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: LOWER LOCUS COERULEUS MRI INTENSITY IN PATIENTS WITH LATE-LIFE MAJOR DEPRESSION
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are some English language errors:
-	Do not use the word “convert” or  “conversion” to indicate progression in disease stage. Conversion is a word used in religious context.
-	 “the more the treatment, the more the LC intensity” : please rephrase as: long treatment was associated greater LC intensity values
-	Throughout the manuscript, the authors describe their effects in terms of “increase” or “decrease”. This falsely gives the impression that there is a change over time. Given that the authors are working with cross-sectional data, they should describe the direction of the association (greater, higher, lower).

Reporting:
-	Please provide exact p-values, degrees of freedom and effect sizes
-	Given that the sample size is so small, the authors should also show the data points on top of the bar plots
-	Raw data is supplied. It is not clear whether the authors will share the code as well, as per PeerJ policy
Experimental design: •	The T1-weighted TSE sequence used in the current manuscript to structurally image the locus coeruleus differs from standard sequences in terms of spatial resolution1-3. Given the rod-shaped nature of the structure, it would be best to image this structure isotropic or by having an in plane cube and larger slice thickness. Here, the authors choose to use 0.42 * 0.68 * 2.5mm^2, which is then reconstructed to the resolution that is normally used. This may induce partial volume effects coming from the fourth ventricle. The authors should provide images of the LC across various subjects. In addition, the sequence lasts 15 minutes, which is very long for these small voxels and the signal may well be impacted by motion artefacts. How did the authors adjust for motion? Where there individuals were the locus coeruleus could not be visually identified?
•	Neuromelanin-sensitive imaging: several quantitative MRI-studies as well as animal studies have now shown that it is unlikely that these contrasts of TSE-scans are reflecting neuromelanin cell density, but rather water content or general neuronal density4. The authors should refrain from the word neuromelanin-sensitive.
•	It is know that TSE sequences suffer from intensify variations across the slices, which can cause variability among subjects that is related to the pulse-sequence rather than representing biological variation. How did the authors deal with this?
•	Furthermore, previous authors have reported that the pontine tegmentum also exhibits an age-effect and this may confound the calculated integrity measure1. Looking at the dataset provided by the authors, there does seem to be a positive relationship between age and signal in the reference region and not for the intensity of the locus coeruleus. This effect seems to be present mainly in the controls and therefore may have confounded the integrity measure and also the group comparisons. This should be discussed.
•	What is the integrity measure referring to? How can it provide information on the function of the LC?
•	Did the authors expect a asymmetric pattern in LC intensity? Why not combine left and right into one measure to reduce the number of comparisons?
•	The new diagnostic framework for Alzheimer’s disease states that the assessment of the underlying pathology of neurodegenerative diseases required biomarker information. For Alzheimer’s disease this involves the assessment of amyloid and tau pathology5. The authors have no information available on these biomarkers (or did not include it) and as such should be more cautious in their claims that these patients are at risk for AD. Likewise, the control group or even the MDD group may have pathology in their brains, which could explain the lack of differences between aMCI and controls.  As such the conclusion that locus coeruleus does not seem to be a biomarker of risk of a neurodegenerative disorders is not supported by the data and should be rephrased.
•	The MMSE indicates that the MDD group is cognitively much more impaired than the aMCI group: does this include individuals with dementia?
•	The range of depressive symptoms of the MDD groups is 0 to 13 (GDS) or 1 to 28 (HDRS). These individuals are no longer experiencing depressive symptoms. Is that due to medication or is there a broad range in variability in these patients?
•	I am confused about the group allocation of medication use. It seems that some patients were taking combinations of different types? How many were only taking SNRIs? How may this effect the results? The authors should also show the group comparisons for the other medication types to fully understand whether this is due to SNRIs or whether this is a spurious observation.
•	Exclusion criteria: what is meant with mental disability?
•	What do the authors mean with accumulated medication dose? How was this accumulation calculated?

References used in this review
1.	Clewett, D.V., et al. Neuromelanin marks the spot: identifying a locus coeruleus biomarker of cognitive reserve in healthy aging. Neurobiol Aging 37, 117-126 (2016).
2.	Betts, M.J., et al. Locus coeruleus imaging as a biomarker for noradrenergic dysfunction in neurodegenerative diseases. Brain 142, 2558-2571 (2019), PMC6736046.
3.	Priovoulos, N., et al. High-resolution in vivo imaging of human locus coeruleus by magnetization transfer MRI at 3T and 7T. Neuroimage 168, 427-436 (2018).
4.	Watanabe, T., Tan, Z., Wang, X., Martinez-Hernandez, A. & Frahm, J. Magnetic resonance imaging of noradrenergic neurons. Brain Struct Funct 224, 1609-1625 (2019), PMC6509075.
5.	Jack, C.R., Jr., et al. NIA-AA Research Framework: Toward a biological definition of Alzheimer's disease. Alzheimers Dement 14, 535-562 (2018), PMC5958625.
Validity of the findings: •	Statistical analyses: the authors should use non-parametric statistics given that some subsamples are becoming very small in size. There was also no adjustment for multiple comparisons, which may have possibly induced spurious correlations. Given the small sample size, the authors should be encouraged to use equivalence testing (TOST) to confirm lack of an effect.
•	In the discussion, there is a lot of speculation on the role of neuromelanin cell density as a possible underlying explanation for the observed effects. Given that several quantitative MRI-studies as well as animal studies have now shown that it is unlikely that these contrasts of TSE-scans are reflecting neuromelanin cell density, but rather water content or general neuronal density4, this should be rephrased and clearly indicated as speculation.
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: LOWER LOCUS COERULEUS MRI INTENSITY IN PATIENTS WITH LATE-LIFE MAJOR DEPRESSION
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.	How did the authors identify brain damages such as white matter change or microinfarct in the brain without T2 images?
2.	The problem of this study is the signal intensity of neuromelanin-sensitive T1 MRI is not absolute quantification of the amount of neuromelanin. So, the authors used the ratio values of LC and reference areas. In order for the reference area to be used as a reference of LC signal change, the reference areas should not be affected by the disease. The authors should provide a valid basis for this point.
3.	The statistical values of LC should be controlled for multiple comparison problem.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This study was tried to evaluate the neuromelanin signal change in the locus coeruleus (LC) in MDD, aMCI, and HC using neuromelanin-sensitive T1 MRI sequences. The authors found decreased LC signal in MDD compared to aMCI and HCs. This study is very interesting in that it is difficult to visualize and quantify LC, and It is also interesting that there is very little research into the role of LC in depression. The neuroimaging methods used in this paper is appropriate.


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: LOWER LOCUS COERULEUS MRI INTENSITY IN PATIENTS WITH LATE-LIFE MAJOR DEPRESSION
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1.	At some instances references should be added to support the claims the authors make (e.g., lines 110; 130, 442 […])

2.	In general, the authors may consider adding why an association between the noradrenergic system and AD can be expected. Even though two conditions that are classified as potentially leading to AD are investigated, the mechanistic relation between LC and AD is not introduced.

3.	Line 96: What do you mean by the sequences are modified to visualize the LC?

7.	Figure 2 / S Figure 4: Is there a duplicated group label?

8.	Please add the statistics and table concerning other noradrenergic medication to the supplementary information.

9.	Please provide references for the claim that standard MT sequences are quantitative measurements.

10.	Please change “T1 signal intensity” to MR (signal) intensity as the basis for the LC contrast is not (only) T1-dependend.
Experimental design: 5.	Related to my concern raised in the first review: I believe that transforming the ROIs from standard space to native space with a nearest neighbor interpolation would be the appropriate approach that would avoid introducing the additional thresholding (cf. line 260). The authors should consider adapting their procedure.

6.	Figure 1 shows that the reference region stretches way more caudally relative to the LC. What was the motivation behind this design?
Validity of the findings: 4.	As MDD participants show particularly low MMSE scores: Is there a relation between LC ratios and MMSE values? Please provide a scatter plot indicating the correlation with different marker symbols for the subgroups (can be added to supplementary information).
Additional comments: The authors addressed most of my concerns and I find the manuscript much improved. A few remaining remarks are listed below


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: LOWER LOCUS COERULEUS MRI INTENSITY IN PATIENTS WITH LATE-LIFE MAJOR DEPRESSION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have done a lot of work to clarify inconsistencies and improve the language.
A few remaining issues:

Line 212: extend should be extent

Discussion: remove "the first study" --> previous work has been done in clinical groups (e.g. Takahashi)

Fig 2B and S4: something went wrong here with the labels?

Please rephrase:"Thus, age-related declines in LCCR have been mainly observed in the rostral part of the LC in healthy subjects (Liu et al., 2019), being such alterations related to disrupted memory (Dahl et al., 2019)." This second part of the sentence is not clear.

S-Figure1: indicate from which diagnostic group the images are taken
Experimental design: I am not convinced by the medication effect, in particular because no difference in MDD patients with or without SNRIs or noradrenergic medication. It seems that the observed differences are simply reflecting the group differences observed earlier. It doesn't seem to be an effect of medication, but rather of group. Are the individuals with SNRIs having worse symptoms, longer disease duration of worse treatment response and isn't that driving the association instead of the type of medication?
In addition (if the above is the case), it would be good to color the dots in box plot in Figure 2B by depression severity
Finally, given the way the groups are made (not pure, but combination of medications) and the sample size, this should also be toned down in the abstract and discussion.
Validity of the findings: Abstract (and conclusion in discussion): the authors need to clearly indicate that these conclusions require more investigation with larger sample sizes.

Discussion: I do not agree that the MT images are only better for absolute quantification of the signal. As previous work has shown MT approaches improve the CNR (Priovoulos et al., 2018; Neuroimage) and at higher field-strength allow for a fast visualisation (with better SNR and isotropic resolution). And in fact, the T1-weighted contrast does contain some MT effect. This should be rephrased.
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: INFLUENCE OF PERIODONTAL TREATMENT ON BLOOD MICROBIOTAS: A CLINICAL TRIAL
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see comments to the authors.
Experimental design: Please see comments to the authors.
Validity of the findings: Please see comments to the authors.
Additional comments: This study aimed to investigate the effects of periodontal treatment on the abundance and diversity of microbiota found in blood microbiota, before and after periodontal treatment. A total of 27 periodontitis patients were recruited and combinations of SRP and glycine air polishing (GAP) were used. Peripheral blood samples were then obtained at baseline and after treatment (6 weeks). Nested polymerase chain reaction (PCR) was used to evaluate the microbiological outcomes. Following treatment, all participants exhibited significant improvements in clinical parameters, but no significant differences were observed between treatment groups. Differences in total bacterial counts and species diversity were observed between treatment groups, whereas Porphyromonas was the dominant genera in one of the treatment groups the next day following treatment. No significant differences in relative abundances and α-diversity was observed between baseline and 6 weeks. The study concludes that local periodontal treatment merely disrupts the stability of blood microbiota in the short term, and that a combination of SRP and GAP is a promising approach to result in limited bacteraemia.
1.	While the study appears to be methodologically well performed, matched data on the effect on this intervention on the oral microbiota is missing. This could have been reflected in saliva or supra/subgingival biofilms from selected sites. If such data is not available, this should at least be discussed as a limitation of the present and references should be made to other works that have investigated the effect of SRP on oral microbiota (saliva or biofilms) using next generation sequencing.
2.	From the methodology and presented data it is evident that 16S rRNA Gene Sequencing was performed, using a MiSeq platform. This is not mentioned in the Abstract, and this important omission prevents the reader from understanding the experimental and analytical processes that have taken place in this work. Therefore, this should be mentioned in the Abstract.
3.	The abbreviation GAP should be defined in the Abstract.
4.	While there are several genera that seem to have been affected (evidenced in Figures 3 and 4), only Porphyromonas is mentioned in the Abstract. More of them should be mentioned accordingly, in order to give a better overview.
5.	At repeated parts in the text, “16srRNA” is written. The correct definition is in fact “16S rRNA”, and this should be corrected accordingly.
6.	Data is presented on the genus, but not species, taxonomic level (Figures 3 and 4). For a higher resolution of the presented data, it would be helpful to provide data also on the species level.


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: INFLUENCE OF PERIODONTAL TREATMENT ON BLOOD MICROBIOTAS: A CLINICAL TRIAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The article meets the PeerJ criteria and should be accepted as is.


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: INFLUENCE OF PERIODONTAL TREATMENT ON BLOOD MICROBIOTAS: A CLINICAL TRIAL
Review round: 1
Reviewer: 3

Output:
Basic reporting: This study had an interesting topic, which is already widely investigated in the past, but this investigation introduced a new procedure (Air-flow), who is nowadays more and more widely used in dental practices. The question whether the use of Air-flow would translocate more or less bacteria in the blood stream is interesting, especially when it comes to treat patients with severe systemic co-morbidities where a massive translocation of bacteria may be harmful.
However, regardless the promising aim of this study, there a couple of substantial issue that make the results of this experiment of difficult interpretation.
Experimental design: -	The first and most important issue is the moment when blood samples are taken. The baseline blood sampling is done at the day of the baseline visit. This is before any procedure is done. Between visit 1 and visit 2 there is no information about the time frame. The most important pitfall is that there is no blood sampling before subgingival scaling (visit 2 in figure 1). This should be the 0-time sample to really test the short term effect of the treatment. Without this is not possible to assess the real effect of the treatment both at short term and long term. Furthermore there is no blood sample taken immediately after treatment (visit 2 in Figure 1). This should be the sample to assess the difference between the groups due to the therapy. The authors took one sample one day after intervention. This is too late to assess the real effect of the treatment. There is literature that showed that already after few hours there is a clearance of bacteria in the blood stream. Furthermore,  we know from previous studies that in inflamed situations brushing and even chewing play a role in the translocation of oral bacteria in the systemic bloodstream. These variables are totally not controlled and may have influenced the translocation of bacteria in the first day following the treatment. In order to be able to assess the role of the treatment, blood samples should have been collected right before and right after treatment. This is the only way to assess bacteremia due to periodontal intervention. A oral supra- and subgingival plaque sample should be also included in order to correlate the type of bacteria in the mouth with the ones found in the bloodstream.
-	Because of this issue, the conclusion of this study are not appropriate because not supported by an appropriate design.
-	A second issue is the use of GAP. This procedure, that should be the positive intervention is used in both B1 and B2 but in different moments. The rationale behind this choice is difficult to understand and it should be widely discussed.
-	A third issue is related to the sample size. This size is clearly underpowered in order to be able to detect a clinical and statistical significant difference between the groups. This is confirmed by the authors themselves in their description of the power calculation. For microbiomic studies, multiple correction for all genera at least should be done if the aim is to identify significant difference in abundance of single genera or species. Furthermore the control group is clearly younger even if no statistical difference is found (because of the low power).
Validity of the findings: Due to the experimental issue, the validity of the findings are jeopardized. Furthermore, -	there is a variation in the treatment result, but the low power was not sufficient to let detect any statistical significant difference. This variation should be used to correct for changing in the bacterial count in the blood.
Additional comments: Dear Authors, your work if for sure appreciable, but the experimental design issues make the data of this experiment difficult to interpret.
There are other issues more related to the lay-out of the manuscript listed below
-	The lay out from tables and figures needs improvement. There are also an amount of abbreviations that are not explained and not consistent with the text.
-	Lay-out references
-	Mattila et al 1989 should be replaced by a recent reference
-	The English language is sufficient but it should be deeply revised.


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: INFLUENCE OF PERIODONTAL TREATMENT ON BLOOD MICROBIOTAS: A CLINICAL TRIAL
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have addressed the comments efficiently.


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: SUGARCANE/PEANUT INTERCROPPING SYSTEM IMPROVES PHYSICOCHEMICAL PROPERTIES BY CHANGING N AND P CYCLING AND ORGANIC MATTER TURNOVER IN ROOT ZONE SOIL
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript compares soil nutrients, enzymes, and microbial dynamics in the sugarcane/peanut intercropping system. The authors analyzed soil nutrients as well as metagenomes to understand nitrogen and phosphorus cycling in the intercropped systems. The work adds new knowledge on the microbial control of N and P cycling. However, there are some issues that raise a question about the work.
Experimental design: The treatment design and sampling plan do not support the title nor objective of the research. It is basically a soil sampling in three rows of intercropping and analysis for differences in root zone soil. Also, it is not clear how long the experimental plots have been established. In materials and methods, authors mention peanut planted on March 10, 2018-2019. Is it established in 2018? If this is single-season work, the discussion and conclusion are too strong and it does not make sense. There is no baseline soil condition and authors did not provide the detail of the experimental field/soil variability. Peanut planted on March 10 and July 25th sampling authors sees an increase in SOC by >8 g/kg, this is unreasonably high, some sampling or experimental biases could have affected the results.
Validity of the findings: This is a single season work. One time sampling in long-term experiments (several years of treatment application) is common but the single-season work may not give the complete picture on how intercropping work,  Figure 1 does not provide a complete picture of the experimental design, it shows they are basically collecting samples from three different rows and tested it for different soil properties. There is no baseline soil condition and authors did not provide the detail of the experimental field/soil variability. Peanut planted on March 10, soil sampled on July 25th, and analysis showed an increase in SOC by >8 g/kg. This does not make sense.
Additional comments: This manuscript compares soil nutrients, enzymes, and microbial dynamics in the sugarcane/peanut intercropping system. The authors analyzed soil nutrients as well as metagenomes to understand nitrogen and phosphorus cycling in the intercropped systems. The work adds new knowledge on the microbial control of N and P cycling. However, there are some issues that raise the question on the hypothesis of this project, design and plan of the experiment.
The treatment design and sampling plan do not support the title nor objective of the research. It is basically a soil sampling in three rows of intercropping and analysis for differences in root zone soil. Also, it is not clear how long the experimental plots have been established. In materials and methods, authors mention peanut planted on March 10, 2018-2019. Is it established in 2018? If this is single-season work, the discussion and conclusion are too strong and it does not make sense. There is no baseline soil condition and authors did not provide the detail of the experimental field/soil variability. Peanut planted on March 10 and July 25th sampling authors sees an increase in SOC by >8 g/kg. However, proportionate changes in other soil properties are not observed.
What was the cultivation, fertility, and other management details for each crop? How many years?
How data on soil physicochemical properties were analyzed? Please provide the detail in the statistical analysis section.
Was metagenomics analysis also done in replicated samples? How the sequence data were analyzed (if replicated). The materials and method section needs a drastic revision with so many changes.

L59-60: remove the sentence “facilitation occurs in other intercropping …………………. systems”
L72: remove “Hense”
L94: was sugarcane and peanut planted on March 10, 2018, and March 10, 2019? This is confusing. How many years of intercropping? Changes in soil properties just after one season of intercropping does not make sense
L121: potassium dichromate
L184: significantly increased “available p” by – the sentence as is incomplete.
L195: if 45.7% decrease is not significantly different, there is a major flaw in your data or data analysis (provide
L201-202: rewrite the sentence, it’s confusing what comparisons were made.
Discussion:
Most of the discussion is focused on what happened but did not interpret why/how aspects. For example, intercropping increased the nitrogenase enzyme, but why?
How intercropping increases p cycling, please clarify. Provide linkages between genomic analysis you did and respond in nutrients in the soil.
L312: changes and differences in what?


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

Input:
Title: SUGARCANE/PEANUT INTERCROPPING SYSTEM IMPROVES PHYSICOCHEMICAL PROPERTIES BY CHANGING N AND P CYCLING AND ORGANIC MATTER TURNOVER IN ROOT ZONE SOIL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is overall understandable, but there is some lack of clarity and unusual phrasing in places.

References seem appropriate, but there are some formatting corrections needed.

Figures and tables are suitable, but several figures lack the field replicates and thereby prevent the reader from seeing the variability within an experimental group.

Raw physico-chemical soil data are shared as a supplemental excel spreadsheet, but the authors need to add the units to each data column in the spreadsheet. Raw sequence data were confirmed available at the Sequence Read Archive, but assembled contigs should be made available as well.

No hypotheses are expressed. The objectives are purely exploratory and could be improved by indicating specific motivations or expectations based upon prior findings in the literature or perhaps from preliminary work within this research group.
Experimental design: The scope is appropriate for PeerJ and is a primary research work.

The research question and motivations for the work could be better stated toward the end of the introduction alongside the objectives.

The data visualization needs further improvement.

There are deficiencies in the methods reporting, in particular citations to the protocols or methods used are needed.

Presently more detail is needed in order to be able to replicate the methods.
Validity of the findings: While there is replication within the study, it is not indicated in several heatmap figures. Significant findings are given in the conclusion section, but need to be expressed quantitatively rather than just in qualitative terms.

The data are provided, and statistical analyses have been conducted. There is a control, sugarcane monocropping group. There is some concern over possible confounding effects of different fertilization regimes between the sugarcane and sugarcane + peanut groups.

As mentioned previously, the objectives/hypotheses and research question need to be more clearly stated and inter-related in the introduction. Clearer linkage that indicates resolution of the research question is needed in the conclusion.

The discussion needs to clearly link leads from the literature to related or contrasting findings in the data from this study.
Additional comments: The researchers compared sugarcane intercropping vs. monocropping to determine whether there was an effect of the cropping on soil fertility parameters including physical and chemical properties and genes responsible for microbial nutrient cycling functions. This is a worthwhile study, but in its present form it requires a number of corrections to ensure that, for example, the data in the figures indicate to the reader the variability within each experimental group. Also, the discussion needs to directly relate findings in the literature to results of this study by citing the values obtained in this study and the supporting figures and tables. This direct linkage is needed throughout the discussion. The writing is understandable, albeit unclear in a few places.

Specific comments:

Abstract background: Although this needs to be kept short, it could perhaps be made more informative--what benefit is there to intercropping these 2 crops in south China? The authors state that it is efficient, but can they be quantitative--for example, what amount of N-fertilization reduction is possible when intercropping the leguminous peanut? Is there greater combined harvest or cumulative economic gain to intercropping?

Abstract results: Can the authors specify by exactly how much the genes and enzyme activities increased?

Abstract conclusion: How do the authors know that it improved the microenvironment? What constitutes an improvement? From an agronomy standpoint, did the soil produce higher yield? If so, is there a way to determine whether the microbial component of soil fertility factors really account for this?

lines 78-81: study objectives are currently stated as purely exploratory. Were there any hypotheses in mind as suggested by prior results in the literature, such as that intercropping would improve specific aspects of the soil in certain ways, or that particular microbial functions would be increased or decreased with intercropping?

line 94: What was the prior cultivation and management history of the site? Briefly state this so as to assure the reader that there are no legacy effects from prior management. Is this really only a field trial measuring the effects of just 1 year of cultivation of these experimental groups? This seems like a short time, but if rhizosphere soil effects are what are being measured, then one could reasonably expect differences to be noted within a single season. If this is the case, it may be worthwhile to state this in the discussion.

lines 102, 103: An aerial photo of the randomized plots with boxes overlaid to show each plot would be useful and provide a clearer picture of the entire field layout. It could be added as an additional panel to figure 1.

lines 103-106: What impact would different fertilization regimes applied to the different experimental treatments have on the results of this study, in particular the N, P, and K measurements?

lines 111, 112: The authors state that both loose soil (do they mean bulk soil here?) and root-adherent (rhizosphere) soil were collected, but were data collected separately for these two soil types? It seems like only rhizosphere soil was analyzed, or were they mixed together? If they were mixed together, then there was likely more influence of the bulk soil than the rhizosphere soil since the bulk soil volume would have been greater. Regardless, the authors need to clarify this in the text.

lines 118-133: The methods section needs citations to methodology articles or handbook protocols for all these methods. Were there any deviations to the protocols? Were there any other missing details of importance like input quantities? How about specifying the equipment models and relevant settings that were used to acquire the measurements?

line 135: Is there need to mention use of special equipment perhaps for a bead beating step not detailed in the manufacturer's protocol? Also, tell whether and how the soil may have been homogenized prior to DNA extraction. Specify the amount of soil used for each extraction.

lines 145, 146: The authors are using a new, less common sequencing platform, and so it would be good to provide a citation indicating that it has been shown to have comparable performance specifications a well-known platform like Illumina.

lines 176-178: Simply give the accession number range since they are consecutive to save space. Write "SRA accessions SAMN15324604 to SAMN15324615".

This reviewer has confirmed that the SRA project is accessible online at the SRA.

But what about the assemblies, can the contigs also be provided online?

lines 137-138: Tell DNA what aspect of DNA quality was confirmed by running on a gel--was it size, fragmentation, relative quantity?

lines 243-264: Give values for the gene abundances.

298-304: The paragraph needs some work to more clearly distinguish what are the findings of other studies vs. the findings of this study.

lines 335, 336: How does the proportion of both bacteria and fungi increase together? It seems that either one or the other should increase in proportion to the other.

lines 374-376: Last sentence--what is the code gene, is that a typo? Also, the linkage between your results of microbial analysis and the plant physiology are unclear. Please reword.

lines 399-404: The last paragraph needs some tweaks to the English and could use overall strengthening.

lines 466-468: It seems most publication titles in the references do not capitalize the first letter of each word in the title, but some such as this one do. Ensure consistent capitalization of titles throughout your reference list. Review all references and correct throughout.

lines 525, 546, 547, 588: Italicize species names throughout the reference list.

lines 594: There is an extra space in "quantity,  and" that needs to be deleted.

line 607: Should "ab initio" be italicized?

Fig. 1: The (IP2) abbreviation is needed at the end of the figure description. It is recommended also add wording to indicate that this is a "field plot schematic". It is not clear whether IP1 or IP2 have just 1 or 2 or even 3 rows of peanut within their respective field plots, and the authors should clarify this here or in the materials and methods section, or perhaps both.

Fig. 2: Use punctuation (a period or full stop) at the end of each sentence in the figure legends. This figure needs to show the data for each of the 3 replicates in all 4 treatments so that the reader has a clear, visual indication of how much variation occurs within treatment; that is, there should be 12 rows in this figure.

Fig. 3: It is unclear how to interpret this network. The data upon which the network is constructed should be indicated in the legend for the figure. Also, when there are multiple occurrences of a circle of a single color (e.g. orange circles), what does each circle indicate, separate samples perhaps? This needs to be indicated in the figure legend.

Fig. 4: Replicates should be indicated as stated in Fig. 2. The legend should detail precisely the normalized unit used for the read counts. The units for the bar plot are difficult to read at the current figure resolution. Vector versions of the figures should be used instead of raster images.

Fig. 5: A legend for circle size is missing in the figure. Tell what the codes on the left correspond to (i.e. are they KEGG identifiers?). Again, replicates are missing as in Fig. 4 and 2.

Fig. 6: Indicate the underlying data for the figure--is it relative abundances for KEGG groups?

Fig. 8: Indicate the type of model; that is, is there a particular statistical modeling technique applied, or is this a conceptual model? The phrase "micro-environmental changes" is uncommon, and thus it is not entirely clear what is meant in the manuscript by this phrase. Do the authors mean microbiota-induced changes in the environment? Correct this phrasing throughout the manuscript.

Table 1: Specify in the tables what is indicated by +/-, either Standard Error or Standard Deviation. Specify this in the legend or as a footnote in the table. Also specify that the combinations of letters a, b, c, and d beside the values in the table indicate statistically significant groups. Also specify the significance cutoff used. Rather than specify n=12 in each column, specify it just once in the legend for the tables stating that each experimental group contained 3 field replicates for each of the 4 treatments for a total of n=12 altogether. For p-values that are below a threshold and presently listed as 0.000, change to, for example, <0.001.


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: SUGARCANE/PEANUT INTERCROPPING SYSTEM IMPROVES PHYSICOCHEMICAL PROPERTIES BY CHANGING N AND P CYCLING AND ORGANIC MATTER TURNOVER IN ROOT ZONE SOIL
Review round: 2
Reviewer: 1

Output:
Basic reporting: The materials and method section is still not clear. Specifically, the authors mentioned it is two years study but two years of intercropping treatments and sequence of planting and intercrop management have not been provided. Other than that they have addressed all comments in the revised manuscript.

The manuscript will benefit from language editing.

Thank you.
Experimental design: Yes, all issues addressed except timeline of two years of study.
Validity of the findings: It looks reasonable
Additional comments: The materials and method section is still not clear. Specifically, the authors mentioned it is two years study but two years of intercropping treatments and sequence of planting and intercrop management have not been provided. Other than that they have addressed all comments in the revised manuscript.

The manuscript will benefit from language editing.


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: QUANTILE-DEPENDENT EXPRESSIVITY OF SERUM C-REACTIVE PROTEIN CONCENTRATIONS IN FAMILY SETS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Pass
Experimental design: Pass
Validity of the findings: Pass
Additional comments: I have only one comment: There is no protein in the serum named as "high-sensitivity C-reactive protein". This is wrong. The protein present in the serum is called C-reactive protein (CRP). Researchers often use a highly sensitive technique to measure CRP since the concentration of CRP in the serum is so low that sometimes it cannot be detected without using a sensitive method. However, if a highly sensitive technique is used to measure CRP, it does not change the name of the protein from "CRP" to "high sensitivity CRP". I recommend that, throughout the manuscript, only the term "CRP" is used; all "high sensitivity CRP" should be replaced by "CRP".


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: QUANTILE-DEPENDENT EXPRESSIVITY OF SERUM C-REACTIVE PROTEIN CONCENTRATIONS IN FAMILY SETS
Review round: 1
Reviewer: 2

Output:
Basic reporting: -	In Line 159 “:” is missing after “Statistics”.
-	In Line 209 “.” is missing after the title and there is no separation between the title and the text.
-	Table 1: There is no description for the values reported in the parenthesis. In addition, the caption underneath the table is incomplete.
-	 Figure 2 is best to be labeled as 2.A and 2.B so that the
-	In Lines 287, 319, 370, 384, 401, 409, 418, 425, 431, 500, 506, 524, 530, 538, 547 and 552,  “.” is missing in the beginning of the lines.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript presents a comprehensive study to investigate whether CRP heritability is quantile specific. This is a well written report with thorough analysis and in-depth discussion.


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: QUANTILE-DEPENDENT EXPRESSIVITY OF SERUM C-REACTIVE PROTEIN CONCENTRATIONS IN FAMILY SETS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Great job, all suggestions are supporting the study and make it available for publication.
1. I suggest the title of the study be according to "PI/ECOS" criteria  (P= population, I= intervention/E= exposure, C= control or compactor, O= outcome, and S= study design), for example, you can check this reference from World Health Organization: https://apps.who.int/iris/bitstream/handle/10665/193040/WHO_HIV_2015.37_eng.pdf?sequence=1
2. The paper requires language editing. For example:
•	Line No. 410, and 412: "estradiol" instead of "esterdiol".
•	Line No. 520: "12 week" should be "be 12 weeks".
•	Line No. 530: you repeated "Ankylosing spondylitis Ankylosing spondylitis" twice.
•	Line No. 565: "These exception" should be replaced by "this exception or these exceptions", and other mistakes, so I suggest language editions.
•      Besides, you should use unambiguous and comprehensible language.
3. References
It is noted that the argument “Quantile-dependent expressivity” was merely supported by self-citations. Although these citations are relevant and were appropriately used, the author(s) is/are advised to support his/her/their argument with the work of other researchers.
Experimental design: 4. I suggest the method section be structured based on Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement: https://www.strobe-statement.org/index.php?id=available-checklists
or as our PeerJ recent publication under the title "The relationship between anti-Müllerianhormone (AMH) levels and pregnancy outcomes in patients undergoing assisted reproductive techniques (AR)", DOI: 10.7717/peerj.10390
5. Table 1. Sample characteristics: Please define in your footnote the following abbreviations: BMI, and CRP
Validity of the findings: 6. I suggest the result section be structured based on Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement: https://www.strobe-statement.org/index.php?id=available-checklists
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: QUANTILE-DEPENDENT EXPRESSIVITY OF SERUM C-REACTIVE PROTEIN CONCENTRATIONS IN FAMILY SETS
Review round: 2
Reviewer: 1

Output:
Basic reporting: none
Experimental design: none
Validity of the findings: none
Additional comments: none


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

Input:
Title: QUANTILE-DEPENDENT EXPRESSIVITY OF SERUM C-REACTIVE PROTEIN CONCENTRATIONS IN FAMILY SETS
Review round: 2
Reviewer: 2

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: Great job,
Thanks for the language edition, all explanations in method and result sections. So, the article should be accepted.


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: IMMUNE FUNCTION OF MIR-214 AND ITS APPLICATION PROSPECTS AS MOLECULAR MARKER
Review round: 1
Reviewer: 1

Output:
Basic reporting: I think the review is not broad since it is only about one microRNA.

I think the review is not cross-disciplinary since it is mainly about immunology.

However, the review is long enough as is, and it might not improve by increasing its scope.

To my knowledge this topic has not been reviewed recently.
Experimental design: no comment
Validity of the findings: Overall, this review provides a catalog of published findings, but the authors do not synthesize a refreshing point of view.
Additional comments: My brief comments and suggestions:

1) The text could benefit from professional editing by a native English speaker. I’ve highlighted a few examples (not exhaustively) in attached, annotated PDF where the English is awkward and could be improved for clarity.

2) Authors should provide more details to their literature Survey Methodology. For example, what exclusion criteria were used? Their PubMed search must have yielded many articles. How did authors choose which ones to cite in their References list? In particular, PeerJ is concerned about selection bias.

3) Since, “miR-214 is located in the dynamin-3 gene…,” does its expression mimic that of its host gene dynamin-3 (perhaps because its transcription is regulated by a shared enhancer)?
Since miR-199a is located right next to miR-214, is it known whether they functionally cooperate (eg. in analogy to miR-17 cluster).

Based on miRbase (http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000290 ), in humans, miR-214-5p is hardly expressed based on 136 published RNA-seq experiments. Perhaps this is an important detail to share with readers. Also, this calls into question the utility of two findings listed in Table 2:

Kook HY et al. (2019) miR-214-5p is down in Ankylosing Spondylitis
Pang J et al. (2018) miR-214-5p is down in Hepatocellular Carcinoma
Could the authors please check the primary data regarding expression of miR-214-5p in those two papers? If the expression of miR-214-5p is low to begin with, it might not be so trivial to assay further down-regulation in a reliable fashion. Is expression of miR-214-3p also down-regulated? If not, why would that be?

4) The central image in Fig 1 looks like it’s copied from miRbase (http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000290 ). If so, miRbase should be cited appropriately.

5) In the Conclusion section, PeerJ requests authors to identify unresolved questions and gaps in knowledge. For example, is there a need to systematically identify direct targets of miR-214?


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: IMMUNE FUNCTION OF MIR-214 AND ITS APPLICATION PROSPECTS AS MOLECULAR MARKER
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a comprehensive review of the literature pertaining to miR-214.  It seems well organized and informative.
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: IMMUNE FUNCTION OF MIR-214 AND ITS APPLICATION PROSPECTS AS MOLECULAR MARKER
Review round: 1
Reviewer: 3

Output:
Basic reporting: In the review article “Immune function of miR-214 and its application prospect as molecular marker” the authors compiled the available data related to miR-214 and its role in immune function.
This review is not at all well written. The literature review is written in pieces and is not well connected with each other. It looks more like notes rather than a review article. There is an absence of critical analysis of the available data from the literature. The review lacks flow and jumps from one information to the other throughout the article which makes it difficult for the reader to focus and understand. The language needs a lot of improvement as sentences are too long and confusing making it difficult for the reader to understand the gist of the review. There are many redundant sentences. The authors have used a lot of incorrect scientific terminology. In some instances, authors have extrapolated interpretation without any scientific evidence.
Experimental design: The review is not well organized. Looks like notes of the available literature.
Validity of the findings: NA
Additional comments: For review articles, authors should summarize and analyze the scientific findings and critically discuss the outcome from their perspective which is lacking in this review.  Language is an issue that makes it difficult to read and understand the review and many incorrect scientific terms are used.
Few examples are
Confusing sentences:  49-54, 114-115
Wrong use of scientific terms:
Line 129: damage anti-tumor immunity
Line 107: what is microbubble structure?
Line 113: what are microcapsules?
Overinterpretation of results
Line 147: how miR-124 plays a role in the polarization pathway of M2 macrophages?
Role of miR-124 in organ transplantation
Line 31: Consistency in the use of the miR-214 or MIR-214
Line 217: Glycogen is repeated twice.


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

Input:
Title: A KINGDOM IN DECLINE: HOLOCENE RANGE CONTRACTION OF THE LION (PANTHERA LEO) MODELLED WITH GLOBAL ENVIRONMENTAL STRATIFICATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article presents original modelled data on the range of the lion (Panthera leo) from the late Pleistocene and Holocene and compares this against the modern distribution using ecological niche models and environmental stratification of palaeoclimate. The article is written clearly and in good English; however, there is significant textual restructuring that needs to be implemented and additional background to be written for the introduction. The authors scientific method appears to be scientifically sound; however, is difficult to follow, and this section often references methods from other papers. This needs significantly more work to clarify. The lack of structure in the methods section is reflected in the results section and needs further clarification. This lack of structure is also present in the discussion section. The conclusions clear.
Experimental design: The methods section is difficult to read and requires most information to be obtained from referenced papers. This section needs much greater clarification and transparency, which should be included within the main manuscript.
Validity of the findings: The findings appear valid; however, are not clearly structured and difficult to tell apart from referenced materials.
Additional comments: Major Comments
L50-118: In the introduction the authors give a well written overview of the background to some of the physical geographical barriers of the lion e.g. vegetation, and rivers, but need to introduce climatic parameters (the focus of the study) in much more detail. Please add a paragraph on climatic parameters of lion habitation with focus on enabling climatic conditions and environmental barriers.
L50-118: The authors also do not make any reference in the introduction to anthropogenic barriers (past or present). A small paragraph referencing the drivers/barriers to lion distribution as caused by people would benefit the introduction (even if dismissed as outside the scope of this study as a subsequent limitation). Please add a small paragraph on anthropogenic impacts to lions.
L127-136: This is part of the background section and not the methods. Please move to the introduction and incorporate into the suggested climatic parameters paragraph.
L137-145: This is a part of the background section; this is not a methodology. While this is a justification for the methodology, it needs to be put into the introduction section to set up the methods chosen.
L272-278: Descriptive and further justification of the methodology. This should be in the Introduction section or Methods section.
L280-291: This paragraph is highly speculative and should make further use of the modelled data generated. References in this paragraph also make the authors contribution and referenced articles difficult to disentangle. Please clarify.
L328-332: This is a key discussion point! Great! Please expand.
L442-448: Really nice and clear conclusions. Please adapt the discussion to reflect these.
L450: Perhaps change “Core findings” to “Key findings”?

Minor/Specific Comments
L27: Italicize “Pantera leo”
L105: Decapitalize “Vulnerable”
L106: Move reference Bauer et al., 2016 to the end of the sentence.
L154: Please define what the suitable bioclimatic conditions are.
L161: Unclear whether the data in table 1 is from Fei & Yu, (2016) or your own collected data. Please clarify the sentence.
L165: This sentence is not supported. Please reference the larger scale of lion home ranges.
Figure 7: Please add time periods to geological periods of time.
Figure 8: Detail illegible. Please split into several figures to make text legible.


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

Input:
Title: A KINGDOM IN DECLINE: HOLOCENE RANGE CONTRACTION OF THE LION (PANTHERA LEO) MODELLED WITH GLOBAL ENVIRONMENTAL STRATIFICATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comments.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: I have read the submitted manuscript entitled “A kingdom in decline: Holocene range contraction of the lion (Panthera leo) modelled with Global Environmental Stratification” by David Cooper and colleagues. I consider that the manuscript is scientifically interesting, important in terms of lion conservation, and well written.

Major comments
In terms of the contents I am concerned by the following five points.

Firstly, as the authors themselves appear to be fully aware it is very likely that lions were widely distributed across the current Sahara during the African humid period in the earlier Holocene (e.g. Yamaguchi et al. 2004, Barnett et al. 2006). Also, I see that the authors have made a substantial effort to overrule the results that the modelling suggested partially because the authors recognised the importance of the lions’ colonisation across the Sahara for their exodus from Africa to Eurasia. I consider the authors’ attempt to overrule the results appropriate to make more bioenvironmental sense concerning the recent colonisation history of the lion especially related to the African humid period (e.g. Adkins et al. 2006, Chen et al. 2020). However, then, it is difficult to avoid pointing out the possibility that the modelling that the authors developed may have major flaws. Such flaws may have resulted in incorrect suggestions (in addition to the Sahara case), which may have been overlooked simply because they are not as well-known as is the case of the Sahara. By any chance, isn’t it possible to make some adjustments to the models to do re-analyses?

Secondly, the similar concerns may apply to the authors’ modelling for the Last Glacial Maximum (LGM). Some modelling appears to suggest that the Sahara was covered by vegetation during the LGM more than it is today (e.g. Shao et al. 2018) consistent with what the authors’ modelling suggests. However, a substantially larger number of references suggests that the Sahara was hyper-arid during the LGM (e.g. Adkins et al. 2006, Ye et al. 2019) although there were some wetter periods during the last glacial period (e.g. Hoffmann et al. 2016). The authors’ modelling suggests results that strongly contradict the idea that the Sahara was hyper-arid during the LGM. Considering the authors’ modelling having suggested incorrect results for the African humid period, the authors may want to carefully re-evaluate their modelling for the LGM.

Thirdly, as the authors appear to acknowledge, there were lions during the Holocene in the southeast Europe substantially larger than the area that the authors appear to have used for the modelling, including the current Bulgaria, Romania, Ukraine, and Hungary (e.g. Daróczi-Szabó et al. 2020). Although Daróczi-Szabó et al. (2020) was published very recently, because the relevant data have been available for a while (e.g. Vörös et al. 1983) I wonder why the authors excluded those areas from the analysis. Is it possible to include those areas into the modelling?

Fourthly, it is biolgeographically very appropriate for the authors to restrict the area concerning lions’ colonisation because similar GIS analyses sometimes overlook the mobility of the focal organisms – e.g. a small mammals in Africa very unlikely colonise Australia even if modelling suggests the existence of very suitable habitats there. However, then, considering the mobility of lions (capable of dispersing > 200km within 1-2 years: e.g. Yamaguchi et al. 2004) and the large distribution range of the extinct cave lions (Panthera (leo) spelaea) throughout the northern Eurasia during the LGM (e.g. Barnett et al. 2009) the authors probably should have set the range of the lions’ possible colonisation substantially larger.

Fifthly, again as the authors themselves appear to be aware, partially due to the nature of the authors’ work concerning ecological niche of the lion, the large distribution of the extinct cave lions throughout the northern Eurasia during the LGM (e.g. Barnett et al. 2009) may cast a shadow over the modelling. It is possible that the extinct cave lion and the modern lion overlap their niches substantially if not completely. The authors state in the line 137 “In this study, we assume that the environmental niche of the lion has been stable”. It’s fine, but, the authors probably need to set the “environmental niche of the lion” much broader than that we estimate based on the geographical range of the modern lion recorded during the historical time. Based on this assumption, one may want to classify a substantial part of the northern Eurasia as potentially suitable habitat for the lion, and develop modelling accordingly. Based on the current modelling it appears that there are few suitable areas for lions to colonise in Eurasia even during the LGM, possibly highlighting the flaws of the modelling. I wonder if the authors may want to do modelling based on this assumption, and, at least, present the results in the Supplementary Materials for comparison.

Minor comments
Lines 181-182
Very briefly explain what those four variables are.

Line 184
Should “(-19°E, 94°W, - 36°S, 50°N)” be “(-19°W, 94°E, - 36°S, 50°N)”?

References
Adkins et al. (2006). The “African humid period” and the record of marine upwelling from excess 230Th in Ocean Drilling Program Hole 658C. Paleoceanography and Paleoclimatology. https://doi.org/10.1029/2005PA001200.

Barnett et al. (2006). The origin, current diversity, and future conservation of the modern lion (Panthera leo). Proceedings of the Royal Society B: Biological Sciences 273: 2119-2125.

Barnett et al. (2009). Phylogeography of lions (Panthera leo) reveals three distinct taxa and a late Pleistocene reduction in genetic diversity. Molecular Ecology 18: 1668-1677.

Chen et al. (2020). Feedbacks of soil properties on vegetation during the Green Sahara period. Quaternary Science Reviews. https://doi.org/10.1016/j.quascirev.2020.106389.

Daróczi-Szabó et al. (2020). Pending danger: Recent Copper Age lion (Panthera leo L., 1758) finds from Hungary. International Journal of Osteology. DOI: 10.1002/oa.2875.

Hoffmann et al. (2016). Timing and causes of North African wet phases during the last glacial period and implications for modern human migration. Scientific Reports. DOI: 10.1038/srep36367.

Shao et al. (2018). Statistical reconstruction of global vegetation for the last glacial maximum. Global and Planetary Change 168: 67-77.

Vörös et al. (1983). Lion remains from the Late Neolithic and Copper Age of the Carpathian basin. Folia Archaeologica Budapest 34: 33–50.

Ye et al. (2019). Multifunctionality debt in global drylands linked to past biome and climate. Global Change Biology. https://doi.org/10.1111/gcb.14631.


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

Input:
Title: A KINGDOM IN DECLINE: HOLOCENE RANGE CONTRACTION OF THE LION (PANTHERA LEO) MODELLED WITH GLOBAL ENVIRONMENTAL STRATIFICATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have carried out a good job in restructuring the article to flow more logically and have addressed most points raised in the previous round of reviews to satisfaction.

A few very minor points:

The authors need to check their referencing throughout as their automated reference manager appears to have malfunctioned leading to many errors. Please also check puctuation (missing full stops on L165; L185).

L630: The authors were asked to change “Core findings” to “Key findings” and stated that they had made this change. This change has not been made. Please make this change.

Otherwise, nice job in making the suggested amendments and well done on writing a great paper.

Best wishes,
William J Harvey


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

Input:
Title: A KINGDOM IN DECLINE: HOLOCENE RANGE CONTRACTION OF THE LION (PANTHERA LEO) MODELLED WITH GLOBAL ENVIRONMENTAL STRATIFICATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: None.
Experimental design: None.
Validity of the findings: None.
Additional comments: I consider that the authors' replies to my comments and revised manuscript are appropriate. 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: NEW DATA ON TAIL LENGTHS AND VARIATION ALONG THE CAUDAL SERIES IN THE NON-AVIALAN DINOSAURS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In some parts the text is a bit colloquially phrased; I have marked a few instances. Please go through the entire MS, not just the marked instances.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This MS was a joy to read, as the structure and language are clear, the relevant literature has been taken into account, the figures are sufficient and clear (exception: in on the species names overlap; if possible this problem should be correct - not essential, though, as the relevance of the individual labelled values is low and the relevant aspect, the curve, is clearly visible), the data is well presented, and the hypotheses are clearly defined and tested.

Aside from some minor text editing, this is in my view ready for publishing!


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: NEW DATA ON TAIL LENGTHS AND VARIATION ALONG THE CAUDAL SERIES IN THE NON-AVIALAN DINOSAURS
Review round: 1
Reviewer: 2

Output:
Basic reporting: See general comments.
Experimental design: See general comments.
Validity of the findings: See general comments.
Additional comments: Thanks for the opportunity to review this manuscript. I think the main observation of the paper, that dinosaur caudal vertebral length does not just uniformly decrease along the length of the tail, and the resulting functional implications, is interesting. However, I have several issues with the structure of the paper and the approach taken that I think need addressing before the paper could be accepted for publication.

1. Introduction and rationale for the study: I do not find the hypothesis to be clearly laid out and the results and discussion do not link really clearly back to the stated hypotheses and predictions. What are you really testing, because it feels like more than one thing, which is fine but should be specified – I feel like questions you are trying to answer are:
• Do caudals smoothly decrease in length, yes or no?
• If no, is there a consistent pattern across dinosaurs or at least some dinosaur groups?
• What do the patterns mean?

Or, are you asking:
• Can you reconstruct body length from tail length alone?
• Can you reconstruct fragmentary tail lengths from more complete specimens?

I think the introduction could be strengthened by a more in depth discussion of caudal anatomy in other tetrapod groups and its functional implications, and how that relates to dinosaur caudal anatomy. Have caudal length patterns been investigated in other groups? What approaches were taken? What were the results?

2. Methods: I’m not sure the methods used are appropriately addressing the hypothesis. I feel like what you want to do is use AIC to determine whether a single segment model or a multiple segment model better fits the data on dinosaur tail lengths, which would test your hypothesis that caudals do not just smoothly decrease in length, but I don’t think that’s exactly what you’ve done.

I’m also concerned that the analysis is not picking up all of the morphology, because I don’t understand the graph presented for Dyoplosaurus. Ankylosaurids have a series of short, ‘typical’ dinosaur caudal centra anteriorly and then those in the tail club are abruptly much longer at about the midpoint of the tail. Surely there should be a break point indicated at the beginning of the tail club, but it doesn’t look like that’s the case in the graph? If so, can you comment on why that might be, and what implications that has for the other graphs and your interpretations?

I think it would also be very beneficial to look at the phylogenetic signal of caudal centrum length patterns or femur to tail length ratios, since one of the points made in the paper is that you can’t use tail length to predict other aspects of anatomy. Would also be helpful for interpreting how easy it is to reconstruct lengths of partially complete tails.

3. General paper structure: Very few results are actually presented in the results section, and a lot are presented in the discussion, which needs rectifying. The results section has a lot of very vague sentences with broad generalizations and not a lot of reporting of statistical results or qualitative patterns. The results section should detail caudal anatomy and the results of your statistical analyses; the discussion should interpret those results in terms of your hypothesis, the functional implications, etc. I also noticed quite a few typos throughout the manuscript, and think it could benefit from a very close read-through for both spelling and sentence readability.

4. Figures: I do not think the first figure adds much to the paper, especially because no anatomical features of note are labelled on it. At minimum, labels should be added to this, but a really useful figure would probably include line drawings of the major clades described here.

The figure detailing the repeated measurements for the hadrosaur specimen vs photograph measurements should be Figure 2, since this is a proof of concept for your use of photograph measurements.

The remainder of the results figures could probably be condensed into fewer individual figures, and it would be helpful for them to be organized in some kind of phylogenetic structure, e.g. all theropods together or at least in a few figures, all sauropods together, all ornithischians together or near each other. The legend describing the break points and transition point should be in the caption text for each relevant figure, not just one. Full species names and specimens should also be included either in the graphic of the figure or in the caption, please don’t make people spend a bunch of energy figuring out which specimen you’re talking about by going through your appendix.

I’m having a hard time parsing Figure 3, is there another way this information could be presented? Or could additional information be included in the caption that makes it easier to understand?

Why does figure 6 appear where it does? Should this be before or after the bulk of the other data figures? Figure 6 is also very difficult to interpret because all of the taxon names are overlapping the data, please clean this up so that it’s more legible.

I have also attached an annotated PDF with some additional notes and comments throughout the text. I hope these comments help to strengthen the revised version of the manuscript.

-Victoria Arbour, 9 May 2020


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: NEW DATA ON TAIL LENGTHS AND VARIATION ALONG THE CAUDAL SERIES IN THE NON-AVIALAN DINOSAURS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript upholds standards for basic reporting, and will be enhanced with just a few improvements to mechanics and content.

Avoid and rework "due to" clauses for slightly more active and direct communication.
Replace "nature of" with specific concepts.
Re-phrase at least two sentences that separate subject and verb with a minor avalanche of (necessary) prepositional phrases.

Consider Gauthier (1986) and Tetanurae when discussing transition points.

For each figure caption, include a take-home message that you want the reader to understand from the visuals.
Experimental design: Other reviewers can better comment on the statistics. For R plots, consider abbreviations rather than long genus names that clutter the chart.

When justifying why centrum length is interesting, briefly acknowledge other dimensions by citing Currie (1978) on centrum transverse radius.

Groups for comparison are well-chosen, but the hypotheses are rather dry. To better frame the question, reiterate "why" after you state the hypotheses.

Impressive venture comparing physical and photograph-based measurements.
Validity of the findings: The most interesting results relate to variation within and between dinosaurian clades and locomotor categories. The exploration was intriguing, and sets up questions of functional morphology.

Clarify earlier which parts of the proximal region of tail are stiff and therefore useful for bracing against m. caudofemoralis longus action. Gauthier (1986) considered the distal region of the tail in theropods (posterior to the transition point) to act as a stiff, unitary dynamic stabilizer.

It's unclear why long centra just posterior to the base of the tail would limit its flexibility in this region. What if joints between these long centra were unconstrained ball-and-socket configurations? Posit how you might further test this flexibility.

The conclusions about applicability to other segmented animals seem dropped in as a hopeful afterthought. Try to tie these better to your methods.
Additional comments: The attached commented manuscript elaborates on points in this review, and includes specific suggestions. Figure 1 is welcome, but more images of actual dinosaur tails, and/or thumbnails of full skeletons, would illustrate the animals behind the uniform or regional regressions.  I suggest at least one figure showing tails in various groups or tail thumbnails in each graph, and a figure with skeletons illustrating locomotor categories.

As mentioned under "basic reporting", elaborate captions with take-home points for each figure. Lots of readers will jump first to the visuals and captions. What do you most want them to notice and learn from a figure? The caption for Figure 1 does this quite well.


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: NEW DATA ON TAIL LENGTHS AND VARIATION ALONG THE CAUDAL SERIES IN THE NON-AVIALAN DINOSAURS
Review round: 2
Reviewer: 1

Output:
Basic reporting: This paper has improved a lot, especially in areas (statistic) outside my area of expertise. Overall, I find the authors' response convincing with regards to the review recommendations they chose not to follow. The grammar has been improved, colloquial phrasing has been removed.
Experimental design: all fine
Validity of the findings: all fine
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: NEW DATA ON TAIL LENGTHS AND VARIATION ALONG THE CAUDAL SERIES IN THE NON-AVIALAN DINOSAURS
Review round: 2
Reviewer: 2

Output:
Basic reporting: See general comments.
Experimental design: See general comments.
Validity of the findings: See general comments.
Additional comments: Thanks for providing the revised manuscript and author comments for a second round of review. In my first review I noted issues with the introduction (ease of understanding the hypotheses, background information from other literature), methods, general paper structure, and figures. I’ve structured my current round of responses into the same categories and note where I feel comments have been satisfactorily resolved vs. where I think additional work is needed. I have extensively marked up the attached PDF and have not repeated all of my comments here, so please review the comments on the PDF as well.

1. Introduction and rationale for the study.
In the response to my comments the authors wrote: “With respect to the point about other taxa, we think this is well beyond the scope of this paper since while clearly relevant, this is an attempt to produce and analyses data on dinosaur tails…Studies of reptile tails we have looked at have focussed on flexibility (joints, articulations) and particular as anchors for key muscle groups and the effect on propulsion and there is almost nothing on total length, or the lengths of centra or patterns of centra.”
I actually think this is an incredibly valuable thing to note because it highlights the novel aspects of this manuscript. I don’t see why you can’t have a sentence that says something along those lines – author X has looked at joints/articulations/flexibility but not about length patterns across multiple families. That is helpful!

Overall I think the hypotheses are a bit more clearly laid out but (as I’ll note in detail shortly) I think the introduction could use another close edit for things like paragraph structure, readability, and conciseness (see general paper structure section).

2. Methods:
I am satisfied with the response to my comments about AIC and Dyoplosaurus. However, a follow-up question is whether or not any other taxa in the dataset should have been treated as discontinuous? I think the answer is reasonably ‘no’, but it would be good to explicitly state why *only* Dyoplosaurus was treated this way, and if you can provide statistical support for one method vs the other that would obviously be beneficial.

In my last review I had noted that it would be helpful to look at phylogenetic signal since a large part of the discussion is about reconstructing caudal anatomy or predicting body size based on tails. While I understand that the data is skewed and it would be extra work to give this a try, I think you need to be very cautious about how you frame your results about reconstructing tail lengths/body size in the absence of phylogenetic context. At minimum, I think you should include a note about why you didn’t look at phylogenetic signal for this – noting that the data is restricted and skewed would be sufficient.

3. General paper structure:

I think the issue with results being reported in the discussion section and such is basically resolved, and that this is much improved from the first submission. However, I think there is still a substantial amount of revision needed in order to tighten up the manuscript overall. For example:
• the Results section does not detail the results of the photograph vs. specimen measurement comparison
• there seem to be contradictory statements at various points in the Discussion about whether you can or cannot (or should or should not, I guess) reconstruct missing elements from partial tails
• there are numerous typos and grammatical glitches that don’t seem to have been resolved from the previous version or might have been introduced when writing new text

In my previous review I noted that the manuscript needed a close read-through for spelling and sentence readability, and unfortunately I do not think there was much improvement on that front in this draft. I found overall that I was having a hard time following the flow of arguments in this paper because there are a lot of parenthetical statements, sequences of sentences beginning with ‘thus’, ‘therefore’, ‘in other words’, etc., and qualifying words like ‘likely’, ‘possibly’, etc. I’ve tried to make some suggestions for where sentences can be trimmed down and made more concise without losing the meaning of the sentence, and which hopefully make the point of the sentence more impactful. I cannot edit the entire manuscript in this way and so I really, really encourage the authors to go back through the ms with a metaphorical fine-toothed comb, make sure all the paragraphs flow well both internally and from paragraph to paragraph, and see if sentences can be trimmed down.

4. Figures:
I appreciate the changes to the figure captions throughout and the inclusion of genus names and specimen numbers. However, I still have several major issues with the figures.

Fig 1: The reviewers note that “We think figure 1 is useful (as reviewer 3 notes) in its current form so have not changed it. Adding detailed drawings of the tails of several taxa would be a considerable amount of work and these are fairly easily accessible in other papers with specialist descriptions (which are already cited).” I note that Reviewer 3 specifically noted “Figure 1 is welcome, but more images of actual dinosaur tails, and/or thumbnails of full skeletons, would illustrate the animals behind the uniform or regional regressions. I suggest at least one figure showing tails in various groups or tail thumbnails in each graph, and a figure with skeletons illustrating locomotor categories.” This is almost exactly the same as my request that a range of tails and their varying morphologies be included in the paper and I frankly find it disingenuous to frame this as though Reviewer 3 and I are in opposition on this point when we are clearly not. (I had written “I do not think the first figure adds much to the paper, especially because no anatomical features of note are labelled on it. At minimum, labels should be added to this, but a really useful figure would probably include line drawings of the major clades described here.”)

I am not sympathetic to the argument that adding labels to this figure, or better yet, revising this figure to include images of other dinosaur tails, is too much work. I think in a paper about the morphology of dinosaur caudal vertebrae and their functional significance that it is entirely reasonable to have a figure showing the range of morphologies of clades mentioned in the study, and to label things like the transition point, extent of the caudofemoralis, and centrum/neural arch/haemal arch/transverse processes. Your readers will obviously include seasoned professionals in vertebrate palaeontology, but will also presumably include nonspecialists and students. Don’t make them go wading through the literature just to know what a whiplash diplodocid tail really looks like, or an ankylosaur tail club, or an oviraptorid pygostyle. I am fairly certain the authors have photographs of specimens they have personally observed that could be compiled into a plate of photos that would be incredibly useful to the readers of this paper. It’s probably the sort of figure that would find its way into university lectures on dinosaurs and vertebrate anatomy, and honestly it’s probably the sort of image that would get tweeted out about the paper and help the paper gain a wider readership.

As another point along these lines, I’d like to highlight the fact that your statistical analyses did not recognize ankylosaurid tail clubs and it was my familiarity with their anatomy that caught this, because I have a mental image of what an ankylosaurid tail looks like. Being able to visualize the morphology of the tail was important for vetting the results of the statistical analysis and interpreting the results. Similarly, having photographs or illustrations of tails at hand in the manuscript would be helpful for readers to be able to associate the abstracted data in the plots with the functional interpretations you make in the discussion.

I leave it to the editor to make the call on this one, but obviously I think the communication value of a figure with some pictures of dinosaur tails outweighs concerns about the amount of effort it would take to create that figure. Reviewer 3 clearly seems to have thought along the same lines.

Fig. 6: I was disappointed to see that there were no changes to Fig 6 and that the species names are still overlapping the data points. As I mentioned previously, this figure needs to be cleaned up so that the plotted data is visible. This is probably annoying and difficult to do in R (which I presume the plot was originally made in), but probably pretty easy in Illustrator or the vector program of your choice.

Fig 3 and overall order of figures: I find it weird that figure 3 (about body size) comes after one of the figures showing break points for sauropods, and then we jump back into break point graphs for other dinosaurs. This is because sauropod results are mentioned in the methods section – I think that text needs to be rewritten so that what is currently Figure 2 appears sequentially along with the remaining break point figures. In my last round of comments I said that the figures should be organized in some kind of phylogenetic structure and this is what I was getting at – currently the figures are, in order: anatomy, sauropods, body size, theropods, hadrosaurs, summary of break points, thyreophorans, ceratopsids, sauropodomorphs, theropods, theropods, theropods, ornithopods. This isn’t an order that really makes sense to me, and in reviewing their appearance in the text I don’t see the pattern either. A figure order that makes more sense, to me at least, would be: anatomy, photograph vs specimen measurements (which is not in the current list but is in the SI), body size, thyreophorans, ceratopsians, ornithopods/hadrosaurs, sauropodomorphs, sauropods, theropods, theropods, theropods, theropods, summary of break points. That’s like walking across the Dinosauria tree, and that’s how I’d look for this data if I was flipping through the figures in the paper looking for specific clades. Alternately you could group results by quadrupedal/bipedal taxa but still at least put sauropods and sauropodomorphs closer to each other within the text (they’re currently separated by 7 figures). You could just have a sentence at the beginning of the section where you talk about break points in your Results that says something like “The results of our break point analyses are presented in Figures 3-11” or however many there are.

I continue to think that the results of this paper are interesting and worthwhile to share via publication and I appreciate the work the authors have done to collate this dataset. I offer these critiques in the spirit of improving the manuscript so that the effort that has gone into collecting and analyzing this data is maximally useful to readers and reaches a wide audience.

-V. Arbour, 27 Aug 2020


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: NEW DATA ON TAIL LENGTHS AND VARIATION ALONG THE CAUDAL SERIES IN THE NON-AVIALAN DINOSAURS
Review round: 3
Reviewer: 1

Output:
Basic reporting: See general comments.
Experimental design: See general comments.
Validity of the findings: See general comments.
Additional comments: I have two remaining comments on the manuscript, both of which are minor, and I do not feel that I need to review this manuscript again:

1. Anatomical features of note should be added to figure 1, such as centrum, neural spine, haemal spine, transition point, etc.
2. I am still catching a number of typos and grammatical errors in this revision, which I've highlighted on the attached PDF.


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

Input:
Title: EVALUATION OF FULL-LENGTH NANOPORE 16S SEQUENCING FOR DETECTION OF PATHOGENS IN MICROBIAL KERATITIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the present study, the authors present a novel molecular approach for detection of pathogens directly from clinical samples, using full length 16S sequencing on the Nanopore platform, in the context of the disease microbial keratitis. The authors investigated several different sample collection methods using an ex vivo porcine model of microbial keratitis to compare relative DNA recovery rates. They also developed a new bioinformatic analysis pipeline for taxonomic assignment. Finally, as a “proof-of-concept”, the authors applied their optimized pipeline and their most effective sample collection method to investigate the effectiveness of full-length nanopore 16S sequencing for pathogen detection from clinical samples. The need for developing newer, molecular approaches for culture-independent investigation of pathogens is immense, and the effort by these authors is commendable. Specifically, their efforts towards optimization of sample collection method seems thorough, and will be a great contribution to the field. However, the introduction and discussions do not help set up the research question very clearly or justify some of the method choices well, and some results need further clarification.
Experimental design: Firstly, the approach presented here is novel compared to standard molecular approaches in that it suggests the use of full length 16S reads, on the Nanopore sequencing platform. The authors however do not clarify why full length 16S reads is required for detection of pathogens in microbial keratitis. Therefore, for readers to better understand both the need and the adequacy of full length 16S sequencing, a discussion comparing the utility of variable region only (maybe V4 since its used here), full length 16S reads and whole genome-based approach for taxonomic profiling of common pathogens involved in microbial keratitis would be useful.
Secondly, the authors do not justify the use of Nanopore technique sufficiently, especially given that they mentioned the relatively high error rate of this platform (lines 118-119), which can be detrimental when discerning between closely related species.Therefore, a detailed discussion comparing full length 16S sequencing on the Nanpore and alternate platforms such as PacBio ccs platforms is essential, to provide required context for the present study. This is especially important because the authors claim that Nanopore sequencing has the potential for rapid, real-tie diagnosis with relatively low cost (lines 409-410), yet provide no information about the time, cost etc.
Finally, while the authors must be commended for including patient samples for validation of their method (and including negative controls), the sample size (n=2) is too small for adding any real value at this time, especially given that there is concordance in one patient and discordance for another, with respect to culture results. A “proof of concept” study might not require as many samples to provide sufficient power for statistical analysis, but as it stands the sample size of 2 is insufficient to draw any meaningful conclusion about the validity of the method. At least another culture positive sample should be included, and Illumina sequencing results for all 3 shown for comparison.
Validity of the findings: In Table 4, for patient B, what were the other species identified by the Illumina method? Did those species match with the Nanopore results, especially since B. subtilis constituted only 13.9% of the sample’s relative abundance in the Illumina results, indicating that it was likely not the most abundant species in that dataset. If the most abundant species is different in the two methods, this might be an issue, especially since the sample is culture negative. Showing top 5 species for each affected eye result would help understand the results better, given the low abundance of top species.
Lines 371-373: Reads as though nanopore results were in concordance with culture techniques for both patients, which is not true, please clarify.
Additional comments: How were the 3 species included in the mock community decided? The authors did not list E. avium as one of the common species found in bacterial keratitis (lines 77-81). Also, the major species detected in the clinical samples were S. marcescens and B. subtilis. Thus, the choice of species in the defined mix needs to be explained further. Including those species found in the clinical samples in the defined mix for bioinformatic pipeline optimization may help generate greater confidence in the method.
The data presented in Table 2 should be presented as bar charts (of relative abundance) for quick and easy comparison, with the counts included in a supplementary table possibly.
While the authors say that filtered reads better represented the mock communities (lines 278-286), details of how the filtering parameters were chosen, is not discussed, either in results or methods. To that end, a figure, maybe in supplementary, with visual representation of the bioinformatic processing steps would be valuable, with more details than in Figure 1.
Line 235-236: The authors claim that the bioinformatics scripts and fastq files are available at the GitHub repository (https://github.com/liyinglow/nanoporeMK), but I did not find any of those at that link (last cloned June 1st, 2020).
Supplementary Figure 1: Please replace with high resolution image for better readability.