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

Input:
Title: COMPARATIVE GENOMICS OF 16 MICROBACTERIUM SPP. THAT TOLERATE MULTIPLE HEAVY METALS AND ANTIBIOTICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written, with few typos and unclear sentences (see general comments) and I was able to access all data as shared/reported.

The introduction does a great job outlining chromium tolerance, and the genes associated with these tolerances, but does not explain what comparative genomics approach adds to the knowledge regarding heavy metal resistance or Microbacterium diversity. Since comparative genomics is the primary method/objective for this study more of the introduction should define and detail what this will provide. Discussions of ecotypes (Hunt et al., 2008 Science; Denef et al., 2010 PNAS; Frangeul et al., 2008 BMC Genomics; Briand et al., 2009 ISME J) and known diversity within the Microbacterium would be a potential place to add needed context (Meyer et al., 2017 PLoS ONE). Additionally, a few sentences identifying the specific knowledge gap this study (or these isolates) fills would provide better context at this point in the manuscript. The nature (metals, sources) of contamination of the study site is not described, which would provided the context for such gaps in knowledge as: Is reduction a proxy for “cleaning up” this environment? Are heavy metals a continued issue, creating a level of steady-state heavy metal stress? Are Microbacterium found in similar contaminated environments in other areas so the knowledge here could inform elsewhere?

I really enjoy figures like Figure 1 because they contain so much information, however, not all readers will be as familiar with An’vio and the way data are presented. If you could expand on the description of what data are contained, and where they are displayed in the figure, more readers would be able to fully explore all the information presented in Figure 1. For instance, what do the dark vs. light portions of the genome plots mean? What does the outermost ring show? Could a scale/legend be added to the tree above the chromate reduction and antibiotic genes?

I was only able to open figure S1 using Adobe Photoshop. Some readers may not have the same access to such software, please consider changing the file format to a more universal type so that all readers can easily view this figure.

Would it be possible to add an additional figure to show the entire pathway, and genes for each step, for the functions discussed in this manuscript? For example in Flores and Herrero (2005 Biochem. Soc. Trans.) Figure 1 helped to show the variety of Nitrogen assimilation and reduction pathways in cyanobacteria. A similar figure in this manuscript could help to show how the chrA and chrR genes would function in relation to one another or independently.
Experimental design: The primary research objective was not entirely clear to me, given that Microbacterium resistant to chromium were previously isolated and studied in this environment (Henson et al., 2015). The inclusion of comparative genomics in the title combined with the focus on heavy metal and antibiotic resistances in the introduction suggest that more focus will be placed on shared (core) and differences (flexible) in genome content. Such differences can better inform population diversity and function and would be filling a knowledge gap. Additionally, Lines 212-214 suggests that “other Microbacterium spp.” were not included the comparative genomics analysis. Why not? I agree that the identification of chrR and yeiF homologues in these other isolates suggest high interspecies diversity, and that diversity would allow for better calculations of core and pan-genome sizes in addition to providing a more complete picture of metal and antibiotic resistances.

The CheckM results suggest that some isolates contain strain heterogeneity, which may be an artifact of mis-assembly of closely related organisms. Hug et al. (2015, Environmental Microbiology) determined that assembly was improved for more abundant organisms’ genomes when subsampled, but the level of subsampling depended on the sample. Testing multiple subsample depths (sequential assembly) could reduce strain heterogeneity in some of your assemblies and could identify multiple isolates from the same sample/culture.

It is not clear if the flexible genome was specifically searched for differences and resistance/tolerance genes (using homology, BLAST, etc.). Perhaps you could add a paragraph outlining the major COG categories of the flexible genome; most will be unknown/hypothetical, but those that have an assigned function could be very interesting as well.

Given that this was a whole-genome approach, I was surprised that the phylogenetic tree was only based on 16S rRNA sequences and did not include Microbacterium isolates mentioned in the text (Lines 183-189). If available these other isolates should be included in the tree as it may highlight subclades with shared function. The use of whole genome gene presences/absence should also be considered to make a more robust phylogenetic tree that takes into account difference in gene content that the authors have access to through their sequencing efforts (as observed in Humbert et al., 2013 PLoS ONE; Meyer et al., 2017 PLoS ONE).

While IMG is a great tool for exploring genomes, it was not clear to me exactly how searches were performed within IMG (Lines 128-135). If using a keyword search, some genes may have different annotations or missing keywords and therefore not show up in search results. Was the COG search a secondary search to verify that all target genes were found or a separate search entirely? If the COG search was separate, please consider double checking that all chromate resistance & reduction and heavy metal resistance genes were found through secondary searches that do not rely on keywords in the annotation.

Please include a description of how you calculated gene homology in your methods. Were the BLAST comparisons bi-directional? Table S3 suggest these were one-way blast comparisons. What informed your decision in choosing cutoff values for homology?  See Albertsen et al., 2013 Nature Biotechnology for such a comparison. Did the Microbacterium chrR genes have a higher homology to one another than to the Pseudomonas reference? I would like to see the data on the interspecies genetic diversity of chromate reductases. Perhaps a table or figure depicting the homology of these specific genes.
Validity of the findings: Your experiments did a good job of testing for and identifying potential genes involved in metal resistance, and then verifying resistance at a range of exposure concentrations. Useful context would be to briefly discuss observed concentrations of the tested metals in natural and contaminated environments. Levels considered to be “contaminated” are not discussed but would be useful in interpreting the different strains resistance to the range of concentration tested. You do a great job provide this context for antibiotics in Lines 245-257.

I like how the introduction outlines the difference in function between Cr(VI) transport via ChrA and Cr(VI) reduction via chrR and yieF and would like the discussion to also touch on this difference since no chrA were identified in your isolates (“Implications to community functions” would be the ideal paragraph). Does this strategy (reduction only) have any broader implications for not only population differentiation, but community composition and function?

You did a great deal of work, sequencing and comparing 16 genomes, and I'd like to see more discussion on differences in gene presence/absence, or even differences in annotated categories of genes (COGs, KEGGs, etc.) between the core and flexible genome. These data will potentially allow for more conclusions on the variation and genetic diversity of the community. It would also lend more weight to the speculation between genomics and physiological diversity and differentiation within this environment.
Additional comments: Multiple In-text references missing in Reference list. For example, Brose and James, 2010; Meyer and Huber, 2014. Please check all references for inclusion and accuracy.

The use of italics for gene abbreviations (ex: chrA) is widely accepted, please double check that all gene abbreviations are italicized throughout the text.

Table 1: Footer ** says “accept for” should be changed to “except”

Lines 75-80: These statements are valid, but seem more suited for the abstract or discussion.

Line 111: Why was 70bp chosen as the new read length for secondary quality filtering?

Lines 122-123: How was Prodigal used to annotate the core genomes? Prodigal provides gene prediction and not annotations; this method description seems incomplete unless “annotation” simply means marking their location(s) across the isolates compared.

Lines 130-132: Was this a nucleotide or amino acid BLAST? Bi-directional BLAST or one-way? Were these BLASTs done within the IMG/MER interface or via command line?

Lines 132-134: At first read the phrase “…were also annotated using KEEG pathway KO identifiers” makes it seem that these genes were annotated by the authors. Was this meant to say that these genes were identified/searched for using KEGG (I believe KEEG is a typo) and KO identifiers? I think just replacing “annotated” with “identified” would be sufficient if that was the intent.

Lines 165-167: I agree that this is a very interesting finding and would like to see this expanded in the discussion. Are there other reports of similar observations?

Line 190: I read the sentence two possible ways: 1- the core genome has a large amount of variation, meaning that shared genes are significantly different among the 16 isolates or 2- the pangenomic analysis succeeded in identifying the core genome and separately found regions that were more variable (flexible genome). Would it be possible to clarify this either though 1- adding metrics for the variation in core genes (SNPs, phylogenetic trees using just the Core or non-core genes) or 2- Removing this sentence as the following line is a great summary of the pangenome initial results for core/pan genes.

Lines 192-193: While this reference is valid, it is also very general. The Bacillus examples presented are from a different phylum. Are there any estimates for core-genome size/percentages for Microbacterium or higher taxonomic levels (Actinobacteria) that could provide better context for whether the 26% calculated in this study is typical for related organisms? Alternately, what about the percent core genome for other organisms from similar environments? Is a larger flexible genome an adaptation for heavy metal exposure? I appreciate this line of thinking but wanted it to dive deeper.

Line 248-249: Awkward sentence. Errant comma after “study” and use of “found” twice seems repetitive. Suggest “A 2015 study found 26% of Microbacteriums isolates from ____ (environments) to be resistant to vancomycin.”

Lines 247-257: You list the three antibiotics in Line 247-248 and have them ordered in the same manner in Table 3 (amp, can, van). However, in Lines 248-254 they are listed in different orders each time. I suggest re-structuring these discussion points to keep that order and discuss one antibiotic at a time unless a relationship between environment/exposure and isolate is more pronounced.

Lines 252-254: This is a very interesting comparison, if only because the tolerances in fish mucus are so much higher. Why so high in fish? What sort of exposure concentrations are typical for fish mucus? Are these isolates included in your phylogenetic tree? How similar are the

Line 254-257: Specific details for the Microbacterium isolated in this study are provided for vancomycin and chloramphenicol, but not ampicillin (even though Isoalte PF3 had a relatively high resistance compared to other isolates). Please include all three results.


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

Input:
Title: COMPARATIVE GENOMICS OF 16 MICROBACTERIUM SPP. THAT TOLERATE MULTIPLE HEAVY METALS AND ANTIBIOTICS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The language used is mostly unambiguous and professional. I have have commented for parts where it is ambiguous in general comments.
Literature references are sufficient but I have suggested some more.
Experimental design: Research question is not well defined. Objective of the study is not clear. It should be mentioned at least in the end of Introduction section.
Validity of the findings: Data is well analyzed and interpreted. There are many conclusions drawn in the Discussion section, However, authours need to conclude their abstract strongly as well.
Additional comments: The manuscript describes the isolation and genome analysis of 16 multi-metal and antibiotic resistant microbacterium species. The authours have determined the metal and antibiotic resistance of the isolates and have sequenced the genomes of the isolates using Illumina Hiseq sequencing. The authours have analysed these genomes looking for presence of possible metal and antibiotic resistance genes and mechanisms and have also done pangenomic analysis. The work described is satisfactory, however, there are certain things that could be make it more interesting and compelling.

General Comments:
Authours should provide a list of abbreviations used in the article.
Cr has been mentioned as Cr (VI) (containing space) at some places and as Cr(VI) elsewhere. Authours should use only one format.
μg/ml should be μg ml-1

Major comments:
Why only Microbacterium species were isolated and focused for this study? The authours should describe it and add related literature in the introduction as well.
Line 32-34: The authours have ended the abstract saying “…was likely required of this population…”. It is already a known fact that certain bacteria/ the bacteria that survive certain toxicants are able to do it due to the presence of such genes and mechanisms. I believe the authous should and can come up with a better conclusion to the abstract. The authours should mention what different they have found in their study and what they can conclude out of it.
Line 74-80: Here instead of summarizing the study, the authours should mention objective of the study.

Minor comments:
Abstract:
Line 25: (Cr[VI]) should be [Cr(VI)]
Line 26: “…were able to tolerate high concentrations (0.1 mM) of cobalt, cadmium, and nickel…”. These are not high concentrations as much higher concentrations have been reported. Moreover, authours should mention chromate tolerance level of the isolates here in the abstract as well here.
Line 28: Authours should mention the concentration of the antibiotics resisted.
Introduction:
Line 46: “Bacteria are able to tolerate Cr(VI)…” should be “Many bacteria are able to tolerate Cr(VI)…” as not all bacteria can tolerate Cr(VI).
Line 58: “…vital role in how bacterial thrive…” should be “…vital role in how bacteria thrive…”
Line 79: “…within microbial populations may help…” should be “…within microbial populations that may help…”
Methods:
Line 87: Was the isolation site already known to have high chromium and other metals concentration?
Line 94: “…with 0.5 mM or 1 mM Cr (VI)…”. Were two concentrations used? Kindly explain.
Line 94 and Line 99: The authours here have said that the isolates were grown with 0.5 mM or 1 mM Cr (VI), and with 0.5 mM or 2 mM, respectively. However, in Line 90 the authours stated “Sixteen isolates were further selected based on their ability to grow on 0.5 mM Cr(VI).” Kindly clarify why they used 1.0 and 2.0 mM concentrations when the maximum tolerance level of chromium checked was 0.5 mM.
Line 104: The auhours should mention how may Hiseq flowcells they used for sequencing. Moreover, in the relevant results they should also mention the coverage they obtained.
Line 128-130: Not clear. Kindly modify.
Line 132: Why the authours decided to compare chrR with that of Pseudomonas putida KT2440?
Line 139-140: Metal tolerance is best determined using broth cultures. Moreover, 10% means 10% nutrients or agar, or both?
Results and Discussion:
Line 160-163. The authours stat that they had 16 isolates showing varied Cr resistance. But here it is confusing as authours are saying that 13 isolates were able to tolerate 2mM Cr(VI), 3 were able to tolerate only 0.5mM Cr(VI), and 5 isolates were able to tolerate 20mM Cr(VI). Kindly clarify here.
Line 164: I think its better to say “…over a 72-hour incubation” rather “…over a 72-hour experiment”.
Lin 175-176: “…all had GC content over 68%”. GC-content is a major character for species identification and classification. Authours should comment on the variability of the GC-content they found in the genomes of their isolates.
Line 200-202: The authours have confirmed the resistance of the isolates against metals and antibiotics through lab experiments. Therefore, the use of the word “may” here is confusing.
Line 207-208: Repetition of chrR ?
Line 243: “Thus, is it possible the presence of these genes…” should be “Thus, it is possible that the presence of these genes…”
Line 261-67: The authours should also cite here doi:10.1080/01490451.2016.1240265 and http://dx.doi.org/10.1016/j.crvi.2017.05.002.
Line 264: “…for metals and antibiotics was found Staphylococcus aureus...” should be “…for metals and antibiotics was found in Staphylococcus aureus...”
Line 280: “All Microbacterium spp. isolates were isolated from…” should be “All Microbacterium spp. were isolated from…”
Line 284-87: the authours should also cite doi:10.1111/jam.13535 for genotypes of Klebsiella pneumoniae, Bacillus subtilis, and Citrobacter freundii.
Line 293: I think “…stressors (e.g. heavy metals) promoted genomic diversity…”  should be “…stressors (e.g. heavy metals) can promote genomic diversity…”
Table 1: Instead of giving * the authours can mention mM alongside Metal tolerance (mM).
Table 3: * should go with Cam and Van as 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: COMPARATIVE GENOMICS OF 16 MICROBACTERIUM SPP. THAT TOLERATE MULTIPLE HEAVY METALS AND ANTIBIOTICS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Edits to the introduction have provided context and objectives previously lacking.

Paragraph: "Bacteria are also know to catalyze the reduction..." ~Line 65
Anaerobic vs. anaerobic reduction of Cr(VI) is not mentioned again in the manuscript. Given the new context, it is not clear to me that discussion of anaerobic reduction of Cr(VI) is necessary. Was it hypothesized that the Microbacterium were using anaerobic pathways? Was the study site hypoxic/anoxic? I can see how demonstrating a diversity in known Cr(VI) reduction pathways is important, but would like to see the aerobic/anaerobic topic to be re-visited in the discussion, if only briefly.

I appreciate the additional text to describe the figures.
Experimental design: Response to specific comments:
Review: The CheckM results suggest that some isolates contain strain heterogeneity, which may be an artifact of mis-assembly of closely related organisms. Hug et al. (2015, Environmental Microbiology) determined that assembly was improved for more abundant organisms’ genomes when subsampled, but the level of subsampling depended on the sample. Testing multiple subsample depths (sequential assembly) could reduce strain heterogeneity in some of your assemblies and could identify multiple isolates from the same sample/culture.

Response: During our assemblies, we tested different subsamples of our reads. In the end, our assemblies with 4 million reads provided the highest quality assemblies. Text has been added to mention that multiple depths of subsampling were tested.

Re-review: Additional text addresses my initial comment, thank you.

----------------------------------
Reviewer: It is not clear if the flexible genome was specifically searched for differences and resistance/tolerance genes (using homology, BLAST, etc.). Perhaps you could add a paragraph outlining the major COG categories of the flexible genome; most will be unknown/hypothetical, but those that have an assigned function could be very interesting as well.

Response: We have added data to show the flexible genome, as it was not previously assessed. We have added additional text about the variability but we kept it focused on metals and antibiotic tolerance as we feel a strength of our work is that it couples both genomics and physiological data.

Re-review: I appreciate the additions and agree the authors place a strong focus on coupled genomics and physiological data.

-------------------------------
Reviewer: The use of italics for gene abbreviations (ex: chrA) is widely accepted, please double check that all gene abbreviations are italicized throughout the text.

Response: We initially tried to use “chrA” for text relating to genes and then “ChrA” when referring to the function, however, for consistency we have changed all to “chrA”.

Re-review: I have no issue with the different formatting being used to differentiate genes vs. functions, unfortunately this was not initially clear to me. I appreciate the authors’ willingness to change all to “chrA” for consistency but did notice many instances of non-italicized gene abbreviations in the Introduction (Ex: lines 68-69).

---------------------------
Other comments have been addressed and been made more clear in the text.
Validity of the findings: Additional commends regarding core and flexible gene content are very informative and I think add to the strength of your paper and highlight the amount of analysis done to produce this data set.
Additional comments: Line 41: “…anthropogenic activities, such as industrial and mining industries” is awkward. Is mining industries an activity? Perhaps a statement “such as industrial manufacturing (items that need heavy metals) and mining (mine types).”

Line 42: Missing period after parentheses. Change “exists” to “exist.”

Line 56: “stressors” not “strossors”

Line 134: Illumina misspelled as “Illumnia.”

Lines 149-153: “A pangenomic analysis…” After edits the sentence reads awkwardly. I understand the intent and context of the edits but recommend re-writing this sentence to improve clarity. Perhaps: “A pangenomic analysis of 20 genomes (from this study and Henson et al., 2015) was conducted using…” or similar.

Line 159: “The IMG systems …of the isolates.” This sentence seems out of place since it is followed by outlining how sequences were downloaded and run through MEGA7. Recommend this sentence is just removed given that the IMG searches are well outlined later in the paragraph.

Line 280: "All did have an annotated..." Suggest, "All possess"


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

Input:
Title: COMPARATIVE GENOMICS OF 16 MICROBACTERIUM SPP. THAT TOLERATE MULTIPLE HEAVY METALS AND ANTIBIOTICS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have made sufficient and necessary modifications to the manuscript and is in acceptable form.
Experimental design: The authors have made sufficient and necessary modifications to the manuscript and is in acceptable form.
Validity of the findings: The authors have made sufficient and necessary modifications to the manuscript and is in acceptable form.
Additional comments: The authors have made sufficient and necessary modifications to the manuscript. I recommend the manuscript for publication. It has been an honour to review it.


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

Input:
Title: THE EFFECTS OF AFFORESTATION ON SOIL BACTERIAL COMMUNITIES IN TEMPERATE GRASSLAND ARE MODULATED BY SOIL CHEMICAL PROPERTIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The following comments pertain to the above mentioned manuscript, in which Wu et al. examine the effects of afforestation on belowground bacterial communities and soil physicochemical properties.  Specifically, this manuscript is written within the context of forest conservation efforts in China which aim to reestablish forested areas in regions that historically were not forests, but grasslands. Wu et al. compare soils in a 27-33 year old artificial forested area to the surrounding native grassland soils, and explore the relationships between soil chemistry and bacterial community structure and estimated function.
       The authors take a unique approach in their analysis, which sets it apart from other literature using 16S rRNA data to examine soil communities.  From this perspective, I found this manuscript to be rather refreshing, and I think it has potential to be a very interesting paper.  However, there is much that needs to be addressed or improved before publication.  Specific comments may be found in the annotated PDF submitted along with this review.  Broader comments may be found in the following sections, as laid out per the PeerJ editorial submission page.

Clear and unambiguous, professional English used throughout:

	For the most part, the language is acceptable.  However, there are sections / sentences / phrases scattered throughout the manuscript that are unclear or vague in their point or meaning (see annotated PDF).  While I only marked a couple of them, this would need to be addressed much more thoroughly before publication.
	I also recommend a thorough check for the use of the word “microbial”, “microbiome”, or similar.  While appropriate in some contexts within this manuscript, there are many where use of this verbiage is not.  These words refers both to bacterial (prokaryotic) and fungal (eukaryotic) communities in soils, and the scope of this paper does not extend beyond bacterial community analysis (16S rRNA), and therefore the authors need to be more careful when using this term.

Literature references, sufficient field background/context provided:

	There are a few confusing sections in the Introduction which are highlighted in the annotated PDF, but otherwise the authors do a good job providing background material and context for the study.  The literature referenced in this study seems appropriate.

Professional article structure, figs, tables. Raw data shared:

	The structure of the manuscript is appropriate.  The figure and table captions need more thorough descriptions.
	One of my major complaints about reviewing this manuscript is the inadequacy of the shared raw data processing and statistical analysis files.  While the authors do provide a spreadsheet which contains counts of sequences at the OTU and phylum level, and subsequent COG and KEGG estimated functional gene counts, as well as reference to where the raw sequence data was uploaded to NCBI GenBank, they fail to provide the Unix code or python and R scripts used for bioinformatics, sequence read filtering, or statistical analysis, making it very difficult to determine exactly what was done, and impossible to check their work or verify their results.

Self-contained with relevant results to hypotheses:

	This manuscript does contain all data that is relevant to the goal of the paper.  If anything, certain aspects of the analysis may be unnecessary, or not directly address the questions put forward by the hypotheses.  Specific recommendations may be found in the annotated PDF.  But briefly, I recommend that prior to publication, the authors consider removing certain portions of the analysis that may be convoluted or unnecessary, and explore alternative, more direct approaches to answer the questions they pose.
Experimental design: Original primary research within Aims and Scope of the journal:

	If sufficiently improved, this manuscript would fit in well with the Aims and Scope of PeerJ.

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

	The main research question posed in this manuscript (i.e. What is the effect of afforestation on soil physicochemical properties and bacterial community structure and function?) are relevant and meaningful to the topic introduced in the Introduction.  Efforts to “re-forest” regions of China may have unintended environmental consequences, or be unsuccessful, if not done properly.  As such, this research highlights potential consequences of such actions, while simultaneously exploring above- belowground interactions,  relationships, and responses which are an important area of current research.
	The hypotheses  would benefit from being more well defined and direct, by stating what results would support or refute the hypotheses.  Currently they are:
(1)	The soil prokaryotic microbiome is indirectly affected by afforestation mediated by soil physicochemical properties, and
(2)	Soil microbial ecofunctions are more sensitive to changes in vegetation type than changes in soil microbial composition.
       In the case of (1), it will be difficult to distinguish if changes seen in the bacterial community are the result of soil physicochemical properties, or instead direct influence of Mongolian pine root exudation (NOT mediated by changes in soil properties)?
       For (2), I am confused as to how this can be tested with the data gathered considering that the microbial ecofuncional data obtained from PICRUSt is directly derived from the microbial composition data?
       Overall, while these hypotheses are relevant to the research question, they are vague as to what is actually being tested.  It is unclear whether the main focus of this manuscript is to identify differences between the two vegetation types (grassland vs. forest), or if it is to examine the relationships between the soil environment and the microbial community, and subsequent functional capacities.

Rigorous investigation performed to a high technical & ethical standard:

	   The overall design of this experiment (10 soils from grassland vs. 10 soils from “forest”) is adequate to answer the research question.  Although there are a few details that require further description (see below), from what I can tell from what is reported, the authors used proper laboratory techniques.
       I would like to reiterate that the usage of PICRUSt is merely an estimation of functional gene abundance, which has not been fully vetted for use in soil environments where there are a great many unknown (unsequenced) and unculturable bacteria.  I recommend that authors acknowledge this somewhere in the manuscript, and use the word “estimated” when referring to ecofunctional data.  Also, be clear that these are functional genetic potentials (or capacities) as opposed to actual measurements or activity.  See line 332 for example.

Methods described with sufficient detail & information to replicate:

       There are many details about the collection (amount of soil collected, equipment used, sterilization techniques, etc.) which are not given in the Methods section (lines 137-144).
       I also have some concerns about the depth of the soil sampling (50cm if I interpreted the methods correctly).  Bacterial communities at 50cm soil depth will be very different from communities in shallower soils where much of the litter decomposition, gas exchange, and nutrient transformations occur.  I fear that the soil samples which were collected do not fully represents the microbial communities in these plots.  While I realize this cannot be corrected, I think it is important to acknowledge this in the methods and discussion sections, or reframe the paper to look only at bacterial communities within a certain soil layer/depth.  It would also help to further characterize the soil profile by identifying the soil layers (O, A, E, B, horizons) and reporting which horizon occurs at 50cm.
       The authors did a very good and thorough job of reporting DNA extraction and sequencing procedures.
       While the actual descriptions of the statistical analyses are adequately described, the authors do not report how the analyses were computed (what software was used).  If done in R, I would greatly appreciate seeing the code that was used.  Also, there were a few inconsistencies in which tests were used (line 196 vs. figure 1), and some confusion as to what analyses data transformations were used on (methods state that all abundance counts were transformed to relative abundance, but Fig. 4 shows count abundance data).
Validity of the findings: Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated:

	No comment.

Data is robust, statistically sound, & controlled:

	Without more descriptive specifics on how the data was handled and analyzed, it is difficult to judge how sound the analyses are.  However, the data that is reported seems reasonable.

Conclusion are well stated, linked to original research question & limited to supporting results:

	The conclusions, while a bit speculative, seem sound.  They are linked to the original research question, but fail to identify key specifics, such as what the potential functional differences are between grassland plots and afforested plots.  The analysis reveals that there is a difference in overall genetic function potential, but doesn’t specify what functional genes are driving this difference.  As such, the authors can not speculate on how afforested regions which were once grasslands might respond functionally to this “disturbance”.

Speculation is welcome, but should be identified as such:

	The language used does a proper job of identifying speculation.
Additional comments: First, thank you for your submission!  I greatly appreciate multiple aspects of this manuscript, including not only the context within which it is set, but the unique approach you take with the analysis.  I recognize the context of afforestation efforts in China to be of great environmental concern, and research such as this is important in understanding the impacts of artificially manipulated natural areas, even if the original intent is based in conservation efforts. Also, your statistical analysis of this data is impressive.  I appreciate your effort to use a variety of statistical methods to untangle the complex interactions inherent in belowground ecosystems.
       There are number of things that can be done to improve this manuscript.  Much of detailed comments can be found in the annotated PDF which I will submit with this review, but the following are the most important:
1)	Improve details in Methods.  More detailed site information as it pertains to soil characteristics and natural history of the region.  More detailed soil characterization and collection description (what horizon was the soil collected from at 50cm).  Provide additional information / raw data on how the analyses were done, including any bioinformatics pipelines, Unix code, R code, QIIME scripts, etc.  The statistical analysis should be able to be repeatable from the raw data files submitted.
2)	Focus hypotheses.  Make sure they are testable and that the data you have gathered can test them.
3)	Focus analysis.  Once you have testable hypotheses, make sure that the statistical tests you use are directly related to testing them.  You use many different analyses, some of which I think are great and useful and informative (cluster analysis, DAPC, dbRDA, CLAM), but others seem to confuse (multiple SLR and MLR models, Rand Abundance Dominance, etc.).  Consider using other methods as well (e.g. PERMANOVA, Mantel tests, differential abundance analysis (DESeq2)).  Be careful not to exclude important players that are most likely biologically important (i.e. most abundant phyla such as Acidobacteria, Verrucomicrobia, or Proteobacteria,  and soil chemical factors such as pH).
4)	Focus writing.  There are many instances where the text is confusing, vague, or contradictory.  Be sure what you are trying to say is clear and has a purpose.


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

Input:
Title: THE EFFECTS OF AFFORESTATION ON SOIL BACTERIAL COMMUNITIES IN TEMPERATE GRASSLAND ARE MODULATED BY SOIL CHEMICAL PROPERTIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.Clear and unambiguous, professional English used throughout.
The presented manuscript has been written in correct English, is clear to understand, unambiguous, and technically correct.
2.Literature references, sufficient field background/context provided.
The manuscript includes a very nice introduction, prior literature and was mention new literature regarding the research topic.
3.Professional article structure, figs, tables. Raw data shared.
The manuscript structure is good. Tables and Figures have a good quality to publish in PeerJ journal. However, according to my comment (.pdf file attached), I would appreciate if the authors could include RDA plots to show the community structure. All appropriate raw data has been made available in accordance with PeerJ "Data Sharing policy".
4.Self-contained with relevant results to hypotheses.
The manuscript also seems to be ‘self-contained,’ and appropriate to ‘unit of publication’, and all results were relevant to the initial hypothesis.
Experimental design: 1.Original primary research within Aims and Scope of the journal.
The manuscript is an original primary research and seems to be within Aims and Scope of the journal.
2.Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.
The research question was defined and relevant.
They hypothesized that the soil microbiome assessed by 16S analysis is indirectly affected by afforestation mediated by soil chemical properties and soil microbial ecofunctions are more sensitive to changes in vegetation type than changes in soil microbial composition.
In a conclusion, they proved the initial hypothesis, and the change in vegetation type was linked, at least in part, to ecofunctional changes in the soil microbiome, despite an indirect impact on microbial composition. However, the authors should do some modifications in Conclusion to improve quality (please see .pdf file attached).
3.Rigorous investigation performed to a high technical & ethical standard.
The investigation has been conducted rigorously and to a high technical standard. The research has been conducted in conformity with the prevailing ethical standards in the field.
4.Methods described with sufficient detail & information to replicate.
In general, the M&M section is good. However, the authors should do some modifications to improve quality (please see .pdf file attached).
Validity of the findings: 1.Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
The manuscript has answered important questions about the effects of afforestation practice on soil microbiome. This type of practice can alter soil fertility, health, and to know the microbial response is very important to environmental sustainability and new research in future. The data is robust, statistically sound, & controlled.
2.Conclusion are well stated, linked to original research question & limited to supporting results.
The conclusions have been appropriately stated, connected to the original question investigated, and supported by the results. Please, see additional comments on .pdf file attached.
Additional comments: Dear authors, I have done specific comments in your manuscript submitted to PeerJ journal. You can find all comments assessing the .pdf file attached. I would be happy if you can make changes to improve your finds. I think after major revisions this manuscript has big potential to be published in PeerJ journal.


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

Input:
Title: THE EFFECTS OF AFFORESTATION ON SOIL BACTERIAL COMMUNITIES IN TEMPERATE GRASSLAND ARE MODULATED BY SOIL CHEMICAL PROPERTIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: None comments.
Experimental design: None comments.
Validity of the findings: None comments.
Additional comments: Dear authors.

Thank you for following carefully all initially requested suggestions. Thus, as per my first criterious review, my final decision is to accept this paper to publication in PeerJ journal.

With best regards.


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: IMPACT OF INTER- AND INTRA-INDIVIDUAL VARIATION, SAMPLE STORAGE AND SAMPLING FRACTION ON HUMAN STOOL MICROBIAL COMMUNITY PROFILES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article by Yeoh and colleagues describes a pilot study (n = 3) to assess the optimal form of stool sample collection for a future population-based cohort study. The article is very well-written and easy to follow. The tables and figures are mostly easy to follow (comments on this below) and the sequencing data used is readily available. Ethics and consent were treated adequately.
Experimental design: The work presents three main weaknesses, two of which are discussed by the authors, namely the small number of subjects involved and the choice of only one preservative for the stool samples. Neither of these weaknesses invalidates the findings presented.

The third weakness, which is not mentioned by the authors, is the very shallow sequencing achieved. In my experience, at least 10,000 reads are necessary to provide a good overview of a stool sample (see, for instance, figure 6 in the pre-print https://doi.org/10.1101/286526). Thus, it is possible that some of the variability observed between different biological replicates is due to stochastic effects of OTU being included or not in a given sample. One way to assess this would be to perform a few random subsamplings from each dataset and assess whether the resulting differences are smaller or comparable to the differences between biological replicates observed.
Validity of the findings: It could be that Subject B does not have a particularly higher diversity or a different pattern of temporal changes in their stool samples, but rather that this Subject’s samples have been sequenced systematically deeper than the other two. A simple way to discard this would be to include a boxplot of sampling depth per subject in figure S1.
Additional comments: While the authors correctly discuss the effect of temperature and overgrowth of opportunistic organisms, I believe the discussion could be improved by considering the role of exposure to oxygen and thus selective killing of strictly anaerobic organisms. Is there any viable way of excluding oxygen from the samples that the authors have considered or could consider? Has this been approached by the existing literature?

Besides the issues discussed above, there are minor issues in the reporting of results that can easily be fixed, namely:
- The centroid sequence of each OTU is not provided. This can be done as an additional column in Suppl File 1 or as an additional fasta file
- The OTU ID in Suppl. Table 1 and 5 doesn’t match the ID provided in Suppl. File 1
- The legend in figure S1 refers to “species”. It would be more appropriate to use OTU or ASV (amplified sequence variant)
- In table S3, please inform the OTU ID, not only the taxonomic annotation
- Figures S2-S4 came with a large portion of grey background around them, probably an artefact from Inkscape
- I couldn’t understand what the different colours used in the text in figure S2 are supposed to mean
- In figure S3, it would be useful to see which of the presented differences are statistically significant


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: IMPACT OF INTER- AND INTRA-INDIVIDUAL VARIATION, SAMPLE STORAGE AND SAMPLING FRACTION ON HUMAN STOOL MICROBIAL COMMUNITY PROFILES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is well structured and well written. The authors use clear and unambiguous, professional English throughout the article. However, in some occasions sentences are unnecessary long and could be simplified, such as in lines 207-210, 315-318, or typing errors occurred, line 240.

In general literature references are up to date and sufficient field background/context is provided. The section on reduction of community diversity in the case of highly abundant taxa in the discussion (lines 301-303) would benefit from a brief explanation of the mechanisms at play. In addition, the finding on dominant Prevotella abundances being severely reduced with storage conditions, is a major one in the light of the importance of this taxon in the determination of gut community types. Referencing to papers describing these gut community types (or enterotypes) would demonstrate how the work fits into the broader field of knowledge. Next to temperature, the possible role of (an)aerobic conditions should also be discussed as diverging factor in the discussion on specific taxa abundance changes.

The authors provide most of the necessary information throughout the text and in figures and tables. All appropriate raw data has been made available in a public database.
At some occasions, information on the statistical tests and its results are missing, such as in lines 180-181 and 283.
Figures and tables are of sufficient resolution, but description and labeling can be improved. Suggested improvements:
•	Figure 1. Reduction in text and better use of symbols to make the study set up easier to understand at a glance. I have attached an example.
•	Figure 2. Place data of Subject A in panel A, B in B, C in C and overall in D. Take legend of PCA overall out of the plot and move it to the side, as it applies to all of the plots. Number all sample points according to the day they were taken, remove text-box. Add info on storage duration (2,5,24h) in individual plots. Add info on the exclusion of samples (failed sequencing, …) in the figure legend – referring to the methods.
•	Table 1: Heading could be turned into ‘all samples’. Sampling occasion might be better formulated as sampling time point. It could be useful to include which kind of levels the parameters include within the table of table legend. (e.g. for sampling occasion: is it all days, or period 1/2/3? Especially to compare with subject:sampling occasion). At this moment it seems like the column names of the data tables used during analysis by the researchers - who are fully accustomed with the research design – are used. Parameters should be turned into easy understandable text and convey all necessary information in an easy way for the reader.
•	Table 2: Idem to suggestions Table 1.
Experimental design: This paper tries to assess the impact of sample storage conditions on stool community profiles of Asian individuals. The research question is well defined, relevant & meaningful. The authors state in the abstract and introduction how their research fills an identified knowledge gap. They performed a rigorous investigation, complying with the ethical standard and using the latest available methods (e.g. application of the Dada2 algorithm for sequence variance identification).

The methods are well described and provide enough detail to repeat the analysis. I would suggest to also include information on the number of unassigned taxa at each taxonomical level at the end of the paragraph ‘16S sequencing data processing’. The authors chose to apply a centered log-ratio transformation for the PCA and state this in the result section, yet I think it is more appropriate to include this in the method section, together with a brief explanation on the reasons to do so.
Validity of the findings: The authors used the appropriate materials and methods and performed appropriate statistical tests, including multiple testing correction where necessary. Some notes:
•	Lines 180-182, statements are not completely supported by the results (I think one cannot state that the specific durations of 2,5 and 24 hours are associated with shifts in community composition but only duration in general).
•	Lines 230-232: A much higher amount of taxa is enriched versus depleted over time, could the authors elaborate on this? This might be a consequence of the relative data.
Importantly, the data on which the conclusions are based are made available for the community.
Additional comments: This work contains some important messages for our field. Thank you for taking the time to look into this subject and report the results of your analysis in a clear way. In order to improve the manuscript further, I would like to make the following suggestions.
•	Include a ranking of the variables according to their effect on microbiota composition.
•	Make optimal use of the longitudinal set up of the study by determining not only the inter-individual variation but also intra individual variation. For instance, using Bray Curtis dissimilarity or another distance metric, variation between samples could be determined between persons (inter-ind.), between time-points of the same time-period (intra ind.), storage methods (for the same person, at the same time point), intra-sample.
•	For the title, ‘delivery conditions’ does not really convey what it should. With the numerous reports on the microbiome of infants, the wrong association with childbirth is easily made. ‘Storage conditions’ might be more appropriate. In order to make the title and text more comprehensible a distinction between biological relevant variation (inter-ind, temporal, intra-sample) and induced variation by storage conditions could be made.
•	To be completely correct, one should make a distinction between the taxonomic units obtained using the Dada2 workflow and those obtained by previous methods (Operational Taxonomic Units (OTUs), defining taxonomic units based on a cut off value for sequence similarity). With dada2 the taxonomic units are of a higher resolution and can be named amplicon sequence variants (ASVs).


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: IMPACT OF INTER- AND INTRA-INDIVIDUAL VARIATION, SAMPLE STORAGE AND SAMPLING FRACTION ON HUMAN STOOL MICROBIAL COMMUNITY PROFILES
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript under review describes work that is relevant and current. Although the work in part represents replication of the previously reported data the rationale for such approach is explained and is convincing. Special value of the work rests in the meticulous description of the sequencing data analysis as well as the appropriate and valid use of statistical approaches.  On the other side, the low initial number of stool donors (n=3) is a statistical power issue which is somewhat compensated with multiple donor sampling timepoints.
There are only several points on which the manuscript should be improved:
1.	There should be original reference added for primers used in 16S amplicon sequencing: Klindworth et al. Nucleic acid research 2013;41(1):e1
2.	In the methodology section describing the amplicon sequencing there should be information added that describes the controls used for verifying the quality of the sequencing runs (positive, negative).
3.	In the discussion section more attention should be given to previous research performed with other preservation buffers (RNA later, OMNIgene gut sysem etc) so to provide broader context of the presented work
4.	The sentence: ”In addition, the findings presented here should also be verified using different sets of universal 16S primers as primer choice is widely known to influence 16S-based community compositional profiles” although in its essence true, should be rephrased in a way to reflect the fact that 16S primers influence 16S-based community compositional profiles but not to encourage other researchers to use different primers. Different studies performed in different laboratories and with only slight variations in the methodology produce sufficiently different results disabling appropriate study comparisons. Studies could only be compared if identical protocols/methodologies are used and the future of any long lasting technology/methodology lies in standardized methodology. The suggested wording would potentially be: “In addition, when comparing the findings presented here with other studies attention should be paid to the primers used for sequencing as primer choice is widely known to influence 16S-based community compositional profiles”.


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: IMPACT OF INTER- AND INTRA-INDIVIDUAL VARIATION, SAMPLE STORAGE AND SAMPLING FRACTION ON HUMAN STOOL MICROBIAL COMMUNITY PROFILES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: All my previous concerns have been addressed. There are only very minor issues left, namely:
- Tables use the term OTU, while the text uses ESV throughout
- In the discussion, the term "stool flora" is inadequate in  a professional setting. While perfectly acceptable in outreach activities, "flora" in a discussion between biologists refers to Viridiplantae. "Microbiome", "microbial community" or, even more precisely, "bacterial community" should be preferred.


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: IMPACT OF INTER- AND INTRA-INDIVIDUAL VARIATION, SAMPLE STORAGE AND SAMPLING FRACTION ON HUMAN STOOL MICROBIAL COMMUNITY PROFILES
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comments.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: The authors sufficiently addressed all outstanding issues in this version. I have no further comments and support the publication of this work in PeerJ.


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

Input:
Title: IMPACT OF INTER- AND INTRA-INDIVIDUAL VARIATION, SAMPLE STORAGE AND SAMPLING FRACTION ON HUMAN STOOL MICROBIAL COMMUNITY PROFILES
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: It is a pitty that no positive controls of sequencing run were included, this would additionally add value and context to the reserach performed and results obtained.


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: ARTIFICIAL LIVER RESEARCH OUTPUT AND CITATIONS FROM 2004 TO 2017: A BIBLIOMETRIC ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This paper performed the bibliometric analysis on publications in artificial liver research. It quantitatively investigated the research progression of an important area, and reported its research trend from different perspectives. The paper also showed the high correlation between citations with author and countries. The paper is well organized and well written, but I still have a few of concerns and listed below.
Major:
1.	I am confusing about the searching criteria. As stated in introduction, the researchers of “artificial livers” are categorized into three groups, one of which is “bioartificial liver”. In the paper searching, both “artificial liver” and “bioartificial liver” were used. I am wondering why the other two terms “blood purification” and “hepatocyte transplantation” were not included. Or I would like to suggest using only “artificial liver” and/or its related words (artificial livers et al).
2.	Also in “search strategy”, which fields the searching were performed on? Is the searching performed on one field (like keywords, abstract, title), or multiple fields?
3.	In “inclusion and exclusion”, criteria (2) is “any type of researches”. I would like to suggest the authors to explain a little bit about the “type” of researches, is it about journal/conference, or abstract/regular paper, or anything else?
4.	I would suggest the authors to explain the “citation index” as it is not straightforward for other readers to get what it is to measure.
5.	In “journals and publication year”, Fig S2 A-B presented the correlation between IF with number of article and number of citation. I have several questions about this subsection “journals and publication year” and this figure listed as below. 1) I didn’t find the information about “publication year” in this paragraph. 2). I am not clear about what information did figure S2 present. There are both 17 points in A and B, each point represent one journal if my understanding is correct? If so, what do the x-axis represent? It is the total statistics for each journal? I don’t think Fig S2 is readable and I also don’t think it is reasonable to get the conclusion that the top 100 publications were published in low IF journals from these two figures, if the number of citation of each journal is calculated by summing the citations of each paper from this journal. Because if using the sum of citations from each publication, the number of papers will affect the correlation. Instead, I would suggest to use the average measurement.
6.	I would like to suggest the authors to add details to the “statistical analysis”, like introducing some terms (e.g., citation index), and present the statistical analysis more precisely (for example, it is not clear to say “correlation between citation and authors”, it would be better if say “correlation between the number of citations and the number of authors in each publication”; and to include more details in figure and table captions, making them be more readable.
7.	In discussion, the author mentioned the possible reasons of decreasing trend of artificial liver. Can the authors help provide literature or evidence for supporting the reasons listed?
Minor:
1.	Line 221: there is an error sentence.


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

Input:
Title: ARTIFICIAL LIVER RESEARCH OUTPUT AND CITATIONS FROM 2004 TO 2017: A BIBLIOMETRIC ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: •	English language:

Actually, I didn’t face any problem in understanding the content of the manuscript. The English language was clear, professional and understandable. It was presented in all parts of the article in a distinct and unambiguous manner.

•	The structure of the manuscript, figures, and tables comply with the standards reported by Peerj. It seems to me that the figures and tables are well designed and labeled and correlate with the content of the manuscript and summarize the key findings and trends in a way that is easily interpreted and comprehended by the reader without returning to the main text. Captions, table headings, and plotted parameters in graphs are described appropriately enabling the reader to understand the data easily.

•	Reference section:
Authors may consider enumerating the full list of authors with their initials in all references based on the format suggested by PeerJ.

•	Title:
The title of this article precisely portrays the main essence of the study. The reader can easily understand what the manuscript is about.

•	Abstract:
The abstract is an essential part of a research paper because it gives the reader a brief review of the study. Herein, the abstract is well written summarizing the main points and findings reported in the main text. It includes all the keywords and the needed information that is easily perceived by researchers outside the specialty of liver transplantation.

•	Keywords:
I think the keywords mentioned in the paper represent the content of the manuscript and facilitate the retrieval of the article.

•	Introduction section:

>>>Positive points:
The introduction formulates the research question that will be addressed in the manuscript. It gives the reader a good definition of bibliometric science, explaining its influence in medical literature and benefits for future researchers. It also clarifies what is citation analysis, which is one of the most important methods in bibliometric analysis, and states how articles are assessed based on this measure. Study aims and objectives are well identified in the last paragraph of the introduction. Furthermore, the authors clearly state the importance and the reasons behind executing this study.

>>>Recommendations/suggestions:
 Obviously, this research article is generally about artificial liver devices. It is apparent that you mention the history and classifications of the artificial liver system. However, to enhance the quality of the introduction, I recommend enclosing sufficient background information about the K. Onodera et al.’s definition of artificial liver used in this analysis, the purpose of this approach and how it works.

Experimental design: The research article conforms to the aims and scope of PeerJ. It is a medical research review study that statistically analyzes the citations and research output for artificial livers publications between 2004 and 2017.

Research questions, aims, and goals are distinctly identified in the introduction section. They are consistent with the main text. Authors explain how this analysis will help future researchers in their work.

•	Methods section:

>>>Positive points:
It is distinctly enumerated the research platform used in performing this study. Authors specify the range of dates and all potential keywords utilized in conducting the analysis. Also, the date of final data retrieval is also written in this part.
Inclusion and exclusion criteria are well listed in the manuscript. Two reviewers with their names were chosen to screen the studies based on titles, abstracts, and full-text versions. Also, a third independent investigator participated in solving any discordances between the two reviewers.
Evaluation of the included studies necessitates an extraction of multiple parameters that are plainly mentioned in this section.
Authors depicted the statistical tools that are applied to carry out the statistical and bibliometric analysis.

>>>Recommendations/suggestions:
1)	My suggestion is to demonstrate which investigators engaged in retrieving the relevant data from the included studies.
2)	References for the statistical and bibliometric methods should be added to the main text.
Validity of the findings: •	Results section:

>>>Positive points:
Prisma diagram is clearly illustrated showing how many articles retrieved from the original search, how many articles screened, the number of articles included, and the number of articles excluded with documented reasons.
The results are well explained and presented in a proper format. The data are robust, statistically significant, and answer the main research question. P value and R2 are calculated when comparing the correlation between the median value of the number of citations and the median value of citation index as well as the correlations between the number of citations and funding, years since publication, countries, and authors.

>>>Recommendations/suggestions:
1)	It would be nice if you calculate the mean values and standard deviations of the number of citations and citation index for both WoS and Scopus Database.

2)	There are some discrepancies between the results in the main text and the results in the tables and figures. For instance;
a)	In lines 227-228, you say that the Human liver tissues and organs topic of human liver domain showed the highest percentage (14.81%), whereas, in Table 5, it is indicated that Human liver technology has the highest percentage (15.74%).

b)	In lines 230-231, Airlift reactor (n=78) in Bioreactor topic should be reported after ‘academic medical center bioartificial liver and before ‘bilirubin’.

c)	In lines 233-234, Galactose (n=37) in bioreactor should be added to the keywords that appeared more than thirty times

d)	In line 171, it is denoted that the range of impact factor of T18 journal of T100 articles is 1.31 to 16.658. While in table 2, the maximum factor is 17.016 for GUT.

•	Discussion section:
>>>Positive points:
Generally, the discussion suits with the aims proposed in the introduction section. Authors conversed about the findings and provide potential explanations and interpretations for data. Also, some results in this analysis are compared to the results of other studies.
Limitations of the study are clearly reported at the end of the discussion part. The authors’ conclusions are properly stated and supported by their evidence and findings.

>>>Recommendations/suggestions:
1)	It seems to me that there are two sharp drops in the total number of publications between 2005 and 2017. I think authors need to touch the second drop (at 2016) and provide possible explanations for it as they did for the first drop (at 2007).
2)	It would be great if authors could state the reasons behind the substantial contributory role of USA, Japan, and Germany to artificial liver research as they did for China.
3)	The results of Zhejiang University in line 262-263 are already mentioned in the results section and I don’t think it is necessary to repeat them in the discussion part.
4)	The sentence about Hepatitis B incidence in China in line 263-264 is duplicate and this information could be added to line 256-257.
5)	In line 260-261, the worldwide influence of USA research is indicated in Figure 4 and not in Figure 2B.
6)	 In line 281, it is pointed out that four factors that impact the citation number have been investigated. However, only three factors (number of authors, number of countries and the number of years since publication) have been addressed. My recommendation is to explain why the fourth factor (number of funding) does not affect the number of citations in this analysis.
7)	The findings of Ahmad et al in line 284 need to be noted and explained how they differ from the authors’ premise.
8)	The impact factor is considered a good indicator for assessing the quality of studies. Authors may consider giving more details about the ineffectualness of impact factor in this analysis.
9)	Authors may need to describe adequately how their findings will affect the future research.
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: ARTIFICIAL LIVER RESEARCH OUTPUT AND CITATIONS FROM 2004 TO 2017: A BIBLIOMETRIC ANALYSIS
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 responded to all my questions clearly. I have no further comments/suggestions.


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: ARTIFICIAL LIVER RESEARCH OUTPUT AND CITATIONS FROM 2004 TO 2017: A BIBLIOMETRIC ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comments. All suggested changes have been performed well.
Experimental design: No comments. All suggested changes have been performed well.
Validity of the findings: No comments. All suggested changes have been performed well.
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: GENETIC DIVERSITY AND ITS CONSERVATION IMPLICATIONS OF VITEX ROTUNDIFOLIA (LAMIACEAE) POPULATIONS IN EAST ASIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: see General comments for the author
Experimental design: see General comments for the author
Validity of the findings: see General comments for the author
Additional comments: Comments:

The ms. of Sun et al. describe the genetic variation of Vitex rotundifolia and V. trifolia in China, Korea and Japan.
The paper is principally well structured and done. Still, some points should be considered before publication.

A major point is the sampling. In many cases only 1 to 3 samples were analysed. This may cause a bias in the interpretation of the data. Low genetic diversity may be due to low sampling. The authors should critically address this problem. I understand that the species is rare, but it should be checked if the sampling affects the conclusions.

The calculation of the molecular clock is not appropriate. Wolfe et al. 1987 is too old. There are much younger papers like Kay et al. (2006). The authors should search for a suitable substitution rate close to Lamiaceae. Otherwise they can run BEAST without substitution rate prior. What is the conclusion or relevance of the outcome of this dating analysis? Pleistocene origin is very likely a priori.

The discussion on the potential refugia should be limited to the studied area.
Due to long distance dispersal this may be redundant.

The taxonomic conclusion is justified based on the presented data. However, to see if both species are really distinct one should include more samples of rotundifolia from other regions than E Asia. (To check if trifolia is nested in rotundifolia.)

The work by Hu et al. 2008 should be more incorporated in the discussion.

Details:

Legend fig. 6 chronograrms => chronograms; indicate 95％ highest…
Fig. 6: Why is trifolia not reaching the present time (right)? It should align to rotund. May be a figtree problem.

l. 58 Drummond AJ et al; is wrongly cited.
l. 64 variety => variation
l. 73 only 2 papers dealing with controversial taxonomy of these 2 taxa?
l. 99 1-12 individuals in each population. One is not sufficient.
l. 111 Liter in small letters (l) for PCR
l. 274/5 What is the relevance of this sentence? Which context?
l. 278 . , delete
l. 292/6 rephrase 2 sentences. Difficult to understand.
l. 297 dispersed => distributed
l. 341 trifolia L & Littoralis Steenis


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: GENETIC DIVERSITY AND ITS CONSERVATION IMPLICATIONS OF VITEX ROTUNDIFOLIA (LAMIACEAE) POPULATIONS IN EAST ASIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Sun et al. used cp DNA and nrITS to study the genetic diversity and structure of two species in Vitex (Lamiaceae). The authors sampled 27 populations and 177 individuals in total. Base on the genetic diversity, divergence time and demographic analysis, they found the two species show genetic differentiation, and low genetic diversity, and may diverge in the Pleistocene, afterwards underwent a potential rapid demographic expansion.

The study provides some new information of the genetic diversity for the populations of the two species in East Asia, and which further can be interpreted implications for practical conservation. However, they choose a regional sampling of the two species to test the demographical history and further try to clarify the delimitation of the two species, which I think it is not very appropriate. I suggest the authors focus on the genetic diversity, structure and its conservation implications of their data.

As the authors motioned in the introduction and discussion part of the two species, one of them are widely distributed in the Pacific coast, and the other is in Indian ocean coast. However, they only collected 27 populations from East Asia to test the species boundary and the demographical history. I do not think such data can reveal the true demographical history and clarify the species delimitation of the two species. Therefore, I suggest the author reorganize the MS.

In all, I think the authors need to pay much attention to the focus of the study and other details of the MS. Thus I will not suggest accepting to publish this MS at the current status.

Detail comments
Title:
I do not think the title can reflect the content of the MS. I suggest to change it like this: Genetic diversity and its conservation implications of Vitex rotundifolia (Lamiaceae) populations in East Asia

Abstract:
The abstract need to further refine according to my other my other suggestions.

Introduction:
The logic is not very well, this part should keep in logic consistent with the other section (such as material and method, discussion part). The last two sentences of the first paragraph are most abrupt.
The second paragraph is also not well organized

I think this part should be rewritten according to the questions they want to discuss.

Materials and Methods:
Four of the sampling site only consist of one individual each. I do not think we can call it population.

How many populations and individuals were sampled should be clearly stated here.

For molecular dating, why the authors used 1.0-3.0 × 10-9 substitutions per neutral site per year (s/s/y) (Wolfe, Li & Sharp, 1987) should be explained.

For demographic history, I do not think they need to do the test for V. rotundifolia and V. trifolia as a whole.

Results:

I cannot see the figure legend. If the Fig. 7 is right for the content in the MS?
The authors used a range of the mutation rate (1.0-3.0 × 10-9), but only one value (0.18 Ma) was shown here.

Discussion
I suggest the author focus on the genetic diversity, structure and conservation implications of the two species. Not too much for the demographical history and taxonomic implications.

Reference:
Some of the references styles are not consistent, which need to be carefully checked and corrected.


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: GENETIC DIVERSITY AND ITS CONSERVATION IMPLICATIONS OF VITEX ROTUNDIFOLIA (LAMIACEAE) POPULATIONS IN EAST ASIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a review on a revised version of the paper by Sun et al. describing the genetic variation of Vitex rotundifolia and V. trifolia in China, Korea and Japan. The ms. is now better than the 1st version. Still, minor changes should be implemented.

Main point: The exclusion of poorly sampled populations should be mentioned in MM and results and shortly discussed in the discussion.

I have marked lots of smaller remarks and changes in the text.

Intro:
‚and also lacks analysis of the genetic variation background of this species‘ explain! The other studies did provide gen. variation!

Results:

0.23  mya (0.94-4.19 HPD) Something worng, probably with the lower HPD, when it is higher than average.

The English should be carefully checked by a native speaker!
Experimental design: see Basic reporting
Validity of the findings: see Basic reporting
Additional comments: see Basic reporting


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

Input:
Title: LOCAL FISHERMEN’S PERCEPTIONS OF THE USEFULNESS OF ARTIFICIAL REEF ECOSYSTEM SERVICES IN PORTUGAL
Review round: 1
Reviewer: 1

Output:
Basic reporting: Generally clear and unambiguous, but there were editorial/grammatical errors throughout.  Most errors were minor / insignificant (e.g. line 25, a word seems to be missing between ‘deployment’ and ‘semi-structured), but at times, grammatical errors obscured meaning (or at least forced the reader to ‘work hard’ to understand content).  Having spent a good part of my life reading material relating to individual and social welfare (utility) functions, I found it particularly hard to understand sections discussing utility.   I could not clearly determine if this was an English-Language problem, or if the author(s) were relatively new to the topic, learning it anew and thus struggling a little when explaining ideas.  I believe the authors should consider having someone take a more careful editorial look at material before submission.

I think the authors were not as clear as they could be about the difference between the frameworks which economists use to categorise values (the aim there, being to guide selection of appropriate valuation methods, since different types of values, require different valuation methods) and other frameworks that highlight the benefits that ecosystems provide to humans (as per ecosystem services).   This is most apparent in Table 1 (also in the associated discussion):  some of the ecosystem services that are listed in column 1, might be described by non-market valuation experts in a different way (e.g. in column 3, the authors do not list any ‘non-use’ values – arguably, habitat refuge and biodiversity preservation generate at least some non-use values). So I think the authors could consider either re-visiting the categorisation, develop a more nuanced comparison, or just stick to one.   Developing a more nuanced categorisation  could be going down a rabbit hole. If using just one framework, I think that Millennium Ecosystem Assessment (MEA) -based frameworks are likely most appropriate (there are many – all slightly different from each other – see for example, the Common International Classification of Ecosystem Services, and more recent material from the IPBES on natures contribution to humans.).

Related to the above - tis very much personal preference, but I tend to think that choice of goods and services for assessment, is a methodological choice – so I would be inclined to move the description of goods and services to later in the paper.   My reasoning is as follows: first, you need to work out what you want to assess, then you need to work out how you will conduct the assessment (the before/after interviews, using likert scales), then you need to work out where you are going to undertake the assessment, and who you are going to interview (the sampling).  Having collected the data, you are then in a position to analyse it.

While I like the idea of developing a conceptual model linking expectations, knowledge, experience, attitudes and behaviour to perceptions of the ‘value’ of various ecosystem services before and after deployment, this model was neither well explained, nor integrated within the paper itself.   I suspect it is used in a larger study, that reports more broadly on the findings in this paper, together with findings that are not discussed here?
Experimental design: I kept wanting to know why/how the authors came up with the list of goods and services to be evaluated?  I think the research would have more credibility, if selected goods/services were linked to published categories.  That would at least ‘ground’ selection of goods and services in known literature.  Whose lead was being followed when devising the list?  If some common goods/services were omitted, why?  If new ones were added, why?

I thought it a very good idea to do pre and post surveys, but it is not entirely clear if data were collected in such a way as to facilitate true comparisons – e.g. finding out how ‘fisher A’s’ values changed over time.   I suspect, from the way results are presented, that this is not the case – hence why the authors are only comparing one sample with another.   If it is possible to ‘match’ responses before and after, then I think the authors should consider analysing data differently (looking at changes in values, rather than reporting values before and after separately).   If it is not possible to match before and after responses, then authors will have to stick with the sample 1 versus sample 2 comparison, but results are much less interesting and perhaps not strong enough to make a paper on their own.  There doesn't really seem to be all that much data to talk about.  It is a relatively small sample, before and after, in two case studies, and it is really only Likert responses to questions about several different ecosystem services.   I wonder about the idea of trying to add more BREADTH – perhaps including other data (obviously collected in the survey), to help flesh out the conceptual model ...?   That could be really interesting.

Figures 4 and 5 could potentially be combined.  All that would be required, is the addition of a * (or similar) to Figure 4, near the names of each ecosystem services, for which there were statistically significant differences in distribution of responses on the Likert Scale, between the ‘before’ and ‘after’ survey (I am presuming here, that authors are comparing entire distributions of responses, not just medians).
Validity of the findings: As noted above,  it is not entirely clear if data were collected in such a way as to facilitate true comparisons across time – e.g. finding out how ‘fisher A’s’ values changed over time.   I suspect, from the way results are presented, that this is not the case – hence why the authors are only comparing one sample with another.

Given the 14 year gap between sample collection, there are likely numerous confounding factors.  These were alluded to in the conceptual model - but not systemmatically reconsidered in the conclusion (e.g. noting that some factors would bias responses in this way, others could bias them in another way, with net impacts being ....?).  Neither were they seriously considered in the analysis.

I am not 100% convinced that the results definitely 'prove' utility or otherwise (people often respond in patterns to Likert-scale questions, so higher scores may mean higher utility for some goods and services).   Moreover, I couldn't work out how to 'ground' the scores --- do they indicate that some Ecosystem services associated with AR's generate more utility than  an ice-cream?  or a meal? or a house? Is the extra 'utility' associated with the AR's 'worth' more than the cost of constructing them?)

I am also not 100% convinced it is possible to draw inferences about expectations if using two different, unrelated samples, 14 years apart. ...........  It might just be a matter of changing language/semantics - my comments are from the economics literature (and utility).   There is a fairly large body of economics literature on expectations, measurement of, and interpretation that is not addressed.   Perhaps there is a need to discuss that literature (and incorporate insights) or change the terminology of the paper, so that readers are not 'expecting' (:-)) to hear about economics, utility, and expectations.
Additional comments: Tis a good topic - and one about which we know relatively little.  Before/after assessments have the potential to contribute enormously to our knowledge.   As presented however, I don't feel that the authors are doing their data credit - my personal feeling is that they have, perhaps focused on too small a subset of data from the whole survey.   Broadening the variables used in the analysis could add some very interesting additional insights.


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: LOCAL FISHERMEN’S PERCEPTIONS OF THE USEFULNESS OF ARTIFICIAL REEF ECOSYSTEM SERVICES IN PORTUGAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: It is interesting to compare the impacts of pre and post artificial reef (AR) deployment for different areas (Nazaré reef off the central coast of Portugal and Oura reef off the Algarve coast),different time periods (pre and post) and nine ecosystem services. The literature review is sufficient for the subject matter covered in the article. The manuscript conforms to PeerJ standards and the discipline norm.  Figures provided are relevant to the title and the subject matter discussed. English language was clear and correct up to the section 3, but section 4 onward professional levels began to drop. Some examples where the language could be improved include lines 303 onward – the current phrasing makes comprehension difficult in several places.
Experimental design: The experimental design is not an original primary research; however, it was within the scope of the journal. Research question was well defined, relevant & meaningful. It is however, not contributing to an identified knowledge gap.

In line 205, it states, “in order to assess AR utility, we developed a conceptual model of how stakeholder expectations and perceptions of ecosystem services provided by ARs change from pre- to post- deployment (Fig. 3)”.  First, this is not a conceptual model of stakeholder expectations and perceptions on ecosystem services.  Ecosystem service is a more complex phenomenon, which cannot be captured through a study of perception, measured using a given 5-point likert scales. Experimental design suggested in the paper is not rigorous enough to perform an investigation on ecosystem services to high technical and ethical standards. The type of investigation on revealing perception of stakeholders using likert scale is not providing sufficient details and information to replicate similar studies elsewhere. Authors have cited papers by Allen & Seaman, (2007) and Dawes, (2008) for previous studies which were used 5-point likert scale as a means to rate stakeholder expectations of ecosystem services. To reveal stakeholder expectations of ecosystem services likert scale is sufficient, however, it cannot use for modelling ecosystem services utility suggested in the paper title.

Two references given below have shown analyses that can be used for modelling ecosystem services.
Katja Schmidt, Ariane Walz, Berta Martín-López, René Sachse, (2017)
Testing socio-cultural valuation methods of ecosystem services to explain land use preferences, Ecosystem Services, 26, 270–288 and
M. Potschin-Young, R. Haines-Young, C. Görg, U. Heink, K. Jax, C. Schleyer, (2018)
Understanding the role of conceptual frameworks: Reading the ecosystem service cascade, Ecosystem Services 29 428–440
Validity of the findings: Section 2 of the manuscript provides detailed description of the total economic value of AR ecosystem services which can be divided into three categories, i.e., (a) direct use value (extractive and non-extractive use), (b) indirect use value, and (c) option and non-use value.
Although, the authors have given detailed description of all 9 ecosystem services considered, their impact cannot be assessed.  There is no novelty of describing these services, but it is useful if these services could be assessed.
Data collection for the purpose seems robust, statistically sound, & controlled.  However, the analysis performed is not novel and it is a very simple approach.
Hypothesis testing of the study is very simple but it was reported wrong. It was stated that a) the null hypothesis that it was equally likely that each of the ecosystem service perception scores given by stakeholders would be the same after reef deployment (H0), against b) the alternative hypothesis that each of the ecosystem service perception scores given by stakeholders would be greater after reef deployment (H1). This is impact true; however, in line 282, it was reported wrong. Instead of “=” sign it was reported as “≥“in null hypothesis.
The conclusion section is well short, linked to original research question but limited to supporting results.
Additional comments: There is no measurement of artificial reef ecosystem services “utility” by local fishermen in Portugal in this study.  I commend the authors for their data collection over many years of detailed fieldwork; however, the results are not robust. The manuscript is clearly written in professional way in some parts but not throughout the manuscript. If there is a weakness, it is in the statistical analysis (as I have noted above) which should be improved upon before Acceptance.


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: LOCAL FISHERMEN’S PERCEPTIONS OF THE USEFULNESS OF ARTIFICIAL REEF ECOSYSTEM SERVICES IN PORTUGAL
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is not adequately written. There are some grammatical errors, and most importantly there are many sentences whose meaning is not clear. For instance: lines 61-62 – stakeholder perceptions of ecosystem services provision by various ecosystems have been done previously. What is the meaning of this sentence?

There are many more examples of sentences whose meaning is unclear. For example, the text between lines 263-269 is totally confusing: we do not understand if interviewees were asked to rate their perception of ES or to rate the change in their perception before and after deployment of the artificial reefs, which is totally different.

Overall, the manuscript lacks clarity. It would be advisable to have the paper revised by a professional English reviewer.

On the other hand, there is a lack of rigor in the use of concepts. There is a confusion between ecosystem services and (economic) values.
Furthermore, some aspects, such as ‘reuse of obsolete structures’ and ‘diversion effect’ are listed as ecosystem services, but they are not ES. They may be important benefits or impacts of artificial reef structures, but they are not ecosystem services.

There is also a confusion between expectations, perceptions and actual provision of ES – these terms are used throughout the text and sometimes it is not clear to which concept the authors are referring to. (see for example text in lines 243-253).
Experimental design: The main contribution of this paper is the idea of studying users’ perceptions before and after the deployment of artificial reef structures in two different locations. There are not many studies that have managed to have a data set that covers both before and after the project.

However, the research question to be addressed is not clearly defined. It is not clear if the aim of the study was to assess the change in perceptions introduced by the project, or if the idea is to assess the contribution of the artificial reefs to the provision of ES.

As the authors acknowledge, multi-functional artificial reefs provide important benefits to multiple stakeholder groups. However, the study has focused only in one particular group: local fishermen. Although this may be understandable from an operational point of view, at least the authors should acknowledge and discuss this limitation of their study.

The methods are generally well described, but some aspects are not totally clear. The same questionnaire was used in both the before and after interviews? What fraction of respondents was the same in both instances in the two areas?

Perhaps the authors could have compared the results obtained in the interviews with the actual values of fishing yields reported in Santos and Monteiro that they cite in the text.
Validity of the findings: The reported findings are not fully grounded on the data that were collected. Again, there is a confusion between, expectations, perceptions and actual value of ES. The results section focused mostly in the changes in perceptions and dispersion of values. However, in the discussion and conclusions the authors try to conclude about the utility of AR and on the justification of using public funds for AR creation and this analysis is not supported in the data obtained.
Additional comments: The paper addresses a very relevant and timely topic. The main added value is the collection of information before and after the development of the artificial reefs structures. However, the paper needs to be substantially revised before being considered for publication, improving the quality of writing and rigour in the use of concepts and methods, as explained above.