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: EFFECTS OF ROOT PHENOTYPIC CHANGES ON THE DEEP ROOTING OF POPULUS EUPHRATICA SEEDLINGS UNDER DROUGHT STRESSES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The study was a pot experiment, which supplied the adaptive regualtion information of root morphology,distribution and architecture under differnter drought treatments. On the whole, the manuscript did not present novel findings in plant grwoth adaption area. There are some mistakes in figs and tables presentation, and the english also need to improve by a native english speaker.
Experimental design: The experiment want to distingush the relative contribution of root morphology, architectue and biomass allocation on root adjustation to drought stress, but it is hardly to address this, due to their autocorrelation. In addition, if the author want to determine the improtance of deep root to suction of underground water, a serial of underground water level shoud be include in the experiment.The define of the relative tap root length is not appreciate.
Validity of the findings: the manuscript distingusished source of variation of deep root under drought, whihc is usful for evaluating plant morphological regulation under unfavoriable environmental change.
Additional comments: This is a simple but valuable study, the author tried to explore the relative contributions of root morphology, architecture and carbon allocation in root adaptation to drougt. Buts this is not a well-written paper, evidencing by unclear organization and figs and tables presentation.


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: EFFECTS OF ROOT PHENOTYPIC CHANGES ON THE DEEP ROOTING OF POPULUS EUPHRATICA SEEDLINGS UNDER DROUGHT STRESSES
Review round: 1
Reviewer: 4

Output:
Basic reporting: Some suggestions were made in this section aiming to improve the manuscript and are described individually in the general comments.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This paper presents a study using Populus euphratica seedlings to compare root phenotypic changes in two drought conditions in relation to a control. This study seems to be relevant for the understanding of mechanisms undertaken by this species to overcome drought effects in the plant. The manuscript is well structured and the results seem consistent. There are some considerations that could help to improve and let the manuscript more clear to the readers. Also, the interpretation of some statistical analysis need to be done more carefully, as suggested bellow.

I recomend to consider the following issues:
1.	A few sentences could be re-written to make more sense or to improve the overall article writing. I tried to re-write some of them, which can be found in the PDF file as comments or marked in red or blue (associated with comment notes). Also, some suggestions were added to the text, indicating where other minor changes should be made.
2.	The legend of figures and tables need to be sufficient for the readers to comprehend the data. So, it is necessary to define the abbreviations and the statistical analysis used in each figure/table and make this very clear.
3.	The authors need to cite the figures and tables when they describe the results and in the discussion, otherwise, the reader won’t follow the ideas.
4.	Figure 1 needs improvements to be kept in the article. For example, the aerial part of the plant shouldn't be the drawing of a heart. In the end of each sentence there is an arrow. Besides that, the arrow from the middle is covering the text. The use of a proper image editor software might be a very useful tool to construct the image again.
5.	The tables 2, 3 and 4 could be combined in one single table, since they show variables in the same treatments and with the same statistical analysis.
6.	For a better organization, the root variables in Table 1 should be described in the order of appearance in the text. Besides that, the overall organization of the table needs improvement. For example, the units shouldn't be in separate lines; sentences shouldn't begin with lower case. The table needs to be reviewed carefully.
7.	Column 1 of the table with the raw data (supplemental material) needs an adequate identification. It is written “numbers”, but do these numbers refer to the seedlings? The authors must let this clear. In column M is it SDRL or SRL? A review of the columns titles is necessary to make a good presentation of the data.
8.	The figure of the supplemental material is very illustrative and helps to comprehend the manuscript. However, it isn’t cited in the text. I think that this figure could be included in the body of the article, not as a supplemental material. A scale needs to be included in the figure and some modifications could be done in the figure legend, as follows: “Representative scan images used in the study to illustrate changes in root system of P. euphratica seedlings with drought treatments”.
9.	The data is statistically controlled. However, the authors need to take more care when analyzing the results and interpreting the statistical significance. Data sharing the same letter aren’t statistically different and the interpretations must take this into account. For example, the lines 203-205, 221-223 and 227 should be re-written according to the statistical significance of the data (see notes in PDF file). In addition, when there isn’t statistical difference this should be indicated with “ns” in the data of the tables.
10.	In the topic “Experimental treatments” of Materials and Methods, the authors need to take a look at the temperature values indicated, because they don’t make sense. About the replications, each experiment was replicated 30 times or 30 seedlings were evaluated per treatment? Also, it is specified that 85 seedlings were used in the total, but there are data for only 81 seedling in the table with the raw data. These numbers need to be corrected also in the legends of the tables, where the authors indicate the “n” of each treatment.
11.	The sentence of lines 153 and 154 is a bit messy and hard to understand. I believe that the authors could write it better.
12.	In the lines 278-280, the authors should state that there is a tendency of change to herringbone, but the data (especially DBI) indicated a dichotomous-like branching in the moment of the evaluations.
13.	Lines 289-290: Is it a title with subtitle? Or is it the same sentence? Please make it clear.
14.	In the line 362 it would be better if the authors could state to what kind of economy they are referring to.


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: EFFECTS OF ROOT PHENOTYPIC CHANGES ON THE DEEP ROOTING OF POPULUS EUPHRATICA SEEDLINGS UNDER DROUGHT STRESSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: - The authors have carefully addressed the issues and have revised the manuscript very well.
- Good revision on the tables, and the legends.
- The work represented by the authors can lead to root-phenotype studies in other plant species.
-
Experimental design: - The authors have revised the experimental design more organized way.
- Easy to understand the experimental design.
Validity of the findings: - The data is good.
- Good representation of the data.
Additional comments: Well representation of the data, and very well revised.  The authors have addressed the issues raised previously by the reviewers, and a great job on revising the manuscript.


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

Input:
Title: EFFECTS OF ROOT PHENOTYPIC CHANGES ON THE DEEP ROOTING OF POPULUS EUPHRATICA SEEDLINGS UNDER DROUGHT STRESSES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised paper has been improved.
Experimental design: The author provided more details of the experiment in the revised paper. It is more clear than the previous one.
Validity of the findings: It would be better if the same data analyses been done on the field investigation.
Additional comments: The author has made a careful revision of the last review opinion, which makes the current text more readable. Because there are few studies on the root architecture of natural plants, this paper may provide some ideas for researchers.


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: EFFECTS OF ROOT PHENOTYPIC CHANGES ON THE DEEP ROOTING OF POPULUS EUPHRATICA SEEDLINGS UNDER DROUGHT STRESSES
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have performed the modifications suggested in the first review of the manuscript and it was substantially improved.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I have only some minor modifications to suggest:

1.	Line 39: Correct the word “acquiring”.
2.	Line 58: What do the authors mean with "except"?
3.	Line 163: I suggest to change this sentence to: "Root phenotypic adjustments and deep-rooting capacity analysis".
4.	Line 168: There is no need to cite Fig. 1 here.
5.	Line 185: Correct the word “representing” to “represent”.
6.	Lines 206 and 213: Correct the position of the “comma”.
7.	Line 220: Correct “(Table. 2)” to “(Table 2)”.
8.	Line 221: Where is the average length of taproot shown in the table?
9.	Line 229: Correct the word “changed” to “change”.
10.	Line 235: MD didn't differ from OW or SD.
11.	Line 254: Correct to: “contributed to approximately only 20%”.
12.	Line 305: It is a good moment to cite Fig. 2 (after “drought conditions”).
13.	Line 310: I suggest to consider the following way to write this sentence: “Additionally, it should be noticed that in our experiment there is a vertical soil moisture gradient in the plant pots. Other studies performed with more actual ground water conditions and different soil texture layers would be an alternative strategy to determine and confirm deep-rooting of P. euphratica seedlings.”
14.	Line 315: Changes to in absolute
15.	Line 333: “thereby leads to a shortening of the root depth”
16.	Line 349: “plants to respond to belowground resources”
17.	Line 355: “functions for seedlings that are suffering with drought conditions.”
18.	Line 357: Remove the word “obviously”.
19.	Line 360: Maybe this sentence could be split in two: “…photosynthates (Muller et al., 2011). Root architecture changes…”
20.	Line 406: Correct the word “colleague” to “colleagues”.
21.	Fig. 1: remove the dots between the words (inside the boxes).
22.	Fig. 2: Correct in the title: (C) & (D) from the seedlings under MD
23.	Fig. 3: Ideally, the legend with the colors should be shown according to the order of appearance in the graph: STRL (dark gray) on the top and TRMF (black) on the base.
24.	Table 2: Title: Correct to: “Mean values of biomass, root morphological and root branching variables of…”


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: TARSYNFLOW, A WORKFLOW FOR BACTERIAL GENOME COMPARISONS THAT REVEALED GENES PUTATIVELY INVOLVED IN THE PROBIOTIC CHARACTER OF SHEWANELLA PUTREFACIENS STRAIN PDP11
Review round: 1
Reviewer: 1

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: The manuscript entitled “TarSynFlow, a workflow for bacterial genome comparisons that revealed genes putatively involved in the probiotic character of Shewanella putrefaciens strain Pdp11”, by Seoane et al., presents an interesting bioinformatic tool (Targeted Synteny Workflow) for comparative genomic analysis. The authors show that the application of such tool for genome comparison between probiotic vs non-probiotic strains is of upmost importance, and might allow to identify probiosis related genes, further detailing current knowledge on probiotics mechanisms of action.
The manuscript is very well written, the methodologies used are adequate and well described, with exception of a few clarifications that authors should do, to improve the overall quality of the manuscript before acceptance for publication.

1.	The authors can add some justification to sequence just six out of the 16 gene amplicons analyzed, and also tentatively comment on the low identity percentage obtained for 2 of the sequences, namely 88.25 and 78.41%.

2.	In the bottom of Figure 4, the protein code, rather than the protein function, should be used to maintain the same nomenclature throughout the paper and facilitate the comparison with Tables 2, 5 and 6.

3.	Line 106: please correct the “dose at which 50% of bacteria die”, since are not the bacteria who die

4.	Line 118: please check the code provided PRJNA510237, since no its can be found at the BioProject database at NCBI

5.	Line 221: please replace the word “goodness” for a more scientific one such as potential, applicability, effectiveness, etc

6.	Line 326: please correct “Nevetheless” to Nevertheless

7.	Line 352: “are” at the end should be deleted

8.	Line 371: “competence” should be replaced by “competition”

9.	Lines 380-382: the authors should add some reference(s) to support the sentence describing Pdp11 strain as being resistant to bacteriocins


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: TARSYNFLOW, A WORKFLOW FOR BACTERIAL GENOME COMPARISONS THAT REVEALED GENES PUTATIVELY INVOLVED IN THE PROBIOTIC CHARACTER OF SHEWANELLA PUTREFACIENS STRAIN PDP11
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is generally well written. The statements and arguments are presented in a clear manner. The figures are clear and easy to follow.
Experimental design: The research question and hypothesis are clearly presented. Deatils are available for this paper to be reproduced. This is particularly important since the manuscript is describing a a method or approach.
Validity of the findings: The method presented here is highly valuable for the progress of probiotics research in fish. The data are presented in a highly understandable manner. The implications are logical and  are supported by the data.
Additional comments: Figure 1. a more descriptive caption would improve this figure. At present, it is too short and vague. The reader needs to scan through the text in order to understand the figure.

Table 4 is best presented through a venn digram.


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: WINTER TEMPERATURE PREDICTS PROLONGED DIAPAUSE IN PINE PROCESSIONARY MOTH SPECIES ACROSS THEIR GEOGRAPHIC RANGE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well structured (standard academic format) and provides new interesting results but requires more explanation in each section for the reader to fully grasp the research. References are well cited and the figures are informative and high quality.

Introduction:

I find the abbreviation of PD distracting and would prefer the authors just write prolonged diapause.

LN 49. “and is generally considered as a way to spread the risk” this sentence makes PD sound like more of a deliberate choice than it is and implies there are other explanations. Could it just be a fluke or mistake rather than a strategy? For readers with little background knowledge of PD it would help to have a little more background.

The intro does not provide the reader with enough background on the life history of PPM. When does it pupate? For how long? What mechanisms cause PD and how do they cause it? Without a thorough understanding of these things one cannot follow the paper or results.

ln 60. what is facultative PD? why does it occur? does this mean all PPM in this range have PD?

ln67. What is the temperature hypothesis? Is this a formal hypothesis established by Demolin? Did the authors just invent it? Describe this hypothesis if it is an established hypothesis in the literature.
Experimental design: The research is original and unique. They take advantage of collaborations with many authors to combine data from a large geographic range.

ln79-83. I do not know why these criteria were chosen since the life cycle is not explained or the proposed mechanisms for PD. This section would benefit from a statement like: Since PPM pupate during this time of year and we predict x to be a mechanism causing PD we only selected studies that include the following criteria….

It would also be helpful to know how the experiments were conducted (briefly). Were larvae collected from trees then reared in the lab? were they kept outside? how can one track a wild cohort for multiple years? I can’t judge the soundness of the criteria without more knowledge of the studies and the PPM.

ln90. Why the average temperature? I thought ppm was affected by minimum temperatures? Many insects are more sensitive to extremes than averages. Please explain this choice.

ln102. How do the author's account for latitude? do they need to? In the introduction they say PD is common or facultative in the northern and southern edges of the range.
Validity of the findings: Results
Would the relationship between temperature and PD or survival be the same if replaced with latitude? For example would figure 2a look the same if the x axis was latitude instead of temperature? From the map it doesn’t look like it but showing this might strengthen the argument that it is temperature and occurs anywhere within the range that temperatures reach the specified lows or highs.

There is no report of the statistics related to clade. This was included in the analyses (methods ln104) but we do not see the results of how much clade influenced PD compared to temperature.

Actually I do not see any statistics reported accept in the figure legends. These should also be in the text.

Discussion
ln148. Again it would be helpful to know how the studies were conducted. In addition I do not understand from this sentence how PD can be assessed from a one year study?
This paragraph should expand on potential mechanisms. For example heat is a stressor but how does this translate physiologically into PD.

The explanations that exposure to heat or cold stress leads to PD which reduces encounters with those stressors the next year is not intuitive. It implies to me that every hot year is followed by another hot year that the animal would want to avoid. I would think an exceptionally hot or cold year is more likely to be followed by an average year. In this case the risk of PD does not make sense. Expand this line of discussion.

Elsewhere like ln152 the authors imply poor nutrition could lead to PD. Is this via the same stress mechanism as cold or a different one. Someone without a vast knowledge of diapause would not be able to follow this.

ln187. Telling us different avoidance mechanisms are important without discussing what they may be is not enlightening and does not allow the reader to ponder hypotheses about this or other species and expand on and cite the work in the future.
Additional comments: The research presented is sound and important but the manuscript needs revision to help the reader get the most from the results and to provide proper context for the results.


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: WINTER TEMPERATURE PREDICTS PROLONGED DIAPAUSE IN PINE PROCESSIONARY MOTH SPECIES ACROSS THEIR GEOGRAPHIC RANGE
Review round: 1
Reviewer: 2

Output:
Basic reporting: I have read with great interest the manuscript (#28902) “Winter temperature predicts prolonged diapause in pine processionary moth across its geographic range” by Salman et al. and the supporting material. The English language is good and understandable, and as I am not a native English speaker, I have no further linguistic comments. Also the Figures are informative and relevant, high quality, and well described. Raw data is (partially) supplied in the electronic supporting material.
The Introduction gives a rather short overview of prolonged diapause, focusing on the role of adverse abiotic factors (winter temperatures) as the selective or triggering factor behind it (lines 61-66) in the Pine Processionary Moth (PPM); in the Discussion (lines 151-154) the potential role of food shortage is shortly mentioned, but I think that the avoidance of parasitoids and diseases should also be discussed. Overall, I think that the paper would benefit from a more thorough introduction to and discussion of the theories of prolonged diapause (to better relate their empirical study to the existing theory); I think that the authors should read and cite the article by Hanski (1988) Four kinds of extra long diapause in insects: A review of theory and observations. Ann. Zool. Fennici 25, 37-53.
Experimental design: The empirical data base of this study consists of published and unpublished studies done during an extended time frame (from 1971 to 2012) in different parts of the distribution area of PPM. The hypothesis tested in the present manuscript has probably not been the main question addressed in these studies, and the data is very heterogeneous (as detailed in the supporting material): both field and field/lab studies, different protocols, etc. Also, it has not been explicitly stated with each data whether the temperatures used are air or soil temperatures; the often repeated statement “Retrieved from the documents” is not informative/accurate enough.
The authors write (lines 106-108) that “It is important to stress that our model tested the effect of spatial variation across the species range, and not the inter-annual variation in temperature within the sites.” However, if I have understood correctly their data (given in supporting material), there are also sites with several observation years and with inter-annual variation in temperatures (e.g., six years for Vielle-St-Girons, France). So, I think that the effects of both inter-annual temperature variation and location effects are studied in this manuscript.
Validity of the findings: The authors state (line 103) that they have “used a linear mixed-effect model”. I think this is not a correct expression, as their model has also a quadratic (non-linear) term. Furthermore, I think that the selection of a polynomial model with only two terms (linear and quadratic) does not use all the information in the data, and leads to (partially) wrong conclusions. On the basis of a visual inspection of Figure 2(a), I think the data should be analyzed as a series of polynomial mixed-effect models with first, second, third and (maybe) fourth order terms, and the performance of these alternative models should be compared with information based criteria (e.g. AICc). Another option would be mixed model ANOVA comparing the groups “< +2oC”, “2-10oC”, and “>10oC”. I don’t agree with the conclusion of the authors that “temperatures .. .. higher than 10oC were associated with higher PD incidence than cohorts exposed to intermediate temperatures”. It seems to me, that ANOVA would not reveal a significant difference in the PD between intermediate temperatures [ “2-10oC”] and high temperature [ “>10oC”] due to the very high variation within the latter group [PDmin = 4.2%, PDmax = 99.6%].
Additional comments: Please, find the above cited article by Hanski (1988) here:
http://www.sekj.org/PDF/anzf25/anz25-037-053.pdf


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: WINTER TEMPERATURE PREDICTS PROLONGED DIAPAUSE IN PINE PROCESSIONARY MOTH SPECIES ACROSS THEIR GEOGRAPHIC RANGE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Basic reporting is OK but as I describe in my review there are many places where text and ideas are confusing and need to be clarified.
Experimental design: Excellent and novel.
Validity of the findings: Data and statistics sound. The authors made changes that help us understand how data were collected. Some aspects of concluding paragraph and discussion can be clarified. Suggestions in review document.
Additional comments: Many good changes made in response to previous reviews were completed. However, some of the new text is confusing and needs rewritten.


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: WINTER TEMPERATURE PREDICTS PROLONGED DIAPAUSE IN PINE PROCESSIONARY MOTH SPECIES ACROSS THEIR GEOGRAPHIC RANGE
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I read the submitted manuscript, the comments of the reviewers, the response of the authors and the modified manuscript. The new version was improved a lot compared with the original version, however I still found manuscript confusing and some of the fair criticisms of the reviewers are no solved in the new version.
The paper deals with the role of the winter temperatures to predict prolonged diapause in Pine processionary moth. Most of the data are from published papers with heterogeneous questions and experiments.  Authors claim that the study range from 1964 to 2015, but in many of the sites the data are from one year, not enough to study prolonged diapause.
With the data that they use they cannot reject other factors that may influence prolonged diapause as limited food resources or parasitism avoidance
The main conclusion is that insect cohorts exposed to average winter temperatures lower than 0ºC were associated with prolonged diapause, but according with data provided in Supplemental Data set 2 there is a single site in the study (of the 46 with data of winter temperatures: Venosta), that reach T below 0 during winter. And, if I understand well the Supplemental data set 2, even in that site diapause in one year was as low as 2.1 (I guess %).
In my opinion the paper address an important question, but the data that they use is not the best option to solve it.


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: RECRUITMENT OF COGNITIVE CONTROL REGIONS DURING EFFORTFUL SELF-CONTROL IS ASSOCIATED WITH ALTERED BRAIN ACTIVITY IN CONTROL AND REWARD SYSTEMS IN DIETERS DURING SUBSEQUENT EXPOSURE TO FOOD COMMERCIALS
Review round: 1
Reviewer: 1

Output:
Basic reporting: See my comment below.
Experimental design: See my comment below.
Validity of the findings: See my comment below.
Additional comments: In this study, the authors examined the neural mechanisms through which the exertion of self-control magnifies subsequent self-control failures. Specifically, the authors tested whether lateral PFC activation during self-control exertion would predict a reduced balance between this PFC region and neural ‘reward’ regions, and would predict increased self-control failure in the form of ice-cream consumption among a sample of female dieters. The authors found evidence that DLPFC recruitment during self-control exertion was subsequently associated with less connectivity between the reward circuit and the lateral PFC.
I’ve been waiting for a study like this (i.e., one that uses fMRI during a reward-based self-regulatory challenge *and* afterwards during an appetitive task) to be run for years, so glad to finally see one! There’s a lot to like here: combining neural predictors with real-world behaviors, examining neural correlates of self-control exertion, recruiting a sample of people in an ecologically-valid chronic self-control challenge (i.e., dieting), and a theory-derived set of predictions on a critically-important and impactful subject. The writing is impeccable and the logical flow of the paper is fluid.
Below, I list some comments and questions for the authors:
•	The second citation in your Intro includes the first author’s first name and middle initial. This also happens in some other parts of the manuscript.
•	In your hypotheses section of your Intro, you should mention the context in which you will be measuring lateral PFC recruitment.
•	At the canonical 80% power level, your sample size of 27 is too small to detect between-subjects correlations smaller than r = .52. We rarely see such large effect sizes in human behavior. As such, the analyses in this paper are almost certainly severely underpowered and this needs to be more clearly stated in the Methods, Discussion, and the N = 27 sample size must be mentioned in the Abstract to engender the appropriate level of skepticism in your readers. As it stands, your reduced sample size is squirreled away in the ‘Analysis of fMRI Data’ sub-section.
•	Unless the authors believe that all participants were 100% successful in not reading the distractor words during the self-control challenge task, their manipulation of self-control engagement is confounded with lexical processing.
•	 How were the DLPFC ROI peak coordinates determined?
•	Please depict the 8 nodes of the frontoparietal network and the ROIs you built around them. Maybe as a supplemental figure.
•	Were the OFC/striatum ROIs spherical or anatomical?
•	Why is one of your p-values labeled as ‘two-tailed p = .005’? Are the other p-values one-tailed?
•	How were the “balance scores” calculated?
•	Some degrees of freedom seem off. You should have 25 degrees of freedom for your bivariate correlations, but I see “r(24) = -0.574” and then when 3 covariates were included, which should’ve reduced your dfs to 22, I see “t(19) = -3.445”. Am I missing something?
•	Please report exact association statistics and p-values, not ranges (e.g., “p’s ≥ 0.536”).
•	Why are 95% CIs reported for some findings but not others?
•	“participants 342 consumed 71.1 grams of ice-cream (SD = 40.8, range: 9–158 grams)” Wow!
•	Were the food commercials equivalent to the non-food commercials in important aspects such as arousal or valence? If not, this may not be a story about food per se, but about arousing/rewarding stimuli more generally.
•	You refer to p-values of .112 and .124 as “trending”. C’mon, that’s way too much of a stretch. Please recall that trending can mean trending *away* from significance just as much as it can mean *towards* significance. I think these just need to be called the null results that they are. I’m bummed that these weren’t significant (and I bet they would be if you had a larger sample). What is the distribution of the ice-cream scores like? If it’s positively skewed, does a log-transformation help?
•	 There’s no way you have the power to accurately test this, but I’m curious of the association between DLPFC recruitment during self-control and ice cream consumption is mediated by balance scores during food commercial viewing.
•	Correlational nature of the study is not mentioned.


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: RECRUITMENT OF COGNITIVE CONTROL REGIONS DURING EFFORTFUL SELF-CONTROL IS ASSOCIATED WITH ALTERED BRAIN ACTIVITY IN CONTROL AND REWARD SYSTEMS IN DIETERS DURING SUBSEQUENT EXPOSURE TO FOOD COMMERCIALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article is well written and contained sufficient background and details.

My main critique of the reporting has to do with the focus on the ego depletion debate in the introduction. The authors acknowledge in the discussion section that “the present study cannot disentangle these two views” (p. 16). While I think it is important to acknowledge this debate, I think the introduction should focus more on the specific questions and gaps in knowledge that this paper can address. I would have been more convinced if the authors spent more time explaining why fatigue/depletion (or whatever you want to call it) of cognitive resources is important to study independent of this debate. For example, spending more time discussing evidence that self-control failure follows earlier attempts at effortful self-control, particularly in the domain of dieting, would be more convincing.
Experimental design: The experimental design is well thought out. Both tasks are more ecologically valid than typical fMRI tasks. My primary concerns are with the small sample size, relatively short initial self-control tasks, and lack of a self-control task in the one week follow-up. If the authors were interested in how initial self-control attempts influence subsequent eating behavior, wouldn't it make sense to include a self-control task before the milkshake and ice cream taste tests? The authors do a good job acknowledging the first two issues and I'm not sure if anything can be done at this point to address the third point.

Finally, I thought the description of the frontoparietal network nodes was lacking some crucial details. Unless I’m mistaken, I believe there are 25 frontoparietal nodes in the Power et al. (2011) parcellation. If the authors only used a subset of these nodes they should explain how they were chosen. It would also be important to note if the results hold when including the full frontoparietal system.
Validity of the findings: The current study compared activation across two tasks and applied a network approach to investigate how activation across multiple brain regions might relate to how dieters respond to food cues. A key strength of this approach is using both task-defined ROIs and a priori system labels. This research takes a novel approach and provides some interesting evidence that recruitment of neural self-control resources may be reduced over time due to depletion/fatigue/reduced motivation. I have two primary concerns with the interpretation of results:

First, I had a hard time following the cognitive efficiency hypothesis as it relates to this data. If weaker activation is actually indicative of better processing/self-control, then why do people who have greater activation in the first task have weaker activation in the second task? Are people getting better at self-control over time? This seems counterintuitive and only makes the results harder to make sense of.

Second, do the results hold if the authors recompute the balance scores excluding ventral striatum? I ask this primarily because striatum is not just a reward region—it is also implicated in response inhibition. It seems problematic to argue that striatum is a reward region when it is also involved in response inhibition.
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: RECRUITMENT OF COGNITIVE CONTROL REGIONS DURING EFFORTFUL SELF-CONTROL IS ASSOCIATED WITH ALTERED BRAIN ACTIVITY IN CONTROL AND REWARD SYSTEMS IN DIETERS DURING SUBSEQUENT EXPOSURE TO FOOD COMMERCIALS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript was easy to follow and clear in its writing. The Introduction covered the relevant literature adequately, although I would have liked to see a more explicit statement on how the current work differs from Wagner et al. (2013). (I was able to piece it together by re-reading, but a more explicit statement of the differences would have saved this reader some time and effort.) The figures were relevant and clear, and the data was made available for review.
Experimental design: This was original primary research, and the research question was well defined, relevant, and meaningful. As mentioned above, a more explicit statement of how this research differs from past research, and how it fills an identified knowledge gap, was lacking.

Methods were described in sufficient detail and the work appears to have been performed to high ethical and technical standards.
Validity of the findings: The data analysis was sound and interesting. I think it is inappropriate, however, to refer to a “positive trending correlation” (lines 342 and 343). The same goes for the “trending” correlation reported in lines 344 to 345. Simpler to say the correlations were non-significant but in the predicted direction (as the authors go on to do in line 389). We don’t know which way they were “trending.”

Further, I thought the Discussion section included a balanced assessment of the strengths and weaknesses of this work.
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: REPEATED ETHANOL EXPOSURE INCREASES ANXIETY-LIKE BEHAVIOUR IN ZEBRAFISH DURING WITHDRAWAL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript by Krook et al. reports on the behavioral effects of ethanol withdrawal on zebrafish behavior in the novel object test (to which the authors refer as a “novel approach test”, without much consequence). They find that zebrafish exposed to 0.8% EtOH, but not 0.4% EtOH, for 21 days, with an exposure of 1 h per day, and later subjected to 48 h withdrawal, appear to either avoid the novel object or display more centrophobia, without motor impairments. Results are discussed straightforwardly in terms of behavioral effects of ethanol withdrawal, as well as of possible neurochemical mechanisms.
The paper introduces a straightforward and relatively selective result in a field that is marked by contradictory findings. Authors introduce a novel approach to study the effects of ethanol withdrawal, building on previous research to test behavioral hypothesis. Although no justification is given for the sample sizes used, they appear appropriate.
Experimental design: 1) There are several issues of experimental design and transparency which make it difficult to understand biases that could have been introduced in the experiment and would be responsible for some of the anomalous findings. These include:
-How were sample sizes calculated? Based on what effect size, power, and alpha? This is crucial to understand whether experiments are under-powered.
-Were experimenters blind to treatment? We know that animals were randomly allocated (lines 129), but the specifics are not reported. Be specific and explain HOW blinding and random allocation was performed.
-Were animals from the control and treatment groups drawn from the same tank/rack? If not, were the racks/tanks positioned at different places in the vivarium?
-Were only males used? Only females? Mixed sexes? What was the phenotype of the fish (e.g., shortfin, longfin)? What was the origin of the fish before they reached the colony? Were they bred in the lab? For how many generations?
-At what time of the day did experiments take place?
2) The dataset provided is formatted in a way that precludes computational reproducibility, as one needs to either format it or copy-paste to another program to make analyses. Please provide data formatted as comma-separated values spreadsheet (CSV), with machine-readable headers (i.e., using dots or underlines as separators), and six columns (group, time in center zone, time in transition zone, time in thigmotaxis zone). Also, please provide raw data for velocity and immobility.
Validity of the findings: The issues of transparency and design described in the previous session need to be addressed in order to judge the validity of  the findings.
Additional comments: 1) In the results, data are presented as means ± SOMETHING; are those CIs, S.E.M.s, or SDs?
2) While the name “novel approach test” is interesting, for the sake of comparison with results from other labs I suggest changing it back to “novel object test”
3) Please provide a full protocol for the behavioral test (it can be posted on protocols.io, e.g.)


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: REPEATED ETHANOL EXPOSURE INCREASES ANXIETY-LIKE BEHAVIOUR IN ZEBRAFISH DURING WITHDRAWAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript depicts the anxiogenic effect of ethanol during withdrawal. Despite the relevance of the study and consider the manuscript to be publishable, however, the presented effect is not supported in literature studies, as mentioned in the manuscript. The concentrations and time of exposure used by the authors do not match those of the literature (see review), therefore, I consider that the observed behavioral effect should also be reproduced in other behavioral protocols. I consider that the breadth of conclusions requires additional experiments. The same occurs when affirming a dose-dependent effect using only 2 concentrations of ethanol, what are the behavioral effects of higher and lower concentrations of those used in the study?

1)	Authors begin and end phrases throughout the manuscript without demonstrating the reference.

2)	In lines 59-63 the authors describe zebrafish positives on genetic manipulation and model neurotransmitter systems provoked by ethanol, however, the reference used (Müller et al., 2017) carried out studies of social behavior and oxidative stress, not those described in the manuscript.

3)	The data presented by the authors show a possible anxiogenic effect of ethanol, however, the concentrations and time of exposure are not the same presented in the literature, referenced and discussed by the authors. I consider that a behavioral protocol is not adequate to affirm the presented effect, since there is no data in the literature that use 21 days of exposure and concentrations of 0.4% and 0.8% therefore, additional studies are necessary to confirm the anxiogenic effect.

4)	In lines 59-63 the authors describe genetic manipulation and neurotransmitter systems induced by ethanol in zebrafish, although the reference used (Müller et al., 2017) carried out studies of social behavior and oxidative stress, not those described. This type of error also occurs in lines 134-135, 227-230, among others. I suggest that the authors review it.
Experimental design: 1)	The manuscript has scientific relevance, however, the data presented in only one behavioral apparatus are not considered enough to confirm the observed effect. I consider the need to include at least one more behavioral protocol proving such an effect, as done by Mathur & Guo (2011) since the concentrations and time of exposure chosen by the authors are not the same as those found in the literature.

2)	The authors mention that Mathur & Guo (2011) do not observe effects on day 1 and 2 after withdrawal in the tests of light/dark and novel tank test, respectively. However, Müller et al. (2017) presented an anxiogenic effect in the social behavior test, also correlating oxidative damage induced by ethanol within 24 hours after withdrawal. Why did the authors opt to perform the experiments only 2 days later?

3)	Were the experiments performed only with male animals? or females? To describe in the materials and methods.

4)	Were the animals fed only once a day (line 120)?

5)	What was the procedure after behavioral analysis? Did the animals return to the dwelling tank? Were they euthanized?

6)	In the description of the exposure protocol, the authors reported their concentrations and exposure times according to Holcombe et al., 2013 (30 min exposure in 0.2% and 1.4% ethanol) and Mathur & Guo 2011 (20 min exposure in 1% of ethanol), in addition to describing that the exposure time was considered 60 min according to a study by Tran, Chatterjee, Gerlai, 2016 (which used 30 min exposure in the protocol). Why did the authors choose to use 0.4% and 0.8% of ethanol for 21 days, since the study on which the time and concentration of exposure was based does not correspond to of the study?

7)	How was the preparation of the exposure tanks, both ethanol, and control? What water was used?

8)	I suggest that the authors add an experimental design showing the arena and the LEGO figurine used, as done in the works cited (Ou et al., 2015, Hamilton et al., 2017).

9)	The behavioral analysis was performed for 5 min, however, the studies with this behavioral protocol do the analysis for 10 min. Why did the authors opt for this time?

10)	Why did the authors decide to exclude animals with more than 300 seconds of immobility from the test?
Validity of the findings: 1)	In the time results in the zones, there was no difference between the groups 0.4% and 0.8%? If the authors say it is dose-dependent, is there no need to show that significant difference?

2)	It lacked to describe in detail what the principle of the behavioral protocol chosen and its effect on the zebrafish. Is not clear the use of LEGO in the protocol, what his role in the test? How does the zebrafish behave with and without the presence of the figurine?

3)	In lines 252-256, the authors state that low concentrations (0.4%) result in less oxidative stress and, for this reason, the group behaves similarly to the control group. Have the authors evaluated the oxidative stress in the study? How can the authors conclude this statement?

4)	A recent study (Agostini et al., 2018, doi: 10.1007 / s12640-017-9816-8) showed exposure to 0.5% ethanol for 7, 14 and 28 days and concluded that the longer the exposure time of the animals to ethanol, the lower the oxidative damage (in 28 days no lipid peroxidation occurs, ROS levels are present only in 7 days of exposure). Considering the difference of 0.1% ethanol between the manuscript and Agostini's study (2018), can the authors conclude that there was less oxidative stress?
5)	The authors conclude that there is a dose-dependent relationship with ethanol, but this was not evident in the graphs at the same time, the concentration curve should be higher for such a conclusion.

6)	During the discussion, the authors mention results from the literature that differ from those found in the manuscript; however, the concentration, exposure time, zebrafish strains and behavioral devices are also different from the present study. For example, between the lines 258-265, the authors describe that the results presented in the manuscript are the opposite of those in the literature, however, reinforcing again the idea that the concentrations (0.2%, 1%, and 1.4%) of the studies beyond the exposure time (30 min) produce fewer anxiety effects. Studies of Rosemberg et al., 2012 (doi: 10.1016 / j.neuropharm.2012.05.009) and Mocelin et al., 2018 (doi: 10.1007 / s11064-017-2442-2) demonstrate opposite effects (anxiogenic in adult zebrafish) on acute exposure for 60 min. A new literature review could support the data found in the manuscript (anxiogenic effect), perhaps demonstrating that even at lower concentrations of exposure and a greater number of days, ethanol is capable of promoting an anxiogenic effect.
Additional comments: 1)	Line 46-47: Which membranes are activated?

2)	Line 54-57: It is confused as described.

3)	Line 71-99: This extensive and confusing. The authors address behavioral issues after mentioning biochemical results. There is no connection between sentences. Rewrite.

4)	Line 78-80: What did the study cited bring about? What is the relation with the study of the authors?

5)	Line 91: Are the authors referring to the study of Mathur & Guo 2011?

6)	Line 145: behavioural tests or test?

7)	Line 220-222: Only concentrations of 1.0% and 1.5% of ethanol were used in the study cited? And the 0.5% concentration that increased time on the light side in the light/dark test?
8)	Line 227: Set reference.

9)	Line 227-230: The study by da Silva Chaves et al. (2018) performs continuous exposure with withdrawals of 60 minutes (in water) for 16 days, and not described in the sentence, is confusing.

10)	Line 240-244: The authors write about the GABAergic system and finally bring studies in rodents emphasizing alteration of sensitivity to hypnotic drugs and steroids, not following sentence line of thought. Do authors find it better to remove?

11)	I do not think it is appropriate to use the term dose-dependent in the abstract and in the text since the number of concentrations used is insufficient for the protocol used. Can you state the behavior of the animals in smaller and larger concentrations of ethanol?

12)	Provide missing references in the text (lines 30, 32, 37, 47, 50-52, 85, 213-215, 240, 242).

13)	Add the parameters analyzed by the authors in line 105.


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: REPEATED ETHANOL EXPOSURE INCREASES ANXIETY-LIKE BEHAVIOUR IN ZEBRAFISH DURING WITHDRAWAL
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is written in good english and with clarity in the presentation of ideas and data from literature. However, the abstract is poor in details. The structure, figures and raw data were presented correctly.
The issue of the manuscript is of obvious importance, since the neurochemical and behavioral aspects of alcohol withdrawal are far from clear, whereas the health and economic burden has enormous impacts around the world. However, the introduction of this manuscript does not cover the main aspects of ethanol withdrawal (eg, dopamine, serotonin, corticotropin-releasing factor levels, etc.) and the recent contribution of zebrafish studies to this issue (eg: Chaves et al., 2018 , Benneh et al., 2017).
Experimental design: The main aim of the manuscript is interesting on the basis of the heterogeneity found in the data available about this issue in the literature. The methodology, while presented as a new approach to test anxiety-like behaviour in the context of ethanol withdrawal, do not add strongest contributions to the area. Some minors experimental points also contributes to the weakness of the manuscript:
(1) the authors discussed the data based in a dose-dependent relationship between behaviour and ethanol withdrawal, but only two doses were tested and just the higher had effect, which is not enought to determine a dose-dependence of factors (Discussion: 209-211; Conclusion: lines 281-282)
(2) The validity of the test should be checked by an anxiolytic drug.
(3)The title of the manuscript enphasizes "repeated ethanol exposure increase anxiety-like behaviour....", however the methodology do not permit to compare to any anxiety-like effect of ethanol.
(4) Also, the age, weight of animals were not detailed. The light/dark period of 12/12 is not common and should be referenced.
Validity of the findings: While the assumption is of interest the results are too preliminary to add novelty to this area. Only one behavioural parameter was altered and no neurochemical aspect was considered (cortisol levels, dopamine, serotonin, glutamate levels, GABA receptor expression,....). The discussion is based in the explanation of how withdrawal occurs, but the experimental design is not focused on that, and any kind of behavioural test could theoretically discuss in this same way even if the methodolgical approach was different.
Additional comments: No additional 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: REPEATED ETHANOL EXPOSURE INCREASES ANXIETY-LIKE BEHAVIOUR IN ZEBRAFISH DURING WITHDRAWAL
Review round: 2
Reviewer: 1

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

The authors responded to the review comments, however, some doubts remain unclear. Problems were also observed in the manuscript and described in the review below.

Initially, disregarding future experiments, I believe that a review has been made in the literature regarding ethanol concentrations, their respective behavioral responses in other protocols, for example using the same 21 days of exposure, I continue to question why use a range of concentrations and not use what is already in the literature?

I suggest that it be described in which animal model these effects were observed, and if this model was withdrawn, intermittent, chronic. And what about zebrafish? and in humans?

Materials and methods:

The authors describe that daily exposure was performed between 8 A.M. and 4 P.M. I suggest describing how this exposure was made, how many fish per tank, in what proportion of males and females?

How were these animals fed? It the authors feed the animals once a day (not described in what time), are there groups received food and were exposed to ethanol at different time intervals, do not the authors consider a possible limitation of the study?

In the preparation of ethanol at the tank, the animals were transferred before or after the add the ethanol?

The authors added in the manuscript that animals of both sexes were used, however, it was not clear the proportion of males and females per group and how these animals were previously housed.

Already described in the description item of the exposure protocol that researchers were not blind to the treatment groups during dosing or testing, I suggest you remove the description in the item of Behavioural testing.

Discussion:

What were the neural changes that occurred? What's the reference?

Line 235-239: The authors did not evaluate neural changes in the experiment, this description is more introductory than discursive. I suggest you remove it from the manuscript.

Line 247-252: The authors describe that the study of Rosemberg (2012) and Mocelin (2018) observed an anxiogenic effect in 20 min of exposure, however in 20 min the effects are anxiolytic (Rosemberg 2012) and were not the time used by Mocelin (2018). It should also be considered that the protocols mentioned above are of acute, not chronic exposure of the authors Mathur and Guo (2011). This discussion needs to be reviewed and best described.

Line 251: Rewrite reference: Rosemberg, not Rosemburg.
The authors should consider that the animal model used by Mathur and Guo (2011) is AB, different from those used in non-study and studies by Rosemberg (2012) and Mocelin (2018) (wild-type). This is an important point to be evaluated since studies show behavioral and neurochemical differences between zebrafish strains.

Using wild-type lineage may be considered more heterogeneous and best represents the high rate of genetic variability, as in humans. I suggest a discussion about the theme regarding the events observed by the authors and the withdrawal model of Mathur and Guo (2011) and Muller (2017).

doi: 10.1186/1471-2202-11-90
doi: 10.1007/s00726-013-1658-y
doi: 10.1016/j.ntt.2018.05.005

Have no studies on zebrafish and rodents assessed the activity of GABA receptors? What do the authors consider similar in zebrafish, the activity of GABA?
-	See Mahabir (2018) 10.1016/j.ntt.2018.05.005

Since the authors describe that the ROS data in the discussion are merely speculative, I suggest that the authors also report in this way the possible involvement of the GABA system.


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: REPEATED ETHANOL EXPOSURE INCREASES ANXIETY-LIKE BEHAVIOUR IN ZEBRAFISH DURING WITHDRAWAL
Review round: 2
Reviewer: 2

Output:
Basic reporting: The overall manuscript was improved. While I am still concerned about the validity of the behavioural test for anxiety, the authors convinced me that the literature present similar works where the anxiety parameters were checked using similar approach.
Experimental design: The experimental design is now precisely detailed.
Validity of the findings: Based on data from this work and literature, the conclusion of the manuscript is acceptable.
Additional comments: The manuscript was improved and probably will encourage further investigation especially on neurochemical aspects of ethanol-withdrawal.


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

Input:
Title: THE NECTAR REPORT: QUANTITATIVE REVIEW OF NECTAR SUGAR CONCENTRATIONS OFFERED BY BEE VISITED FLOWERS IN AGRICULTURAL AND NON-AGRICULTURAL LANDSCAPES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Pamminger et al present an extensive dataset on nectar concentrations in 322 species of wild, cultivated and weedy plant species from across the major world continents. The paper is well structured and well written throughout, with only a few areas that need to have their phrasing polished up which I detail below.

The results are interesting (a very consistent average nectar concentration found in insect pollinated plants across all regions) and highlight particular crop genera that may benefit from additional nectar sources in the surrounding landscape to benefit bee populations.

The figures and tables are clear and the statistical techniques used are well described. The literature is reasonably well covered, but I would like more discussion of the evidence base as to why the sugar concentration in nectar is important, and which species of bee this evidence is drawn from (predominantly social bees). I go into this point in more detail in section 3.
Experimental design: The research question is well defined (the importance of sugar concentrations in nectar) but as detailed in the next section I would like some more discussion of whether or not this is valid to apply to all bees.
Validity of the findings: I have a few more major comments that I would like to see addressed. The major point is that the manuscript talks about the importance of nectar to 'bees', but almost all the evidence we have that nectar concentrations are important comes from social bees (Bombus, Apis, etc). I think the data presented is really interesting, but I find it hard to argue that nectar concentrations are quite so important for solitary bee species (the vast majority of bee species) that have mainly had their fitness linked to the type and quantity of pollen available to them. I think a slight rewrite that shifts the focus (and justification for choosing nectar as the response variable) onto social bees would make the manuscript more representative of our current understanding of how important nectar is to bees, given the substantial knowledge deficit that exists for solitary bee species.

L69-71. I would argue strongly that maintaining a diverse bee community has not been the focus of agri-environment schemes targeted at bees. Early work in the UK focused strongly on bumble bees and their foraging requirements Carvell et al 2004; 2006; 2007 leading to currently incarnations of flower-rich field margins containing predominantly bumble bee food plants (e.g. Trifolium, Centaurea, Vicia and so on). Until recently, few pollinator-focused schemes were appraised for their ability to provide resources to a wide variety of wild bee species e.g. Scheper et al 2015 JAE

L72-75. I think you need to be careful here. Most of the work that has be conducted on floral resource quality (especially nectar) has been on social bees. Next to nothing is known about how nectar quality affects solitary bee fitness, and if it even does at all. Pollen quality has been more extensively studied, but still predominantly on social bees (Vaudo, recent work on polyfloral/monofloral pollen diets for B. terrestris colonies from the Michez lab). For solitary bees, the limited amount of work suggests that it is the quality of the landscape (i.e. density of floral resources) that determines bee fitness e.g Persson et al 2018 Ecology and Evolution. I would lay out a couple of these issues in a little more detail in the introduction to highlight that most of the nectar work comes from social bees, which are of course very important as pollinators due to their abundance in most ecosystems.

L152. This dataset from Kim et al 2011 is restricted to a narrow selection of bees in Apinae, e.g. Bombus, Apis, Meliponini and Euglossini, all but the Euglossines being social bees. These bees, whilst capturing a lot of the social diversity, are not necessarily representative of the other 19,000+ bee species and so statements such as “However, all evidence suggests that bees avoid foraging on nectar sources below 20% sugar concentration, likely because the caloric intake cannot support sustained foraging activity with potentially detrimental effects for the bee colony” are not necessarily true for solitary species. An earlier discussion of the ecological differences between social and solitary bees seems warranted. If the paper was to state that the focus on nectar was to better inform management for social bees (as is implied by the focus on nectar and references to colony health) this would resolve many of these issues. As it is currently written, the paper refers to ‘bees’ but focuses mostly on social bee examples and on a problem (nectar concentration) that has not empirically been demonstrated to be an issue in solitary species.

L326-327. I was interested to read this reference to see what evidence the authors present, but it is incomplete and gives only the title of the book without publisher information. Looking it up online, the subtitle is ‘the most important food sources for the honey bee’. As written, the current sentence “While it is well-known that pears (Pyrus) are not considered attractive to bees due to their low nectar (Fig.3) quality (Maurizio and Grafl 1980)” is misleading, since the evidence is for honey bees only, and pear is known to have attractive pollen for spring-flying wild bees, particularly Andrena (Chambers 1946 J Animal Ecology; Westrich 1989 Die Wildbienen Baden-Württembergs). Focusing the manuscript more explicitly on honey bees, bees known to be sensitive to nectar concentrations, would seem more appropriate.
Additional comments: Formatting of references – I have not seen the first and second author et al citation structure before. Please check against PeerJ standards

L11. “some bee populations are in decline,” add comma
L14. “associated with this trend, nutritional stress resulting from a mismatch between bee nutritional needs and plant community provisioning has been suggested as one potential driver.” change placement of commas
L19. “this information gap,” add comma
L42. Are in decline, or seem to be declining
L44. rephrase to ‘are declining in only a few geographic regions and over certain time periods’
L48. correct to “intensification, habitat loss- or fragmentation and emerging pathogens” check use of commas in lists
L60. cornucopia – perhaps a more scientific and slightly less flowery word? (no pun intended)
L61. Discreet – perhaps change to relatively short?
L65. I’m not quite sure what you mean by ‘selective’ in this context. Is it the habitat that is selective, or the criteria used to determine the kind of habitat type that is selective? ‘Specific’ may be a more appropriate word choice if you intend the latter meaning
L82-83. Add comma ‘developing bees, sugar concentration’
L86. Check second quality
L126-133. Check formatting e.g. double parentheses, underlining etc
L147-149. Repetition of ‘for bees’
L213. Check capitalisation of genus
L223. Check PeerJ formatting for Tab. or Table 1
L248. Check spelling of variety
L249. Check spelling of hybrid
L254. Difference singular
L273. Check spelling of Pyrus
L308. Repetition of findings, consider rephrasing
L331-335. Repetition of high-quality, consider rewriting. In fact, both sentences could be rewritten for clarity.
L347. Check capitalisations

Figure 2. Check spelling of Wilcoxon
Figure 3 legend. Check spelling of border
Figure 3. Hoffmannseggia and Hoffmanseggia seems to be listed twice, are these different genera?


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

Input:
Title: THE NECTAR REPORT: QUANTITATIVE REVIEW OF NECTAR SUGAR CONCENTRATIONS OFFERED BY BEE VISITED FLOWERS IN AGRICULTURAL AND NON-AGRICULTURAL LANDSCAPES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is generally well-written and well structured in professional English and easy to read. The introduction is convincing and presents the background information necessary to understand the objectives of the paper. Additionally, the figures are relevant and well described.
However, the raw data has not been supplied. It is necessary to include a list with all species for which nectar sugar concentrations were found and the references. This list could be in the appendix.
Experimental design: The study is well designed and the approach is appropriate for the research questions. However, some important information is missing, for example the exact search terms for the search in ISI web of knowledge and google scholar. Additionally, all data bases that were searched should be mentioned to make it possible to repeat the study.
In the method section on the statistical analysis some important information is missing, for example which test was used to test which hypothesis. Moreover, I think that it is difficult to compare the variability of nectar sugar concentration between crops, weeds and wild plants. The authors write themselves that the higher variability in wild plants is probably due to the fact that this group includes more taxa. Unless the authors do not account for the number of taxa included in the analysis I don’t think that it makes sense to test for these differences.
Validity of the findings: The authors have compiled a large data set of reported nectar sugar concentrations from the published literature. Therefore, the findings are robust and the conclusions are supported by the data. However, I wonder how important the sugar content really is for bees. For example a mass flowering crop might provide such a high nectar availability that the sugar content might be completely unimportant. These constraints could be more emphasized in the discussion. Additionally, I miss some more concrete ideas how the data is useful for pollinator conservation. For example, I like the idea very much to combine this data set on nectar quality with the timing of flowering in plants. Why was that not done in this study? It could be very useful to know when most crop/wild plant species are not flowering to support plants that provide nectar of high quality in times of scarcity.
Additional comments: L21 insert "from the literature"
L80 compiled from the literature
L81-82 Did you really only study plants from agricultural landscapes? I understood that you integrated all published reports of nectar quality including those from wild plants. Are some of these plants also growing for example in forests and not in agricultural landscapes?
L85 replace "this data base" by "the data base that we compiled"
L97-98 You should state the exact search codes to make the study replicable.
L99-101 How was that done? Which literature was searched?
L126-127 You should state all data bases that were used.
L131-133 Which data base was used for Europe? You should give the details on all data bases used.
L141-144 Is there any data how important each of those variables is for nectar quality?
L152 Can you explain why the highest sugar concentrations (above 65%) are not optimal for bees? Another questions that I have is why plants would produce nectar concentrations that are above the optimum for bees. Do they attract other species?
L158 You write about bee colonies, so are these results only about honey bees?
L176 replace "possible" by "possibility"
L182 I do not understand what was "paired". Irrigated vs. non-irrigated fields?
L199-206 More details are needed. Which test was used for which hypothesis using which data set?
L203 heterogeneity?
L215 replace "the lest" by "and we found the lowest"
L235 replace by "we had only a limited number of species with multiple..."
L238 the three different groups?
L248 In line 179 you write that there were 52 different varieties?
L264 See comment on agricultural landscapes above.
L273 Pyrus
L294-296 This hypothesis should be stated in the introduction.
L309 "in ab"?
L325-329 Do you know of any evidence that this low nectar quality is balanced by high quality pollen resources?
L333-335 I think this is important for every pollinator dependent crop, not only orchards.
L341-343 You should give some references.
L356 See comment on agricultural landscapes 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: THE NECTAR REPORT: QUANTITATIVE REVIEW OF NECTAR SUGAR CONCENTRATIONS OFFERED BY BEE VISITED FLOWERS IN AGRICULTURAL AND NON-AGRICULTURAL LANDSCAPES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is well written, and the revision has improved the format and structure of the references. There are a couple of areas where the English could be polished that I have notes in the general comments to the authors.

Following a comment from Reviewer 2, the data are now accessible via a download link.

The overall thrust of the article is clear: to describe global patterns of nectar quality and to use this information to propose suitable plant species for management interventions to support farmland bee populations.
Experimental design: Unchanged from the first review, meets all PeerJ requirements for scope and clarity of research question.
Validity of the findings: The authors have now added more discussion to all parts of the manuscript on the subject of sugar concentrations in nectar and how this is important for solitary bee species, as opposed to just social bee species. It is a difficult area to discuss since most of the empirical work has been conducted on social bee species, but I am happy that the manuscript now sets this knowledge gap in a wider context for readers. Overall, I am happy for this manuscript to be accepted with only the very minor changes I detail below.
Additional comments: I just have a couple of minor places where the English could be tightened up a little for readability.

L17. (during flower-scarce periods)
L21. suggest rephrase as “as well as outside fields (wild species)”
L21-22. suggest rephrase as “Social bees are particularly sensitive to”
L23-24. suggest rephrase as “We find that the total sugar concentrations in nectar generally do not differ between the three plant communities studied”
L24-25. In contrast,
L25. Increased variability in nectar quality
L32. Database is one word; do you mean data set?

L353. resources could be located close by, which in turn
L354. change to “which use pear flowers as a source of pollen”


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

Input:
Title: THE NECTAR REPORT: QUANTITATIVE REVIEW OF NECTAR SUGAR CONCENTRATIONS OFFERED BY BEE VISITED FLOWERS IN AGRICULTURAL AND NON-AGRICULTURAL LANDSCAPES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The basic reporting is good.
Experimental design: The experimental design is sound and now described in more detail. I would just like to know whether the second experiment of Simidtschiev et al. (1988) with the irrigation treatment was also conducted in two regions or only in one. In case it was also performed in both regions, region should be included in the model either as a fixed or random effect.
Validity of the findings: The findings are valid.
Additional comments: The manuscript has improved compared to last version. Many parts are now much more clear and I only have few minor comments:

L79-80 replace by "taken into consideration when developing management plans for complementary and high quality nutritional bee resources."
L138 replace by "in case they were not mentioned in one of these data bases"
L171 replace by "below 20% as low quality"
L195-196 Was this second experiment also conducted in two regions? In this case region should be included in the analysis either as a random or fixed effect.
L203 "pre-defined categories"
L209 add citation of R software
L210 delete "used" in this line, otherwise it is confusing which test was used for which question
L215 make clear what you mean by "communities"
L216 make clear what you mean by "local habitat"
L229 You should mention at this point where your data can be found.
L260 I don't think that "local habitat" is the right term here, because I would assume that the plants were grown in different habitat types. Maybe better "Regional effects on nectar quality..."?
L267 replace by "varieties"


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: IDENTIFICATION OF A NOVEL POTASSIUM CHANNEL (GIK) AS A POTENTIAL DRUG TARGET IN GIARDIA LAMBLIA: COMPUTATIONAL DESCRIPTIONS OF BINDING SITES
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are abundant grammatical errors, and English revision is required. See examples below, with many others throughout the text.
52: “The ionic” channels should be changed to simply “ionic channels”
70: “Others, emphasizes…”
72: “In another hand…”
163: “…we found that GiK has a 34.6% relation with potassium channels, and its taxonomy is related to bacteria in a 63%.”

Many phrases need references, i.e. lines 47-50.
Experimental design: The present work by Palomo-Ligas et al. describes the generation of a homology model of and screening of potential ligands of a potential potassium channel, which is a potential target for drugs against Giardia lamblia infection. The research appears to be timely and of sufficient importance, however, the manuscript is very terse (results is <50 lines), and should be expanded to include further comparisons to experimental data of similar ion channels. Further, the impact of the work would be higher if the authors evaluate the binding thermodynamics and stability of the best ranked poses (e.g. as in Martins et al. JCAMD, 2018, 32: 591-605), for instance by MM/GBSA or MM/PBSA, and Molecular Dynamics of the channel embedded in a membrane with different ligands, possibly followed by free energy calculations.
Validity of the findings: Introduction:
76-77: “However, the structural characterization of ionic channels in this

protozoan and its possible role as drug targets is poorly understood.” Should be elaborated to mention what is known about them, or similar receptors.

Results:
148: “showed 24-50% identity and 50-90% positivity”. Positivity or similarity?

The receptor has a large number (>1400) of residues, and it is not clear if the entire protein, or a specific domain was modeled. Alignments of domains and sequences suggest that only the transmembrane section was included, which, depending on what is known about the importance of other segments, may be acceptable. Figure 1 should be updated to highlight the relevant section, and perhaps reduce the representation of the region not included in the model.

A disorder prediction of the sequence highlighting the modeled region should be included. If it includes disordered segments, the modeling approached used, which is unlikely to handle such regions well, must be justified.

Please describe how the tetrameric form was obtained in Figure 4.

Was a specific region of the channel specified in the docking, or the whole receptor? This needs to be clarified.

The docking section needs to be elaborated, for instance, to include a visual comparison of the binding modes found to experimental data of ligand binding to similar receptors. Do the poses localize to known pockets? What affinities are expected for the different binding pockets (based on experimental data for similar channels in the literature)? Which pocket is typically targeted in these channels?

Discussion:
Do the most promising ligands identified have similar functional groups, properties, etc as those known to bind to similar to channels? A figure showing binding site and molecule similarity to homologous K channels would be helpful.
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: IDENTIFICATION OF A NOVEL POTASSIUM CHANNEL (GIK) AS A POTENTIAL DRUG TARGET IN GIARDIA LAMBLIA: COMPUTATIONAL DESCRIPTIONS OF BINDING SITES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The Introduction provides sufficient background for the readers to understand the research problem. It will be better if the authors can briefly describe the molecular basis of the standard therapy (i.e. nitroimidazole compounds) of giardiasis, and how targeting potassium channels is a novel/different therapeutic approach.

The most important issue, however, is the lack of details in the Materials & Methods, and the Results sections make it hard if not impossible for the future readers to reproduce the analyses described in this work.
Experimental design: In this work, the authors used protein sequence analyses and molecular modelling to construct a three-dimensional model of a putative Giardia potassium channel GiK. Based on this structural model, the authors then performed docking virtual screening to search for potential binding sites of potassium channel-blockers.

The Materials and Methods section needs a lot more details. For instance, the authors used multiple web tools to perform sequence analyses of GiK, however, the parameters used in these analyses were not described. I suggest to include the details of how these analyses were setup in the supplemental materials so that readers who are interested in can reproduce the analyses. Also, the authors should state what information they intended to obtain from each web tool.

In Line 94, the authors mentioned that 51 potassium channel genomic sequences of different species were used to identify potential K channel in Giardia lamblia.  The authors should list all 51 sequences used.
Validity of the findings: The results from bioinformatic sequence analysis, molecular modeling, and docking were summarized in the Results section, however, more detail analyses should be provided.

1. In Figure 2, the authors should clearly state the source of each result presented.  For instance, ion_trans_2 is the result from NCBI Conserved Domains (?).

2. In Line 163-164, the authors mentioned that "......by the Protein Data Bank
 platform, we found that GiK has a 34.6% relation with potassium channels, and its
taxonomy is related to bacteria in a 63%". It is unclear how these two numbers were obtained from PDB.

3. Based on the UniProtKB accession number of GiK, it seems that the protein has 1416 amino acids.  The author should state clearly which domain(s) or region of the protein they modelled.

4. The authors used multiple modelling programs to construct the three-dimensional models of (part of ) GiK. They then used different web tools to verify the quality of their models.  Even though multiple approaches have been used to generate 13 structural models (listed in Table 4) of GiK, the authors only showed one of them in Figure 4. In order to demonstrate the reliability of this modelling approach, the authors should perform a detail comparison of the structural models they obtained from different programs and templates. One would expect that if this approach is robust, the models obtained should be similar.
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: IDENTIFICATION OF A NOVEL POTASSIUM CHANNEL (GIK) AS A POTENTIAL DRUG TARGET IN GIARDIA LAMBLIA: COMPUTATIONAL DESCRIPTIONS OF BINDING SITES
Review round: 2
Reviewer: 1

Output:
Basic reporting: General remarks:
-The revised manuscript addresses most of the concerns associated with the first version
-It should be suitable for publication once the items below have been addressed
-Another round of revision is not required

Issues:
-Figure 2 is of low resolution
-Labels for S5, S6, etc should be added to figure 7
-Line 72: Remove comma after "Following"
-Line 73: Use micro symbol, µ, and add space between value and units
-Line 242: Ley117
-Line 245: Remove extra space (Ile 411)
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Even if the additional analyses of pose stability and free energy by molecular dynamics simulations are to be left for a future study, it would be worthwhile to mention in the discussion section that this is a logical next step before experimental investigations, accompanied by a reference or two: (1) Martins et al. JCAMD, 2018, 32: 591-605 and (2) Kokubo et al. J. Chem. Inf. Model., 2017, 57: 2514-2522.


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: IDENTIFICATION OF A NOVEL POTASSIUM CHANNEL (GIK) AS A POTENTIAL DRUG TARGET IN GIARDIA LAMBLIA: COMPUTATIONAL DESCRIPTIONS OF BINDING SITES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Even though the authors mentioned that the manuscript was edited for correct English language, there are a large number of grammatical errors in the revised version,
Experimental design: The authors have included more details in the Materials and Methods section. Unfortunately, it is still not clear how the tetrameric form of GiK was generated. Did the authors generated it based on the monomeric form? If that is the case, details on how this was done should be included.
Validity of the findings: The authors have used multiple approaches to generate 13 structural models of GiK, In Table 4, they reported the quality of individual models assessed by different web tools.  However, there is no comparison between the models.  For instance, what are the RMSD between the models?
Additional comments: The authors have revised the manuscript based on some of the reviewers' suggestions/questions. Unfortunately, I found that some of the questions were still not properly addressed. In addition, there are many grammatical errors in the manuscript.


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

Input:
Title: TRENDS IN FRAGMENTATION AND CONNECTIVITY OF PASPALUM QUADRIFARIUM GRASSLANDS IN THE BUENOS AIRES PROVINCE, ARGENTINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript describes trends in fragmentation and connectivity of grasslands in the Buenos Aires province, Argentina using 5 satellite images from 1974 to 2016. The introduction is well developed and sets the scene with adequate references. Figures are relevant but could be improved with more detailed captions. I appreciate providing the raw data to the readers so they can replicate the steps described in the paper.
Experimental design: The design setup is within the scope of the journal, research questions are well defined and relevant. The method section provides many details on the data preparation but very little detail on the actual processing of the data, esp. for connectivity. Since the topic of the paper is about trend analysis, the reader expects to find sufficient details describing how exactly that trend analysis was conducted but right now there is not much information about this topic.
Similar to Connectivity, the notion of fragmentation is a very wide field and the authors should explain why they have chosen MSPA for their analysis, or which features of MSPA are considered to provide more appropriate information compared to typical Fragstats indices, as used in the Lara & Gandini paper 2014 (in the supplemental data). For example, I could imagine that the MSPA-exclusive information on core-areas, islets and connecting pathways and their direct relation to network theory analysis used to calculate the degree of network connectivity, was a decisive argument.
Validity of the findings: The findings are sound, relevant and to my knowledge new in the field. However, I think the paper would benefit from additional information and/or reasoning on the chosen settings for scale, MSPA-settings, and connectivity assessment.

a) In the introduction you mention the importance of spatial scale, which is good. But where and how is this topic addressed in your analysis?

b) MSPA analysis:
- you seem to have used an older GTB version of the 2.6 series. Please note that a small bug in the MSPA statistics calculation has been fixed and more recent versions also provide information on different type of perforations within Core area, a topic which is of interest when talking about fragmentation. You could reprocess the 5 images (only takes some seconds) to get updated and enhanced MSPA information for your 5 images.
- you use EdgeWidth=3, why did you opt for a 3-pixel edge width, because that corresponds to roughly 100m (90m to be precise)?
- you selected to divide core-areas into small/medium/large using the default settings of 1000 and 4600 pixels. Can you explain why you choose to show the core-area distinction and why you used the two thresholds and not others, for example 1000 and 10000? Maybe you use the distinction to visually highlight the locations of large/medium/small core areas and/or analyse how these types have changed over time?

c) connectivity analysis
You may add some comments on connectivity. Similar to fragmentation, connectivity is not good or bad, and it strongly depends on the selected species. The same habitat can be well connected for one species but not connected at all for another species. The ECA as calculated via GTB looks at the overall structural connectivity between the set of network components (Core + bridges but excluding all the other MSPA classes) accounting for their area and the number of nodes and links within each component. Because of this definition, ECA provides a measure which is not species-specific and as such an independent overall measure of connectivity.

The supplemental raw data supplied is somewhat confusing:
- Binary images are provided for 1974, 1988, 2000, 2011, 2016: all images should be provided in byte format instead of integer to allow reproducing the analysis within GTB.
Looking in the subfolder "frequency" we find that the actual image for 1974 is actually from 1975 with the name "pajonal1975_r", which must be in byte format. Maybe better to provide the actual data you used in your analysis instead of the integer formatted image "1974.tif"
- The information in the subfolder "GToolbox" is superfluous as it is identical to the one in the subfolder "frequency"
- the purpose of the paper in the subfolder "Paper" is unclear to me, or please add an explanation why it is needed as supplemental material.
Additional comments: The paper describes dynamics in Pq grassland fragmentation and connectivity. But the title puts a strong focus on MSPA without mentioning fragmentation and connectivity. If the importance is MSPA then maybe there should be a dedicated section on MSPA, highlighting why this and not any other methodology was chosen. Or the maybe the title could be changed to better reflect the actual focus of the paper, something like:
Trends in fragmentation and connectivity of Paspalum quadrifarium grasslands in the Buenos Aires province, Argentina.

Specific remarks at line number:
25: correct: Guidos Toolbox -> GuidosToolbox (GTB), then any later reference can be: GTB

27: We found a loss in total coverage area, number of habitat nuclei and a reduction in connectivity. The number of large Pq....

38: GTB is not a forest fragmentation software but instead a generic image analysis toolbox, which can be used for various analysis schemes, fragmentation is just one of them. Either delete the last sentence at line 38 (because you do not talk about forest fragmentation in the paper) or you could say:
On the methodological side, the generic design of the fragmentation assessment scheme employed here for grasslands could be applied equally to assess and monitor dynamics of any other land cover type, i.e., fragmentation of forests or agricultural land.

100: add a reference for the statement "the numerous ES they provide".

115-137: Morphological Spatial Pattern Analysis (MSPA, Soille & Vogt, 2008) is a customized sequence of mathematical morphological operators targeted at the description of the geometry and connectivity of image objects. Starting with a binary representation, MSPA will conduct a segmentation of the image objects into various morphological feature classes, including core, islet, loop, bridge, perforation, edge, and branch. A summary description and application examples can be found on the MSPA website.
[MSPA website: http://forest.jrc.ec.europa.eu/download/software/guidos/mspa/
Below the animated image on that website you can find links to papers where MSPA was used]
If you want to go into details...
The generic setup of MSPA has been used to identify and map forest patterns, both, structural (Vogt et al 2007a, 2007b) and functional (Vogt et al., 2009), identify key connectors for habitat suitability (Saura et al., 2011), riparian corridor conservation studies (Clerici and Vogt, 2013), or evaluation of the US green infrastructure (Wickham et al., 2010) up to classifying zooplankton (Schmid et al, 2016).

142: maybe delete the sentence "In this way,..." and just keep the last sentence of the introduction

185: .. using MSPA within GTB (Vogt and Riitters, 2017).

187 - 191: provide details how you did the connectivity analysis. Explain the reasons why you think the selected method is appropriate for your project.

192 - 195: provide details how you calculated ECA, with GTB or with Conefor, or how? Explain the reasons why you consider ECA to be a meaningful measure for your project.

192: In addition, we calculated the Equivalent Connected Area (ECA, Saura et al 2011), defined as ...

201: To simplify interpretation we only show the start and endpoint of the observed period in the main body of the text, all results can be found in the supplemental material.

212: "A drastic reduction in connectivity is also evident." I can not see that in Figure 2, maybe you refer to the table 1?
A suggestion: In addition to Figure 2 it might be interesting to show the area change (morphological change detection) from 1974 to 2016, showing where, and how much, grassland cover was lost/gained and where it remained unchanged. This can be done quickly in GTB (File -> Change -> Morph.Change), please find the result here:
https://www.dropbox.com/s/0837puze26r9uh7/morphdiff.tif?dl=0
With these statistics:
MCD (A->B) FG: -43.0117, FGi: -47.5600, Elasticity: 1.10575
Gain [pixels]: 2113195
Loss [pixels]: 5147462
Indicating that the loss has predominantly taken place within existing grasslands and overall 43% of grasslands were lost since 1974. This result is more sound than a visual comparison of the two images in Figure 2.

213: where can I find the number of large fragments (> 50 ha)?

219: Table 1 does not suggest that Edges remain stable and what are "curls"?

226: %ECA did not drop but has increased after 2011.

229: why is the loss of connectivity evident in Figure 3? Compared to 1974, in 2016 the total number of components has increased and the total area has decreased => more fragmented => less connected.

References:

376: Vogt P, Riitters KH (2017) GuidosToolbox: universal digital image object analysis. European Journal of Remote Sensing, 50, 1, pp. 352-361, DOI: 10.1080/22797254.2017.1330650

Vogt P, Ferrari JR, Lookingbill TR, Gardner RH, Riitters KH, Ostapowicz K, 2009. Mapping Functional Connectivity. Ecological Indicators 9: 64-71. DOI: :10.1016/j.ecolind.2008.01.011

Figure 2: replace "References" with "MSPA classes", add a bar outlining the actual area of the image shown in km in x/y direction. This figure shows the study area as a quadratic image but the actual image in the supplemental data clearly is rectangular having the dimension 5168 x 3120 pixels.


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: TRENDS IN FRAGMENTATION AND CONNECTIVITY OF PASPALUM QUADRIFARIUM GRASSLANDS IN THE BUENOS AIRES PROVINCE, ARGENTINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: - English writing could be improved throughout. Specifically, many paragraphs include many fragments of ideas, jumping from one theme to another. Keep paragraphs focused on a single idea.

- Length of the introduction was far too long compared to overall length of manuscript. Specifically, > 50% (147/257 lines of text) of the manuscript text was in the introduction. The manuscript needs to be rebalanced with a tighter introduction and more detail in the methods & results and a more thorough discussion.
Experimental design: - The image classification methods employed seem standard, but these could be discussed in much more detail.

- There was inadequate detail in the methods about the MSPA method, and the subsequent connectivity analysis. Some of this appears in the introduction, this could be moved and improved.

- What is the relevance of the perfectly square study area?
Validity of the findings: - The classification results seem appropriate for the study.

- The results need to be elaborated upon in more detail in the text. Specifically, the numerical results in the table need improved reporting. It would be beneficial to separate the results from the discussion.

- The data was sourced over multiple years, then only results for 1974 and 2016 are shown. Why not all years?

- What can be gleaned from these maps exactly, it is difficult to tell. Needs more explanation about what is being seen. MSPA is a useful method but needs to be contextualized and analysed further, simply producing these maps without providing a more detailed summary analysis (e.g., what do the numbers in table 1 mean?) is not really useful.
Additional comments: - The manuscript does not provide enough detail about the methods or explanation of the results.

- The discussion is intertwined with the results and needs to be separated. Specifically, I would like more here about how the results compare to previous studies in the area, or similar studies elsewhere.

- Study area map could be improved greatly. Specifically, I’m not sure what projection was used but this seems like an odd choice for Argentina, also, Argentina is not an island. The coloring of the agroecological zones could be better suited to assist the reader in understanding what all these zones represent. More info on the study area is warranted in the text for those  not familiar with the region.