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: WHAT IS AN ARCHAEON AND ARE THE ARCHAEA REALLY UNIQUE?
Review round: 2
Reviewer: 2

Output:
Basic reporting: The basic reporting expectations are met by this paper. Nonetheless, I continue to believe that the paper would be much better received (and more likely to be cited) if it did not attempt to lay out every argument for the author's pet theory, all of which he has already published, and instead stuck to what is new. The narrative structure would be simple. "As I have showed previously [refs], the presence/absence of protein domains provides a robust method for the inference of rooted trees. Thus, provided one accepts that there has been no major chimerism - i.e., that the endosymbiotic theory for the origin of eukaryotes is incorrect [Harish & Kurland 2013] - this approach is well suited to resolving the overall structure of the rooted tree of life. Here I analyze a larger/better data set of domains and, using improved methods, re-evaluate my prior conclusion that eukaryotes are sister to "akaryotes" (=bacteria+archaea). I find...."

It is very important that the reader be told in the introduction that the entire method assumes that mitochondria arose autogenously. It is an attempt to better root the tree of life, under this (controversial) assumption.
Experimental design: I stand by my prior concerns regarding chimerism. First, to clarify I used a 10:1 step matrix and did indeed find a root within bacteria (I just checked). Second, it all turns on the author's MAJOR assumption that the endosymbiotic hypothesis is wrong. I went back an looked at the key paper, Harish and Kurland (2013), and am not at all convinced! Let me provide a quick critique.

The paper makes the major mistake of equating the mitochondrial proteome with the endosymbiont. To be sure we would expect the mitoproteome to be ENRICHED for endosymbiotic genes, but we need to remember that under the endosymbiotic hypothesis, (a) very many bacterial genes were transferred to the nucleus and acquired distinct functions outside of the mitochondrion, and (b) some proteins of archaeal ancestry would have acquired mitochondrial target sequences and mitochondrial functions. With than in mind lets look at their data, bearing mind that for some reason Archaea seem to have  fewer domains than the other groups (ignoring those shared by all groups, the numbers are: Arch = 156; Bact = 419; Euk = 738): In the mitoproteome, seven times as many genes are uniquely shared with bacteria (and not archaea) than the reverse. This contrasts with only 2.5X enrichment for the non-mitochondrial proteome.  To my mind this over-enrichment of bacterial genes in the mitoproteome is fully consistent with the endosymbiotic model!

I would also ask the author to explain away the many gene trees with robust support for eukaryotic genes that originated from within the proteobacteria - including some where the analysis used non-stationary models as the author favors (e.g., Wang and Wu, 2015).
Validity of the findings: At the end of the day I believe the author is wrong in his rejection of the endosymbiotic model. This invalidates the method as it stands.
Additional comments: There are three choices:
a) Push ahead with this method and present a result that will be completely ignored by anybody who shares my view - and that is pretty much all biologists.
b) Accept that the endosymbiotic model could be correct, in which case the method could be used to evaluate competing models for where eukaryotes sit and where the root goes. Such an analysis would be of broad interest. You would need to split the eukaryotes into two taxa, for example one taxon being composed only of domains found anywhere in bacteria and anywhere in eukarya, plus a random half of the eukaryote-specific domains, and the other being composed only of domains found anywhere in archaea and anywhere in eukarya, plus the other half of the eukaryote-specific domains. But there may be other strategies.
c) Do both in the same paper.

I recommend option b or, failing that, c.


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: THUMBS UP: MOVEMENTS MADE BY THE THUMB ARE SMOOTHER AND LARGER THAN FINGERS IN FINGER-THUMB OPPOSITION TASKS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English needs significant editing.

As the study focuses on movements of the fingers, it is strange that there is not a single graph showing mean or representative kinematic data. It is essential that this data be included to allow the readers and reviewers to evaluate the claims made in the paper.

The motivation of the task is not clear - opposition of distal phalanges is commonly used in day-to-day life (albeit usually with an object in between), while opposition of the thumb with the other phalanges is much less frequent. Can you give examples from day-to-day life when these movements are used?
Experimental design: The sample size (N=8) seems small, and no sample-size analysis was performed. Please justify why this sample size was selected.

The measure E_F is redundant-  it always equals 1-E_T. So there is no need to analyze it separately. When you want to check if E_F and E_T are different, just compare E_F to 0.5

The smoothness measure is unclear - are you measuring smoothness of a particular phalange, or the whole finger? joint angles or position?

The methods are unclear, I do not feel that I could replicate the calculations:
- Is any filtering performed? Calculation of velocities without filtering is very problematic
- In the calculation of R_D and R_V, it is not clear what is the base of the coordinate system. Are separate calculations performed for the thumb and finger? which phalange? How is movement onset and end determined (it is written based on maxima and minima, but how are these defined?)
- The specteral arc length (SAL) used is difficult to understand intuitively - what should be considered a meaningful difference in smoothness in terms of SAL?
Validity of the findings: Due to the problems described above, I found it difficult to determine the validity of the findings.

Regarding the smoothness, I would like to see comparisons of the velocity profiles of the finger and the thumb (graphs) so I can judge whether one looks more "smooth" than the other. Having different smoothing from the two effectors performing a coordinated action seems sub-optimal.

Regarding the exertion index, one major factor has been ignored, namely the range of motion of the fingers. The biomechanics of the hand do not allow arbitrary movements of the fingers. Hence the differences observed in "exertion" between the thumb and fingers made be purely due to biomechanical constraints. I would like to see this taken into account in some way (e.g. by comparing movements to the edges of the range of motion of the finger joints)
Additional comments: Minor corrections:

line 51: phalanx positions is mentioned here in abstract with no previous reference
line 65: this claim seems hard to believe
line 73: forces coordinate with each other -> forces are coordinated
line 80/81: shape, contour of the hand. This is unclear what you mean
line 99: has -> have
line 100: healthy from unhealthy. these terms are not precise
line 110: The first hypothesis
line 113: The second hypothesis
line 117: This hypothesis needs justification, it is not obvious why this is true
line 134: An electromagnetic
line 134: Describe which system, not just the company (Liberty?)
line 152: at a comfortable speed
line 152: how were they instructed? verbally? on the screen? did they have to start moving in response to a beep?
line 156: delete for
line 160: The experimenter monitored
line 163: this sentence is redundant (was explained previously)
line 171: I have difficulty understanding what R_D is. From your equation, it seems like it is the distance from the origin of the coordinate system (the transmitter). This seems like a strange choice. Shouldn't it be the distance between the fingers, or the distance from the palm of the hand?
line 174: R_D has a bell-shaped profile - this is not obvious why - later you investigate smoothness, if all movements are bell-shaped they presumedly have similar smoothness values
line 192: The reference to your previous studies for calculating a standard measure (the z-score) seems self-serving
line 213: I assume you mean H-F was used when sphericity was violated\
line 215: This paragraph is redundant, no need to redefine the measure
line 227 and elsewhere: units are missing!
line 250: Why 12 one-way ANOVAs and not a single repeated measures? Running this number of ANOVAs without corrections is problematic
line 257: redundant paragraph
line 278: "wonderfully" - not scientific language
line 316: The thumb
line 326: This is not an explanation, rather just states a fact
line 332: What do you mean by flex? Some joints must flex, but not all joints.
line 375: anatomical architecture - this a vague term, it is not clear what is meant


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: THUMBS UP: MOVEMENTS MADE BY THE THUMB ARE SMOOTHER AND LARGER THAN FINGERS IN FINGER-THUMB OPPOSITION TASKS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a generally well written and presented manuscript.  The authors make appropriate references to the literature.

At the moment the raw data for the work is not available.

There are two hypotheses, the first hypothesis has two parts but there is no rationale for the second part of the hypothesis.
Experimental design: The experimental design is good.

There are three primary issues with the Methods,
1) Reference is made in to segment angular orientations, but these are never used.  Reference to the angular orientations should be removed.

2)  Velocities are analyzed, but no information is provided on data filtering and differentiation.

3)  The authors present a metric, the "Exertion Ratio", but what is quantified only indirectly related to exertion.  This ratio should be more appropriately named, for example the "Movement Ratio".
Validity of the findings: No Comment.
Additional comments: Line 33 - delete second "the".
Line 34 - "...activities of the hand.".
Line 37 - "...has not been well understood.".
Line 49 - "The Exertion Ratio...".
Line 51 - better in what sense?
Line 55 - "...made a greater...".
Line 61 - "...of the opposite...".
Line 67 - delete "to".
Line 78 - replace "this" with "that".
Line 101 - please provided references for these comparisons of novices and experts.
Line 108 - "...of the thumb.".
Line 160 - "An experimenter monitored...".
Line 201 - this in text citation should be "...et al. (2012)...".  This should be corrected elsewhere in the manuscript.
Lines 216-221 - this is not a results but details which should be in the Methods section.
Line 316 - "The thumb showed...".
Reference 40 - this reference has a different format to the other references.


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: THUMBS UP: MOVEMENTS MADE BY THE THUMB ARE SMOOTHER AND LARGER THAN FINGERS IN FINGER-THUMB OPPOSITION TASKS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have improved the presentation of the data by adding the graphs and more details.

The presentation of the graphs is fine, but it would be more useful to combined figures 1 & 2, and figures 3 & 4 (e.g. used dashed line for thumb). In this way, it makes it easy to observe the claims made in the paper (regarding the relative size of the movements, and the differences in velocity profiles).

I still have a problem with the equation for R_D. The current equation implies that you take the distance relative to the base of the hand coordinate system. I assume you actually take the displacement from the start of the movement. So the equation should be something like: R_D = sqrt( (x - x_0)^2 + (y-y_0)^2 + (z-z_0)^2))

Also, in the equations for D_T and D_F, please use the notation of R_D you defined above.
The authors have improved the presentation of the data by adding the graphs and more details.

The presentation of the graphs is fine, but it would be more useful to combined figures 1 & 2, and figures 3 & 4 (e.g. used dashed line for thumb). In this way, it makes it easy to observe the claims made in the paper (regarding the relative size of the movements, and the differences in velocity profiles).

I still have a problem with the equation for R_D. The current equation implies that you take the distance relative to the base of the hand coordinate system. I assume you actually take the displacement from the start of the movement. So the equation should be something like: R_D = sqrt( (x - x_0)^2 + (y-y_0)^2 + (z-z_0)^2))

Also, in the equations for D_T and D_F, please use the notation of R_D you defined above.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I found the comment about traditional south Indian music / counting interesting, and a good motivation for the study, I would add these lines to the introduction.

Regarding speculation on the differences in the exertion index - in the discussion when you bring this topic up, I think you should at least mention that the differences observed may be due to the biomechanics of the hand. Although you did not measure range of motion of the fingers, this data is readily available, and seems to present the most parsimonious explanation for your findings.


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

Input:
Title: THUMBS UP: MOVEMENTS MADE BY THE THUMB ARE SMOOTHER AND LARGER THAN FINGERS IN FINGER-THUMB OPPOSITION TASKS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised manuscript is much better than the previous version in all of these aspects.
Experimental design: The manuscript is good in these aspects, and with use now of the term displacement index is superior to the term term used in previous submission.
Validity of the findings: The research provides some new insights into the function of the thumb.
Additional comments: Specific Comments (line numbers refer to the version with tracked changes)
Line 40, Comment – change “has not been well understood” to “ is not well understood”.
Line 91, Comment – in what sense special?
Line 120, Comment – “…of the thumb.”.
Line 140, Comment – “…using the Edinburgh…”.
Line 187, Comment – “…only onward movements were analyzed.”.
Line 195, Comment – reference needed for stencil.
Lines 215-217, Comment – the process outlined here does not find the first time when 5% of the maximum velocity was crossed.
Line 402, Comment – “e.g.” means in Latin exempli gratia, which means “for example” therefore there is redundancy in this sentence.
Line 420, Comment – “The second hypothesis…”.
Line 521, Comment – “…smoothness of finger movement…”.
Line 524, Comment – here and elsewhere what do you mean by “superior”?
Line 534, Comment – “…case of the thumb.”.
Line 537, Comment – “…in terms of movement smoothness.”.
Line 556, Comment – not “day-to-day” to “daily”.
Line 559, Comment – “…compared with the fingers.”.


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: THUMBS UP: MOVEMENTS MADE BY THE THUMB ARE SMOOTHER AND LARGER THAN FINGERS IN FINGER-THUMB OPPOSITION TASKS
Review round: 3
Reviewer: 1

Output:
Basic reporting: I am satisfied with the changes made by the authors. In particular, combining the graphs makes it much easier to understand the central claims of the paper.
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: THUMBS UP: MOVEMENTS MADE BY THE THUMB ARE SMOOTHER AND LARGER THAN FINGERS IN FINGER-THUMB OPPOSITION TASKS
Review round: 3
Reviewer: 2

Output:
Basic reporting: Acceptable.
Experimental design: Acceptable.
Validity of the findings: Acceptable.
Additional comments: The manuscript is much improved in this submission.  I have no further comments.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF THE CNGC GENE FAMILY IN MAIZE
Review round: 2
Reviewer: 1

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: The author describes first the identification of Cyclic nucleotide gated channels (CNGCs) from maize and subsequently characterized for structural analysis of the protein and other in-silico parameters. The work is unique since the current study reports this family of genes from maize. Yet the manuscript is deficient with many sections. Some of them are mentioned below.
Major points:
a)	The work is unique since the current study reports this family of genes from maize. All the 12 ZmCNGC genes should be renamed in this manuscript to better understanding of the following analysis.
b)	For identification of the ZmCNGCs, you should concider about potassium AKT /KAT channels (Shaker type) as homologs of CNGCs, due to the presence of transmembrane region, CNBD and ion transport domains and additional ankyrin repeats. Please re-confirm this part, and you’d better to list the conserve domain as a table in this manuscript or in supplementary material.
c)	Please re-write the phylogenetic analysis section. To get the accurate model, Maximum likelihood (ML) phylogenetic tree should be used. In the Figure 1, the bootstrap value must be shown in the figure. On the other hand, the branch of group III and group IVb you separated was strange. There is an inverse relationship about the group III and group IVa in this manuscript and the other CNGC family papers. Finally, the annotation of the rice and Arabidopsis CNGCs should be renamed in this figure.
d)	In Figure 2, GRMZM2G129375 and GRMZM2G066269 did not show the upstream part in the gene structure part, and the length of those two genes is shorter that other ZmCNGCs. Please confirm the full length and the structure of those two genes.
e)	In Figure 3, you should highlight the PBC and hinge region in this figure.
f)	CNGC gene families were welled studied in a lot of animals and plants, and no need to do the GO annotation of this gene family. Please remove this part.
g)	In Figure 5, Please improve the name of those genes.
h)	The expression profiles are important part in this manuscript. Please put the figure s4 into the manuscript. In addition, please explain why the heatmanps of the embryo, endosperm and seed only have 10 ZmCNGC genes, and submit the FPKM date as a supplementary material.
i)	Discussion pertaining to conclusion should be made, at many places its mere repetition of introduction or the results. No new hypothesis has been generated, making this part of the manuscript very weak. It very interesting to think about evolutional aspecs of this gene family. The numbers of ZmCNGCs is less than other CNGC gene families (20, 16, 21, 18, and 47 CNGC genes were identified in Arabidopsis, rice , pear, tomato, and wheat). Please discuss more about the Gene duplications and evolutional part, and re-write the conclusion section.

Minor points:
a)	there are some grammatical problems (English). I listed some of them here. And I recommend having editorial corrections by a native speaker.
Line 148-152 “Subcellular localization analysis indicated that all of ZmCNGCs localized in the plasma membrane except for GRMZM2G066269 which was localized in the nuclear fraction, this result is consistent with Arabidopsis, for example, previous studies have revealed that CNGCs are majorly localized in the plasma membrane (Lemtiri-Chlieh & Berkowitz 2004).”
Line 182 “is use to evaluate”
Line 222 ”associated with associated with”
b)	the figures in this manuscript should be more stringent. Figure 2 must mark the A, B, C if you mentioned as Figure 2A,2B, and 2C. Figure 1,3,5 is unfriendly to understanding.


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: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF THE CNGC GENE FAMILY IN MAIZE
Review round: 2
Reviewer: 2

Output:
Basic reporting: This manuscript aims to characterize the CNGC family in maize. The manuscript is poorly written (requires English editing) and the authors do not use references properly. The introduction  does not cover the literature well. There are many wrong statements or wrong citations associated with statements (see below for some examples).

-References:
Harada et al. 2003   I don't think this paper refers to CNGCs at al
L67	KW et al. 2006	          what is that?
L67: Wang et al. 2013	this paper is about guard cells not pollen
 Mosher et al. 2010)  	this paper is about the chimeric CNGC11/12, not CNGC6, 10, 19, or 20

these are just a few examples
Experimental design: There are no experiments. Everything is just computational analysis. There is no research question.

The investigation is shallow with no followup.
Validity of the findings: The novelty of the findings is extremely limited and no experiments were conducted to add any meat to this story.

Furthermore I have doubt about their phylogenetic tree. The authors are very sloppy and mixed up group IVb and group 3 (Maser et al., 2001, Navaz et al., 2014).

The presentation of their “bioinformatics” analysis range from uninteresting to bizarre statements:

-Abstract: Synteny analysis showed that 2, 2 and 1 ZmCNGCs had homologous genes in Sorghum, rice and Brachypodium, respectively

What kind of a statement is that? Of course there is homologs in all other plant species!

-Gene ontology (GO) analysis demonstrated that most of the ZmCNGCs are involved in various biological processes including cellular processes, establishment of localization, and transmembrane transport.

-Is that all they could come up with? A known ion channel gene is membrane localized?

-L161 The physiological and biochemical properties of these 12 ZmCNGC genes are listed in Table 1.
There is no information about any physiological properties.

-L162 The protein lengths ranged from 326
that would indicate either a truncated pseudogene or a wrong sequence. A functional CNGC can not be that short.

-L282: Fig4 is completely uninformative. It does not make sense to do the GO analysis for the whole family. And the conclusion of a “bioinformatics” analysis of a known ion channel family to be involved in transmembrane transport is more than obvious.

-Further, cellular component analysis revealed the localization of ZmCNGCs in the cell and membrane, may be the reason why the subcellular localization of most ZmCNGCs localized in the plasma membrane.
What do the authors want to say here?

-L296 based on the orthology-based predictions following the network in Arabidopsis were constructed.
This seems to be extremely far-fetched and does not provide any useful information

-Suppl 4 seems to be the most interesting. Why it is a supplement? Where is the figure legend?
Additional comments: The authors did not make a lot of effort and did not study about CNGCs at al. The final conclusion is only that maize also has CNGCs, which was obvious from the start.


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: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF THE CNGC GENE FAMILY IN MAIZE
Review round: 3
Reviewer: 1

Output:
Basic reporting: There are very serious issues. See point 4
Experimental design: There is only one experiment. I can not comment about the validity of the statements about evolution.
Validity of the findings: The data has to be connected and discussed properly (see point 4)
Additional comments: The revised manuscript by Hao and Qiao has addressed some of the criticisms of the reviewers and I acknowledge that the authors now included their expression data in figure 5. This data is the most interesting in the manuscript and should be analyzed and discussed in much more detail.

The two major problems of the manuscript is a) it appears the authors just do not make much effort to do a thorough analysis of the maize CNGCs. The text is full of platitudes without any significant content.
The second major problem is the English is just not adequate. This may in part be the reason for problem #1. Editing by a service that is specialized in SCIENTIFIC WRITING is required.
In my opinion the manuscript still needs major revisions –especially in writing. There are so many either wrong or badly worded statements. A small selection is in Minor points below.

Besides the writing, major issues for me are:
Fig 2C. What are those motifs??? This looks really strange to me. It is necessary to define those motifs, otherwise the reader can not know which parts CNGC2 and 3 are lacking. Which are the TM domains, the pore and what other motifs are there? If ZmCNGC2 and 3 (and potentially (ZmCNGC9 and 12) are lacking many major domains, then it is questionable whether these are functional channels at all. Or as seen in the Xu et al. 2017 UsCNGC13 paper, CNGCs with truncated C-termini may function as dominant negative isoforms. Also, the fact that no EST hits for ZmCNGC3 were detected could indicate that this I a non-expressed pseudogene.

The results have to be discussed in a more comprehensive manner in the discussion. The data from the phylogenetic analysis (homologs from known Arabidopsis and rice genes) have to be connected to their network analysis (Fig4). What are the genes in the network and how could that connect to the function of the included CNGCs? Why only 5 of the maize CNGCs are in the network?
Most importantly, the data from the expression analysis in Fig 5 needs to be connected and discussed in much more detail. This is the valuable part I see in the manuscript.
Finally, how does the expression and network data connect to the promoter element analysis (SI Fig 4)?

Since the paper is very short on experimental data, at least we can expect a much more thorough analysis of the bioinformatics data presented.

The conclusion should concentrate on some real finding, rather than technicalities like the well-known PBC.

Regarding the expression analysis: Does this mean that ZMCNGC3, 5, and 7 are not expressed in any tissue analyzed?

Minor points/not acceptable statements:

Abstract:
Furthermore, the co-expression network analysis of ZmCNGC genes may establish the importance of better understanding ZmCNGC transduction pathways in maize

Additionally, expression profiles of ZmCNGC genes were shown to express in a tissue-specific pattern

Main text:

Most CNGCs have been characterized by genetic 52 methods and found to be related to plant physiological and molecular functions, including playing 53 vital roles in multiple physiological processes which are involved in signal pathways, plant 54 development, and environmental stresses.

- This sentence does not really have any content, besides the obvious statement that genes have functions.

As a molecular switch, secondary messengers such as cyclic nucleotide monophosphates (cNMPs; 3’,5’-cAMP and 3’,5’-cGMP) and Ca2+/calmodulin (CaM) can regulate 37 CNGCs, those messengers are activated by directing binding of cyclic nucleotides as well as are 38 inhibited by binding of CaM to the CaM binding domain (Saand et al. 2015b; Borsics et al. 2007; 39 Defalco et al. 2016; Kaplan et al. 2007).

- The messengers are not activated by binding, but the CNGCs are activated by binding of the messengers

In plant CNGCs, they are composed of six transmembrane (TM) domains and one pore region 41 between the fifth and sixth TM domains.

- This is not only for plant CNGCs, but all CNGCs (and as reviewer 1 mentioned also AKT/KAT channels). And they also have cytosolic portions at the N and C termini

CNGC16 and CNGC18
56 participate in the pollen development (Tunc-Ozdemir et al. 2013b; Frietsch et al. 2007

- Have to add Gao et al 2016 PNAS

is involved in jasmonic acid induced apoplastic Ca(2+) influx in epidermal cells (Lu et al. 2015).

- I am not sure if Lu et al talk about apoplastic Ca2+. Have to cite Wang et al 2016 for apoplastic Ca2+

Line 59. The Fortuna 2015 paper is talking about flowering time, not abiotic stress.

Subcellular localization analysis showed that all of ZmCNGCs localized in the
154 plasma membrane except for ZmCNGC3 localized in the nuclear fraction

- These are just predictions! The wording sounds like experimental data.

Fig1: Where is AtCNGC3?

Fig2B upstream/downstream refers to UTRs?

182 showed that 2, 2 and 1 ZmCNGCs had homologous genes in Sorghum, rice and Brachypodium, 183 respectively (Table 2).

- The authors mean ZmCNGC1 and 2?   Still, this statement is odd. The phylogenetic tree clearly shows many more homologous genes. Do they mean these homologs are only present in maize and sorghum, rice, Brachypodium and maize, not in others?

Suppl Fig 2: I have no idea what this table is supposed to show.

The CNBD identified in CNGCs but no other proteins, 204 hence was recognized as a tool to identify plant CNGCs

-They mean PBC?

Fischer et al. (2013) have showed that IQ as a functional motif within CaMBD and 232 downstream of the CNBD domain, also conserves among plant CNGCs and enhances the 233 changeable of Ca2+-dependent channel control mechanisms in plant.

_ what is changeable?

- cis element analysis: In my opinion, as presented without any additional analysis or experimental validation or any kind of meaningful connection of this prediction to expression data, this analysis is pointless!

-Fig 4. This analysis without further explanation or validation or connection to any other data is pointless. See above. It should be discussed in the context of all other data available, and maybe compared to CNGCs in rice.

Line 297 Compared to other ZmCNGC genes, the gene expression of Group IV genes were more intense

- what is more intense?

suggested that these three ZmCNGC genes obtained gene functions from other plants after 299 duplication.

- They probably mean they obtained novel gene functions that are different from other plants.

These results further illustrated that Group IVb gene play important role in maize 302 gene duplication, evolution and expression.

- What should that mean??? CNGCs have a role for gene duplication?


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: SCLEROSTIN PROMOTES HUMAN DENTAL PULP CELLS SENESCENCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: SOST immunochemistry: controls have to be presented at least as supplementary material.
 - In Fig 1 the image pixel definition seems  a bit low.
The authors should precise with initiale letters the localization of pulp, dentin,
predentin, and odontoblasts on the immuno figures.

- Fig 2: same comment as for fig 1 images, the pixel definition is low.
Validity of the findings: Data is robust, statistically sound, & Controlled
No speculation
Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: -	The beginning of the introduction, aiming to point out the importance of the dental pulp complexe preservation ,could be ameliorate ( cf pdf notes).
The others aspects ( Sost and its implication in bone formation, aging and dentin regeneration) are very well described.
-	Except for the “legal aspect” ( see below “ custom check note as demanded by the editors) , the materiel and method part is particularly tidy and will be really helpful for the readers.
-	The results are clearly stated by the authors and answer point by point to their initial questions and objectives
-	The discussion provides well-documented informations to the reader and proposes perspectives  for further research works.

CUSTOM CHECK:
- Ethical approval and consent form are in Chinese so can’t be verified. An empty form translated in English should be provided for the informed consent as well as for the signed page of the Ethical approval.
- The authors have included a statement in their material and methods part concerning the Declaration of Helsinki and the information consents but no approval reference numbers are mentionned ; neither the name of the granting organization.

-Identifiable info have been removed from all files: OK
- Were the experiments necessary and ethical? YES


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: SCLEROSTIN PROMOTES HUMAN DENTAL PULP CELLS SENESCENCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the reporting is good. There are a few minor typos and needs careful copy editing.Eg; Line 67 – Elderly
Experimental design: The manuscript #28635 entitled “Sclerostin promotes human dental pulp cells senescence” by Ou Y et al is appropriate for this journal audience. The authors use primary dental pulp cells and both loss of function (sh RNA) and over-expression to elegantly dissect SOST function.

A few minor issues can be clarified to improve interpretation and clarity of this work.
1. Were the Alizarin studies done at the same time points? This should be explicitly stated.

2. Details of serial sub-cultured are necessary. As per the traditional 3T3 protocols, cell surface area per cell spilt must be carefully documented to account accurately for population doubling.

3. Figure legends and labels are very confusing, they should be redone. Eg; simply label HDPC/PCDH as  ‘Control’ and HDPC/SOST as ‘SOST’. This will significantly help the reader follow the data.
Validity of the findings: No comments
Additional comments: The manuscript #28635 entitled “Sclerostin promotes human dental pulp cells senescence” by Ou Y et al is appropriate for this journal audience. The authors use primary dental pulp cells and both loss of function (sh RNA) and over-expression to elegantly dissect SOST function.

A few minor issues can be clarified to improve interpretation and clarity of this work.

1. Were the Alizarin studies done at the same time points? This should be explicitly stated.

2. Details of serial sub-cultured are necessary. As per the traditional 3T3 protocols, cell surface area per cell spilt must be carefully documented to account accurately for population doubling.

3. Figure legends and labels are very confusing, they should be redone. Eg; simply label HDPC/PCDH as  ‘Control’ and HDPC/SOST as ‘SOST’. This will significantly help the reader follow the data.

4. A minor typos must be corrected. Eg; Line 67 – Elderly   Careful revision of the complete manuscript should be performed.


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: SCLEROSTIN PROMOTES HUMAN DENTAL PULP CELLS SENESCENCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The present article passes the requirement for basic reporting topics.
Experimental design: The present article passes the requirement for experimental design topics.
Validity of the findings: The present article passes the requirement for validity topics.
Additional comments: The present study described the role of SOST on human dental pulp cell senescence. The experimental designs are straight forward and several approaches were employed to identify one research question, confirming each other data. Overall manuscript was well-written. However, the discussion on the teeth samples should be added. Authors may obtained teeth from elder group due to the potential extraction from periodontal diseases. It has a possibility that dental pulp of periodontal affected teeth may be compromised. Hence, the observation of SOST expression and p16 p21 p53 expression may also be influence. The discussion on this issue would help to clarify the results.


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

Input:
Title: COMPARATIVE SYSTEMATICS AND PHYLOGEOGRAPHY OF QUERCUS SECTION CERRIS IN WESTERN EURASIA: INFERENCES FROM PLASTID AND NUCLEAR DNA VARIATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors present an impressive amount of work on a challenging but very interesting question about the relationships among Sect. Cerris oaks of western Eurasia.  Presenting the many detailed and variable results among markers and species presents difficulties for the reader in fully appreciating where the data tell the story and where the authors interpretation of those data are driving the story. The length and readability of the manuscript presented the largest challenges to me. The questions are very interesting, the analyses are all justifiable, and the figures are extremely helpful in clarifying the interpretation of the results. The colors are nice and consistent but If the publication allows, individual photos of each species (close up of 1 leaf) may be helpful in keeping track of them as well.
Experimental design: Starting at line 130, I’m not sure if the challenges presented are specific to your system/seciton or the entire genus where a lot of that type of work has already been done. It also seems like a list of general challenges that seemed to slow the momentum of your introduction section. Maybe these methodological details could be broached in the Methods section or in the Discussion? Just a thought as I found lines 130-147 to be a little less clear than the rest of the introduction.

Generally, it seems you should keep the focus on Sect. Cerris and focus less on the broader patterns in the the genus Quercus unless there are similar phytogeographic patterns that might serve to highlight expectations or contrasts with this study. This might help focus the manuscript a bit as well as allow for the elimination of some of the more tangential information about broader Quercus
Validity of the findings: I understand the challenges in finding appropriate molecular markers but it should be emphasized that most studies rely on more than 1 nuclear and 1 plastid sequence region to infer relationships to the degree that authors set out to in this study. An upfront explanations of potential caveats about inferences made from so few markers should explain the degree of uncertainty in drawing conclusions from only these markers.

The Discussion is very long but does a good job of identifying all the additional hypotheses that are raised by the results presented here. The use of subheadings is helpful to the reader but I encourage the authors to try to find sections that could be more concise by reducing the amount of speculative interpretation.

Overall, this manuscript seems like an important first step in many future more detailed studies of these oaks.
Additional comments: INTRODUCTION
86  "Section Cerris (Cerris oaks) currently comprises 12 or 13 species (Table 1) occurring from the"
In the abstract you say 15 species, I think I understand why this difference but it seemed odd reading these different numbers so close together without clarifying information.

64 "Oaks (Quercus L.) are a worst case and, at the same time, an ideal model for comparative
65  phylogeographic studies."
Clarify this statement by saying immediately following it with "because they . . . " or start the next sentence with “They are ideal because  . . . but challenging because . . . “

89  "with different precipitation regimes (Cs, Cf, Cw, Ds, Df, Dw; Kottek et al., 2006;"
Readers may not be familiar with codes for precipitation regimes  - could you summarize the range with words: arid - mesic?

METHODS
I am unfamiliar with the Goker and Grimm (2008) method the authors used so I cannot comment on the appropriateness of each step but I appreciate the transparency describing the steps taken.

I didn’t follow the rational for reversing the inverted sequences?

RESULTS

I may have missed the explanation for why or how the root of the tree was chosen?

294  "sections in the Aegean and the western Mediterranean regions. As shown in Fig. 2a-b, plastid

295  diversity is largely decoupled from species identity, and related to geography; the least derived

296  haplotypes within the ‘Cerris-Ilex’ lineage (H1 and H2) occur across the whole distribution

297  range of the investigated group, except the Levant and the western Mediterranean (H2). All other

298  haplotypes are more circumscribed, concentrated in Anatolia (H3–H5, H7–H8), the Levant (H6,

299  H9–H10; the latter two showing single occurrences in Italy), and Iberian Peninsula + Morocco

300  (H11-H12)."
 This is a cool result and it seems to be a major outcome of the study! It might help the reader to hear more about nuclear-plastid incongruence in the Introduction to set some expectations and may provide an opportunity to explain

Was there any thought of trying to better characterize the putative hybrids using methods like those summarized in the Twyford and Ennos 2012 Heredity paper “Next generation hybridization and introgression” (Heredity volume 108, pages 179–189): "The genomic contribution of the parental lineages in each hybrid individual can then be estimated (the ‘hybrid index’ or ‘admixture proportion’; maximum likelihood implementation in HINDEX; Buerkle, 2005) as well as the hybrid class (for example, F1, F1 backcross, model-based Bayesian implementation in NEWHYBRIDS; Anderson and Thompson, 2002). Moreover, where detailed genomic data are available, genomic clines of introgressed alleles can be identified (using INTROGRESS; Gompert and Buerkle, 2010)."

Although most of those methods do require broad marker coverage throughout the genome. Or maybe what you have in Fig. 4 does something similar to this?

DISCUSSION

456  "Quercus cerris has the largest range and broadest climatic envelope (from perhumid Cfa, Cfb via"
 This section of the discussion that brought in the climatic and ecological data was interesting and the reader could be more prepared for these results with clearer expectations about the role these factors could potentially play in shaping genetic diversity.

506   "This oak grows isolated from

507  Q. trojana on the Greek island of Euboea and differs morphologically by its coriaceous leaf

508  texture and the conspicuous white tomentum of the abaxial leaf surface that is made up of stellate

509  trichomes (T. Denk, pers. observ.) In addition, Q. euboica is characterized by special edaphic"
Can all these morphological and ecological characteristics of the species/populations be summarized in some kind of clustering approach that might allow comparisons between genetic variables and morpho/ecological variables?

515  "grouped (mostly) in a specific sublade (Fig. 3, File S4). However, more (morpho-ecological) "
  should be “subclade” I think


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: COMPARATIVE SYSTEMATICS AND PHYLOGEOGRAPHY OF QUERCUS SECTION CERRIS IN WESTERN EURASIA: INFERENCES FROM PLASTID AND NUCLEAR DNA VARIATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: Simeone et al

This paper nicely presents the genomic landscape of section Cerris in western Eurasia based on impressive sampling of plants, two genomes, and more or less standard sets of analyses.  The substantial break between Q. acustissima and Q. variabilis at the plastid level is interesting and a bit underplayed in the paper.  It would have been nice to have that end of the diversity within Cerris included in the the 5S-IGS study.  If those sequences are available, I encourage their inclusion.  The introduction could use some structure that would help the reader understand the research objectives associated with Cerris, but also to clearly lay out the possible zones of contact with Ilex species.  As these are potentially known, the added sampling here is useful in uncovering other areas of historical contact.  The previous study of Ilex is important, but it seems that the goals for the study of Cerris are somewhat obscured in the Introduction.  The main point is unpack the Introduction a bit, and to let the questions related to Cerris oaks drive the study.

Abstract:

Line 31 – “species coevolution” – not really what is intended, so change to -- reflected phylogeographic patterns originating from likely interspecific cytoplasmic gene flow within…
Line 33 – “siblings” -- change to descendent species, related species or sister clade or whatever might work better.

Introduction:

Line 64 -- “worst case” – don’t agree in this context, but if you want to convey that idea, it should be justified.  In the past, the “worst case” qualifier has been applied to oaks based on failure to meet the criteria for particular species concepts, but I don’t think this section was going there.

Line 90 – you might use leaf persistence or leaf lifespan, eg, Leaf lifespan is quite variable, ranging from…

Lines 127-129 – Consider turning these couple of sentences into a general objective statement for the study.

Lines 141 – 35S rDNA?  Is 35S correct?

Lines 146 and on – this is an important paragraph but it seems to focus mostly on Ilex phylogeography.  Consider using this as opportunity to better set-up the study of Cerris. For example, what are the main issues?  Will related species show regional patterns of distribution?  Where are the main contact zones with other Cerris and Ilex species?  Is the signature of that contact detectable in both data sets?  Perhaps add something about the hybrid origin of Q. afares (suber x canariensis), which is of broad interest as the only case of a suspected naturally occurring intersectional hybrid species.  It’s good to put that concept to rest.  These are just off the top of the head, so there could be more.

Line 150 – phylogenetic quality?  -- each main plastid group has a distinct geographic distribution that encompasses parts of the taxonomic breadth of sect Ilex and Cerris…not sure if that gets to what you mean?

Line 152 – “WAHEA”  -- define this acronym  -- perhaps by describing the main component bio regions that contain plastid lineages etc.

Materials and methods:

Consider clarifying the amount of new data that is being presented relative to the proportion of previously published data. This would provide a better understanding of how the new sampling and data fill the holes needed to address Cerris and interactions with Ilex.  I see the details in the Results, so perhaps consider this an opportunity to develop a sampling design statement.

For example, it seems that comparing east Asian diversity of Cerris is challenging at the plastid level, eg, divergent, and no shared haplotypes, and unfortunately not sampled for the 5S-IGS study.  That seems like a weakness in the sampling design.

Results:

Figure 2/3.  Nice.  Consider crossing referencing the biogeo areas (occidental, Vallonea, etc.) referred to in Figure 5 within the inset for the 4 groups in panel C.  Perhaps do something similar with haplotypes if possible.

Discussion:

Mostly good and informative.  It could be shortened by transferring certain descriptive aspects to the Results.  I encourage the authors to consider streamlining this section if at all possible.

Line 437 – “confirmed their status as most variable”  -- I get that but is this source of variation the best choice?  I’m not so sure given the most of inheritance, need to clone, etc.  I guess I’m thinking that 1000s of single copy genes would be preferred because of the potential to look at allele trees from many loci using a range of species tree gene tree reconciliation methods. I also wonder whether analyses designed to reveal levels of admixture, like STRUCTURE, would be appropriate.  The approach used here to identify hybrids appears to be somewhat ad hoc and visual, based on shared presence of gene copies that share network edges etc.

Line 442 – “Combined data” -- this sounds like an analytical process – Perhaps try something like, Insights gained from the combination of data sources were concordant.  It’s important to recognize that these insights are not based on a formal correlation analysis, but It would be interesting to consider a spatial auto-correlation approach to the two sets.

Line 608-618 – the writing in this section could use some work.

“derivedness” maybe try divergently derived in another sentence structure?

“nor” – and there was no obvious phylogenetic linkage between the Q. afares and the Q. crenata-suber lineage.

619-630 – this is a great point that will no doubt require looking at 500 or more loci and examining parental contributions at the allele tree level.  And now, with multiple oak genomes available, the possibilities for fine scale analyses using gene tree species tree reconciliation methods is possible.  The ability to discriminate current vs historical patterns of introgression is another way to possibly frame the interaction zones described throughout the paper.


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: INTRASPECIFIC PHENOTYPIC VARIATION IN LIFE HISTORY TRAITS OF DAPHNIA GALEATA POPULATIONS IN RESPONSE TO FISH KAIROMONES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an experimentally sound paper and will be of interest to readers because it explores the effects of population and clone on responses to predation. I commend the authors for taking up this topic—it is important to include more than 1-2 genotypes in studies such as these!
Please see below for my general comments. I think the Results and Discussion should be streamlined and reorganized so that the readers can more easily follow along. The authors also should more clearly report their statistical methods in the Methods so that the results can be more easily interpreted.

Good job in Intro with reporting background, though I do think that it would be useful to use more specific examples (see General Comments). The literature is well referenced and relevant.

In general, the language is clear, but the paper should be reviewed by native English speaker. E.g., Abstract line 18-19 isn’t structured properly, word choice in lines 362 (should be notably) and 394 (unfolds isn’t correct).

Figures:
Figure 1: put axes
Figure 3: could be supplemental
Figure 4: Why are the data for the treatment shown as dots and data for the random effects shown as bars? The legend description is also confusing (first paragraph).
Figure 5: put axes
Table 3: why is there a matrix of p-values? Is this the results of pairwise posthocs?

Raw data are supplied.
Experimental design: Within scope of journal.

Well-defined research question and clear statement of its usefulness. You are right that exploring one genotype within a population is problematic.

Line 108-109: curious what happens when you use “time since lineage established” as covariate. This is just a suggestion, I don't think it's necessary.

Line 200-201: what were your treatments and random factors? In general, you need more detailed description of actual models so that reader can properly evaluate your stats.

I cannot fully evaluate the validity of the findings because the statistical methods are not fully described. It seems like a glm should be used for all analyses, with treatment, population and clone as fixed effects and block as a random effect.
Validity of the findings: Difficult to assess validity because statistical models not reported. See above.

Conclusions need to be more clearly stated. In general, the organization of the Results and Discussion make it hard to keep track of the patterns. You have a lot of results to present, so organization is key. As stated throughout, I recommend organizing these sections by response variable rather than treatment.

However, I agree with the authors’ interpretations that there is inter-population and clonal variability.
Additional comments: Abstract line 12 – not sure conditions is the right verb
Introduction
Line 40-41: Plasticity is favored in variable environments and is important because it helps organisms in heterogeneous environments. It would be worth saying that here to distinguish it from general phenotypic variation.
Line 51: why not just call them kairomones instead of infochemicals?
Line 51: helpful to discuss the specific predator induced defenses (e.g. morphological defenses or behavioral shifts) rather than just saying that predation triggers phenotypic changes.
Line 73: how are these changes adaptive? E.g. smaller size would not seem to be adaptive at first glance.
Methods
Line 103: is “large lakes” an ecotype? I am not a Daphnia researcher, so this may be something that people in your field recognize, but if not, it should be clarified what this means.
Line 127: good job standardizing the number of generations
Line 131: I am confused about which individuals you are talking about for the remainder of this paragraph: the breeding and experimental individuals?
Line 214-217: would be better to give effect sizes rather than raw changes
Line 218-223: why aren’t p-values given for population and clone effects? Were population and clone treated as fixed factors?
Results, general comments: I find the organization of the methods confusing, but this could be because the models weren’t described in the Methods. I assume the fixed factors and their interactions were analyzed in the same statistical model. Why then are they divided into separate sections? I suggest re-organizing by response variable rather than fixed effect, for example, like your description of body shape.
Line 258-261: This interpretation should be saved for the Discussion.
Line 278: There was no description of the PCA in your Methods.
Discussion
Line 323: be more specific than “different organizational levels”
Line 326-327: Do you mean the mean of the trait vs the variance of the trait?
Line 340-342: worth briefly summarizing here even if briefly discussed before.
Line 344: be specific about why more bacteria.
I like how the “driving forces of phenotypic variation” section is organized. It is easy to understand. I recommend replicating this throughout the Discussion.
Line 351: Why was genotype a random factor if this is one of the main factors you are examining in this experiment?
Line 391: Confused by the word “variation” because it implies differences in diversity. Do you mean the identity of the genotype is more important?
Line 397: I like this section of the Discussion. It is well written and gives a good overview of the Discussion so that the reader has general take aways. Consider replicating throughout Discussion.
Line 451: If being slow is beneficial, why would Daphnia in the fish treatment not be bigger?


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: INTRASPECIFIC PHENOTYPIC VARIATION IN LIFE HISTORY TRAITS OF DAPHNIA GALEATA POPULATIONS IN RESPONSE TO FISH KAIROMONES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript presents data on the responses of differed Dapnhia clonal lineages from 4 different populations to predation risk (chemical cues). Although it is always interesting to see these kind of experiments showing variability in plastic responses to predation risk I feel that the study lacks some focus. It is well known that Daphnia clones exhibit variance in response to chemical stressors also to biotic stressors like predator kairomones.
In this sense is not clear how the authors are integrating results with their hypothesis. Also a better review on the relevant literature on this would help.
Experimental design: The results of this study come from laboratory tests and I have some questions about the experimental design and analysis of data that should be addressed before publication. Some justification of the chosen experimental design is also lacking. See comments below
Validity of the findings: The results show variability in the responses of different clones but I feel that they lack contextualization since no information is provided concerning the different predation pressures of the origin sites of the different clones. Moreover, some effect of the block design somewhat hampers the validity of findings. I also think that the discussion section lacks depth. See comments below
Additional comments: Although the manuscript is in general well written authors should make an effort to include some more relevant literature on this especially concerning all the plastic responses that have been observed in Daphnia exposed to predator chemical cues. In fact, responses such as feeding inhibition or increased metabolic costs have been also described for Daphnids exposed to fish kairomones and should be referenced. However, authors should integrate their results better. As such it is well known that There are genetic and population differences in the plasticity and in the magnitude of induced defences. The specific response of a population to predator’s chemical cues is influenced by the evolutionary history with respect to the presence of a particular predator in a particular habitat. Therefore, populations which co-occur with predators show local adaptation to habitat-specific predation regimes and stronger responses towards predators than naïve prey. In this sense it would be critical for authors to present some info of the different predation pressures from the different lakes?
At the same time, it is well known that different genotypes can also respond differently to the presence of predators, and this difference relies not on the presence/absence of a response but also on different combinations and magnitudes of the different traits showing that different responses can be uncoupled and evolve independently.
The discussion section and the important role of phenotypic plasticity and evolutionary potential of daphnid populations would have greatly benefited from an in-depth discussion on these important issues. Also a better justification and discussion of the following:

1-	Authors state that “The Jordan Reservoir is an artificial inner city water reservoir,
used for recreational purposes such as fishing since 1900 (Kubecka and Bohm 1991) and had been regularly stocked with fish (Seda et al 2000). Therefore, D. galeata of Jordan reservoir had the possibility to adapt to a high-predator environment for more than a century.” This is all fine but without some information concerning the predation pressure of the other lakes we cannot conclude that popJ was “better adapted” to fish predation pressure.
2-	Also on this why was Leuciscus idus used as a model? Any evidences that this fish species exists in any of the lakes where these populations were taken from? This is important again since different Daphnid responses have been shown towards kairomones of different fish species.
3-	Since Responses to fish predation risk is stronger when alarm cues from conspecifics are present and if the objective was to show plasticity and variability is response in a number of different traits why weren’t they used?
4-	Why didn’t authors prepared a single stock kairomones solution to be used throughout experiments? This would be the only way to guarantee that the kairomones concentration is the same and would eventually allow for testing all clones simultaneously. In fact, the significant block effect is hard to neglect.
5-	 I don’t understand why the different clonal lineages were maintained in control medium AND in kairomones solution for 2 generations prior to testing. If the induced responses are plastic, all clones should have been maintained in control medium (so as to remove any maternal effects) and only exposed to kairomones in the actual test. If I understood correctly the life history data reported correspond to a 2 generation exposure. Do authors think this might have affected the results?
6-	Also responses to fish kairomones are known to be concentration dependent, why did authors decide to use only one kairomones treatment? I think that using a lower kairomones concentration would have helped in comparing the magnitude of responses of the different clones.
7-	Why did authors addressed fitness without taking into consideration Age at first reproduction. This parameter is critical isn the response to predation pressure ( in natural conditions with predators present  Daphnids don’t usually get to the third , fourth broods so easily…). A better Proxy for fitness with these kind of experiments might be the calculation of R (intrinsic rate of population increase. Which is influenced greatly by age at first reproduction and size of first brood
8-	I am not sure if the number of genotypes within each population is sufficient but wouldn’t these results be better analysed using quantitative genetic analyses?
By analyzing the norms of reaction of the different clones, different micro-evolutionary paths can be analysed by correlation measures of fitness traits across environments. Maybe mean values and variances for each trait can be calculated and genetic variation of a trait among treatments (environments) will be compared using the genotypic coefficient of variation (CVG). Wouldn’t this be better to evaluate the evolutionary potential of each population?
9-	Authors state that males were eliminated from analyses. Shouldn’t the whole replicate be eliminated? Did those genotypes produced males in control medium?
10-	I think that the investigation of morphological responses interesting but it is strange to see that the size of neonates within the different treatments was not presented. This is important for the discussion (L451-455). Also measuring size at maturity could also give important clues.
11-	Authors state that ( L 357-362) “The main effect of ‘Genotype’ on the traits offspring and SGR implies that the presence or absence of genotypes within one population might have an effect on overall population survival. If the clonal diversity within one population is reduced and the majority of genotypes which produce relatively less offspring are present in a predator environment, the result could be of an overall low number of offspring in the following cohorts which would threaten the persistence of the whole population.” I don’t undertand this and it should be rephrased and discussed with the plastic responses and the chemical cues that induced them in mind. In principle these plastic responses are adaptive so we can expect that the genotypes showing more plastic responses (reproducing earlier at a smaller size and producing more and smaller neonates would be better adapted in an environment of higher fish predation pressure. As it is the sentence is too vague, too confusing.
12-	Finally, authors should be coherent throughout the manuscript and refer predation risk or fish kairomones when referring the results of their experiments and not predation or predator presence since no actual predation was tested nor were other cues ( visual , hydrodynamic) . (e.g. L21-22) . Also avoid the term vertebrate kairomones (L397). Again be careful when using the term “maturity” or early maturation since you didn’t assess age or size at maturity.


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: INTRASPECIFIC PHENOTYPIC VARIATION IN LIFE HISTORY TRAITS OF DAPHNIA GALEATA POPULATIONS IN RESPONSE TO FISH KAIROMONES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The introduction section is in need of focus and improved clarity. The information I would like to see in the introduction is:
1.	What is phenotypic plasticity (a definition – maybe something involving an interaction between genotype and the environment). Why is phenotypic plasticity important for evolution? (as opposed to selection on existing phenotypic variation)
2.	What do we know about how predators can influence life history traits and morphometric traits – how much is due to selection vs. plasticity? (do we know?)
3.	What is the main question the researchers are addressing that leads them to the hypotheses they make in the final intro paragraph

Related to these ideas, I have these thoughts:
-	The first paragraph is confusing. What is the main point the authors want to make? This is where it will be important to both define phenotypic plasticity and why it is important (from an ecological/ evolutionary standpoint) to know more about it. It feels backward to begin by talking about the importance of plasticity without first explaining what it is.
-	Lines 67-75: I’m not quite sure what main points the authors want to make. Line 72-73 is strange – plasticity is not necessary for adaptation. But maybe the issue is the meaning of “adaptation”. My expectation is that adaptation means evolution, which does not need to involve plasticity. Though plasticity may certainly evolve. I think the authors should explain what they mean by adaptation and why plasticity is necessary for adaptation to take place. Lines 73-75 are also not very informative.
-	Lines 76-87: This paragraph should define the research question inspiring your work. Right now, it’s pretty vague, about comparing clones in different populations and the major focus is on replicate numbers. That’s great that this study has greater replication at the clone level than other past studies; however, six clones still isn’t a lot to base an entire population on. What question(s) are you addressing? Beyond having more clones per population than anyone else? What specifically are you interested in examining? Why do you want to look at within population and across population variation in phenotypic plasticity? This paragraph should lead naturally to the next one where you describe your hypotheses/predictions, but right now the predictions come out of nowhere because there’s no research question.
Experimental design: This article needs some work in defining the research question and in describing the methods in sufficient detail.

There are some important details that are missing in the methods section.
1)	Line 105: These clones come from sediment cores from the four lakes. Were these sediment cores dated? Do you know the time span of the sediment slice that the clones come from? This is important for understanding the variation observed for each population. If the clones span 25 years from one lake but 4 years for another, this could be an issue to consider.
2)	What do you know about variation in predation pressure among lakes and over time in the lakes? Why do you expect to see variation in predation responses among these populations? This important ecological aspect is barely addressed in the study. The first mention of the predation environment in any of the lakes is line 409 and I don’t see any mention of the other three lakes. I find this very perplexing. How can you study GXE interactions with so little discussion of the environment that the populations come from?
3)	Lines 199-206: What were the actual statistical analyses performed to test the various hypotheses? As is, the statistical methods are completely insufficient to understand the analyses that were performed and then interpret the results and assess the discussion section.
Smaller methodological details
4)	Line 115-116 – Why do you starve the fish to avoid the Daphnia receiving alarm cues from the kairomones? Isn’t that the point of the study, to make the Daphnia feel the threat of predation and respond to it?
5)	Line 167-169 – Were the Daphnia measured alive or preserved in ethanol?
Validity of the findings: I am unable to assess the results and conclusions section because there is so little information available in the methods on what was done (statistical methods in particular). I also want to know about the predation regime in the lakes to know how to interpret the variation seen in plastic responses to fish kairomones.

Based on what I understand from the results tables, one question I have is about why so many measures of fecundity were included as response variables. Wouldn’t there be an issue of these variables being  highly correlated with one another (e.g. size of brood, number of broods, total number of offspring)?
Additional comments: The authors have undertaken an impressively large study. They have rigorously measured fitness and morphological traits of four populations of Daphnia galeata in a control and predator cued environment. The major issue I see with this manuscript is lots of detail in some areas (e.g. figures and results) but little attention to the big picture (e.g. important background information, what is plasticity and why does it matter for evolution, what ecological settings do the four populations experience, why did the authors measure what they measured). I also see an important lack of detail in key aspects of the methods (what statistical tests were performed??).  While I think this study could be very interesting, at present there are some major issues that need to be addressed.


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: INTRASPECIFIC PHENOTYPIC VARIATION IN LIFE HISTORY TRAITS OF DAPHNIA GALEATA POPULATIONS IN RESPONSE TO FISH KAIROMONES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript presents data on the responses of different Dapnhia clonal lineages from 4 different populations to predation risk (chemical cues).

The authors have reviewed the manuscript addressing most of mine (and other reviewers ) comments.
Experimental design: The results of this study come from laboratory tests  and authors have satisfactorily replied to my questions/comments. Most details missing are due to the size and workload necessary, which I fully understand and other analysis are being prepared for different papers. Details of fish presence in the different collection sites are now provided.
Validity of the findings: I continue to feel that effect of the block design somewhat weakens authors findings but the statistical analysis section was improved and these results deserve to be published
Additional comments: I agree with other reviewers that this study is important to show variability within Daphnid populations towards predation risk. The results are interesting and deserved to be published. Authors did a good job reviewing the manuscript and correcting some details while adding the necessary information on methods and statistical analysis. Main concerns/ comments and suggestions made by reviewers were addressed in this version which I think now meets the standards for publication in PeerJ.

 Since authors were not certain of having found the right studies concerning metabolic / feeding responses of daphnids to fish predation risk I leave some suggestions of papers that can be cited :

Rose, R. M., Warne, M. S. J., and Lim, R. P. (2003). Exposure to chemicals exuded by fish reduces the filtration and ingestion rates of Ceriodaphnia cf. dubia. Hydrobiologia 501, 215–217. doi:10.1023/A:1026284314506

Beckerman, A. P., Wieski, K., and Baird, D. J. (2007). Behavioural versus physiological mediation of life history under predation risk. Oecologia 152(2), 335–343. doi:10.1007/S00442-006-0642-6

Pestana JLT, Baird DJ, Soares AMVM (2013) Predator threat assessment in Daphnia magna: the role of kairomones versus conspecific alarm cues. Mar Freshwater Res 64:679. doi: 10.1071/MF13043

Pijanowska, J. (1997). Alarm signals in Daphnia? Oecologia 112(1), 12–16. doi:10.1007/S004420050277


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: INTRASPECIFIC PHENOTYPIC VARIATION IN LIFE HISTORY TRAITS OF DAPHNIA GALEATA POPULATIONS IN RESPONSE TO FISH KAIROMONES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The overall structure (introduction, methods, results, discussion) is appropriate, but I feel this article needs a good deal of revision to be ready for publication. The lack of clarity, abundant detail in some cases and lack of detail in others hampered my ability to read, understand, and evaluate this manuscript.
Experimental design: I am not totally sure the experimental design is appropriate now that I know more about the study. The nature of the predation regime in the study lakes is a really important thing to understand here.

I still see very limited information about the predation regimes in the four study lakes. I believe the new information is in the supplemental table S1. There is a row showing the number of kg/ha of fish in each lake. This tells us very little about the predation regime from the perspective of the daphniids in the lakes. When were the fish surveys conducted? At the same time of year for each lake? What kinds of fish are these? Are they planktivorous? Also, are any of these the type of fish used for the kairomone assays?

If you want to know why you see variation in predation kairomone responses, it seems to me that the predation regime that the population experiences is important for interpreting that variation. Based on the response to Reviewer 2's comments It sounds like the fish used to produce kairomones is a model species but maybe not a species that lives in any of the lakes? I would find this problematic if this is true. But this should also be made clear in the text of the paper.

I am disappointed that a study focusing on the plasticity of predation responses in natural populations gives so little attention to describing or trying to understand the selection pressures in the lakes. How can you or the reader hope to interpret the variation that you observe?

In addition, I still do not know the time periods when the Daphnia were hatched. The authors mention they’re from the same 5 year time span in each lake. What time span is that? Does it correspond with when the fish densities were calculated? Just citing the studies that used these cores previously is not enough information. Also, from having done paleolimnological work in multiple lakes myself it seems kind of unlikely that there was a sediment slice for each lake that was deposited during the exact same 5 year time span. Is this really what the authors mean?
Validity of the findings: It s still a bit difficult for me to assess the findings since I don’t know enough about the environmental pressures these Daphnia populations experience.

I am also concerned with the way the authors have discussed the block effect. If the block effect is significant I think you just need to take that variation into account while seeing if other variables are significant. You can’t just ignore the fact that it’s significant. I totally understand why blocking needs to be done, but you can’t ignore a significant block effect (lines 411-413)
Additional comments: I appreciate the authors providing some additional information in response to the reviewers’ comments. However, I am really struggling to read through the manuscript and understand what the authors have found. I think the main issue is that a lot of details are reported but without a coherent structure or story. There are also important things that are not shared in enough depth (e.g. predation regime).

My recommendation is to have several colleagues provide feedback to help shape this manuscript and make it easier to understand and have a clear message. It is just too much work right now to figure out what the authors are trying to get across and I cannot do my job as a reviewer to assess the strength of the science. I would also suggest focusing on the results that support your key finding rather than listing all the results in the order they were entered into the models.

I recently learned of this blog post that gives some great tips for scientific writing. You might like to take a look! https://dynamicecology.wordpress.com/2016/02/24/the-5-pivotal-paragraphs-in-a-paper/


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: MACHINE-LEARNING-BASED QUANTITATIVE ESTIMATION OF SOIL ORGANIC CARBON CONTENT BY VIS/NIR SPECTROSCOPY
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1) Clear, unambiguous, professional English language used;
2) Both the introduction and the background are well presented and the references listed are also relevant.
3) Structure conforms to PeerJ standard.
4) Figures are relevant, well labelled & described. It is suggested that the author needs to use different colors and different line types to better describe the sample data, preferably color figures.
5) Raw data supplied.
Experimental design: 1) Within Scope of the PeerJ journal.
2) Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap.
3) Structure conforms to technical & ethical standard..
4) Methods described with sufficient detail & information to replicate.
Validity of the findings: 1) Data is robust, statistically sound, & controlled. 2) Conclusions are well stated, linked to original research question & limited to supporting results.
3) Speculation is welcome.
Additional comments: I would like to acknowledge the authors for the tremendous amount of work they have put into developing the manuscript. This article uses feature selection algorithms and multivariate modeling methods to estimate SOC content in arid regions. The overall level of English expression is very good, but some detailed parts need to be improved, especially in the section of Material and methods. I recommend acceptance of the manuscript after revision following the specific comments listed below.

Some specific suggestions:
Line 49: please replace “efficacy” with “feasibility”.
Line 52: please replace “SVM-RFE” with “RF -SVM”. Same as below.
Line 54: please replace “mainly were in” with “were mainly within”.
Line 58: The sentence of “For the testing set, Rt 59 was 0.79, RMSEt was 0.19 %, and RPD was 1.61.” is not very clear. It's best to connect with the last sentence.
Line 85: please add “, and ” before “therefore”.
Line 86: Who does “it” refer to? Please revise.
Line 90: replace “conteny” with “content”.
Line 91: please add “including ” after “mainly”.
Line 110: please replace “partial ordinary least square regression ” after “partial least square regression”.
Line 133: please replace “this context ” with “these backgrounds”.
Line 164: please replace “content” with “property”.
Line 172: please replace “selected” with “collected”.
Line 173: please replace “screen” with “mesh”.
Line 189: please remove “also”.
Line 201: better remove “using”.
Line 204: please replace “if” with “is”.
Line 204-206: “PLS if often employed to address heavy multi-collinearity among the variables, which is very common in spectral datasets due to the small increment of wavelengths.” Please rewrite this sentence, it is difficult to follow.
Line 253: Please add a period to the end of the sentence.
Line 262: Please tilt Y.
Line 263: Please revise the sentence, “The algorithm was implemented in were implemented with the …….”.
Line 276: Please revise the sentence, “…… will be used to split the tree each time a split is to happen”.
Line 278-279: Please revise the sentence, “it is hence very possible the influence of the strong predictor(s) is avoided and different trees can be produced.”.
Line 319: please replace “content” with “contents”.
Line 330-331: Please revise the sentence, “……the performance of model could be under threat.”.
Line336: Please add a space in the sentence of “Rt was0.83”.
Line348: please replace “spectra” with “spectral”.
Line 367: Please add a space in the sentence of “we could learn thatthe”.
Line398: please replace “Compared” with “comparing”.
Line398: please replace “concentrate” with “concentrated”.
Line 401-402: Please revise the sentence, “in the spectral wavelengths Hou et al. (2014).”.
Line 453-455: Please revise the sentence, “All three algorithms consistently identified the wavelength segments of 745-910 nm and 1911-2254 nm the most effective spectral regions to detect SOC content.”.
Figure 2: It is better to distinguish five curves by using different colors and different line types.
Figure 4: Please revise the sentence “SVE-RFE”.


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: MACHINE-LEARNING-BASED QUANTITATIVE ESTIMATION OF SOIL ORGANIC CARBON CONTENT BY VIS/NIR SPECTROSCOPY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper has grammatical errors, and unclear uses of English in places.  Thorough review for language and readability needs to take place before it can be seriously considered for publication.

References are sufficient and thorough and field background is provided.

The raw data for the spectra is shared, but the SOC contents were not provided. Those are required for anyone to validate if the methods proposed by the authors produces consistent results with this data set.

Statistical methods should be simplified, too much detail currently and could be made cleared with cleared language.

Tables and Figures contain duplicate information and could be simplified and merged to reduce the number of tables and figures. This would make interpretation of the results easier.
Experimental design: The research question is well defined. However, the authors do not justify sufficient why the methods used were selected for this study. They do establish that limited work has happened with these soils, which does merit examination. However, they are other statistical tools that have been used in recent (last 5 years) literature (which I provide in the general comments section) that are not included. The authors need to justify why they used the methods they did.

Additionally, the rationale for the hyperion simulation is not provided. This analysis is not meaningful for a number of reasons. The first is that direct soil measurements require bare soil and the authors did not establish of bare soil with no vegetation cover occurs in this region. Second, satellite measurements have additional factors such as atmospheric effects, varying levels of solar radiation and angle, calibration effects etc. None of these factors were simulated in their lab analysis, which makes their simulation a poor representation of reality. Additionally, new tools such as EnMAP from DLR are planned, and the rationale for simulating hyperion is not established. Hyperion was a test program, and a important one, but it is not the future of hyperspectral satellite imaging. A study designed for the newer proposed tools would be more meaningful.
Validity of the findings: The analysis is robust and the analysis is sound. Particularly the use of cross validation and an independent 50-50 training and test data set selected by the Kennard-stone algorithm was defensible.

However, the conclusion I not well stated and is written more as an abstract. A clear conclusion needs to be written.
Additional comments: Line 56: indicate that Rt is the testing set results
Line 68: Carbon stocks not stock
Line 69: Can delete “Despite its small coverage, the wetlands store nearly 1/6 of all the carbon stock on Earth. “ already cleared stated with previous sentence
 Line 77: This sentence is poorly worded and confusing
Line 84: Delete unreasonable, that indicates a value judgement that should not be stated
Line 89: in arid regions, not in the arid regions
Line 90: SOC content not conteny
Line 94: Confusing grammar
Line 95: Have reported that
Line 96: change eco-friendly to non destructive
Line 111: This is true of the historical literature, but not for publication over the past 5 years. There have been a number of machine learning focused papers i.e. (Rossel and Behrens 2010; Chun et al. 2012; Doetterl et al. 2013), some of which are cited in the next paragraph. Need to state that initial approaches for VIS/NIR spectroscopy included the methods stated
Line 117: these papers and others have used a variety of other machine learning tools: ex. Cubist models, ANN, gradient boosting etc. Some of which have had more success than random forest and SVM. Need to justify where SVM and RF were used specifically.
Line 132: Why is this the case? This is an unsubstantiated assertion
Line 139: This seems like a very bold claim. Can the authors provide a reference to justify this statement?
Line 159: Report soil types according to an international soil system, either the WRB or USDA systems
Line 192-194: Justify why these smoothing methods were selected
Line 197: Good justification for training sample selection, and 50-50 split is sufficient and defensible.
Line 199: Need to justify why these particular models were selected
Line 234: How were p and Q selected? Was it is based on RMSE minimization?
Line 271: Mutange reference is wrong, please site correct reference.
Line 284: Should be a new subsection, and explained earlier in the methods.
Line 292: Gao et al 2011 is not the original reference for this convention. See (Chang et al. 2001)
Line 296: I don’t see the justification for the hyperion simulation. It is not a true simulation as this study does not have all the atmospheric and lighting condition variation that comes with satellite data. Additionally newer proposed satellite programs such as EnMAP from DLR will be the future of satellite hyperspectral imaging.
Line 441: Conclusion is too long. Needs to be simplified to one paragraph with clear conclusions and statements from the research. Currently reads like an abstract.

All tables: Caption needs to completely explain the table, including what the abbreviations mean.

Figure 1. Include source for geographic data
Figure 2. Add more explanation about what types of soil samples were used in the caption
Figure 3. Not needed. This information could be merged into table 2
Figure 4. Not needed. Duplicates info in table 3.
Figure 5. Not needed. Duplicates info in table 4.


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: MACHINE-LEARNING-BASED QUANTITATIVE ESTIMATION OF SOIL ORGANIC CARBON CONTENT BY VIS/NIR SPECTROSCOPY
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The author has made revisions to the questions I have listed. In general, after the revision, the article’s quality has been greatly improved. There is only one suggestion below that needs to be revised:
Line 215-218: “Due to its advantage of dimension reduction, synthesis, and solving multi-collinearity problems among independent variables, PLS has been adopted as a robust and reliable approach in spectral quantitative research.” Please rewrite this sentence.


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: MACHINE-LEARNING-BASED QUANTITATIVE ESTIMATION OF SOIL ORGANIC CARBON CONTENT BY VIS/NIR SPECTROSCOPY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article is clearly written, and the number of unclear sentence is limited.

Sufficient literature has been cited.

The article is professional structured. Currently there is duplication in the information in Figures 3, 4 and 5, and Tables 2 to 6. The figures and tables should be simplified to avoid the duplication of information.

The results and hypotheses are relevant and self-contained.
Experimental design: The research is original primary research. The research question is well defined and meaningful but more work is still needed to explain how it fills a knowledge gap. The following needs to be clearer:

The authors needs to explain more clearly why these soils are different any why techniques applied to Alfisols, Entisols and Ultisols and Molllisols won’t necessarily apply to wetland soils. What is different about how wetland soils will interact with the methodology used in the paper needs to be explicitly stated and explained. Wetland soils are under investigated in reflectance spectroscopy, and in soil science in general, the authors need to make the case of what is different about them more explicitly.

Additionally, the rationale for the ACO-iPLS methodology inclusion needs to be discussed and the rationale for the hyperion simulation need should be in the introduction.

The methods are described in sufficient and clear.
Validity of the findings: The findings are valid and clearly explained, and the data is robust and statistically sound. A 50/50 training/testing split is very robust and defensible, and is more robust than typical, so excellent work by the authors in that regard.

The conclusions are more clearly stated, and the manuscript has been improved. One outstanding question for the discussion is how might the signal:noise compare between the field spec 3 and the simulated hyperion data. How does that influence accuracy and detection limits.
Additional comments: The manuscript has been improved. The main outstanding issue is more improvement is needed in how the introduction sets up the rationale for all aspects of the paper.

Also the authors have not provided sufficient justification for continuing to include Figures 3, 4 and 5. The information is duplicated amongst the information with the Tables. The quality of the paper is reduced by duplicating information in the tables and figures as it makes the paper more complicated to read and review.


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: MORPHOLOGICAL CHARACTERIZATION OF VIRUS-LIKE PARTICLES IN CORAL REEF SPONGES
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript by Pascelli et al describes virus-like particles observed within coral reef sponges.  This is clearly an initial, observational study of these viruses, but does provide a first look at the possible viral morphotypes inhabiting the sponge holobiont.  I have a few comments regarding issues I think need to be addressed within the manuscript, mostly to increase clarity regarding what the results represent.

General Comments:

1) In the Abstract and Introduction especially, the use of “host” is a bit confusing at times regarding whether it means “species of sponge where the virus is found” versus “organism that the virus infects”.  Can the authors find a way to standardize this language throughout the manuscript to avoid confusion?  For example, in Line 76, the use of the term “host-specific” to refer to the unique community of viruses within sponges is misleading (or incorrect).  To determine if a virus is “host-specific” there needs to be evidence showing that the virus only infects one host.  I know that the title of the Laffy et al paper uses that term, but they are correct later in the paper when they use the terms “habitat-specific” and “host species specific”.

2) I think there should be some statement in the Discussion regarding the possibility that some of these VLPs might not be viruses.  Yes, that is inclusive in the definition of “VLP”, but this nuance may be lost on non-specialist readers, and thus an explanation is in order.  For example, Figure 3K shows an obvious virus (and it is a very nice image!), but Figure 2D could be some type of inclusion bodies.  This is a limitation of using TEM to identify viruses; obvious head-tail structures are easily identified, but it is difficult to distinguish other morphologies because there are many other things in cells and in the environment that are within the same size range of viruses and have dark staining in TEM images.

3) Within the Abstract, the phrases “varied in prevalence” (lines 27-28) and “abundant” (line 34) are a bit misleading and should be changed/removed because the authors do not use quantitative methods to investigate the relative or absolute abundances of viral morphotypes in the samples.  I appreciate the authors not describing their results as quantitative throughout the remaining part of the manuscript, but I also think that the authors can insert a statement in the Discussion regarding (i) the fact that the dataset is observational and (ii) suggesting that future studies be conducted in a quantitative manner (i.e., analyzing a known area for each thin section sample, using a known/consistent volume from the mucus and aquiferous samples, not using CsCl gradients because this can remove specific virus types that fall outside of the density range selected such as lipid-containing viruses, using ultracentrifugation to deposit viruses onto grids instead of adsorption, and observing a fixed number of viruses per sample such that the percent of each morphotype can be determined – see Brum et al 2013, ISME J, for example).

Minor Comments:
Line 78:  The paper by Laffy et al shows that most of the *identifiable* virus sequences were Caudovirales.  Like most environments, most of the viral sequences in that study were taxonomically “unknown”.

Line 139: Please describe the method for sample preparation for “TEM analysis”.  I assume it is the adsorption method described in the next section (lines 147-148).

Lines 147-148:  Please add a few more details regarding the TEM grid preparation.  What method was used to render the grids hydrophilic?  How long was the mucus sample allowed to adsorb to the grid?  Was the grid then rinsed a few times with purified water?  I also assume that the grid was rinsed after staining with UA.

Line 214: A beaded virus that varies in the number of beads would be a completely new discovery (as far as I know).  There should be some discussion of this, regarding whether this is a new virus family, or possibly a part of the sponge itself.  For example, these look a bit like chains of magnetosomes (e.g., Lohsse et al, 2011, PLoS One).

Lines 235-240: The significant loss of virus tails through deposition of viruses onto grids via ultracentrifugation was shown *not* to occur in seawater samples (Brum et al, 2013, ISME J).  And in fact the non-tailed viruses dominated the viral communities throughout the oceans.  Recently, a new family of non-tailed bacteriophages has been discovered (Kauffman et al, 2018, Nature), further supporting the hypothesis that many marine viruses may be non-tailed bacteriophages.  The authors may wish to amend this section of the manuscript to state that their specific preparation methods (i.e., CsCl, centrifugal concentration devices, etc.) may have resulted in damage to the viruses, as this has not yet been tested.

Lines 288-289:  Most bacteriophages studied have a wide variety of host ranges (see Flores et al, 2011, PNAS, for a metaanalysis of this).

Figure 2B:  It is a bit difficult to distinguish the arrow heads in this image.  At first I thought they were part of the virus.  Can the authors make these larger, or make them stand out from the image in some way?