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: DISTRIBUTION OF BACTERIAL COMMUNITIES ALONG THE SPATIAL AND ENVIRONMENTAL GRADIENTS FROM BOHAI SEA TO NORTHERN YELLOW SEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the article is clear and well written. However, there are enough small grammatical errors that I think the authors would benefit from having it edited by a native English speaker in order to avoid any ambiguity. The figures are well constructed and add to the overall understanding of the text. I do think, however, that a figure perhaps in the form of a few section plots of important environmental parameters across the sampling region would greatly benefit the understanding of the text and give the reader a better sense for the study area. The supplemental data and figures are accessible and clear to understand. I find Figure S4 highly valuable, however, it is slightly confusing because the color scheme used to identify each of the three regions are the same colors used repeatedly in the Venn diagrams to represent different stations. Consider using a different color scheme to identify each region.
Experimental design: The study goals are clearly stated in the introduction as well as the motivation for undertaking the study specifically in this region. The methods are well described and within the current standards for the field. I think seeking to understand the factors that determine the composition of bacterioplankton in the marine environment as well as factors that may limit their dispersal are important factors to consider. One of the main questions posed by the authors is whether geographic distance/depth contribute more than environmental factors to the structure of bacterial communities. However, truly separating the effects of these factors is difficult as many of them co-vary together. And the authors acknowledge in their results that their statistical analysis indicates they are at least partially dependent on each other, making their question difficult to clearly answer. I think the authors could benefit by basing their analysis on the physical structure of the water column across their study region and using a water mass analysis rather than simply distance/depth to determine what may be more important in structuring bacterial community composition and potentially limiting dispersal. Because the study region contains three distinct bodies of water (the Yellow River, the Bohai Sea, and the Yellow Sea) I think this would be feasible and allow the authors to make clearer conclusions.
Validity of the findings: The authors perform a rigorous statistical analysis of their data to help support their findings and very clearly address their first posed question. As stated above, I think their analysis for the second question posed could be strengthened by basing their analysis on a physical structuring of the water column and investigating the influence of various water masses and mixing processes rather than geographic distance and depth which co-vary with many environmental factors.
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: DISTRIBUTION OF BACTERIAL COMMUNITIES ALONG THE SPATIAL AND ENVIRONMENTAL GRADIENTS FROM BOHAI SEA TO NORTHERN YELLOW SEA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The writing has been properly clarified in response to most of my comments. The authors have done well at improving the English writing and generally increasing the clarity and depth of explanation for many sections.
Experimental design: Thank you for including multiple test corrections in your statistical analysis.
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: INTRA-COLONIAL DIVERSITY IN THE SCLERACTINIAN CORAL, ACROPORA MILLEPORA: IDENTIFYING THE NUTRITIONAL GRADIENTS UNDERLYING PHYSIOLOGICAL INTEGRATION AND COMPARTMENTALISED FUNCTIONING
Review round: 1
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: In this study, the authors examine nutritional gradients within coral colonies finding distinct heterogeneity in symbiont cell number, protein, lipid and energy content across the colony.  The rationale for carrying out the study is strong.  The authors state that this knowledge could contribute to coral nursery restoration practices where fragmenting colonies is key to propagation and increasing survival of fragments increases yield.
Overall the work is strong.  The experimental design and methods are well done and described.  The results are backed up with appropriate statistical rigor.  I believe the study is a good contribution to coral physiology and restoration science.
I do feel, however, that the manuscript could be much stronger than it is currently with some work on the writing.  To me, the message of this work is fairly straightforward and could be told in a much, much shorter manuscript.  Succinct, but important, results call for a relatively short paper.  I put this work into that category.  The Introduction and especially the Discussion are overly long.  The authors can tell a more succinct and better story.  In the discussion, in every section, the authors recap what they have already said in the results.  Get rid of these!  You don’t need to say things twice.  If you are struggling with this, then combine the results and discussion together.  The discussion desperately needs subheadings.  The discussion of the lipids is way too detailed and strays from the central theme of the paper.  I believe that much of this could be omitted.
Another problem that makes the lipid section of the results remote to the average reader (me) is the lack of any background on lipid chemistry.  The authors need to think about how to bring the uninitiated up to speed in short order before they dive into their data.  I would suggest a simple table describing the lipids discussed, whether they are structural or storage etc.  This could go in supplemental materials.  Without this background it is very hard to follow this section.
Minor concerns/suggestions
1.	Figure 1 is very important to understand how the sampling was done.  But the authors could add a section somewhere – intro, or results and figure legend? – that describes in words how A. millepora grows – as multiple finger-like branches arrayed roughly in 2 dimensions.  This will help the reader understand how the branched were sampled.
2.	Put N numbers in the figures
3.	Discuss the parts of figure 2 in order.  If you keep the text the same, then you need to rearrange the figure.
4.	What does proximate analysis mean?  I am not familiar with this term
5.	Stony corals are not soft corals – they are distantly related.  There are multiple citations in the manuscript where soft coral findings are lumped with stony coral findings.  These either need to be called out as different or removed.  For example, whereas there is ample evidence that symbionts are transported around soft coral colonies, there are no data in hard corals to suggest this.  Very different biology between the two.  Compare apples to apples.


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: INTRA-COLONIAL DIVERSITY IN THE SCLERACTINIAN CORAL, ACROPORA MILLEPORA: IDENTIFYING THE NUTRITIONAL GRADIENTS UNDERLYING PHYSIOLOGICAL INTEGRATION AND COMPARTMENTALISED FUNCTIONING
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript’s main idea and general focus are good. It has been a long-standing goal in coral biology to understand the relationships between polyps within a colony.  Specifically, It has been a major focus to understand if and when there are nutritional allocations within the colony that allow for subfunctionalization of different parts of the colony.

The authors do a good job summarizing the motivation of their experiments in the introduction. However, some of the statements asserted as fact in the introduction are not supported by the references provided. For example, the reference provided in line 75 refers to a mass-spec experiment trying to determine the proteins that accumulate in the skeleton. Unless I am mistaken, this paper does not speak to whether or not “protein fuels tissue growth, organogenesis, and calcification.” The same can be said about line 272 and 273. The authors should take more care in writing the introduction and discussion not do overstate conclusions or observations made by other authors.

Generally, the data presented here suggest some significant differences in the nutrition allocation across the coral. However, many of the differences are marginally significant. Given the variance in the data (all error bars in figures are SE) and the degree of multiple testing within the paper, it is hard to interpret the biological significance of the findings.
Experimental design: The experimental design attempted to be replicative both technically and biologically. The authors conducted well motivated experiments. However, they saw high variance in most of their measurement precluding robust statistical significance.

Major comments:

The authors aimed to determine if there is differential nutritional compartmentalization along across coral colonies of two different size classes. The authors deploy a sophisticated set of analytical approaches to address this question. However, it is not clear based on the paper how many of the stated conclusions are supported by significant statistical tests (Examples of this are discussed below). It is important for the reader to know, which results are significant in both the original ANOVA and post-hoc tests. Please report the both of these P-values for each statement in the text to help clarify this for the reader. For example, the end of Line 205.

I would like to see raw data points plotted over the SE plots for Figure 2 and 3.

In general, it is not clear how meaningful discussing the trends in the data are when the underlying differences are not significant (especially when contradicted by other corals in a different size class).

For example:

Line 196-198: "Within the size classes, there were no significant differences in total lipid (P>0.05) (mg g sample-1) (Fig. 2a), although both edge positions contained lower lipid compared to the next, immediate position."

Line 198- 200: "A similar trend was apparent in zooxanthellae densities for the larger
colonies (cells cm2 -1) (Fig. 2e), whereby the density at the edge was again lower than the next immediate position."

Line 206-208: "The caloric enthalpies showed that, for both size classes, the edge and next immediate position contained the highest energy contents, while the S-centre and L-centre and L-33 positions were low (Fig. 2f)."

There are many examples of this within the text, and they should be removed or modified to avoid confusing the reader into thinking these trends are significant.
Validity of the findings: As stated above, more effort should be made to ensure the reader can easily determine, which statements and claims came from significant statistical analysis. These limitations of the study should be addressed directly in the discussion.

Minor comments:

Line 67: 'reasonable to hypothesise that this' - spelling

Line 129: Elaborate on air-spraying method.

Line 135-137: Redundant with next paragraph

Line 142: Not clear how to crush something in a mortar and pestle under a french press. Can you please elaborate?

Line 153: Is four digits the appropriate number of significant figures for this measurement?

Line 212: "The proportion of storage lipid in the larger colonies followed a similar.." It is not clear what this proportion is similar to based on the text.

Figure 1: It would be helpful to reorder the figure panels to match the order in which they are discussed.
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: INTRA-COLONIAL DIVERSITY IN THE SCLERACTINIAN CORAL, ACROPORA MILLEPORA: IDENTIFYING THE NUTRITIONAL GRADIENTS UNDERLYING PHYSIOLOGICAL INTEGRATION AND COMPARTMENTALISED FUNCTIONING
Review round: 2
Reviewer: 1

Output:
Basic reporting: All looks good.
Experimental design: Looks good.
Validity of the findings: Looks good.
Additional comments: The authors have addressed my concerns/suggestions.  I would still ask that they simply define 'proximate' in the manuscript - they don't have to remove it, but just define it for the uninitiated.


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: INTRA-COLONIAL DIVERSITY IN THE SCLERACTINIAN CORAL, ACROPORA MILLEPORA: IDENTIFYING THE NUTRITIONAL GRADIENTS UNDERLYING PHYSIOLOGICAL INTEGRATION AND COMPARTMENTALISED FUNCTIONING
Review round: 2
Reviewer: 2

Output:
Basic reporting: No Comment
Experimental design: No Comment
Validity of the findings: The authors did a fine job indicating significances in the updated manuscript.

However, I am still worried about discussion "trends" in data as "biologically meaningful." In many cases, there is no support that the means are actually different (i.e. not statistically significant).

I suggest removing these discussions from the paper. because if these experiments were done again, these trends could go in the opposite direction. Illustrating this, many of these trends, are not supported across the different coral size classes.
Additional comments: No Comment


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

Input:
Title: ESTABLISHMENT OF AN IMMORTALIZED MOUSE DERMAL PAPILLA CELL STRAIN WITH OPTIMIZED CULTURE STRATEGY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Relevant prior literature on human immortalized human dermal papilla cell line should be referenced. For example, Shin et al., Establishment and characterization of an immortalized human dermal papilla cell. BMB Rep. 2011 Aug;44(8):512-6. Also, Won et al., The establishment and characterization of immortalized human dermal papilla cells and their hair growth promoting effects. J Dermatol Sci. 2010 Dec;60(3):196-8.
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: ESTABLISHMENT OF AN IMMORTALIZED MOUSE DERMAL PAPILLA CELL STRAIN WITH OPTIMIZED CULTURE STRATEGY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors have clearly referred to relevant literature.  However, sometimes the prose is confusing.  For example in the introduction the phrase 'when the culture system was changed to 2D' is misleading.  This suggests that intact DP which are being placed in 2D are in a culture system as well, which is not the case.  The idea that intact DP are in a 3D culture system is also suggested within the paper, which again is incorrect.  Cells within intact DP are in a 3D environment, but this is not a culture environment.  The formatting and structure of sentences should be checked to ensure they are not misleading.
There are also numerous typos in the manuscript-eg Western Bloting rather than Blotting.
Experimental design: The aim of the paper is to 'optimise isolation and culture' methods of DP and 'establish an immortalised line'.  The reason for establishing an immortalised line is not clear-suggestions as to why an immortalised line would be beneficial over a primary line would help here.
One of the reasons for performing this work is that DP cells are difficult to isolate, however the method of isolation in the paper appears the same as that described within the literature.  Please explain how the isolation method is changed to make DP isolation easier?
n numbers should be included-i.e. how many times were the immunostaining protocols performed on cells.  How many times was the immortalisation reversing protocol attempted.  Did it work on multiple iDP lines?
The control media used is dMEM with 10% FBS, however this is media commonly used for human DP cells.  Mouse DP cells (which are being assessed here) are usually grown in dMEM with 10% FBS, with additional growth factors such as FGF2.  Why did the authors not have murine DP media as the control medium?
Temperatures used for PCR should be indicated.  Product size should also be indicated in Table 1.
Validity of the findings: One of the main results of the paper is that the authors optimised a media to culture DP cells.  Optimisation suggests that several media were attempted, but these are not shown.  How many other medias were attempted, and what enabled the conclusion that the selected one was the best.  In addition, please explain how the optimised media is different from that which is usually used.
Another finding in the paper is that iDP6 is the optimal induced line as it has similarity to primary DP cells.  However, the profile of primary cells is not shown.  To make this argument more convincing, the PCRs and staining (Figures 4/5) should include DP cells which are not immortalised.
What do the authors mean by 'agglutination'?  Please add a sentence to explain.
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: ESTABLISHMENT OF AN IMMORTALIZED MOUSE DERMAL PAPILLA CELL STRAIN WITH OPTIMIZED CULTURE STRATEGY
Review round: 2
Reviewer: 1

Output:
Basic reporting: N/A
Experimental design: Methods are much better, with n numbers and more detail on the experiments performed.
Validity of the findings: N/A
Additional comments: It seems from the expanded methods, and the response to reviewers that the main addition to the media, was sodium pyruvate and non-essential amino acids, and FGF2 had little effect by itself.  This should be reflected in the discussion, especially page 6, lines 12-16.

page 6, line 28-add the following reference after 'But exactly how many kinds of cells are in DP remain to be discovered'
Epithelial-Mesenchymal Micro-niches Govern Stem Cell Lineage Choices.
Yang H, Adam RC, Ge Y, Hua ZL, Fuchs E.
Cell. 2017 Apr 20;169(3):483-496.e13. doi: 10.1016/j.cell.2017.03.038.

I wouldn't call primary DP cells 'wild-type'.  This makes it seem like the others were from a transgenic/mutant mouse.  Just call them primary.  Update text and the figure legends for Figure 4/5 accordingly.


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: REMOTE COLLECTION OF MICROORGANISMS AT TWO DEPTHS IN A FRESHWATER LAKE USING AN UNMANNED SURFACE VEHICLE (USV)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The report is well written. Objectives, methods, discussion and conclusions are clear. Some minor editing is needed as described below.
Experimental design: Experiments utilized a custom designed sampling system to successfully collect water for microbial analyses at two different depths. Need some minor additional information to include approximated depth of the lake at the sample sites. Add this to Table 1 reference line 88.

end of line 133....How was the system sterilized between sample set collections

line 144....250ml.....spell out as it starts a sentence

line 162.....how many colonies......10 as the table indicates?....but several samples recovered less than 10cfu.......specify here and correct the table that states 10cfu sampled for these few samples

you should refer to the Pseudomona cfu's as presumptive Pseudomonas....as you ice nucleation experiments show that many were not.....this is common - colilert will indicate positive with certain species of Vibrios and Pseudomonads
Validity of the findings: Looks OK with addressing above comments. Check references of genus/species italics and standardize as one or a few use caps for many title words while the most use all lowercase
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: REMOTE COLLECTION OF MICROORGANISMS AT TWO DEPTHS IN A FRESHWATER LAKE USING AN UNMANNED SURFACE VEHICLE (USV)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Language used throughout the manuscript is clear and well written. Only a few comments on this topic. There is a sentence in line 221 that may be correct, but sounds like it might has been a mistake during typing: “to collect samples and multiple depths and locations”, probably meant to be “to collect samples at multiple depths and locations”. Also lines 255 – 256, repetition of terms (location, due) should be avoided. In line 279, repetition of the preposition “in” should be corrected. Revision of sentence from line 297 to 304 is recommended.
Bibliography cited along the manuscript is extensive and recent, showing the authors are updated in their research field. There are however, corrections that have to be made to this section. Several papers cited in the References section are not mentioned in the text. Authors should include those citations where needed in the text or remove them from the References section. There is also one reference: Pietsch et al. (2007) (Line 44) which is not in the Reference list and it seems to be an error for what it should be Pietsch et al. (2017), which is widely cited along the manuscript. Although PeerJ Journal do not have a strict policy regarding the References section, they do have some guidelines. Authors should revise the entire section to comply to those guidelines, e.g. provide the full list of authors.
Article structure follows PeerJ Journal format. This paper contains 4 figures, which are helpful to the reader for the understanding, not only of the scientific results, but also for the development of the technology used to obtain them. They are presented in good resolution formats and well described. Table 1 and the supplemental table with all raw data are also well presented and clearly labeled.
Work presented in the manuscript is well organized and follows the objectives derived from the initial hypothesis stated by the authors in the introduction.
Experimental design: Research described in this manuscript fits within the Aims and Scope of PeerJ Journal. As mentioned before, it states a clear hypothesis with its specific objectives in the introduction that are fulfilled along the article. At first sight, it may seem a simple research work, with only 4 different samplings using culture-based methods, in a world where papers with big amounts of data are imposing. But the technology developed by the authors has a great potential in all kinds of research approaches and sometimes small research initiatives are needed in order to optimize that technology and/or methodology that could later be applied to more complex objectives. It is also known among the scientific community that environmental studies, although increasingly more frequent, haven’t provided the information needed to completely understand them. The use of USVs in environmental studies allows not only to reach possibilities beyond the human range, but also to those that could represent a risk to them.
Description of methodology allows replication of the experiments. Figure 3 and the ones included in the Supplemental files, plus the explanation in the Material and Methods section are elaborate, providing the capability of reproduction. Data in lines 186 – 192 from the Results section seem to repeat information already described in Material and Methods. Only new information related to the results should be included in this subheading.
Validity of the findings: Results obtained in the work presented add knowledge on the aquatic microbial community of a freshwater lake and the presence of ice nucleation active bacteria is a very important feature for the water cycle, as describe by the authors.
As considered by the authors in the discussion, collecting samples in different seasons as well as in different locations of the lake, would contribute to increase the understanding of how microbial communities change throughout the lake and are influenced by different environmental factors. Regarding this last topic, authors state in lines 90 - 91 of Material and Methods that “Though water temperature is an environmental variable, it was assume to be constant across the distances and times within and among samples collections in this study”, although no references or hypothesis that would back up this consideration are given. But later in the discussion section, lines 236 – 237, they acknowledge that “Other environmental factors, such as seasonal temperature variation and pH, are additional factors that could have an impact on bacterial distributions (Lindströem et al., 2005)”. Future efforts to complement this work should consider all environmental variables and authors may contemplate the possibility of including in the USV platform different sensors that would allow collecting that kind of information on site during the sampling process.
No conclusions are made in this manuscript, but speculations based on previous publications and the hypothesis of the authors. This is considered to be a wise decision regarding the small amount of samples collected and the statistical diversity of the results obtained.
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: REMOTE COLLECTION OF MICROORGANISMS AT TWO DEPTHS IN A FRESHWATER LAKE USING AN UNMANNED SURFACE VEHICLE (USV)
Review round: 2
Reviewer: 1

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


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

Input:
Title: REMOTE COLLECTION OF MICROORGANISMS AT TWO DEPTHS IN A FRESHWATER LAKE USING AN UNMANNED SURFACE VEHICLE (USV)
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Authors have addressed reviewers and editor's comments. Changes made and text added have improved comprehension of the manuscript. They have also adjusted bibliography format to the journal guidelines, and corrected those citations that were missing in either the main text of reference section.


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

Input:
Title: IDENTIFICATION BY SHAPE-BASED VIRTUAL SCREENING AND EVALUATION OF NEW TYROSINASE INHIBITORS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The overall data in manuscript is interesting and informative.
Experimental design: Good experimental approach was used in manuscript
Validity of the findings: Authors tried to verify their results using various approaches. However, further techniques are required to validate their results.
Additional comments: Comments
The present manuscript screen the possible inhibitors of mushroom tyrosinase. The manuscript is well written and the findings are interesting. Therefore, article can be consider for publication after revision.

•	The manuscript title “Identification of new tyrosinase inhibitors with shape-based virtual screening” is just emphasize on in-silico based study, whereas authors also performed in-vitro experiments. Therefore, manuscript title must be rephrased. Moreover, add few studies such as (Eur. J. Med. Chem. 141: 273-281; DOI: 10.1007/s12539-016-0171-x; Comput Biol Chem. 2017 Jun; 68:131-142; Chem Biodivers. 2017 Sep;14(9); Drug Des Devel Ther, 2017, 11:2029-2046; Bioorg Chem, 2017, 74:187-196) in introduction part.
•	Authors used Neorauflavane as a template, is it possible to use Kojic acid and Arbutine as a template for screening as you used in in-vitro experiment as a positive control.
•	Author did not discuss about size of protein structure, domain and binding pocket. I would suggest to make one graphical image of all inhibitors or your potent inhibitor against tyrosinase to show binding pocket and ligand conformation.
•	Authors did not discuss how many conformations he selected in docking experiments and on what basis he selected best pose of among all either just energy value or binding interaction pattern. In docking figure authors did not mentioned the binding distances of interactions. Moreover, authors claims “One hydroxyl established a hydrogen bond with the side chain of Asn260, while the other hydroxyl interacted with the copper ion” in docking results. However, it’s not clear from graphics either it is hydrogen bond or some other type of interaction. In discovery studio mostly the hydrogen bonds are represented by green dotted lines. Moreover, authors did not mentioned docking energy value for 5186-0429 in the results part.
•	Moreover, I would suggest (if possible) to run MD simulation using any software to check the stability of target protein.
•	For in-vitro analysis Table 1, Authors only mentioned IC50 value for 5186-0429, however, it’s better to calculate IC50 values for all other compounds in μM. It would help in comparison amongst all screened compounds with control.
•	In MM section, authors mentioned “Vmax and Km values (for Michaelis-Menten kinetics) were obtained with Graph Pad Prism 5.0 from the nonlinear regression of substrate-velocity curves”. However we could not found Vmax and Km values in manuscript file. Moreover, also provide Ki value (Ki is the EI dissociation constant).
•	Authors used two substrate (L-Tyro and L-Dopa) for inhibition purpose as mentioned in MM section, while in kinetic study only L-Dopa was used to generate Lineweaver-Burk plots. Therefore, I would suggest to use both substrates and make separate graphs for both substrate.
•	Furthermore, I would suggest (if possible) to use Zebrafish model for melanin quantification along with toxicity assay for your most active compound.


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

Input:
Title: IDENTIFICATION BY SHAPE-BASED VIRTUAL SCREENING AND EVALUATION OF NEW TYROSINASE INHIBITORS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: *In which solvent the L-DOPA, Tyrosine and test samples were prepared?

"0.9 mL of sodium phosphate buffer solution (0.05 M, pH 6.8)" This volume of PBS was fixed for all samples? If this is true, we will have difference in the concentrations after the addition of the test samples.
Validity of the findings: Some of the data are without error bars. For example: Fig. 3A-B and Fig. 4. How about the reproducibility of these experiments? At least two independent experiments, preferably three with error bars, are required.

In graph 3C determine the values of R-saquared and P value and Calculate the values of Ki,

The major point is: the discussion of the results needs to be reviewed, relating the theoretical data with in vitro
Additional comments: The design of the study appears appropriate and most of the data are satisfactory. However, there are some points that need to be improved, as summarized above.


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

Input:
Title: THE OLDEST ARCHAEOPTERYX (THEROPODA: AVIALIAE): A NEW SPECIMEN FROM THE KIMMERIDGIAN/TITHONIAN BOUNDARY OF SCHAMHAUPTEN, BAVARIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The description of the new specimen is thorough, both in text form and as documented photographically.

I was somewhat concerned about the legality of and potential access in posterity for the specimen. However, the authors seem to have found an effective and appropriate means of making the specimen permanently available to scientific study.
Experimental design: The paper is essentially all descriptive, not analytical. Thus there is no significant part of this particular study which involves experimental design.

The relevant specimen data, however, all all provided. Thus, future workers would be able to independently evaluate the morphological observations of the author, and could potentially study the original locality in search of additional specimens or new stratigraphic/sedimentological data.
Validity of the findings: The morphological observations presented are sound, and measurements of the skeletal elements are provided.
Additional comments: Additional comments and corrections:
p. 9, line 62:  Origin of Species should be in italics, not quotation marks.

p. 9, lines 69: “1970ies” should be “1970s”.


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

Input:
Title: THE OLDEST ARCHAEOPTERYX (THEROPODA: AVIALIAE): A NEW SPECIMEN FROM THE KIMMERIDGIAN/TITHONIAN BOUNDARY OF SCHAMHAUPTEN, BAVARIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.

Sufficient field background/context provided. Additional references could further enhance the paper but not essential (see reviewer PDF).

Professional article structure, figs, tables. Some minor suggestions to the figures. Suggestions for two non-essential additional supplementary tables (see reviewer PDF).

Self-contained with relevant results to hypotheses.
Experimental design: Original primary research within Aims and Scope of the journal

Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.

Rigorous investigation performed to a high technical & ethical standard.

A paragraph of the UV imaging methodology should be added as their are a range of UV imaging techniques currently available. The methodology is otherwise sufficient in detail and information to replicate the results.
Validity of the findings: Valid, well-supported and impactful findings. Data are robust. Conclusions are clear and speculation is stated as such.
Additional comments: This is an impressive amount of work, well done! I am actually supportive of publication as is (with a few typo and grammar corrections and a short paragraph on your UV methodology), but it would be great if you can take my minor corrections on board as I think they will improve the quality and value of the paper.

Importantly, I think the manuscript should not be shortened. In fact my suggested corrections would involve a very slight lengthening of 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: THE OLDEST ARCHAEOPTERYX (THEROPODA: AVIALIAE): A NEW SPECIMEN FROM THE KIMMERIDGIAN/TITHONIAN BOUNDARY OF SCHAMHAUPTEN, BAVARIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Meets PeerJ's standards. The authors addressed the comments from my first review.

- Clear and unambiguous, professional English used throughout.
- Literature references, sufficient field background/context provided.
- Professional article structure, figs, tables. Raw data shared.
- Self-contained with relevant results to hypotheses.
Experimental design: Meets PeerJ's standards. The authors addressed the comments from my first review.

- Original primary research within aims and scope of the journal.
- Research question well defined, relevant & meaningful. It is clear how the research fills
  identified knowledge gap.
- Rigorous investigation performed to a high technical & ethical standard.
- Methods described with sufficient detail & information to replicate.
Validity of the findings: Meets PeerJ's standards. The authors addressed the comments from my first review.

- Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful details for replication provided (rationale and benefit to literature clearly stated)
- Data is robust.
- Conclusions are well stated, linked to original research question & limited to supporting results.
- Speculation identified as such.
Additional comments: Oliver, Christian and Helmut,

Thank you for taking my comments on board. Here are some final items to address. I do not need to see the manuscript again and fully support its publication.

- line 139: 'artificial' not needed
- lines 165 and 166: replace '.' with ',' in '50.000' and '8.000' so it is clear you mean fifty-thousand microwatts and eight-thousand microwatts.
- typos in the marked changes on lines 1166, 1327, 1381, 1419 and 1564.
- line 1921: the potential cartilaginous pad shown in the LSF image in Fig. 4b of Wang, Pittman et al. 2017 is visible as a orange-coloured feature under white light. You could perhaps change the text to support the fact that soft tissues can preserve differently in the same specimen e.g. the orange-coloured pad vs. the white-coloured areas that reveal patagia. This might mean that some 'absent' soft tissues could be possible to reveal in the future using a wider suite of imaging techniques.

I look forward to seeing the published paper.

Best regards,

Michael Pittman


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: LINKING PANGENOMES AND METAGENOMES: THE PROCHLOROCOCCUS METAPANGENOME
Review round: 1
Reviewer: 1

Output:
Basic reporting: The Authors show how pangenomes and metagenomes can be linked and provide proof-of-concept of how this metapangenomics provides unique insights.

The English should be improved to ensure text is clearly understood. For example:
1/ Line 27 to 32. In the abstract, the authors give two statements, “Rapidly growing number of ...	…of populations across microbial genomes.” The first statements is a general statement that is followed by a second statement that is supposed to provide more clarity of which aspects of the general statement is the key focus of this manuscript. However, the current phrasing makes comprehension difficult.
2/ Line 65 to 68. Also rephrase these statements to make comprehension easy.
3/ Line 274. Rephrase these statements to make comprehension easy.
4/ Line 411 to 415. Rephrase these statements to make comprehension easy.
5/ The entire document needs to be proofread.

Most of the references used are from the Nature Journal but some references are old and newer published manuscripts with impacting findings have not been included. For example:
1/ Line 69:  include after reference (“Lorenz & Eck 2005; Thies, Stephan, et al. "Metagenomic discovery of novel enzymes and biosurfactants in a slaughterhouse biofilm microbial community." Scientific reports 6 (2016): 27035.)
2/ Line 70: include after reference (“Tringe et al., 2005; Al-Amoudi, Soha, et al. "Metagenomics as a preliminary screen for antimicrobial bioprospecting." Gene 594.2 (2016): 248-258)
3/ line 71-72: include after reference (“Tyson et al., 2004; Haroon, Mohamed F., et al. "A catalogue of 136 microbial draft genomes from Red Sea metagenomes." Scientific data 3 (2016): 160050; Delmont et al., 2017)

Overall, I commend the authors for the thorough data analyses and on conciseness of style of writing. If there is a weakness it merely is with respect to making comprehension easier (as I have noted above).
Experimental design: Research question well defined and meaningful.
Validity of the findings: Conclusion are well stated, linked to original research question & limited to supporting results.
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: LINKING PANGENOMES AND METAGENOMES: THE PROCHLOROCOCCUS METAPANGENOME
Review round: 1
Reviewer: 2

Output:
Basic reporting: I am putting my entire review in this section, as nearly all, if not all, of my comments are related to basic reporting.

Overall:

This is a nice contribution by Delmont and Eren describing the utility of a new software pipeline in the existing Anvi’o tool, along with a few new insights into Prochlorococcus ecology. The pipeline links genes from isolate genome sequences to their abundances in the environment via metagenomic read mapping, and it can identify specific genes (or protein clusters) that exist in isolate genomes but may be very uncommon in the environment. I have few, if any, scientific criticisms, but I found a lot of the text confusing, mostly due to undefined terminology and some long, confusing sentences. I think that this is a relatively straightforward study that would benefit from some streamlining of the text. Specific comments are below.

Abstract:

-The abstract is somehow both well written and deeply confusing. Please simplify the language. I am left not really understanding what the main question(s), methods, and results are. I know intuitively what both metagenomics and pangenomics (or at least pangenomes) are, but it would be helpful for the authors to explain these terms in the context of how they were considered for this study. Is the sentence starting with “While pangenomics offers …” meant to define both terms? If so, please restructure it along the lines of “The pangenome of a population (or genus?) consists of both core (shared) and accessory genes and genomic features …” or however you want to define it, and please similarly define metagenomics and its use in this study. If metagenomics is being used for abundance estimates (abundance estimates of what – SNPs, populations, and/or genes within pangenomes?), then consider calling it something more direct (metagenomic abundance estimates?). To me, the term metagenomics primarily evokes community predicted functional profiling and/or population genome assembly and metabolic reconstruction. Even though I have personally also made abundance estimates from metagenomes in much the same way as the authors, that would not be what first comes to mind. [edit: that is almost exactly how this is described by the authors in ln 69-74, so this needs to be much more clear in the abstract]

-Along those lines, I do not think of metagenomics and pangenomics to be inherently different. I would assume that metagenomics could be used both to define the pangenome (i.e., to find core and accessory genes in metagenomic assemblies) and to determine the abundances of subpopulations and/or specific genes or regions of the pangenome (through read mapping to metagenomic assemblies, SAGs, isolate genomes, or any combination of the above). Which, if any, of these possibilities apply to this study is not clear.

-Metapangenomics is not defined, and I do not find it to be a helpful term. Consider removing it from the manuscript, including the title. Based on my reading of the abstract alone, it looks like metapangenomics is meant to describe an abundance-informed pangenome, and if so, why not call it something like that, with some useful meaning in the term itself?

Main Text:

-ln 32: complementary

-ln 38-42: What does this sentence mean? Consider splitting it into two sentences. How does a metapangenome correlate with something? What is it correlating with? I am not sure that “traits” is an appropriate word – are the authors describing core and accessory genes here? What are “sub-clade demarcations”? How would these results differ from phylogenetic analyses (wouldn’t phylogenetics by definition separate sub-clades?)? Do the authors mean some specific phylogenetic analyses that are typically performed at a coarser resolution? If so, please provide more context on the phylogenetic analyses.

Main text:

-ln 54: “have been” should be “has been”

-Throughout: consider changing “shared” to “core” in the context of core genes across pangenomes, as this is more common in the literature and therefore more intuitive. If the authors mean shared among some but not all populations, then that should be explicitly mentioned, but I assume that they mean core genes shared among all populations.

-ln 74-76: What does this mean? Wouldn’t metagenomic assembly + read mapping do that too? It might help if you explain the particular utility of isolate sequences here.

-ln 77-78 (or earlier): Please define functional traits in the context of a microbial genome or pangenome. I think that you also mean isolate genomes here, so please change the end to “… mapping of closely related isolate genomes.”

-ln 80-83: This sentence is a bit long and confusing and can probably be broken down. The authors can rework it for clarification as they see fit, but here are some examples of what I find confusing: What are “well-established practices in pangenomics”? Please give a few examples. What are “emerging opportunities from metagenomic data”? Is this just using metagenomic read mapping for abundance data? If so, just say that. What is a genome-centric framework (I assume that this involves the use of closed, isolate genomes), and how does that differ from what you would get from a combination of metagenomic assembly and binning to identify populations + read mapping across a number of metagenomes to get abundance estimates? What are “pangenomic traits” and how do you define which ones are “key”? Are “key” traits just those that are linked to “niche partitioning” and “population fitness”, and if so, how do you determine that?

-ln 85: please state exactly what you mean by integrating pangenomic and metagenomic data. Again, what is “pangenomic data” and what is “metagenomic data”? Are there better terms for these types of data in this context, for example, “… integrating population pangenomes from multiple isolate genome sequences with their abundance profiles across environmental samples from metagenomic read mapping?” That might not even be correct, but the point is that I do not understand.

-I think that the focus of both the abstract and introduction and maybe even the title should be on the need for and development of this software pipeline, as that seems to be the key novel result of the study, tested on Prochlorococcus as an example, right? [[later edit: wait, but the tool is Anvi’o, which is fabulous but not new; please use the introduction to very clearly walk the reader through what is known vs. what is new in this study, both in terms of the visualization software pipeline and the Prochlorococcus biology]]. It seems dangerous to imply that metagenomics has never been used to identify the ecological niches of specific subpopulations (for example, the Banfield lab has worked in that general area, at least in AMD systems; how would isolate pangenomes add further information in that context?) and much safer to say that your software and visualization pipeline can help to identify and show these differences more clearly.

-ln 93: How many genomes? That number seems important if all of these genomes are going into your downstream analyses.

-ln 95: Were these 16S rRNA gene amplicon surveys or otherwise not metagenomic studies? That seems like a worthwhile point of clarification to help make the case for the current study that links isolate genomes to metagenomic data.

-ln 96: dynamics plural

-ln 97-98: Correlations between the “genomic traits” of isolates … are these just groups of genes that correlate with environmental variables? Were there correlations to variables other than HL and LL? If so, maybe call these “other environmental variables.”

-ln 98-104: This is a long sentence, and I got lost halfway through. Do “these two groups” refer to the core and accessory genes? How many metagenomes? What does “independently” mean here, and what is “their differential occurrence”? Does “in Prochlorococcus populations” mean in the same 12 as at the beginning of the sentence? If so, change to “in the 12 Prochlorocuccus populations,” otherwise define these populations. I don’t understand the last part of the sentence at all. Maybe summarize these three studies in three separate sentences and explain which part(s) of each are being included in the current analyses, and then explain clearly how the current study will expand on what is already known from these previous studies.

-ln 104-105: This seems like an important distinction. To this point, the general implication is that this is the first time that anybody has thought of exploring niche partitioning in pangenomes or metagenomes, yet here the authors say that the difference is that previous studies have not had resolution at the level of protein clusters. Again, please dispense with the fancy sentences and terms and use the introduction to tell the reader what has been done in the past that is relevant, both in terms of biology and visualization/software, and then explicitly state what knowledge gap will be filled by this study. For example, it would be useful to explicitly say why monitoring protein clusters is useful.

-ln 106: Again, what are “pangenomic traits”? Maybe I am just stuck on “traits” as an ecological term and the authors just mean similarities and differences across populations?

-ln 106: How do these 31 Prochlorococcus isolates relate to the 12 (or more?) populations described from previous studies above?

-ln 108: Please give an exact number for billions

-ln 110: Define ecological niche; is this just HL vs. LL here?

-ln 109-115: These are results that do not belong in the Introduction. Consider either reworking to frame these as hypotheses (or similar) that will be explored in this study, or remove this.

-ln 113 and 117: These are the first mentions of SAGs. This seems to be of abstract-level importance in how you are defining your pangenomes (i.e., a combination of SAGs and isolates). Or am I not understanding how your pangenomes were defined? After reading more of the results, I do not see much in the way of SAGs there, so how did you decide when to use isolates and when to use SAGs in your analyses? SAGs are presumably less complete genomes, so if a particular gene is not detected in a SAG, it does not necessarily mean that it is actually absent. The authors know this, I am sure, but if this is part of the rationale for using only isolates for some of the analyses, it should be mentioned.

-I think that Anvi’o can also be called out specifically in the Abstract and/or Introduction. It is not clear to me what aspects of the Anvi’o workflow are new in this study, though the Introduction suggests that this is a novel pipeline. Based on the text to this point, I was expecting the presentation of a novel workflow, and this needs to be made more clear. I think that a paragraph in the Intro with Anvi’o background would be appropriate – how has Anvi’o been used in the past, and what specifically is the new application here? It seems like more than just plugging new data into the software, so maybe a flowchart figure would help? There is a section of the methods dedicated to this, which is good, but I wonder if at least some of that should be moved to the main text, given that the pipeline is one of the key outputs of the study and not just an ancillary method.

-ln 134: Has phylogenomics not been done on these 31 genomes before?

-ln 176-199: Is this the new part? If so, you could start with something along the lines of “The Anvi’o pangenomic workflow developed for this study consists of …”

-ln 179: What is a “genome of interest”? Is this just every genome that will be considered for a given analysis, i.e., 31 Prochlorococcus isolates for this study? [I see later that this is the case, so please rephrase to make this more clear]

-ln 234 and 264: What about the SAGs?

-ln 234-243: Has this been done already for Prochlorococcus in any of the TARA Oceans publications? I would guess so, but maybe not all 31 isolates were included. It would be worth clarifying what parts of this analysis are new vs. what just needed to be done again here to feed into the Anvi’o pipeline.

-ln 244-256: These specific clades have not been described anywhere. I realize that a description of each could get tedious, but is there something general that you could say along the lines of, e.g., “All LL lineages come from low-light niches and include subclades I-IV defined by x, y, z” Otherwise, the description of these clades is not particularly useful. The figures just say that these are “literature-defined” lineages, which is fine, but the authors could briefly elaborate on these clade distinctions in the text.

-ln 277-285: Cool!

-ln 298-299: ECGs and EDGs -- do we really need more acronyms? I saw these again a couple of pages later and had to dig back to this section to remind myself of what they are. [and again when I came back to the manuscript after a break] When these acronyms appear again a couple pages later (ln 356), the next sentence has four different acronyms occurring eight times …

-ln 313: Okay, metapangenomics is finally defined! I still don’t fully understand the utility of this term. Maybe it is just me, but I do not find the introduction of new terms and acronyms in nearly every new manuscript in this field to be helpful.

-ln 313-320: What is the result here? The result cannot just be the figure; there has to be some interpretation or guidance for what the reader should be seeing.

-ln 354-356: This seems like an important contribution, and it is buried near the end of the Results.

-ln 366-367: Change to plural

-ln 370-371: Please rephrase this sentence for clarity. What is “they”?

-ln 420: Is this really only a little effort?!

-ln 464-466: Okay, the authors have confirmed the obvious application that I mentioned above, which is that this can also be applied to metagenome-assembled genomes. Why were those not considered here? I don’t think that this is a hole-in-the-paper offense, but I am puzzled, as it seems like a relatively easy addition that would boost the size of the available pangenome significantly.

Figures:

These are nice. If the authors insist on keeping the ECG and EDG acronyms, please define them in each figure legend.
Experimental design: See above (Basic reporting section) for a few specific comments related to better defining the research question and knowledge gap(s) filled by this study in the Introduction.
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: LINKING PANGENOMES AND METAGENOMES: THE PROCHLOROCOCCUS METAPANGENOME
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has been extensively reviewed and English use is clear and unambiguous.
Experimental design: In the current form, the manuscript results is relevant to the hypothesis and it is now clear how the work fits into the broader field of knowledge.
Validity of the findings: The findings are appropriately stated, and connected to the original question investigated.
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: LINKING PANGENOMES AND METAGENOMES: THE PROCHLOROCOCCUS METAPANGENOME
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I thank the authors for addressing my concerns and making the manuscript much more clear. 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: MOLECULAR AND SEROLOGICAL DYNAMICS OF CHLAMYDIA PECORUM INFECTION IN A LONGITUDINAL STUDY OF PRIME LAMB PRODUCTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Chlamydia pecorum is an important, and globally significant livestock pathogen causing pathology and production losses. The authors in this study applied molecular and serological tools to investigate the age-dependent distributions of C. pecorum in a lamb production. The manuscript is well written, and conclusions are well supported by the data. This is an important and well-designed study, and significantly improves our understanding on the C. pecorum dynamics.
Experimental design: Well-designed study.
Validity of the findings: The findings are novel.

Comments:

For table-3, the data are shown as mean,max and SD. Normally, we show the data as mean with SD, or mean with range (max, min). The authors may consider to modify the data.
Additional comments: 1. Beyond qPCR and CFT used in this study, we have also culture and ELISA approaches for diagnosis of Chlamydia.  The authors may address briefly the rationale to use only PCR and CFT in this study.

2. The authors should address clearly the specificity of CFT against C. pecorum alone; In the lamb production, it is not unusual to see the presence of other Chlamydia species;

3. Please reduce the length of the Statistical Analysis if possible;

4. The readers will benefit if the results for statistical analysis can be provide in the figures or legends of Figures 1 and 2;


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

Input:
Title: MOLECULAR AND SEROLOGICAL DYNAMICS OF CHLAMYDIA PECORUM INFECTION IN A LONGITUDINAL STUDY OF PRIME LAMB PRODUCTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Well written, but a little too many self citations in the introduction.  Would be nice to see corrected.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Nice, clear, well written manuscript.  No doubt about the validity and importance of the findings.


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

Input:
Title: MOLECULAR AND SEROLOGICAL DYNAMICS OF CHLAMYDIA PECORUM INFECTION IN A LONGITUDINAL STUDY OF PRIME LAMB PRODUCTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors have presented a longitudinal study comparing detection of Chlamydia pecorum in lambs from 2 to 10 months of age via qPCR and CFT and discussed the relative merit of both techniques in terms of monitoring the presence of Chlamydia pecorum over time and as tools to guide the management of disease within a flock.

Longitudinal studies are hugely beneficial to the study of diseases especially those caused by intracellular pathogens. The study design has been well thought out and executed. The structure of the article is satisfactory and the literature cited is appropriate and sufficient and supports a strong knowledge of the topic.

The abstract, introduction and material and methods are well written. However, material and methods needs more detail (see below). The results, including tables 1 and 2, need a major revision. I think the authors have attempted to extrapolate more from their data than is available. It is clear that the authors have established interesting patterns within in and between their tests but I think they have overstated the interpretation of these results with respect to the progression of disease

Tables 1 and 2 are extremely confusing and should be reconsidered and reformatted. The authors have presented to much data in each table and should consider revising to make it easier for the reader. For example:

Table 1. A total number of animals qPCR+ at each time point, regardless of repeat positives from different samples, would aid in making sense of the comparative columns. Also, the 68 at the bottom of the second last column is a number not a percentage and equates to the 96% positive by both tests stated in the text. The totals at the bottom of columns 2-4 and the # footnote make no sense.

It might be worth having two tables, the first with the expanded explanation of the qPCR results and then these summarised to number of animals and compared to CFT in a second table.

Table 2. As with Table 1, this table is very confusing and should be revised as described in Table 1. The combination of numbers and percentages, time points and new detection permutations is not easy to follow. Once again, consider two tables as described for Table 1.

I am also concerned about the terminology used to describe the inferences from the qPCR results. Infectious load should be bacterial load as there are no references to how infectious load has been determined in the paper. A qPCR positive result is an indication of shedding but how does this correlate to the progression of disease without other information. The authors have also indicated the presence of “Supershedders” in the flock and animals that were qPCR positive but CFT negative. Could these animals have chlamydia simply passing through theire system following ingestion of faecal material from the “Supershedders” The “Supershedders” were eluded to a source of infection but not passive transmission.

I would encourage the authors to revise and simplify their results and the interpretation of results in accordance with what is achievable with qPCR and the progression of disease in the absence of other information, most notably pathology. At the very least some indication on the quantitative nature of the qPCR results needs to be correlated to disease if they choose not to revise.

Suggest revising the use of the term detections throughout

Suggest standardising infection load" "qPCR load" and "qPCR copy load" throughout

Line 52; Suggest Point-of-Care (POC) first and then POC throughout

Line 100: Has qPCR been correlated any better

Line 191 Delete, qPCR and CFT detection, with

Line 193: Revise sentence "meaning these lambs tested C. pecorum DNA qPCR positive and also seroconverted and developed a detectable antibody response measured by CFT"

Lines 197-200: "Interestingly, 3.9% (3/76) of lambs were qPCR positive and CFT negative throughout the study period and 2/3 of these lambs had recurrent C. pecorum infection as measured by repeated qPCR positivity" Surely this raises questions about the correlation between immune response, infection and shedding? Or the accuracy of the CFT test.

Line 273: unapparent

Line 474: Delete second use of word detections

Line 477: qPCR+ not PCR+
Experimental design: I think this is a new and valuable contribution to this field of research and would be valued by readers when revised.

The aim is well defined and the results, when revised, will make a valuable contribution to field of research and progress to contributing to the gap in knowledge.

In describing how qPCR was performed the authors refer to Marsh et al. 2011. There are several sets of primers in this reference and the set used here should be either shown in full or referred to by name with respect to the original reference. No mention is made of the qPCR reagents and should be included.

A description of how the bacterial load was determined quantitatively and what this means should also be included.

Generally speaking it would appear that the study has been undertaken to an acceptable standard and that all ethical requirements have been met.
Validity of the findings: Overall the study is quite straight forward. The data when revised will be a sound foundation to build on with future research in this area, particularly the application of qPCR and CFT, for monitoring the presence of chlamydia in research trials and as management tools on farm. this should be emphasized.

The discussion has been adequately written but will also improve with the revision of the results and their interpretation. As will the conclusion.

Whilst a revision of the results and toning down of speculation of these with regard to the progression of disease is warranted. The results do support differences in the dynamics of qPCR and serological dynamics of sheep exposed to Chlamydia pecorum.

The study should be published and will contribute to the knowledge of Chlamydia pecorum in prime lamb production
Additional comments: You have a great story, don't over complicate it. Be consistent in the use of terminology and be mindful of the use of terminology with respect to presence/absence of pathogens and how far this can be used to speculate on the progression of disease. Disease studies usually integrate pathology and histopathology to confirm 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: CHARACTERIZATION OF SYMPATRIC PLATANTHERA BIFOLIA AND PLATANTHERA CHLORANTHA (ORCHIDACEAE) POPULATIONS WITH INTERMEDIATE PLANTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Authors need to improve the flow and the clarity of the text in  introduction and discussion parts.

Literature is almost sufficient; information of the reproductive strategies of Platanthera in American (where there are more diverse) would be added.

Line 68. P. bifolia is also largely present in calcareous grasslands
Experimental design: Line 104: In the end of introduction, the main research question is curiously presented “In order to determine whether intermediates are indeed hybrids, ...”. What are the alternative hypotheses? This part needs to be better explained.

Why intermediate individuals are investigated in only two populations while mixed populations between these two species are frequently observed (as said line 71). Why scents are investigated in only one of these two mixed populations? And why scents are not investigated in two control population? Why autogamy is investigated in only one population and with so few number of individuals (see table 1)? Why morphology is investigate on the base of only four traits? Why authors did not justify the selection of these morphological traits? What is the interpretative value of the number of days of co-flowering between only two populations of two species and during only one year? All these points strongly reduce the explanative power of this study. This situation renders the results very poorly convincing. All interpretations at species level are then based to a comparison between individuals from only two (and sometimes one) populations!
Validity of the findings: See before. Method used explained that results are poorly convincing.

In the figure 2A, how authors explain that sympatric and allopatric populations of P. bifolia are significantly different?

In the table 4, authors have to add both the retention index for each compounds, and the occurrence of each compounds of each species (and repeated the number of sampled individuals per species). The last information about occurrence is a key indicator to determine the evolutionary stability of each compound, especially when the number of compounds is very low.

The sentence Lines 435-436 “The results of this analysis show a significant similarity of chemical patterns in floral scent composition with P. bifolia of all intermediate morphotypes sampled” is indeed in contrast with the opposite values of relative proportions for 3,7-Dimethyl-1,3,6-octatriene and 1,2-Hexanediol-2-benzoate indicated in table 4. How authors explain this contrast?

Several parts of discussion are too speculative, like the involvement of mimicry system (549) or genetic drift (L 575). For example, how mimicry can occur if morphology and scent are different?
Additional comments: The resuls presented here mainly concern one population (for scents and pollination experiment) and only two for morphology and AFLP analyses. The absence of pollinator observations is a pity. Thus the generalisation of these results is quite low. Several points may be improved by additionnal experiments in order to reach the scientific value of PeerJ.


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: CHARACTERIZATION OF SYMPATRIC PLATANTHERA BIFOLIA AND PLATANTHERA CHLORANTHA (ORCHIDACEAE) POPULATIONS WITH INTERMEDIATE PLANTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a generally well written manuscript on the genetic, chemical and morphological characterization of plants of the terrestrial orchid species Platanthera bifolia and P. chlorantha with intermediate morphologies occurring in Belgium to investigate the possibility that hybrids are evolving in mixed populations in response to local pollinator-selected selection.
Experimental design: My main concerns are with the fact that it is not clear whether the genotypic frequency classes were assumed or experimentally verified and that the maternal effect is not mentioned and discussed to explain the results obtained.
Validity of the findings: Adding more information about the assignment of the genotypic frequence classes and the maternal effect is recommended.
Additional comments: Abstract line 25: The sentence ‘The results of the genetic analyses showed the same genetic patterns of morphologically intermediate individuals with the P. bifolia group’ is inclear so I recommend rephrasing it into ‘Morphologically intermediate plants had similar genetic patterns as the P. bifolia group’.

Introduction line 68: What is meant with a ‘fresh’ meadow? Please rephrase.

Materials and Methods line 120: ‘… for their attachment to the base of the pollinator’s head’. Please rephrase into ’… to various organs on the pollinator’s head’ to make the text more compatible with the information provided two paragraphs further down on attachement of pollinia to either the proboscis or eyes.

Materials and Methods lines 156-158: Was this done noninvasively?

Materials and Methods lines 263-264: The authors state that they ‘used six genotypic frequency classes to classify the analyzed individuals: pure parental species, F1, F2, backcross to each parental species. It is unclear how the authors assigned these classes. Were F1, F2 and backcrosses made by the authors themselves? If not, the authors should make more explicit here that they assumed the individuals to belong to these classes but that this was not verified experimentally.

Results line 335: Please remove ‘the’ in ‘… traits explained the 89%...’

Results line 420: Please change ‘the honey bee colony’ into ‘a honey bee colony’.

Discussion line 439: In the sentence ‘… individuals with intermediate column…’ is not clear what exactly is intermediate here so please add a specification such as ‘shape’ or ‘size’ here.

Discussion line 464 and further: I agree that the hypothesis that P. chlorantha evolved from P. bifolia is plausible. The authors could provide a bit more information about the timeframe in which this happened, though, for instance by referring to Inda et al. (2012) Annals of Botany, in which a molecular clock analyses is presented for a.o. the divergence of P. chlorantha and P. bifolia. According to these analyses, this divergence started only quite recently (i.e. in the Pleistocene), which could explain the fact that lineage sorting is still ongoing.

Discussion line 483: What is meant with the word ‘expanded’? Elongation? Widening? The authors should make this more clear.

Discussion in general: What I missed is the possible influence of the so-called maternal effect in plants (Roach and Wulff, 1987 Annual Reviews), where the mother contributes most to the phenotype of the offspring. It might very well be that the plants with intermediate phenotypes are genetically, chemically and morphologically most similar to P. bifolia because this species received most of the pollinia from B. chlorantha! The authors should mention this and the possibility to verify this by carrying out man-made crosses and investigating the resulting phenotypes.

Discussion line 571: Please add that this particular study was carried out ‘in Austria’ as most readers will not know this and assume that Durka et al. (2017) also studied plants in Belgium. Likewise, please add ‘in Belgium’ to the sentence ‘… our observed populations of intermediate plants…’

Acknowledgements line 606: please provide information on permits obtained for the experimental crosses by hand pollination carried out in the field to determine the level of compatibility between the species investigated.


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: BREEDING AND MULTIPLE WAVES OF PRIMARY MOLT IN COMMON GROUND DOVES OF COASTAL SINALOA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is fine and relatively clearly written in English but would benefit from a widening of focus to explore a wider view of the scope of the difficulties in scoring molt in a field study were there will always be unknowns related to seasonality or lack of, lenght of breeding/non-breeding period, molting/non-molting periods, multiple broods related to all of the former.  Two additional references not included I noted in general comments.

Both authors have extremely strong backgrounds and are leaders in this field of research

The paper is structure professionally

The paper is self contained for the narrow look at stepwise molt given the limitations induced by unknown is this type of field work.
Experimental design: Methods could use some clarification and checking of numbers of cited in text based on supplemetal data tables, see General Comments.
Validity of the findings: Data are intriguing and the paper provides unique data that will become a much cited reference for future research.  I spent several hours checking and proposing what if scenarios and I'm sure others will do the same.  With this in mind, the authors should consider a more open framework to outline alternatives rather than forcing data into Stafflemauser and arrested molt for breeding scenario.  Consider patterns from arrests of molt from seasonality, for breeding, combinations of the two, age structure of populations, and arrests with or without re-initiation because feathers have a latency to molt.   And perhaps most interesting, why juveniles are replacing primaries so soon after growing them.  But I'd like to stress that this is my opinion and should be construed as necessary.
Additional comments: First task is to proof and spell check supplemental data table and verify numbers.  For example, the first sentence of Results indicate there were 50 juveniles when there are 44 in the supplemental table.  Methods indicate 175 birds when there are 182 in supplemental table.  See comments. These are minor inconsistencies perhaps reflecting birds included or excluded for various reasons but clarification would be helpful.   One colorful addition is the conditional formatting option in Excel that color codes molt values - I left this in on the Supplemental Table sent to the Associate Editor (I couldn't attach the Excel sheet as a pdf).  When completed, move Table 6 to Table 1 adding ages to provide an introductory summary of data.

Data in the manuscript are crucial to further understanding patterns of molt.  However, data were subjected to narrow interpretation and need a more careful and thorough analysis and summaries without preconceived notions regarding Stafflemauser.  There seems to be sufficient info in Stresemanns’s, Pyle, BNA, especially Rivera-Milan, F. F. 1996. (Nest density and success of columbids in Puerto Rico. Condor no. 98:100-113) to assume molt is slow sequential descendant with arrests possible, but it is useful to verify this as the ms does.  Ground-dove data seem similar to woodpigeons (cited in ms) except for unknown impacts of the length of seasons on ground-doves.  A relevant paper to include is Ashmole's (1968) study of Fairy Terns stepwise molt, near year-around breeding, and arrests for breeding.

There may be an inconsistency in consideration of Stafflemauser.  The discussion of omissive molt and the multiple replacement of proximal primaries (or Ps) once or twice while setting up Stafflemauser are said to be maladaptive in the paper.  The last sentence of abstract suggests the latter is the “single greatest flaw”.  If so, then is Stafflemauaser simply an artifact of incomplete P molt?  Alternatively, if Stafflemuaser is an adaptation, an incomplete P molt is a prerequisite to setting up waves.  Thus the retention of outer Ps for a year or two are putative costs that immatures incur in order to benefit from Stafflemauser as adults.  As for multiple replacement of inner Ps, I believe this refers to yearly replacement which birds would do typically so not seeing how it is a flaw or cost. Once waves are set up, benefits are speculative depending on how many Ps are replaced as adults.  With complete replacement, benefit might be more efficient replacement with 3 waves than in a single wave of molt.  With incomplete replacement, the outer Ps can be replace every other year.  The discussion of omissive molt and multiple replacement of proximal Ps need some minor rewriting to reflect this overview.

However in revising consider that if data are filtered to only show the 13 adults that are molting P10, the interpretation of Stafflemauser is suspect.  A prediction from Stafflemauser is that waves are synchronized more or less.  If so, if P10 was molting and appears to be a nodal feather (proximal neighbor was old (0=old), then proximal and medial waves would also be re-initiating with a similar number of Ps renewed. However only two wings, UWBM 82497 and CWS 9167 appear as predicted with similar numbers of new (=1) Ps in proximal and distal waves suggesting a lack of synchrony on re-initiation.  In others the proximal wave has progressed many more Ps than would be expected by an assumed roughly synchronized re-initiation of waves.  Perhaps this results from proximal waves abutting more distal leaving a continuous sequence of similar wear class Ps as discussed in the ms?  Alternative, P10 is molted asynchronously from proximal  Ps or as Broody for woodpigeons.

P1 appears to be similar.  Of 9 growing P1, 5 appear to initiate at P2 so it appears P1 molted independently or perhaps with secondaries?

So for ground-doves, is the molt observed a result of a slow (150 days in ms, 200 days in Rivera-Milan 1996) simple sequential  descendant pattern that takes place yearly with some arrest & re-initiation or an adaptation/modification worthy of being considered Stafflemauser similar to Fairy Terns (Ashmole 1968).  Data indicated that only 30% of ground-doves have multiple waves suggesting the pattern might be an artifact.  This might be a better way to frame hypothesis testing rather than the idea that there are distinct series in primaries (similar to secondaries).   Add that since 70% of adult have a single wave, multiple waves may be SY and TY (second or third year) birds ironing out the wrinkles to set up a single slow descendant wave as adults.

For clarity, redo the direction summary using pairs of growing primaries for P2-9, excluding P1 & P10 as focal feathers because they lack prox and distal neighbor Ps, respectively. This will affirm (or reject) that molt is directional.  Then summarize waves more simply than Table 3 & 4.   The hypothesis to disproof appears to be that are set nodes, e.g. P1, P4, P8 and therefore node and terminus of waves would be predictable.  The difficulty here is that this is only true if all Ps are replaced in which case the Stafflemauser and the hypothetical set-node Stafflemauser would be indistinguishable in adults.  With incomplete replacement, assuming re-initiation, there would be no predictability of either accept perhaps a node at P1.  Most the set-node patterns in primaries start in middle or proximal Ps and go both ways.  Owls, not mentioned, likely have the record for slow molt. Regardless of direction, terminal and nodal Ps are predictable only if all Ps were replaced.  Incomplete molt results in unpredictability.

On constraints from breeding and crop milk for feeding young.  Milk is fed to young nestlings but then I believe adults shift to regurgitated seeds as young grow so not sure that an active crop signals an energetic constraint from feeding young and manifest as a molt arrest.   If it takes 150-200 days for adults to molt all Ps, and there are multiple broods, seems more likely the extremely slow molt is an adaptation to prolonged window when breeding & molt is possible.  And assuming molt is extremely slow, there is likely a latency in some wings to completion of one P and dropping of the (distal?) neighbor, which might appear to be an arrested molt (a check for molting secondaries might provide a clue here) (also mentioned by Boody for woodpigeons). One could turn the discussion around and suggest that so many were molting (~80%) that they molt continually while breeding and most successful adults do both without arrest.  Leaving multiple waves an artifact of incomplete molt or a rudimentary Stafflemauser.  A latency to molt hypothesis might be presented as a contrast to Stafflemauser in highly seasonal species.  Once renewed a feather will not be physiologically ready to molt until it is X months old thus stopping or merging waves and introducing the idea for what was observable in P1s and P10s.

An interesting offshoot is why are juveniles molting primaries so early?  If the poor feather quality, then the juvenal feathers should be able to be identified and if no obvious different wear categories, then Ps probably aren't juvenal.  E.g., 5 "juveniles" had class 0 (old) primaries in the wing distal to J primaries.  These would have to be scored as "J" or "J0" if the birds were juveniles.  Perhaps they SYs (second year) that have gone through one non-molting period.  A complication, briefly mentioned is that if a pair had multiple broods over a breeding window, the first fledged would likely complete molt while there would be a progressively fewer completing molt in subsequent broods with the last fledged only molt few Ps.  Following a non-molting period, if it exists, the SY birds would likely be classed as adults unless juvenal plumage hints were retained.  This would create a scoring nightmare with birds in all stages of complete, incomplete, and some erratic molt patterns with the underlying pattern of sequential descendant molt.  To further non-temperate molt studies introduce molt latency as a controlling factor and provide a simple figure showing what P wear categories might look like with staggering introduction of juveniles that undergo a period of arrest.  Depending on the age structure of population of ground-doves and similar species, SY birds might be most common.  Also suggest as Ashmole did that researchers might punch primaries if they had an opportunity to recapture the birds.

Figure 1. Number of feathers between the outermost growing primaries in the 15 adults that had two active waves of primary replacement. (in supplemental table there are 19 adults with 2 active waves, 17 scored and 17 shown in figure)

Figure 2 legend.  "sailed population" an autocorrect for "Sinaloa population"?

Table 1 Totals 43 41 95% should be 44 total sampled and 42 replacing primaries

Table 2 check counts of growing, correct except P1 looks like it is 14 rather then 13

Table 3 delete simultaneously as it isn’t possible for a single feather

Table 4. Summary table for 100 adult Common Ground Doves that were growing primaries. But there are 112 in supplemental table, some were not included? Previously noted UWBM 90871 incorrectly designated no molt but P1 is growing so make P1 growing 9 rather than 8, check others growing, most are correct but there may other discrepancies depending on how many are included

Table 6 132 adults in table 138 in supplemental data, 28 not molting (one –UWBM 90871 - molting at P1 designated not molting in the supplemental table)

Throughout change Ground Doves or Ground-Doves

Some images of wings showing various wear classes of primaries would be useful for future reference

A few comments on the Supplemental data table and second worksheet summarizing growing P1 and P10 were sent to the Associate Editor because formatting was lost while saving as a pdf.  I can forward these to the authors on request.


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: BREEDING AND MULTIPLE WAVES OF PRIMARY MOLT IN COMMON GROUND DOVES OF COASTAL SINALOA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is written clearly and overall well referenced, and the introduction provides good background on the subject.  The structure conforms the journal’s standard.  Raw data were provided but need more descriptions of symbols they used (for example, I was not sure what “r” means in the primary molt score columns).  Also, I felt that some of the conclusions were not well-supported by the data and/or analyses (figures and tables) they provided.
Experimental design: The research was within the scope of the journal and performed to a high technical/ethical standard, and research questions were well defined in the Introduction. As the authors stated, the molt sequences and cycles of tropical species are poorly studied, and this study fills a knowledge gap and describes interesting molt patterns that have never been previously examined. Methods were described in sufficient detail and in a replicable manner.
Validity of the findings: Figures and tables are well described in the captions and text but lack associated statistics to support the authors’ conclusions.  The conclusions are linked to the original research questions but not rigorously supported by their data and analyses, and the authors need to present alternative hypotheses.  Please see the comments in the “General Comments” section for details.
Additional comments: L. 102–103: It was not clear why juveniles “should” initiate primary molt at the same loci as adults if they replace the primaries in multiple series.  Could they not have different nodal points?

L. 134–137: Need more explanation for the relationship between aggression and retainment of bursa.

L. 162–164: If the sample collection was not random, the data would still not be “unbiased.”  Obviously, the samples were not collected randomly throughout breeding and molting seasons.

L. 212–217: The fact that only 17%–31% of adults replacing primaries in two or more waves appears to indicate that this is not their dominant molt strategy.  Is it possible that only younger individuals (especially second-year birds) that have not  completed the mot of distal primaries in the previous year molt in two waves?

L. 214: It is stated here that two groups of primaries were separated by one to several older primaries, and Fig. 1 shows that the number of feathers between two molt waves are less than 6 for more than 1/3 of individuals.  Because of the sample size, I am not sure if the distribution shown in Fig. 1 is significantly different from the normal distribution. This seems contradictory to the later statement that the molt of P1 does not usually start until the distal wave reaches to about P6, which is the mechanism to reassure the fresh inner primaries are not replaced redundantly.

L. 221–228: I didn’t follow the logic here.  If P1 is growing and P2 is new, should the direction of molt be ambiguous as P1 is the first primary?  Same for P10.

L. 222: The phrase “P1 being too short to be compared for wear and fading with its adjacent inner primaries” does not make sense as P1 doesn’t have adjacent inner primaries.

L. 254–258: The overlap of breeding and primary molt appears to occur within a small window based on the supplemental raw data provided (mid July to early August).  Most of birds do not molt during the most intense period of breeding season.  I understand that the molt may be arrested during that time, but based on the data author presented, I am not convinced that the arrests during the breeding season “cause” the multiple waves of the primary molt.

L. 256: The authors do not show statistically that the arrested primary replacement was “strongly” associated with having an active crop gland.

L.271: Regarding the sentence starting “Two birds directly support…,” I feel the data did not support their conclusion exclusively as data on other birds showed that molt on P1 started before the distal wave reached P6 (Supplemental data). Since there are many birds that do not molt primaries in two waves, it seems that these examples instead support that the molt arrests during the intense breeding period do not “cause” the two waves of molt.

L. 282: Need to show the statistics if the authors are claiming “significantly positive.”

L. 282–289: These two flat lines are clearly the artifacts of sampling in July and September intensely.  I agree that their molt is little synchronized, and it would be difficult to estimate molt duration reliably.  However, the figure seems to indicate that most birds were in the latter half stages of molt by September.  Does this indicate that their molt cycles designed to complete their primary molt before winter for most birds?

L. 324: Need statistics if you are stating that molt arrests are “strongly associated” with active crop glands.

L. 369–371: The “surprisingly” greater frequency of growing feathers in the outer than in the inner primaries in adults (Table 4) corresponds well with the greater frequency of growing feathers in the inner primaries in juveniles (Table 2).  This made me think that some of the “adults” replacing primaries in two waves are second year birds that failed to replace the outer primaries in the hatch year.

Figure 2: It would be helpful if you could mark the individuals that were molting primaries in two or more waves.