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

Input:
Title: HIGH-THROUGHPUT METABARCODING REVEALS THE EFFECT OF PHYSICOCHEMICAL SOIL PROPERTIES ON SOIL AND LITTER BIODIVERSITY AND COMMUNITY TURNOVER ACROSS AMAZONIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The review of the article is well written and well-structured. The authors have considered all the comments from the reviewers, particularly the comments on the description of the state of the art, missing information in the methods section and new data analyses.
Experimental design: The authors have considered the comments from the reviewers and have improved the description of the methods, particularly providing a detailed description of ‘soil’ and ‘litter’ sampling in section 2.1.
Validity of the findings: The authors have reworded the conclusions section and they are now connected to the original questions investigated.
Additional comments: The research questions are well defined, and their introduction has been meaningfully improved. Methods are described with sufficient detail. Discussion is well written and has also been improved. Therefore, I would suggest accepting the paper for its publication in PeerJ.

One minor comment: It is mentioned that freezing soil samples was not feasible (line 168). If that was the case, how were samples stored/treated after soil sampling until DNA extraction? It would be good to specify this in methods section.

Some very minor details are showed below:
Abstract, line 47: I suggest modifying “…diversity of in one…” to “…diversity in one…”.
Lines 162-163: Organic Matter abbreviations are inconsistent between these two lines.
Table 2: Change “besence” to “absence”.


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

Input:
Title: HIGH-THROUGHPUT METABARCODING REVEALS THE EFFECT OF PHYSICOCHEMICAL SOIL PROPERTIES ON SOIL AND LITTER BIODIVERSITY AND COMMUNITY TURNOVER ACROSS AMAZONIA
Review round: 2
Reviewer: 3

Output:
Basic reporting: The text is well written. It contains a few minor mistakes that have to be solved.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: AN ADAPTIVE SCALE GAUSSIAN FILTER TO EXPLAIN WHITE’S ILLUSION FROM THE VIEWPOINT OF LIGHTNESS ASSIMILATION FOR A LARGE RANGE OF VARIATION IN SPATIAL FREQUENCY OF THE GRATING AND ASPECT RATIO OF THE TARGETS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general the article is not particularly well written.   There are places particularly in the introduction and methods where things are inconsistent and confusing. With some effort the writing of the manuscript could be greatly improved making it much more readable.

For example, in the introduction there are too many unnecessary abbreviations. I recommend cutting 'Human Visual System' HVS altogether.

Here is a truly remarkable sentence from the introduction:

" This they conclude because the White’s illusion occurs irrespective of the aspect ratio of the test patches in Figure 1, which means that although it so occurs that with respect to the two flanking bars, the lightness of the column on which the test patch is located becomes dominating beyond a certain aspect ratio as is evident from Figure  3 and Figure 4, still the direction of lightness induction does not change in such a situation."

The reference to de Weert and Spillmann misstates 'the pincushion illusion'. The use of the term 'pincushion' in their paper its to describe the shape of the target field. The Pincushion Illusion is one of illusory contours unrelated to the effect reported in their paper.

There is great inconsistency throughout the manuscripts in referring to the components of the White's Illusion stimulus. I recommend: Background (uniform grey field present across the display), Inducing Grating, and Grey Target.

A description of the phenomenology that the authors seek to explain and how/why existing models fail to account for it is kind of given in the introduction, but it is disorganized and confusing. With some work this can be made more systematic,

Spatial Frequencies are reported in cpd but target size is reported in pixels.

Luminance values should be reported, not % or RGB levels.

Ambiguity in number of bars used to define the inducing grating: text indicates a minimum of 5, the figure appears to only show 3. Clearly the authors are counting the white bars as well.

Explanation of red-indicator bars being at either top or bottom of display comes too late.

Unclear whether or not red-indicator bars would interfere in Experiment 2 with the 2/4 pixel targets. How much of the target is visible in the presence of the indicator?

2AFC: typically the stimulus that is held constant across trials would be called the standard and the one that varies the comparator. Here the gray target is held constant. Whereas the patches in the stripe are varied.

If the experiment was conducted as described then Lines 316-321 are incorrect.

Line 336 is redundant.

If methods and parameters are spelled out more clearly, table 1 is unnecessary.

Experiment 2 should show examples of the stimuli as was done for Experiment 1.
Experimental design: Aside from clarity in description, the experiments seem fine. I recommend showing at least one set of actual subject data with error bars indicating the variability observed in the results. As is, only fit-curves are shown.

The application of models appears fine; however, this sort of modeling is not really my domain of expertise and I defer to other reviewers whether the selection of parameters and how they are applied is truly appropriate.
Validity of the findings: My primary concern has to do with implicit assumption that the effects of the Gaussian Model apply only to the Gray Target without also applying to the Inducing Grating. I can simulate the Gaussian model to some degree by blurring my eyes while looking at the various figures. Indeed, doing so results in one light target and one dark one. The issue is that they appear 'solid'. That is to say that in addition to the grey target appearing either lighter or darker , the inducing grating appears darker/lighter as well. Presumably, this would be apparent if the authors included a figure similar to Figure7 showing the output of the Gaussian Model.

I note that to me I sometimes do get a filling-in of the gray target such that it does look like a solid (i.e. in front of and occluding the inducing grating). Which would be along the lines of what I would expect from the Gaussian Model. That said, the solid appears 'crisp' with sharp edges, whereas I would expect to experience some blurring with the Gaussian Model. Importantly though, I do not always experience the stimulus this way and frequently experience White's Illusion while the inducing gratings seem just as black or white as when distal to the Target.

I also note that the deWeert and Spillmann papers reveal asymmetries between induced lightening versus induced darkening. This is something that should be explored/addressed in the current paper. Perhaps their example is not a good one to use in the introduction as it may be distinct from the White's Illusion.
Additional comments: Maybe it is just me but when blurring my eye just a little bit, I note that when viewing Figure 4 the upper targets appear to get progressively lighter whereas the lower targets appear to get progressively darker to the point where for rightmost (smallest) targets, the top one looks lighter than the dark one.


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: AN ADAPTIVE SCALE GAUSSIAN FILTER TO EXPLAIN WHITE’S ILLUSION FROM THE VIEWPOINT OF LIGHTNESS ASSIMILATION FOR A LARGE RANGE OF VARIATION IN SPATIAL FREQUENCY OF THE GRATING AND ASPECT RATIO OF THE TARGETS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This paper reports two experiments and a model of White’s illusion (WI), which involves perception of gray targets embedded rectangular gratings. The results showed that the strength of the illusion (different appearance of targets belonging to black and white portions of the grating) increases with increasing spatial frequency of the gratings (and thus the width of the targets), but that it does not change much with the change of the length of the targets. The model involves filters (simulated neural units) with gaussian receptive fields.
This paper is similar to a paper from the same team that I reviewed last year but is improved and involves a number of changes, including a new experiment. Furthermore, it is interesting to learn how a particular neural model reacts to WI stimuli. Some changes seem to reflect and address, in part, some of my comments. However, a few of my original concerns apply to the new paper as well.
1. The model is based on neural units with purely excitatory receptive fields, with gaussian fall-off. However, generally receptive fields of neurons in the visual system have both excitatory and inhibitory portions. Mitra et al. speculate that inhibitory portions could be suppressed by high frequency displays, such as WI stimuli, and cite some indirectly relevant literature. However, I am not aware of physiological evidence directly supporting the actual existence of such neurons, or the crucial role of high frequency stimuli in generating their receptive fields. This is relevant for assessing the import of the model, and also in light of the fact that Mitra et al. criticize other authors (Blakeslee & McCourt) on the grounds that their model involves a feature which has not been physiologically confirmed (p. 24).
2. The simulations of results for different stimuli involved gaussian filters with different parameters. Inspection of Table 2 indicates that the size of one parameter increased with spatial frequency of the gratings and decreased with the length of the target, for a total of about 30 different numerical values used in the simulations, varying approximately within a 1:16 range. No principled reason for the particular choices of these sizes for particular stimuli was offered, so that the criterion was probably goodness of fit. This makes engineering sense, but I wonder about the physiological plausibility of such a scheme. One possibility is that neurons with gaussian receptive fields with all these parameter values do exist in the visual system. If so, the question is why would, for a given stimulus, only a small subset of them which share a particular value of a parameter be activated, and none of the others? Another possibility is that the same neurons can change their receptive field parameters for different stimuli. Then the question is whether such changes could be as drastic as in the proposed scheme.
3. The authors call their model ‘multiscale’, since it uses units with receptive fields with different scale parameters. However, for any particular stimulus, they use units with a single scale to model the results, but different for different stimuli. Usually, the term ‘multiscale’ is reserved for models in which units of several different scales are all applied to all stimuli, such as in the Blakeslee&McCourt model. Such genuine multiscale models have advantages over the authors’ model, since they don’t have the problems described in the previous paragraph.


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: AN ADAPTIVE SCALE GAUSSIAN FILTER TO EXPLAIN WHITE’S ILLUSION FROM THE VIEWPOINT OF LIGHTNESS ASSIMILATION FOR A LARGE RANGE OF VARIATION IN SPATIAL FREQUENCY OF THE GRATING AND ASPECT RATIO OF THE TARGETS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is greatly improved!
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: Thank you for the revisions you have made to 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: AN ADAPTIVE SCALE GAUSSIAN FILTER TO EXPLAIN WHITE’S ILLUSION FROM THE VIEWPOINT OF LIGHTNESS ASSIMILATION FOR A LARGE RANGE OF VARIATION IN SPATIAL FREQUENCY OF THE GRATING AND ASPECT RATIO OF THE TARGETS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have generally taken into account my comments and incorporated them into the revised version. I have no further 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: PLURALITY IN MULTI-DISCIPLINARY RESEARCH: MULTIPLE INSTITUTIONAL AFFILIATIONS ARE ASSOCIATED WITH INCREASED CITATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Okay, some refinement with table illustration (i.e., table 1) would make it even better
Experimental design: Research question is well defined and is a relevant one. Basic statistical methods were followed for research analysis.
Validity of the findings: The result is not robust. In fact, it is significantly biased. The authors need to use a normalised value for the citation count. An article published in 2005 (for example) will obviously get more time to be cited compared to another article published in 2015.
Additional comments: Without considering the normalised value of citation count the entire findings of this research will be completely misleading. I would recommend the authors to consider the following article regarding how to normalise a citation count value:

The impact of author-selected keywords on citation counts
By S Uddin, A Khan, Journal of Informetrics 10 (4), 1166-1177


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: PLURALITY IN MULTI-DISCIPLINARY RESEARCH: MULTIPLE INSTITUTIONAL AFFILIATIONS ARE ASSOCIATED WITH INCREASED CITATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Background/context could be improved (see suggestions below).
Experimental design: This article looks at the correlation between author numbers, affiliation numbers and citations. In doing so it fits into the broader range of articles looking at the importance of network size on citation impact.

I have several comments:

1. The title is misleading and should be changed: a) The title refers to ‘biomedical research’ but the authors analyse multi-disciplinary journals which cover a wide range of different subject areas, not just biomedical. b) The notion of ‘improved research output’ is misleading. The article looks at citations, and this should be reflected in the title. c) Author numbers are more consistently associated with citations, while multiple affiliations only under limited circumstances show such association. Thus, the title gives a very wrong impression of the findings.

2. The paper is poorly motivated. I understand that it wants to contribute to the study of the importance of teams in knowledge production. It does this by adding the dimension of multiple affiliations to the analysis of co-author numbers. This is all fine, but the whole introduction about digital revolution and mobility is entirely misplaced. More effort should also be made to tell us what the papers that are cited found, such as the increase in team sizes [1] and increase in multiple affiliations [4], as well as the growth of international teams and their association to citation numbers [2]. At present the research question and knowledge gap are not clear.

3. The paper does not mention how citations are counted. Are they looking at all citations received by 14/06/2017? Having citations at one point in time means that 2010 publications had 7 years to accumulate citations, while 2014 publications had just 3 years. This truncation may influence the findings if the number of authors or affiliations has increased over the same period (something that the references [1] and [4] suggest). This truncation problem is normally addressed through the use of fixed citation windows. Moreover, the regressions analysis (supplementary table) should control for year and journal as both may affect citation counts.

4. I find the discussion of the results somewhat confusing, especially as no complete descriptive statistics and correlation tables are provided. Table 1 specifically should provide percentages in addition to paper numbers and the table caption should include a definition of ‘lowest’ and ‘highest’ citations. Table 3 in turn is difficult to read as the average number of citations is not known. This makes it difficult to understand what the citations difference actually means (i.e. whether it is small or large).
Validity of the findings: 5. The article uses the language of causality, when all they provide is correlations. For instance on lines: 97, 119, 135 phrases such as ‘influence’, ‘effect’, ‘affect’ are used. This is plainly unacceptable as no conclusions regarding the direction of this correlation can be made. It is not clear whether better, more cited author attract more affiliations and co-authors or whether more affiliations or coauthors automatically lead to better, more cited research.

6. The conclusion that multiple affiliations ‘may also play a positive role in the production of high-impact science’ (152-153) is thus very tentative and can hardly said to be supported here. Longer time-series and an ex-ante/ex-post comparison of citations would be necessary to approach this topic. The one interesting finding one can read from your analysis is that team size and multiple affiliations seem to be complementary. This is what we can learn here and what could be stressed in the conclusions.
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: PLURALITY IN MULTI-DISCIPLINARY RESEARCH: MULTIPLE INSTITUTIONAL AFFILIATIONS ARE ASSOCIATED WITH INCREASED CITATIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: I have a few initial concerns upon reading the abstract and introduction:

1.	First, the background suggests there is no prior research regarding multi-institutional research teams and output productivity. But yet the social sciences (e.g., team science), and particularly network sciences, have been publishing citation/bibliometric analysis for quite some time. I think your claim is that they haven’t analyzed multi-institutional teams as a factor, yet the Jones et al [2] and Hottenrott [4] papers have indeed. Specifically, there are over 80 citations available from a Science of Team Science research community reference list on this issue, which I have attached ( “Authorship, Contributorship, Publishing Issues” folder of the Science of Team Science (SciTS) group in Mendeley).
2.	Second, the use of the term polygamous behavior seems inflammatory and suggestive of illicit research.  Why this choice of term?
3.	Third, the suggestion that multi-institutional research is shunned by IRBs is generally not true (though it may be true on some campuses).  It complicates the review process, but I don’t think there is evidence that IRBs prevent this kind of research.  What is the basis for your suggestion that IRBs shun multi-I research? This is not discussed or reviewed at all in the paper,

[2] Jones BF, Wuchty S, & Uzzi B (2008) Multi-university research teams: shifting impact, geography, and stratification in science. in Science (American Association for the Advancement of Science), pp 1259-1262.
[4] Hottenrott H & Lawson C (2017) A first look at multiple institutional affiliations: a study of authors in Germany, Japan and the UK. in Scientometrics (Springer Netherlands), pp 285-295.
Experimental design: 1.	Jones et al used at dataset that included “4.2 million papers published over three decades.” Why did you choose 27,000 papers in a 4-year period?  What was the justification of this smaller dataset?
2.	Given the data ends 3 years prior to analysis, is that sufficient time to realize citation impact differences?
3.	Why the choice of the 4 journals chosen (e.g., these are the 4 highest impact journals in biomedical research)?  Does that choice skew the results in any way?
4.	Given only 6% of the articles in the data were multi-institutional, are you concerned about the possible insufficiency of data available to assess the contribution of multi-institutional affiliation?  Why not create a larger data set and focus only on multi-institutional papers?
Validity of the findings: 1.	According to Table 1, 74% of articles in the dataset were authored by a single institution, and 20% by two institutions. Thus only 6% of the dataset was more than 2 institutions.  Given your literature review of growing multi-institutional research, was this surprising?
2.	Table 3 (and in text) – what are the units in the average change in citation count column?  Assuming percent change, but it could be absolute value as well.
3.	Table 3 – one of 2 main findings is that “increasing the number of authors on a paper tends to results in more citations irrespective of the number of affiliations held.” This is how I interpret this as well, which is not included in the Discussion.
4.	Supplementary table – is the author number here the categorized sets (2, 3, and 4), or the raw number of authors? I think the former, but it’s not clear from the table presentation
5.	Regression (and supplementary table) suggests that the significant predicting value is still the number of authors, not the breadth of institutional affiliation.
6.	The overall finding is that when papers have fewer than 6 authors, the number of institutions doesn’t matter, but that for larger authored papers it does enhance the impact of the paper.  What does this reflect?  Is it the diversity and breadth introduced to large research teams, or that papers generated from much larger research teams tend to reflect quality and prestige in other ways?
7.   The Conclusions section needs to include more substantial discussion of the results here. (see questions below)
Additional comments: 1.	Were you surprised that the majority of articles in that dataset were NOT multi-institutional?  How does this compare to the suggestions from [2[ and [4] about a growth in multi- institutional research?
2.	What is different in the findings of your study compared to what Jones et al and Hottenrott found?
3.	What is the importance of 5+ authors being more likely to be multi-institutional and in turn have higher citation impact? Is this an artifact of highly-funded institutional research (e.g. NSF collaboration grants) that are highly competitive and therefore likely to generate high quality publications? Or is there something important about broadening the institutional representation to the quality of knowledge generated (which presumably leads to higher impact)?  Neither of these possible explanations are discussed, and so we’re left with the question of what to do with the findings.


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: PLURALITY IN MULTI-DISCIPLINARY RESEARCH: MULTIPLE INSTITUTIONAL AFFILIATIONS ARE ASSOCIATED WITH INCREASED CITATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: okay
Experimental design: In the rebuttal letter, the authors mentioned that they considered the normalised citation count values. They also mentioned that they followed the approach proposed by Uddin and Khan (2016). However, I did not find any details of that approach in the main manuscript file. On top of that, I did find any correct reference of this kind of normalisation. They just mentioned they used normalised citation count value but did neither explain nor provide reference regarding how did they do it.
Validity of the findings: Please see my comment in the previous section
Additional comments: In the rebuttal letter, the authors mentioned that they considered the normalised citation count values. They also mentioned that they followed the approach proposed by Uddin and Khan (2016). However, I did not find any details of that approach in the main manuscript file. On top of that, I did find any correct reference of this kind of normalisation. They just mentioned they used normalised citation count value but did neither explain nor provide reference regarding how did they do 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: PLURALITY IN MULTI-DISCIPLINARY RESEARCH: MULTIPLE INSTITUTIONAL AFFILIATIONS ARE ASSOCIATED WITH INCREASED CITATIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The background section still has too many unreferenced sentences. For example, the authors talk about 'expectations' but do not back this up.

It is not clear what 'roaming' is to mean in this context.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: VARIATION OF SOIL BACTERIAL COMMUNITIES ALONG A CHRONOSEQUENCE OF EUCALYPTUS PLANTATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see general comments
Experimental design: Please see general comments
Validity of the findings: Please see general comments
Additional comments: Li and colleagues have analysed soil samples from a chronosequence of Eucalyptus forests, as well as a secondary forest, to determine the changes that occur in bacterial community structure and inferred function. The authors found that the soil communities under two and 10 year old eucalyptus plantations were significantly different to those found under a five year old eucalyptus plantation, and a secondary forest. This difference could be linked to differences in soil chemistry, and was also reflected in differences in the inferred function of microbial communities.

This paper has the potential to provide useful insights into the microbial communities residing in Eucalyptus forest soils. However, the description of the sampling design (lines 84-92) is very confusing, and does not clearly indicate how many sites were sampled, or the spatial scale covered. While 12 samples in total is a small dataset regardless, if these 12 samples are from separate sites (meaning n=3 replicates sites per forest type), the data could be meaningfully interpreted. However, if only four sites were sampled in total (meaning n=1 per forest type), this would be inadequate to draw the conclusions presented in this manuscript. The authors must clarify this important point.

Additionally, classifying the 2YR and 10YR plantation forests as ‘impacted’ and the 5YR and SF as “recovered” does not make sense (lines 177-185; 225-227). How can five year old Eucalyptus plantations be considered recovered if they are then considered “impacted” again five years later? The terminology used for these two groups should be changed, and accordingly the word “recovery” should be removed from the title of the manuscript. Additionally, since it is unclear if each sample within a forest group is from the same site, or from different sites, it is difficult to judge whether adequate sampling has been done to determine when a Eucalyptus plantation is different to a secondary forest.

The manuscript can be further improved in these areas:
Lines 58-65: There are examples of other relevant studies that do use NGS techniques and these should be mentioned here. For example Lan, G. et al., (2017) Soil Bacterial Diversity Impacted by Conversion of Secondary Forest to Rubber or Eucalyptus Plantations: A Case Study of Hainan Island, South China. For. Sci. 63: 87–93.
Line 73: “nutrient cycling and metabolic etc.” Should this say metabolism? The use of etc. also makes it a vague statement and should be better defined as it is a main research question.
Lines 84-85: How far apart was each replicate plot and what is meant by “S” shape? 20 x 20 cm is a very small “plot” size, how many samples were collected within each of these plots? Perhaps a figure showing the sampling design could be useful.
Line 87-88: How have six replicate plots, in four forest types resulted in 12 composite samples? How much distance separated each forest site? How many replicates were sampled for each plantation stage/secondary forest?
Line 106-108: These primers amplify the V3-V4 region, not just V4. Please correct this.
Line 110-113: Please indicate the sequencing length, and number of sequencing cycles that were used.
Line 119: Please explain what is meant by sOTU, as it is not commonly used. My understanding is that the output from DADA2 is a table of ASV (amplicon sequence variants) so why has this terminology not been used instead?
Lines 131-132: Please clarify if a rarefied OTU/ASV table has been used only for alpha-diversity analyses, or for all analyses.
Lines 140-142: The justification of the variables included for this analysis does not make sense. Both AP and AK were not significant according to the Mantel test, so why are they included? Furthermore, Mantel R values should be presented along with the p-values, or direct the reader to Table 2.
Lines 150-159 and lines 252-271: The detailed descriptions of differences in soil chemistry, without relating these directly to bacterial data, seems unnecessary as it was not a main research goal. I would remove these two passages.
Line 165: The number of OTUs per sample seem very low compared to what is normally reported for soil bacterial communities. Were low abundant OTUs/ASVs removed, or any other filtering steps included? This should be noted in the methods.
Lines 177-185; 225-227: As discussed above, the use of “recovered” and “impacted” does not make sense for the data presented, please use alternative terminology.
Line 205: The table that is referred to here (Table S1) was not included in the review material.
Lines 215-222: For the specific taxa that are discussed, please include the histogram of relative abundances that is part of the LEfSE output (i.e. the plot in Figure 1c in the LEfSe paper: http://doi.org/10.1186/gb-2011-12-6-r60.) This should be in the supplementary information, but is important to determine the “biological relevance” of these results, and ensure they are not due to outliers.
Figures 1-2: The manuscript has a lot of figures, some of which are barely referred to. I recommend moving figures 1 and 2 into supplementary. ‘(P<0.05)’ should also be removed from Figure 2’s legend or put into context.
Figure 5: It would be helpful if the legend mentions that it is showing the abundance of different phyla for different forest ages/secondary forest, and how many samples were combined for each ‘bar’. There should also be an X-axis title.
Table 1: Please indicate what statistical test was used to obtain p-values, either in the table heading or in the methods. Please include standard error values to show variability among samples from the same forest type.
Table 3: Please indicate what statistical test was used to obtain p-values, either in the table heading or in the methods.
Figure S2: Some of the bars have no taxon names showing.
There is no data deposition statement in the manuscript, please provide this. The EBI accession number provided with the manuscript submission details is not yet accessible, but raw data were available for review through figshare.

There are several grammatical and spelling errors throughout the manuscript, along with referencing issues; some of these are listed below, but this is not an exhaustive list and the manuscript could benefit from further proof reading:
Line 42: The reference includes the lead author’s first and last name, and this is also incorrect in the reference list
Line 51: remove the word “which”
Lines 52-55: The two statements in this sentence are somewhat contradictory. Please explain what is meant by not “negatively affected” if there is evidence of differences in community structure
Line 78: Should read “on June 8th” not “in June 8th”
Line 133: Incorrect spelling of Kruskal-Wallis
Line 139: Reference is in all capitals.
Line 198: remove “relative to SF” at end of sentence
Line 216: overrepresented, not “overpresent”
Line 304: ‘of evenness’ should be deleted
Line 316: Remove “of” after “Since”
Line 323-325: This doesn’t make sense, please clarify
Line 327: The reference includes the lead author’s first and last name, please correct.


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: VARIATION OF SOIL BACTERIAL COMMUNITIES ALONG A CHRONOSEQUENCE OF EUCALYPTUS PLANTATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript by Li et al. entitled "Recovery and variation of soil bacterial communities
along with a chronosequence of Eucalyptus plantation" describes the effects of Eucalyptus plantation on soil and in bacterial soil inhabitants. In short, the authors study how the community would change and recover in the chronosequence of Eucalyptus plantation. In conclusion, the authors show that soil bacterial community change after 2 years of Eucalyptus plantation, recover after 5 years and change again after 10 years. Overall, the manuscript is average written, descriptive, and although contains results of interest, the conclusions drawn are not supported by the results.
Experimental design: While reading and evaluating this manuscript, a variety of questions and remarks came regarding the experiment. More information is needed related to sampling site and just a few replicates are available to draw strong conclusions related to the recovery and variation of the bacterial community. However, it is an original research and the molecular, bioinformatic and statistical analysis was described with sufficient details.
Validity of the findings: The results are well described but I am not convinced about the recovery and change of the bacterial community in the consequence. To have this conclusion, soil samples should be sampled from the same area and needed to be monitored over time. There is a novelty in the manuscript and data are robust (although from only 12 samples). However, the conclusions from the results are not supported.
Additional comments: the subject is relevant, however, the authors did not succeed to answer the research questions with the experiment. To track recovery of bacterial community, the community should have been assessed by comparing the soil samples before and after Eucalyptus plantation in the same plot. Their conclusions are not supported by the experiment.


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: VARIATION OF SOIL BACTERIAL COMMUNITIES ALONG A CHRONOSEQUENCE OF EUCALYPTUS PLANTATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: The revised manuscript submitted by Li and colleagues is a significant improvement on the original draft. I thank the authors for their careful consideration of my previous comments, and their responses.

The revised description of the sampling design highlights that this study was conducted by sampling only one plantation per group (or age). Therefore, the results from this study cannot be extrapolated beyond what is happening in these four sites, and it cannot be determined if the results hold true for other Eucalyptus plantations. While for the most part the authors do not over-state their results, I feel that there is not enough evidence in this paper alone for the recommendation in lines 359-362 and would recommend removing this to not “over sell” the results presented in the manuscript.

In my opinion, a few minor changes are required before the article is ready for publication:
Please specify the number of soil cores taken per plot. I.e. how many soil cores went into each composite sample (lines 87-89).
The authors have misunderstood my original comment asking them to include the sequencing length/number of sequencing cycles used. Illumina MiSeq chemistry can either sequence 2 x 250 bp or 2 x 300 bp fragments, depending on whether there are 500 or 600 cycles. Please remove “The number of sequencing cycles was 28” in lines 111-112, as this is the number of PCR cycles, and add the correct information to lines 112-114 (again, 468 bp is the average length of your PCR product, not the sequencing length).
Lines 129 – 131 claim that the results from the QIIME 1 pipeline are similar to those from the QIIME 2 pipeline, however the location of the samples on the PCoA plot are quite different. Either remove this statement or provide statistical evidence for the similarity between the two beta-diversity outputs.
Line 135 should say “number of sOTUs”
Line 244 states that PICRUSt has a “predicted minimum accuracy of 85-90% for soil”. Please explain where these values came from. The Langille et al 2013 paper does not make this claim; the closest statement to this within the paper is that the predicted metagenome correlated to an actual metagenome with a Spearman’s’ R of 0.81.

The manuscript still contains grammatical errors which at times make it difficult to read. Further proof reading would greatly increase the readability. Several examples, as well as a few typos, are highlighted below.
Line 17: “comparing with” should say “compared to”
Line 56: “Other relevant study that…” should say “Another relevant study of” also tropical is misspelled.
Line 59: What is meant by “artificial forests”
The research questions in lines 68 and 70 should either have the question marks at the end removed, or start with “How do”
Line 79-80 is hard to understand
Line 96: K2Cr2O7 should have a capital K
Line 123: the “and” should be removed
Line 137: Replace “Equally, according to the” with “Using”
Line 187: Use “Impacted” instead of “impact”
Line 221: Remove period after Overall
Lines 248-251: There are multiple unnecessary capitals in this paragraph
Line 292-293: Again, this sentence is hard to understand, please clarify
Line 309: Replace “even though alterations” with “even though there were alterations” and remove occurring
Line 315: Remove “of”
Line 321: change “tolerance” to “tolerate”
Line 323: “an indicators of” should either be “indicators of” or “an indicator”
Line 334: Change “illustrates that low ability” to “illustrates the low ability”
Line 344: “suggested a decrease in overrepresented” overrepresented compared to what?


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: DIFFERENT ENVIRONMENTAL GRADIENTS AFFECT DIFFERENT MEASURES OF SNAKE Β-DIVERSITY IN THE AMAZON RAINFORESTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study is interesting and a good contribution to the field community structure at regional scales applied to snakes. The manuscript is well written and does not need to much revision in professional English. I made some suggestions in order to improve the quality of the manuscript, I have some criticisms related to some definitions in ecology, and also some criticism applied to the experimental design of this study. But it is something that could be easily tackled by the authors.

When I read the title of your manuscript I noticed you were studying the effect of “ecological gradients” on different measures of beta diversity. Nevertheless, I noticed that there are ambiguous uses to the term “gradient” in your manuscript. Sometimes you used the term gradient meaning “filter” (along the discussion), sometimes you used the term gradient meaning “scale” (e.g. line 275 – “the gradients geographic distance”, along the abstract,…). Gradient means a gradual variation of abiotic or biotic factors through space or time. By definition, a spatial scale cannot be considered a gradient like an environmental gradient is. The term “ecological gradients” used in the title might be being misused if you are considering space or time as gradients. Otherwise, the term “scale” or “filter” could be applied to variables other than the environmental. Scale could be applied to space, time, environment and a biotic factor. Filter could be applied in a similar way. As already there are a more efficient terminology that incorporates space and time and it is not the term gradient, I recommend some changes in the text in order to remove conceptual ambiguities applied to the term gradient.

Line 74 to 77. It seems to me that the mechanisms used to explain PD changes along landscapes are more associated to a population genetic/phylogeographic level (in other words, to an intraspecific level) than to a interspecific/community level. As the estimate of PD you used in your study is applicable to a community level, I recommend you to rewrite this part of the paragraph including explanations of changes in PD applied to a community level (e.g. extinctions, migrations, selection of lineages due to, habitat complexity, source and sink models in a metapopulation and metacommunity perspective,…) and remove explanations more applicable to a population genetics level (e.g. disruptive selection). I noticed more appropriate explanations for variations of PD at a community level in some paragraphs of your Discussion section and could be used in the introduction as well.

I tried to find the paper Magnusson et al. 2005 cited in lines 130-131, but I did not find It at the references. I’m afraid that some citations in the body of the manuscript are not at the references section. So I suggest revision of the references section in order to ensure that all of them are appropriately included.

I strongly recommend you to take the Fig. S1 off from the supplementary material and include it inside the body of the manuscript. This figure is really important for the comprehension of the study you conducted and for the RAPELD concept and experimental design. The inclusion of the figure inside the body of the paper would be more interesting for the comprehension of the readers.

HAND is a good acronym, but you included more than one sentence applied to it. Sometimes you used “vertical distance from drainage”, sometimes you used “height above the nearest drainage” (HAND). Please, use only one term and correct it along the text.
Experimental design: L. 156-157: “The sampling expeditions were distributed throughout the year so that samples are independent of seasonal variables, such as rainfall.” Have you statistically tested that the samples are independent to seasonal variables? It should be tested before the analyses you conducted.

Actually, this is an important issue to be considered in your analyses. As the title suggests, you tested different “ecological gradients” (meaning scales?) in order to test if there are changes in beta diversity estimates at a regional scales in the Amazon and I suppose you aimed to infer which variables/gradients where the most important for community structure . However, you only considered two ecological scales in your analyses: spatial and environmental. Why did you not include a temporal scale into your analyzes. If you did not include it because of what you mentioned in lines 156-157, you should conduct an analysis to test if there is a temporal effect in your data. As you made samplings from 2007 to 2014, I think a temporal scale should be somehow included into analysis.

About the explanatory variables you used into your multiple regressions: why you chose these variables and consider it good enough to explain TD, PD and FD differences among communities? For some of them I find good reasons (e.g. geographic distances, tree cover). For other ones, I think that other variables might be considered more important than those you used. For example, why you did not include climatic data as well (precipitation, average temperature, seasonality, or some bioclimatic variables from worldclim)? As the worldclim project provides several data for the Amazon and these data could be extracted in R through the package raster as you did with other variables, I think some bioclimatic variables could enrich your analyses. I have noticed that you do consider other variables (average temperature, total precipitation and seasonality) as important variables in your discussion (lines 330-333). So, why not to include them into your analyses?

Actually, I have one criticism to the inclusion of geographic distance as an explanatory variable into your multiple regressions, and not as a covariate. The title of your manuscript suggest that you were evaluating the influence of ecological gradients on beta diversity estimates applied to snakes. Therefore, the most important variables are those involving gradients.  Space is not related to gradient, but it is applied to a scale. The same conclusion is applicable to time. I think you should reanalyze your data considering space and time as covariates, removing the effect of them from environmental variables. Doing so you could separate the effects of space, time and environment variations in beta diversity measures.

L. 163: You should include geographic distance as a covariate and reduce its effects in order to notice just the effect of environment variables structuring the beta diversity of snakes. You need to separate the spatial effect from the environmental effect of your analyzes.

L. 148 – savannah. It was mentioned that there are some savannah fragments nearby some RAPELD modules and it was also informed that they were not sampled. This is an important aspect to be considered, since it can increase habitat complexity and might influence TD, FD and PD. Could you include this information as an explanatory variable to your data? Have you already included information of habitat complexity and the presence of savannas in your modules with one of the variables you used in the study?
L. 216-230. Mitochondrial 12S sequences have fast evolution rates and provide too much variation to your data, even more at an interspecific level. Actually, it is interesting at some point. Nevertheless, at an interspecific level the evolution rates of 12S are so fast that uncertainties about mitochondrial evolution emerges. Evolutionary models are alternatives to tackle this problem, but there are more recommendations to improve the assessment of the PD. One of it is to include more than one marker (such as nuclear markers) into analysis, reducing artifacts by the evolution of “one simple gene” compared to the evolution of multiple markers in a consensus tree or species tree. Therefore, I recommend you to include more than one marker to calculate PD. How did you select the Tamura-Nei model of evolution as the best for your data? Have you run several models and selected it as the best? If so, please inform it at material and methods.
Another question to the same issue: why did you not use the branch length from a tree generated by other studies (e.g. Pyron et al. 2013. A phylogeny and revised classification of Squamata, including 4161 species of lizards and snakes. BMC Evolutionary Biology 13:93–146) to calculate PD? Piron et al (2013) tree already includes many markers an taxa, so the value of PD by this study seems better than those you generated only with 12S. At lines 231-233 you cited some studies (including the above mentioned) with apparently better phylogenetic hypotheses of snake evolutionary relationships just for comparison. Then, why did you not estimate the PD for each module by their studies instead of just citing them for comparison?
Results
You have conducted three separate multiple regression models, one for each diversity index (Taxonomic, Functional and Phylogenetic), but you did not include any correction to the α you used. Actually, you also did not specify which value of α you used for your analysis (I suppose that was 0.05). As you conducted three different analyses with the same basic data, I suggest you to include a correction index (such as Bonferroni or other one) to control your α previous to analysis and reduce type I errors. The same thing must be applied to the correlation analyses for the pairs of Beta diversity estimates (TDxFD; TDxPD; PDxFD) you conducted.
Validity of the findings: In conclusion, the paper presents a few things that need to be revised or redone to correct some conceptual and analytical misinterpretations. However, in general, the manuscript is well written and can be a good contribution to the study of community structure at the regional level, especially for snakes.
Additional comments: The study is interesting and a good contribution to the field community structure at regional scales applied to snakes. The manuscript is well written and does not need to much revision in professional English. I made some suggestions in order to improve the quality of the manuscript, I have some criticisms related to some definitions in ecology, and also some criticism applied to the experimental design of this study. But it is something that could be easily tackled by the authors.


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: DIFFERENT ENVIRONMENTAL GRADIENTS AFFECT DIFFERENT MEASURES OF SNAKE Β-DIVERSITY IN THE AMAZON RAINFORESTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper describes the beta-diversity of snake assemblages in Amazon rainforests using different dimensions of biodiversity (taxonomic, functional and phylogenetic). The introduction is clear (I suggested only minor changes) and well referenced. Because I am not a native speaker, I can not judge the English quality. The figures and legends are clear and also need only minor changes. The authors provided the raw data which allowed me conduct some tests that I specify below.
Experimental design: The study is well conducted and the sampling effort is impressive. The authors performed a standardized sampling of snakes in a large area of the Amazon. I really appreciate that they care and consider the limitations of snake detectability in their analyses. The authors provided two field study permits but I think they could provide a bit more information about these documents to check their appropriateness. The research question is well defined. I only suggested to specify a little bit the hypothesis to not be so general. I also suggested to the authors some additional tests to confirm their results, especially applying a multiple regression on distance matrices to confirm the results of the summarized multiple linear regression. I got some results slightly different from those reported in the text when reproducing the analyses, and I suggested to the authors to check if the raw data available is correct (if it is exactly the same used in the paper) or if there is any method description missing (see details below).
Validity of the findings: In analyzing the data, I observed collinearity among some predictor variables. Such collinearity may be inflating the explained variance of the taxonomic beta-diversity and altering its result. The result that I got after removing one collinear variable is similar to that observed after applying a multiple regression on distance matrices. I recommend to the authors check if the raw data available is correct to confirm such effect. If the collinearity is real, great part of the discussion as well as the conclusion of the paper will need to be altered (see details below).
Additional comments: Major comments:
- Lines 126-127: The hypothesis is interesting but it seems too general. Consider thinking in a more objective hypothesis, something like evaluate if all the three measures of beta-diversity respond to the same environmental gradient or, alternatively, each measure has its own environmental predictor.
- The first field study permit provided is actually an operating license for an energy company. The document does not seem describe permission about snake sampling. I am not familiar with this kind of document. Can you clarify this permit? Additionally, its date of emission seems posterior to the dates of collection (2016). Is that right? The number of second field study permit I think is incomplete. Would it be 13777-2/2008? It is necessary an authenticity code and date of emission to check the appropriateness of the document. Can you, please, provide some additional information?
- You summarize each measure of beta-diversities by applying an ordination method and using one of the resulting scores as response variable in a multiple linear regression. But you did not inform how much each of the used scores is summarizing the respective measure of beta-diversity. For taxonomic beta-diversity, for example, it seems that you used the second score instead of the first (TD2 in the raw data). Is TD2 more informative then TD1? I think that would be interesting provide a table in the supplementary material informing for each measure of beta-diversity how much of its variability is summarized in each (two or three) score and indicate which score was used in the analyses. In addition, using scores necessarily implies loss of information. Especially depending on how much information is summarized by the used scores. Therefore, I think that would be interesting repeat the tests using a multiple regression on distance matrices just to confirm the results.
- In the lines 231-234 you report that there was low support for several branches in the phylogeny, but that the overall phylogeny did not conflict significantly with other existing and well-supported phylogenetic hypotheses. Do you think that would be interesting repeat your tests using these well-supported phylogenetic hypotheses available just to confirm the robustness of your results?
- In the line 267 it is affirmed that the gradients showed little collinearity. However, using the raw data available I found strong correlation between geographic distance and clay content (r = 0.7). The collinearity was confirmed using the VIF analysis and the Klein’s rule (see the function imcdiag in the mctest R package). But I may have overlooked any method to extract the scores from geographic distance (I calculated the Euclidian distance based on the coordinates available in the raw data and used the first axis of the PCoA) or the raw data available may be slightly different from that used in the paper. Please, check the collinearity in your data to confirm that they are not affecting the results. If the collinearity is real it will have great impact in your results (see below).
- I reproduced the analyses and found collinearity between geographic distance and clay content. When I removed one of these variables the result for TBD changed. The results showed that no environmental variable explain taxonomic beta-diversity (score TD2). The closest variable was HAND, with p = 0.0697 after removing clay content. Curiously, applying the multiple regression on distance matrices (which not reduce the information to one score) returned the same result. This may indicate that HAND is the best (only) predictor variable for all three beta-diversity measures in the area analyzed.
- Lines 319-321: You state that different measures of beta-diversity such as FBD and PBD are affected by different ecological gradients. However, these two measures were equally affected by the same environmental variable: HAND. Do you think that the additional effect of geographic distance on PBD (which must be revised; see below) is enough to affirm that both measures are affected by different sets of ecological gradients?

Minor Comments:
- Line 31: “Snake assemblage turnover”? You did not mention the taxonomic group in the hypothesis.
- Line 31-33: It is not clear in the abstract why variables such as clay content or HAND could influence beta-diversity of snakes. Consider provide some additional information first, something like: “…are influenced by gradients of geographic distance, as well as by environmental variables associated with… (the reason why you chose these three variables)”.
- I think that would be more interesting abbreviate the beta-diversity measures as TBD, FBD and PBD instead of TD, FD and PD to avoid confusion with measures of alpha diversity.
- I think you should avoid using turnover to report your results and use just beta-diversity (e.g., lines 31, 303). As you surely know, beta-diversity is composed by two distinct components (turnover and nestedness), and you did not partition the overall beta-diversity to state about the turnover component.
- line 62: Please, check if it is missing a comma after “resources”.
- It is not essential but consider moving forward the paragraph starting at line 80. That is, experiment change the position of the fourth and fifth paragraph to make your introduction more linear.
- Consider moving the lines 134-137 to the next paragraph (line 154). This way you will have one paragraph describing the modules and another paragraph detailing the sampling.
- Line 152: include the expression “(see details below)” after “prior to flooding”. Just to notify the reader that information about sampling date will be detailed soon.
- Just for curiosity. Do you think that the period of sampling (nocturnal; line 154) may have any influence in the period of activity trait (diurnal, nocturnal; line 180)?
- Lines 231-234 discusses about the phylogenetic tree. I think they should be moved to the line 226, before describing the phylogenetic beta-diversity.
- Line 237: Are these coordinates the centroid of the modules? Please, clarify.
- Coordinates information has some differences between files provided. Please check and standardize the data.
- HAND and Tree cover were log transformed but this is not mentioned in the text.
- Line 279-others: You provide the R-squared of the regression. But I think you should provide the adjusted R-squared since you are calculating a multiple regression.
- Please, check the p-value numbers. In some cases the number does not seem rounded correctly (e.g., the p-value for effect of Tree cover on PBD is 0.958 and not 0.957).
- Line 288: Geographical distance contributed to explain PBD (p = 0.05). However, my analyses returned p = 0.066. Please, check if the raw data available is correct.
- Line 297: The relationship between TBD and the two others beta-diversity measures informed in the text are different from those calculated with the data available. For example, the test for relationship between TBD and FBD returned r = -0.39, p = 0.08, and not r = 0.1, p = 0.1 as informed. Please, check if the data available and your results are correct. You could also think about applying a mantel test using the dissimilarity matrices.
- In the figures legend sometimes is wrote beta in others used the Greek letter. Please, keep a pattern.
- I could not reproduce exactly the graphics involving PBD. But just some points (e.g., the upper left points of the TBD x PBD plot), nothing affecting the relationship informed. In addition, the axis of the PBD in the figure 2 (ranging from -0.4 to 0.2) are different from the data provided (0.5 to 1.3). Please, check if the PBD score provided is the same used in your analyses.
- Line 302: “Our results indicate that…”
- Line 429: “multidimensional”?


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: DIFFERENT ENVIRONMENTAL GRADIENTS AFFECT DIFFERENT MEASURES OF SNAKE Β-DIVERSITY IN THE AMAZON RAINFORESTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I analyzed the response letter from Fraga et al. and I also read the new version of the manuscript. In general, the authors attempted to solve the problems presented in the first version of the manuscript, which made them rethink the introduction and analyses. They made a good job reanalyzing data and incorporating the suggestions from the first draft. Nevertheless, I still have some small suggestions to the authors, prioritizing cohesion and fast reading.

It seems that there are some words missing along the text. Please, make a revision mainly at lines 33, 88, 135 (“spaces”).

I noticed that you used the term ecological gradient (abstract) and environmental gradient (along the text) with the same meaning. Please, choose one of them to uniformize the text.

The second paragraph seems dislocated in the introduction, since you already expose your objectives, and not the general question to be studied. I think it would be more appropriated if it was placed at the last paragraph of the introduction. Similarly, part of the last paragraph of the introduction might be more useful if it was dislocated to the second paragraph.

One of your conclusions is not associated with your results. At lines 455-458 you conclude: “Our findings highlight the importance of investigating different diversity measures and ecological gradients in ecological studies and conservation actions, because multidimensional approaches are clearly more informative than simple metrics, such as the number of species that occur in a site.” First, I do agree with this sentence in a broad vision. However, your results (lines 337-339) indicate that all diversity measures were positively related to each other. Moreover, it was indicated at the results that the three measures of diversity used in the study were associated with HAND or clay content and three cover. These results make me conclude that, due to the strong association among diversity measures, the inclusion of just one or two of them would bring similar results you got at your study. Moreover, of the variables you selected, just three of them were considered significantly important. Therefore, your results are not related to this conclusion. Actually, I suggest you to make a discussion paragraph about it, taking it off the conclusion.

Table 1. Change precipitation and temperature to the variables you properly used (temperature of the coldest month and precipitation of the wettest month).
Experimental design: The issues emerged about the material and methods in the first draft were properly tackled by the authors. I do not have any criticism applied to this new version.
Validity of the findings: In general, I do not have anything important to comment about the results of the study. Just that one of your conclusions is not associated with the results of the study. I made a comment about it in the basic reporting section.
Additional comments: I analyzed the response letter from Fraga et al. and I also read the new version of the manuscript. In general, the authors attempted to solve the problems presented in the first version of the manuscript, which made them rethink the introduction and analyses. They made a good job reanalyzing data and incorporating the suggestions from the first draft. Nevertheless, I still have some small suggestions to the authors, prioritizing cohesion and fast reading. These suggestion can be easily solved by the authors, reason why I recommend minor revisions to 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: DIFFERENT ENVIRONMENTAL GRADIENTS AFFECT DIFFERENT MEASURES OF SNAKE Β-DIVERSITY IN THE AMAZON RAINFORESTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revisions adequately addressed my concerns.
Experimental design: The revisions adequately addressed my concerns.
Validity of the findings: The revisions adequately addressed my concerns.
Additional comments: A few minor comments below.

- Line 359-361 may give the impression that diversity measures strongly correlated are affected by the same environmental gradients, which is not the case (e.g., TBD and PBD). Consider replace “weakly correlated” by “different”.

- The acronyms used in the Table 1 are different from those used in the text (BTD x TBD). Standardize it.

- The legend of the Fig. 3 must be revised. For example, the plot B, which show the relationship between PBD and TBD, is labeled as “FBD = Functional B-diversity”.

- Just an observation. I understand that you used the residuals of the environmental variables to remove the effects of geographical distance. In this case you are analyzing only the pure environmental factor on community beta-diversity, and it does not seem wrong since it is explicit in the text. However, keep in mind that environmental variables are spatially structured and by using this technique you are reducing their power of explanation. What I mean is that environmental predictors will have spatial autocorrelation, but that is not a problem. The problem only occurs if there is spatial autocorrelation in the residuals of your model. I believe that a more appropriate solution would be run the model with your environmental predictors and check the residuals. If there is no spatial autocorrelation in the residuals (which is your case, I checked) you don’t have to worry about geographical effects on your 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: A SECRETORY HEXOKINASE PLAYS AN ACTIVE ROLE IN THE PROLIFERATION OF NOSEMA BOMBYCIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comments
Additional comments: Huang and co-workers studied the location of hexokinase and its impacts on the spore proliferation. This is an interesting topic and the result could be used to control N. Bombycis infection. The experiment is well designed and the results are robust. I have only few comments:
1. Line 37: microsporidia can also infect healthy human.  the sentence sounds odd.
2. Line 55: need reference
3. Line 67: consider replace “significant function” with “significant impact”
4. Line 90: use the full name
5. Line 122: has the off-target effect been controlled?
6. Line 143-145: it is confusing. infected samples mean cells or individual silkworm? the infection silkworms were immediately stored or later?
7. Line 204: has the off-target effect been controlled?
8. Line 209: revise “the infection of level the two groups”
9: Line 236: is there any reference?


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

Input:
Title: A SECRETORY HEXOKINASE PLAYS AN ACTIVE ROLE IN THE PROLIFERATION OF NOSEMA BOMBYCIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The text is clear, unambiguous and technically correct. However, I am not a native speaker and can not professionally evaluate it. The MS include sufficient references to demonstrate its actuality. The structure of the paper is correct and consists of standart sections. It is self-contained and all results are relevant to the research topic.
Experimental design: Submitted article represent original primary research within Aims and Scope of the journal. It clearly defines the research question and knowledge gap which the study has filled. The investigation was carried out with the use of technically advanced methods and with observance of ethical standards. The most part of methods are described with sufficient information to reproduce them. Some remarks are listed below.
Validity of the findings: The data are robust and statistically reliable. They are base for conclusion which is limited to supporting results, well stated and connected to the original research question.
Additional comments: Since 2012 (Cuomo et al. 2012) hexokinase has become one of the most interesting enzymes of microsporidia  because of (1) its secretion into an infected cell and (2) accumulation in host nuclei.
This paper  focuses on the hexokinase of microsporidia Nosema bombycis - a dangerous pathogen of silkworms, that emphasizes its practical significance. In this study, the authors performed heterologous expression of hexokinase in E. coli, produced  immune serum against  recombinant protein and for the first time showed that parasite enzyme is secreted into  cytoplasm of  infected cell and accumulated in host nuclei  during microsporidia maturation. In addition, the authors showed  NbHK expression at the early stage of infection and relatively  stable transcription  level during all Nb proliferation stages.
Very interesting result of this research is the suppression of Nb development by RNAi.  It not only emphasizes the functional significance of hexokinase for the parasite, but also opens up new approaches for creating microsporidia-resistant insects.
At the same time I would like to make some remarks.  I think,  after consideration of the listed proposals, the article can be accepted for publication.

Lines 46, 47, 65 (Wiredu Boakye et al., 2017) or (Wiredu et al., 2017) is correct ref. ?

Line 56 Plasmodium instead of plasmodium

Line 83 “the recombinant bacterial was induced” or “the recombinant bacteria was induced”?

Line 86 Please note which (complete  or incomplete) Freund's adjuvant  was used?

Line 115 KpnI site is GGTACC instead of GGGTACC

Line 113 – 119 In contrast to the plasmid for bacterial expression (lines  72-80) the construction of vector  for HK-G3S-RFP expression in insect cell is described not fully enough.
All information that can be obtained:
1. Forward primers as well as pIZ/V5-His MCS contain KpnI site.
2. 5’-end  of Reverse   primer tgaccctgagcctcc (ggaggctcagggtca) encodes    GGSGS  linker instead of G3S.
Please describe the strategy of chimeric gene construction . It would also be interesting to know the reagent and the effectiveness of lipofection, and whether zeocin was used to select the transformants.

Lines 170 -174 Since “The identified pET-32a-NbHK vector was sequenced by Sangon (Shanghai, China)” and “The sequencing results showed a 1,287-bp fragment encoding 428 amino acids, which was consistent with the  data from the genomic sequence of N. bombycis in SilkPathDB  (Fig 1 C)”, images of agarose gels may be omitted from the results -  It rather refers to materials and methods.

Lines 173-179 in the text: “ which was consistent with the data from the genomic sequence of N. bombycis in SilkPathDB (https://silkpathdb.swu.edu.cn/) 174 (Fig 1 C).  SDS-PAGE analysis showed that rHK was expressed at a molecular mass of ~60 kDa, which was consistent with the predicted size (Fig 1 D). The purified protein was used to prepare the antibody. The titer of the HK antiserum was determined by ELISA (Fig S1). Western blot 178 indicated that the HK antiserum can recognize a 50 kDa protein specifically in the infected cells 179 proteins (Fig 1 E).
Fig. 1 legend: “(C) Purification of recombinant HK. Recombinant HK was eluted by elution buffer containing different concentration of imidazole and analyzed by SDS-PAGE. (D) Specificity of the HK antiserum. Proteins extracted from infected Sf9-III were subjected to western blot using polyclonal antibody against HK. M: Protein maker (Transgene, China).”
(E ) - is absent!

Line 186 I do not like the phrase “in meront stage” It looks like localization in microsporidia meronts.

Line 202 “The transcript profile of NbHK implied it functioned during all proliferation stage” or “stages”?

Lines 215-216 “The location (cytoplasmic?) of NbHK during the early stage of N. bombycis infection implied it may play a role in regulating and controlling the host’s energy metabolism” .
Only localization of parasite  enzyme in host cytoplasm  is not  sufficient reason to assume its participation in regulation of host metabolism.  For example, protein may be accumulated in host cytoplasm simply because it is secreted into this compartment by microsporidia meronts and sporonts (in contrast to late stages and spores). At the same time,  secretion of  key enzyme of  glycolysis (regardless of in which compartment it will accumulate)  implies itself  such important role.

Line 217-218 “while nuclear localization signal during the late stages of infection suggested it functions in apoptosis (Bryson et al. 2002; Gottlob et al. 2001).
In both of the cited articles there is no mention of the nuclear localization of hexokinase. I think,  so far there is no link between the nuclear localization of hexokinase and apoptosis regulation.

 Lines 223-224.
 “The cytoplasmic localization of NbHK revealed that NbHK may regulate energy metabolism and the apoptosis of host mitochondria.”
Why  localization of parasitic enzyme in  host cytoplasm indicates the possibility of its participation in regulation of apoptosis of host  mitochondria ?

Lines 230-231.  Why Goldberg et al., 2008? First paper about  microsporidial mitosomes -  Williams BAP, Hirt RP, Lucocq JM & Embley TM (2002) A mitochondrial remnant in the microsporidian Trachipleistophora hominis. Nature 418: 865–869.

Line 232.  “The unique NTTs of microsporidia allow for a simplified energy metabolism pathway (Dean et al. 2018).”
I would add here Tsaousis, A. D., et al. 2008. A novel route for ATP acquisition by the remnant mitochondria of Encephalitozoon cuniculi. Nature 453:553–556.


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

Input:
Title: A SECRETORY HEXOKINASE PLAYS AN ACTIVE ROLE IN THE PROLIFERATION OF NOSEMA BOMBYCIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: Microsporidia are a fascinating phylum of obligate intracellular pathogens that infect many animal species. They cause both death and disease in humans and agriculturally important animals and there is both fundamental and practical reasons for understanding how they can interact with their hosts. Microsporidia likely use many host-exposed proteins to interact with and reprogram hosts to a beneficial state for the pathogen. Here, the authors study a hexokinase from Nosema bombycis, one of the few proteins that microsporidia secrete with known molecular function.  Using antibodies and heterologously expressed protein, the authors show the protein is localized to the nucleus and the cytoplasm in host cells. Impressively, the authors also provide a convincing example of using RNAi to knockdown hexokinase and demonstrate that it reduces the amount of the microsporidia. If this approach to RNAi is generalizable to other genes, this would provide a powerful approach to study N. bomybisis proteins and to potentially control the disease it causes in silkworms. There are several points to address before publication:

Major points
1.	There needs to be data to demonstrate that the antibody used against hexokinase is specific in both western blots and microscopy. Line 177-179 mentions figure 1E, which doesn’t exist, but is stated to include western blot data where the band of the expected size is only observed in infected cells.  This should be included, as well as microscopy data showing that the antibody doesn’t bind to uninfected cells.
2.	For Figure 5 there are statistical tests performed on the RNAi treated samples, but no description of the test is provided.  This needs to be included.
3.	Lines 235-236 state that secreted hexokinases from microsporidia increase glycolysis in hosts.  All though this is likely, to the best of my knowledge this has yet to be demonstrated.  Also lines 253-254 state that hexokinase controls host energy metabolism. These statements need to be changed to indicated that although it is likely that hexokinases from microsporidia are involved in altering host metabolism, it is not yet known that this is the case and thus provides avenues for future research.

Minor points
1.	Figure 4A would look better if presented as a log scale on the X-axis, rather than broken up as it is currently.
2.	There is work on describing hexokinases from Nematocida microsporidia that would be helpful to cite. Namely Cuomo et al 2012, on describing that microsporidia have hexokinases with signal peptides, and Reinke et al 2017 demonstrating using biochemical experiments that hexokinases from two Nematocida species are observed in host cells.
3.	The Y-axis units on Figure 5B could be changed from “starting quantity” to a more informative unit.


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

Input:
Title: A SECRETORY HEXOKINASE PLAYS AN ACTIVE ROLE IN THE PROLIFERATION OF NOSEMA BOMBYCIS
Review round: 1
Reviewer: 4

Output:
Basic reporting: A reasonable study of the role of hexokinase in the microsporidia Nosema bombycis.  The authors have used a standard approach of cloning the gene and immunolocalization to provide some insights into the potential role of this protein. However, they need to provide appropriate controls for the immunolocalization studies.  In addition,  they provide some data on RNAi in Nosema bombycis and this is important, as genetic manipulation of this group of pathogens has been difficult and has not previously been confirmed.  To that end, it is critical that they carefully document the RNAi experiment to prove that they are inhibiting the gene of interest.  The data are suggestive that the RNAi has worked but additional controls are needed.

The paper could used redaction to improve English syntax.

Overall, this is a reasonable paper, but it needs control data in the figures to accept the conclusions that were presented in the discussion.
Experimental design: The antibody was raised to Nb Hexokinase cloned using standard techniques.  The data confirm expression of NbHx and the production of an antibody.  What is missing is data on the cross reaction of this antiserum with uninfected host cells.  Hexokinase is a relatively conserved gene and the antibody was raised to the Hx domain and thus is likely to cross react with other Hx.   The figure should contain a lane for uninfected host cells as well as lane of purified spores of Nb.   This would help confirm the specificity of the antiserum.  In addition, the images on IFA need uninfected controls as well.  It is important to demonstrate that the anti-NbHx serum does not react with the cytoplasm or nucleus of uninfected cells.

The RNAi experiment is interesting but more details are needed as the authors Line 245 to 247 indicate a system has been developed for Nb for this, but it is not referenced and a search of PubMed did not identify an article on this topic from this group.   The experiment Fig 5A shows that all of the data is controlled for RNAi using EGFP (set to 1) and thus there is no error bar (if so then the graph of EGFP is meaningless as it is fixed at 1;  The issue being does the control level vary over time cannot be obtained from the data as presented).  It would be useful to show a control where EGFP is suppressed as well as a readout to demonstrate that the RNAi indeed works.   The growth assay in FIg 5B needs to include growth in the absence of RNAi as a separate control (e.g. does RNAi nonspecifically suppress growth).  Finally, if growth is reduced and the Hx level is lower is this due to the fewer numbers of organisms or to the RNAi.  Data should be expressed as the RNA content for the gene per organism to demonstrate that the RNAi suppressed the transcript.  Fig 5A has a problem in that if the EGFP increases in number then the relative transcript of Hx will be lower.  Protein data would be useful to show the RNAi decreased the expression of Hx in the cells (or the amount of Hx found in host cells).   Controls to demonstrate that the RNAi constructs are not having their effects to changes in the host cell (leading to less growth of the microsporidia) are also needed. (e.g. does the dsRNA suppress host cell Hx; what is the data that the microsporidian Hx is being suppressed).  Overall, a very interesting and potentially exciting experiment, but more data is needed to make this compelling.
Validity of the findings: See section 2.  The data needs more controls to clearly demonstrate the findings

Another issue if the comment that localization of Hx to the nucleus is due to apoptosis.  There is not data presented to support this.  There are many ways to confirm (by staining) that a cell is undergoing apoptosis and if the authors want to make this argument for the localization of Hx then they need to do cell staining to demonstrate that these cells have apoptotic markers.
Additional comments: Overall, there are some very interesting experiments presented and with appropriate controls this would be an exciting paper.  The RNAi observation is extremely important due the absence of genetic techniques for these organisms and this needs to be controlled and presented so that it is evident that the RNAi technique is working on the microsporidia.


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

Input:
Title: A SECRETORY HEXOKINASE PLAYS AN ACTIVE ROLE IN THE PROLIFERATION OF NOSEMA BOMBYCIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The text is clear, unambiguous and technically correct. However, I am not a native speaker and can not professionally evaluate it. The MS include sufficient references to demonstrate its actuality. The structure of the paper is correct and consists of standard sections. It is self-contained and all results are relevant to the research topic.
Experimental design: Submitted article represent original primary research within Aims and Scope of the journal. It clearly defines the research question and knowledge gap which the study has filled. The investigation was carried out with the use of technically advanced methods and with observance of ethical standards. The most part of methods are described with sufficient information to reproduce them. Some remarks are listed below.
Validity of the findings: The data are robust and statistically reliable. They are base for conclusion which is limited to supporting results, well stated and connected to the original research question.
Additional comments: In my opinion, the authors have considered and corrected all remarks and suggestions. Thus,  the article can be accepted for publication. Please only check some minor mistakes like
L 105-106. “…cells were prepared by the glass-bead breaking method as reported” instead of  “…cells were prepared by the glass-bead breaking method as report”
L 138-139. “X-tremeGENE™ HP DNA Transfection Reagent” instead of “X-tremeGENE HP DNA Transfecti Reagent”.
L 256. “(Ferguson & Lucocq 2018)..”


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

Input:
Title: A SECRETORY HEXOKINASE PLAYS AN ACTIVE ROLE IN THE PROLIFERATION OF NOSEMA BOMBYCIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: The authors have adequately addressed all of my concerns and I now enthusiastically support the publication of the article.


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: DOES TWITTER LANGUAGE RELIABLY PREDICT HEART DISEASE? A COMMENTARY ON EICHSTAEDT ET AL. (2015A)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well written and enjoyable to read. For the most part, the arguments in the paper are supported by theory and sufficient references. Some comments:

1. I think the paper overstates how much Americans move away during their lives, and doesn't provide sufficient citations when discussing this (lines 123, 310). For example, this article suggests that it is fairly rare to move far from home: https://www.nytimes.com/interactive/2015/12/24/upshot/24up-family.html (sorry I don't have an academic reference, but the article links to the original data source).

2. The paper insinuates (lines 248+) that Psychological Science violated their standards by giving the award to a project that did not share their code, but this is not plainly true given only these quotes. It can be argued that a paper can provide enough detail to be reproducible without sharing their code, so it would strengthen this argument to state that the Eichstaedt article was unclear without code, and/or to cite a perspective that code is necessary (for example, Gundersen and Kjensmo (AAAI 2018) define code sharing as a necessary component of reproducibility in computational research).

3. Line 177: it's not obvious why misspellings would invalidate the use of these words as a measurement.
Experimental design: The methods are well justified, following Eichstaedt et al. as closely as possible.

One comment:

1. I was fascinated by the discussion of the variability of how AHD is measured. It's a strong point to make. It made me wonder (I don't have the domain expertise to answer myself), are there other ICD codes besides I25.1 that would capture similar conditions? This study used a range of codes for suicide, so I wonder if a range of codes could similarly be used for cardio causes of death.
Validity of the findings: There is at least at least one place where the article states that the problems of Eichstaedt et al. apply to all social media research. (544: "It appears that we are some way from being able to generate reliable estimates of health outcomes based on tweets.") Of course, this article does not go far enough to make this claim; a lot of the issues are specific to this study.

For example, detecting flu at the national level from tweets is a very different problem, which relies on a straightforward observable construct, and does not have the issues of counties/communities. This article does cite the failure of Google Flu Trends, but those problems were due to poor practices on the part of Google, and do not necessarily apply to newer systems that have been more carefully validated (see e.g. Santillana, PLOS CompBio 2015, e1004513).

If the authors do want to make generalizations to other social media research, a more helpful way to do it would be to add discussion about what lessons can be learned from this analysis to be applied to social media research more broadly. While I can infer interesting takeaways throughout the analysis, it would strengthen the paper to make them more explicit.
Additional comments: Overall, I think this paper makes a strong argument that the findings of Eichstaedt et al. have been misinterpreted. There are interesting lessons to be learned here for using social media to study health outcomes. I think the paper could do a better job of making these lessons explicit, and I have a few suggestions for improvement in my specific 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: DOES TWITTER LANGUAGE RELIABLY PREDICT HEART DISEASE? A COMMENTARY ON EICHSTAEDT ET AL. (2015A)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The two authors put the claims in Eichstaedt et al. 2015's work to test. In particular, they test whether there is a relationship between the use of language in tweets and US county-level mortality rates from atherosclerotic heart disease. I commend the authors for their extensive work in verifying the claims made in Eichstaedt et al.'s article. The manuscript's topic is timely, its methodology is robust, and the results are compelling. Also, the manuscript is clearly written,  yet a few sentences should be rephrased to avoid reporting personal opinions, jokes and ironic statements.
Experimental design: The design is robust. One small thing: the choice of "mortality rates" as outcome variable should be further motivated.
Validity of the findings: The findings are valid.
Additional comments: Please check the following statements and modify them accordingly:

- 127, pls add citations supporting the following sentence, "there is another caused of death for which the existence of an association with the victim's immediate psychological state is widely accepted, namely suicide."

- 149, please clarify what you mean by randomly and why the random nature of the selection is important here, "randomly-selected tweets"

- 231, replace "apparently chose not to include " with "did not include"

- 363, i would remove "before any program .. to be undertaken." no need for it unless Eichstaedt et al.'s work states that as the main aim of their study

- 472-73, remove "as they might have preferred". you don't know that.

- 559, remove "unless the transmission of this characteristic ... telepathic level." it s unnecessary

- 608, rephrase "slight wrinkle", irony does not contribute to this manuscript's quality (on the contrary!)

- 636-7, remove "variations in chakra energy or UFO activity"