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

Input:
Title: ANTS CONTRIBUTE TO POLLINATION BUT NOT TO REPRODUCTION IN A RARE CALCAREOUS GRASSLAND FORB
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: 12 December 2017: Referee's report for PeerJ on
Michael Rostás, Felix Bollmann, David Saville, Michael Riedel
"Ants contribute to pollination but not to reproduction in a rare calcareous grassland forb "
This an interesting and well-conducted study following a clear logic. It confirms in the example of an Euphorbia species that ants are no effective pollinators of plants having no special adaptations to ant pollination. This message is not new but still valuable. However, and this is one of the merits of the paper, the authors also observed seed germination and showed that ants have a clearly deleterious effect on that forb species. The authors also made credible that the high micro-site fidelity of Formica cunicularia should lead to geitonogamy which adds to the well-known deleterious effect of pollen sterility caused by metapleural gland secretions.
Demonstration of pronounced micro-site fidelity of Formica cunicularia was surprising for a myrmecologist. These ants, as many other species of the subgenus Serviformica, do not establish territories, are subordinate to most other, even smaller, ants when encountering them at a food source and were believed to explore their home range in search for food opportunistically and at random. At least the assignation "random" is has to be deleted now – the activity of individual foragers is apparently directed to rather narrow sectors just as in the foragers of the dominant Formica rufa group species (subgenus Formica s.str.) which exploit and defend rich and very long-lasting food sources.
I recommend acceptance of the paper with minor changes or additions. I suggest the following
(a) The authors have stated " We conclude...that ants, although the most frequent visitors, play a negligible role in the reproduction of E. seguieriana." Integrating all facts we know, the authors should better say that ants definitely play a deleterious role in reproduction of Euphorbia segueriana the more as one other point was not addressed by the authors: If one considers that Serviformica ants occupy the flowers for a considerable time and that they are predatory on diverse insects, they will reduce the visiting frequency of the flowers by the true (flying) pollinators – even if it is probably a rare exception that the F. cunicularia can successfully catch such a pollinator on the very top of the plant. The pollinators are chased off at least (see also discussion of this issue in Claessens & Seifert 2017).
(b) There is a rather weak literature survey for sources referring to the area of observation: the Holarctic and Central Europe in particular. The only plants in Central Europe that are specially adapted to ant pollination are the two orchid species Coeloglossum viride and Chamorchis alpina (Baumann & Baumann 2010, Claessens & Kleynen 2011, 2016; Schiestl & Glaser 2012, Claessens & Seifert 2017). As this strongly contrasts the situation described here, the issue should be commented by more than a single sentence.
(c) The message of Fig. 5 is poor. Delete?
Keeping the anonymity of the referee is not necessary.
With best wishes
Bernhard Seifert
Suggested literature:
Baumann, B. & Baumann, H. (2010): Pollination of Chamorchis alpina (L.) Rich. in the Alps by
worker ants of Formica lemani Bondroit: first record of ant pollination in Europe. – J. Eur.
Orch. 42 (1): 3-20.
Claessens, J. & Kleynen, J. (2011): The flower of the European orchid - Form and function. – :
Jean Claessens & Jacques Kleynen, Geulle: 440 pp.
Claessens, J. & Kleynen, J. (2016): Orchidées d'Europe, fleur et pollinisation [Orchids of
Europe, flower and polination]. – Biotope Éditions, Mèze: 448 pp.
Claessens J, Seifert B 2017: Significant ant pollination in two orchid species in the Alps as
adaptation to the climate of the alpine zone? – Tuexenia 37: 363–374.
Schiestl, F.P. & Glaser, F. (2012): Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants. – Alpine botany 122 (1): 1-9.


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

Input:
Title: ANTS CONTRIBUTE TO POLLINATION BUT NOT TO REPRODUCTION IN A RARE CALCAREOUS GRASSLAND FORB
Review round: 1
Reviewer: 2

Output:
Basic reporting: excellent
Experimental design: creative
Validity of the findings: very convincing
Additional comments: This study examines a rare Euphorbiaceae plant species with flowers that are most frequently visited by ants. The investigators conduct some creative experiments, marking individual ants with different colors on the plants on which they were observed, and saw on subsequent days that the ants never seemed to visit other plants, indicating that all their movements were likely within individual plants (and presumably back to their nests). From this they reasonably conclude that the ants may transfer pollen but only for geitonogamous pollination.  Exclusion experiments were employed to demonstrate that plants need visitors to their flowers for fruit set, but that flying insects provide most of the effective pollination (not significantly different from open controls), whereas plants with only ants visiting their flowers set less than one-third of the fruit of other plants.  Furthermore, the seeds from those ant-only plants did not germinate well, suggesting there is some inbreeding depression operating.
There were two ants species observed visiting flowers, and one carried much more pollen than did the other. However, a larger proportion of the pollen grains taken from the bodies of the first species were inviable. The scientists implied that the pollen grains were inviable due to the antibiotic secretions of the metapleural glands of the ants, but I wonder: How long is pollen viable when not in contact with ants? And what proportion of fresh pollen is viable? Maybe they know, and did not include this info in the paper.  If not, I suggest that it may also be a function of the amount of time spent on the body of the individual ant, for in many plants pollen is viable only for one or two days, and it is possible that pollen stays on an ant so long it may simply be old.
This elegant study was quite comprehensive, but only the pollen viability question was left it my mind.  Perhaps the authors can comment on that possibility or provide some more evidence that it was the ant secretions that rendered the pollen inviable.  Actually I see from the data table on pollen viability, they evidently present data on the natural proportions of viable pollen from flowers – is that correct? And so just a little more comparison and explanation would be helpful.
In Fig. 1 caption - I think the term ‘cyathium’ should be used as it is one only that has the ant on it.
It may be reasonable to eliminate Figure 5.
Fig. 8 caption – if * means that, what do *** mean? Maybe better to put different letters above the bars.


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

Input:
Title: CHIMPANZEES (PAN TROGLODYTES) DISPLAY LIMITED BEHAVIOURAL FLEXIBILITY WHEN FACED WITH A CHANGING FORAGING TASK REQUIRING TOOL USE
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: I have one major concern (pertaining to the authors’ objectives and hypothesis-testing framework… or lack thereof) that needs to be addressed before I can recommend this manuscript for publication.
A couple of minor specific comments should also be addressed.
Validity of the findings: No comment.
Additional comments: This study aimed to investigate the capability of chimpanzees to alter their behaviour in response to an artificial foraging task (i.e., liquid-retrieval task) in which viable solutions became gradually restricted in captive chimpanzees originating from two separate subgroups with different experiential histories, and after providing them with limited exposure to scaffolding towards a novel tool technique.

Overall, this is a well-written manuscript on an interesting topic with key implications for our understanding of the evolution of cumulative culture in our hominid ancestors. The Methods, Results, and Discussion sections are strong and well laid out.

However, I have one major concern (pertaining to the authors’ objectives and hypothesis-testing framework… or lack thereof) that needs to be addressed before I can recommend this manuscript for publication.
A couple of minor specific comments should also be addressed.

MAJOR CONCERN:

I think the “study aims” section (Line 120-134) should take a more robust hypothesis-testing approach. In the present version of the manuscript, this section is much too general, descriptive and vague (“This may be anticipated to impact their problem-solving abilities”; “this study also considers the impact of subgroup membership upon task performance”; “we examined whether chimpanzees could acquire novel tool behaviour through exposure to favourable affordances”).
After providing a very interesting background review of the literature on behavioral flexibility and conservatism in tool-use and tool-making tasks, I am wondering why the authors did not use some of the theoretical aspects they introduced (e.g., the “copy-if-dissatisfied” social learning strategy) and the potential asset of their study conditions (i.e., a group of subjects originating from two different subgroups with different experiential histories) to generate at least two competing/alternative hypotheses (e.g., behavioral flexibility versus behavioral conservatism… or functional fixedness), each of them being associated with a series of testable and mutually exclusive predictions. Without this framework, I started reading the Methods, Results, and Discussion sections with no specific expectations in mind, which, I believe, may seriously weaken the impact of the study.

MINOR CONCERNS:

To what extent the authors’ results could be interpreted in terms of “functional fixedness”, which is not mentioned in the paper?
As a reminder, functional fixedness is the disinclination to use familiar objects (including tools) in novel ways, and as such, is a likely inhibitory factor in tool innovation? (cf. Brosnan and Hopper, 2014).

Line 381: “although we are not aware of any direct experimental evidence for such hypothesised effects.” – Please read the fascinating observational and experimental studies of the developmental processes and indirect social influences (via behavioral artefacts) underlying the acquisition tool use and tool-making techniques (Pandanus tools) in wild New Caledonian crows, and showing that juvenile birds learn these complex behaviors by manipulating parents’ (and others’) discarded tools and using counterpart artefacts (i.e., negative template of the tool after removal from the thick and enduring Pandanus leaf) as easier starting points for tool-making (e.g., work by Jennifer Holzhaider, Gavin Hunt and Russell Gray).


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

Input:
Title: CHIMPANZEES (PAN TROGLODYTES) DISPLAY LIMITED BEHAVIOURAL FLEXIBILITY WHEN FACED WITH A CHANGING FORAGING TASK REQUIRING TOOL USE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Dear authors,
This is an interesting study on the ability of individual chimpanzees to change their behavior when a task changes.
Experimental design: There are two main points I’d like more information on throughout:

1) Examine within-subject changes in behavior of effective vs not effective solutions depending on which phase the response was recorded in (see more details below).

2) Say something about why you tested them socially in the introduction and how you predict this to influence your measurement of flexibility and innovation.
Validity of the findings: My more detailed comments follow below.
Additional comments: L33: define “conservatism” or just say what it is rather than using jargon

L39: explain a bit how flexibility supports problem solving

L41: define “culture” and “cumulative culture”

L42: say something about the tension in cumulative culture between modification of the trait through flexibility and needing to have high copying fidelity. The latter is often thought to be the main facilitator of cumulative culture (e.g., Hunt & Gray 2003).

L46: how do you predict these traits (flexibility, innovation, social learning) limit cumulative culture in chimpanzees?

L52: Logan 2015 and Logan 2016 are the same paper. The 2015 version is the preprint at bioRxiv and the 2016 version is the peer reviewed published version. You can delete the Logan 2015 citation.

L59: did the differences in flexibility support or refute predictions?

L60: why are “individual differences” important to cumulative culture and flexibility? A bit more explanation will help place these results in context so the reader can follow your argument.

L64: were individual differences not found in corvids and great apes because they weren’t investigated? Why the switch to interspecific for these taxa and what does this level of explanation contribute to furthering your argument about flexibility and cumulative culture?

L71: define conservatism

L73: point out that it might reflect the flexibility of the innovating individual(s) (but not those that subsequently used the innovators’ technique/material choice).

L82: I’m not sure how you consider innovation distinct from flexibility. It would be good to define innovation in the first paragraph of the introduction.

L101: indicate whether the great apes were successful on the multi-access box task.

L102: explain how the juice retrieval task works.

L113 and 117: in the discussion, you don’t mention what your results indicate with regard to whether chimps copy if dissatisfied or explore if dissatisfied. If you didn’t intend to test these predictions, I would exclude the discussion of them from the introduction.

L120: given your study aim, what were your predictions?

L129: explain how the different life histories might impact their problem solving abilities.

L133: explain what the favorable affordances were and how they differed from scaffolding.

L137: of the 18 individuals, how many were from which subgroup?

L148: were chimps tested individually? It looks like it was a mixture of individually and with others present. If others were present when an individual was being tested, did you account for their opportunity to learn about the task socially?

L159: transparent or opaque tubes?

L161 and 162: inner or outer diameter?

L172: how much previous experience did the chimps have with the different tool options? If some tools were novel, did you check to see if they were afraid of them? It sounds like some individuals had been previously rewarded for using sticks. It would be good to mention their reinforcement history with each material and discuss how this might influence their tool preferences in the tasks.

L175: was the juice reachable using their hands/lips/tongue at a depth of 7cm? Which of the tools provided were functional in stage 1?

L176: was the “bait” the juice? How many attempts could occur during each session or did the session end after the juice was reached the first time? Were the number of different tool choices/attempts per session accounted for in analyses?

L177: how did you determine the end of a session? Why is it important that the tube was emptied of liquid at the end of each session? Did the chimp observe the emptying?

L180: depth 7cm again?

L185: explain which tools were functional and which were not.

L189: I’m not sure how the number four was arrived at for the inserted stick sessions.

L195: why were chimps allowed access to the empty tube and tools between sessions? Did you record whether they attempted to use any of the tools on the tube between sessions? If they did, in the absence of a reward, they may have learned not to prefer certain tools.

L209: kudos for citing R packages!

L214: could the technique also be “not effective”?

L227: did all 18 individuals observe another interacting with the task?

L237: novel to them in terms of this was the first time they had used the technique, but not novel to them because they had seen another use this technique? It is important to separate when individuals acquire information versus when they use information if you are testing something about innovation. I would think innovation is when an individual invents a new solution to a problem without having seen others perform that solution.

L243: what does “potential across phases” mean?

L244: since the functional techniques differed according to phase, effectiveness should be classified only with regard to the phase that the response variable was recorded in. So a hand response in Phase 2 would be Not Effective, whereas it would be classified as Effective in Phase 1. This will be really important for determining whether individuals changed their behavior according to the phase. If this wasn’t already accounted for in the model (it looks like it wasn’t), I would recode the response variable for effectiveness (yes/no) by phase and tool type and rerun the analyses. In the case of the stick, you could break them down into two categories: long (functional only in phase 1) and short (functional in both phases?). Categorizing the response variable in this way will allow you to determine whether individuals changed their behavior according to the phase. The within-individual behavior change will be the key to determining whether individuals exhibit flexibility.

L268: because of what I brought up in the previous comment, it’s not clear whether individuals are rarely choosing Always Effective tools in phase 1 because they are effectively getting the juice using the Partially Effective tools, which is why they then increase their use of Always Effective tools in phase 2. I think categorizing tools as either effective or not according to which phase they are in will be key for understanding the results.

L284: why were only 4 individuals in this phase? How were these individuals chosen?

L286: if Frek and Kindia didn’t retrieve the reward even though it just needed to be licked off of the leaves they pulled out of the tube, were they motivated to participate in the task? It appears that there was no food deprivation prior to testing to increase their motivation in the tasks. How do you know that juice is a high enough reward to motivate them to try hard in tests?

L287: which individuals are “the four individuals who encountered the leafy stick solution first-hand”? It’s not clear whether these are Kindia, Edith, Frek and Pearl or not.

L308: what reward was Manrique et al (2013) using in their tests and was there food deprivation prior to testing? Could this help explain the differing chimp results on a problem solving task? Or perhaps the multi-access box is easier to solve than the test you gave?

L327: in at least a few individuals? At the species level? Specify at what level you think flexibility is occurring.

L331: which study lacked social information, the Davis et al study?

L332: if I understand phases 1 and 2 correctly, they had the opportunity to socially learn about the functionality of the tools by watching conspecifics. These individuals would serve as “demonstrators”. I’m having trouble following “Had the chimpanzees in this study failed to discover ‘Always effective’ techniques, this lack of social information would be a plausible explanation for the relatively diminished behavioural flexibility observed”. Your chimps did have access to social information about effective techniques and they discovered many effective techniques.

L393: explain a bit more about “cultural biases” - the preexisting behaviors in the group, which vary among groups, will be the ones more often used?

L399: explain what “favourable affordances” are.

L483: what factors would you recommend exploring?

Table 1 caption: mention that all individuals observed others attempting/solving the test using a variety of techniques (if this is true).

Table 2: break “Effective in all phases” into 2 columns, one for each phase so readers can see which techniques were effective or not in each phase.

Table 2 caption: note at what hour phase 2 started.

Table 4: excellent that you provided the full model results! Could you note which effects were fixed and which were random? If random effects aren’t included in the table, please add them.

Edinburgh_Dipping_data.xlsx: rows 330, 602, 806, 910, 911, 972, 1106, 1225, 1271, 1496, 1497, 1552, 1553, 1671, 1672, 1804, 1805, 1836, 1837, 2058, 2059, 2123, 2239, 2491, 2769, 2770, 2799, 2819, 2892, 2922, 2936, 2962, 2976, etc are empty. Were they excluded from the analyses? If they were, they should be deleted from the data sheet to avoid confusion. Were the Unknowns excluded from the analyses?

Supplemental Article S1:
- Fig S1: y-axis should say “Always effective” I think.
- Table S1: add random effects to the table. Indicate in the table that Phase refers to Narrow condition and that the intercept includes the Wide condition.

I hope these comments have been useful!

All my best,
Corina Logan
University of Cambridge

References
Hunt, G. R., & Gray, R. D. (2003). Diversification and cumulative evolution in New Caledonian crow tool manufacture. Proceedings of the Royal Society of London B: Biological Sciences, 270(1517), 867-874.


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

Input:
Title: CHIMPANZEES (PAN TROGLODYTES) DISPLAY LIMITED BEHAVIOURAL FLEXIBILITY WHEN FACED WITH A CHANGING FORAGING TASK REQUIRING TOOL USE
Review round: 1
Reviewer: 3

Output:
Basic reporting: This study by Harrison and Whiten investigates behavioural flexibility in chimpanzees by providing them with different apparatuses that afford different solutions. In the first experimental phase (“Wide Tube”) the juice contained in an apparatus tube could be accessed by using either both bare hands alone or else by recourse to a wide assortment of materials available beside the apparatus. In a second phase (“Narrow Tube”) the opening of the tube was made narrow, thereby rendering inefficient some of the tools and impeding previous solutions. In a third phase, an attempt was made to investigate the structure of their behaviour by observing whether the four chimpanzees might recognize the appropriateness, for accessing the juice, of a suitable tool placed inside the tube, thereby affording a clue that could guide their choice in future.

The results showed a moderate amount of behavioural flexibility because even when chimpanzees’ use of an invariably effective technique increased by 50-fold in the ‘Narrow Tube’ phase, as compared to the previous “Wide Tube” one, the apes were reluctant to relinquish previously-employed albeit now inefficient strategies. Nevertheless, in the “Wide Tube” phase those strategies still comprised 50% of all individuals’ attempts and “no individual that made more than one attempt used “Always effective” techniques for a majority of their attempts”. The “scaffolding” provided by the experimental design did not seem to exert any significant change on behaviour. The high percentage of “Partially effective” attempts in the new “Wide Tube” phase owes to the prepotence that those behaviours rewarded hitherto had acquired in the first “Narrow Tube” phase.

Both the Introduction and Discussion need refinement and clarification. The authors talk of cumulative culture, behavioural flexibility, individual innovativeness, copying of behaviours by conspecifics, etc. It is understood that cumulative culture can emerge only when some individuals are able to innovate solutions that spread horizontally and are passed from one generation to the next. All the topics included in the Introduction and Discussion are appropriate and relevant, yet in the interest of clarity it would be helpful to organize the information in separate sections or paragraphs: for instance, clustering studies that investigate individual innovativeness in order to differentiate them from those that address social learning of newly-acquired behaviours. Also, it would be convenient to separate clearly those studies in which the changes of the apparatus task wholly prevented subjects from using responses rewarded previously, from those which did not prevent them from using old albeit less efficient techniques. In contrast to previous reports, the flexibility observed here could be simply because the old strategies had become so inefficient as to provide no reward at all (cf. Manrique and Call, 2015). Literature on innovation in chimps seems to indicate that they are reluctant to abandon a previously-rewarded response in so far as it had seemed to work: flexibility may be just what we should expect to see whenever a former response was found later to be utterly fruitless.

The authors discuss the results by considering the effects of subgroup yet fail to report how factors such as age (see Manrique and Call, 2015) or hierarchy (Kendal et al., 2015 ) may influence or prevent the acquisition and/or copying of new solutions. In Manrique and Call (2015) it was shown that youngest and oldest individuals were extremely perseverative and failed to abandon old and inefficient responses once the reward contingencies were reversed. Also, another study by the same authors (Manrique and Call, 2011) showed that the oldest orangutans needed considerably more time than younger ones in order to change from the less efficient technique of dipping technique to more efficient sucking in order to drink from a juice container. Interestingly, the only chimpanzee who discovered the most efficient, straw-like technique was a juvenile male. Therefore, it would be interesting for the authors also to analyse their data by focusing on whether age influences behavioural flexibility. Furthermore, because Hopper and colleagues reported a bias towards copying by higher- ranking hierarchy members when individuals were tested in group, it is also necessary to take into account the degree of behavioural flexibility in terms of an individual’s rank: were lower ranking individuals to be those likeliest to change responses, their “innovation” could go unnoticed by conspecifics more readily than were higher-ranking individuals to hit upon the more efficient solution. In lines 328 to 339 the authors refer to the role of an expert model as a constraining factor, which reinforces the need to take into account the hierarchical structures of the two subgroups and the possibility of preferential access by higher-ranking individuals to the apparatus and/or their willingness to change behaviours.

Specific comments:
Lines 45-46. I am not sure that there is evidence of cumulative culture in chimpanzees at all, yet the authors’ statement implies that there is, albeit limited. Given the rarity of the phenomenon it would be convenient to explain briefly what this evidence is.

In line 55 “such as reversal learning paradigms” the work by Manrique and Call (2015) should be added as they also employed a reversal learning paradigm to measure cognitive flexibility as a function of age.

References used:
Kendal, R., Hopper, L. M., Whiten, A., Brosnan, S. F., Lambeth, S. P., Schapiro, S. J., & Hoppitt, W. (2015). Chimpanzees copy dominant and knowledgeable individuals: implications for cultural diversity. Evolution and Human Behavior, 36(1), 65-72. DOI: 10.1016/j.evolhumbehav.2014.09.002
Manrique, H.M., Call, J. (2011). Spontaneous use of tools as straws in great apes. Animal Cognition 14, 213–226.
Experimental design: The main weakness of the study stems from the design: specifically from the fact that individuals were tested in group. Were the authors to have been investigating social learning and transmission of a newly- acquired behaviour, then group testing would have been a mandatory requirement. However, because the stated aim was “to investigate the capability of chimpanzees to alter their behaviour in response to an artificial foraging task” and the method chosen was to test the flexibility of the behaviour and/or innovativeness of the individuals, it might have been preferable to test individuals in isolation and to avoid confusing individual behavioural flexibility with social learning. A more appropriate methodological approach would be first to investigate behavioural flexibility of individuals while tested in isolation with administration of the three apparatus “phases”, and then to select successful individuals and introduce them to the group in order to find out whether or not conspecifics choose the new behaviour after watching how success can be achieved.

When presenting new tasks to a group, factors such as rank, social dynamics, etc., may affect an individual’s attempts to solve problems and thus confound interpretation of the results, particularly if comparisons are to be drawn with previous studies undertaken with isolated individuals. Nor yet is generalization of findings helped by using two different subgroups of chimps with different life histories.

Many factors in the study (social testing, previous history, etc.) could have impinged on the observed behaviour, thereby rendering it difficult to tease apart the specific influence that each of them could exert on the solving abilities of the apes. Now (Given) that the final results of the study have been arrived at, thereby precluding any modification of the experimental procedure, the authors should go the extra mile by discussing how the variable factors mentioned above might impinge on the purported results.
Validity of the findings: Statistical analyses and appropriateness of the interpretation of the results:
The statistical analyses employed are suitable and the conclusions reached are derived appropriately from the data provided. I see no problems there. Notwithstanding the fact that results would have been clearer were subjects to be tested alone.
Additional comments: No additional comments


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

Input:
Title: CHIMPANZEES (PAN TROGLODYTES) DISPLAY LIMITED BEHAVIOURAL FLEXIBILITY WHEN FACED WITH A CHANGING FORAGING TASK REQUIRING TOOL USE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: Dear authors,
You did an excellent job of clarifying terms and concepts in your revision. Your treatment of testing in a social context and how this relates to behavioral flexibility was also well done and provides a nice base for future studies to build on. Additionally, because you now provide information on the effectiveness of each technique, I understand how you treated individual effects in your models. Since there was very little individual variation in your study, I can see why it makes sense to discuss the results in the context of the whole group rather than at the level of the individual.
Validity of the findings: No comment.
Additional comments: A few additional thoughts/comments came up as I read your revised manuscript (line numbers are from the tracked changes Word document):

100 - “cumulative progressions rather than unconnected innovations”.

Couldn’t cumulative culture result from both cumulative progressions and unconnected innovations? The assumption would be that only those unconnected innovations that were more functional would end up being incorporated into the repertoire.

110 - “Social learning, innovation and behavioural flexibility are expected to work in concert to support cumulative culture – an individual innovates an improvement to a behaviour or tradition, and this improvement is passed on via social learning to other group members.”

It’s not clear where flexibility is in this example. It seems from later sentences in this paragraph that flexibility involves the ability to choose the more functional behavior, which keeps culture adaptive?

165 - the western scrub-jay is now called the California scrub-jay…I’m not sure if you should update the name in your paper even though you are referring to the name used in the paper you discuss, but I thought I would mention it.

257 - didn’t the orangutans abandon some solutions and solve some, but not all, of the new solutions in the Manrique study? If so, I would rephrase the sentence to indicate the orangutans didn’t completely fail this task, they just performed more poorly relative to the other species.

320 - “scaffolding in this manner”
Please clarify what “in this manner” means because I don’t quite know, which makes it unclear how it differs from learning through exposure to artefacts.

480 - “Note that all techniques (see Table 2) were effective in the ‘Wide Tube’ phase, in which there were few constraints upon potential solutions.”

It sounds like there were no constraints in the Wide Tube phase. If there were “few constraints” list what they were.

758 - I would change “cognitive flexibility” to “behavioural flexibility” because the latter is the term used throughout the paper and it is the behavior that is measured.

761 - “and so the age of many individuals in the current study may have impaired their performance”

Because most of the individuals in your study were over 27 years of age?

All my best,
Corina Logan
University of Cambridge


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

Input:
Title: HIGH-RESOLUTION MODELING OF THERMAL THRESHOLDS AND ENVIRONMENTAL INFLUENCES ON CORAL BLEACHING FOR LOCAL AND REGIONAL REEF MANAGEMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript “High-resolution modeling of thermal thresholds and 1 multiple environmental influences on coral bleaching
for regional and local reef managements” by Kamagai and Yamano provides a new and useful modeling approach for coral bleaching integrating citizen science, sophisticated statistical and machine algorithms and satellite data. Introduction and background shows well the context of the research. Literature is well referenced and relevant. Structure conforms to PeerJ standards, discipline norm, or improved for clarity. Figures are relevant, high quality, well labeled and described. Raw data and some codes are supplied. To improve the manuscript to be accepted authors must improve professional English language used throughout. I also recommend authors making available all R codes (including figures/maps plotting).
The manuscript is relevant and may be accepted to publication after the minor modifications suggested bellow.
Experimental design: The original primary research is within Scope of the journal.
Research question is well defined, relevant and meaningful. It is stated how the research fills an identified knowledge gap.
Rigorous investigation performed to a high technical and ethical standard.
Methods described with sufficient detail and information to replicate but I suggest including all codes (including for plotting all figures).
Authors chose GLM and Random Forest for modeling bleaching but not justify why these algorithms were chosen. Please add a sentence explaining the motivation for this choice.
Validity of the findings: Data is robust, statistically sound and controlled.
Conclusions are well stated, linked to original research question and limited to supporting results.
Additional comments: Abstract:
Authors refer coral reefs as corals. Corals are animals and coral reefs the environment.

Material and Methods:
The first paragraph of this section should be considerable shortened.
In line 110 I found: “An observer who attempted to submit an observational record to the Sango Map Project was requested to provide the following information as mandatory fields for quality control”. All this information is really necessary or authors can site the monitoring program?

Lines 140-146: this section should be considerable shortened

Lines 215-217: It is not clear why authors used this data/information. If it is relevant for models building please add a sentence explaining.

Line 233-249:
Why authors chosen RF and GLM? Please add a sentence explaining the motivation for this choice.
What R package was used to perform GLM, please add a citation.
There are some assumptions for GLM, what about RF? Please add a sentence explaining if there are some or not.

Results:
Line 299: “worst” does not sound scientific for variable evaluation. Please add a more technical term such “did not explain well…”. Please see line 367 in Discussion section, this comment extends also for the term “good”.

Table 3 – It is uncommon to see tables divided by letters. I suggest to reformat this table (all lines should have values/observations for each column) or to split in four tables.

Discussion:
Lines 385-387: I really think using RF in to model coral bleaching is very interesting and innovative, please add a sentence explaining how RF is being using for ecology modeling.


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

Input:
Title: HIGH-RESOLUTION MODELING OF THERMAL THRESHOLDS AND ENVIRONMENTAL INFLUENCES ON CORAL BLEACHING FOR LOCAL AND REGIONAL REEF MANAGEMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: Kumagai and Yamano took a bold move to challenge NOAA’s predictive model of coral bleaching. By optimizing thermal thresholds and using different environmental parameters and modeling methods, the authors describes the potential of their high-resolution bleaching predictive model to improve local reef management. This interesting insight may be very helpful for small coastal countries where potential bleaching incidents may be overlooked by general models. One concern is that the screening experiment is considered a trial and should not be emphasized in the abstract. The paper is generally easy to follow. However, it will also certainly benefit from proof reading to correct the numerous typographical and grammatical errors. Tables and figures are important and it is critical to lead your audience on how to read or use the information.

Abstract:

1.	Abstract can be more concise and seems to be very similar to the conclusion section of the paper.

Introduction:

1.	The authors need to have supporting citations even for general information. For example, in P.3, line 41: “Extreme rising sea temperature … reef-building corals (REF, REF).

2.	P.4, line 78: “Observations in local areas of research interest … Database. Can the authors name some areas covered?

3.	P.4, line 80: Can the authors name some areas where records are limited?

4.	P.5, line 100: Figures and tables should not be mentioned in Introduction.

Materials and methods

1.	P.7, line 149: Table 1 is an important information. Authors should describe and introduce Table 1 appropriately in the text such as
“Steps to analysed the data were summarized in Table 1”.

2.	P. 10, line 212. Similarly, for table 2, authors should explain how to use and read table

Conclusion:

1. Conclusion is very similar to abstract, authors should consider rephrasing either segments.

Table and figures:

1. Table 1: Caption should be more refined and proper. For those without references, are those steps created by the authors? If so, please include ‘This study (2017)”.

2. Table 2: For those terminologies without references, are those terms created by the authors? If so, please include ‘This study (2017)”.
Experimental design: Materials and methods

1.	P.6, line 112 to 124: What about coral area surveyed?

2.	P.10, line 204: Were the climatologic data from July to September obtained from 1997 to 2016?

3.	P.13, line 270. Suggest to review the sentence to “In total, we evaluated 22,650 and 36,150 models … RF model respectively”.

Discussion:

1. P.20, line 443-449: “Our study showed that reducing UV radiation by increasing screening significantly reduced bleaching risk as … warming”. An actual study of coal screening was not described in materials and methods. Authors should include the description of the experiment if they want to include this point.
Validity of the findings: Results:

1. Results can be more qualitative than descriptive. The authors have all the numbers in tables and figures, use them to reinforce the results segment. For example, the improve scores can also be included in results segment.
Additional comments: Abstract:

1.	P.1, line 3: Suggest to replace “managements’ with ‘management

2.	P.2, line 17: Suggest to remove  ‘living’

3.	P.2, line 18-20: I don’t understand this sentence. Can the authors try to rephrase this or combine with the previous sentence?

4.	P.2, line 31-32:  Suggest to review the sentence to “Prediction based on the best explanatory model revealed that coral reefs in Japan are experiencing bleaching in many areas recently. A practical method to reduce bleaching frequency by screening UV radiation was also demonstrated in this paper.”

Introduction:

1.	P.3 line 47:  Suggest to revise phrase to “Degree Heating Weeks (DHW).

2.	P.3, line 50: Citing one significant paper from the author should suffice.

3.	P.4, line 70-75: Suggest to revise the sentence to “Furthermore, there are potentially interacting environmental stressors such as ultraviolet (UV) radiation (Hoegh-Guldberg, 1999; West & Salm 2003; Maina et al. 2008; Yee at al., 2008) and variables such as water turbidity (REF), topography of the sea floor (REF), … that can affect coral bleaching.

Materials and methods

1.	P.9, line 195: Can the authors explain what is “MODIS-Aqua and Terra”?

2.	P.13, line 270. Suggest to review the sentence to “In total, we evaluated 22,650 and 36,150 models … RF model respectively”.

Discussion:

1. P.20, line 429: Replace “coral bleaches’ with ‘coral bleaching’.


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

Input:
Title: HIGH-RESOLUTION MODELING OF THERMAL THRESHOLDS AND ENVIRONMENTAL INFLUENCES ON CORAL BLEACHING FOR LOCAL AND REGIONAL REEF MANAGEMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors had greatly improved the manuscript after revision. I recommend this manuscript for publication in PeerJ in the present form.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: HIGH-RESOLUTION MODELING OF THERMAL THRESHOLDS AND ENVIRONMENTAL INFLUENCES ON CORAL BLEACHING FOR LOCAL AND REGIONAL REEF MANAGEMENT
Review round: 2
Reviewer: 2

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


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

Input:
Title: SEROPREVALENCE OF HEPATITIS B VIRUS IN TAIWAN 30 YEARS AFTER THE COMMENCEMENT OF THE NATIONAL VACCINATION PROGRAM
Review round: 1
Reviewer: 1

Output:
Basic reporting: In introduction section, author shew context of hepatitis B infection and prevalence globally. There are some data need to be updated.
1. Author mentioned that hepatitis B virus infection is a major cause of acute hepatitis, this is not precise because most of the acute infections become immune and the major problem is chronic infection, since majority of chronic infections are lifelong.
2. Author cited literature and  said  “More than 350 million people worldwide remain chronically infected with HBV”, which should be updated by the WHO new published document (Hepatitis Report 2017, WHO).
Experimental design: Suggest to make close linkage between birth cohort and policies change
Validity of the findings: 1. What makes difference of HBsAg prevalence is not the birth cohort, but the policies change. Therefore, author should have more discussions about the policies rather than the birth cohort.
2. The HBsAg positivity rate decreased from 9.7% among patients born before June 1994 to <1.0% among patients born after 1992. There is a cross time, please ckeck.
3. See Table 2. Author had clear criteria for age of each group, why there is an overlap for age range. Please clarify.
4. Table 1 needs to be restructured in a more standard way.
5. Figure 2, Can author provide more information and explanation why there is a stepwise decline for anti-HBs?
Additional comments: This manuscript is written in simple and common English language, need more professional.


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

Input:
Title: SEROPREVALENCE OF HEPATITIS B VIRUS IN TAIWAN 30 YEARS AFTER THE COMMENCEMENT OF THE NATIONAL VACCINATION PROGRAM
Review round: 1
Reviewer: 2

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: Hu et al. report seroprevalence of hepatitis B virus (HBV) infection in Taiwan, after 30 years of the initiation of the national HBV vaccination program, since 1984. To explore the long-term efficacy of the HBV vaccination program, the investigators analyzed available data on the presence of HBV surface antigen (HBs Ag) and anti-HBs antibody in the sera of a total of 17,611 students enrolled into a Taiwanese university, during 2005-2016. I have some comments that might help to improve the manuscript, below:

-The title and other places in the manuscript: the term 'hepatitis B infection' should be clearly stated as 'hepatitis B virus infection'.

-The term 'patients' used in the abstract and the main text may not be accurately defined, since the subjects here are students with no diseases.

-Line 67: duplicate words (that that).

-Line 67-68: four- or three-dose plasma-derived vaccination? please check the number of time points for vaccination, mentioned in the sentence.

-Line 74, 77, 78, and other places: "hepatitis B (HB)" should be clearly stated as 'hepatitis B virus (HBV)'.

-Line 99-111: the Methods '2.1. National HBV vaccination schedule' - This section seems to be more appropriate to be incorporated in, as a part of, the introduction.

-Line 130: titer or level?

-Line 130: Please provide a reference for the criteria "anti-HBs Ag antibody < 10 mIU/mL is non-protective". Is it possible to have the immuno-protecitve state against HBV with the detectable anti-HBs Ab level < 10 mIU/mL?

-Line 131-132: Please check the criteria for setting 'reactive' VS 'non-reactive' for anti-HBc antibody. Should 'S/CO > 1' be considered as reactive (not non-reactive nor just 1-3)?

-Should not begins a sentence with number, i.e., Line 168 and 170.

-Line 196: please consider to delete 'due to sampling restriction'.

-Line 198-203: This paragraph, as it currently stands, is more like discussion. The APC model analysis seems to be the key information of this manuscript, so please revise this section to report clearly the facts/results (with some details). This includes brief descriptions of some key parameters used in Table 3 (perhaps as footnote), so that the readers who are not familiar with this kind of APC model analysis can follow.

-Line 175-176 mentioned the HBV carrier state in males > females, and in contrast, Line 181-182 mentioned the anti-HBsAg antibody level in male < female. Since the male-to-female ratio < 1 (i.e., as shown in Table 1), what is/are reason(s) behind this paradoxical observation, in regard of gender and HBV carriers?

-Line 183-186: Anti-HBc antibody can be used to discriminate the HBV protective immunity from natural infection and vaccination. The anti-HBc antibody dropped from 4.3% (in 2008) to 1.7% (in 2012) and 0.6% (in 2016). What does it mean? Rate of natural HBV infection is decreased? What about the other markers (i.e., HBsAg and anti-HBsAg) in this groups of students?

-The discussion could be shortened, by for example, removing repeated statements (i.e., the first sentence of the first paragraph), and perhaps shortening/removing the statements that are considerably non-relevant to the results of the current study (i.e., Line 284-296).

-Regarding the persistence of anti-HBs antibody, Cohort C (students were born during 1986-1992, and received the plasma-derived vaccine), the mean anti-HBs antibody level was observed to be higher in the 1990-92 birth year group, compared to that of the 1986-88 and 1988-90 groups. This observation can be expected, because the 1989-92 group received the vaccine in later time point than the other 2 groups. However, for Cohort D (students were born during 1992-99, and received the recombinant vaccine), I wonder why the mean anti-HBs antibody level of the 1996-99 group was observed to be lower than the students born and received the vaccine earlier (i.e., 1994-96 and 1992-94).


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

Input:
Title: SEROPREVALENCE OF HEPATITIS B VIRUS IN TAIWAN 30 YEARS AFTER THE COMMENCEMENT OF THE NATIONAL VACCINATION PROGRAM
Review round: 2
Reviewer: 1

Output:
Basic reporting: All fit, no comment.
Experimental design: No additional comment.
Validity of the findings: No additional comment.
Additional comments: Author responsed: We have updated this text accordingly in lines 66–67 on page 3: “more than 350 million people worldwide have chronic HBV infection, with the majority of the infected people living in Africa and Asia (WHO 2017).” However,
I checked The Global Hepatitis Report 2017(WHO), it says that:
WHO estimates that in 2015, 257 million persons, or 3.5% of the population, were living with chronic HBV
infection in the world.
Hence, 257 million should uodate 350 million.


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

Input:
Title: SEROPREVALENCE OF HEPATITIS B VIRUS IN TAIWAN 30 YEARS AFTER THE COMMENCEMENT OF THE NATIONAL VACCINATION PROGRAM
Review round: 2
Reviewer: 2

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: No additional comments


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

Input:
Title: BODY MASS INDEX INFLUENCES THE PLASMA GLUCOSE CONCENTRATION DURING IATROGENIC HYPOGLYCEMIA IN PEOPLE WITH TYPE 2 DIABETES MELLITUS: A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: In this paper, the authors demonstrate that in individuals with T2DM who experience severe iatrogenic hypoglycemia, BMI influences plasma glucose levels at hospitalization. The paper is susceptible for publication, but I have some comments that need to be addressed before by the authors:

1.	Since some patients are young and treated with insulin, the authors should specify how type 1 diabetes has been excluded;
2.	Patients affected by any organic disease impacting on BMI must be excluded;
3.	Methods for the determination of  glucose, HbA1c, creatinine, and potassium should be indicated, as well as quality of the analytical performance (precision).
4.	By analyzing the file data, it emerges that all continuous variables have a non-normal distribution. Thus, non parametric tests must be employed;
5.	In linear regression analysis, the impact of BMI must be adjusted for other confounding factors.
6.	In the discussion: line 180, “glycemic therapy” is not clear.
7.	Line 204: The Authors should briefly indicate the role of fat tissue in the production of adipokines, whose role is important in insulin sensitivity and inflammation. (Waki H, Tontonoz P, Annu Rev Pathol 2007; Fasshauer M, Bluher M, Trends Pharmacol Sci, 2015).
8.	Line 205 could be better expressed as follows: “Weight loss improves insulin sensitivity by decreasing free fatty acid mobilization, and by changing adipokine profile in obesity (Schenk et al., 2009; Greco M, et al, Mediators Inflamm 2014), and intentional weight loss in T2DM, correlates with lower fasting plasma glucose concentration (Wing et al., 2011)”.
9.	References should be listed in alphabetical order.


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

Input:
Title: BODY MASS INDEX INFLUENCES THE PLASMA GLUCOSE CONCENTRATION DURING IATROGENIC HYPOGLYCEMIA IN PEOPLE WITH TYPE 2 DIABETES MELLITUS: A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written and associates BMI with the nadir of hypoglycemia upon admittance to the hospital for hypoglycemia-care. The study is observational and not an intervention, however, great care was taken to adjust for confounding variables where applicable. The main finding of this work is that individuals with lower BMI tend to have lower plasma glucose levels (30 vs 38 mg/dL) and that this relationship is quite strong (B-coefficient of 0.72). I have a few minor suggestions that may improve the presentation of the data and the discussion of the results, however, overall I find this manuscript to be of good scientific merit.
Throughout the manuscript, the authors refer to “energy deficit and glycogen depletion.” It should be noted, however, that unlike rodents, humans do not experience glycogen “depletion.” In fact, even after days or weeks of fasting, glycogen is still present in the liver. Furthermore, Hendrick and Cherrington (1990, AJP) showed that in dogs, glycogen is present in the liver after a 7d fast and that when their livers were exposed to glucagon, a “normal” amount of glycogen was mobilized, despite the liver glycogen concentrations. Therefore, in the context of your conclusions, I am of the opinion that the proposed mechanism whereby “BMI is associated with chronic energy deficit and glycogen depletion” would cause “more severe hypoglycemia” is slightly inaccurate. On the other hand, in the paper by Winnick et al., that you cite, they observed that low liver glycogen, such as what you are suggesting is present with lower BMI, is associated with diminished secretion of the counterregulatory hormones glucagon and epinephrine. By reducing glucagon and epineiphrine secretion, it would be expected to lower hepatic glucose production and make the glucose nadir lower than what was occurs in the high-BMI group. I am of the opinion that in order to provide a speculative mechanism of action, this is the most consistent with your data.
In the discussion, the use of the phrase “tolerance of hypoglycemia….” is of interest. Perhaps the authors mean to say “ability to counterregulate hypoglycemia….” Or perhaps “tolerance to hyperinsulinemia….” Would be a better description?
Experimental design: In their statistical analysis, the authors report the relationship between the blood glucose level and age, BMI, A1C, creatinine and diabetes duration. The only variable that correlated with the blood sugar was BMI. I do not have a problem with the analysis or the presentation of the data, however, did the authors probe further to determine if a multivariate analysis would account for a greater proportion of the variance in plasma glucose? I expect that it would not, but the authors might want to add this explanation somewhere in the results section.
Validity of the findings: Having diabetes at a lower body weight can often mask other illnesses an individual possesses. I comment the authors for controlling the cause of hospital admission in the cohort, but do the authors have any indication of any differences between the low- and high-BMI? For example, some of the low BMI individuals may have undergone weight loss surgery (e.g., RYGB or sleeve-gastrectomy), which can lead to “dumping” and overt, severe hypoglycemia. Do the authors know if any of the low-BMI subjects had previously undergone hypoglycemia? If the data are available, it would be helpful to report it in the results section (i.e., the number of subjects who had previously undergone weight loss surgery). Likewise, the dose of insulin and sulfonylurea each patient was on would be informative to the reader. However, I understand if they are not available.
In the section where the authors note the limitations of the study, it should be included that they did not measure 1) liver glycogen or 2) counterregulatory hormones. The inclusion of these data in the manuscript would have markedly improved the merit of the manuscript. The reasons why you did not have these data, however, is understandable.
Additional comments: My comments are covered by the first three sections.


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

Input:
Title: BODY MASS INDEX INFLUENCES THE PLASMA GLUCOSE CONCENTRATION DURING IATROGENIC HYPOGLYCEMIA IN PEOPLE WITH TYPE 2 DIABETES MELLITUS: A CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: OK
Experimental design: OK
Validity of the findings: OK
Additional comments: The authors addressed all my concerns.


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

Input:
Title: BODY MASS INDEX INFLUENCES THE PLASMA GLUCOSE CONCENTRATION DURING IATROGENIC HYPOGLYCEMIA IN PEOPLE WITH TYPE 2 DIABETES MELLITUS: A CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revisions are well done, just a couple of minor things.
Experimental design: Good.
Validity of the findings: Good.
Additional comments: In the abstract, it would be more accurate to say “This study investigates the relationship between body mass index….” Instead of “This study investigates the effect of body mass index….”

At the end of the Introduction (lines 110-112), this statement is not completely accurate. A lower BMI per se does not reflect lower liver glycogen. For example, young people with lower BMI have higher liver glycogen than older people with type 2 diabetes. I think the statement “People with T2DM who have a lower BMI are also likely to have lower hepatic glycogen stores and this can diminish the secretion of glucose counterregulatory hormones during hypoglycemia.” is more accurate.


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

Input:
Title: A NEW PYTHON LIBRARY TO ANALYSE SKELETON IMAGES CONFIRMS MALARIA PARASITE REMODELLING OF THE RED BLOOD CELL MEMBRANE SKELETON
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall good: readable, very clear, well-structured and appropriately contextualized.
Experimental design: I the the comparison with prior art is well designed and thoroughly explored. I wonder, is Fiji’s Analyze Skeletons plugin the only comparable software?
Validity of the findings: Looks good.
Additional comments: This paper was a pleasure to read. It's exciting to see another scientific application of Numba (indeed, there aren't very many yet). I appreciate the use of real experimental data and the detail-oriented comparison to existing software.


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

Input:
Title: A NEW PYTHON LIBRARY TO ANALYSE SKELETON IMAGES CONFIRMS MALARIA PARASITE REMODELLING OF THE RED BLOOD CELL MEMBRANE SKELETON
Review round: 1
Reviewer: 2

Output:
Basic reporting: Nicely written, objective, and to the point. The software is available and is licensed under a FOSS model, but the authors should also include a tutorial, for users to be able to both test their installation and learn how to use the software. I see that they have included test data, which is great. But it is useless to most users, as I will expand on below.
Experimental design: PeerJ "considers articles in the Biological Sciences, Environmental Sciences, Medical Sciences, and Health Sciences", and this article, being bioinformatic in nature, makes it. But as the software package is right now, it is more of a mathematical work than something usable by the PeerJ audience.

A significant flaw of this work is the complete lack of any documentation whatsoever for the software. There are no instructions on how to install or use it. No tutorial on how to use the program or its alleged GUI, no screenshots of it in action.

Thus, very few, if any, biologists will use this program as it is right now. I have at least some experience programming in three languages (not Python though) and I have no clue what to do to get Skan up and running. So I didn't, and have to take the authors at their word that the program works. Because I know very little Python. Other biologists typically know even less than I do. Time is limited, people will go use Fiji instead, I am afraid.

The authors say Skan is a library, with an extensive API. However, an API is not much use without documentation --don't think many people will go read your code to find out how to access your functions, what they return, etc.
Validity of the findings: No comment.
Additional comments: The article itself seems OK to me, although I thought the discussion was a little thin. But, OK, no use in belaboring the point if there is not more to say.

What I believe the authors must do in order to avoid this apparently nice piece of software becoming yet another example of "abandonware" littering the scientific softwarescape is to write a point by point tutorial that:

1) Explains to the general biologists, who I believe are the main target user of Skan after all, how to get the program to work;
2) Explains to the general biologists how to use the program (GUI or not), by employing the test data set included (it is advisable to have also included the expected output in a directory, so the users can compare what they got to what was expected).

Besides this "general user" guide, in order for the Skan API to be usable by other programmers, there must also be some documentation on how to use the API (e.g., what should be imported to begin with?) and of each of the available functions. Save the many user a significant amount of time later by spending a bit of time now and they will be very grateful to you.


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

Input:
Title: METAGENOMIC ANALYSIS OF ORANGE COLORED PROTRUSIONS FROM THE MUSCLE OF QUEEN CONCH LOBATUS GIGAS (LINNAEUS, 1758)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Occasionally the writing is little hard to get through (although usually one can understand its intent). Some examples are:
L30 “explaining the nature of a plausible parasite infection”  - awkward.
L50 overfishing would seem to be included in the detrimental human activities adduced later in the sentence.
L111 “lowest common ancestor” Unsure what this means.
L211 “taxonomical identification”… what other kind of identification is there?
L254 to say trematodes "have been reported" in mollusks reads like a strange sort of understatement (all digenetic trematodes infect mollusks).
L271-274 quite awkward.
L279-280 a reference is needed for the 40 million infections mentioned, as well as the “previous studies.”
L501-502. Fig 2 legend is awkwardly worded. Also, some of the nodes lack numbers, and circle size is not truly proportionate to hit numbers, as it seems the same for “no hits” (n=3000) and “not assigned” (n=900).
I am not an expert, but should this conch be referred to as Lobatus gigas?
Experimental design: The research gap (identity of the orange colored protrusion in conch) is well defined. However, I am puzzled by the methods chosen to address it, even if they are impressively multidisciplinary.

Few macroparasite infections can be reliably identified histologically (those that can are well studied taxa in well studied hosts). So, the use here of histology as primary identification method is surprising. It is also a relatively costly in terms of sample usage, which is a concern in a case like this, when there are only a few isolates in hand (L100).

As for pyrosequencing, the results speak for themselves. It does not seem surprising that the reads yield little information on the pathogen (the hits with bacteria may be of some interest, but I am not a good judge of that. However, I do find the reference to contamination for some of the more surprising bacterial hits a little understated at L249-250). Essentially, if I take a bottom-line look at the 454 results, they seem almost misleading, given the authors’ eventual conclusion: some are subject to possible contamination, and only a tiny minority are related to Trematoda.

Sanger sequencing could have been a potentially valuable approach, but by choosing CO1, the authors are hoping for a very specific match. Which would be great, but is a faint hope, given the largely unknown, unsequenced nature of most marine invertebrate taxa. For assignment to higher taxa – probably the most realistic, best-case outcome here – CO1 is a poor choice, because saturation of the third codon often makes deeper phylogenetic placement unreliable. For example, the authors cite Moszczynska et al, who recommend using ribosomal markers for higher taxonomic placement. 18S could well yield something more specific than Trematoda (even if it is not a great choice for identification at the species level). Moszczynska et al also reported sequencing individual metacercariae that were very small – on par with the specimens here. Pooling the specimens (L100) is unfortunate.

The analysis of the CO1 data is surprising. An NJ tree seems less appropriate that one based on character changes, such as ML, BI or MP.

It is disappointing that no specimens were examined in toto, stained, mounted on slides. This could have led to a more specific identification (in addition to being less work) and would have clarified the developmental stage of the trematodes. There is a reasonable chance these are metacercariae, not sporocysts or rediae. The authors seem to be assuming  the latter at L275-277. However, it is metacercaraie that are responsible for foodborne trematodiases, which are discussed at several points.
Validity of the findings: The results appear valid, although as stated above, methods slightly different from those chosen might have yielded more satisfactory results (given the stated goal of the study).

At L291. Not very clear to me how any particular identification would yield any kind of practical preventative or control measure. Suggest elaborating with specifics or removing.
Additional comments: I don’t insist, but I feel like if the authors really want to identify this, they should sequence 18S and possibly ITS. Better chance of a more-informative near-match. Also, even if the data are left as is, NJ is not be best analytic method for the CO1 sequence.


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

Input:
Title: METAGENOMIC ANALYSIS OF ORANGE COLORED PROTRUSIONS FROM THE MUSCLE OF QUEEN CONCH LOBATUS GIGAS (LINNAEUS, 1758)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting was relatively clear; although, contextual and word usage corrections were made throughout the paper. I have made detailed corrections in my attached pdf of the article.
a)	L26:  Please replace ‘organisms’ with ‘taxonomic groups’ as no definitive species
                 identifications were made in this study.
b)	L29: Please replace ‘class Trematoda’ with ‘subclass Digenea’.
c)	L32: Please replace ‘meat’ with ‘muscle’.
d)	L45: Please add ‘marine’ after endangered and remove ‘mollusk’.
e)	L47-L49: The 2 conservation organizations that were cited are not named correctly.
                        Please rewrite with the correct names as follows: ‘Convention on
                        International Trade in Endangered Species of Wild Fauna and Flora’
                        ‘Red List of the International Union for Conservation of Nature’
f)	L56: Please replace ‘the parasitology and microbiology of this species’ with
                ‘;however,  the parasitological and microbial studies of this species’.
g)	L61: Please replace ‘phylum’ with ‘phyla’.
h)	L66-69: Rewrite as follows: Moreover, the etiological agent of orange-colored
                     protrusions in the muscle of S. gigas remains unknown. These orange-
                     colored protrusions were observed sporadically in queen conchs from the
                     Columbian San Andres archipelago. This observation is characteristic with
                     digenean infection found in other marine gastropods.
i)	L70: The phrase ‘orange/lemon-colored lesions’ is actually referring to the larval
                 digenean stage known as sporocysts. Replace the word ‘lesions’ to ‘sporocysts’.
j)	L75: Please replace ‘lesions’ with ‘sprocysts’.
k)	L80: What is the brand of the automated DNA sequencer used?
l)	L82-84: Rewrite to clarify that the ‘marine samples’ are more specifically the
                     microbiome of orange-colored protrusions found in the muscle of S. gigas.
Experimental design: The experimental design was thorough with regards to the metagenomic analysis, however, this study was lacking in the morphological aspects of the digenean parasites and number of genes used for the genetic analysis with Sanger sequencing. Strombus gigas specimens could have easily been examined for the shedding of cercariae using non-destructive sampling methods by placing a live S. gigas specimen with orange-colored protrusions (sporocysts) in a container with purified seawater overnight.  Morphological description of cercaria could then be compared to the other studies you cited (Stunkard, 1950; Galaltionov & Skirnisson, 2000; Werding , 1969; Hechinger & Miura, 2014). For gene sequencing a 740bp region of the mitochondrial COX1 gene was sequenced in this study. 28S rRNA gene sequence (AF023113) exist for Renicola
roscovita and other Renicola species. The ITS2 region would have also been helpful in
determining the taxonomic position of this unknown digenean species reported in the study.

a)	L89:  Please clarify whether orange-colored muscle is describing an anatomical
                 character of the queen conch or are you referring to the sporocysts. Where
                 these three samples of orange-colored “muscle” examined from a single
                 specimen or 3 different individuals?
b)	L117-118: Please clarify if you intended to write ‘100 nucleotides or amino acids’.  If
                         not, please replace with ’300 nucleotides or 100 amino acids’.
c)	L132: What is the brand of the automated DNA sequencer used?
d)	L150-151: Please explain why 22 sequencing assigned to the Trematode category
                         is remarkable, while 19 sequences assigned to the fungi group is
                         downplayed?
e)	L254: It is a stretch to suggest cestode infection just because the histological
                  approach shows membrane consistent with tegument, especially since no
                  mention of cestode sequence came up in the metagenomic analysis. Please
                  remove ‘cestode’.
f)	L263: Please replace ‘lesion’ with ‘protrusion’.
g)	L523: I recommend that the cestode sequences should be removed from the
                  Neighbor Joining tree analysis and replaced with additional Digenea
                  sequence. The outgroup should be a specimen from the subclass
                  Aspidobothrea, which is the sister group of Digenea.
Validity of the findings: The validity of the findings with regards to evidence of Digenea infection of Strombus gigas could be much more robust with a non-destructive sampling method to recover shedding cercariae for the histological/ morphological analysis and use of additional nuclear gene sequencing if 18S, 28S, and ITS2 for the gene sequencing analysis.
Additional comments: I find the title of the article somewhat misleading. Based on the title of the article, I’m expecting a parasitological study that would provide solid morphological and molecular data to show evidence of digenean infection with data on larval stages (sporocysts) within and juvenile stages (cercaria) exiting Strombus gigas. Remarkably, roughly half of the results section and half of the discussion is dedicated to bacteria. This research seems to be more in line with a metagenomic study which is focused on the microbiome of “orange-colored protrusions” found in the muscle of Strombus gigas, then a study of digenean parasites infecting Strombus gigas.


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

Input:
Title: METAGENOMIC ANALYSIS OF ORANGE COLORED PROTRUSIONS FROM THE MUSCLE OF QUEEN CONCH LOBATUS GIGAS (LINNAEUS, 1758)
Review round: 1
Reviewer: 3

Output:
Basic reporting: This study reports on the apparent presence of trematodes and bacteria in the tissues of Strombus gigas. Although the work is technically spectacular, the findings are so minor that I cannot recommend publication.
The approach used has demonstrated, probably convincingly, that the conch or conchs (not many infections and the samples were pooled) had a trematode infection. However, the molecular data are so generalised that there is no real identification beyond that of "trematode". This is my problem with the paper. Ultimately, probably every marine mollusc acts as intermediate host to trematodes at some stage. Without better identification or prevalence information we are really not significantly advanced by this study. Notably, the Discussion (Lines 275-6) states that the typical outcome of trematode infections in molluscs is partial or complete castration. The authors betray their lack of familiarity with these parasites here. Castration is associated with infection as a 1st intermediate host. The current infection is consistent with infection as a second int. host and likely to be relatively insignificant in the biology of the conch.
A great deal of the ms is devoted to issues associated with bacteria about which I know nothing. However, I do note that this makes the title of the paper rather misleading.
According to the WoRMS database, the name Strombus gigas is outdated - it should be Lobatus gigas; this does not inspire confidence.
In my view this study has not advanced knowledge of parasitism in this conch and I cannot therefor recommend publication
Experimental design: As above
Validity of the findings: As above
Additional comments: As above


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

Input:
Title: METAGENOMIC ANALYSIS OF ORANGE COLORED PROTRUSIONS FROM THE MUSCLE OF QUEEN CONCH LOBATUS GIGAS (LINNAEUS, 1758)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English writing has improved. It is still not fluent but there is no real barrier to understanding or communication.

I think the workflow used by the authors would be better represented, and their decisions more understandable, if they mention the specifics of their attempts to sequence 18S and 28S, as they do in the response (see paragraph above gel image in response), rather that making it sound like the attempt is a bad idea to begin with, as it comes across in the ms (L132-133). Also, the details of just how little tissue was available (only in the response) would be good to include in the MS proper.
Experimental design: I BLASTED the CO1 sequence and I think the fact that its highest similarity (82-83%) is to Synthesium, another member of the Xiphidiata, not mentioned in the MS because (presumably) it only overlaps at about 320 bp, is also worth mentioning in support of the eventual conclusion that the lesions are caused by a digenean belonging to the Xiphidiata.
Similarly, in addition to the statistical support of the clade membership, it would be relevant to state at L208-211 what ranges of similarities the sequences had with others in the tree.

I think the fact that 2 different translated contigs of endonuclease-reverse transcriptase indicate the specimens belong to the Schistosomatidae needs explanation. Either the method of Monzoorul Haque et al is flawed in this, or there is some other problem. The schistosomes are very evolutionary distant from the Xiphidiata.
Validity of the findings: I think the findings were, and remain valid.

I think the statement that the specimens are sporocysts (L202) (rather than metacercariae) requires justification. Some discussion of the known life history of related taxa might be pertinent.
Additional comments: Can the authors comment or speculate on why, if a digenetic trematode is what is causing these lesions, so many of the reads are from other taxa? If the sample's biomass is predominantly worm, than why are there few or no worm rDNA reads, but so many from fungi?


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

Input:
Title: METAGENOMIC ANALYSIS OF ORANGE COLORED PROTRUSIONS FROM THE MUSCLE OF QUEEN CONCH LOBATUS GIGAS (LINNAEUS, 1758)
Review round: 2
Reviewer: 2

Output:
Basic reporting: Since most of the corrections were made on the manuscript the basic reporting is very clear
and reads well. There is one correction that need to be made in the manuscript with the tracked changes as shown below.

a) L64-L67: This sentence needs to be rewritten as it is a run on sentence that
                    is awkwardly written.
Experimental design: Since most of the corrections were made on the manuscript the experimental design is more detailed and reads well. There are still a couple of corrections that either need to be made or were not addressed in my first review. The lines listed below are based on the manuscript with the tracked changes

a) L89-L90: Were these three samples of orange-colored "muscle" examined from a single
                    specimen or 3 different individual queen conch specimens?

b) L122-123: "Only contig alignment lengths above 100 nucleotides or 100 amino acids
                       were included in the assignment analysis." This still does not make sense
                       to me as 100 amino acids are equivalent to 300 nucleotides, but this is not
                       reflected in the text you have written in the text shown above.
Validity of the findings: The validity of the finding are now acceptable with regards to evidence of Digenean
infection of Strombus gigas as this is not the main focus of the paper as previously
written. This is especially true since changes were made to the title of the paper.
Additional comments: I'm happy to see that the authors took into to consideration the comments and corrections that
I previously made. The paper now reads much better and is clear and to the point. I believe that
this manuscript should be accepted with the minor revisions that I have listed in the above sections.


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

Input:
Title: METAGENOMIC ANALYSIS OF ORANGE COLORED PROTRUSIONS FROM THE MUSCLE OF QUEEN CONCH LOBATUS GIGAS (LINNAEUS, 1758)
Review round: 3
Reviewer: 1

Output:
Basic reporting: In the first review I mentioned that the writing had some problems. For the most part, these did not prevent comprehension but were distracting. I can see that the authors attempted to address this issue by making all the changes I specifically mentioned, but I’m afraid the problem remains. For publication purposes, a native speaker should be recruited to go over this closely. For what it's worth, I can sympathize: where I work the linguistic environment is not my first language, and often have to write/teach in a second language. ¡no es fácil!

Other than this, I have no major comments on the ms (one minor one, below), and I commend the authors on their work.
Experimental design: no comment
Validity of the findings: I think the authors could explicitly state the rationale behind their opinion about the developmental stage involved.
Additional comments: No additional comments


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

Input:
Title: LIFE HISTORIES PREDICT GENETIC DIVERSITY AND POPULATION STRUCTURE WITHIN THREE SPECIES OF OCTOPUS TARGETED BY SMALL-SCALE FISHERIES IN NORTHWEST MEXICO
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are some areas, particularly in the Introduction where the language should be improved to make it more clear for all audiences.  Some of the grammar is confusing and ambiguous and a native English speaker should double check the wording. (See my comments on the PDF)
Otherwise, the references, background, figures, data, etc are clear and the paper reads well.
One table (Table 1) could benefit from a delineation between the section on data and the hypotheses.
Experimental design: The experimental design and methods are clearly defined and thorough.

It might be beneficial to have a short section providing the reasoning behind using a subset of samples for the mitochondrial analysis.

The morphological differentiation between species is stated in a slightly challenging way.  Since only one individual from each species was identified through morphology, it might not even be necessary to include this; especially if the genetic information on each species already exists to indicate reproductive isolation between the three.
Validity of the findings: You could also discuss the literature that suggests size at sexual maturity is extremely variable depending on food availability and temperature in multiple species (Forsythe + Hanlon, 1988 Marine Biology)
Additional comments: The findings and conclusions are sound and well stated.  Implications in octopus fishery management are clearly indicated and discussed.  This paper adds to the growing literature on octopus fishery concerns and management recommendations.


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

Input:
Title: LIFE HISTORIES PREDICT GENETIC DIVERSITY AND POPULATION STRUCTURE WITHIN THREE SPECIES OF OCTOPUS TARGETED BY SMALL-SCALE FISHERIES IN NORTHWEST MEXICO
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear, unambiguous, professional English language used throughout. Mostly – some odd phrases and occasional wrong words – suggestions added to the pdf.

Intro & background to show context. - yes

Literature well referenced & relevant. - yes

Structure conforms to PeerJ standards, discipline norm, or improved for clarity. - yes

Figures are relevant, high quality, well labelled & described. - yes

Raw data supplied (see PeerJ policy). - yes
Experimental design: Original primary research within Scope of the journal. - yes

Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap. - yes, clearly defined in the Abstract, revisited in the Introduction.

Rigorous investigation performed to a high technical & ethical standard. - not sure - see the issues raised in General Comments
As only 316 individuals across 3 species, 20 localities and 5 years are used, suitable rigour could be questioned? But, because of fairly clear distinction of species by molecular typing, and the subsequent use of pooled analysis to assess general species-wide parameters, then the dataset is suitable.

Methods described with sufficient detail & information to replicate. – yes – nice explanations linking hypotheses and predictions to tests employed.Two mitochondrial genes and even nuclear microsatellite loci are suitable markers for the objectives
Validity of the findings: The Conclusions are clearly stated, and on the whole justified. There is some speculation, but this is identified.

However, regarding whether the data are robust, statistically sound and controlled there may be a potentially major issue with the identification to species of individuals and their incorporation to subsequent analyses.

What is done with the individuals in Fig. 2B that are not clearly assigned to one of the three clusters? For example, a number of individuals in the Puerto Penasco locality appear to be more assigned to O. hubbsorum rather than O. bimacularus, but this is not indicated in the species distribution map? This is acknowledged in the text (l.291-294), but the colour coding of the figure does not convey this, and so is slightly misleading. Could be rectified by cross-hatching the sample localities that contain individuals from two species?
This then leads on to the question as to what was the cut-off level in the assignment to decide on which species to assign an individual to? Or was one employed? There is no mention of this in the text, but after this point the reader is presented with a series of tests that are calculated from location-based genetic data which presumes that either all individuals within a location have been assigned to a single species and pooled (which would be wrong as Fig.2B appears to indicate that many samples contain individuals that assign to species different from the one predominant in that locality), or that some individuals have been excluded from the analyses because they did not assign to the predominant species in that locality (which is not mentioned or indicated, and so cannot be presumed).
This is a serious potential flaw in all subsequent analyses, because it is not clear whether some individuals that may be from a different species have been included in the within-species tests, which would introduce substantial random variance in the microsatellite genotype and allele frequencies, and so generate high and random values of statistics such as Fst (which might explain the high values in the pairwise tests – see below?). If the authors can confirm to the Editor that suitable steps were taken to identify the species assignment of each individual before subsequent analyses, and then that individuals were partitioned appropriately so that only single-species datasets were used, then the ms can be adjusted with relatively minor changes (a section in the Methods or Results to explain the ID and partitioning of individuals more thoroughly, and adjustment of Fig.2C to reflect the mixed allocations within localities). If robust ID and removal of individuals was not carried out then the dataset needs re-analysis and re-presentation. I am presuming the former for this review.

Also, you might comment on what the “mixed allocation” individuals represent in Fig.2b?

I’m not convinced by the values given (l.327-334) for pairwise between-locality estimates of Fst based on the 7 microsatellite loci. Many of these tests are based on sample sizes in single digits, and so will suffer from random sampling variation. I think that this section, along with the supporting Tables should be removed, as it is pushing the dataset too far and so detracts from the other better-supported sections.
Additional comments: No additional comments


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

Input:
Title: LIFE HISTORIES PREDICT GENETIC DIVERSITY AND POPULATION STRUCTURE WITHIN THREE SPECIES OF OCTOPUS TARGETED BY SMALL-SCALE FISHERIES IN NORTHWEST MEXICO
Review round: 2
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: The authors appear to have taken on board the concerns expressed and have adjusted their analyses and/or interpretation and presentation of data to account for this.


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

Input:
Title: CODOMINANT GRASSES DIFFER IN GENE EXPRESSION UNDER EXPERIMENTAL CLIMATE EXTREMES IN NATIVE TALLGRASS PRAIRIE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article was for the most part easy to understand.  There are a few items that could be clarified in order to be more understandable to a broader audience.
Experimental design: As the authors acknowledge, the sample size was a low and microarrays are not as detailed as an RNA seq study, but the study is important and provides an important narrative toward understanding how species may respond to climate change.

I have never used microarrays, so I will defer to another reviewer on those statistics.
Validity of the findings: No comment.
Additional comments: Great article!  We're starting an RNA-seq assisted migration study on cool-season grasses in North Dakota, so your article was of particular interest to me.


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

Input:
Title: CODOMINANT GRASSES DIFFER IN GENE EXPRESSION UNDER EXPERIMENTAL CLIMATE EXTREMES IN NATIVE TALLGRASS PRAIRIE
Review round: 1
Reviewer: 2

Output:
Basic reporting: It is well written, clearly stating the results and the implications for this field of research.
Meets all the guidelines of PeerJ wrt references etc.
Experimental design: Good
Validity of the findings: Good
Additional comments: Review of PeerJ manuscript;
Codominant grasses differ in gene expression under experimental climate extremes in native tallgrass prairie.
Ava M Hoffman, Meghan L Avolio, Alan K Knapp, Melinda D Smith
This is a small but worthwhile analysis of the differences in gene expression between two grass species grown under environmental stress. It is well written, clearly stating the results and the implications for this field of research. I recommend acceptance and publication of this manuscript.
However, I do have a number of minor comments that should be addressed.
•	Line 37 – Cite IPCC 2013 but there are no details in the reference list under IPCC. So add Stocker et al 2013 to the text or put the details under IPCC

•	Following sentence is incomplete -  improving our understanding of what and there is no full stop.
52	"However, most molecular studies of plant responses to drought and heat stress are focused on
53 	model organisms with limited ecological relevance (Leakey et al. 2009), although awareness and
54 	sequencing costs are improving our understanding (Voesenek et al. 2014; Ellegren 2014) "

•	In the site description (line 99) the plot sizes are given as 3 x 6m but in the legend to Online resources Supplementary methods line 3 it states 2 x 6 m;

•	line 276 – 'both histone acetyltransferases were upregulated" implies only two exist in the species. Add that the two histone acetyltransferases identified as differentially expressed in A.gerardii  were upregulated.

I do have two comments that could be taken into account for future studies;
1. The study is limited in that only two individuals were sampled for each species under each of the treatments but I agree with the authors arguments that this is sufficient for this preliminary study.
2. Hybridisation of cDNA synthesised from leaf RNA against maize ear tissue may have limited the type of genes identified.
However, I also recognise that there is a limit to the resources available for a study such as this and despite the above reservations I think these results add to the information available in this area of research.