=1e-2 for models allowing dN/dS>1 vs models with fixed or variable dN/dS<=1), two of which correspond to kunitz toxins, in-line with what is reported by the authors, but also including the family containing the bogus ORFs of lycotoxins. Although the authors have not documented how they obtained the plot in Figure 2 (alas, it would be better if they do), I parsed the *mcl files and found 15 and 5 sites with dn/ds>1 in each of the kunitz families (I suppose corresponding to "kunitz 1" and "kunitz 2", respectively, in the figure). Validity of the findings: The "validation" and simulated datasets are not adequate to test the performance of FUSTr. The amount of data in both cases is unrealistically small for a typical transcriptome study, let alone for a tool intended to analyze several transcriptomes at once. Additional comments: Suggestions for improvement - Perform the "validation" on a relevant dataset, this should include potentially conflicting sequences (e.g. multiple isoforms derived from a single gene) and identifiers. One alternative would be to generate Trinity/cufflinks transcriptomes from publicly available SRAs known to be rich on multi-gene families - Document all the steps performed by the pipeline (the more detailed the better) - Include the raw fasta files for the validation datasets in the git repository - If authors choose to maintain the current "validation" dataset as an additional proof of the performance, the dataset must be corrected for the several inconsistencies detailed below and the inconsistencies in the original dataset described - Provide the results of the simulated dataset in graphical form Main issues 1. "Validation" dataset is not adequate for testing the features of the pipeline The dataset used is a manually curated set of full precursor sequences originally analyzed in PLoS Genetics 11:e1005596 and contains less than 600 sequences. This is an unrealistic size for transcriptome based studies, where thousands of transcripts would be predicted from a single experiment. 2. Insufficient description of the methods The pipeline is not properly documented, with several steps not even described (e.g. the construction of new headers and the processing of the codeml outputs), non described third-party options (e.g. blastp evalue and SiLiX minimum overlap). See extensive comments in the "Experimental Design" section. Problems with the "validation" dataset I will comment on the several inconsistencies of what I consider very problematic "raw data" (in this case the dataset used for the validation part), but it should be kept in mind that, as described above, the dataset used for validation is not adequate for testing the purposed use of FUSTr. - The number of sequences analyzed here is reported to be 624 in the text (marks 100-101), but numbers in Table 1 add up to only 618, whereas there are only 583 accession numbers in the supplementary file. I would have expected the authors to identify this not minor inconsistency before, but I understand it may be related to the not well structured presentation of the original publication (PLoS Genetics 11:e1005596, 2015). For the sake of this review, I have downloaded the relevant files from https://doi.org/10.1371/journal.pgen.1005596.s005 (including the reportedly accession numbers reportedly used) and https://doi.org/10.1371/journal.pgen.1005596.s006 (reportedly including the codon based alignments used for testing selection). Whereas the total number of analyzed sequences is not reported in the original publication, there are nine named "spider toxin families" in the supplementary files (alas somehow ten are plotted in figure 1of that publication), including 708 sequences in the alignments (four sequences appear in two different alignment files) and only 686 in the accession number tables. I regret these inconsistencies were left unattended in the original paper, but here the authors have the chance to correct the public record. I could map 567 identifiers shared between this report and the original publication, the remaining 16 identifiers correspond to the "FUSTr family 5" (omega hexatoxin in Supplemental Figure 4) for which the IDs in the original publication were not-standard. - The number of "highly curated spider venom toxin protein families" analyzed is said to be seven in the text (mark 100), all of which are said to be correctly recovered by FUSTr (mark 103). The eight bars in Figure 2 reflect the fact that "kunitz toxins" were split into two groups by SiLiX's step of FUSTr). Further inspection of the Supplemental Table 1, reveals that the 583 sequences are actually classified into 23 similarity clusters by the pipeline, nine of which contain 10 or more sequences and including five cases in which several FUSTr-derived clusters mapping to a single "family" as (ill-) defined in the original publication. - The dataset analyzed also has problems regarding the assumption of phylogenetic independence (identical sequences, unknown biological sources, erratic phylogenetic breadth, 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: FUSTR: A TOOL TO FIND GENE FAMILIES UNDER SELECTION IN TRANSCRIPTOMES Review round: 1 Reviewer: 3

Basic reporting: no comment Experimental design: It is not very clear to me how the simulated data generated in validation section (Starting from line 106). Also, in the implementation part, it seems that FUSTr performs many processing steps for input data. However, one question I have is that is this a data-driven model? For example, does the output purely come from the clustering results? Is FUSTr aimed to identify how many gene families exist in the input sequence? 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: FUSTR: A TOOL TO FIND GENE FAMILIES UNDER SELECTION IN TRANSCRIPTOMES Review round: 2 Reviewer: 1

Basic reporting: I agree that the new real life dataset provides an "ideal case use for FUSTr", however is not really a benchmark. In the original publication, more than 2000 homologous loci were identified in six specimens from two species. In that publication, less than 70 loci were identified as being under selection for color-changing or colonization phenotypes. I think it would be interesting if the authors double check if the families identified as under selection by FUSTr are the same. It would be also interesting to see if other multi gene families are also under selection. Experimental design: Although I'm happy with authors responses to the previous round of revisions, in particular the with the new validation dataset, and that I celebrate the almost complete re-engineering of the pipeline, which now relies on Docker as container and snakemake for workflow management, I would encourage the authors to further document the potential problems their intended users could face. For instance, Docker requires root privileges for running commands or to create a group of users that also needs root privileges, which many users might not have. Also is not clear for me if the setup_docker should be run every single FUSTr run (i.e. with different data folders). This time I was not able to reproduce authors' results, due to conflicts with my system configuration. Yet, I can follow the rationale in the Dockerfile, Snakefile and FUSTr wrapper and I do not think there should be any problem to reproduce the analysis step by step. In my test, the docker daemon fails while trying to "apt-get install -y wget git build-essential cmake unzip curl" (all of which are part of the any linux distribution) and exits with a non-zero code. When I tried tweaking the FUSTr wrapper to run the pipeline, I faced an "snakemake: error: unrecognized arguments: --use-conda" which leas to another non-zero exit status 2. $ conda -V conda 4.3.30 $ snakemake -v 3.13.3 My suggestion at this point is that the authors document all the programs needed to run the pipeline and offer an alternative customizable wrapper that the user can modify to point to the local installation of the programs (do the user really needs to re-install everything every time?). Validity of the findings: nothing to add Additional comments: nothing to add

You are one of the reviewers, your task is to write a review for the article. You will 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: DISTRIBUTIONAL DYNAMICS OF A VULNERABLE SPECIES IN RESPONSE TO PAST AND FUTURE CLIMATE CHANGE: A WINDOW FOR CONSERVATION PROSPECTS Review round: 1 Reviewer: 1

Basic reporting: Initially I was a bit concerned about the use of English but this was quickly dispelled. The article is generally well written however I request that the earlier sections, up-and-till the Results, be re-checked for use of English, particularly sentence construction. Experimental design: The reader may not be familiar with Pseudolarix amabilis hence it is important to provide a bit more useful background but relevant information. State that it is a tree, occurs family Pinaceae, and provide a few additional plant traits such as it has cones with winged seeds, pollination by wind, longevity, etc. Provide its full IUCN red list status. Sentence in Lines 145-147. Please reword ending as approach is not clearly articulated. Figure 3 (A). The arrow direction should be reversed as it currently shows migration from expansion areas to contraction/no change areas. (B) is correct. "Figure 4. Main limiting climatic factors for the distribution ..” add word “climatic” Validity of the findings: Lines 408 to 411. I am uncomfortable with this statement. The model shows sensitivity to climate change and not necessarily the species, given the limitations discussed. Additional comments: There is no mention of the likelihood that some populations occurring during the LIG and LGM periods could have gone extinct and therefore their locations and influence on Maxent model could not be included (and therefore the models may be slightly more conservative, failing to include extinct populations).

You are one of the reviewers, your task is to write a review for the article. You will 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: DISTRIBUTIONAL DYNAMICS OF A VULNERABLE SPECIES IN RESPONSE TO PAST AND FUTURE CLIMATE CHANGE: A WINDOW FOR CONSERVATION PROSPECTS Review round: 1 Reviewer: 2

Basic reporting: Overall the paper seems fairly sound, and should eventually be published I believe. It uses well established methods to predict past, present and future suitable climate areas for one particular plant species It has various issues that should be addressed before it is published, as detailed below. Literature references and sufficient field background/context are provided in most places, with some exceptions noted below. The structure of the paper is ok, but some revision of Discussion is required (Discussion too long, first paragraph should focus on your results, contains some material that should be in Results, see below for details). I would say the paper just represents an appropriate 'unit of publication'. SDM studies like this are becoming so easy to do, with the general availability of data on species presence and climatic variables. It would seem relatively easy to do the same study done here for another species, and another, and another and so on. I wonder whether each one would really deserve its own publication? I think studies that focus on several species at once are already becoming the norm (eg. Yates et al and Hamer et al below). Perhaps these could be referenced as examples of what such studies could try to encompass in future? Nonetheless, I think this study just meets the requirements for an appropriate 'unit of publication'. Yates, C.J., McNeill, A., Elith, J., Midgley, G.F., 2010. Assessing the impacts of climate change and land transformation on Banksia in the South West Australian Floristic Region. Diversity and Distributions 16, 187–201. https://doi.org/10.1111/j.1472-4642.2009.00623.x Hamer, J.J., Veneklaas, E.J., Poot, P., Mokany, K., Renton, M., 2015. Shallow environmental gradients put inland species at risk: Insights and implications from predicting future distributions of Eucalyptus species in South Western Australia. Austral Ecology 40, 923–932. https://doi.org/10.1111/aec.12274 The English language should be improved to ensure that an international audience can clearly understand your text. In many places the choice of words, or tense or expression is a real barrier to understanding the science. For example, in the abstract: “To avoid potential biodiversity loss, biological conservation is significant,” should be “To avoid potential biodiversity loss, biological conservation is important,” “While future modelling discovers a significant migration north of the potential climatically suitable areas of this species relative to current ones.” is not a sentence. Lines 65-66 121 ‘literatures’ is not correct The tense used seems incorrect at times. For example, the present tense is used in Materials and Methods for things that were clearly done in the past. This has real consequences for understanding the meaning of the text. These is sometimes an inappropriate mix of tenses. Further examples are provided in the general comments below. Experimental design: The study seems to follow a very established standard procedure for MaxEnt species distribution modelling and uses well-established software and methods. There is little that is original in the approach, beyond applying the established methods to a new species, but the flip-side of that is that there is little to criticize since the methods are so well established. The authors do also acknowledge some of the limitations of this kind of approach in the Discussion, which is good to see, but this should be better referenced, including references to more mechanistic approaches that are trying to overcome some of these limitations (see below for more details). Validity of the findings: There is very little detail given on the climate projections used, and yet obviously all the results depend intimately on the climate assumptions used. The climate layers for the past climates and especially the one for the future climate must be very suspect, in terms of accuracy. This is somewhat acknowledged in the Discussion, but I would like to see some more details in the Methods as well, on how these climate layers are generated and how confident we can be in them (see details below). Additional comments: The use of the word ‘window’ in the title seems strange to me. What does it mean?? Abstract, line27 “The response mechanism revealed,” It is unclear what the response mechanism is as it has not been mentioned previously in the abstract. 98-99 not clear to me how reconstructing range dynamics can reveal actual mechanisms… can you explain or provide a reference? 110 you cannot say that MaxEnt always has excellent predictive performance. It will always depend on the data, whatever the modelling approach used. 98-112 Overall this paragraph is not convincing. Maybe this is because of the references to response mechanisms. MaxEnt and most climate envelope methods are NOT mechanistic… they are purely statistical/descriptive. 101-112 is a reasonable summary of this kind of modelling, but its justification at the start is not convincing to me. 115 Following from the previous paragraph, the overall aim is not clear. ‘revealing the response’ – ‘revealing’ sounds like you are finding the actual response, but rather you are ‘predicting’ or estimating or ‘modelling or foracsting/hindcasting’ I think. “provide a theoretical foundation for future conservation strategies” – this sounds too vage to me to be a real scientific aim, or at least it needs more detail on how the distribution predictions will help future conservation strategies 124: I think some more information is needed on these climate layers, especially the past and future ones. How are they generated? How confident are we in them? For the future one, I think there are many different models and methods and emission scenarios that can be used to generate such layers… which were used in this case? How do your results depend on these choices and would conclusions be different if different climate models and emission scenarios were assumed? 127 – model overfitting is model overfitting… I would say that it cannot be exacerbated in certain conditions. However, the impacts of overfitting will be seen when the data is applied to new conditions. 129 – you don’t remove all environmental predictors with high pairwise correlation I think… you remove all except one?? 131 – what is “the physiological role of P. amabilis”?? I think the word choice is wrong. 131-132: what is a low percentage contribution? How low is low? 135: “too many correlated parameters” – no, it is not the parameters that are correlated, it is the explanatory variables, right? 144: These two sentences seem unconnected, so I do not understand why ‘however’ is used for contrast here. How is the first sentence related to the rest of the paragraph? 149: why is it a bias FILE? Is file the right word choice? 151: what layers? ‘layers’ hasn’t been mentioned in this paragraph, or the one before. Is this something to do with the bias ‘file’? 153: the use of present tense here makes it sound like the authors are claiming this is the only way to assess predictive performance, but it is definitely NOT. I think they mean that this is how they chose to assess it?? 159: and here past tense its used (was). The mix of past and present tense is very confusing, as it strongly impacts the meaning, as per the example above. 165: “by excluding the 10% most extreme data points in the modelling” – I don’t think you are excluding data points? Rather you are excluding predictions???? In either case, 10% most extreme in what sense/direction??? 168: so why include the 10% TP at all? 169: ‘survey’? I don’t think you are actually surveying anything? 172: again the use of ‘file’ is confusing. I think you mean you calculate the vector? The fact that you store this information in a file is incidental surely. 176: “finally, we evaluate the potential migration from one period to the next” – too vague. How did you actually do this based on the centroids explained above?? 181: “We focus on the limiting factors affecting the contraction and expansion of climate suitability.” – again, this is vague. What do you mean by this? 186: No, perfect would be AUC=1 ?? 188: well predicted rather than well described I think 189: Do these values mean anything? Are they low or high or good or bad? 217: acronyms not useful – explain in full Discussion. 222-233The content of this paragraph does not refer to results at all. It reads more like general background. It should be in the Introduction, or come later in the Discussion when discussing caveats on the study. The first paragraph of the Discussion should focus on the most interesting results of your study!! 236-241: This is not Discussion, this is Results! The Results section is for presenting results, like this. The Discussion section is for saying why they are interesting, important, relevant, applicable, expected/unexpected, etc etc 255: what’s TS and AP? Overuse of acronyms, especially for Discussion which may often be read in isolation. 258: “the regional heterogeneity of climate baseline and the different amplitude of the variable’s variation” I don’t understand – should be clarified. Overall the Discussion has many interesting points, but it is much too long and unfocussed. I think it should be substantially rewritten to much better focus on the most important points of YOUR study. For example, 283-290 has no reference to your results at all. In fact 246-290 has only very occasional and brief reference to your results. The final paragraph on limitations could be much better referenced. The limitations of MaxEnt-style correlative SDMs have been well discussed by many others (eg Fordham below, but many others as well). People are starting to address some limitation using more mechanistic modelling approaches and these should be acknowledged. The CSIRO CLIMEX model is one example, as are the papers below, and there are many others. Some of this work should be acknowledged. Fordham, D.A., Resit Akçakaya, H., Araújo, M.B., Elith, J., Keith, D.A., Pearson, R., Auld, T.D., Mellin, C., Morgan, J.W., Regan, T.J., Tozer, M., Watts, M.J., White, M., Wintle, B.A., Yates, C., Brook, B.W., 2012. Plant extinction risk under climate change: are forecast range shifts alone a good indicator of species vulnerability to global warming? Glob Change Biol 18, 1357–1371. https://doi.org/10.1111/j.1365-2486.2011.02614.x Renton, M., Shackelford, N., Standish, R.J., 2012. Habitat restoration will help some functional plant types persist under climate change in fragmented landscapes. Global Change Biology 18, 2057–2070. https://doi.org/10.1111/j.1365-2486.2012.02677.x Tomlinson, S., Webber, B.L., Bradshaw, S.D., Dixon, K.W., Renton, M., n.d. Incorporating biophysical ecology into high-resolution restoration targets: insect pollinator habitat suitability models. Restor Ecol n/a-n/a. https://doi.org/10.1111/rec.12561

You are one of the reviewers, your task is to write a review for the article. You will 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: DISTRIBUTIONAL DYNAMICS OF A VULNERABLE SPECIES IN RESPONSE TO PAST AND FUTURE CLIMATE CHANGE: A WINDOW FOR CONSERVATION PROSPECTS Review round: 1 Reviewer: 3

Basic reporting: This paper presents a modelling-based study of the historic, present and future distribution of Pseudolarix amabilis, a conifer with a restricted distribution in eastern China. My personal field of expertise is ecology, not modelling, and thus I have appraised the paper on the bases of its ecological relevance and interpretation rather than the technical strength of its modelling approach. Firstly, the flow and content of manuscript could be improved to help the reader along the journey of the study. At present parts are not concise, particularly the introduction – at four pages long it is a rather rambling background to the study, and several sections could perhaps be removed as they are not intrinsically relevant (LL31-46) or should be consigned to the methods (L101-112). Similarly, the discussion at 11 pages is far too long and reads somewhat like an unfocussed essay. The first paragraph of the discussion makes no reference to the results of the study, which again makes interpretation difficult. Additionally, some elements of the discussion (e.g. L236-241) belong in the results. I was not left with a clear understanding of the study outcomes, how these related to other international literature, or whether the study had achieved its aims of informing future conservation. In fact, the last paragraph seems to suggest that the available data are not sufficient to achieve this aim and that future studies using ENMs might be sufficient. As the reader I need to be led through the study of the study and left with no uncertainty about what was undertaken and why, and the significance of the findings need to be clearly articulated. I am afraid this was not the case – from the summary paragraph (L408-414) I took the message that P. amabilis responds to climatic change exactly as one would expect, and I am afraid I disagree with the statement that “investigating the response mechanism of P. amabilis to past climate change and predicting future climate suitability results in an effective conservation strategy”. This study doesn’t examine the response mechanism of P. amabilis (rather it examines a modelled response underlain by many, many assumptions), and effective conservation requires implementation of actions rather than simply predicting a future scenario. Fig S1 – very difficult to distinguish between symbols for historical distribution given colour and overlap. Table S1 – Bio variables should be arranged numerically to match the caption I think overall, the paper fails this section in its current form. Experimental design: The paper presents something of a case for conservation, although I feel this basis for the study is rather weak – particularly as the authors don’t really background how threatened the species actually is. The IUCN Red List assessment for P. amabilis notes “this species is very rare in the wild and occurs in a few remnants of primary forest on isolated mountains. Most locations are not within protected areas and loss of habitat is still continuing in this densely populated part of China.” Area of occurrence is estimated at <500 km2, and threatening processes continue unabated. With this in mind, it seems the species requires more pressing conservation initiatives than future modelling – predictions would suggest it may be extinct in the wild long before the 2080 scenario modelled. Following on from this point, why was the “future moment (2080, HadCM3 A1B)” chosen? There is no justification provided in the text. Is this widely considered to represent the future climate of the region? Does only one modelled snapshot of the future provide an adequate assessment of future conditions and the variability in future climate predictions? My main issue with the paper is this: the authors provide little discussion of the ecology of the species, which is in my opinion crucial information for the reader in interpreting their results. For example, in the introduction the only non-distributional information about the species (which is only introduced in line 86) is that it belongs to a monotypic genus. We only find out that P. amabilis is a tree as a side note on line 245 in the discussion, and nowhere is it stated that the species is a long-lived conifer restricted to acidic soils between 180-1000 metres altitude (all information provided in the IUCN Red List assessment that is critical to interpreting species distribution). How long-lived is the species? Many conifers are long-lived and take many years to reach reproductive maturity; is 80 years sufficiently far in the future to account for multiple reproductively successful generations? 80 years seems like a very near snap-shot of the future compared with the past in a physiologically adaptive sense (particularly for long-lived individuals): the species had ca. 120,000 years to adapt to changing climate between the LIG and LGM, 21,000 years to adapt between the LGM and present, but only 80 years between the present and the future scenario. I imagine if they are not logged, many individuals alive today will still be alive in 80 years which would imply no change in distribution from climatic factors; rather perhaps a change in the suitability of climate for seedling germination to occur if we work off the assumption that seeds possess a narrow thermal/hydrothermal envelope and exhibit thermal suppression at temperaures 1-2 degrees greater than the present (unlikely based on the global literature). Additionally, there is also no information provided on the dispersal strategy of the species (though there is significant conjecture about past distribution, dispersal, and habitat occupancy). The authors acknowledge that they do not include soils data in their analyses due to a lack of available data (and note that this omission may limit the efficacy of the model). This is unfortunate given the species has known soil affinities, but is perhaps understandable if such current or historical data is not available. However, the authors also make no mention of altitude or elevation in their manuscript. This to me seems like a terminal oversight given the literature notes the species to be elevational restricted. If the chosen future climate scenario is one in which conditions are warmer than present (this is not clear), then surely an analysis of geographic area incorporating elevational data is required to assess the altitudinal shift in distribution – and this would surely make the situation of the species far more dire. Validity of the findings: See many of my comments in section 2. Additional comments: No general 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: DISTRIBUTIONAL DYNAMICS OF A VULNERABLE SPECIES IN RESPONSE TO PAST AND FUTURE CLIMATE CHANGE: A WINDOW FOR CONSERVATION PROSPECTS Review round: 2 Reviewer: 1

Basic reporting: The authors appear to have exhaustively addressed the comments of the reviewers in their response document, and provided significant background to these responses in their rebuttal. The quality of English throughout the manuscript has been improved somewhat. The introduction is to me still rather long, but this is not a huge concern. The aims and hypotheses are much clearer, and the significance and outcomes of the study are presented with much greater clarity. The addition of ecological information on the species is appreciated and adds to the manuscript, although I feel “branchlets are dimorphic: long branchlets (leading shoots) with helically borne leaves and short branchlets (brachioblasts) with fascicularly arranged leaves.” (L84-86) Is probably unnecessary as morphology is not relevant to the study. Experimental design: The methods appear well-structured and are presented with a high level of detail. With the additions made after the initial review, I feel these are now satisfactory. Validity of the findings: I still feel to some extent that this study lacks ecological significance; that is, the data presented are heavily constrained by the absence of soil and altitudinal data given the relevance of these factors on the species distribution. As far as I can see, there is also little attention paid to the highly fragmented nature of remaining natural habitat (very little of which remains in the study region). The authors have acknowledged some of this, and explained why they are not included in the rebuttal, and while I accept this explanation I do wonder how meaningful the conclusions that have been drawn are in their absence. The identification of (for example) 100 km2 of climatically suitable habitat is not particularly useful if half of this is unsuitable soil type, three quarters of the suitable soil type is developed land, and only a fraction of the remaining area is altitudinally suitable. All of these factors are critical for seed germination and seedling establishment, which the authors have noted is the primary life cycle stage they are modelling. As a side note, proper examination and modelling of these factors would require an understanding of seed dormancy, seed ecology, and the thermal niche required for germination to occur, as well as looking at shifts in seasonality which may impact upon moisture availability during critical life cycle stages. My concern, essentially, is that the authors may be significantly overestimating the future distributional envelope of this species. However, I understand their argument that some of this critical information is not available. I have deliberated on this for some time and cannot decide how to appraise this; I will defer to the opinion of the editor on whether this issue is significant enough to hinder publication. 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: PHYLOGENETIC RELATIONSHIPS AND GENETIC DIVERSITY OF THE POLYPEDATES LEUCOMYSTAX COMPLEX IN THAILAND Review round: 1 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: Buddhachat and Suwannapoom conduct a phylogeography of frogs belonging to the Polypedatus leucomystax complex occurring in Thailand. There is a basis for an interesting paper here, but in current form I find it unfocussed. I find it difficult from the present paper to get a good idea about this complex, not only in general, but also the situation in Thailand. Particularly relevant for this study is that it is not clear how many species are supposed to occur in Thailand and if they are all included. The aim of the paper is not clear. Obviously the presence of cryptic species in the complex and potential co-occurrence of such species is a big part of it. However, the paper suffers from equating mtDNA clades with species. I suggest to tone this way down and merely suggest that mtDNA clades might suggest additional species and sympatry of species. You have much more work to do before you could talk about species (morphological, bioacoustics, nuDNA analyses) and this should be acknowledged in the paper. The mtDNA data help raise testable hypotheses, but they don’t solve this complex of species. Furthermore discussion on the difficulties of mtDNA-based species hypotheses is missing. What about mtDNA ghost lineages? What about mtDNA introgression? This paper is quite light on references and this can be explained by lack of discussion of such evolutionary processes. Finally I think the biogeographical aspect of this paper should be fleshed out. Obviously in the set-up of the paper you want to explain why there is such a richness of (cryptic) species in the region and furthermore the data provide insights on biogeography. The authors seem to not have realized that the Istmus of Kra is an important biogeographical boundary and there is much digression in the discussion that can be ditched. These frogs once again validate this boundary and that is something to discuss in a wider context. I think the presentation of the mtDNA data can be better. The paper is quite well written but I do note some obvious issues. I give more details below. The set-up of this work in the introduction is not optimal. This concerns the first and lines 81 – 83 from the last paragraph. These need to be combined as currently the flow of the introduction is cut up. I suggest first para the greater context of this study, second para the system (so as it is now) and third para the aims of the study (so lines 83-87). Regarding the set-up, I think it makes sense to start about SE Asia as a hotspot for amphibians. However, lines 50-53 are just a list of taxa with references and the link with the present work is not clear. This could be fleshed out however. I feel that this work is mostly about cryptic species and that is a good angle. It also makes sense to give some more background on the historical biogeography of the region to explain why there are so many species and to use new phylogenies such as created here to inform on historical biogeography. About the study system: it is very difficult for me as a reader without knowledge on this system to grasp this. Are all the species you listed members of this complex and are all other known species of Polypedates not members of this complex. If so how was this determined? How are the relevant species distributed (perhaps provide a map with ranges and type localities?). Line 45: remove substantial as hotspot already suggests biodiversity is substantial Line 79: a complex is not a cryptic species, but a complex may contain cryptic species. Line 49: remove “in the current period” or change to “observed today” Line 54: Change “The clarification of ambiguous species” to “Accurate species delimitation” Line 91: It is mentioned in the introduction that five (described) species of the complex are found in Thailand. But why are not all species included in this study? And which species are included? It is not clear to me how you chose the sampling that you did. Is it perhaps the case that some described species cannot be distinguished based on mtDNA? Line 129: It seems like you directly provide a result here. In the Methods you should say you used Barrier 2.2 and why, but not what the outcome was, that should be in the Results. Line 130-131: THE minimum spanning network >>> A minimum spanning network Line 143: to assume should be assuming? Line 144: Multiple should be multimodal? Lines 161-164: It is not clear how you go from clades to species names. Do you base that on included sequences that were ascribed to a species? You need to be explicit. Of course you run the risk now of equating mtDNA with species while that is a dangerous interpretation without other data such as morphology and nuDNA. So you need to bring these names in carefully. It needs to be explained where the use of Polypedates sp. comes from. Does that mean this clade cannot be ascribed to a particular species while the others can? And if so, is that because you simply did not include relevant species or is it likely to be something new? It is not clear. I think it is not a good idea to talk in the Results about species rather than clades, for example describing the distribution of species based on mtDNA clades. If you want to interpret clades as species you would do that in the Discussion, but of course you need to be very, very careful there because mtDNA alone is not a good basis for species delineation. Line 161: “Bayesian analysis model with MrModeltest” this phrasing makes no sense. You also did a Maximum Likelihood analyses. Don’t repeat methods. Line 162: could consists >>> either just say consists or something that you recognize four clades. Line 165: I guess you mean inference? Line 174: incomplete sentence Line 174-175: this is interpretation and does not belong in Results. Line 176-177: This sentence is gibberish. Line 181-186: there seems to be circularity here. You have allocated clades and hence haplotypes to species and now you say haplotypes belong to species. And what do you mean with unique haplotypes? Line 202-203: Remove “one of the most notoriously challenging” as I can think of a million other difficult groups. Line 204: You do not SOLVE anything, you get a little bit closer to the solution or at least to a hypothesis. Line 205-208: These sentences should not be here. They just show that you should have used another marker than COI. But that is the marker you have so that is what you have to stick with. These studies may provide some insight into the system (I am not going to read them) and might be worth to integrate into the introduction of the study system, but there is no point of mentioning this here. Lines 208-214: What you do here is just not right. You cannot just assume that these four clades are species and that these species occur sympatrically. You could only do that if you had independent data to delineate species, such as morphology or nuDNA. What about mtDNA introgression or simply the occurrence of distinct mtDNA clades in a single species? I am familiar with European newts and all these issues occur here. Related to this and hinted to in Line 221-222, apparently morphology could give some insights and you have looked at morphology but only as an aside? I noticed you have collected whole individuals so either you would do a morphological analyses of all these samples to see if you could come up with a morphological based species delineation to compare your mtDNA dataset to, or you do not make larger claims than that the mtDNA supports and can support. Line 223-225 & 228-229: again, how do you link these names to these clades, it is not clear. Is it based on and ID given to a sequence taken from GenBank? If so you need to be clear on this (and what if the ID on GenBank is wrong?). Lines 227-228: This is a strange sentence, it seems to suggest that somebody already looked at the exact same frogs and found the same pattern, but that is not the case. You mention another taxon where this pattern was found. Be more clear, so say it was stated in another taxon. However, there is much more to it than just a single other taxon, this is a known biogeographical boundary (also applies to lines 233-238). Isn’t this the official boundary of the Sunda region? This needs to be worked out because it is this kind of historical biogeographical inferences that are juicy results. Lines 243-247 seem to suggest that nobody before knew that this was a biogeographical barrier… Lines 239-243: I don’t see what is the point of this section? So they could be moved by people (but I guess islands would also have been connected to the mainland during glacial times?) but how does that relate to the current work? You find a clear biogeographical boundary despite any artificial movement. Lines 248-265: I don’t see the point of this section. There are two biogeographical regions and these frogs seem to show a divide as well, that is the bottom line (and for all the sympatric lineages you have no idea if they even are species and if so what separates them). Lines 266-284: again you talk about a particular species without justification. Why would you need to invoke human-mediated translocation to explain homogeneity? Why not a recent population expansion from a small source population? In the conclusion you talk about putative species and clades and this is the proper route to take. Fig. 1 This legend is a mess. It makes MUCH more sense to show clades instead of haplotypes in Fig. 2. This way the clades in Fig. 1 can be directly linked to the map and that is way more insightful. The present use of colours in Fig. 2 is poor, I cannot distinguish different shades of yellow etc. In Fig. 3 colouring the haplotype network according to populations is pretty pointless and actually very confusing. This haplotype network is better presented as part of Fig. 1. Just use the colours of clades for the haplotypes belonging to clades.

You are one of the reviewers, your task is to write a review for the article. You will 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: PHYLOGENETIC RELATIONSHIPS AND GENETIC DIVERSITY OF THE POLYPEDATES LEUCOMYSTAX COMPLEX IN THAILAND Review round: 1 Reviewer: 2

Basic reporting: The grammar of the manuscript is not acceptable. There are many sections where I did not understand what the authors were trying to communicate. As an example, consider the first sentence of the abstract. Do that authors mean to imply that the taxonomic uncertainty in this group makes it challenging to infer its biogeographic history? I think so, but the wording is unclear and ambiguous in the first sentence. There are many additional examples. The figures are clear and easy to interpret. The references are sufficient and appropriate. Experimental design: The research is clearly within the scope of the journal. The research question is not clearly define, due in large part to the ambiguities in the written presentation. The methods description for the molecular biology are adequate. The methodology for the data analysis is adequate in general. However, there are several small issues that should be addressed: i. It is inconsistent to use a model of sequence evolution to infer the phylogeny (i.e., lines110-115), but to then calculate genetic distance for use in the other analyses using p-distances (line 128) because substitutions are not corrected in the p-distance as they are in the phylogeny inference. Thus, the same data can produce two different distance matrices. ii. The Mantel test has been questioned on several levels for inferring isolation by distance. Better options are available (e.g., Legendre P, Fortin, MJ. 2010. Comparison of the Mantel test and alternative approaches for detecting complex multivariate relationships in the spatial analysis of genetic data. Mol. Ecol. Resour. 10, 831-844. AND Diniz-Filho JA, et al. 2013. Mantel test in population genetics. Genet. Mol. Biol. 36, 475-485.) iii. It is in poor form to use a software package such as DNAsp to conduct an analysis such as the mismatch distribution without citing the original justification for this test [in this case Rogers, A. R., & Harpending, H. (1992). Population growth makes waves in the distribution of pairwise genetic differences. Molecular biology and evolution, 9(3), 552-569.] Validity of the findings: The general findings appear valid. The authors appear to have interpreted their results in a reasonable manner. Additional comments: I believe that this work has a substantial amount of potential. However, I cannot recommend that it be published without a clear improvement in the presentation, and a correction of some of the analyses.

You are one of the reviewers, your task is to write a review for the article. You will 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: PHYLOGENETIC RELATIONSHIPS AND GENETIC DIVERSITY OF THE POLYPEDATES LEUCOMYSTAX COMPLEX IN THAILAND Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: Buddhachat and Suwannapoom revised their submission on a phylogeography of frogs belonging to the Polypedatus leucomystax complex occurring in Thailand. They have managed to improve the clarity of their MS but I still thought it somewhat long-winded in places. Below I suggest some cuts to streamline it more. I also have some language/style issues. Why is the list of five species in line 54 not a subset of the six species mentioned in 50-51? Based on everything you wrote before that should be the case! Also, please keep the order of names consistent (e.g. alphabetical). Line 51: delimited from >>> distinguished in Line 56: remove redundant words “A study by” Line 64: remove “highly” Line 67-69: remove these sentences, they are out of place here. Line 76: remove redundant words “In this study” Line 90: remove “independently” Line 91: genus name should be abbreviated, it seems you are inconsistent with this throughout the MS Line 115: remove “population genetics software” Line 142: could consist >>> consists Line 149-150: the clade C population >>> clade C Line 157-158: remove this sentence Line 162-163: Change sentence to: Haplotypes F and G of clade D are considerably divergent from the rest of clade D and we partition it in two subclades: D1 and D2. Line 184 and elsewhere: it is quite confusing to call this Northern B rather than Northern D… Lines 190-198: These sentences are irrelevant, remove. I think spelling out a phylogeny rather than simply referring to the figure that summarizes the info is a bit much for Results already, but there is definitely no place for that in Discussion. Line 198-199: Although mtDNA seemed to offer cleanly split clades with a unique haplotype in each clade, mtDNA introgession/hybridization between the closely related species can occur. This can occur particularly in sympatric populations or at the boundaries of species distributions resulting from incomplete reproductive isolation. >>> Although the mtDNA phylogeny reveals the presence of distinct clades, mtDNA introgression is frequently observed where closely related species are in secondary contact (CITE: (1) Toews DPL, Brelsford A. 2012. The biogeography of mitochondrial and nuclear discordance in animals. Mol. Ecol., 21:3907-3930. (2) Zieliński P, Nadachowska-Brzyska K, Wielstra B, Szkotak R, Covaciu-Marcov S, Cogălniceanu D, Babik W. 2013. No evidence for nuclear introgression despite complete mtDNA replacement in the Carpathian newt (Lissotriton montandoni). Mol. Ecol., 22:1884-1903. (3) Wielstra B, Burke T, Butlin RK, Arntzen JW. 2017. A signature of dynamic biogeography: enclaves indicate past species replacement. Proceedings of the Royal Society of London B: Biological Sciences, 284:20172014. (4) Wielstra B, Burke T, Butlin RK, Avcı A, Üzüm N, Bozkurt E, Olgun K, Arntzen JW. 2017. A genomic footprint of hybrid zone movement in crested newts. Evolution Letters, 1:93-101.) Line 202-205: For instance, some populations of European newts Triturus montandoni have an mtDNA haplotype of Triturus vulgaris in an area where their distributions are connected, most likely as a result of historical or ongoing hybridization and multiple introgression of mtDNA from T. vulgaris to T. montandoni (Babik et al., 2005). – I think this sentence is too much detail and can be removed. The taxonomy used is outdated and a more recent and more detailed study is a more appropriate citation. See the rewritten sentence I propose above and I suggest to add some (newt) citations relevant citations there. Line 187-189, 201-202 and 205-207: Merge these sentences and place them after suggested rewrite of lines 198-199 as “Therefore, the use of mitochondria DNA data alone is not sufficient for species delineation. An integrative approach, consulting additional data such as morphology, bioacoustics, ecology, and/or nuclear DNA, would be required to more accurately assess the taxonomy of the Polypedates leucomystax complex (CITE: Padial J, Miralles A, De la Riva I, Vences M. 2010. The integrative future of taxonomy. Front. Zool., 7:16.)”. Line 207: remove “A recent study by” Line 207: THE P. leucomystax complex Line 217: remove “expansion” Line 212-213: “forms a significant region separating them” >>> “corresponds to a considerable phylogeographic break” Line 219-220: “we assumed that the northern B clade and the Southern clade might be P. megacephalus and P. leucomystax, respectively” we ascribe the northern B clade to P. megacephalus and the southern clade to P. leucomystax” Line 221: remarkable >>> considerable Line 223: to suggest a hypothesis >>> to suggest OR to hypothesize Line 231: remove “A study by” Line 236-238 “Additionally, data on rainfall in Thailand by the Thai Meteorological Department indicates a difference in the amount of rainfall between areas to the north and south of the isthmus (Fig. 3).” This does not add anything, remove this sentence and remove the Fig. 3 (which is not convincing). Line 238: Based on this, we believe that climate may be a >>> We suggest climate was a Line 240-241: remove this sentence Line 245: Where does this molecular dating come from? I presume a previous study? Cite it! Line 247: may be >>> is Line 250-262: Remove this paragraph. You have linked the phylogeographic break at the IOK to correspond to distinct climate zones, that is enough. This paragraph is highly speculative and confusing and does not help. Line 272: remove “great”

You are one of the reviewers, your task is to write a review for the article. You will 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: SICEGAR: R PACKAGE FOR SIGMOIDAL AND DOUBLE-SIGMOIDAL CURVE FITTING Review round: 1 Reviewer: 1

Basic reporting: See "General comments for the author" Experimental design: See "General comments for the author" Validity of the findings: See "General comments for the author" Additional comments: The manuscript „Sicegar: R package for sigmoidal and double-sigmoidal curve fitting“ by Caglar and coworkers describes an R package that conducts sigmoidal curve fitting and model selection to choose between “sigmoidal”, “double sigmoidal”, “ambiguous” (no decision can be made) and “no signal” (no growth characteristics) curve structure. The current implementation is based on the following steps: i) normalizing the data within [0, 1], ii) checking for minimum and maximum threshold levels, iii) fitting a sigmoidal and double sigmoidal model by maximum likelihood (nonlinear least-squares) using the minpack.lm package and a grid of starting estimates for robustness, and finally iv) model selection based on lowest AIC. This algorithm is tested by an extensive simulation regime, in which increasing noise is added to perfect sigmoidal/double sigmoidal models (“ground truth”). It could be demonstrated that with the exception of extremely high noise levels, the implementation succeeds in identifying the underlying true model. The manuscript is well-written and the package works well, however there are some important points the authors should address before rendering it suitable for publication. 1) It is not really clear - and nowhere mentioned - in which scenario (biological, pharmaceutical or clinical question) the selection between sigmoids/double sigmoids is essential. This information (maybe discriminating growth from growth with subsequent decay) should be supplied in the “Introduction”; else, the readership will see no real-world application. 2) AIC is not explained and not written in unabbreviated form. 3) When drawing the starting parameters from a uniform distribution (line 49), does it make sense to draw random values from it? Functions such as runif might miss some essential values. Would it not be better to create a sequence from within this window, i.e. seq(lower, upper, length.out = 100) to ensure that the complete parameter space is employed? 4) The authors should at least verify that the quality of model selection is also achieved when i) the predictor values are not an equidistant sequence (3 to 30, in 0.5 increments) and ii) the noise setup is heteroscedastic, i.e. noise is a function of the magnitude. This must not necessarily be included as a figure, but the outcome would be interesting as most of the real-world data does not have constant (homoscedastic) noise. 5) It would also be interesting to know how the model selection performs when the authors use a 5-parameter (Richards) sigmoidal model, which often fits better when the curve is asymmetric. 6) In lines 172ff, the authors describe a decreasing performance of model selection in a high-noise regime. In this context, the authors should cite the work from Spiess & Neumeyer (https://bmcpharma.biomedcentral.com/articles/10.1186/1471-2210-10-6), which investigated model sigmoidal model selection and resulting goodness-of-fit measures at varying noise levels. 7) I strongly believe that Fig. 1 and Fig. 2 could be merged into one concise Figure, i.e. the different phases of the double-sigmoid could be denoted in Fig. 2B. 8) The authors have completely neglected the drc package, which also offers a wealth of functionality for sigmoidal dose-response curve fitting and model selection. It should be cited.

You are one of the reviewers, your task is to write a review for the article. You will 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: SICEGAR: R PACKAGE FOR SIGMOIDAL AND DOUBLE-SIGMOIDAL CURVE FITTING Review round: 1 Reviewer: 2

Basic reporting: 1) The introduction would need to be improved: it is simply too short (one (self) citation is not enough). The authors need to provide background paragraphs on 1) existing (statistical) methods for fitting (nonlinear) s-shaped data (e.g., generalized linear and nonlinear regression) and 2) (more importantly) existing software for fitting s-shaped data. There are several R packages that should be mentioned: drc, drfit, grofit, qpcr, some of which are mentioned in the later subsection in lines 188-205; these lines should be integrated into the introduction. Note that the R package "drc", which can also fit biphasic patterns just like the proposed double sigmoidal model, is widely used for analysis for many different types of data. 2) There are several claims throughout the manuscript that would need to be substantiated by suitable references, e.g., lines 21-23, 25-26, 64 (Why these thresholds?), 132 (Fermi?), 159-160 (Any reference for this definition as it's an unusual definition. Usually such a range would be defined based on a range on the y axis, e.g., EC5 to EC95). 3) Definitions and methods are placed in the Results section. Please consider re-arranging parts of the material in the manuscript, e.g., lines 107-164 should be placed in the Methods section. On the other hand lines 124-131 are very relevant in the Discussion. 4) Please do not repeat definitions and methods used in the Results section, e.g., lines 182-183. Typo: "insure" should be "ensure". Experimental design: The manuscript addresses the problem of fitting high-throughput s-shaped data by providing an R package that allows classification of such data into 4 categories (including two types of nonlinear patterns) depending on the signal in the data. This is an interesting but also challenging problem. Moreover, it seems that a novel multiplicative double sigmoidal model has been proposed although there could be links to models for independent action of mixtures. However, some claims should be omitted: In lines 123, 132 it's claimed that any sigmoidal curve is fully determined by 3 parameters. This is simply not correct in general. There exist both four and five-parameter models for s-shaped data. Please look into the literature on logistic or log-logistic models. Actually, the authors define a four-parameter model in the appendix! Confusing. In lines 59-60: Perhaps I don't understand, but why should the AIC be larger than -10? Depending on the data AIC can take on arbitrary values I believe. A sensible rationale (a reference?) need to be supplied for such a non-standard rule. It would also be helpful if the authors could specify the probability distribution that is assumed when fitting models using maximum likelihood (line 47). Is it least squares estimation? Validity of the findings: The proposed R package performs as would be expected: the more noise the more difficult the classification. This is nicely shown in Figure 3. Would it be possible to include a real application? It is always to good to see a methodology used in practice. The authors need to provide a short conclusion summarizing their findings and, possibly, experiences using the R package. Also, if no real data example can be provided, it should be mentioned as a limitation (as then it would seen that the package has not been used much in practice; good to know for potential users). Note also that high-throughput data may be very heterogeneous, being a mixture of data with various noise levels. It seems that this realistic setting has not been explored for the R package "sicegar". 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: SICEGAR: R PACKAGE FOR SIGMOIDAL AND DOUBLE-SIGMOIDAL CURVE FITTING Review round: 2 Reviewer: 1

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: No comment Additional comments: The authors have adequately addressed all raised points and the manuscript is now suitable for publication. I especially liked the now included thorough analysis of the different noise regimes. One small grammar issue: Line 280: Unlike sicegar, ...

You are one of the reviewers, your task is to write a review for the article. You will 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: SICEGAR: R PACKAGE FOR SIGMOIDAL AND DOUBLE-SIGMOIDAL CURVE FITTING Review round: 2 Reviewer: 2

Basic reporting: The manuscript now complies with rules of basic reporting. Experimental design: More details and explanations have been included. The manuscript reads well now. Validity of the findings: A data example has been included, providing added value to the manuscript. All results (real data and simulations) fit together. Additional comments: The manuscript is much improved (a detail: make sure poliovirus is written the same way throughout the manuscript). No further 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: GIANT ANTS AND THEIR SHAPE: REVEALING RELATIONSHIPS IN THE GENUS TITANOMYRMA WITH GEOMETRIC MORPHOMETRICS Review round: 1 Reviewer: 1

Basic reporting: 1. Clear, unambiguous, professional English language used throughout. Yes. 2. Intro & background to show context. Yes. 3. Literature well referenced & relevant. Yes. Only few items can be added. (See "General comments" №№ 2, 3, 5, 21) 4. Structure conforms to PeerJ standards, discipline norm, or improved for clarity. Yes. 5. Figures are relevant, high quality, well labelled & described. In general, yes, but I detected missing data in one figure and ambiguous labels in another one. (See "General comments" №№ 4, 10, 14, 15, 17, 19) 6. Raw data supplied (see PeerJ policy). Yes, presented completely. Experimental design: 1. Original primary research within Scope of the journal. Yes. 2. Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap. Yes. 3. Rigorous investigation performed to a high technical & ethical standard. Yes. 4. Methods described with sufficient detail & information to replicate. In general, but some parts need correction, another ones have to be reformulated. (See "General comments" №№ 7, 8, 11, 13, 14, 18, 19) Validity of the findings: 1. Data is robust, statistically sound, & controlled. In general, but I suppose, that some of the data can be analyzed additionally for more supported results. (See "General comments" №№ 10, 11, 14, 17, 19) 2. Conclusions are well stated, linked to original research question & limited to supporting results. i. In general, conclusions are well stated, linked to original research question & limited to supporting results. However, I suppose, that some of the data can be analyzed additionally for more supported results. (See "General comments" № 14, 19) ii. It would be better if authors provide diagnostic keys for the determination of the investigated fossil species. Additional comments: Comment № 1-(lines:107-108)-You should not use “genus” for the collective group name (Formicium) Comment №2-(lines:109-110, 116, 117, 119)-Check taxa names. According to International Code of Zoological Nomenclature (article 51.3): «Use of parentheses around authors' names (and dates) in changed combinations. When a species-group name is combined with a generic name, other than the original one, the name of the author of the species-group name, if cited, is to be enclosed in parentheses (the date, if cited, is to be enclosed within the same parentheses).»For example, Titanomyrma gigantea (Lutz, 1986), Formicium berryi (Carpenter, 1929) Comment №3-(lines:119, 121)-Check taxon name. Formicium mirabile, not mirabilis (according Bolton, 2017. An Online Catalog of the Ants…) Comment №4-(lines:121-Authors write: “The single specimen of F. mirabilis is not included in the analyses.” Why? Please, give reasons. Comment №5-(lines:195-198, Fig. 1A)-Authors write: “classical nomenclature is slightly modified to be shorter and more stringent”. In my opinion, this modification is not accurate. Following to the Fig. 1A: Cu1 (or R1) – is the name of the longitudinal vein (it means that it is first branch of cubital (or radial) vein). But M1, M2 etc. look like previous names, but their mean different. M1 and M2 are only parts of longitudinal vein (first or second part (fraction) of medial vein), etc. Moreover, some numbers look like indexes, but not systematically applied, and indexes never appear in the text. I recommend to turn back to nomenclature of Brown and Nutting (1949) or to use actual nomenclature for Hymenoptera (see, for example, works of Denis J. Brothers, Hasan H.Basibuyuk, A. P. Rasnitsyn, K. S. Perfilieva, also at site of American Entomological Institute http://www.amentinst.org ) Comment №6-(lines:213)-Put “reduction cross vein 1r” instead of “reduction first radial vein 1r” Comment №7-(lines:343-344)-Authors write: “…20 PCs (highest dimensionality in MorphoJ), of which PCs1-6 describe more than 5 % variance and PCs1-3 describe more than 10 % variance.” Obviously error. The sum of PCs1-6 can not be less than the PCs1-3. Comment №8-(lines:357-363)-Use here and in the whole text “left and right side” to name forewings of one specimen. Because a part and counterpart in paleoentomology, are the matching halves of a compression fossil, a fossil-bearing matrix formed in sedimentary deposits. Therefore, it may be a confusion. Comment №9-(lines:380)-Put “connection between M+Cu and anal veins” instead of “connection between the medial and anal veins” Comment №10-(lines:388-390, Fig. 2A)-Better for understanding, if objects of observation will be labelled in Fig. 2A Comment №11-(lines:416)-Obviously error: “male wings being narrower” should be “female wings being narrower” Comment №12-(lines:447)-Misprint “0” Comment №13-(lines:450)-centroid size, not “size” Comment №14-(lines:457-459, Fig. 4, 551-552)-“Five Eckfeld specimens fall in the range of small T. simillima females but are nevertheless assigned as males: PE-1990-582_Mir, PE-1992-258_Mir, PE-1992-506_Mir, PE-459 2000-15_Mir, and PE-2000-18_Mir.” Further explanation of this decision is not supported by the calculations. I think that authors can present a range of wing lengths for these specimens and mark specimens in Fig. 4. Moreover, to support this decision (all Eckfeld specimens are males), it will be better to analyze only Eckfeld sample. Authors analyze Messel sample separately (topic “Shape discrimination between Messel species” Fig. 3), but not Eckfeld sample. Comment №15-(lines:465)-Fig. 4A, not 5A Comment №16-(lines:467)-Misprint “0” Comment №17-(lines:Fig. 4C)-Missing two objects Formicium berryi and F. brodiei Comment №18-(lines:499)-“The negative loadings of PC1 that separate males of T.simillima…” I’m sure, that authors mean “values” instead of “loadings”. In this case, it is not the same. Loadings in PCA are related with variables (parameters, features) but not with objects. Please, check this terminology in the whole text. I see correct application in the lines 504 - 505. Comment №19-(lines:517-518, Fig. 7)-“The shape-similarity between other specimens from Eckfeld with PE_1998_17-LS in PC1 and PC2 leads to the assumption that they are conspecific.” Sorry, but I don't see shape similarity in figure 7. Why PE_1994_167_LS absent in Fig. 7? In general, two specimens (PE_1998_17-LS and PE_1994_167_LS) needed to be discussed more clearly. Comment №20-(lines:537-539)-See the comment № 19 Comment №21-(lines:687-689)-See Perfilieva, K.S.[Variability of quantitative characteristics of wings by the example of some ant species (Hymenoptera, Formicidae)] in Russian // Uspekhi sovremennoi biologii. — 2007. — Vol. 127, no. 2. — P. 147–156.

You are one of the reviewers, your task is to write a review for the article. You will 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: GIANT ANTS AND THEIR SHAPE: REVEALING RELATIONSHIPS IN THE GENUS TITANOMYRMA WITH GEOMETRIC MORPHOMETRICS Review round: 1 Reviewer: 2

Basic reporting: Authors show an interesting study concerning Geometric Morphometrics of ant’s wings that include fossil specimens with the aim of discriminate sexes and species. In general, the literature references and field background are enough to contextualize this study. The English language should be improve to ensure that the international audience can clearly understand the text. I suggest to use professional and unambiguous language. Some examples where the language could be improved include lines 300, 304, 355, 427-428, 495. The current phrasing makes comprehension difficult. Experimental design: This study represent an original research concordant with the aims and scope of this journal. The research question is well defined and the information obtained may help to the discrimination of these species. The methods described are conceptually detailed but they lack of some relevant information to estimate the robustness of the analysis. Validity of the findings: Results found could represent a valuable help in the entomological studies of this taxonomic group. However, the statistical analysis, the results as well as Discussion should be improved. Additional comments: Material & methods should be improved to estimate the robustness of the study. The results as well as the Discussion should be also improved. I suggest the following changes: • • Please, review the sample size since it is inconsistent in the abstract and the lines 168, 169, 170. Please consider exchanging the current table 1 by a table that condense the number of individuals of T. gigantea, T. simillima, unclassified specimens, classifier used, sex, type of wing (right or left), origin or source of the wing (Messel, Eckarft, …). • Summarize the analysis. Several analysis of the 12 subdata set are reiterative. • Include technical information of the pictures (camera, focal distance, so on) or drawing, to estimate possible optical distortion. • Include the repeatability of digitalization and indicate the number of persons that made the digitalization, which will allow estimate the precision in the digitalization and the role of technical causes on the differences found. • Please, summarize the idea between the lines 269-275. • The table 2 evidences variations in the patterns of wing venation in different species and samples. Please, explain how this situation affect your analysis considering the missing landmarks. • Include the statistical significance of the difference of Procrustes/Euclidian distances provided by the multivariate analysis. • Please consider exchanging the linear regression analysis by multivariate regression analysis to test the allometric effects. • Lines 340-363: Please consider include in the MS, figure(s) or table(s) that allow to follow your description in this section. Indicate the percentage of variance explained by the number of PCs included in the text and the P-values. • Lines 373, 385: Cite figure(s). • Lines 388-389, 392-393, 399-401: Please, shown the data in the figure. • Lines 407-410: These results suggest allometric effect. Please, explain why you did not test the hypothesis of common allometric slopes and make correction for size after testing. Include the statistical significance. • Lines 606-608: In GM, the mixture of data from different persons that makes the digitalization, introduce biased measures. Instead, it is recommended to publish the pictures that may be used by different persons. • Lines 634 – 636: In this case, it is recommended to compare statistically the magnitude of sexual shape dimorphism. • Figures 2, 3(C,D), 4, 6, 7(A,B): Please, include the percentage of explained variance in each axis or in the legend of each figure. • Please consider exchanging the current figure 8 by information included in the text. • Please consider delete the figures 9 and 10. • Focus your discussion to supporting 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: GIANT ANTS AND THEIR SHAPE: REVEALING RELATIONSHIPS IN THE GENUS TITANOMYRMA WITH GEOMETRIC MORPHOMETRICS Review round: 1 Reviewer: 3

Basic reporting: Although English is not my native language, I believe the manuscript is very well written, with an enormous amount of details that are usually only described in reviews or textbooks. I think this detailed description very important. Literature is well used and the article structure is ok.The results are very interesting and important to the field, especially for ants. The applied methods are correct, and the results support the discussion and the most of the conclusions. Experimental design: I have two significant concerns regarding data acquisition that may compromise the results and, therefore, a large part of the results. The first concern is related to the use of the two wing drawings of holotypes. It is well known that Geometric Morphometric techniques are very sensitive to detect differences in shapes, even in small scale. The use of drawings introduces a new source of variation other than the operator plotting the landmarks, that are the small distortions that a drawing may contain that are not in the original piece that was used as a model. That said, I would not recommend the use of these specimens in the analysis. If authors decide to maintain the drawings in the analysis, the results must be interpreted cautiously, due to the reasons exposed above. My second concern is related to the inclusion, in the analysis, of the wings with missing landmarks, using a statistical function to estimate the relative position of these landmarks. Again, it introduces a non-natural variation that may influence the results, minimizing the natural variation. I understand that, for paleontological studies, the introduction of this variation is acceptable, due to the rarity of the samples and the different preservation status of them. However, again, these results should be interpreted cautiously. Validity of the findings: see comment above. Additional comments: no comments

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

Title: POPULATION GENETIC STRUCTURE OF PATAGONIAN TOOTHFISH (DISSOSTICHUS ELEGINOIDES) IN THE SOUTHEAST PACIFIC AND SOUTHWEST ATLANTIC OCEAN Review round: 1 Reviewer: 1

Basic reporting: The manuscript spends considerable time discussing geographic and hydrological 'seascape' features that end up having no influence in the data. In particular, these features were not mentioned with an specific hypothesis for how the results would be expected to appear if any particular feature was important, which leads to post-hoc narration, especially considering the authors find no population structure along the South American continent, so these features simply contribute to unsubstantiated theorizing. I think the description of the seascape needs to be condensed. The English is passable (intelligible) in most cases, but it needs to be edited by a native speaker for grammar, syntax, and spelling. There are a few cases where I was not able to follow what the authors meant, but mostly just errors that distract, some of which I will note below (not comprehensive). The data have been included as a supplemental file. I do not know if this conforms to journal policy or if they need to be transferred to Dryad. Experimental design: The authors use six microsatellite loci, which to some degree are affected by null alleles or other genotyping errors, to examine the population structure of Dissostichus eleginoides along the coast of southern South America (including Falkland Islands, FI) and South Georgia Island (SGI). They discover significant population structure between the continent and SGI, and claim to find “subtle and cryptic genetic difference” among locations along the continental coast although I see no evidence of significant structure in their results. The discovery of structure between the continent (including FI) and SGI is not new: it has been shown since Shaw et al. (2004), as the author’s acknowledge, while the examination of structure along the continental coast is novel and of greater interest. I do not believe that the lack of structure precludes these new data from being of general interest, and there is simply no solid evidence that there is structure among the continental and FI sampling localities. While I agree that there is a phantom pattern in the Structure data, 1) it has not been quantified and tested for significance and 2) it seems more likely to be to be a result of using the locprior with the uncorrelated allele frequencies model, which may often introduce artifacts*. In contrast, the IBD test** and pairwise Fst show no significant divergence between the northern and southern localities. Moreover, the largest continental Fst value (albeit with no confidence interval beyond just ‘not significant’) is between GP and PW, while between the most distant localities (NP and FI) the Fst is 0. I also find it disconcerting that the magnitude of Fst found between SGI and the other localities is almost an order of magnitude lower than found by the other studies to include these localities (Shaw et al. 2004, Rogers et al. 2006). I wonder if this may be a result of null alleles in the data. This also may be affecting the inference of population sizes and past changes in population size. First, the inference of population size from gene diversity is totally different from linkage disequilibrium, except that they are generally larger in larger populations. One (gene diversity) measures long-term population size, while the other (LD) measures contemporary effective number of breeders. As the authors point out, it would not be surprising for the SGI population to have a smaller population considering the smaller available area and habitat compared to the continent. Do these values correlate to catch data? However, I do not see any other trend towards low population sizes from north to south, as the authors claim: in fact, the smallest continental population sizes were the middle localities, so there appears to be no “trend.” Also, considering that there are several instances of null alleles in the data, including at SGI, I would be very wary of interpreting the apparent heterozygote deficiency at SGI as a history of population expansion. Also, I do not even see heterozygote deficit in the data; according to Table 1, observed heterozygosity is larger than expected at 4 of 6 loci! In addition, regarding null alleles, the authors mention that they examined the data for genotyping errors (e.g. with Microchecker), but it is unclear to me how actually this was done. It is my recollection that Microchecker only checks for null alleles (putative violations of Hardy-Weinberg equilibrium), not genotyping error. This is generally done by repeated genotyping of the same sample followed by comparison of inferred genotypes. If this was done here, please state, and if not, please describe how genotyping errors (and their overall rate) was identified. More broadly, just the sheer variation in the number of individuals genotyped at different loci at each localities is worrisome to me. The authors need to explain why this variation is so great. Further, the authors need to more thoroughly justify why (if) their results are robust to null alleles since no correction was made and the only analysis that supposedly accommodates null alleles in Geneland. With regards to the inference of connectivity, it is clear based on the Fst values that 1) the connectivity between SGI and the continent should be very low, and 2) that the connectivity among FI and continental localities could/should be non-zero. While the observed values for SGI appear low (2-3%), and ARE low with respect to demographically meaningful values (e.g. for fisheries), they are actually quite high given the magnitude of population divergence (Fst) between these areas. 2% per generation immigration would rapidly erode population divergence. Given that Geneclass is known to give spurious results when the data are weak, I find these values hard to believe. For example, how is it possible that 100% of individuals can be assigned back to the continental cluster but 2.9% are putative migrants? Moreover, first-generation migrants should show up as clearly identifiable in the Structure results, but I see none. *why was the uncorrelated frequencies model used? These populations with recent common ancestry, no doubt experiencing gene flow, almost certainly have correlated allele frequencies (and admixture). The authors need to justify the use of this model or reanalyze their data with the correlated model. **Why was this correlation used rather than a Mantel test? Validity of the findings: see above comments Additional comments: Please address the following issues: Figure 1 is interesting, but at the very least needs to have the sampling localities shown. Moreover, I would have preferred to have seen a figure that showed the native range of Dissostichus eleginoides and summarized previous findings as far as genetic population structure is concerned. Considering that so few of the features depicted in Figure 1 are actually implicated in the results (and some of which are unlabeled, e.g. CHC), it seems kind of a waste. Also, “a)” is superfluous if there is no “b)” Line 83: what is “low fecundity”, what is this relative to? Whales have low fecundity. Most broadcast spawning bony fishes do not. What sort of fecundity limitation are we talking about? Line 88: taxon is singular, taxa is plural. Line 129: please correct “can act like barrier promoting genetic divergence as it have been showed in several researchers” Line 144 DID not instead of DO not Table 1. I don’t think most of these data (for individual loci) need to be included in the main body of the text. I recommend including coordinates, sample sizes, and multi-locus measures of observed and expected heterozygosity in Table 3 and moving Table 1 to supplemental. Line 296: Estimating Ne using LD cannot be used to estimate the number of clusters directly, and is only effective indirectly in limited circumstances (e.g. when confidence intervals are fully bounded and not overlapping). This does not apply in with the current results Line 345 and Figure 3 I don’t understand what the authors mean by this statement (i.e. “subtle similarity of its genetic diversity”), and if I interpret it correctly, I do not agree with that conclusion. Yes, when an additional cluster is added, it largely is found in the northern localities, but also in the Falkland Islands, which is not mentioned. A more appropriate way to examine structure within the continental cluster would be to remove the SGI individuals and re-run Structure (although the Evanno et al. method generally fails when the true K=1). However, given the Fst results, I would surmise that no significant structure will be found. Line 349 and Figure 4. While what the authors say about PCA is true (that it provides an analysis free of the constraints of HWE and LD), it generally only provides a more easily interpretable rendition of what structure and Fst tell. And the present case confirms this. Thus I think that this analysis and figure are redundant with other analyses, and only serves to clutter the manuscript. I recommend moving it to the supplemental or eliminating it entirely. Figure 5. Does not show anything of particular note. Move to the supplemental. Line 405: as I mentioned, there is no test to substantiate this trend, and without the SGI point (which is in a separate cluster and may be spurious anyway), there is no trend to speak of. Line 407-413: I find this result rather incredible given the observed vs. expected heterozygosity for this locality in Table 1. Moreover, the result may partially derive from modeling with a stepwise mutation model, so I would ask the authors to substantiate how well an SMM model fits these data? Do the alleles form a continuous distribution or is it multi-modal, suggesting that the SMM is inappropriate (and favors a two-phase model). Figure 6. I don’t find this figure particularly informative beyond a textual description of the results (as has been included, questionable though they are). I recommend moving it to supplemental. Line 431: I find this statement to be unsubstantiated in the results Line 432: Even if there were structure (which I do not see), this is post-hoc narration of the data, since to explicit test of any of the mentioned geographical features was made. This would require predicting the form that population structuring would take in response to a given feature, then testing fit to that model (aside from the ACC, which, given previous studies, should have been a foregone conclusion). Line 438: “-in a conservative way-“ is confusing; I assume that authors mean that ‘…Dissostichus eleginoides showed, conservatively, two well-differentiated…”. Also, general practice is for a genus name to not be abbreviated the first time it is used in each paragraph. Lines 454-460: see above comments. Lines 461-483: Post-hoc narration based on an unsubstantiated pattern; needs to be removed, unless the authors wish to describe why they EXPECTED to find a pattern that did not it present itself, at least with these data (there is always the possibility that further data collection will find significant structure). Lines 492-510: I found this section hard to follow. The authors skip from four populations (which the data seem to support, the continental plus FI population being one of them) to six, including a division of the continental population into two, without any explanation. The continental division is unsubstantiated, but couldn’t follow where the remaining structure comes from. Again, a figure summarizing previous results (including Toomey et al. 2016 Antarctic Science, Volume 28, Issue 5) would be useful. Line 550: see above comments regarding this “trend”. It would be interesting to compare abundance or catch data from SGI versus continental localities to know if these values are reflected there also Line 567: I think this is spurious, and the authors make no explanation (a priori or post-hoc) for why an expansion would be expected for these fishes. If anything, given increasing fishing pressure, I would have expected a decline. Line 573: sentence fragment

You are one of the reviewers, your task is to write a review for the article. You will 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: POPULATION GENETIC STRUCTURE OF PATAGONIAN TOOTHFISH (DISSOSTICHUS ELEGINOIDES) IN THE SOUTHEAST PACIFIC AND SOUTHWEST ATLANTIC OCEAN Review round: 1 Reviewer: 2

Basic reporting: # MAJOR COMMENTS Canales-Aguirre et al. present a study of the population genetic structure of Patagonian toothfish (Dissostichus eleginoides) populations from sub-Antarctic waters over continental shelf habitats around the South American cone eastward to South Georgia Island. The goal of this study was to use genetic markers to infer potential causal mechanisms responsible for population genetic structuring in marine fishes of these sub-Antarctic waters including part of the Southern Ocean, using D. eleginoides as a study system. The authors obtained a large sample of D. eleginoides from a small number of populations across part of the geographical range of the species and evaluated population structure and demographic history using traditional population genetic analyses of microsatellite markers. Overall, the authors generate a molecular dataset that contributes to our knowledge of genetic variation and geographical structure of economically important D. eleginoides populations. But, while the paper could become suitable for publication in PeerJ in the future, significant improvements need to be made first. My recommendation is therefore Reject with encouragement to resubmit after a major revision. The paper is fairly well written, considering that English is not the authors’ first language. However, a number of changes and improvements to the written English will be required before the paper can become acceptable for publication. These include many minor edits to improve grammar and orthography, as well as more difficult passages that need to be restructured or completely re-written. I have outlined a number of these changes below, but my comments may not be exhaustive and there may be other errors I have not identified. The authors should carefully inspect their paper so that changes are made following these recommendations throughout, where necessary. *Please have a native English speaker edit the manuscript prior to resubmission to avoid any further English issues.* There are other minor issues with notation, tables, and figures that I also address and suggest improvements for below. However, I find the following three issues more troubling: #1) the authors demonstrate problems of focus from the Abstract forward and poorly formulate and test their hypothesis; #2) the authors do not accurately and straightforwardly present existing knowledge of genetic structure in the focal taxon, based on previously published studies, or properly cite other studies in the Methods; #3) the paper is too long, relative to what I would consider warranted by the content and size of the dataset/sampling (though this can be partly alleviated by fixing #1 above). ## Issue #1: Focus and Hypothesis Statement Within the first two lines of the Abstract, the authors need to set their focus on the study region/system of interest; as written, these two lines are too vague (though this can be OK for an opening sentence). The Abstract is weakened by not zeroing in on the question and region of interest (continental shelf waters of the southeastern Pacific and southwestern Atlantic around South America, plus part of the Southern Ocean) soon enough and leaving the second sentence too open since. For example, the study area is not the only region with deep-sea habitat. The entire ocean (~>90%) is largely made up of deep-sea zones, with only a minor percentage being comprised of shallow photosynthetic or aphotic zones. *The section on Lines 72-76 echoes with the same problem of lack of precision seen in the opening lines of the Abstract. I don’t think using the focal species as a model system for addressing such a general question will give results that will be broadly applicable to all deep-sea fishes.* You are simply not going to be able to do as you propose and “generate an overall microevolutionary scenario for how the [deep-sea] species… have originated and changed over time” by looking only in this area of the world’s oceans. In fact, the authors’ conclusions are unlikely to be general to any of the three oceans that they have sampled, because they have only sampled a very small portion of each one. They haven’t even sampled the Southern Ocean except for one sampling locality at South Georgia--the Southern Ocean extends all the way around Antarctica! *As a result, the authors would do well to focus on setting up a hypothesis for their study area, principally around continental shelf zones of South America over to South Georgia, and then test that hypothesis(es), and then only attempting to generalize to that area. The authors should re-write this section and the next paragraph to take up this focus (e.g. using the available literature to propose and test a biogeographical hypothesis for the study area).* Going further along these lines, *the section at Lines 123-137 seems to contain the hypothesis the authors really want to test, as well as potential mechanisms driving population structure in the focal taxon. However, the hypothesis could be stated more clearly, and this section also suffers from redundancy, as paragraphs above it.* Specifically, sentences on Lines 128-133 are redundant and should be unified to a single, cohesive (non-redundant) whole. These lines, coincidentally, also show **very poor** English. This section has not been carefully proofread, and this and surrounding sentences must be rewritten. Please let a native English speaker review this section after re-writing, prior to next submission. Looking down in the Discussion, there are two other sections that it might help the authors to move up into the Introduction, in a new section formulating a better hypothesis(es) (and predictions); those sections are found at original Lines 441-454 and Lines 467-478. Moving some of this up to the Intro could help improve the hypothesis statement, and it could also reduce some of the redundancy in the text. Importantly, bringing some of this material up to the Introduction might allow the authors to form a priori hypotheses about expected patterns of population structure/demography in the southeastern Pacific Ocean area of their sampling. This is **very important** as this is the main area of novelty this study brings in terms of sampling and the advance it makes in our understanding of genetic variation in the focal taxon--apparently, no other studies have ever evaluated genetic structure in D. eleginoides in this region using suitable genetic markers. ## Issue #2: Flawed Literature Review and Citing The literature review presented in the Introduction is flawed in several ways, some related to the poor formulation of the hypothesis. First, I do not recommend that the authors base their study on an essentially unaccessible book or white paper from Oyarzún et al. (2003), as discussed on Lines 98-105 and elsewhere. Perhaps this is the only resource available. However, it is not possible for the reader to quickly or easily verify these claims, because the paper is not available in the primary literature or online. Fortunately, I can offer a solution. In order to avoid the reader simply having to take your word for it, I recommend, especially if the Oyarzun et al. (2003) data belong to the fourth author (who goes by a similar/same name), that the authors reprint a figure or table from Oyarzun et al. (2003) as a supplementary file showing that D. eleginoides in fact shows no population structure in the southeast Pacific Ocean. Second, the authors present a rather disingenuous and inaccurate accounting of previous genetic studies of the focal species, D. eleginoides, by Shaw et al. (2004) and Rogers et al. (2006). These two studies are only tangentially mentioned, if at all, in the Introduction, are not used to formulate any hypotheses a priori. Much later, however, they resurface during the Discussion, where the authors state they recover some of the same patterns of population structuring as Shaw and Rogers, on Lines 489-491. This trend must be reversed, and a clear and accurate review of these studies must be provided before I will look at the paper again. The authors would do well to formulate a priori hypotheses based on results of Shaw et al. and Rogers et al., and then test those hypotheses using unlinked genetic markers. In addition to the above issues, the authors have not properly provided citations for the microsatellite loci that they studied. Although they cite one previous study by Appleyard and colleagues on Lines 194-197, this is not the study in which the loci were developed, which are not cited. The authors should cite the following two papers (citations given below) in which the loci were developed. This is open-access publishing, so we have room for more citations (but the paper is still too long, overall, as noted under Issue #3). Citations to add: Reilly A, Ward RD (1999) Microsatellite loci to determine stock structure of the Patagonian toothfish Dissostichus eleginoides. Mol Ecol 8(10):1753–1754 Smith P, McVeagh M (2000) Allozyme and microsatellite DNA markers of toothfish population structure in the Southern Ocean. J Fish Biol 57(Suppl A):72–83 ## Issue #3: Paper Is Too Long The paper is far too long, at >30 pages and probably >10,000 words, given the number of loci and only partial sampling of the species geographic range, in addition to partly negative results. There are some sections where the authors cut cut the most, especially the Discussion, which is about 1-2 pages too long. The first paragraph of the Discussion section is a brief outline/overview of what is discussed below it, without strongly stating support or rejection of the hypothesis. SO, the authors could simply delete this paragraph. However, in that case, I think the description of congruence between genetic differentiation and province and ecoregion boundaries should be kept, only moved further down in the Discussion. The authors can also cut length by cleaning up the writing so it is not so redundant, and by focusing in on one or two very specific hypotheses in light of the biogeographical context of the study area (only). Experimental design: Given other studies of D. eleginoides have been published based on combined analyses of 5-7 microsatellite loci and mtDNA gene sequences (e.g. 12S) (Shaw et al. 2004; Rogers et al. 2006, cited in main text), it is somewhat puzzling that the authors do not follow suit here and include data from mtDNA sequences as part of their study. Moreover, given that >10-12 years have elapsed since these previous studies, I was suprised to find that the authors are still using a smaller number of loci is used than I find to be common in most such studies, which often employ at least 10 microsatellite markers. I was also very surprised that mtDNA genes were not included, even from a partial sampling, given that fish mtDNA genomes are readily and cheaply sequenced today. These are not damning issues, but I wonder why the authors have designed their study this way, and I would like to hear a response or discussion on that, for example why 6 microsatellites is rigorous enough to address the questions that they are interested in. Two aspects of the experimental design that partly overcome the limited number of loci are 1) that the loci are informative and 2) that the authors sampled many individuals within each population, apparently from 24-71 individuals. So, this should give them good sampling properties for inferring allele frequencies etc. The methods, as written, also seem quite replicable, and I am pleased to see that the data are included in an appropriately formatted supplementary file (avoiding their placement in online repositories, although this might also be a good idea; for example, the authors could store the data in Dryad or Zenodo). Nevertheless, the authors do a poor job of discussing the need for mtDNA or other marker types to be integrated with their data to improve their inferences. In particular, I felt the statements on Lines 482 to 483 are right but could be expanded upon. For example, what do these other data types offer that microsats do not? Validity of the findings: The novelty of this paper is partially undercut by the fact that previous studies that used more spatially and numerically (in terms of nucleotide / locus) extensive sampling also arrived at similar conclusions (e.g. Rogers et al. 2006). However, this paper contains what appear to be statistically valid analyses, and thus is consistent with Aims & Scope of the journal. See other sections where I have made recommendations about the hypothesis-testing approach used in the paper, and how to improve on it. Additional comments: # MINOR COMMENTS BY SECTION ## ABSTRACT See Major Comments above. After these are fixed, I will thoroughly review English in the Abstract; however, here are a couple suggestions: Line 27 – Causal mechanisms “form” or “are responsible for” or “underlie” phenomena observed in nature, they are not “of” those phenomena. Change “of” on this line to something like “potentially underyling…” Lines 28-29 – Your study area isn’t really these regions, but rather is confined to continental shelf waters around the southern tip of South America and only one site within the Southern Ocean proper. Change the wording here so that it doesn’t sound like your study area is larger than it really is. Line 35 – Change “Georgia” to “South Georgia” in order to make your writing more specific. Georgia is a state in the USA and a small country in the Caucasus of Eurasia; don't leave any possibility of being confused with these, not even for a millisecond. ## INTRODUCTION Line 48 – This should say, “…that populations of deep-sea animals with broad geographic ranges had low genetic variability.” That’s what you really mean. Otherwise it might read as though you meant deep-sea animals should also have broad ranges. Line 49 – Change for improved readability. This should say “it has often been assumed” or “workers have often assumed…” This line would also be improved by saying “speciation in the deep sea”; there is a worrying pattern of breaking sentences with commas. You are not speaking; you are writing a scientific paper, so it’s better not to have so many of these. Line 50 – I think you mean “geographic isolation by distance”, rather than “geographic distance”. Please change this. Line 51 – Change “that marine organisms have” to read, “of marine organisms.” Line 53 – You really want to contrast the preceding sentences with the information that is given starting on this line. So, why not start this sentence with “However, during the past…”? The authors also need a comma after “decade” here. Line 54 – Don’t use so many little words. Delete “the” wherever it is not necessary. We should strive for pithy, concise scientific writing, and doing this will help you achieve this goal. Delete “the” before the word “increasing” on this line. Line 55 to 56 – Change “In addition” to “For example,”. Besides that, this is too wordy. Delete “other”, change “the organisms” to “organisms” (delete little words that don’t matter!), and simplify the end by changing it to read, “may restrict population connectivity”. There is also a level of imprecision here—what “physical changes” are you referring to? Change “physical changes” to “environmental change” or something along those lines. Line 59 – If a species has population structure, then that is obviously a phenomenon that occurs within its range! Delete “in the geographic range of the species.” Line 61 – Delete the comma after (Ne) and change “Ne” so that the “N” is in italics (it is an estimated parameter) and the “e” is lower case. Do this *throughout*. Line 63 – Here, we find a sentence that is completely out of place. This should be the third-from-last sentence in the preceding paragraph. Move this sentence up to the end of Line 55, just after the Etter et al. (2005) reference. Also, I would recommend shortening it to focus only on how these gradients influence genetic structure, since the first part of it (“At present, deep-sea environments are regarded as highly complex ecosystems”) is redundant with Lines 54 to 55. Continuing on the theme of redundant sections that need to be revisited, I realized at Line 67 to 68 that the authors were re-writing the same thing over and over, that bathymetric gradients promote population structure. This is highly redundant, so this section needs to be cleaned up to avoid being redundant. Line 67 to 68 – No comma is needed after the closing parenthesis (delete that comma). Line 68 – Make this clearer. A better antonym for sessile is “vagile.” I would suggest changing “some studies of mobile vertebrates” to read, “some studies of vagile marine vertebrates”. Note that you should change “on” to “of”. Also, change “fish” to “fishes” or “fish species,” because “fish” is often used to indicate a single species (the other terms are more inclusive, referencing multiple fish species, and also sound better in my opinion). Line 71 – change “and potentially increase” to “, potentially increasing” (note I added a comma and you should too). Line 77 – Add a comma after “study”. Line 79 – Don’t call it a “commercial resource”, it is a fishery. Also, please don’t undersell this species. The Patagonian toothfish fishery is the most productive and lucrative (i.e. economically important) fishery in the entire Antarctic, Southern Ocean, and southern portions of the oceans around the southern South American cone. As far as I am aware, there is no fish or invertebrate that is more popular in the region or as an export to Europe and North America. Line 81 – Also due to its life history, including relatively small numbers of eggs and delayed onset of reproductive maturity—I know, you say the latter in the following sentence but you omit the small clutches (add them please). Line 85 – Instead of “found”, I think you mean that this species is typically fished at these depths. The Patagonian toothfish spends most of its life cycle in less than 300 m of water, then larger juveniles and adults move into deeper water to reproduce. It’s wrong to say individuals of the species are typically found in the deep sea. It’s mainly in the epipelagic seas, in terms of total time throughout the lifetime of an individual. Line 86 – Be consistent with hyphens. Use “deep-sea environment” throughout (as you have been doing), or just “deep sea environment”—use one, but not the other. Line 92 – Avoid vagueness, which can confuse the reader. You just referred to two species in the genus. So, you can’t just use “it” here. Change “it” to read “D. eleginoides”. Lines 92 to 98 – This is a long and *confusing* sentence!! Readers may struggle to find the second, complementary statement relative to “While”, because the sentence is too long. Please fix this by breaking this into two sentences. Line 93 – Again, delete unnecessary little words. Delete “its”. Line 101 – Change “challenges” to “challenge.” Line 104 – Be specific. Start this sentence by referring to isozymes, not “These molecular markers.” Lines 106 to 107 – You’ll want to focus on what really matters here—genetic, demographic, and geographic processes, not simply “environment and the biology of species.” Line 124 – Change “be” to “present”. Line 134 – What does “South American distribution” mean? It’s not distributed in South America, but along the coast/continental shelf waters there. This appears to be a prediction of the authors’ hypothesis and it is poorly stated. ## MATERIALS AND METHODS Line 144 – “do not” and “obtain” are present tense; however, M&M should almost always (with few exceptions--see below) be written using past tense. Please fix this. Line 154 – You will want to add that this species is not listed under CITES. Lines 157 to 158 – Sorry, not true! You did not sample “the entire distributional range” of the species. You sampled the portion of the species range around South America and South Georgia, but you conveniently fail to mention that the species has an extensive distribution across the southern Atlantic Ocean and Indian Ocean, which you didn’t sample. ... Lines 302 to 303 - This is one of the exceptions to the past tense rule for M&M sections I mentioned above: here, you need "corresponds" instead of "corresponded", because the symbol/abbreviations were, are, and will remain representations of specific parameters. Lines 304 to 305 - You need to change µ to "mutation rates". Next, delete the next instance of mutation rates and replace with "of which." So, I think the sentence, as rewritten, should look like this: "We used two mutation rates for μ: a) 2.5 x 10-4 and b) 1.0 x 10-5 mutations / locus / generation, both of which were based on Bos et al. (2008)." The real problem with this however is that you do not provide sufficient explanation of why you chose these rates. Please provide additional detail, including a justification of why you believe these rates are valid for the loci and taxon you studied. ## RESULTS ... ## DISCUSSION Line 425 - Remove little words that clutter your writing. You could improve this sentence by deleting the "a" before "strong." You also need to fix the same error at other points in the text, including Line 468. Lines 427 to 428 - Keep it simple. You could change "analyzed geographic area" here to simply "study area." Lines 429 and 430 - Need to refer to authors with year in parentheses here, in both cases... as in, "Camus (2000)... Spalding et al. (2007)." Line 431 to 432 - Change "subtle" to "subtly" and do not refer to "locations," because you are referring to the populations not the areas in Chile. Line 434 - Look at the present-tense word "generate" here. This should be past tense ("generated"), but also remember that there is a distinction between processes that have generated geographic population structure, and processes that maintain that structure. Line 438 to 439 - Change the dashes/hyphens for commas. This format is unusual and very, very sparingly used in academic writing, and usually avoided instead. Also change "differentiate" to "differentiated" to correct verb tense. Lines 439 to 440 – There are number and tense problems here. Change “genetics” to “genetic”, and also change “described” to “describe”. Check verb-tense agreement in this section. Line 454 - Why is Camus 2001 cited here? Probably should remove this citation. Line 457 – “STRUCTURE software does not revealed(space)” looks like an unfinished line. Please complete this sentence. Line 459 – Things are not separated “for” a distance, they are separated “by” a distance. Change this to read something like, “separated by over 2,900 km from the nearest sampling locality…” Line 461 - You don't mean the entire southeast Pacific Ocean, you mean the portion of that ocean in your study area; change the text to reflect this. Lines 462-464 - It is my opinion that the end of the sentence that starts the paragraph needs to be fixed to say that the upwelling pattern has been correlated with changes in species composition and genetic isolation of marine taxa, so as not to imply that you know that it caused these, to have proof of mechanism (we work in a probabilistic framework). Also, remove the comma after the closing parenthesis on Line 463. Lines 466 and 471 – You do not need to hyphenate cold water, unless it is written to modify the word that follows the phrase (e.g. cold-water fusion). Thus, remove the hyphen between “cold” and “water”. Line 467 – Delete “their” before “populations.” Line 468 - Delete the comma after "inodorus," which is unnecessary. Lines 472-474 – It looks as though translating from Spanish to English did not work well here. As a result, the order of the clauses in the sentence is jumbled. Please rewrite this sentence to improve clarity by placing "as a barrier promoting genetic isolation" after the word "suggested." Line 483 - Need to define abbreviations at first point of use. Here, since you don't really use this abbreviation much in the paper (and not after this point of use), you should simply write out the meaning of SNP, rather than using the abbreviation. Change "like SNP" to ", such as single nucleotide polymorphism data, "... Line 492 - Simplify and clarify the writing by changing, "of D. eleginoides, its," to read, ", D. eleginoides." Line 514 - The populations don't "have" connectivity per se; instead, the populations appear to be connected, or exhibit evidence of connectivity, based on your results. Lines 529 to 530 - Again, avoid using these hyphens to separate clauses. I suggest that you place the clause between hyphens inside of parentheses instead. ## TABLES AND FIGURES Tables are pretty good, as constructed; however, I'm not sure they adequately note where p-values have been given Bonferroni's correction. I think this information should be included/referred to in the table captions. The figures are beautiful and mostly well designed and appropriately labelled. However, K is an estimated parameter, not a known quantity, so it should be italicized in mathematical notation. Thus, go through the figures and text and make sure K is in italics throughout (see similar comments elsewhere (e.g. Table 1 comments below) for similar recommendations for Ne and other popgen summary statistics/parameters). This was a striking mistake, since "Q" was appropriately placed in italics in the figures and captions (e.g. caption to Fig. 3), while "K" was not. Figure 1 - The Cape Horn Current (CHC) is not labelled on the diagram. Please add the abbreviation in the appropriate place. This figure could also be improved by adding country names for South American countries shown, as well as the name for nearby islands. Table 1 Locality column – It is not appropriate to only given latitude and longitude coordinates to the level of detail of degrees followed by minutes. This is insufficient. Coordinates should be given as degrees, minutes, seconds, or in decimal degrees format. Please change this. Table 1 Parameter column – Here, and throughout the entire manuscript, for NA, Ho, and He, these parameters should be placed in italics because they are estimated parameters rather than known values. For Ne, for effective population size, it’s the same thing: place the “N” in italics, as I mentioned above. Table 2 caption - Bold values do not represent p-values at all. Fix this by stating that the Fst values with p-values of P < 0.001 are shown in boldface. Table 3 - "MIG_PUT" is not an appropriate abbreviation for this variable. Use just "Mig." or "PPM" or something logical, not a variable name with an underscore. Also at line 392 within the caption, I think you meant to refer to populations instead of localities, and also you should state that these localities are on the South American coast or continental shelf. Figure 5 - The "F" in "Fst" and the "p" in "p-value" must always be italicized because these are estimated parameters. Change this throughout the manuscript (e.g. also in the text on Lines 352, 353, 407 and 408, and others). Figure 6 - The dots/symbols for data points in this figure are small and hard to see or differentiate. Please make the symbols more legible by making them larger and filled in. Also, following along the same lines as above, the "H"s in "He" and "Heq" should be given in italics in the figure and caption.

You are one of the reviewers, your task is to write a review for the article. You will 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: POPULATION GENETIC STRUCTURE OF PATAGONIAN TOOTHFISH (DISSOSTICHUS ELEGINOIDES) IN THE SOUTHEAST PACIFIC AND SOUTHWEST ATLANTIC OCEAN Review round: 1 Reviewer: 3

Basic reporting: #The text is in general very clear but need some minor adjustments as provided to the authors. #The authors showed a large biological background on the species studied and also a good background on abiotic features afecting the phenomenon studied. #Figure 1 could be improved. My suggestion is to provide the sampling sites on in. All other Figures are ok. #The raw data (msats genotyping) was not provided. #One table could be moved to Suplementary material as mentioned to authors at General comments for the author. #The defense of the hypothesis is the critical point in the text. But I am sure the authors can offer a better and focused view on the phenomenon detected. My suggestions are also provided at the General comments for the author. Experimental design: #The research is very original and is fitted to the journal. #The questions are clearly posed. #The investigation was well performed. #Methods were tiresome describe. The authors need to provided a table with the loci genotyping among individuals. Validity of the findings: #The findings are very interesting an important for fishery management. #The data are very robust. #Clear conclusions and hypothesis are the critical points in the text. But I am sure the authors can offer a better and focused view on the phenomenon detected. My suggestions are also provided at the General comments for the author. Additional comments: Sugestions: INTRODUCTION LINES50 -53: It is important to comment briefly that currently there are lots of evidences that marine environment is not so homogeneous in terms of genetic discontinuities. It is necessary to include around 3 papers from 2014, 2015 and 2016 on this issue. LINE63: "At present????…(Chase et al. 1998; Etter et al. 2005; Zardus et al. 2006)." The youngest citation with 11 years. It needs to be modified. LINES63-76: The authors need to update the references used. For example, why not to use AMY R. BACO, RON J . ETTER, PEDRO A. RIBEIRO, SOPHIE VON DER HEYDEN, PETER BEERLI and BRIAN P. KINLAN (2016). A synthesis of genetic connectivity in deep-sea fauna and implications for marine reserve design. Mol.Ecol 25, 3276–3298???? The antiquity of the papers is repeated throughout the text and it must be altered. LINES 133-134: The phrase "Therefore we expected find that D. eleginoides populations are genetically structured in its South American distribution" must be removed. The main hypothesis of the study is very clear and the authors do not need to improve it with a sub-hypothesis as posed. M&M LINES152-154: The phrase "No specific approval of this….." is unnecessary and might be deleted. LINE208: Please correct "…only one loci in the location GP…" to "…only one locus in the location GP…". LINE210: Please correct "Although (Chapuis & Estoup 2007)) proposed…" to Although Chapuis & Estoup (2007) proposed…" LINE215: Please substitute "sibship" to "kinship". LINES215-216: Please substitute "full-sibs" to "total kinship" in the whole text. LINE219: Please substitute "outcomes" to "results" LINES236-240: It is important to measure the RST parameter because it indicates specific differences for msat loci. LINES275-277: The phrase "This kind of multivariate method…" is unnecessary. Important: The authors must use the Rst parameter. This is specially addressed to a msat dataset. The Rst values can be obtained easily in Arlequim. The authors can also improve the table 2 offering the Rst values up of the diagonal. In general the text can be optimized by excluding lots of method explanations. Most of the text offered by authors in M&M refers to method explanations. In general the community knows the general basis of those methods used. RESULTS LINES410-413: Results must have only the description of the obtained results. The phrase comprising these lines indicates a conclusion. Explanations, conclusions, and new hypothesis are expected to be in the Discussion section. Table 3 must be better edited. It was almost impossible to understand it. Figure 6. I suggest this figure to be moved to supplementary materials. DISCUSSION Line 457. Please change from "cryptic genetic" to "incipient genetic". Lines459-460. Change the phrase "…and are separated for more than 2,900 km of distance from the next sampled locality (i.e. Quellon)." by "…and are separated for more than 2,900 km from one region (Northern and Southern Peru) to another (Iquique)." Line 477. After Wares et al. 2001, delete the word "these". Lines 478-481. The authors are a little confused to explain the genetic structure observed between the northern and southern pacific samples of Patagonian toothfish. In one moment they argument in favor to a IBD (isolation by distance…2900km in distance). However they provide the IBD test that failed to support it. In another moment of the discussion the upwelling zone would be the support for the genetic division within those demes. Based on the evidences provided by authors I suggest they argument in favor to incipient allopatry by upwelling zone. Line 540, 541. change "infinity" by infinite" The topic "Contemporaneous demographic history" is very confuse and must be revised. Previously authors showed high migration rates and in the line 546 is written "In our study we can discard immigration given the scarce number of estimated migrants,…". I suggest the authors to be focused. At several points in the discussion the authors insist to explain methods. This is not adequate. Another feature of this text is the double-hand stile. The authors start explaining something in a way of thinking and immediately after that they change the way of thinking offering another explanation. It is also inadequate. The authors need to defend what they actually believe. The evidences are very clear and I do not understand why the text is sometimes so confuse. Lines 557-563 This veer long phrase "Consequently, the South Georgia cluster has a higher probability of being affected by stochastic processes (e.g. environmental changes, demographic or genetic changes) and therefore, has a higher risk of extinction (Palstra & Ruzzante 2008) and loss of genetic variation (Frankham et al. 2003). Stochastic factors are commonly initiated by deterministic factors (e.g. natural selection or harvesting; Palstra & Ruzzante 2008), like fishing efforts that could affect and reduce the population size of D. eleginoides in its Southeast Pacific and Atlantic Ocean distribution." is not correct. I did not understand the relationship between habitat availability (at this case a large area) provided by authors. Species with small habitat size in general are more susceptible to those mentioned stochastic events. If not, why conservation biologist as us are so concerned o understand the genetic structured populations? Closed or limited gene flow in terms of habitat availability has more impact than in those species occurring in large habitat availability as showed by authors. Lines 567-569: "Finally, we suggest that D. eleginoides has recently experienced a population expansion in the Georgia cluster, based on the significant heterozygote deficiency obtained in BOTTLENECK"… is this conclusion correct? Based on another methods regarding tests on historical demography it is expected that populations experiencing expansions has more variation than contrary. Please revise it.

You are one of the reviewers, your task is to write a review for the article. You will 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: POPULATION GENETIC STRUCTURE OF PATAGONIAN TOOTHFISH (DISSOSTICHUS ELEGINOIDES) IN THE SOUTHEAST PACIFIC AND SOUTHWEST ATLANTIC OCEAN Review round: 2 Reviewer: 1

Basic reporting: I consider this paper to be a marginal "pass" in the category of "Basic reporting," but this "pass" score is conditional on the fact (and _required future outcome_) that basic presentation needs to be improved before the paper could become suitable for publication. I give a more detailed list of suggestions for helping with this below, under the "General comments for the author" section; here I explain my score for Basic reporting. Under the category of Basic reporting, I appreciate that the literature references and background information provided in the revised manuscript are much better than that of the initial submission at the beginning of the year. Also, given that the authors have followed my suggestions to re-write the paper to have a clearer focus on the study organism/area and to clearly state an a priori hypothesis of no genetic structuring within the focal taxon, I would generally agree that the paper is now mostly self-contained, with results that bear directly on the hypothesis. However, the English has a number of orthographic errors, and exhibits unprofessional aspects of content in writing and the table and figure captions. Second, and more importantly, the paper as written does not adequately describe or discuss the results, with problematic sections mainly being the Abstract and Discussion, but also part of the Introduction. Experimental design: No comment. Validity of the findings: No comment. Additional comments: # MAJOR COMMENTS Canales-Aguirre et al. present a study of the population genetic structure of Patagonian toothfish (Dissostichus eleginoides) populations from sub-Antarctic waters over continental shelf habitats around the South American cone eastward to South Georgia Island. I reviewed the first submission of this manuscript, in which the goal of the study was to use genetic markers to infer potential causal mechanisms responsible for population genetic structuring in marine fishes of these sub-Antarctic waters including part of the Southern Ocean, using D. eleginoides as a study system. This goal was overstated and over-generalized, and I suggested that the paper needed to be re-written due to this and other issues including a number of glaring problems with the written English. After pointing out these shortcomings, I summarized three major concerns in my previous review, as follows (paraphrasing): *Issue #1:* problem of focus and poor formulation and testing of the hypothesis(es) of the study; *Issue #2:* inaccurate and inadequate review of existing knowledge of genetic structure in the focal taxon based on previous studies available in the literature; and *Issue #3:* paper length not warranted by content and size of the dataset/sampling and scope of study (paper too long; related to #1 as well). Regarding Issue #1, I commend the authors for re-writing the paper to align with some of the main aspects of my suggested focus—i.e. focusing on the focal species alone, and the study area within which that focal species was sampled. I have also stated this in my assessment of the paper under the Basic Reporting category of the review form. Moreover, the authors have improved the paper by explicitly stating the hypothesis in the Abstract and Introduction. In response to Issue #2 that I raised previously, the authors have re-written parts of the paper, especially in the Introduction, to provide a more suitable account of the state of knowledge of genetic variation and population structure within D. eleginoides at the onset of their study, and I appreciate that aspect of the revised manuscript. Also, as a result of my initial comments and those of Reviewer #1, the authors have deleted a number of problematic sections (containing potentially spurious analyses or weak conclusions/weakly supported patterns) from the manuscript, and to an extent this has alleviated issues revolving around my point raised under Issue #3 above. The length of the paper is now suitable for publication. Nevertheless, I still find several issues that keep the revised manuscript from being suitable for publication at this time, and, in particular, issues arise because (1) I find the authors’ handling of Issue #1 has been less than satisfactory—they need to better relate their results (e.g. in Abstract and Discussion sections) back to the hypothesis, and (2) there are a number of issues with the written English and presentation that preclude the paper from being suitable for publication. Quite frankly, I am slightly angered that the authors would state multiple times in their rebuttal letter that “Matthew Lee native speaker and Marine Biology Ph.D.” has proofread the new version of the manuscript, so that “this new version should not have any typography error. Other issues associated with notation, tables, and figures were also checked.” This is just *_ridiculous_*. As noted below, I found _two errors with the English in the first line of the Abstract alone_. If Matthew Lee has given the final revised manuscript draft an in-depth read through and actually provided corrections, then only one of three possible scenarios could be true: (1) the English in the revised draft is poor because he did a _terrible job_ (I can assure you, his performance was really awful in this case, and you should *never use him again for proofreading*); (2) Matthew Lee did a good job, but you didn’t follow his recommendations (shame on you); or (3) Matthew Lee did an OK or good job, but you chose to go on your own and edit the paper substantially afterwards, and many errors crept into the manuscript during this period. The next time that someone recommends you to take your manuscript and have it fixed by a native English speaker, please do a better job and present a better final draft than what you have provided in this revision! And it’s probably best that you _not_ use Matthew Lee!! Due to these issues, I consider this manuscript to warrant a decision of Reject with encouragement to resubmit a paper with Major Revisions. The only reason I feel that this will be a Major Revision, and not a Minor one, is that I agree with the journal description of this type of Recommendation, in that I would “prefer to re-evaluate any revised version,” and my suggested edits for correcting the English (below) are extensive. # MINOR COMMENTS As a general suggestion, never submit a Word document to an English journal that has the language setting set to Spanish. This indicates to the Editor and Reviewers that your experience during writing was one in which the Spanish spell checker, and not the English spell checker, was used during writing. Moreover, this has obviously caused you to miss several mistakes that a spell checker in English could have provided you with. So, when writing, and when revising papers, *always* perform the following procedure when creating the document: (1) select all text/content in your .doc or .docx file (i.e. press ctrl + A); (2) click on Tools > Languages…; (3) select English (US) if the paper is destined for an American journal (one that uses American English conventions) and select English (UK) if the paper is destined for a British journal (or one that uses British English spellings); and (4) then save the file and go back through it looking at possible errors highlighted with underlining in the text (possible misspellings or grammatical errors). **This is very easy to do, and everyone should take the responsibility to do it.** Another **general issue** is that the authors have managed to screw up the italics and subscripting throughout the manuscript. The big letters in parameter names like He, Ho, Ne, etc. are always italicized. Fix throughout. For He and Ho, it is probably more common that the subscripts be in uppercase; so make that change too. Also, these are not just "parameters", they are "summary statistics" or "statistics of DNA polymorphism." ## ABSTRACT Abstract Line 1 – How can the authors, in their rebuttal letter, possibly claim that a native English speaker has proofread the manuscript, when the _first line_ of the Abstract contains two errors?? The errors are as follows: - You need to change “population genetics” to “population genetic” - Also, the name of the focal species is misspelled as “Disosstichus eleginoides”. Please fix to read “Dissostichus eleginoides”! Abstract Lines 3 & 4 – Problems here with tense and the word “distribution” that must be fixed. The word “focus” should be replaced with “have focused” and the authors shouldn’t be discussing distribution of D. eleginoides as the focus of previous studies—they should be stating that these were genetics studies. I strongly recommend the following rewording, “…studies have focused on the genetics of D. eleginoides in the Southern Ocean… knowledge of their genetic variation along the South American continental shelf.” Other Abstract changes: - Change “structure on the South American” to read “structure along the South American.” - Make next sentence read, “We hypothesized that this species would show zero or very limited genetic structure…” - The very next sentences should read as follows: “We used Bayesian and traditional analyses to evaluate population genetic structure, and we estimated the number of putative migrants and effective population size. Consistent with our predictions, our results showed no significant genetic structuring among populations of the South American continental shelf, but supported two significant and well-defined genetic clusters of D. eleginoides between regions (South American continental shelf and South Georgia clusters).” - Change “…these clusters was 11.3%” to read “…these two clusters was 11.3%”. - Finally, add “D. eleginoides” before “populations along the South American continental shelf” in the last sentence of the Abstract. ## INTRODUCTION Line 2 – Change “which” to “that”… as in “that remain stable.” Line 5 – change “isolation by distance” to “isolation-by-distance” with the words separated by hyphens, and also provide the abbreviation for this term here in parentheses. This should read “isolation-by-distance (IBD; Wilson & Hessler 1987).” 2nd Paragraph – I have helped you by re-writing the middle section of the 2nd paragraph of the Introduction to have several minor fixes that it would be complicated for me to write out and explain. Please make the section starting with “In broadly distributed benthopelagic fishes…”, and continuing several lines, read as follows: “In broadly distributed benthopelagic fishes, considerable gene flow has been reported among populations. Scarce genetic divergence is therefore mainly the result of the availability and continuity of their habitats (e.g. continental slopes, the slopes of oceanic islands, slopes on seamounts), facilitating gene flow (Smith & Gaffney 2005; Jones et al. 2008; Lévy-Hartmann et al. 2011; Varela et al. 2012). In addition, biological features such as highly vagile and/or pelagic adults and long-duration planktonic egg, larvae, and/or juvenile stages are associated with low intraspecific genetic differentiation (Shaw et al. 2004; Rogers et al. 2006).” 3rd Paragraph – First, the correct family name for rockcods of Antarctica is “Nototheniidae” and NOT “Nothothenidae” or "Notothenidae"; please fix this spelling here and throughout (fix elsewhere as necessary). When referring to Nototheniidae at the middle of this paragraph, this would read better as, “…belongs to the Nototheniidae family, a diverse clade of Antarctic and sub-Antarctic origin.” 3rd Paragraph – Change “This continuous distribution along…” to read “The continuous distribution of this species along…”. Also you should probably say that the continuity or connectivity of the populations could facilitate gene flow homogenizing their population structure. I suggest that the first part of the last sentence of this paragraph be changed to read, "The continuous distribution of this species along the South American continental shelf in the Southeastern Pacific Ocean could facilitate gene flow homogenizing their population genetic structure..." 4th Paragraph – First line: population studies are conducted on or of species, not "in" them. Change this line to read, "Population genetics studies of D. eleginoides to date..." 4th Paragraph – Second sentence: before citing "Smith & McVeagh", move the mention of data type to the start of the sentence. Change sentence to read, "Using allozyme and microsatellite loci, Smith & McVeagh (2000) showed that D. eleginoides ..." 4th and 5th Paragraphs - I have too many edits to suggest for this section. In fact, I have more edits than I care to write out line by line. Here I provide a highly edited version of the last two paragraphs that I suggest the authors simply copy and paste into their manuscript: "Population genetics studies of D. eleginoides to date have been mainly conducted in the Southern Ocean. Using allozyme and microsatellite loci, Smith & McVeagh (2000) showed that D. eleginoides has restricted gene flow between the Falkland Islands, and zones south of the Antarctic Polar Front (i.e. Heard Island, Ross Dependency, Prince Edward Island and Macquaire Island). Later, Shaw et al. (2004) showed that populations to the north of the Antarctic Polar Front (i.e. Patagonian Shelf, North Scotia Ridge) and to the South of the Antarctic Polar Front (i.e. Shag Rocks, South Georgia) have stronger genetic differentiation in the mtDNA genome than the nuclear genome, based on microsatellites and mtDNA sequences. In a study conducted in the West Indian Ocean sector of the Southern Ocean, Appleyard et al. (2004) investigated mtDNA and microsatellite loci but found no evidence for among-population genetic differences associated with islands. Subsequently, Rogers et al. (2006), surveying samples from islands in the Atlantic, Pacific, and Indian Oceans, found genetic differences based on microsatellite and mtDNA data. Specifically, Rogers et al. (2006) indicated that toothfish populations from around the Falkland Islands were genetically distinct to those from around the South Georgia Islands. Recently, Toomey et al. (2016) studied DNA from otoliths and found differences between populations around the Macquarie Islands and other locations surveyed in the Southern Ocean. All previous studies discussed above have focused mainly on islands of the Southern Ocean, leaving a distinct gap in our knowledge of the genetic structure of D. eleginoides populations across their Southeastern Pacific Ocean distribution. The only study carried out in the Southeastern Pacific Ocean was developed by Oyarzún et al. (2003) based on allozymes and was restricted to a small geographic area. Oyarzún et al. (2003) did not find population genetic structure among samples collected in south-central Chile (c. 37ºS to 43°S). Sampling across a wider geographical area of this region while using more sensitive molecular tools that have higher levels of detection of DNA polymorphism, such as microsatellite loci, could aid in determining whether or not significant population genetic structure exists among D. eleginoides populations across their Southeastern Pacific Ocean distribution. In this study, we used a panel of six microsatellites previously developed for D. eleginoides to test whether this species shows population genetic structure on the South American Plateau. We hypothesized that D. eleginoides will not show genetic structure due to the continuity of suitable habitats along the South American continental shelf, from Peru in the Pacific Ocean southward and eastward to the Falkland Islands in the Atlantic Ocean (Fig. 1). " ** Please compare with original text and consider taking all of my suggested edits here. ** ## MATERIALS AND METHODS Ethics Statement – This paragraph would read better if parts of it were changed around, reordered to improve the readability and flow of the writing. In particular, it would be a good idea to mention the point about CITES, and how the sampling strategy obviated the need for collecting permits/government approval, near the beginning of the paragraph. Here is a re-write for you with the suggested change and some fixes to the English: “Dissostichus eleginoides has not yet been assessed for the IUCN Red List and is not listed under CITES. Samples used in this study were collected in accordance with legislation of the corresponding nations. In fact, no governmental approval of this vertebrate work was required since the Patagonian toothfish individuals sampled in this study were obtained from scientific and commercial fishing activities. We did not kill fishes for the purpose of this study; instead, we obtained tissue samples from individuals that were fished by authorized commercial vessels using long lines. Tissue samples of Patagonian toothfish used in this study were obtained from the Peruvian exclusive economic zone (EEZ) in collaboration with the Instituto del Mar del Perú (IMARPE). Tissue samples from the Chilean EEZ were obtained during scientific research programs with the permission of the Chilean Fishery Government and obtained by the Instituto de Fomento Pesquero (IFOP). Additional tissue samples from the…” Molecular and pre-processing genetics dataset - Please call the ABI 3730 an "automated sequencer". Also, you should say that you "estimated" or "measured" allele size, not that it was obtained. One fix for a problematic sentence: "Ultimately, we obtained a total data set of 357 individuals that we used in subsequent analyses." Further down... instead of saying to "identify total kinship," you should say "To estimate total kinship..." Another fixed sentence: "Results from the total kinship identification analysis did not show evidence for putative total kinship in the data set; therefore, we proceeded with data analysis without excluding any individuals. " Genetic variability and structuration index - I don't like the authors' use of "structuration" in the title here. But another issue is the vague use of the term "parameters" here. Please refer to number of alleles and heterozygosities etc. as population "summary statistics". Further down in this paragraph, Markov chain is misspelled... this should be "Markov chain" (chain is lowercase) and not "Marcov Chain". I also fixed another sentence in this section: "No pair of loci in our data set exhibited significant LD, which indicated that all the loci used in this study were independent from one another (unlinked). " Another issue in this and all sections: FST and RST should always have "ST" shown as subscript text. Fix throughout. Number of clusters and isolation-by-distance - Here, I have too many edits to write out. Please re-write as follows: "To infer the most likely number of genetic clusters (K) present in our data set, we used two Bayesian clustering methods, one in the program GENELAND v1.0.7 (Guillot et al. 2005a; b, 2008) and the other implemented in STRUCTURE v2.3.4 (Pritchard et al. 2000; Falush et al. 2003). GENELAND uses a Bayesian statistical population algorithm to model a set of georeferenced individuals with genetic data, while accounting for the presence of null alleles in the sample. The number of clusters was determined by 10 independent Markov Chain Monte Carlo (MCMC) searches, which allowed us to estimate K using the following parameters: K from 1 to 8 (which is equivalent to the number of sampling locations surveyed in this study), 5 x 106 MCMC iterations, a thinning interval of 1,000, the maximum rate of process Poisson fixed at 357, and the maximum number of nuclei in the Poisson-Voronoi tessellation fixed at 1071. Following recommendations of Guillot (2008), we ran the analyses using the uncorrelated frequency allele model, the spatial model, and the null allele model because the value of K in the study area was unknown. Finally, we plotted a map of South America over the output of GENELAND, in order to visualize the results in the context of geography. Although STRUCTURE does not account for null alleles and uses a non-spatial model based on a clustering method, it is useful for quantifying the proportion of each individual genome from each inferred population in K. The number of clusters was determined by performing 10 runs in STRUCTURE with 50,000 iterations, followed by a burn-in period of 5,000 iterations, for K = 1–9. All STRUCTURE runs were carried out with an admixture model of ancestry, an independent allele frequency model, and a LOCPRIOR model (Hubisz et al. 2009). We incorporated Evanno’s index ΔK (Evanno et al. 2005) in order to identify the best K value for our data set, using STRUCTURE HARVESTER (Earl & VonHoldt 2012). Then, we plotted ‘consensus’ coefficients of individual membership (Q) in R, followed by cluster matching and permutation in CLUMPP (Jakobsson & Rosenberg 2007) to account for label switching artifacts and multimodality in each K tested. We summarized the genetic diversity using a Principal Components Analysis (PCA) in ADEGENET v2.0, which does not make assumptions of HWE or LD (Jombart 2008; Jombart & Ahmed 2011). Finally, we conducted a Mantel test to evaluate isolation-by-distance (IBD) using the standardized genetic distance (FST / 1 - FST) and the logarithm of the geographic distance among sampling sites. To identify significant correlations, Pearson’s correlation coefficient, r, was calculated in the software program ZT (Bonnet & Van de Peer 2002), which it is a program specifically designed for conducting the Mantel test. We used 10,000 permutations to obtain a p-value and we plotted the correlation among all locations, excluding the South Georgia Islands, the most differentiated location (see Result below). We performed Mantel tests in order to test for two processes that can arise in an IBD pattern: a) a continuous cline of genetic differentiation or b) the existence of well-differentiated and disjunct populations (Jombart & Ahmed 2011). " Recent migration and effective population size - Looking at the references to methods by Paetkau et al. (2004) and Paetkau et al. (1995), these are _really old methods_. _Are these methods still valid, or have they been replaced by more recent methods that improve on them or fix errors?_ If so, or if newer methods are available for doing this, then why did the authors use older methods? The authors should provide a brief answer and explanation here. Also in this section, the authors use the term "confidence intervals" for the first time. Later in the paper (e.g. in table or fig captions), the authors abbreviate this as "IC"; however, this is the Spanish abbreviation and is **incorrect** for an English journal. _No one will intuitively know what this means, or shouldn't be expected to do so_. The authors should change this abbreviation to "CI", and it should be given here and used throughout. I suggest changing the sentence mentioning NEESTIMATOR to read as follows, "Values of Ne within corresponding 95% confidence intervals (CI) for each population were estimated using NEESTIMATOR (Do et al. 2014) ..." Another fixed sentence: "No mutation rates for microsatellites within the D. eleginoides genome have been estimated in the literature; therefore, we chose these broad ranges of mutation rates reported for marine, freshwater and anadromous fishes in DeWoody & Avise (2000) as useful approximations of appropriate rates for D. eleginoides." Recent migration and effective population size - Fixed sentences in this section: ". Each cluster showed a high percentage of self-assignment, with 89% of the SGI cluster including individuals from the South Georgia Islands and 99.3% of the South American cluster composed by locations from South America; this clearly supported patterns of genetic structure indicated by GENELAND and STRUCTURE. The same pattern of genetic structure was also supported when analyses were performed based exclusively on sampling locations (Table S3)." ALSO "Conversely, using the formula of Nei while assuming the stepwise mutation model (SMM; Ohta and Kimura 1973) and either of the mutation rate values discussed above, the northern Peru location had the highest Ne values and the South Georgia cluster showed the lowest Ne (Table 1). The maximum calculated Ne value, for northern Peru, was 2.73 times greater than the minimum calculated value for South Georgia Islands." In the above fixed sentence, I suggest that the authors write out the full name of the abbreviated model, referred to as "SMM", using its full name, "stepwise mutation model." Here, I believe the correct citation is Ohta and Kimora 1973. But someone please check this before adding. ## DISCUSSION The first paragraph needs numerous fixes. Here they are: "Overall, our results support a lack of genetic structure among the populations of Dissostichus eleginoides inhabiting the South American continental plate, but we infer strong population genetic structure between populations of this area and those of the Southwest Atlantic Ocean. We conclude that the continuity of deep-sea habitat along the continental shelf and the biological features of the study species are plausible drivers of intraspecific population genetic structuring across the distribution of D. eleginoides on the South American continental shelf." Compare this text to the original text to see that I have made several changes here. Genetic diversity and genetic divergence - Here, I would highlight how an array of "complementary analyses" were conducted on the 6 microsatellite loci, not just "different" analyses. ## TABLES AND FIGURES & THEIR CAPTIONS Table 1 - Caption could be improved by changing title to, "Mean summary statistics for genetic variability, percentage of putative migrants, and effective population size by location and cluster inferred for Dissostichus eleginoides." Also caption has errors. Try: "Locality abbreviations: NP, Northern Peru; SP, Southern Peru; IQ, Iquique; GP, Gulf of Penas; PW, Puerto Williams; DRI, Diego Ramírez Islands; FI, Falkland Islands; SGI, South Georgia Islands. Locality code SAC refers to the cluster including all locations that are on the South American continental shelf. The SGI cluster included individuals from the South Georgia Islands. Other abbreviations: CI, confidence interval; Inf, infinite; Lat, Latitude; Long, Longitude; N, Number of individuals sampled; NA, average of the number of alleles per locus, HO, average of the observed heterozygosity; HE= average of the expected heterozygosity; NA, not applicable. Effective population size (Ne) was based on Linkage Disequilibrium (LD) (Waples & Do 2010) and Nei (1987) formula. The migrants (M) column shows the percentage of putative migrants from the first generation. ( * Estimated using a mutation rate of 1 x 10-2 (refs. in DeWoody & Avise 2000) ** Estimated using a mutation rate of 1 x 10-4 (refs. in DeWoody & Avise 2000). " Table 2 - Don't use the word "index" (singular). Change the title to, "Table 2. Pairwise FST and RST indices estimated between sampling locations for D. eleginoides." Also, I suggest that the caption be re-written as follows: "Here, Fst values are shown below the diagonal and Rst values are shown above the diagonal, with estimates corresponding to p-values of p < 0.001 shown in boldface (after Bonferroni correction). Abbreviations: NP, Northern Peru; SP, Southern Peru; IQ, Iquique; GP, Gulf of Penas; PW, Puerto Williams; DRI, Diego Ramírez Islands; FI, Falkland Islands; SGI, South Georgia Island." Fig. 1 – Nice figure. I like the distribution polygon. However, please make several changes. Consider changing the first line to mention this is a map of sampling localities that has all of this other detail. For example, you could change this line to start as, “Map of sampling locations used in the present study…” (highly recommended). Another issue with Fig. 1 is that the distribution polygon is not clearly explained. Please change the next-to-last sentence to read something like, “The geographical distribution of D. eleginoides on the South American continental shelf was obtained from Aramayo (2016) and is shown in transparent gray shading.” Fig. 2 - The caption to this figure _must be completely re-written!_ I fixed it for you. Again, there are way too many errors or inconsistencies between fig/tables/text to write out all of the suggested changes here. Here is the fixed version: "Figure 2. Results of Bayesian clustering analyses used to infer the number of genetic clusters (K) within Dissostichus eleginoides. A) posterior probability isoclines denoting the extent of genetic landscapes inferred in GENELAND. Clusters indicated by GENELAND included the South American cluster (left panel), and the South Georgia cluster (right panel). Black dots represent localities analyzed in this study and regions with the greatest probability of inclusion are indicated by white, whereas diminishing probabilities of inclusion are proportional to the depth of color (increasingly darker red colors). B) STRUCTURE results showing the estimated population admixture coefficients (Q) for each individual, in each cluster. Each vertical bar in plot B represents a single individual, whose genome is broken into colored segments representing the proportion of that individual’s genome derived from each of the K inferred clusters. Abbreviations: NP, Northern Peru; SP, Southern Peru; IQ, Iquique; GP, Gulf of Penas; PW, Puerto Williams; DRI, Diego Ramírez Islands; FI, Falkland Islands; SGI, South Georgia Islands."

You are one of the reviewers, your task is to write a review for the article. You will 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: POPULATION GENETIC STRUCTURE OF PATAGONIAN TOOTHFISH (DISSOSTICHUS ELEGINOIDES) IN THE SOUTHEAST PACIFIC AND SOUTHWEST ATLANTIC OCEAN Review round: 2 Reviewer: 2

Basic reporting: The new version was improved and attended the suggestions offered. Experimental design: The data analyses was improved and attended the suggestions offered. Validity of the findings: No coments. Additional comments: The contribution was reallly improved. The phenomenon tested was now better interpreted given the new analyses provided. The paper will contribute for a better understanding on the phenomena explainning the historical population history of marine organins living around South America.

You are one of the reviewers, your task is to write a review for the article. You will 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: POPULATION GENETIC STRUCTURE OF PATAGONIAN TOOTHFISH (DISSOSTICHUS ELEGINOIDES) IN THE SOUTHEAST PACIFIC AND SOUTHWEST ATLANTIC OCEAN Review round: 3 Reviewer: 1

Basic reporting: Despite many improvements, I find a number of minor inadequacies in the written English and so I provide some suggestions for correcting those under "General comments for the author" below. However, I am confident that after correction the paper could become suitable for publication. Experimental design: No comment. Validity of the findings: No comment. Additional comments: # MAJOR COMMENTS Canales-Aguirre et al. present a study of the population genetic structure of Patagonian toothfish (Dissostichus eleginoides) populations from sub-Antarctic waters over continental shelf habitats around the South American cone eastward to South Georgia Island. I reviewed the first and second (revised) submissions of the manuscript, and have been pleased to see that this next and 2nd revision is very close to publication quality. However, I feel that the manuscript is a minor revision, as a number of minor issues with the English and other aspects of presentation preclude it from being publishable in its present form. # RECOMMENDATIONS Line 28 - Change "their" to "in this species." Lines 31 to 32 - Change "if" to "whether", and then also change "genetic structure on the South American continental shelf" to "genetic structuring in this region." There is too much redundant mention of "South American continental shelf" in the Abstract. Line 32 - Change "structure" to "structuring" for added variety and easier reading. Line 41 - Change "America continental shelf compared with the" to "American continental shelf as compared with". Don't call South Georgia Island "the South Georgia Island", unless island is plural. Lines 43 AND 691 (check throughout) - Your Spanish speaking roots show through here! Change "intraespecific" to "intraspecific". Line 109 - several minor issues here. Please re-word text in parentheses to read, "(e.g. slopes of continents, oceanic islands, and seamounts)." One issue was a missing period after the "g" in "e.g." Line 115 - change "also" to "as well as". Line 116 - Change "like" to "such as." Some will disagree that this is not an error or inappropriate; however, I would intensely disagree. The over-use of the word "like" in daily English conversation is a common issue with millennials, and many if not most authors and editors of scientific papers have switched to avoid this word so as to keep with technical style. Search a major paper in a high quality peer-reviewed journal such as Molecular Ecology or Systematic Biology (e.g. Near et al. 2011 Syst. Biol. 60(5):565–595) and you will find that the word "like" is usually absent. Instead, it is more common to use "unlikely" or "likely" or "likelihood." And the phrase "such as" is more commonly used in place of "like." Line 136 - move the "(Bialek, 2003)" reference to the end of this sentence (next line) where it belongs. Line 142 - Please say "family Nototheniidae" and not "the Nototheniidae family." Lines 155 AND 177 - Please change "Macquaire" to "Macquarie" in both places here. I think the authors meant to refer to the World Heritage Site island, Macquarie Island, between New Zealand and Antarctica. Line 169 - Please change "nuclear genome" to "the nuclear genome." Line 171 - Change "among-populations" to the singular form, "among-population." Line 172 - Need to add a comma after "(2006)." Line 189 - Change "will not show" to "would show limited." Line 233 - No need for page breaks between sections. Please delete this break. Line 245 - Delete the comma after "Additional," so that this reads "Additional tissue samples." Line 251 - Please stop adding comma splices to your sentences. Delete the comma after "eleginoides" on this line. Lines 310 to 311 - Capitalization errors. Several expanded abbreviations are capitalized but their definitions need to be given in lowercase. Fix this by changing "Not" to "not", "Confidence Interval" to "confidence interval", and "Infinite" to "infinite". Lines 315 and 316 - Both lines should read, "a mutation rate of..." Also, the double parentheses, created by putting "2000" in parentheses, are inappropriate. Simply delete the parentheses immediately around the year 2000. Lines 422 and 425 - The authors failed to completely fix their misspelling of "Markov chain", which I believe that I pointed out in my review of the first revision of the manuscript. I know you've had to do a lot of edits, but please be a little more careful here. It is correct to put "chain" in lowercase; however, Markov must also be spelled correctly. Thanks. Line 443 - Add "the" before "Bonferroni." Line 447 - Change "cluster number" to read simply "clusters" here. Line 448 - Here, you need to correct the sentence for number agreement. Since you are referring to two methods, then "methods" must be plural. Change "clustering method" to "clustering methods." Line 452 - Here and several other places in the manuscript you have double period marks, as a result of your editing the manuscript. Please correct this here and throughout by searching and replacing all double periods ("..") with a single period mark ("."). Line 461 - Change "account for" to "include a." Line 510 - Change "Mantel test" to "Mantel tests," making it plural. Line 512 - Misspelling. Change "disjunt" to "disjunct." Line 571 - Change "FST" to "FST values." Line 607 - Need to make the word "posterior" uppercase here because it is the first word in the sentence. Line 661 - No need to define the abbreviation for SSM again here; it is already defined at Lines 542 to 543 above. Line 744 - Change "are" to "is." Line 773 - Change "well differentiated" to "well-differentiated," with the two words separated by a hyphen. Also change "i.e" to "i.e.", with a period after the "i" and the "e". Line 782 - Change "the knowledge" to simply "knowledge." Line 788 - Change "spent" (past tense) to "spend." Line 793 - Change "migration" to "migration pattern." Line 801 - Please state this as "...migrants that we identified..." Lines 802 to 803 - Please state the species name rather than saying, "...in this species." Line 828 - Change "visceversa" to "vice versa." Line 868 - Change "which" to "who." That's all. 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: DIFFERENTIAL RESPONSE TO STRESS IN OSTREA LURIDA AS MEASURED BY GENE EXPRESSION Review round: 1 Reviewer: 1

Basic reporting: BASIC REPORTING Major considerations The manuscript is generally well-written and the language clear and generally unambiguous (specific problems are addressed further in this review.) There are some minor typos and occasional grammatical errors that can easily be caught in revision. The structure of the manuscript conforms to the PeerJ standard. The Intro & background give the reader context and are well-referenced. However at times this section seems to delve in to what may be too much detail given the rather general scope of PeerJ. The hypothesis is well-constructed and supported based on the background information, but commentary on reproductive cycle specifics may not be required based on the exact gene expression data collected and the discussion later in the paper. The authors set out to test the hypothesis that one geographically separate population may have a more pronounced stress response than the others and mention relation to the Baldwin effect and reproductive performance in the discussion. There is mention of local efforts to restore O. lurida and reproductive performance is obviously a consideration for such efforts. For the generalist reader more explicitly mentioning these models and their relevance to our understanding of biology in general as well as their relation and application to restoration efforts may help the reader better understand the intentions and relevance of the research and the rationale for the general research design. Indeed, the rationale is in the manuscript, and understandable to the reader, but scattered and not emphasized in an obvious way. Currently the authors are under-selling the value of their work and relevance. A bit of reorganization and focus on greater biological questions at hand or potential application of the knowledge would remedy this. The figures are all relevant and generally well-presented. The few concerns I do have are mentioned below. Minor considerations It would help if the reader is referred to Figure 1 in the introduction describing the different populations rather than in the Material and Methods section. Experimental design: EXPERIMENTAL DESIGN The specific experimental design directly tests the hypothesis that oysters from Dabob Bay will demonstrate a more pronounced response to stress via changes in gene expression compared to oysters from other geographic locations. This is done through comparison of mRNA levels of a battery of different O. lurida genes in animals taken from three different geographic locations that were placed under temperature or mechanical stress. The mRNAs studied were predicted to be involved in stress response based on sequence similarity to genes with well described functions in other systems. The methods with respect to the biochemistry are described in sufficient detail for replication by investigators experienced with RT-qPCR although minor clarifications are required. The data analysis, however, would benefit if the authors were more explicit with how changes in gene expression were calculated and why. This is especially true with respect to what a sample (n=1) represents in the data sets. It would be appreciated if the authors would clarify the following: Major considerations Line 117-127: Would the authors please clarify if tissue was pooled from animals. Explicit descriptions of what one data point represents would assist with interpretation of data and assessment of its robustness. Further, were technical replicates performed, or was only one RT-qPCR reaction performed per unique animal per gene? Clearly “stating n= X animals per condition” for example in the results section or in the figure legend would make this crystal clear to the reader. Line 174: This table is useful, but please comment on how gene function was inferred from sequence. Is this from GO terms based on closest known homolog? Is this manual or semi-automated curation? Additionally, are there potential paralogues for these genes and why was one picked over another? The authors even suggest that the HSP70 transcript they chose may not actually be the true HSP70-like protein in other organisms given the lack of transcript change in response to heat stress. In the last paragraph of the introduction the authors state “A suite of genes was selected based on their predicted function (gene regulation, immune response, and growth).” Binning the genes in to these categories in the table helps connect the experimental design to the hypothesis and may make discussion of the results easier. Line 187-201: Even though Real Time Quantitative PCR as a commercially available tool has been around nearly longer than some of the youngest current grad students have been alive, data analysis and reporting is still inconsistent between groups and multiple methods for absolute quantitation based on normal curves or relative levels to a reference gene are in use. Statistical analysis of these data represents even more of a “Wild West” situation. The authors reference the Schmittgen and Livak, 2008 paper. Schmittgen and Livak offers a number of different analysis methods. The fold change=2−ΔΔCt method where 2−ΔΔCt = [(Ctgene of interest - Ctactin)treatment - (Ctgene of interest - Ctactin)control] is often used by other groups and could also be appropriate for this type of data as best I can tell. Please explicitly state why the normalized mRNA levels are calculated as ΔCt= (target Ct – actin Ct) as well as why a logΔCt were used for statistical analysis. In the results section the y-axis is labeled as the data being plotted at a ΔCt value. The statistical calculations being performed on logΔCt value but the graphs showing just the ΔCt can be a bit confusing. It’s not so much that there’s an inherent problem with how the data were analyzed, but rather just being clear why it was done this way. Overall, the treatment of the data as ΔCt values, given the way the experiments were designed and run, is still better than doing fold-change calculations and statistics with the fold-change without efficiency correction and linearity controls as many do. Kudos to the authors for avoiding this pitfall. Line 204-207: These data are mentioned in the discussion but it’s unclear from the introduction as to why these experiments were done in the first place. What direct relevance does this have on the rest of the data and or with respect to how this work falls within the greater body of the literature? Minor considerations Line 126-127: Was the tissue simply stored in RNAzol or was it lysed in RNAzol and one of the intermediary steps used for storage at -80oC? The manufacturer’s protocol has a number of convenient stop points but storage of whole tissue in RNAzol is not in any protocols I’ve seen or performed myself. Line 148-149: Please comment on which primers were used to detect genomic DNA via PCR. Were primers targeted to intronic or mRNA non-coding regions? Or were primers generating an amplicon spanning an exon junction used? Line 152-157. Awkward phrasing. Line 161: Reference renders and links strangely in PDF. This may be something the editor needs to deal with. Lines 159-168: Please comment on whether the priming sites for the primers were known to contain SNPs that may have affected hybridization efficiency. This should be available since the Authors’ lab appears to have made its own transcriptome and have access to raw sequencing reads. Since the authors are not using highly inbred populations and these populations are originating from different geographical locations where some degree of genetic drift, founder effects, selective pressures, etc. may have generated SNPs between groups there may be variability in the mRNA sequences. A single SNP in a primer binding sequence is enough to dramatically affect replication efficiency and the downstream relative abundance calculations. Even within highly inbred lab strains of fruit flies or C. elegans SNPs are observed within a lab’s own stocks and between labs- even in protein coding sequence or regions of high evolutionary conservation! Validity of the findings: VALIDITY OF THE FINDINGS Specific comments on the Results section If n=8 animals as the methods suggest then the data appear to be generally robust, statistically sound, and controlled. No concerns there except for those raised above in terms of data representation and explicit statement of what n=1 constitutes. Interpretation of two-way ANOVAs can be tricky, but I think the authors have done a generally good job here. Lack of significant difference is also noted and is relevant to the hypothesis and the discussion of the results. What may make the results section easier to interpret for a more generalist reader is if full gene names were given as they are in the discussion. Possibly the table currently in the methods section could be split up in to one table just for primer design and another put in to results for gene name and inferred function from sequence similarity or existing literature. Specific comments on the Discussion section In general, connecting changes in mRNA level of the battery of genes studied with existing literature is well done. The inferences are sane and based in literature findings without speculation that raises any flags. However, many of these genes have been very well-studied in model genetic organisms like mouse, Drosophila or C. elegans. It may be worth citing some of that literature as well. In some places the authors already do this. Are there potentially more overarching biological phenomena you’re observing here that are worth noting? Given some of the literature cited in the discussion, I get the feeling that some of the mRNA targets were chosen based on what was already known in other marine organisms. That’s an understandable way to pick targets. If that’s true, maybe this should be brought up in the methods or results section when discussing what targets were chosen and why. This would also help with proposing gene function from sequence similarity arguments. Lines 226-237: Again, the discussion on mortality seems disconnected from the specific hypothesis of this study. More needs to be done to connect this to the rest of the paper. Lines 258-267: The lack of change in the HSP70 orthologue is a bit worrisome. In many model systems the HSP70 transcriptional response should be in the timeframe of the experiments performed in this manuscript (minutes to hours.) There is some speculation as to isoform specific responses. From the transcriptome available, are different isoforms of this gene observed? Do the RT-qPCR primers target all or only a subset of them? Specific comments on the Conclusion section The authors conclude that the Oyster Bay population appears to have the greater ability to effectively respond to stress. This runs opposite to their initial hypothesis that oysters from Dabob Bay will demonstrate a more pronounced response to stress via changes in gene expression. Lines 303-312: Conclusions are supported by the data - well done. Lines 319-335: Maybe a little too speculative. The experiments done here don’t really directly test these very general models. Although, this discussion does put the results in a greater context. Depending on how the authors restructure the introduction some of this section could be put in the introduction. Perhaps the style of writing and papers I’m used to reading in my field is different, but front-loading this in the introduction would make the general relevance and aims of this study much more clear. The line: “For long term restoration of O. lurida populations in Puget Sound, understanding the phenotypic plasticity of individual populations will help determine proper supplementation procedures for existing and historic habitats,” succinctly sells me the rational for the research and experimental design. Additional comments: Overall Reviewer’s Report The hypothesis proposed is well-developed and the experiments to test the hypothesis are well designed and directly test the hypothesis. The authors’ discussion and conclusion are grounded in the data for the most part and made relevant with respect to the existing state of the field. Overall this work is of good quality on the raw science side of things. However, lack of clarity and consistency in some parts obfuscates the interpretation of the data as well as the general intentions and objectives of the work. These can be solved with revisions to the text of the manuscript. No additional experiments are required other than some low level bioinformatics with existing tools. In the following sections I have made recommendations where clarification or reorganization would fix these problems. I would happily re-review the manuscript upon revision.