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: MESQUITE BUGS, OTHER INSECTS, AND A BAT IN THE DIET OF PALLID BATS IN SOUTHEASTERN ARIZONA
Review round: 1
Reviewer: 1

Output:
Basic reporting: It is not clear the importance of this study.

The authors should show the number of collected insect parts in Table 1.

It may be better to describe the habitats of the identified prey in Table 1 (mesquite bosque or outside it).

“Toxic pheromone” or “noxious pheromone” is funny. Pheromones are compounds that are involved in intraspecific interactions. Pheromones are signals to conspecifics, and basically not toxic or noxious to conspecifics. On the other hand, defensive compounds are toxic, deterrent or repellent to predators. “Toxic pheromone” is funny because the term for intraspecific interactions is mixed with one for interspecific interactions.
Experimental design: I think the data in this study are preliminary.

The authors said that the mesquite bugs were immobile below 21 °C. How did they measure the mobility of this bugs?
Validity of the findings: Most of the discussion are not based on the results in this study. Discussion is too long.
Additional comments: This manuscript written by Czaplewski describe the diet of pallid bats by collecting inedible parts of prey. The authors found that the bats predominantly eat adult mesquite bugs in the experimental period. Because the mesquite bugs appear in a limited season, it is difficult to understand what the bats eat regularly. It is not clear what is the preferable prey of the bats in an area where the mesquite bugs are not distributed. Because the lack of these points, it is not easy to understand the importance of this study.


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: MESQUITE BUGS, OTHER INSECTS, AND A BAT IN THE DIET OF PALLID BATS IN SOUTHEASTERN ARIZONA
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: "Mesquite bugs and other insects in the diet of pallid bats in southeastern Arizona"

This is a natural history note describing three samples of the diet of pallid bats from insect parts at night roosts.

The most interesting part to me was the first evidence that a wild pallid bat ate another bat. This finding should probably be in the title.

What is the evidence that a pallid bat ate the Choeronycteris and that this bat was not eaten by something else?

I have no major problems with this paper. It’s nice to see people writing natural history papers like this one.

The writing is very thorough, but it could be greatly improved by deleting unnecessary words. It could easily be cut in half. But this is a minor issue. To give one example, consider this randomly chosen sentence:

“Hind leg parts (hind femurs and hind tibias) allowed determination of the sex of the individual mesquite bugs eaten. Males have inflated hind femurs with projecting spines while those of females are not inflated and lack spines, and males have hind tibias ridged with a central bend and spur while females have a straight hind tibia without a spur (Schuh & Slater, 1995)”

This sentence could be:
“We inferred sex using sexually dimorphic hindlimbs.”

Many of the adverbs and adjectives could be deleted. Another way to omit words is to reduce some of the speculation. For example, line 540, “The uncommon occurrence of C. gloriosa remains (one elytron) beneath the bat roosts indicates that the pallid bats move away from the mesquite bosque at times to hunt along the riparian vegetation of the creek and on adjacent uplands among the occasional junipers.” Can we really infer all that from a single beetle wing?

Much of the information is also stated several times.

The authors should avoid providing links to websites. These links will change with time, e.g.  Lines 111, 531, 447, 454.

Other minor comments:
Lines 17 and 19  need revision. There are words missing.

Line 32 should maybe say “possible predation” or say why it’s certain to be the pallid bat who ate the other bat.

The level of precision on numbers lines 316, 368 is too high for such a small sample.

Line 558. What is the sentence saying? In a refuging species like the pallid bat, efficient and rapid dispersal and the ability to exploit patchy food resources is probably essential. Maybe delete.


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: COMPREHENSIVE TISSUE-SPECIFIC TRANSCRIPTOME PROFILING OF PINEAPPLE (ANANAS COMOSUS) AND BUILDING AN EFP-BROWSER FOR FURTHER STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: See below #4
Experimental design: Major problem is lack of biological replications for each stage and tissue.
Validity of the findings: replication is an issue though if addition pineapple transcriptome  tissue data can be added to the eFB then this manuscript has merit
Additional comments: The difficulty in the results are the absence of biological replication making analysis a challenge.

ABSTRACT:
Pineapple is a tropical plant and can only be grown in the warm subtropics
Line #26 - briefly the stages of fruit growth when fruit were sampled: early flowering, late flowering and mature green.
Figure 1A claims that Stage #3 is when the fruit is starting to ripen however the picture show no yellowing between the eyes at the base of the fruit. The manuscript gives no details as to the variety of this fruit though it is not a Smooth Cayenne Clone possibly “Queen”.

INTRODUCTION
Line #39 - Pineapples do not grown on trees - the plant is a herbaceous perennial. Replace tree with herb.

Line #42 - The whole fruit is not consumed, only the fleshy coalesced floral parts as shown in Figure  5.

MATERIALS and METHODS
The word “fruits” implies different types of fruit (lizhi, apples, oranges) when refering to one type of fruit such as pineapple “fruit” is both singular and plural. Please correct usage in the text.
Line #81 give the name of the variety used in this study.
Line 92-92 not sure of the relevance of this last sentence.
Line 101 - “Tau method” is this the Kendall-tau rank correlation coefficients - clarify or is it Yanai et al, 2005 approach?
Line #109 - First use of TPM (Transcripts per Kilo base Million) as normalization method.

RESULTS
Line 135 - “fruits” should be individual “florets”
Line 167 - TAU methods this ranges from 0 to 1 if I recall broad to specific expression may want to explain to reader.
Line 214 - Pattison et al., 2015 did there research on tomatoes not pineapple - correct.
Line 248- Strawberry is not an aggregate fruit, more correctly it is a aggregate accessory fruit as we eat the swollen flower receptacle,


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: COMPREHENSIVE TISSUE-SPECIFIC TRANSCRIPTOME PROFILING OF PINEAPPLE (ANANAS COMOSUS) AND BUILDING AN EFP-BROWSER FOR FURTHER STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper titled “Comprehensive tissue-specific transcriptome profiling of pineapple (Ananas comosus) and building an eFP-browser for further study ” reports a transcriptomic resource that has the potential to be beneficial to the subtropical fruit research community. The authors generated RNA-sequencing data at three stages and 14 different tissues of early fruit (and development (and flower parts) in order to identify stage and tissue-specific genes.  The authors use the Tau method to identify tissue specific genes. Additionally, WGCNA was used to establish clusters of co-expressed genes. This work is important for researchers interested in fruit development and the inclusion of the eFP makes these data much more attainable. However, the transcriptomic work is limited by single biological sampling at each stage-tissue.

(1)	Issues with biological replicates
The authors spend a large part of their paper discussing the different stages of development sampled. It is clear from Figure 1 that stage 1, stage 2 and stage 3 are drastically different from each other in terms of development. Unfortunately, no differential expression was conducted across these three stages possibly due to only one biological replicate at each tissue and time point.

Instead the authors treat these three stages of the same tissue as three replicates in order to identify tissue-specific expression. This treatment may result in unsuitable representation for genes only expressed at early or late stages of fruit development. For example, gene Aco001279 in the browser has a standard deviation of 62.79 in the floral disk; this high standard deviation indicates the data points are spread out over a very wide range of values. Collapsing such diverse datasets makes these data difficult to interpret and valuable stage-specific information is lost.

(2)	The authors concluded that there were 1,051 tissue specific genes but did no perform other analysis on categorizing these genes or their potential role in fruit development. Was GO enrichment done on these tissue specific genes? Do the function of these genes corroborate with their tissue specificity? The authors also concluded that they classified 18 gene modules. They did GO annotation on these genes but they didn't seem to have looked any further.

(3)	The legends in many of the figures are unclear. Specifically, I found the legend for 2A difficult to understand and a redundant representation of Figure 2B.  The distance metric used for Figure 2C should be included. In Figure 3, there is no mention of what the values in the heatmap actually represent (tpm, z-score, rpkm?). There is also no labeling on the scales on within the supplemental figures. The color scheme for the supplemental figure heatmaps is red and green which would be unacceptable for readers with red-green color blindness.

(4)	Some small notes:
On line 70, it should be “eleven” not “eight” pineapple fruit samples
On line 65 you only cite the strawberry eFP but also mention the tomato and grape eFP
On line 98 Trimmomatics should be Trimmomatic
On line 111 defaults should be default

In conclusion, a lack of stage-specific differential expression limits the utility and value of the work, including eFP. The lack of replicates makes this data statistically unsound to accomplish these necessary components. Additionally, a more in-depth analysis is needed for the Tau and WGCNA segments.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: COMPREHENSIVE TISSUE-SPECIFIC TRANSCRIPTOME PROFILING OF PINEAPPLE (ANANAS COMOSUS) AND BUILDING AN EFP-BROWSER FOR FURTHER STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have improved the presentation and removed the ambiguities in the first version. The major concern is still the absence of biological replication, though quantitative PCR does give support to their conclusions.

The pineapple eFP browser is an excellent development from the non-replicated data presented and provides a structure to add results from future transcriptome profiling.  The author may want to consider a statement how additional data can be added to build upon this effort..
Experimental design: Biological replication is still the major problem
Validity of the findings: Biological replication is a problem
Additional comments: None


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: COMPREHENSIVE TISSUE-SPECIFIC TRANSCRIPTOME PROFILING OF PINEAPPLE (ANANAS COMOSUS) AND BUILDING AN EFP-BROWSER FOR FURTHER STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed most of previous criticisms except the issue with replicates.  A lack of funding or limited funding is not the reason for sacrificing the quality of the research. Given that eFP will be a useful resource for the community, I am OK to let this pass as long as the authors provide warnings (in their website and paper) to the eFP users concerning the lack of replicates and suggesting the users to validate the data using qRT-PCR.

There is a recent publication in Horticulture Research on Pineapple Genomics Database (PGD) by Xu et al., 2018. The authors of this manuscript should cite the PGD paper and discuss what sets this manuscript apart from PGD.

There are still issues that should be address:
(1)	Is pineapple an accessary fruit or botanical fruit or both? The statement “All fleshy coalesced floral parts of the pineapple inflorescence” does not make any sense to me. Does the fleshy fruit consist of an inflorescence plus all flowers? Which part of the flower? Ovary wall (the botanical fruit) or the entire flower?
(2)	Please label each dissected floral part in Figure 1B.
(3)	Please also label Figure 1A, indicating which is bract, which is sepal, and which is leaf.
(4)	Is stage 1 pre-fertilization or post-fertilization? If pre-fertilization, fruit should not expand at stage 1 unless pineapple is different. Please make this clear in text and in figure legend.
(5)	There are grammar mistakes in many areas, especially in figure legend and in the newly added Discussion. For instance, in Figure 2B legend: “Darker the color denotes, higher similarity the global gene expressing patterns between two samples”. You mean “The darker the color, the higher the similarity between two samples”?
(6)	In many places (legends and Discussion), you use “genes preferentially expressing….”, “genes specifically expressing…”, It should be “genes preferentially expressed….”.
(7)	In Figure 3 legend, “constitutively-expressing genes…” should be “constitutively expressed genes…”.
(8)	In Figure 3 legend, “log-transformed”, you mean log10 or log2 transformed?
(9)	Figure 3 legend, “normalized-scaled by row..”, you mean “normalized by row”?
(10)	In Figure 4 legend, “interesting expressing pattern” should be “interesting expression pattern”.
(11)	In Figure 5 (eFP), the scale label is too small to see.
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: SAPROXYLIC DIPTERA ASSEMBLAGES IN A TEMPERATE DECIDUOUS FOREST: IMPLICATIONS FOR COMMUNITY ASSEMBLY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is mostly fine, but needs a few adjustments to the text:
L53: It's unecessary to list all trophic modes occurring in dead wood, I suggest to delete this parenthesis.
L55: Speight 1989 and Grove 2002 are sufficient references for the definition of saproxylics. The rest can be deleted, no reason to overdo it for such a basic statement.
L101: change from "American beech bark has smoother bark" to "American beech has smoother bark".
L258-263: The meaning of this section seems unclear to me. First of all, there are several Coleoptera associated with specific tree species, as there are references for in the Stokland and Siitonen (2012) chapter cited here. An example is Jonsell et al 1998 in Biodiversity and Conservation. Furthermore, even though the cited papers (Irmler, Rotheray, Persson) might present results in contrast to those of this manuscript, it is unclear why the following sentence stating that few Diptera feed directly on wood would explain that tree species might have a greater influence on Diptera, as opposed to on the many Coleoptera that do feed on wood. One would rather expect the opposite.
L273: The authors state that there were four years between young and old logs, but in the methods section they say decay class of logs was determined by visual inspection. How could the authors know the exact age of the logs?
L293-L295: This sentence is poorly connected with the rest of the section. Also, starting with "even though" one expects the two parts of the sentence to contradict or at least contrast each other, but they rather support each other, so "even though" should be deleted.
L296-L298: This sentence is also poorly connected with the rest of the section. It is also a sentence describing the findings of this paper, which should be clearly stated. Furthermore, I don't understand what is meant by spatial segregation in this context, nor what results are supporting this claim. And I do not see what results support the claim that deterministic community assembly for Dipterans in late decay stages is any less driven by tree species than in earlier decay stages.
L308: Again, what is meant by spatial segregation among species in old logs, and what data supports this statement?
L320-328: This entire section does not belong in the conclusion. It could be moved to another part of the discussion or to the introduction. L311-315 could rather be moved to the conclusion.
I couldn't find the full reference for Persson et al. 2013 in the reference list.
Experimental design: No comment
Validity of the findings: Conclusion is clearly stated in the abstract, but not in the conclusion. See comment under basic reporting.
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: SAPROXYLIC DIPTERA ASSEMBLAGES IN A TEMPERATE DECIDUOUS FOREST: IMPLICATIONS FOR COMMUNITY ASSEMBLY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is overall written in professional English and all statements are clearly and unambiguously expressed. There are but a few issues that I want to point out:

Abstract – Line 1: This definition of saproxylic insects is a bit too narrow. Why not use the definition that is given in Line 53-54? The word limit of the abstract should allow this.
Line 44-48: The transition from the 1st to the 2nd paragraph is a bit sudden. There should be at least another sentence on why the authors are interested in the communities of forest dwelling invertebrates.
Line 65: “diversity patterns” would be better and shorter than “patterns of diversity”.
Line 77:  The mention of response to disturbances can be omitted since this is not what is examinined in this article
Line 87: “Some” should be replaced with another word, like “clear” or “distinct”. Since this is where the hypotheses are stated, the expectations on stochastic and deterministic processes should also be addressed.
Line 101: This “bark” is redundant – there is another in the next line.
Line 148: Replace “were” with “was”.
Line 158: Insert “a” at the beginning of the sentence.
Line 215: Insert “the” in front of “GLM”.
Line 233: Even though “dipterans” is correct, it should maybe replaced with “Diptera” which is used in all other instances in the text.
Line 269: The author Persson does not need to be named twice within two sentences. Just write “he”.
Line 270: Maybe replace “other” with similar since this study is not focused on Coleoptera or midges
Line 317-328: I would probably change the entire conclusion section. In the current state it almost exclusively discusses stochastic and deterministic processes. This part should rather be put in the section of the discussion where this point is first brought up. The conclusion could instead contain something like the consideration on conservation measures that were put at the beginning of the discussion.

An appropriate number of literature references is used to give necessary background and to discuss the results in light of previous findings and they are all implemented correctly into the text. There is, however, the case of the authors “Persson (2013)” and “Diamond (1975)” who are cited in the text but do not appear in the reference list.
It also seems as if the by PeerJ required citation format has been switched between the text and the reference list: In the list all names should be stated without “&”, while in the text “et al.” should only be applied if there four or more authors – if there are less all are to named with an “&” before the last. This seems not to be case in all citations in the text.

The article is professionally structured in accordance with the requirements of PeerJ. The figures and tables effectively display the results of the study and are correctly labeled and cited in the text. However, Figure 1 would likely look better if the y-axis would at least extend the same level as the highest bars.
I would also maybe do something different with Table 3. An entire table that only shows why the best fitting model was used is not particularly interesting. Instead, the table should be used to describe the model itself – thereby showcasing how the predictors influence the response variable.
Furthermore, it seems to me the supplementary map of the research area has a rather low resolution.
The raw data has been made available clearly structured in an appropriate format.

All in all, the article is an appropriate unit of publication and contains only the results that are relevant to the hypotheses that were posed in its introduction.
Experimental design: The article describes original research in the fields of ecology and zoology and is thereby fully within the aims and scope of PeerJ. Within these fields it poses a relevant research question: Studies on saproxylic invertebrates focus indeed heavily on beetles and therefore this study on Diptera that focuses not on taxonomy, but instead on the mechanism which from their communities, fills a gap in current knowledge. Furthermore, the attempt to discern between stochastic and deterministic processes in communities is not made often and constitutes an interesting approach in this field.

The field experiments have been conducted rigorously and adhere to the technical and ethical standards in this field. A higher number of replicates may have led to more robust results but the sampling effort can still be deemed sufficient for the posed research questions.
All methods have been described in adequate detail to make the experiment replicable at any time.
Validity of the findings: The data is in my opinion robust enough for the statistical analyses that were performed in this study. These analyses are also an appropriate way to test the acquired data with regard to the posed research questions.
In terms of the results it is a bit curious that the individual-based rarefaction curves are that steep in a temperate habitat. However, Diptera are known to be a very species rich group. It is also surprising that only the GLM of abundance lead to significant results, but this might be due to the limited number of replicates.
The conclusions drawn from these results seem sound to me and are competently discussed and compared to similar experiments citing a variety of other authors. At the same time, the limitations of this study are discussed and suggestions for future experiments as well as diversity conservation measures are made. All these conclusions and suggestion are clearly discernible as such.
Additional comments: Overall this manuscript describes an interesting approach and furthers the knowledge on saproxylic communities outside of beetles. The investigation on the importance of stochastic and deterministic processes also adds considerably to this.
With a few minor changes – as suggested above – I would very much approve of this study being published.


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: SAPROXYLIC DIPTERA ASSEMBLAGES IN A TEMPERATE DECIDUOUS FOREST: IMPLICATIONS FOR COMMUNITY ASSEMBLY
Review round: 1
Reviewer: 3

Output:
Basic reporting: See comments
Experimental design: See comments
Validity of the findings: See comments
Additional comments: I found this to be a useful paper, with only a couple of serious problems:

Major points:

l 35 - I don’t think that this opening paragraph is a particularly useful characterization of this debate, nor do I think that this issue is addressed at all in this paper.  The debate it is not deterministic vs. stochastic per se, but rather focusses on to what extent individuals of different species can be thought of as more or less identical (e.g., lack of competition or lack of differences in dispersal abilities, etc.).  Even those that firmly ascribe to the importance of competition (and niche differentiation) in structuring communities would never deny the importance of stochastic processes (think of islands for example).  This paragraph needs to be reworked. Perhaps most importantly, neutral theory is meant to be applied within a guild (trophically similar species), which is not the case here (the various dipterans show huge trophic variation).

line 317 -  Similarly, I find the whole deterministic and stochastic aspect in the conclusion to be highly speculative; it does not deserve to be front and centre in your conclusion.  You don’t actually have any evidence one way or the other.  Instead, focus the conclusion on your actual contributions to the literature (log decay and species).

l 55 - "There is evidence based…." - this sentence needs to be expanded to a full paragraph given the importance of this literature for this paper (there are many more studies to be mentioned).  Indeed, this literature should be the focus of the introduction rather than "stochastic" vs. "deterministic" processes!  The latter is interesting, but the authors have not collected data in a way that can be used to provide useful tests of the neutral theory.

This paper would be strengthened by downplaying this stochastic-deterministic aspect.  I found the discussion on deterministic vs. stochastic processes to be interesting enough to include, but this aspect should be more-or-less removed from the introduction and conclusion.  Stay with your strengths (log decay class and species).

l 105 - How were these log ages (approximately 2 and 6 years approximately) determined?  From my experience, they appear to be very inaccurate.  I was just in a maple stand logged 3 years ago and the bark was intact, and often still firmly attached to the downed wood (not 20% gone).  From my experience, the bark will not be 80% absent at six years (it will take considerably longer).  Also, what is meant by "the heartwood had been infiltrated with moisture and decay"?  How was this determined?  This raises a key point: a key objective of this paper is to investigate different decay stages, hence we need accurate and reliable determination of the decay stages (i.e., more than the vague and possibly inaccurate age information provided here).  I suggest that the author characterize the logs according to one or more of the "decay class" systems found in the literature (which tend to focus primarily on wood firmness) and provide ages based on empirical information (either their own, or from the literature).  At the moment, all we have is bark information, which does little to help use distinguish among latter decay stages (for example, decay stages 3-5 all are characterized by little bark).  You return to these ages on line 272, indicating their importance.  You need to support them empirically.  I do not believe that an estimate of 6 years for the older decay stage is even close to correct (80% bark gone).

More minor points

l 19 - this is not the standard definition of saproxylic, which is more along the lines of "species of invertebrate that are dependent, during some part of their life cycle, upon the dead or dying wood of moribund or dead trees (standing or fallen), or upon wood-inhabiting fungi, or upon the presence of other saproxylics".  (SPEIGHT, M.C.D. (1989). — Saproxylic invertebrates and their conservation. Nature and Environment Series, No. 42. Council of Europe, Strasbourg.).  You actually have the right definition in the sentence starting on line 51; use it here also.

1 27 - indicators of what?  Be explicit.

l 28 - "Both stochastic and deterministic…." Delete the sentence - it is devoid of information (what other kinds of processes are there?).

l 32 - add Diptera to the key words.

l 48 - to characterize bark or wood inhabiting species as having a "significant role in wood decomposition" again is a mischaracterization I think (termites?).  See my comment above about the definition of saproxylic as well.  Saproxylic organisms do many things and many are reliant on wood decomposition, but whether they have a "signficant role" is more up to debate (for example, do they influence the abundances of white and brown rot fungi?).

l 94 - It is standard to leave a space between numbers and SI units (i.e., use 500 n, not 500m).

l 95 - this sentence implies that old growth forests are the most common type in southern Quebec, which is not true (maple forests, on the other hand, may be common).  Clarify.

l 98 - had beech bark disease begun to affect beech trees in the study area at the time of sampling?  This may affect subsequent rot processes.

Figure legends -  I do not find these sufficiently detailed.  Some basic information about the study should be included.  The idea is that if someone found the figure and legend on the floor, they would be able to understand what it is about.  This can be resolved by adding a sentence or two.  Also, in Fig. 1, the light blue looks almost white.  Use a better colour (or a pattern).

In Fig. 1, you need to indicate what YM, OM, etc. are.  Also, add ± 1 standard error of the mean abundance (across the 5 logs per treatment).

In Fig. 3, it would be better to use standard errors of the mean, so that the statistical significance can be better judged.  You note that they are not significantly different, but this is hard to access with just standard deviations.

Please add the actual sample points to Fig. 4.  The polygons are good, but it is better with the actual points as well.

line 207 - "no tendency".  Why not statistically test this?  Did indicator values differ significantly among the trophic groups? (P value?)

line 209 - Why are the observations reported in this paragraph not tested statistically?  You have five replicates per treatment after all, and hence can do statistical tests (log-based abundance, richness, diversity, Simpsons).  The central limit theorem applies, and hence you may be able to do ANOVAs (or non-parametric if need be).  You list AIC values, but have no indication of significance.  Are any of the AICs meaningful?

line 215 - you note a "combined effect" (interaction).  But, what was the effect?  Interaction indicates that the effect of decay class differed among the species.  How so exactly?

line 232 - use "relatively distinct" (the polygons show much overlap, and it was only 22% explained).


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: MOLECULAR EVOLUTION AND EXPRESSION DIVERGENCE OF THREE KEY MET BIOSYNTHETIC GENES IN PLANTS: CGS, HMT AND MMT
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: Minor comments:
Line 177: “From algaes to angiosperms” should be “From algae to angiosperms”
Line385: The references listed here mentioned the rosette leaves convert Met to SMM, which is then transported to the reproductive tissues to support the synthesis of Met in the developing seeds, but none of them mentioned “most” of the SMMs are transported to the reproductive tissues.
Line387：The reference (Cohen et al., 2017b) could not support your statement: “the transported SMMs are reconverted into Met by the seed-specific expressed HMTs”.  AtHMTs are expressed in all plant tissues in Arabidopsis in Cohen et al., 2017.
Table S3B in File S3: what are the red numbers and blue numbers mean?
Additional comments: This study is nicely done but the conclusion can be revised or re-written.
The expression patterns data of HMTs in soybean could not support the conclusion in Fig6” blue HMT were specific expressed in seeds”. If the author want to concluded from the previous paper：” Molecular Evolution and Expression Divergence of HMT Gene Family in Plants” (Zhao et al., 2018), all the data shown in Arabidopsis, soybean, rice, tomato, and Medicago did not help interpret the results” HMT were specific expressed in seeds”.


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: MOLECULAR EVOLUTION AND EXPRESSION DIVERGENCE OF THREE KEY MET BIOSYNTHETIC GENES IN PLANTS: CGS, HMT AND MMT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is not clear and unambiguous.

Literature references appear to be appropriate.

The figures and tables are well structured.

There does not appear to be a hypothesis being tested. The paper is entirely descriptive. The summary model shown in Fig 6 does not show more than what has been known from the literature for a decade already.
Experimental design: The work appears to be original.

The research question is not well defined and/or explained.

No justification is given for choosing these specific methionine biosynthesis enzymes, or why other plant methionine biosynthesis enzymes were not included in the analysis.

Analyzing the structure of unrelated genes (e.g. CGS compared with HMT and MMT) is sure to give the result that was found- the genes evolved differently. What is the relevance of such a finding?

Methods are described in sufficient detail.
Validity of the findings: Rationale and benefit to the literature is not clearly stated.

The data is robust and in the case of gene expression analysis, statistically sound and controlled. For most of the data, robustness and statistical analysis is no relevant.

Conclusions are not well stated. Conclusions appear to be trivial.

There is not much speculation that I could find.
Additional comments: The writing, which is in reasonably good English and sentence structure, is full of statements whose meaning is ambiguous and misleading. I present a few examples that highlight the ambiguity, but the following examples are not an exhaustive list and many such instances would need to be re-written. Lines 27-29 states that “CGS, MMT, and HMT genes have been studied, yet how they cooperate with each other to synthesize methionine during evolution in plants is unknown.” It is far from clear what this sentence means. How would the presented gene structure analysis reveal “cooperation with each other”? Also, it isn’t clear what is meant by “methionine biosynthesis during evolution.” Lastly, the sentence ends with “in plants,” but no justification is given for focusing solely on plants. In fact, all three genes evolved prior to the divergence of the kingdoms of life so it isn’t clear what evolutionary answer is being sought by analyzing only the plant lineage. Also, the authors overlook describing how methionine is synthesized in animals, fungi and protists, which is quite relevant given that the enzymes are present in all of these kingdoms of life. In lines 29-31 it is stated that “In the present study, a reconstruction of the evolutionary relationship of CGS, MMT and HMT gene families showed that they inconsistently diverged.” It isn’t clear what “inconsistently diverged” means. Throughout the abstract and text the terms “conserved”, “basically conserved”, “relatively conserved” are used without being defined.
The authors haven’t explained and justified why they analyzed expression of the genes in soybean. Why would soybean provide the evolutionary insight the authors were seeking? They also don’t explain why they chose the selected species to analyze in PLEX. Line 265 “AthCGS1 and AthCGS2 were fluctuant in all of the tissues” suggests that the expression was not consistent, meaning expression pattern showed a great deal of variation. I think what the authors mean is that the expression of the genes differed in different tissues.
Lines 329-330 “Their phylogenetic relationships were distinct,” not clear what this means.
Lines 334-335 “Therefore, the evolution of CGS, HMT and MMT gene families was
335 inconsistent.” What does this mean?
Line 195 “The divergence of genes is reflected in their structures to a certain extent.” What do the authors imply by using the words “to a certain extent”?
In the exon-intron analysis, lines 194-215, the authors analyzed exon-intron number and size. Notably missing is an analysis of intron position in the coding sequence. Intron position is an important aspect of the evolution of genes.


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: MOLECULAR EVOLUTION AND EXPRESSION DIVERGENCE OF THREE KEY MET BIOSYNTHETIC GENES IN PLANTS: CGS, HMT AND MMT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors made a reasonable effort to address reviewer concerns. They didn’t convince me of the significance of their study. The data aside, a large part of the problem is in the writing, which is convoluted and equivocating. I don’t know a way around this other than to advise the authors to hire a writing service.
The abstract needs to be clear and concise because most readers will read only the abstract. So miost of my comments focus on the abstract.
To claim that “The biological functions of CGS, MMT and HMT genes have been studied, respectively, yet both their evolution processes and the evolution pattern of Met biosynthetic pathway in plants are unknown” is a gross overstatement. In fact, quite a great deal of information is known about the evolution of these genes. See doi:  10.1186/s40064-015-1163-8 and doi: 10.1042/BJ20140753, and there are more published studies.
The manuscript fails to mention, particularly so in the abstract, the S-adenosylmethionine repair cycle that plant HMT participates in. The omission is notable because the authors do mention Met biosynthesis and the SMM cycle, which are equally as important as the AdoMet repair cycle. In this regard an important reference is missing on evolution of plant HMT. Bradbury et al. 2014, doi: 10.1042/BJ20140753.
What is meant by “…the divergence of grass and seed plant levels in plants”? Grasses are seed plants.
In the abstract, “…but the CGSs of Class 2 diverged.” Up to this point in the abstract there is no mention of classes of CGS. The authors need to explain what the different classes are, especially in the abstract, because most readers will not know about the classes.
In the abstract, “Nevertheless, all of the genes were under strong negative selections during evolution.” The evidence for this statement needs to be stated. The prior sentence claims that MMTs and HMTs are conserved, but CGSs are not. So what constitutes strong negative selection?
The meaning of the following sentence of the abstract is not decipherable: “The expression patterns of the MMTs were consistent, in which the expression levels in leaves were higher than seeds, but the CGSs and HMTs had diverged in seed plants, which were higher in leaves or seeds, or fluctuated.” I’ll make an effort to rewrite this sentence to reflect the results that are presented. How about this: “The expression of MMT mRNA was consistently higher in leaves than in seeds of Arabidopsis, rice and soybean. Expression of CGS and HMT was not consistently higher in leaves of these three seed plants.” (I have difficulty understanding how the authors decided to generalize their study to “seed plants” when in fact, they analyzed three species, a very small sample of the seed plants, and also they studied on one species, the others were taken from electronic databases.)
The authors should justify how the present work differs from their own recently published paper doi:  10.3390/ijms19041248. This paper seems partly duplicative of the present Peer J manuscript.
The choice of spelling “sulfphur” is not the convention of the International Union of Pure and Applied Chemists, the governing body for chemical nomenclature. The IUPAC convention is “sulfur”, notwithstanding the policy of a few British journals.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: see basic reporting


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: COMING OUT IN A HARSH ENVIRONMENT: A NEW GENUS AND SPECIES FOR A LAND FLATWORM (PLATYHELMINTHES: TRICLADIDA) OCCURRING IN A FERRUGINOUS CAVE FROM THE BRAZILIAN SAVANNA
Review round: 1
Reviewer: 1

Output:
Basic reporting: A new genus and species of Geoplaninae are proposed on a morphological basis. Disagreement between descriptive text and figures are main concern. Literature is well explored. Article structure is correct. English is acceptable; it is not my mother language but some errors suggest a second revision by a native English speaker.  The MS is valauble for publication after revision.
Experimental design: Manuscript meets Aims and Scope of the Journal. Research question is well stated. Methods are adequately detailed.
Validity of the findings: Impact and novelty are valuable and expand knowledge of morphological and systematic diverisity of Geoplaninae. Data are robust. All figures are welcome; Fig. 5 might be improved.   Conclusion is supported by the results although it includes as speculative reflection on the habits of the species.  The manuscript is valuable for publication.
Some organs (eyes, sensory pits, male organs) of the species are not described in detail and should be rechecked. Even the 'eversible penis' is a diagnostic feature of the genus but there is no mention to it in the descriptive section. Text on male organs matchs only partially the figures.
Additional comments: In the manuscript the authors provide a description of a new species and genus of a Geoplaninae land planarian. I made some minor corrections to the text. I also requested clarifications on ambiguos or incomplete statements of the text.

RESULTS
Adequate but incomplete in some sections.
Telegraphic style is used inconsistently along the text, although this does not affect understanding.
Some sentences are unclear and need a revision.
It is not stated whether sensory pits are present at the anterior tip of the body (or whether it could not be checked  due to the condition of the histological sections).
Diagonal dorsal mesenchymal muscle layer is not reported; might it have gone unnoticed due to the sectioning plane?
The 'eversible penis' is a diagnostic feature of the genus but there is no mention to it in the descriptive section. Text on male organs matchs only partially the figures. See detailed comments and corrections along the revised text.
Fig. 5: Instead of gray, blue (or red, green etc.) color would facilitate to observe the structures represented.

DISCUSSION
Adequate.
CONCLUSIONS
Supported by the Results. Furthermore, speculative statements on the habits of the species are presented.

I recomend publication  after revision.

I attached a PDF version of the MS. If requested, I'll provide the DOCX version too.


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: COMING OUT IN A HARSH ENVIRONMENT: A NEW GENUS AND SPECIES FOR A LAND FLATWORM (PLATYHELMINTHES: TRICLADIDA) OCCURRING IN A FERRUGINOUS CAVE FROM THE BRAZILIAN SAVANNA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article is well written, understandable and with good scientific English. The structure is correct as expected in a scientific article and conforms to PeerJ standards. The figures are correct, although the quality of the photographs of the specimen described can be improved since this is the only individual that exists.
Experimental design: The experimental design is correct in terms of histological methods and morphological description. However, given the importance of describing a new species, and even more, a new genus, the need to have molecular data of this individual should have been taken into account. Nowadays the integrative taxonomy is crucial, and especially in these cases where there is little available material, it is very important to have this type of data for a possible future identification of new individuals as well as to be able to locate the new genus in a molecular phylogeny.
Validity of the findings: no comment
Additional comments: The article: "Coming out in a harsh environment: a new genus and species of land flatworm (Platyhelminthes: Tricladida) occurring in a ferruginous cave from the Brazilian savanna" is a good contribution to the increase in the known diversity of terrestrial planarians. It also describes a new genus, something that should not be underestimated. The habitat where the specimen has been found is also peculiar, so it is endowed with characteristics important enough to be considered for publication. However, I have some concerns about whether it is ready for publication or not. The difficulty in finding terrestrial planarians is well known, especially in recondite habitats, such as caves, but the danger of describing a species and a genus from a single individual is notorious. In addition, there are no molecular data that certify the phylogenetic position of the new genus, so it is difficult, without a good morphological discussion, to phylogenetically place the new individual described. Following I write my comments on the manuscript that I think should be taken into consideration.
Introduction
- Bearing in mind that not all the readers of the article have to know the terrestrial planarians, I would dedicate a part of the introduction to describe them, talking about their most important characteristics, which later could be of help to understand the characters that are described in the results and discussion sections.
-The last sentence in the introduction section is a bit rare, please, rewrite.
Material and Methods
-More information about the external appearance of the specimen would be appreciated. Colour pattern is needed for the description. If it is not available, it must be explained in the text and the reasons why.
Results
- In the remarks of the genus, only a comparison is made with the genus Xerapoa, based on a characteristic, narrow creeping sole, which could be due to the early stage of development of the individual studied, as the authors state, or to the small size of the specimen which could respond to an immature individual. A discussion comparing the new genus with others of the group would be advisable to try to locate, at least morphologically since there is no molecular data, Difroehlichia within Geoplaninae.
- The presence of a secondary male copulatory organ is something very peculiar. Is there any other case like this in Geoplaninae? If not, have the authors considered if this individual could belong to another subfamily that is not Geoplaninae? The position of the testes (typical of Geoplaninae) could be a rare character within another subfamily that presents a cylindrical body, narrow creeping sole and other characters of this individual ...
- As a suggestion, I would place the Ecology and distribution section after Etymology, and not so towards the end of the results section. It would help to understand some characteristics, that of the way in which it is ordered now are described above.
- Has the authors or collaborators sampled in the regions near the cave to try to find more individuals? It could be possible that the cave was only a refuge for this animal and therefore does not present some typical characteristics such as depigmentation or reduction of the number of eyes.
Discussion
-A comparative discussion is NEEDED, comparing the new species with other species of the group.
- Have the authors considered the relationship of the ferruginous character of the cave and the characteristics of the animal? Maybe in the dark coloration of the specimen?

In my opinion, the article is publishable but doing a previous rethinking, and rewriting some sections before, such as the discussion. Or maybe it would be good to wait until authors have another individual available to corroborate the described characteristics and do molecular analyses. Although I understand that this may take a long time given the circumstances of the abundance of the species.


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: COMING OUT IN A HARSH ENVIRONMENT: A NEW GENUS AND SPECIES FOR A LAND FLATWORM (PLATYHELMINTHES: TRICLADIDA) OCCURRING IN A FERRUGINOUS CAVE FROM THE BRAZILIAN SAVANNA
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: The source of the geographical coordinates provided for the location, such as whether they were derived from a GPS or map, is not indicated.
Validity of the findings: No comment
Additional comments: An interesting paper that is a most welcome addition to the body of knowledge resulting from extensive research on land planarians in South America.


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: COMING OUT IN A HARSH ENVIRONMENT: A NEW GENUS AND SPECIES FOR A LAND FLATWORM (PLATYHELMINTHES: TRICLADIDA) OCCURRING IN A FERRUGINOUS CAVE FROM THE BRAZILIAN SAVANNA
Review round: 2
Reviewer: 1

Output:
Basic reporting: OK
Experimental design: OK
Validity of the findings: OK
Additional comments: OK


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: COMING OUT IN A HARSH ENVIRONMENT: A NEW GENUS AND SPECIES FOR A LAND FLATWORM (PLATYHELMINTHES: TRICLADIDA) OCCURRING IN A FERRUGINOUS CAVE FROM THE BRAZILIAN SAVANNA
Review round: 2
Reviewer: 2

Output:
Basic reporting: All the conditions required in this section are satisfactorily fulfilled.
Experimental design: Given the limitation due to the lack of available material, it is accepted that only one individual has been worked with and that there are no molecular data, hoping to have this data in the future when it is possible to find more animals of the genus and species described in this article.
Validity of the findings: No comment.
Additional comments: -The authors have added a section in the introduction about terrestrial planarians, as I suggested so that it now is more understandable for a non-planarian expert what these animals are.
-“However, it could not been assigned to any known genus, and thus, we provide the description of a new genus and species to accommodate this flatworm.” Please, change “been” by “be” in this sentence.
- All my comments have been answered satisfactorily in the responses to the reviewer. I would have appreciated some change or clarification in the text including some of those justifications.
A beautiful and interesting contribution to the increase of knowledge about the terrestrial planarians in a habitat where they have been little studied, the caves.


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: COMING OUT IN A HARSH ENVIRONMENT: A NEW GENUS AND SPECIES FOR A LAND FLATWORM (PLATYHELMINTHES: TRICLADIDA) OCCURRING IN A FERRUGINOUS CAVE FROM THE BRAZILIAN SAVANNA
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In my opinion, the authors have adequately addressed the constructive points previously raised by the reviewers, by either modifying the manuscript or by clarifying their course of action in the rebuttal.


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 RETROSPECTIVE STUDY OF SURGICAL TREATMENT AND OUTCOME AMONG WOMEN WITH ADNEXAL TORSION IN EASTERN TAIWAN FROM 2010 TO 2015
Review round: 1
Reviewer: 1

Output:
Basic reporting: Dear Editor,
	Thank you for the opportunity to review the interesting manuscript entitled ‘A retrospective study of surgical treatment and outcome among women with
adnexal torsion in eastern Taiwan from 2010 to 2015’.

The authors reviewed the outcomes of 42 female patients with adnexal torsion managed in eastern Taiwan between 2010 - 2015.
The paper is interesting and has merits to be accepted after minor revision.
Experimental design: Major concerns:
 - Methods: Please use non-paratemtric tests or otherwise prove that data are normal distributed using a statistical test (Kolmogornov Smirnov for example). Please report the data as median median and range or IQR if they are not normal distributed. Chi-sure test should be Fischer s test in some cases - please detail.
Validity of the findings: Minor concerns:
In Abstract - please detail more clear what mean eastern Taiwan.
In Abstract - pleas present the data as median and range or IQR.
Table 5 should be moved to Discussions section, not Results.
Please discuss the recurrence of the torsion after surgical treatment in the literature and the fertility rate after these surgical procedures.
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: A RETROSPECTIVE STUDY OF SURGICAL TREATMENT AND OUTCOME AMONG WOMEN WITH ADNEXAL TORSION IN EASTERN TAIWAN FROM 2010 TO 2015
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study -A retrospective study of surgical treatment and outcome among women with adnexal torsion in eastern Taiwan from 2010 to 2015 is an importent issue among gynecology emergency.
Unfortunately this study cohort is small and cast represent truly the study subject.
The main idea is important and well disigned  but again too small.
Experimental design: well desigh
Validity of the findings: There is no innovation in this study
Additional comments: When you have a small cohort -try to fined a special point to designate it from other big studeis


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 RETROSPECTIVE STUDY OF SURGICAL TREATMENT AND OUTCOME AMONG WOMEN WITH ADNEXAL TORSION IN EASTERN TAIWAN FROM 2010 TO 2015
Review round: 1
Reviewer: 3

Output:
Basic reporting: Comments to PEERJ 29482 entitled A retrospective study of surgical treatment and outcome among women with adnexal torsion in eastern Taiwan from 2010 to 2015

The authors retrospectively evaluated women who underwent surgical management for ovarian torsion and finally a total of 42 women were enrolled into the current study.
Among these, 27 women received laparoscopic approach and the remaining 15 underwent exploratory laparotomy. Conservative surgery was done in one-third of patients (35.8%), suggesting that the majority of patients with ovarian torsion were ended by oophorectomy. Although the authors did not comment this part, it is rational to be explained by only 64.4% of patients who had supposed ovarian torsion preoperatively. However, it is interesting to find that the time interval between the onset of symptom and the final operation is not long, suggesting that the ability of urgent operation is relatively good in the authors’ institute. Some comments are shown below.
1.	it is relatively useful to read the difference between radical surgery and conservative surgery. If possible, the audience might be much more interested in this topic. I am wondering why the authors separate their patients into two groups based on the different operation approaches. Tumor size was significantly larger in the laparotomy group, and simple cyst was dominant in the laparoscopy group. Furthermore, fibroma and fibrothecoma were only found in the laparotomy group. All suggested that selection of operation might be biased. In addition, did it mean the surgical approach might be dependent on the “fear” of the malignancy? If the answer is yes, what was the value of the comparison between laparoscopy and laparotomy?
2.	If four cases of fibroma and/or fibrothecoma are noted, did ascites exist?
Experimental design: Comparison between radical surgery and conservative surgery might be much more valuable.
Validity of the findings: If possible, please evaluate the relationship between parameters and radical surgery and provide the risk estimation for the radical surgery.
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: A RETROSPECTIVE STUDY OF SURGICAL TREATMENT AND OUTCOME AMONG WOMEN WITH ADNEXAL TORSION IN EASTERN TAIWAN FROM 2010 TO 2015
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors made all the suggested modifications.
However, in Tables 2, 3,4 contrary to the results section, the authors did not report the data as median and range (still mean and std dev). Please change that.
I recommend the article to be published after this minor change.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In Tables 2, 3,4 contrary to the results section, the authors did not report the data as median and range (still mean and std dev). Please change that.


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 RETROSPECTIVE STUDY OF SURGICAL TREATMENT AND OUTCOME AMONG WOMEN WITH ADNEXAL TORSION IN EASTERN TAIWAN FROM 2010 TO 2015
Review round: 2
Reviewer: 2

Output:
Basic reporting: Clear and well written
Experimental design: acceptable
Validity of the findings: age is the final dependent factor for radical surgery, suggesting that complete family might result in the radical surgery by authors.
Additional comments: significant improvement and acceptable


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 RETROSPECTIVE STUDY OF SURGICAL TREATMENT AND OUTCOME AMONG WOMEN WITH ADNEXAL TORSION IN EASTERN TAIWAN FROM 2010 TO 2015
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors made all the suggested modifications. I recommend the paper to be published in the current form. Congratulations!
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: No 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: REAL-TIME LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR RAPID DETECTION OF ENTEROCYTOZOON HEPATOPENAEI
Review round: 1
Reviewer: 1

Output:
Basic reporting: 'no comment'
Experimental design: The authors in this paper were aimed to establish an effective diagnostic method for the detection and prevention of Enterocytozoon hepatopenaei (EHP), which has emerged as a serious pathogen reported to be associated with retarded growth in cultured shrimp. The real time loop-mediated isothermal amplification (real-time LAMP) for rapid detection of EHP has been developed. This method was compared with the conventional LAMP, showing less time and cost consumption. Its application greatly aids in the surveillance, prevention, and control of EHP. Thus the method for EHP presented might mean a contribution to the cultured shrimp industry. For benefit of the reader, however, before acceptance for publication, I would recommend minor changes to be taken into account in the manuscript.
Validity of the findings: 'no comment'
Additional comments: Recommendation: The paper is probably publication, but the paper should be reviewed again in revised form before it is accepted.
Additional Comments: Firstly, regarding the EHP, I think the following article should be cited in the chapter introduction:
Tourtip, S., Wongtripop, S., Stentiford, G.D., Bateman, K.S., Sriurairatana, S., Chavadej, J.,Sritunyalucksana, K., Withyachumnarnkul, B., 2009. Enterocytozoon hepatopenaei sp. nov. (microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): ﬁne structure and phylogenetic relationships. J. Invertebr. Pathol. 102, 21–29.
Secondly, the labeling in Figure 4 should be lowercase.
Thirdly, I think that there are words more appropriate other than “primers are preferred” in the result in line 165.
Fourthly, the paper also has some typos and language issue which needs to be checked and corrected in the revision.


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: REAL-TIME LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR RAPID DETECTION OF ENTEROCYTOZOON HEPATOPENAEI
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: Please provide the data about practical application to clinical samples.
Validity of the findings: no comment
Additional comments: Enterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth. In this study, the authors established and evaluated a rapid real-time Loop-Mediated Isothermal Amplification (real-time LAMP) for detection of EHP. It is important and necessary to develop a specific, rapid and sensitive EHP diagnostic method to identify this parasite. However, I think the authors should consider the following comments:

(1)	EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination. Then in the field, in order to control or eliminate EHP, how were samples collected from the shrimps without clinical signs? Does sampling cause enormous damage to shrimp?
(2)	In this study, a real-time LAMP for detection of EHP were developed. But in the parts (Materials and methods, and Results), there is not data about the practical application of the real-time LAMP to clinical samples. Please provide the relative data.
(3)	On lines 209-210, are you sure that the equipment for the LAMP is not expensive?
(4)	The real-time LAMP is helpful to identify the EHP infection. But it is difficult to control parasite in the shrimps using this method. Some drugs or vaccines might eliminate EHP. Please revised 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: REAL-TIME LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR RAPID DETECTION OF ENTEROCYTOZOON HEPATOPENAEI
Review round: 1
Reviewer: 3

Output:
Basic reporting: The Enterocytozoonhepatopenaei(EHP) becomes more prevalent pathogen since the pathogen was associatedwith retarded growth. Therefore an effective detection method for the pathogen is necessary. This MS have developed real-time loop-mediated isothermal amplification (real-time LAMP) for EHP, which is very helpful for survey and detection.
Experimental design: no comments
Validity of the findings: the findings of the MS is already validated.
Additional comments: 1. The introduction at the first of the paper is too simple for the readers, as a newly microsporidian parasite, it is the characteristics of this article. I believe that  it will be helpful if the authors can make a more detail.
2. EHP is a pathogen, not a disease, for the word “EHP diagnostic method”in line 36, so maybe should add a word “infected” to make it more clearly, “EHP-infectedshrimp diagnostic method”.
3. There are four pathogens used in the paper, so the word “pathogen” in line 40 should be “pathogens”, also the “was” should be “were”correspond.
4. The word “EHP diagnostic” in line 91 should be “EHP-infected shrimp”.
5. The first sentence in the part “The established real-time LAMP is highly sensitive’ in line 182 should be adjusted to “Sensitivity analysis was performed…”.
6.For the legend of Figure 2, there are many repeated descriptions“Amplification curve” in line269-272, the legend about different curve could short for “(A)EHP3.(B) EHP


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: EXPLORING MECHANISMS THAT AFFECT CORAL COOPERATION: SYMBIONT TRANSMISSION MODE, CELL DENSITY AND COMMUNITY COMPOSITION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Writing is excellent. Ideas are well introduced and contextualized with the literature. Figures and tables are clearly presented.
Experimental design: Study is well designed and statistical analyses are rigorous.
Validity of the findings: I have two comments which may impact the results and interpretation:
1) The constraint that Symbiodinium sequence variants were only retained if present in >3 samples may exclude too much data, especially since some species were only comprised of a single sample (e.g., Amil). Therefore, sequence variants specific to Amil may be excluded. This should be corrected or the approach justified.
2) There appears to be variation in the dominant symbiont among colonies of Gast. If these are separated in data analysis (or this variation accounted for statistically, i.e. with dominant symbiont added as an additional predictor), might some of these results change? This needs to be addressed in some way. There is also the possibility that A. millepora samples contained different dominant symbionts, but since samples were not sequenced individually this is difficult to test. The gels provide some indication that they are all C-dominated and that D is present at lower levels, but this could be addressed also in the text.
Additional comments: This study investigates whether a coral’s mode of symbiont transmission influences the degree of ‘cooperation’ (measured as proportion of fixed carbon translocated) and bleaching in response to thermal stress. While the transmission mode was not a significant predictor of these traits, the authors explore how they are related to both symbiont density and diversity. With these data, the study supports a relationship between high symbiont density and bleaching susceptibility, but does not support a shift to parasitism among symbionts as a trigger of the bleaching response. The paper is very well written, and the results are presented clearly and contextualized well with existing literature. I commend the authors for conducting rigorous statistical analyses and making them reproducible by providing all data and R code.

Abstract
-Briefly define vertical/horizontal transmission in Abstract for readers unfamiliar with the terminology

Materials and Methods
-Were all corals collected from the same reef? How close together? Was depth the same? (i.e., were these corals likely acclimated to similar environmental conditions?). What was the temperature at the time of collection? I see now some of this information is in the Supplement, but important bits could be brought into the main text. The depth indicated is less than 10m, leaving room for significant potential variation among species?
-What type of lights were used? Was light ramped up and down each day? Light levels are relatively low in the treatment tanks.
-Line 135: Change Waltz to Walz
-Line 137: Interpretation of declining effective quantum yield as onset of bleaching response?
-Line 153: Change holobiont to symbiont?
-Line 170: add “sequencing” between “for” and “at”
-Line 195: Since samples from individual corals were pooled by species, does the removal of sequence variants appearing in less than three unique samples mean that they had to appear in at least three unique species? If so, this seems inappropriate as many species-specific sequence variants may be expected. Since for some species additional individual samples were sequenced, does this cutoff favor the retention of sequence variants found in these species?

Results:
-Line 245-246: Here a non-significant effect of transmission mode on symbiont density is reported. However, the statistical analysis of transmission mode as a predictor is not described in the data analysis section. Was a separate model fitted replacing the species predictor with transmission mode?
-Figure 1: It would be helpful to include ‘HT’ or ‘VT’ after each species name in the facet titles to remind the readers which species are horizontal or vertical transmitters... Picky point: the abbreviation for M. aequituberculata should probably be ‘Maeq’ instead of ‘Maqe’, and likewise G. acrhelia should probably be ‘Gacr’ instead of ‘Gach’.
-Line 304: Change Fig. 4A to Fig. 4? Doesn’t appear to have multiple panels...
-Line 307: Add references to Fig. 5A, Fig. 5B, Fig. 5D.

Discussion:
-Line 373: I would suggest adding another sentence or two expanding on the idea that nutrient enrichment may be necessary to observe a shift to parasitism, i.e. a decrease in the proportion of fixed carbon shared with the host. Indeed, I think this idea is a key component of the Baker et al. 2018 findings. If symbionts are able to access more nitrogen, they may be able to retain more of their fixed carbon to build biomass, thereby becoming more ‘parasitic’. Nutrient enrichment therefore favors this scenario, and the variation among hosts observed here could potentially be explained by variation in the ability of the host to limit the symbionts’ access to nitrogen, as also discussed by Wooldridge (2009 Mar Fresh Res) and Cunning et al. (2017 J Theor Biol).

Supplementary Materials:
Figure S5: Could the colors be labeled with the closest blast hit instead of the OTU id? This figure indicates that within some species (Gast), variation in the dominant symbiont existed among colonies – how could this have influenced the results? Would separating these samples by dominant symbiont be valuable and potentially reveal trends that are masked by pooling these samples? Some of the samples presented here were removed from data analysis as indicated in the Methods section – could this also be indicated somehow in the figure to remind the reader of this?


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

Input:
Title: EXPLORING MECHANISMS THAT AFFECT CORAL COOPERATION: SYMBIONT TRANSMISSION MODE, CELL DENSITY AND COMMUNITY COMPOSITION
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	All the derived variables lack clear justification and calculation information in the methods. The entire paper is set on the premise of transmission strategy, which is never actually tested.
2.	How are these physiological measures related to fitness and cooperation? The authors are discussing the results as fitness and cooperation, but have measured proxies without a clearly articulated linkage. They also have derived variables that are not clear, such as degree of symbiont parasitism.
a.	fitness and cooperation - need to define and justify the proxies used for this upon first or early mention (e.g., Line 97), given that these are reported as primary results in lines 111-118 and cannot be interpreted without further detail. These lines cannot be interpreted without further information.
3.	The discussion is much more of what the introduction should be. The authors are not testing vertical transmission, so why not just contextualize the paper with the hypotheses of the derived variables, given they are well defined and justified at the start.
Experimental design: 1.	All pairs of species were collected from different reefs at different times. Thus all pairs had different thermal histories and acclimation conditions. This should be discussed.

2.	Methods for respirometry are not fully detailed and result in problematic data. There is no indication of oxygen was measured at the initiation of the experiment. No light or temperature data are provided.  No mixing or stirring information is provided. It is not clear if the rates accounted for the volumes of the chambers and differential displacement due to nubbin and stand sizes. There is no information on if the water was filtered.  The authors cannot count on the supplementary data of Strahl et al to make their case for the single endpoint measurements used in the current study. This is all apparent in the photosynthesis data themselves, where the rates seem to be an order of magnitude from the cited protocol (Strahl et al 2015) and at least an order of magnitude off from the literature in general (e.g., Anthony et al 2008 www.pnas.org cgi doi 10.1073 pnas.0804478105).

3.	If protein was measured using the BCA kit following manufacturers instructions, then the measure that is reported is not total protein, but soluble protein. This can problematic to normalize to, as the BCA kit can also quantify lipids and lipoproteins, which would likely differ by species. I would recommend also normalizing cell density to surface area to check if patterns remain the same.

4.	Sequencing was done very haphazardly with 2 different primer sets, at 2 different facilities, with samples pooled across all replicates, and contamination and mislabeling in some samples, reducing the confidence in the data.  It is unclear when the samples were collected in the experiment and how the community at that single time point relates to the response variables measured at different time points. This confounds the statistical tests.

5.	Line 139: This is an oversimplification of EQY and not always indicative of subsequent bleaching.
Validity of the findings: 1.	The major hypothesis was not actually tested statistically. The authors state “We compared physiological components of cooperation and fitness between horizontal and vertical transmitters in a phylogenetically controlled design using three pairs of related coral species exhibiting different strategies.” They however only used “Species” as a main factor, which ignores the factor of symbiont transmission that the experiment was supposedly designed to test. With this approach they are essentially saying species differ. The authors report “No differences were detected between focal species pairs by symbiont transmission mode.” But this analysis was not included anywhere. Similarly lines 257-259.

2.	There are a multiple references to derived variables that are not clear in calculation or definition. For example the authors need to define degree of parasitism and bleaching intensity, cooperation, fitness, etc clearly with rationale.

3.	Every response variable has some issues with it. Respirometry methods are not up to standard and rely on others data. Cell density data normalizations are not Total protein. Sequence data are a grab bag of approaches and represent pools of unknown timepoint. Derived variables are not clearly defined in terms of calculation or rationale for the calculation.

4. No raw data or analytical scrips from the study are provided, only references.

5.	Due to all of these factors, it is not possible to judge the veracity or validity of the authors’ conclusions.
Additional comments: The authors have examined several physiological responses of corals to thermal stress in the context of exploring mechanisms that contribute to the symbiotic function. While the hypothesis that vertical transmission contributes to enhanced cooperation and fitness is solid and a timely issue, the work does not actually appear to test this hypothesis. Primarily, there are issues with the methodologies of all response variables that generate issues with data quality and capacity to compare statistically. Further, there is a lack of clarity in the terminology and derived variables used for additional analyses and a lack of all physiological data and analytical scripts.

Other general comments:
line 53: replace suggesting with indicating
Line 91: clarify species of what, coral-Symbiodinium pairs?
Line 96: Baird et al 2009 more comprehensive ref (Annu. Rev. Ecol. Evol. Syst. 2009. 40:551–71)
Line 128: PAR units are unclear.  Also what is meant by peaking at 130-160? Was this a fluctuating light cycle? Include light data in supplement.
Line 146: substitute “fraction of autotrophically derived carbon” for resource
Line 152: separated
Line 154: What is the need for skeletal volume? Measured, but not used again in manuscript.
Line 154: This is not total protein, but soluble protein if only the manufacturer’s instructions were followed.
Line 198: Include reference database a supplementary file.
Line 221: Now have switched to calling responses thermal tolerance and cooperation, but have still not defined them
Lines 232: Clarify how exactly each of these were calculated to include in the statistical analysis. Also check the sentence for missing information.
Lines 276-278: why was the full interaction not in the final model? The stats section Lines 223 indicates all possible interactions were included.
Supp Mat Lines 8-14 include collection dates for each reef location.


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: EXPLORING MECHANISMS THAT AFFECT CORAL COOPERATION: SYMBIONT TRANSMISSION MODE, CELL DENSITY AND COMMUNITY COMPOSITION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is generally well written and follows a construction that adheres to the typical formatting guidelines required of original research. Raw data and the scripts to reproduce the outputs are all provided which is excellent. I agree with the overall premise and intent of the manuscript and it is clear that symbiotic corals are undoubtedly important model systems for the study of host:symbiont coevolution and the spectrum of mutualism:parasitism (although your assumption regarding the tendency towards specificity/fidelity in vertically transmitting species is questionable given recent studies showing cryptic horizontal transmission in vertically transmitting coral species: starting with Byler et al 2013 PLOS ONE and more recent examples, such as Reich et al 2017 PLOS ONE). Indeed, considering the global degradation of coral reefs and research initiatives such as the progressive management of coral reefs (restoration, guided evolution, etc), research themes such as presented here, primarily related to the functional biology of coral/Symbiodinium species combinations under stress, are more timely than ever. However, the manuscript could have been presented better and more care must be taken during the preparation and process of submission. For example, a number of cited references are missing from the reference list (e.g. Green et al 2014), there are multiple instances where the formatting of genus/species names is incorrect (e.g. L189, L424), informal abbreviation is used without explanation (e.g. “ctrl” and “NAs”, nit-picky but it is important to be explicit), and important parts of the methodology are needlessly moved to the supplementary sections. In addition to these minor problems, I have several major concerns with how the data have been collected and analysed that likely preclude the publication of this text in its current form.
Experimental design: I have few issues with the design or replication overall (although it is unfortunate that a number of samples had to be pooled). My primary concern is with the processing of sequencing data, which I describe in the sections below. The methods used to assess symbiont diversity are simply not rigorous enough.
Validity of the findings: The most significant problem with this study is the analyzing of the ITS2 amplicon data. The authors have used an amplicon sequence variant (ASV) approach, rather than the well established OTU approach to analyse the ITS2 region (although confusingly the R scripts in the supplementary files contain references to “OTUs"? Recycling of old scripts?). While the case for the usefulness of ASVs has been made (Callahan et al 2017 ISME), it has been quantitatively demonstrated that ITS2 amplicon sequencing massively over-inflates diversity estimates for Symbiodinium. A significant aim of this study is to “investigate whether symbiont community composition could explain patterns of fitness and cooperation among horizontal and vertical transmitters at a species level”. Furthermore, the primary conclusions in the closing paragraphs of this manuscript are centered around relationships between cooperation and Symbiodinium ‘diversity’. These are important themes both in relation to the fundamental biology of the coevolution of host and symbiont(s) and also for researchers who need to strategize/prioritise certain hosts/symbiont species combinations in the anticipative management of corals reefs. However, it is widely known and recognised that the ITS2 marker is multicopy in nature and single genomes are comprised of ITS2 variants that are in various stages of homogenization due to convergent evolution (See Smith et al 2017 ISME for an ITS2 + psbAncr approach). The distances between intragenomic ITS2 variants will likely vary lineage to lineage, but if we take an example from the present study, Symbiodinium ITS2 type C1 (S. goreaui), Arif et al 2011 (Molecular Ecology) found that amplicon sequencing of a monoclonal C1 cell line (rt152) returns a multitude of intragenomic ITS2 variants. This is completely ignored by the present study. It was found that the vast majority of these C1 intragenomic variants can be collapsed into a single biologically meaningful “OTU” using the 97% similarity threshold. The approach in the present study finds up to eight ITS2 type C1 amplicon sequence variants (ASV) that may simply represent intragenomic variants of a single species. If we assume that this is at all possible, including Symbiodinium diversity as a factor in your mixed effect model makes little sense and simply does not evaluate the proposed aim. Diversity and performance/function are not reconciled in any biologically meaningful way. I would suspect that using the suggested clustering approach of Arif et al will collapse all of the presented diversity down to < 10 OTUs. At the very least these data must be explored using this approach in tandem with the present analysis if the goal of comparing diversity as a function of cooperation is to remain as part of the study.

Regarding the physiological measurements.

Please clarify at what time the “peak irradiance” occurs (line 118)? Photosynthetic parameters such as fluorescence transients respond strongly to light but photochemistry also follows endogenous rhythms (i.e. including non-photochemical quenching which is where much of the drop in EQY comes from during the day).

For the photosynthesis rate measurements, were the corals removed from their aragonite plugs before moving them to the incubator chambers? If not, did the “blank” incubations contain coral-free aragonite plugs? From experience the aragonite plugs quickly become covered in crustose coralline algae which also will produce a significant amount of O2 during the incubation. This would need to be accounted for.
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: EXPLORING MECHANISMS THAT AFFECT CORAL COOPERATION: SYMBIONT TRANSMISSION MODE, CELL DENSITY AND COMMUNITY COMPOSITION
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: My concerns have been addressed in this careful revision. I commend the authors on a solid, transparent dataset and analysis.

Suggestions:

Line 30: Replace “metrics of fitness” with “performance”

It would be helpful to more explicitly define “cooperation” in the abstract. The sentence beginning on line 28 implies the meaning of cooperation as translocation of photosynthate, but there are quite a few words in between “translocation” and “cooperation”, so this connection does not immediately jump out at the reader. I suggest restructuring this sentence to make this clearer, and/or including “(i.e., translocation of photosynthate)” after “cooperation” on line 32.


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: EXPLORING MECHANISMS THAT AFFECT CORAL COOPERATION: SYMBIONT TRANSMISSION MODE, CELL DENSITY AND COMMUNITY COMPOSITION
Review round: 2
Reviewer: 2

Output:
Basic reporting: Fitness is used in many different ways throughout the analysis. Consistency and discretion is necessary as actual fitness is not measured and fitness and cooperation are used synonymously at points and not others. Address throughout the manuscript.
e.g., Line 281: remove the word fitness here

Raw data and code are now shared in supplement.
Experimental design: Statistical design is still not clearly rationalized.

If testing the hypothesis of transmission mode and specifically choosing 3 “replicates” of each mode, provide rationale as how it is conservative to use species to test this in the analysis, which inflates the df available for the test.
Lines 277-280
Validity of the findings: The respirometry data have severe issues that are not addressed by the revision and cannot be addressed by simply stating light levels were lower.

1)	PI curves were not completed in the current study and it is assumed the same light levels apply in the same way to multiple coral species from multiple reefs. Therefore measurements were done most likely as the authors indicate at pre-saturating irradiance and at different places on different species PI curves, which means they are potentially comparing apples to oranges. It is precisely because “phosynthesis-irradiance curves are logarithmic, showing a marked increase between 0-500 μmol quanta m-2 s-1, but plateauing at higher irradiance levels.” that the authors should have done their own PI curves and chosen to work at a saturating irradiance for all their species. Their data would then be comparative and more likely would match that reported in the literature from values measured at Pmax.

2)	These light and photosynthesis issues (lack of comparison at Pmax) then carry over to the quantification of photosynthetically fixed carbon translocated to the host and this needs to be discussed as it has implications for the entire discussion/conclusions.

3)	The two-point method described and validated in Strahl et al. (Strahl et al. 2015) apply only to the conditions (physical, biological, and experimental) of Strahl et al. 2015. This validation should also have been done in the current study. Also it is a two point method n Strahl et al, not an end-point method.

4)	The photosynthesis values may be calculated incorrectly. It looks like the volume of the chamber was only accounted for 1 time. It appears the authors may need to convert both blank and sample to mg by multiplying by their respective volumes prior to subtracting them.

5)	The photosynthesis measurements appear to be gross, not net. Dark respiration measurements were not reported.
Additional comments: The authors have responded positively and in detail in review, but some issues/weaknesses remain that I am not sure can be addressed without new measurements. As it is unlikely that new measurements can be made at this time, it is critical that the authors remove the data or place strong caveats clearly up front with regards to the respirometry data.

Other Comments:
Line 97 – fitness proxies
Line 114 – proxy for holobiont fitness
Lin 166 -171 – place caveat here for single endpoint measurement of rates
Line 176 – what are the light conditions here? It is not clear to say identical to the experimental conditions as light was fluctuating and would have changed over the 5 hour incubations. Add what time of day these were done and the average light level estimate for this time and clarify that this is an estimate.
Line 182: host protein (as assessed below)
Line 293: include citation for inverse Simpson index, or otherwise clarify


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: EXPLORING MECHANISMS THAT AFFECT CORAL COOPERATION: SYMBIONT TRANSMISSION MODE, CELL DENSITY AND COMMUNITY COMPOSITION
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have provided satisfactory responses to my comments and I recommend this manuscript for publication.
Experimental design: The authors have provided satisfactory responses to my comments and I recommend this manuscript for publication.
Validity of the findings: The authors have provided satisfactory responses to my comments and I recommend this manuscript for publication.
Additional comments: The authors have provided satisfactory responses to my comments and I recommend this manuscript for publication.


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: EXPLORING MECHANISMS THAT AFFECT CORAL COOPERATION: SYMBIONT TRANSMISSION MODE, CELL DENSITY AND COMMUNITY COMPOSITION
Review round: 3
Reviewer: 1

Output:
Basic reporting: The basic reporting is clear
Experimental design: Point 3 in response

The light level issues are not fully addressed by looking over time, within a species. PI curves can change through time under thermal stress and for accurate rigorous results PI curves should have been run prior to all time points to determine if the curves have shifted and light levels were within the saturating part of the curve and not sub-saturating or inducing photoinhibition. The authors should also include this caveat in their tests of the onset of the bleaching response and clarify that they are lacking for this study and should be study specific. E.g., “but data on species-specific photosynthesis-irradiance curves are lacking in this study. Consequently, these differences among species may be influenced by variation in species-specific photobiology and differences within species may be influenced by shifting photophysiological performance within the experiments."

Also this argument of testing within species through time is counter to the approach or conclusions between species or transmission mode.

Point 4 in response

The light levels absolutely impact carbon production and thus carbon translocation potential. This should be stated.
Validity of the findings: The validity of the findings are impacted by the methods, specifically light levels.
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: COMPATIBILITY BETWEEN OBJECT SIZE AND RESPONSE SIDE IN GRASPING: THE LEFT HAND PREFERS SMALLER OBJECTS, THE RIGHT HAND PREFERS LARGER OBJECTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall very clear and professionally written. Only a few minor comments (mostly English use) as follows:

73,90 - 'spatial aspects', 'motor aspects' - not clear what is meant. 'parameters'?
82 - 'trajectories shifts' - should be 'trajectory'
86 - 'movements'
113 - spell-out 'S-R'
121 - add ',' after 'specifically'
129 - 'movement latencies' - reaction times or overall movement times? Perhaps use 'lower' and 'higher' latencies to distinguish from object size?
135&136 - 'stronger' - compared to what?
146 - was this '20%' criterion set in advance? what is specific about 20% - is it, for example, the cut-off for chance performance?
149 - add 'years' after age stats
159 - add 'the' before 'object'
160 - change 'weighted' to 'weighed'
163 - 'rapidly' - how rapid? I am aware of similar technology (Plato goggles) which is very rapid (<ms)
165 - '1mm' - this must be at a specific distance from the camera
182 - insert 'as' before 'possible'
205-206 - change 'to' to 'too'
232 - remove 'considerably'
294 - please repeat 'mental number line (MNL)' here, as I'd forgotten what this was after reading results (retroacronym interference!)
317 - 'objects'; add 'than the non-dominant (left) hand'
339 - add 'the' before 'presence'
365 - remove [we demonstrated...] 'not only...artificial stimuli' - the authors did not present any new evidence showing this! Just make conclusions about the current data set, and refer to others' results for the symbolic stimuli

Figure 1 - Does left and right panel need A), B) labels? No need for colour really; Otherwise, very nice!
Figure 2 - (see Fig 1 commentS). Why not present the mean differences in Panel B as well - this the (second) critical effect?
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Clear, concise, well-reported and very well analysed, really nice figures showing (most or) all of the necessary data.


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: COMPATIBILITY BETWEEN OBJECT SIZE AND RESPONSE SIDE IN GRASPING: THE LEFT HAND PREFERS SMALLER OBJECTS, THE RIGHT HAND PREFERS LARGER OBJECTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Literature references provided is extend and well discussed. In my opinon, beside the studies of Lindemann et al. 2007 and Andres et al. 2008 (see lines 95-101), some references should be added: e.g. Badets and Pesenti (2010, Cognition) and Ranzini et al. (2011, Frontiers in Human Neuroscience).
Experimental design: The research question is clearly define and the knowledge gap being investigate, first of all in relation to the previous study of Wühr and Seegelke (2018), is identified and well discussed.
Since the present study, compared to Wühr and Seegelke (2018), added kinematic parameters, in my opinion it could be very relevant for the reader to understand the rationale behind these measures. In other words, I suggest authors to specify better the hypotheses expected as regard the specific kinematic parameters detected.
This could be very helpful also for understanding the findings and the respective discussion.
Validity of the findings: If I understand right the results' section, findings demonstrated a compatibilty effect only for RTs and for the time to maximal grip aperture.
I think that authors should explain why in the other measures (MTs, peak velocity, time peak velocity, maximal grip aperture) the overall compatibility effect did not emerged and, in some cases, the effect is reversed for the left hand.
Additional comments: In my opinion this study is a very interesting and meaningful replication of previous studies (Wühr & Seegelke, 2018; Ren et al. 2011) in which authors added two important aspects: a visually-guided grasping task and the measure of kinematic parameters.
While the first aspect is motivated (authors hypothesized that "interaction effects between different magnitudes might be stronger in a visually guided motor task...") and the finding is well discussed in the general discussion, the second aspect is less deepened (see my comments in the previous sections).