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: INCIDENCE OF HEMOPARASITIC INFECTIONS IN CATTLE FROM CENTRAL AND NORTHERN THAILAND
Review round: 2
Reviewer: 1

Output:
Basic reporting: The scientific names still need correction in Lines 72-88. Write the full genus name in the first citation (e.g. …Two species, Babesia bovis and B. bigemina, are known to be extremely ….., such as B. divergens, B. major, B. jakimovi, B. ovata, .....).
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: -	Line 222 and 224 correct to spp.
-	The style of the Journals names still not unified (see Ref. 1, 9, 12, 15, 20, 22, 23, 29, 31).
-	Incorrect or incomplete reference details (copied from ResearchGate??): Ref. 6, 34, 38.
-	Ref. 9, “trypanosomes” is not a scientific name.
-	Italicize scientific name in Ref.23.
-	Ref. 32: add, (the article language is Polish and [English Abstract] was used).


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

Input:
Title: EVALUATION OF COLONIZATION AND MUTUALISTIC ENDOPHYTIC SYMBIOSIS OF ESCHERICHIA COLI WITH TOMATO AND BERMUDA GRASS SEEDLINGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Present study titled Evaluation of colonization and mutualistic endophytic symbiosis of Escherichia coli with tomato and Bermuda grass seedlings- describe about the successful colonisation of E. coli in tomato and Bermuda roots. Bacterium has also shown some plant growth promoting activity also. With confocal microscopy author have tried to confirm its endophytic colonisation in roots of tomato and Bermuda. Study is interesting since E. coli is generally animal gut bacterium and reported as water bodies’ contaminant. Paper is written well some of the comments and questions are given below which need to be addressed by the authors.
Experimental design: 1. In the material method section authors have not given details about the microscopy procedure-
This section needs revision. The introduction part may also be elaborated.
2. Why have the authors measured only photosynthetic pigments from Bermuda leaf not from tomato, for any specific reason?
3. Authors have shown that E. coli inoculation induces the root growth in tested plants but no effect on shoot- why. It must be discussed appropriately.
4. Did authors examined the shoots or leaves of the plant for the endophytic colonisation of bacterium? It would very interesting to add it.
5. Authors may check for typose error at some places in the manuscript.
Validity of the findings: Line 137-138- This should be in the materials and method section.
Line 143-144- The bacterium is producing more auxin in tryptophan less medium when compared to tryptophan amended medium. Can there be a reason as it’s an interesting result?
Line 194-196- The authors mention that presence of cellulase and pectinase activities might help in the successful colonization of E. coli inside the host. However, the authors did not check whether these activities are present in their strain or not. Since the authors have shown successful colonization as well as rhizophagy, therefore it would have been interesting to see
whether they possess these traits or not?
Line 197- The authors have used the term disinfected, but in my opinion, it should be surface sterilized as the presence of endophytic bacteria cannot be ruled out by chlorox treatment. Reports have shown in vitro activation of endophytic bacteria after surface sterilization
(Refer to Shaik and Thomas, 2019 “In Vitro Activation of Seed-Transmitted Cultivation-Recalcitrant Endophytic Bacteria in Tomato and Host–Endophyte Mutualism”).
Line 205-206- The growth effects of the strain are seen mainly on roots and not on shoots. However, the authors might have checked the dry weight of shoots or the whole seedlings as better root architecture generally leads to better plant growth.
Line 226-228- The authors attribute better mineralization and mobilization of nutrients contributing to healthy E. coli treated seedlings but the strain showed no phosphate solubilization activity while potassium solubilization, nitrogen fixation, siderophore production was not checked. The authors did mention the rhizophagy cycle; however, a detailed study of the cycle is required to confirm its role in nutrient mineralization and solubilization.
Additional comments: Authors may check for typose error at some places in the manuscript.


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

Input:
Title: EVALUATION OF COLONIZATION AND MUTUALISTIC ENDOPHYTIC SYMBIOSIS OF ESCHERICHIA COLI WITH TOMATO AND BERMUDA GRASS SEEDLINGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study is interesting but some revisions are needed
 Line 51 rhizophagy
239 reference is wrongly written
272 acknowledge
Some references are missing
Figure 3 looks old school style
Figure 4 is horrible, please delete it or do it again by reducing aperture or by decreasing the time of talking the photo
Figures 4-8 can be combined in a single figure, and it will be better for the reader.
Figure 8 confocal microscopic observations, where
Experimental design: Do the authors always used gfp trasnformed cells? Normally we compare to wild type strain for effects on plants, but is also ok.
Validity of the findings: The study is interesting but some revisions are needed
 Line 51 rhizophagy
239 reference is wrongly written
272 acknowledge
Some references are missing
Figure 3 looks old school style
Figure 4 is horrible, please delete it or do it again by reducing aperture or by decreasing the time of talking the photo
Figures 4-8 can be combined in a single figure, and it will be better for the reader.
Figure 8 confocal microscopic observations, where
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: EVALUATION OF COLONIZATION AND MUTUALISTIC ENDOPHYTIC SYMBIOSIS OF ESCHERICHIA COLI WITH TOMATO AND BERMUDA GRASS SEEDLINGS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The text is clear but can be improved.
The article is conforming to professional expression standards.
The work is simple in methodology, reaching an important contribution by demonstrating that Escherichia coli can establish as an endophyte in at least two types of plants, becoming a plant-symbiont by stimulating the host development.
Literature references are sufficient in this field background and well contextualized.
The article includes sufficient introduction and background to demonstrate how the work fits into the broader field of knowledge. Relevant prior literature is appropriately referenced. Nevertheless, text needs edition from a fluent English-speaking microbiologist or microbial ecologist.
Experimental design: The adopted methodology is correct and well described, with the necessary and sufficient details to be repeated. The experiments were well planned and met the assumptions of casual accuracy. Statistical analysis is missing in M&M section of results. Nonetheless, statistical values are described in the discussion section (line 208)
Validity of the findings: The results are conclusive and supported by the appropriate methodology, including statistical analysis. The hypotheses have been tested and proven.
Conclusions are well stated, linked to original research question & limited to supporting results.
Nevertheless, authors failed describing the statistical analysis used method.
Additional comments: The article is valuable, demonstrating that Escherichia coli can establish as an endophyte in at least two types of plants, and become a plant-symbiont by stimulating the host development. Thus, this study has great importance and applicability.
Nevertheless, text needs edition from a native English microbiologist or microbial ecologist. It is recommending to refer in the text, first the older reference and the newer references at the end, taking in consideration also the alphabetical order. Per example in line 40, change “(Walker et al., 2013; Nautiyal et al., 2010; Sabat et al., 2013)” by “(Nautiyal et al., 2010; Sabat et al., 2013; Walker et al., 2013).
Throughout the text, many “commas” are missing.


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

Input:
Title: EVALUATION OF COLONIZATION AND MUTUALISTIC ENDOPHYTIC SYMBIOSIS OF ESCHERICHIA COLI WITH TOMATO AND BERMUDA GRASS SEEDLINGS
Review round: 1
Reviewer: 4

Output:
Basic reporting: 1. Please homogenize your English. Some places e.g., colonise (L47), mobilise (L48), colonisation (L49), etc. use British English, and other places, e.g., colonize (L21) and internalize (L51), use American English.

2. L12. Please spell “E. coli” out.

3. L15. Please show the scientific name of tomato and Bermuda grass here.

4. L95. A total of.
Experimental design: 1. Please provide the objectives of this study in the introduction.

2. The authors concluded that effects on seedling root growth could in part be explained by IAA production. IAA indeed is able to promote plant growth, but this conclusion drawn based on a culture-based study is speculative.
Validity of the findings: 1. This study has a significant limitation: no statistical analyses of data were present. Without statistical analyses, the conclusions you reached are not convincing.

2. L69-72. Please add the refs for the surface sterilization you used.
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: EVALUATION OF COLONIZATION AND MUTUALISTIC ENDOPHYTIC SYMBIOSIS OF ESCHERICHIA COLI WITH TOMATO AND BERMUDA GRASS SEEDLINGS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors improved their manuscript as suggested. It is ready for publication. just a minor mistake: Line 72 seeds instead of seed
Experimental design: Authors improved their manuscript as suggested. It is ready for publication. just a minor mistake: Line 72 seeds instead of seed
Validity of the findings: Authors improved their manuscript as suggested. It is ready for publication. just a minor mistake: Line 72 seeds instead of seed
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: EVALUATION OF COLONIZATION AND MUTUALISTIC ENDOPHYTIC SYMBIOSIS OF ESCHERICHIA COLI WITH TOMATO AND BERMUDA GRASS SEEDLINGS
Review round: 2
Reviewer: 2

Output:
Basic reporting: This is the reviewed version. Authors addressed each question and comment correctly.
Manuscript is approved.
Experimental design: In the M&M section, significant value for differences is missing. I suggest to insert “(P ≤ 0.05)” after “significance differences” (line 147)
Validity of the findings: The results are conclusive and supported by the appropriate methodology, including statistical analysis. The hypotheses have been tested and proven.
Conclusions are well stated, linked to original research question & limited to supporting results.

In results section, change “P≤ 0.001 and 0.003 respectively” by “P < 0.001 and P < 0.003, respectively” (line 220); and “(P≤0.000, 0.009, 0.001 respectively).” by “(P < 0.001, P < 0.009, and P < 0.001, respectively).” (line 224)

In Figure 3 legend, Change “P≤ 0.001 and 0.003 respectively” by “P < 0.001 and P < 0.003, respectively”; and “(P≤0.000, 0.009, 0.001 respectively).” By “(P < 0.001, P < 0.009, and P < 0.001, respectively).”
Additional comments: Be aware that the worlds in manuscript and book chapter titles, the first letter should not be written in capital letter (such as in Tharek et al. and Walker et al. references). That is only for book title.


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

Input:
Title: EVALUATION OF COLONIZATION AND MUTUALISTIC ENDOPHYTIC SYMBIOSIS OF ESCHERICHIA COLI WITH TOMATO AND BERMUDA GRASS SEEDLINGS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript is improved a lot compared to the previous one.
Experimental design: Looks good.
Validity of the findings: Looks good.
Additional comments: Fig. 3: Please show the significant differences between controls and treatments directly using letters or asterisks.


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: DEEP EVALUATION OF THE EVOLUTIONARY HISTORY OF THE HEAT SHOCK FACTOR (HSF) GENE FAMILY AND ITS EXPANSION PATTERN IN SEED PLANTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript identified HSFs in some species which were sequenced more recently, and investigated the evolutionary relationship of some HSF subfamilies. The HSF subfamily classification should also be induced from the phylogenetic tree, with a comparison to the annotation from HEATSTER website. Thereafter, some results should be thoroughly investigated and verified according to new classification results.

Major issues:
1. The scientific question should be more clearly and detailed (lines 80-81). Contrary conclusions, or insufficient evidences reported by previous studies should be indicated.

2. The author should precisely show how the HSF was identified using transcriptome data, with or without RNA-seq assembly?

3. The subfamily classification in table 1 is derived from the HEATSTER. The classification should be induced from the phylogenetic tree, instead of the HEATSTER. This is may be the reason that your result that HSFs from Chlorophyta were divided into HSFA (I didn't find these HSFs in figure 1). The previous study reported the HSFs from Chlorophyta were more closely related with HSFB in plants and HSFs in animal.

4. The phylogenetic tree in figure 1 has no bootstrap values supporting the robust of the tree. Other phylogenetic trees constructed by different model or methods should be included to support your classification and evolutionary results. So, the first result “The phylogeny and evolution of HSFs in land plants” should be verified and reconstructed.

5. In result parts of “Divergence time analysis”, why were only HSFA2 and HSFA9, and HSFC2 and HSFC1 included? How about the other subfamilies?

Some Minor issues:
59 line, “Thermo tolerance” should be “Thermotolerance”

60 line, “They (HSF) functionalized as molecular chaperones in protein folding and assembly to protect cells”. This is the HSP’s functions, not the HSF.

68 line, “transcription active region” of the HSF should also be included.

160 line, a total of 46 species in Table S1 and S2, instead of 44, were used for HSF identification. In addition, why were these species divided into two supplemental tables? The species which were not used in previous study should be indicated.

185 line “The deep divergence of HSFA1 in pteridophyta and gymnospermae, indicated that HSFA1 diversified before the radiation of seed plants” showed be exhibited as a figure. I think the separated clades of HSFA1 should be classified as other subfamilies (not evolutionary divergence) based on a robust phylogenetic tree, instead of HEATSTER. See the major issue 3.

254 line “The results indicate the ancient HSF gene duplications were not easy to be detected, because most duplicates have been lost”. The author should give the evidences or references supporting the “most duplicates have been lost”

In table 2, the sequence identities of the duplicated HSF should be exhibited to help readers for the understanding of duplication.

In table 3, the definition and the identification methods of the orthologous were not clearly discribed.

402 line, no evidences were provided to support the “gene loss and sub- or neo-functionalization during the evolution and diversification of land plants”

The resolution of the figures 2 and 3 should be improved.


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: DEEP EVALUATION OF THE EVOLUTIONARY HISTORY OF THE HEAT SHOCK FACTOR (HSF) GENE FAMILY AND ITS EXPANSION PATTERN IN SEED PLANTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting of the article is ambiguous with several unclear statements, which often confuses the actual findings. The word 'deep' in the title itself is ambiguous. Moreover, the expansion of HSFs was inferred in 'seed plants' as appears from the title, but the sampling of seed plants analyzed by the authors may not be sufficient to make a blanket statement. In the article, authors have often mixed Results with Discussions, though a separate Discussion section existed. Some crucial literature references were missed out by the authors, which might have changed their perception of the evolution of HSFs in seed plants. There are also problems with the representation of Tables. The contents were not very clearly understood as far as the paralogs and orthologs are concerned. As far as the writing is concerned, the authors were very casual in checking the typos and English usages in the sentences throughout the manuscript. That results in so much annoyance that I could barely read beyond the Results section. I am sorry, though it sounds a bit harsh, I could not stop making this comment for a scientific review for a reputed journal like PeerJ. This critical assessment might enrich authors to improvise on the sections where they need to strengthen.
Experimental design: The experimental design though is okay, but somewhere falls short. Broadly, authors have compared the HSF proteins from a few groups of plant taxons, which could have expanded further with a large number of sequenced genomes available. Though it has been often mentioned by the authors as a comprehensive account of HSF, it is merely more than 600 HSF sequences, when there are 2.4 K sequences from 103 species already available in the Pfam database. The research question on the evolution of the HSF genes was also not well defined. Why do authors need to reanalyze the evolution of HSF genes may clearly be stated. Additional methods and approaches may need to be supplemented in order to really provide valuable and novel information on HSFs evolution and functionalities in plants.
Validity of the findings: The findings are not very novel and aren't explicitly validated with robust experimental results. The theme of research and conclusion looks okay, but without sufficient data to support it, the conclusions perhaps appear weak and overstated.
Additional comments: The manuscript may be checked thoroughly. If required, it can be checked through professional editors to submit a manuscript with minimal flaws. The image qualities and information depicted can be improved further.


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: DEEP EVALUATION OF THE EVOLUTIONARY HISTORY OF THE HEAT SHOCK FACTOR (HSF) GENE FAMILY AND ITS EXPANSION PATTERN IN SEED PLANTS
Review round: 3
Reviewer: 1

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


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

Input:
Title: SPATIO-TEMPORAL DISTRIBUTION OF OSTRACOD SPECIES IN SALINE INLAND LAKES (MANSFELD LAKE AREA; CENTRAL GERMANY)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The structure of the manuscript conforms to PeerJ standards.  The introduction provides useful and appropriate context for the research; below I suggest the inclusion of more explanatory text about certain aspects, and clearer identification of research questions, aims and objectives. The text is written in good English, but it can and should be improved in the interests of clarity and completeness. I recommend that once revisions are completed the authors should seek the help of a native English-speaking scientist to revise and improve the English.  Figures are well designed and informative but in some cases very complex and it takes time to understand what they are showing (more informative captions would help); some are of poor resolution and look fuzzy or pixellated when magnified sufficiently to study them closely (I am looking at the separate figure files, not just the figures in the compiled pdf).  This issue affects Fig. 1 especially which includes some symbols and names that are practically illegible.  Literature is well referenced and relevant; I have suggested (below, under General comments) some additional references that I think would be useful. Raw data are supplied as supplementary spreadsheets.
Experimental design: The submission presents original primary research that is within the scope of the journal. As noted above and in more detail below, research questions are more implicit than explicit and need to be defined more clearly; nevertheless, the research is certainly aimed at filling a significant research gap. The execution of the research appears rigorous and of a high technical standard. Some methods are not described in sufficient detail to facilitate replication, but this problem should be easy to rectify.  See below (under general comments) for detailed criticisms and suggestions.
Validity of the findings: All underlying data have been provided as supplementary information; they appear to be robust and statistically sound; the acquisition of such a comprehensive dataset is impressive and commendable. However, in my opinion the analyses, interpretation and conclusions do not always do justice to the impressive data and I offer some suggestions (below) for ways of exploiting the data more fully. “Conclusions” could be better linked to original research questions (once these are made more explicit); some acknowledgement of limitations of the research design, methods and results could usefully be included in the “Conclusions”, together with suggestions of how future research design might be improved. In spite of criticisms, the overall research findings are significant and valuable, and I trust that the authors will not find it too onerous to address the detailed critical points and suggestions made below (under general Comments)..
Additional comments: This submission represents a valuable contribution to understanding the ecology and taphonomy of assemblages of ostracods, a crustacean group valuable for palaeoenvironmental reconstruction. The research is underpinned by extensive fieldwork and laboratory analyses. The longer I spent exploring the authors’ raw data, analyses and interpretation, the more I was impressed by what they offer, but at the same time I developed a strong feeling that the dataset was under-utilised and could be analysed and interpreted in greater detail. Although there are shortcomings in various aspects of the submission (and these are inevitably the focus of my detailed comments below) which should be acknowledged, I believe they can all be addressed positively in ways that will result in a more robust and useful publication.

Figures and tables

Figure 3, running over two pages, presents a lot of useful data and is very informative once you have worked out what it all means. It needs a fully explanatory caption, however; it took me some considerable study of the figure and relevant main text sections to work out that the small pie charts show % abundance in taphocoenoses and the coloured blocks show % abundance in the biocoenoses (working this out was not helped by the fact that some comments in the main text do not make it clear whether they apply to biocoenoses or taphocoenoses). The caption should explain all abbreviations; I could work out that a = adult and j = juvenile, but this should be made clear, and the abbreviations for Sample ID (OT2, OT etc.) should be explained or at least cross-referenced to a look-up table, e.g., the caption could state “Sample IDs as in Table 1”. It is actually quite confusing that the figures and tables refer to “Sample-ID” but the main text refers to “water bodies”, in both cases matched with the same abbreviations (OT2, OT etc.).  The use of full stops and commas in numbers is confusing, as it shows total numbers of valves as 1.019, 1.452, 1.263 etc., which makes the full stops look like decimal points (these numbers should be 1,019, 1,452, 1,263 etc.). The salinity values on this figure, on the other hand, feature commas where decimal points are required: 0,7, 4,4 etc. should be 0.7, 4.4.... (as is done correctly in the main text).  Practical salinity units actually have no units of measurement (confusing, I know) and should really be reported as simple numbers (perhaps with a statement in Materials and Methods to the effect that “all salinity values are in practical salinity units”), but the use of “psu” as an apparent unit of measurement is now so widespread it is probably not worth worrying about.

Figure 5 needs more explanation in the caption. In this case the caption states “Abbreviations of waterbodies as in Table 1”, but they are actually given as sample IDs in Table 1 (also in Fig. 3), although of course they match sampling site names, which are the names of waterbodies (but some waterbodies have more than one sampling site). More informative site/waterbody descriptions (especially in the “habitat type” column) would be helpful. For example, “lake” might be any of several different lacustrine habitats (littoral, sublittoral, profundal, mud, sand, stones, aquatic macrophytes etc.); is “Teufe 2”, described as “lake temporary”, an ephemeral/temporary lake or a marginal part of a permanent lake that dries out when lake level falls? Is “Quelle im Wald” a name or a description (e.g., “a spring in the woods”)? English versions of the names, as descriptors, could be helpful.  The figure caption tells us that it shows “Ratios of adult and juvenile ostracod species in the water bodies”; I think it shows adult:juvenile ratios of ostracods and I would assume that the black part of each bar represents % adults and the grey part % juveniles (this needs to be clarified in the caption), but no species are named, so do these bars represent combined data from all species collected at each sampling site (i.e., these are adult:juvenile ratios of assemblages, not of individual species)? This is not really made any clearer in the relevant section of main text (lines 455-462), and it needs to be clarified.

Fig. 9 needs to be adjusted (redrawn if necessary) so that all letter codes are clearly visible and associated with, but not overlapping, the data points, lines or each other (some are so tightly clustered and overlapping as to be illegible). The figure also needs a clearer explanatory caption; it states “species code similar to Fig. 3” but they should be exactly the same as in Fig. 3 (e.g., “Cn”, not “CN” for Candona neglecta) so as to distinguish them more clearly from sample ID codes. Preferably different symbols should be used for the species and the waterbody/sample ID data points.

Materials and Methods

It is evident from “Materials and Methods” and “Results” that the study differentiated between live (with soft parts) and dead (empty valves and carapaces) ostracods; exactly how this was achieved is not very clearly explained (lines 145 -152), however. Were ostracods ever observed alive, or were they assumed to have been alive at the time of collection because their carapaces contained soft parts (appendages etc.)? That would be a perfectly reasonable assumption, but it needs to be stated.  Line 145: “The samples were fixed with 70% Ethanol to preserve living specimens.” Was this done in the field at the time of collection, or after the samples were returned to the laboratory? If the latter, how long were samples kept, and in what conditions, before ethanol was added?

In “Materials and Methods” it is stated (lines 151-152) that “Empty carapaces and valves were sorted by juveniles and adults. Different juvenile stages were not distinguished.”  Although it does not seem to be made explicit in “Materials and Methods”, references to, e.g., “...biocoenoses which are also characterized by very variable adult/juvenile ratios” (lines 460-461) and the data shown in Fig. 5, as well as the raw data, show that specimens collected alive were also sorted into adults and juveniles. Perhaps this could made clearer.

I also wonder about the way in which the adult:juvenile ratios reported here were calculated, if the specimens were picked separately from two different size fractions (retained on the 500 and 250 micron sieves; lines 147-148). As long as each sample’s size fraction was picked in its entirety then the counts could simply be added together, without need to consider proportional adjustments; I am fairly certain this was the case, but it needs to be stated clearly to inspire confidence in the data.  Additional picking of ostracods from the >125 micron fraction might have yielded smaller juveniles of many species, although they would probably be difficult to identify to species level. In the case of D. stevensoni and Cyprideis torosa there is the added complication that these species have brood care, the eggs and the earliest hatched instars being retained in the brood chamber of the adult female (something that does not seem to be considered in the submission).

Salinity

It is stated in “Materials and Methods” that electrical conductivity was measured in situ in the waterbodies sampled (line 141), along with temperature, pH and oxygen concentration. In the “Results” section it is stated (line 184) that “Conductivity values range between 1.3 mS/cm (OT2) and 26.7 mS/cm (Fig 2 B)”. Elsewhere (e.g., lines 300-313) there are numerous references to salinity (values presented as practical salinity units), both for the waterbodies sampled (lines 304-305: “Water bodies were also classified in ascending order according to salinity values.”) and in terms of the salinity tolerances of the species recorded. The relationship between salinity and electrical conductivity should be explained; the two are not simply synonymous, and salinities reported in the literature may have been measured in different ways and therefore not always be directly comparable (especially true in non-marine waters).

In the “Conclusion” section there is a statement (lines 757-760) that “The water bodies show differences in ostracod species assemblage composition which cannot explained by measured ecological parameters. Especially, salinity has a comparably small influence on the ostracod species distribution.”  Other possibly influential factors are mentioned (lines 765-766) such as “vegetation types, composition or texture of substrate, nutrient input, hydrologic conditions”, but the authors seem unaware that the solute chemistry of waterbodies is well established as a significant factor affecting species distributions. For example, different species have preferences for Calcium-, Sulphate- or Chloride-dominated waters (see Fig. 6 in Pint et al., 2015, already cited in this submission), or for bicarbonate-enriched or bicarbonate-depleted waters (see, e.g., Smith, A.J. & Horne, D. J. 2002 Ecology of marine, marginal marine and nonmarine ostracodes. In Holmes, J. A. & Chivas, A. R. (eds), The Ostracoda: Applications in Quaternary research, AGU Geophysical Monograph Series, Vol. 131, 37-64). To equate water chemistry to salinity (lines 611-612: “water chemistry (i.e., salinity)” is an over-simplification; the influence of solute chemistry needs to be acknowledged, at least.

The collection of abundant living Cyprideis torosa inhabiting inland waterbodies (especially some with very low salinity/conductivity) is of considerable importance and deserves greater prominence in the text; it could also be highlighted in the abstract and in “Conclusions”. Such apparently anomalous occurrences are quite frequently mentioned in the literature but as far as I am aware this is the first confirmation of living specimens in German inland waters. The discussion of these occurrences with respect to salinity (lines 718-728) is useful but it could be expanded and considered more critically. Cyprideis torosa has been studied extensively and is far more than just “an indicator species for brackish water”, since variable morphological features (carapace size, sieve pore shape, phenotypic noding) and shell geochemistry (stable isotopes, trace elements) have been calibrated for use as proxies for palaeosalinity. See the special 2017 issue of Journal of Micropalaeontology devoted to papers on Cyprideis torosa, starting with: De Deckker, P. & Lord, A.R. 2017. Cyprideis torosa: a model organism for the Ostracoda? Journal of Micropalaeontology 36, 3-6.

Biocoenoses and taphocoenoses

I very much agree with the authors’ view that ostracod research has often differentiated insufficiently between living ostracods (biocoenoses) and their empty valves (taphocoenoses) (lines 102-103). However, these terms should be explained, not only to ensure that the reader understands them, but also to demonstrate what the authors understand by them, because they have often been applied incorrectly in ostracod studies (this was explained by Boomer, Horne & Slipper, 2003, a reference cited only in a more general context in this submission). Assemblages of ostracods represented by empty carapaces and/or valves (resulting from moulting or death) may often be taphocoenoses (transported, sorted and mixed before final deposition) but there are other possibilities (for example in a very small waterbody with little or no potential for transport, sorting and mixing, an autochthonous thanatocoenosis may accumulate). Many natural assemblages probably comprise both thanatocoenosis and taphocoenosis components. Even after burial of an assemblage there may be taphonomic processes such as diagenesis to take into account.  Pint & Frenzel (2017: p. 118) (a reference already cited in this submission) have understood these issues well: “Analyses on fossil ostracod associations rely mainly on taphocoenoses. Using preservation and population structure as indicators, allochthonous elements may be identified... but this criterion is hard to use for well-preserved specimens of rare species. Hence, some portions of the studied associations have to be accepted as unidentified allochthonous elements. This problem is especially serious in estuaries, where inflowing water from the mainland and also the sea carries ostracod shells to the sites. Similarly, athalassic saline lakes and coastal lagoons with abundant C. torosa may be affected by transported limnic taxa from rivers or marine taxa from the sea. The produced taphocoenosis is, of course, not completely an assemblage of animals living together or in the same location over the seasons but a mixture of autochthonous and allochthonous elements. This makes palaeoecological interpretation more complicated but tells us something about provenance and, hence, the character and interconnectedness of the studied water body. We confess that we are not able to judge the autochthonous character of every individual registered in our count, but use its presence for the characterization of the waters inhabited by C. torosa, thus integrating ecological and taphonomical information”. I welcome the present authors’ apparent intention of addressing such issues by studying ostracods in the “natural laboratory” of the Mansfeld lakes area waterbodies. As submitted, however, these intentions are mostly only implicit; the only exception seems to be the final sentence of the Introduction (lines 120-122): “By repeated sampling, we also wanted to investigate if there is a seasonal effect on the distribution and abundance of the ostracod species related to the specific life-cycle of the species”. Knowledge gaps are identified with reference to relevant literature, but the authors need to clarify how the research was designed to fill those gaps; the purpose of the research really needs to be made more explicit by means of clearly stated research questions and aims. That being done, it would then be helpful to refer to aims and objectives when explaining the methods; explain not only the method but also its purpose (e.g., “In order to determine seasonal variations in ostracod species occurrences and environmental factors, three sampling campaigns (April, June, September 2019) were carried out in the Mansfeld lake area”).  A limitation of the research is that juvenile valves were not differentiated according to moult stages; doing so could have been very informative in assessing which components of each assemblage had been most subject to transport and sorting (again, see Boomer, Horne & Slipper, 2003); I appreciate that time constraints mean that you cannot do everything, but it would be helpful if the authors themselves would acknowledge such a limitation rather than leaving it for readers to recognise and criticise.

Lines 597-598: “An indication of valve transportation is the extremely high number of adult valves compared to juvenile valves in most water bodies”. I agree, but this is an observation that could usefully be explained, in terms of processes, in more detail. Make it clear that this is an indication of differential transport (removal by winnowing, probably) of juvenile valves, leaving behind the larger/heavier adult valves. In an enclosed environment (e.g., an aquarium) juvenile valves should greatly exceed adults in number, because ostracods typically moult eight times before reaching maturity and thus each adult individual has already contributed sixteen juvenile valves to the sediment.  Transport could be by currents or wave action, for example. Wave action around the shallow margins of a lake will most easily resuspend the smaller juvenile valves which are then redeposited in deeper water; assemblages in shallow marginal waters are thus dominated by adults and depleted in juveniles. It is a pity that juveniles were not differentiated and assigned to different juvenile stages, because such data could be very informative about transport and size-sorting (see, e.g., Boomer et al. 2003, already cited in this submission). Nevertheless, the simple counts of adults and undifferentiated juveniles in the raw data could be used more effectively, in combination with published information about the life-cycles of species.

Life-cycles

Although life-cycles of ostracod species are quite frequently mentioned in the “Introduction” and “Discussion” sections, I could find few indications of any real consideration of the known life-cycles and seasonality of any named species in the context of the research reported here (not even under “Inferences on species life-cycle”; lines 473-492) The only exceptions are perhaps the mention of Darwinula stevensoni having a life-span of <1 to 4 years (line 710) (but no mention of the fact that this species has brood care), and salinity-related differences in life cycles of Limnocythere inopinata (lines 711-713) (citing Geiger, 1998, which is missing from the reference list). The latter most likely comes from Geiger et al. 1998, who found that, in L. inopinata, parthenogenetic clonal lineages respectively adapted to saline water and to fresh water never co-occur, and the saline water populations overwinter as adults and juveniles while the freshwater ones disappear in winter; this implies that freshwater populations overwinter as eggs. The present authors’ data is uninformative in this respect since they did not sample in the winter months.  It is worth noting that L. inopinata is a Holarctic species that also has sexual populations, for example in North America where the parthenogenetic and sexual populations have different though overlapping distributions that are probably related to water chemistry.

Therefore, despite the concluding comment of the Introduction (lines 120-122) that “By repeated sampling, we also wanted to investigate if there is a seasonal effect on the distribution and abundance of the ostracod species related to the specific life-cycle of the species”, it seems that this aspirational investigation was not pursued to any significant extent. There could be many good reasons why this aspect of the intended research could not be addressed; at least some brief explanation of why it could not be achieved should be included in discussion. Alternatively, if the authors would like to pursue it and take into account what is known about the life-cycles of the species they identified, Claude Meisch’s (2000) monograph (which they have already cited a few times) would be a good starting point as it  includes summary life histories of many species, for example Candona neglecta (a permanent form with two generations per year), Sarscypridopsis aculeata (two or three generations per year, overwintering mainly as eggs) and Cyprideis torosa (with brood care; one generation per year, overwintering as adults and juveniles).

I wondered: were juveniles as well as adults in the biocoenoses and taphocoenoses identified to species level?   I looked at the Supplementary spreadsheets and found that they were. For example, the April TS biocoenosis has Darwinula stevensoni with 48 adults and 33 juveniles, April TE biocoenosis has Cyprideis torosa with 68 adults and 46 juveniles. These impressive datasets seem to have been under-utilised, with only a few comments in the main text, such as (lines 481-483): “Almost all species show the same pattern between adults and juveniles in the biocoenoses. For example, adult D. stevensoni are most abundant in summer, juveniles as well”. It would be useful to at least make greater use of the graphic presentation of seasonally changing adult:juvenile ratios of selected species (summarized for all waterbodies) shown in Fig.7, but ideally to also consider data for selected species and selected waterbodies, and to attempt to match these with known life-cycles. I think this would be quite straightforward to do, and not particularly time-consuming. For example: a;j ratios of Heterocypris salina at TE (lake, mud substrate) were 2:61 in April, 12:50 in June and 1:5 in September, and at BS (lake, sand substrate) they were 5:1 in April, 13:12 in June and 13:14 in September. This species can produce two or three generations per year, taking only 45 days to complete a generation’s life-cycle, and overwinters as eggs (Meisch, 2000). I suggest, tentatively that the dominance of juveniles in the April and June collections might represent two successive generations. The equivalent taphocoenoses data seem to support this idea, and it is interesting to note the significant numbers of juvenile carapaces recorded, because these imply juvenile mortality (moulted carapaces are typically disarticulated into separate valves) which could be indicative of an adverse perturbation of their environment.  The lower numbers at BS compared to TE (in both the biocoenoses and the taphocoenoses) might be explained by the different substrates sampled at these two sites, and it is tempting to infer that the BS assemblages (from sand, a higher-energy substrate than mud) have been more affected by transport. I may be over-interpreting the data, and the authors should do their own analyses; this example is simply intended to demonstrate that much more could be done with the available raw data.

ref.: Geiger, W., Otero, M., Rossi, V. 1998. Clonal ecological diversity. In: Martens, K. (ed.) Sex and parthenogenesis. Evolutionary ecology of reproductive modes in non-marine ostracods, 243-256. Bakhuys Publishers.

Conclusions

It is apparent from the “Discussion” section that the multitude of potentially influential variables makes it difficult to determine the factors that may drive the differences between biocoenoses and taphocoenoses. In “Conclusions”, some attention might usefully be paid to lessons learned from the present study and suggestions for improving the design of future field collecting campaigns in ways aimed at reducing the number of variables (for example, seeking out and sampling the same kinds of substrate or habitat in each waterbody).

Other comments

lines 251-252:
Family: Notodromatidae Kaufmann, 1900
Subfamily: Notodromatinae Kaufmann, 1900

Correct spellings are Notodromadidae and Notodromadinae

Please check throughout for spelling errors, especially in taxonomic names incorrect endings of names are commonly introduced by autocorrecting software). For example, in the caption for Fig. 3 “Potamocypris arcuate” should be “Potamocypris arcuata” and the same error is quite frequent in the main text.  Similarly “S. aculeate” should be “S. aculeata” (line 393).  Fig. 4 caption: “thaphocoenoses” should be “taphocoenoses”.

David J. Horne 15th February 2022


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: SPATIO-TEMPORAL DISTRIBUTION OF OSTRACOD SPECIES IN SALINE INLAND LAKES (MANSFELD LAKE AREA; CENTRAL GERMANY)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Despite the English text is of high quality there is still room for improvements. I indicated mispellings, stylistic problems etc. within the annotated version of the manuscript. I have to stress, however, that I am not a native speaker, so check carefully. The use of the term abundance is sometimes confusing because it is not always clear for me if abundance, i.e. the number of individuals in a given volume/area, or relative abundance [%] is addressed.
The literature used gives a good background for the discussion of data obtained. An exception is the largely missing description of the study area. I suggest to provide a chapter Study Area using some information from Introduction and M & M and adding data about variability of salinity and maybe pollution. Some of the water bodies investigated are monitored, so information could be gathered. The variability, especially in salinity, is crucial for the interpretation of associations from taphocoenoses. The lack of this type of information is a shortcoming of the manuscript under review.
The results chapter and, in a lesser degree, the discussion could be better structured enabling the reader to follow more easily. This is not an easy task with the wealth of data obtained and ambiguous patterns of distribution. I know this from my own painful experiences with similar datasets. Maybe focusing and a little shortening of the text could help. Furthermore, as suggested within the annotated manuscript, the authors could add a table with their classification of sites as reference for the reader. Clear research questions and focusing discussion to them could also help to foster a clear structure.
The diagrams and other figures are mostly well prepared, but their captions are often too short. Details are given within the annotated manuscript. The diagrams of Fig. 7 are too complex and should be split.
Experimental design: The study clearly adds to our understanding of ostracod associations from athalassic saline water bodies, a field not well investigated so far, and compares living assemblages with taphocoenoses what is crucial for applying Recent data to palaeoenvironmental reconstructions. I highly appreciate this. I feel, however, the authors could point out the relevance of their paper more clearly by focusing on selected research questions.
There is an important information missing but needed for interpreting distribution patterns: The area/volume of sampling is not given. Based on the text I conclude that not all samples have the same size, so it would need standardisation of abundance data to compare abundance. If abundance is standardised within the manuscript, it should be mentioned and explained how.
Using the adult/juvenile ratio is important not only for taphonomic processes but also for evaluating seasonal population dynamics as shown by the authors. Please, be always aware that this ratio is highly taxon-specific in your size fraction >250 µm.
Validity of the findings: The first finding of living Cyprideis torosa in athalassic waters of Central Europe shoud be stressed as highlight. Many researchers were hunting for such material before but without success. This is mentioned but not recognisable as a remarkable finding.
The living/adult ratio was calculated but not used for taphonomic interpretation. This should be added, a diagram plotting the sites with standardised abundances in both groups could be helpful by displaying productivity and preservation potential of all sites.
The conclusions are too negative in implications for palaeoenvironmental analyses by my opinion. The data set is rather large in terms of efforts but probably too small and hererogenous to discover all patterns lying below. For instance, the statement of salinity not being a driving factor of distribution of species is based on the missing of the brackish water taxon Cyprideis torosa at highest salinities, but this is not diminishing its worth as brackish water indicator because it is an opportunistic holeuryhaline species. I am convinced, the heterogenous distribution pattern of other species is not hampering their use for salinity reconstructions, what could be proven with applying the salinity tarnsfer function used by Pint et al. (2015) to taphocoenoses of the present study.
The seasonal occurence is not discussed referring to literature (e.g. Meisch 2000).
Habitat descriptions exist but are not used for discussion of distribution patterns, at least not recognisable for me.
Additional comments: See scan of annotated manuscript for more details.


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: SPATIO-TEMPORAL DISTRIBUTION OF OSTRACOD SPECIES IN SALINE INLAND LAKES (MANSFELD LAKE AREA; CENTRAL GERMANY)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript titled “Spatio-temporal distribution of ostracod species in saline inland lakes (Mansfeld lake area; Central Germany)” submitted by the authors Marlene Hoehle and Claudia Wrozyna is written in clear, scientifically sound manner. The abstract is concise and text is well organized within required units of IMRAD structure. Appropriate taxonomical nomenclature is used throughout the text and the technical and scientific quality of the illustrations is acceptable. Apart from few corrections suggested, the literature cited is adequate and it is appropriately listed. The quality of the English language is satisfactory. Comments on the references and some technical details in particular parts of the text and on figures are given in the additional PDF.
Experimental design: The manuscript contains valuable and new information on the subject of ostracod ecological preferences and dynamics of biocenoses and taphocenoses, with a critical analysis important for the implications in palaeoenvironmental reconstruction. The experiment and research questions are well set, and appropriate methods have been used to obtain and accurately analyze the data.
Validity of the findings: The conclusions drawn from this research are well stated and supported by the results, and adequately corroborated with literature citations.
Additional comments: A minor revision is needed and the authors are asked to take in consideration the more detailed comments marked in the particular parts of the text, and given in the annotated PDF of the manuscript draft.


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: SPATIO-TEMPORAL DISTRIBUTION OF OSTRACOD SPECIES IN SALINE INLAND LAKES (MANSFELD LAKE AREA; CENTRAL GERMANY)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article is written in comprehensive way, with professional scientific style. All required sections are included from Introduction to Conclusions, and the Abstract is in coherence with the manuscript content. Illustrations are well labelled and referred in the text.
Experimental design: This work presents an original and valuable study on the complex relationships of ostracods with their environment, tackling an important topic of taphocoenoses that are the only available material in paleoecology.
The description of methods is significantly improved comparing to the previous version.
Validity of the findings: The data was processed with a lot of care in the previous version already. Now, the considerable improvements in the Material and methods sections and the results enabled bettering the explanations in the discussion and conclusions. The new literature sources chosen to refer in the discussion are well selected and connected to the topic. However, there are some remarks to consider in the particular comments given in the additional file.
Additional comments: Additional, mostly technical details that need to be corrected are given in the PDF.


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

Input:
Title: THE USE OF SYNTHETIC AGONISTS OF QUORUM SENSING N- ACYL HOMOSERINE LACTONE PATHWAY IMPROVES THE BIOLEACHING ABILITY IN ACIDITHIOBACILLUS AND PSEUDOMONAS BACTERIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Caicedo and Orlandoni describe the application of Acidithiobacillus and Pseudomonas bacterial species in leaching copper and gold, implementing N-AHLs to enhance metal leaching by quorum sensing. The bacterial species discussed in the paper have been recognized and characterized in copper/gold leaching previously. The addition AHL homologues facilitates metal bioleaching is also consistent with published paper. Authors discussed possible underlying mechanisms in the discussion session, but the paper doesn’t include mechanism study data to support the discussion, which weakens the novelty and profundity of this work.
Experimental design: no comment.
Validity of the findings: no comment.
Additional comments: Please provide high resolution images for figure 2 and 3.
In figure 5, A.ferrooxidans and A.ferrivorans are monitored under absorbance 562 nm. Please modified the “Material and Methods” session accordingly.

And the English language need to be improved to ensure international audience can understand the context clearly. Here are some examples where the language can be modified for easier understanding.
1.	The second sentence in abstract. “However, the lack of complete understanding about microbiological processes involved in bioleaching reaction lead to drop in metal solubilization when using large amount of e-waste.
If “lead” is the verb of the sentence, then “leads” is the correct form.
If the original meaning is there is not enough understanding about how bioleaching reaction leads to drop in metal solubilization, then the sentence is not finished.
2.	In the last sentence of abstract.
“Bio leaching reactions using bacterial quorum sensing modulation, it could represent a valuable tool… “     please remove “,” and it.
3.	Similar issue in line 196-line198. Line 196-line 197 is not a sentence, please combine the line 198 with it.
4.	There are many minor grammatical mistakes in the manuscript including:
Line 42: as well as it is also responsible for ….
“As well as” cannot be followed by a sentence.
Line 55-56: “… it leading to…”
“which leading to” is grammatically correct.
5.	Many sentences start with ‘So,’ ‘Afterwards,’ ‘After,’ ‘Whoever,’. There is no need to add comma after these prepositions and adverbs.  And sentences start with “it which” are grammatically incorrect.

Please have someone who is proficient in English and familiar with the subject of the manuscript to proofreading and editing the paper.


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

Input:
Title: THE USE OF SYNTHETIC AGONISTS OF QUORUM SENSING N- ACYL HOMOSERINE LACTONE PATHWAY IMPROVES THE BIOLEACHING ABILITY IN ACIDITHIOBACILLUS AND PSEUDOMONAS BACTERIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The author reported the article according to the journal standard while there is some suggested improvement.
Experimental design: The experimental design is good however some of the improvement is needed in methodology section. I provided the comment in t
Validity of the findings: Novelty, rationale, conclusion are clearly defined
Additional comments: Following changes must be incorporated before publication in the journal
1.	In Introduction section give more detail about bioleaching property of Acidithiobacillus and pseudomonas genus. Give detail of these bacteria on their source, living environment, genetic makeup,  also state that these are pathogenic or non pathogenic (this is important when your research related to industry).
2.	In methodology: Biofilm formation assays, How you observe the biofilm formation as the standard method for biofilm detection as scanning electron microscope. You can clear this point and include a picture of biofilm in the results section if applicable.
3.	Include a statistical analysis at the end of method section under heading data analysis or statistical analysis
4.	The author use Acidithiobacillus for copper bioleaching and pseudomonas for gold bioleaching. Explain why you not tested both the bacteria for copper and gold. If it is exclusion or inclusion criteria, mention it in methodology.
5.	Previous studies are available on Acidithiobacillus and pseudomonas bioleaching. It should be better to explain the variation and your study strength compared to other studies in the Discussion section.


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

Input:
Title: THE USE OF SYNTHETIC AGONISTS OF QUORUM SENSING N- ACYL HOMOSERINE LACTONE PATHWAY IMPROVES THE BIOLEACHING ABILITY IN ACIDITHIOBACILLUS AND PSEUDOMONAS BACTERIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is well-written with professional English, and the experiment is clearly designed, and conclusion is solid.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: N/A


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 USE OF SYNTHETIC AGONISTS OF QUORUM SENSING N- ACYL HOMOSERINE LACTONE PATHWAY IMPROVES THE BIOLEACHING ABILITY IN ACIDITHIOBACILLUS AND PSEUDOMONAS BACTERIA
Review round: 2
Reviewer: 2

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


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

Input:
Title: ARBUSCULAR MYCORRHIZAL FUNGI ENHANCED THE GROWTH, PHOSPHORUS UPTAKE AND PHT EXPRESSION OF OLIVE (OLEA EUROPAEA L.) PLANTLETS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: minor questions:
1, the author mentioned the boron in the abstract and results, but the material  and methods were not mentioned boron. N, P and K are major elements and boron is minor element, why choose boron as representative of minor element for Oliver ?
2，dose the author measured the N, P, K in the roots and shoot? why just measure the leaf?
3，is there any plant pictures ? it will be more direct evidence for the promotion of plant growth by AM.


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: ARBUSCULAR MYCORRHIZAL FUNGI ENHANCED THE GROWTH, PHOSPHORUS UPTAKE AND PHT EXPRESSION OF OLIVE (OLEA EUROPAEA L.) PLANTLETS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the manuscript is quite well written and structured and as a result I was able to read it very smoothly and quickly. Literature discussed along the text is quite comprehensive and hypotheses were clearly stated. However, I would add to the introduction section a greater focus on the effects of AMF and soil microbiota on olive tree health, as these information were already afforded in the very recent literature.
As a further comment I would recommend authors to better define the rationale of the study concerning some detailed aspects. E.g. for the quantification of Boron: why this element was quantified? It has to be mentioned that Boron is crucial for olive tree flowering and fruit-set stages, for readers who do not known detailed Olive tree physiology.
Moreover, some sections needs to be re-organized. E.g. the paragraph at lines 206-211 should be anticipated as it would be preparatory to the performed expression analyses.
Finally, I would smoothen the first results section in which growth response results were described in too much detail showing the percentage of each ratio.
Experimental design: The experimental design was very clear, and lot of attention was devoted to compensate nutrients level across the treatment, which is really appreciable. However, if most of experimental procedures were described in greater detail, some other fully lacks or further need to be integrated. As an example, the soil sterilization protocol, which is often a critical step, was not provided as well as the geographic coordinates of the sampled soils (I would report them as supplementary materials). Similarly a phylogenetic analysis was reported in Results but methods used to generate alignments and the final tree were not reported at all.
Concerning this point it is not clear how Pt transporter homologs were initially identified (lines 138-139) and further named. At lines 198-205 it would be useful to detail if PT genes numbering was introduced here arbitrarily for olive tree or if it was already present in the genome annotation.
I saw that a pylogenetic tree with the putative PT transporter genes was performed. However, it is not clear if orthologies were tested (e.g. by specialized software which compares proteomes, or at least by the ‘best reciprocal hit’ method). For the sake of clarity I would put in brackets, at the first gene mention, the corresponding homolog genes of a mycorrhiza Eudicots model species such as Medicago or tomato.
More in general, methods should be better referenced, e.g. at line 152 (qPCR analysis methods), and significance threshold levels adopted in statistical tests should be stated (e.g. lines 156-159).
Validity of the findings: Unfortunately, I have not found anywhere the number of biological replications used in the study. It would be crucial to assess the strength of results showed. Moreover, in gene expression analysis differences among means should be tested using a proper statistical posthoc test after ANOVA(in Figure 3 no differences among conditions were reported).Beside these two aspects I found results solid and the performed analysis convincingly sound.
As a conclusion I found that these results well-supports what is known from literature about olive tree and AM symbiosis. For this reason I would further stress in conclusion section the applicative framework of the research, e.g. to obtain healthier plantlets which potentially hold higher survival rate after transplanting from nurseries to the field.
Additional comments: Detailed points:
- line 219: please define how do you mean with “effectivenenss of plantlets”.
- lines 231-232: here it is not clear if the authors means “inconsistent” or the opposite..
- lines 276-277: please update tomato scientific binomial to the currently accepted name Solanum lycopersicum.


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: ARBUSCULAR MYCORRHIZAL FUNGI ENHANCED THE GROWTH, PHOSPHORUS UPTAKE AND PHT EXPRESSION OF OLIVE (OLEA EUROPAEA L.) PLANTLETS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Reviewer’s comments
BASIC REPORTING
•	Clear, unambiguous, professional English language used throughout. [Yes, it is ok]
•	Intro & background to show context. Literature well referenced & relevant. [ Fairly good]
•	Structure conforms to PeerJ standards, discipline norm, or improved for clarity. [Ok}
•	Figures are relevant, high quality, well labelled & described. [ok]
•	Raw data supplied (see PeerJ policy). [Supplied & checked]

EXPERIMENTAL DESIGN
•	Original primary research within Scope of the journal. [Yes]
•	Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap. [Some limitations are there which have been mentioned in the reviewed version]
•	Rigorous investigation performed to a high technical & ethical standard. [Fairly good]
•	Methods described with sufficient detail & information to replicate. [Needs improvement]

VALIDITY OF THE FINDINGS
Impact and novelty not assessed. Meaningful replication encouraged where rationale & benefit to literature is clearly stated. [To be fine-tuned]
All underlying data have been provided; they are robust, statistically sound, & controlled. [these are ok]
Experimental design: Ok.
Some suggestion made in the M&M.
Validity of the findings: Fairly good, however, some suggestion have been made which needs to be done.
Results:
1.	Nicely written based on the hypothesis and raw data also provided.
Discussion:
Discussion part is ok, however, I didn't find the requirement of study of B in the growth performance of plantlets and significance of the B in the study. Please justify.

Suggestion to add: Future scope of research
Additional comments: •	The article is good and having relevance.
•	It needs revision in the direction as suggested.
•	Can be considered for publication after revision.


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

Input:
Title: ARBUSCULAR MYCORRHIZAL FUNGI ENHANCED THE GROWTH, PHOSPHORUS UPTAKE AND PHT EXPRESSION OF OLIVE (OLEA EUROPAEA L.) PLANTLETS
Review round: 1
Reviewer: 4

Output:
Basic reporting: The topic of this experimental article is well-chosen, timely and within the scope of PeerJ. However, Manuscrit needs revision before it is accepted for publication in PeerJ.
The information presented is indeed not complete and needs to be updated. More attention should be paid to the following points:
- The current version lacks a critical evaluation of the results achieved and a treatment of the already known literature, on the subject. A good discussion should not only report what has been written in the past but should critically evaluate the results presented, especially where there are conflicting reports.

The organization of the manuscript is somewhat cluttered and numbering would be helpful. The review should be more concise and the synthesis more elaborate.
I recommend that the Figs be cleaned up and redesigned - they often lack post-hoc tests and clear significance - e.g. Fig4. In addition, some are in colour and others in black and white. All in colour or convert to clear tabs as well.
References should be double-checked and formatting should be done strictly according to journal rules. Authors constantly alternate between citing titles in upper and lower case.
Furthermore, typos were consistently found in the ms and authors are asked to read their ms carefully before submitting them.
Below, I will address some of the other points I came across while reviewing the ms:
Experimental design: It is not clear when the experiments were carried out?
r. 113 - add country
HSD test is not clear from figs.
Figure captions must be self-supporting.
Validity of the findings: Wrong statistics, homogennous group etc. - needs clarification. Experimental design is missing. No clear schema of experiment, Pht1; Pht 11? ? AMF 6? Fert3?
Additional comments: My suggestion regarding this MS is to accept after revising with the following points:
1. Authors presented a good introduction, but they could explain more about molecular mechanisms of plant phosphorus tolerance and potential mitigation from phosphorus toxicity.
2. Introduction and discussion sections authors could involve also new aspects - hormonal regulation of the phosphorus tolerance (interaction with SA, K etc), including new references.
3. Add more view on photosynthetic parameters/discuss, which kind of parameters are more sensitive and why.
4. Add some recent references in the MS. It is important to discuss the plant regulation mechanisms.
Paper brings many new aspects and the novelty of the paper is OK, but I would like to invite authors to discuss also more eco-physiological aspects using new references: DOI: 10.1016/j.scitotenv.2021.147943; 10.1016/j.scienta.2020.109712; 10.3390/agriculture11030194; 10.1002/jsfa.9196; 10.3390/microorganisms10010051; 10.1016/j.agwat.2020.106635; 10.1016/j.apsoil.2020.103520

Final COMMENTS
- The manuscript is useful and innovative, it contains original data.
- This study presents the relevant matter in more depth than some of the other related publications, therefore, I recommend its publication after MODERATE  changes.


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: ARBUSCULAR MYCORRHIZAL FUNGI ENHANCED THE GROWTH, PHOSPHORUS UPTAKE AND PHT EXPRESSION OF OLIVE (OLEA EUROPAEA L.) PLANTLETS
Review round: 1
Reviewer: 5

Output:
Basic reporting: The introduction needs more logic. I suggest that you improve the description of the relationship between Olive and P for your study as this was not sufficient to support your story. The reason why you focused on the topic of your manuscript was not well expressed to us. For example, Prof. Gu Feng of CAU is an expert in the study area of AMF and P, his group has some findings on your topic, however, it is lacking in your introduction.
Experimental design: M&M: methods are important for any experiments, it is suggested that supplement some literature of yours or others about M&M, especially of Methods in L75-L115.
L76, the references of 'Koroneiki was selected since it is one of the most common olive cultivars in Yunnan province' should be supplemented.
L131, concentration should be content.
L153, what is the statistical software?
Validity of the findings: Results: actually, I can not find that the results support can the conclusion and the tile of Arbuscular mycorrhizal fungi enhanced phosphorus uptake, which should be given more information.
Additional comments: In figure 4, the same italic style of Pht1;11, Pht1;4, Pht1;2 and Pho3, is suggested to express in the abscissa heading of the figure.


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: ARBUSCULAR MYCORRHIZAL FUNGI ENHANCED THE GROWTH, PHOSPHORUS UPTAKE AND PHT EXPRESSION OF OLIVE (OLEA EUROPAEA L.) PLANTLETS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is written and structured well. I didn't find any problems with the English written. However, there is still some results mentioned Boron in the results (lines 226-235). Please check and revise. As the author response, it should be comprehensive described and conducted in another article.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: ARBUSCULAR MYCORRHIZAL FUNGI ENHANCED THE GROWTH, PHOSPHORUS UPTAKE AND PHT EXPRESSION OF OLIVE (OLEA EUROPAEA L.) PLANTLETS
Review round: 2
Reviewer: 2

Output:
Basic reporting: This is a revised version of a manuscript I previously reviewed. Compared with the first version I found it largely improved since authors integrated all the comments raised by all the five reviewers. However, I still found many missing information and paragraphs that can be largely improved. My further comments here below refers to the “track-change” *.docx version.
Experimental design: The manuscript should be corrected and integrated according to the following points:

- Please check all scientific names are in italics (see e.g. lines 178, 179).
- information on how protein/nucleotide sequences were aligned, manipulated to perform phylogenetic analysis remains still unknown. There are plenty of methods to do so which can results in profoundly different outputs. I would require authors to add these information to ensure reproducibility of results, including all the parameters set to truncate alignments, selects evolutionary models and perform phylogeny.
- I notice that in M&M section authors detail the composition of AMF communities/inocula used. This is especially relevant to understand differences among treatments. However, it is not clear if these data comes from already published studies or it they newly characterized these material. In this latter case they should provide methods used to do so, otherwise a bibliographic reference is required. If available it would be great if they will be able to provide a detailed AMF communities characterization as supplementary material.
- differently to what they stated in their rebuttal letter, the Boron story is still present in the manuscript (see lines 244 and 31). I do not have any issues against it since in my view also negative data (i.e. no effects on B uptake upon mycorrhizal inoculation) is data and can be relevant and useful to other researchers. However, as I already reported previously, it should be better contextualized to guide the reader who does not known its relevance in olive tree.
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: ASSOCIATION BETWEEN LUNG FUNCTION IMPAIRMENT WITH URINARY HEAVY METALS IN A COMMUNITY IN KLANG VALLEY, MALAYSIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, this is an interesting research. The authors aim to explore the prevalence of lung function impairment as well as its association with urinary heavy metal levels and other influencing factors among the community in Klang Valley, Malaysia. However, there are points should be improved.

The article included sufficient introduction and background to demonstrate how the work fits into the broader field of knowledge. Relevant prior literature appropriately referenced.
Experimental design: 1. The statistical used in this is not clear. Due to the aim of the study was to explore association between the lung function and urinary heavy metal levels. Thus, the multivariable regression analysis must be used. The others pollutant such as PM10, PM 2.5, O3, CO, NO etc. must be included as confounding factors in the analysis.
	2. The results were not correlated with the objective of the study, For example, Table 5, 6. Why you present the mean differences in lung function by education level and mean differences in lung function by gender, ethnicity, and employment. The rational for presenting these results must be provided. I recommend including these parameters (education level, gender, ethnicity, and employment) as confounding factors in the multivariable regression analysis.
	3.The inclusion and exclusion criteria is not clear. Why the subjects with history of chronic respiratory diseases including asthma and COPD not excluded from the study. Please clarify.
Validity of the findings: The results were not correlated with the objective of the study, For example, Table 5, 6. Why you present the mean differences in lung function by education level and mean differences in lung function by gender, ethnicity, and employment. The rational for presenting these results must be provided. I recommend including these parameters (education level, gender, ethnicity, and employment) as confounding factors in the multivariable regression analysis.
Additional comments: Abstract
	- mean age of 38.4, the SD of age should be provided.
	- “full vital capacity” should be changed to “forced vital capacity”
	- “force expiratory volume at 1 second” should be changed to “force expiratory volume in the first second”
	- “force expiratory flow between 25-75% FVC” should be changed to “force expiratory flow between 25-75% of FVC”
Introduction
	- The full term of Cd and Pb should be provided.
Methods
	- line 96, sample size calculation, please provide the parameter that account for sample size calculation
	- line 112, “CHESTRAPH” should be changed to “CHESTGRAPH”
	- line 117, please provide the reference of ATS guidelines.
Statistical Analysis
	- You mentioned that “Pearson's correlation test were used to analyze the effect of continuous variables on lung function”. I think the Pearson's correlation showed only the correlation but not effect.
 Definition
	- “Forced Expiratory Volume in one second” should be changed to “Forced Expiratory Volume in the first second”.
Results
	- line 186, please check the unit of PEF, L/hour is correct?
	- in the table 2,3, and 4. The decimal should be consistency.
Discussion
	- In overall, the discussion section was not consisted with the main findings of this study. Thus, I recommend editing the discussion section.


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

Input:
Title: ASSOCIATION BETWEEN LUNG FUNCTION IMPAIRMENT WITH URINARY HEAVY METALS IN A COMMUNITY IN KLANG VALLEY, MALAYSIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: •	Regarding phrases of “Urinary Cd (or Pb),” you used “Urinary” and “urinary” inconsistently in the manuscript. Please use the same expression. I would like to recommend that authors use “urinary.”
•	In the explanation of Table 10 (e.g., lines 256 and 259), multivariate analysis models were described as the “final model,” but it seems unclear. Please make those explanations clearer with more specific expressions.
Experimental design: •	The authors used urinary concentrations of cadmium and lead as biomarkers to assess exposure levels. However, urinary lead concentration may not be suitable for the assessment, especially in non-contaminated settings. This is a very important limitation in this study. Also, although urinary lead concentration did not show any correlations with lung function parameters, the null results might be because of the limitation (Lines 312–319).
•	Lines 78–80: if the authors wanted to study other sources of heavy metal exposure, all analyses should be performed separately by smoking status (smoker/non-smoker).
•	Did you correct urinary heavy metal concentrations for urine dilution effects? In usual, urine specific gravity or urinary creatinine concentration is used for the correction.
•	You apply log-transformation for urinary Cd concentration, while you use square route-transformation for urinary lead concentration. Please add explanation of the reasons why you used two different methods of transformation separately.
•	Line 250: Could you describe which variables you considered as confounders in the multivariate models.
•	Line 252: Could you more deeply explain the results of interactions?
Validity of the findings: •	Line 48–50 in the Abstract: the explanation seems unclear. Urinary cadmium concentration must be a biomarker of cadmium exposure level.
•	Lines 299–311: The authors only discuss the comparison of results of associations of cadmium exposure and lung function parameters between the present and previous studies. Please add the explanation of the possible biological mechanisms of the effects of Cd exposure on lung function.
Additional comments: This paper discusses the associations between cadmium and lead exposure and lung function impairment. The paper provides very interesting data but it still needs a considerable revision to be acceptable for the PeerJ. Please see my comments and suggestions below, which would contribute to improving your paper.


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

Input:
Title: ASSOCIATION BETWEEN LUNG FUNCTION IMPAIRMENT WITH URINARY HEAVY METALS IN A COMMUNITY IN KLANG VALLEY, MALAYSIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: The author has to explicitly mention how the study fills the gap in the introduction section
Validity of the findings: The author need to revise the conclusion of the article
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: ASSOCIATION BETWEEN LUNG FUNCTION IMPAIRMENT WITH URINARY HEAVY METALS IN A COMMUNITY IN KLANG VALLEY, MALAYSIA
Review round: 2
Reviewer: 1

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


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

Input:
Title: ASSOCIATION BETWEEN LUNG FUNCTION IMPAIRMENT WITH URINARY HEAVY METALS IN A COMMUNITY IN KLANG VALLEY, MALAYSIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: I think authors have improved the manuscript well based on the comments from reviewers.
Before acceptance, please confirm the manuscript overall to avoid any typos (e.g., "mitrchondrialm"=>"mitochondrial").
Experimental design: No additional comments.
Validity of the findings: no additional comments.
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: ASSOCIATION BETWEEN LUNG FUNCTION IMPAIRMENT WITH URINARY HEAVY METALS IN A COMMUNITY IN KLANG VALLEY, MALAYSIA
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: See attached PDF


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

Input:
Title: APPLICATION OF PLASMA METABOLOME FOR MONITORING THE EFFECT OF RIVAROXABAN IN PATIENTS WITH NONVALVULAR ATRIAL FIBRILLATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this paper, Zhao et.al. conduct plasma metabolomic analysis for monitoring of rivaroxaban therapy in patients with nonvalvular atrial fibrillation. They find a panel of metabolites that are changed in the control vs NVAF patients; and NVAF patients before and after rivaroxaban treatment. They additionally use this panel to validate remaining set of NVAF patients and controls.
Although they have a found many metabolites to be differentially regulated in both the comparisons, my main concern is they have not carried out experimental validation of the same in this study.

Additionally these are some of the minor concerns.
1. The ethical review document is in Chinese with no translated document provided.
2. The emails of the corresponding authors should be from an official account
3. The whole manuscript should be checked for grammatical errors and ease of understanding. Some examples are
Line 59: individuals who are overweight
Line 76 needs rewriting
In places wherein 'plasma metabolomics' is mentinaled should be changed to 'plasma metabolome'
Experimental design: These are my concerns in the experimental designs:

1. Line 114: Before collection, rivaroxaban was taken once orally for at least four days. This line is not understandable. It is not clear from the statement if the patients were given the drug before sample collection.

2. Line 183: It is not clear which groups are being compared, controls vs NVAF (before or after rivaroxaban)

3. Figure 2C and 3C: Authors should mark which metabolites belong to which pathway and whether the pathway metabolites cluster together

4. The authors should mention as to how were the patients divided into experimental and validation groups

5. Have the authors monitored rivaroxaban levels in the plasma

6. The figure legends description is incomplete. Please specify what is 1 and 2 in Fig 4A and C
Validity of the findings: These are my concerns for validations:

1. It would be helpful if the authors can carry out an analysis of the selected metabolites using the metadata from the patients and associate the two together.

2. Can the authors carry out validation experiments for some of the metabolites that were differentially regulated in the groups and exploring how they affect the patients.
Additional comments: No comment


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

Input:
Title: APPLICATION OF PLASMA METABOLOME FOR MONITORING THE EFFECT OF RIVAROXABAN IN PATIENTS WITH NONVALVULAR ATRIAL FIBRILLATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Fig 1 workflow has to be improved to make it clear to the readers
Experimental design: Most of my comments are minor and require revision
1.	Line 131: gradient is water or buffer; please clarify
2.	During the metabolomics studies, did the researchers use internal standard? If not, explain why?
Since the plasma has interfering compounds, they may strengthen or reduce the peak or AUC detection, leading to erroneous results.
3.	Please include the HPLC file in the supplement and peak extraction figs of all three significant metabolites.
Validity of the findings: The impact has to be included in the discussion section.

The authors have to include a small paragraph in the discussion, what are the Strengths (robustness of the method, analysis) and Limitations ( like the population group under study belongs to one particular country and other variables in the patient group, the obtained results may not be globalized) of this study.
Additional comments: This is a well-written and competently performed study by investigators that provides information about Plasma Metabolomics in patients with nonvalvular atrial fibrillation.


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: APPLICATION OF PLASMA METABOLOME FOR MONITORING THE EFFECT OF RIVAROXABAN IN PATIENTS WITH NONVALVULAR ATRIAL FIBRILLATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have considerably revised the manuscript and I am happy with the revisions.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: APPLICATION OF PLASMA METABOLOME FOR MONITORING THE EFFECT OF RIVAROXABAN IN PATIENTS WITH NONVALVULAR ATRIAL FIBRILLATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: No Comments
Experimental design: No Comments
Validity of the findings: No Comments
Additional comments: The authors tried to address most of the comments raised by the reviewers. The manuscript could be accepted in the current form for publication.


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

Input:
Title: EARLY MIOCENE REMAINS OF MELISSIODON FROM MOKRÁ-QUARRY (MORAVIA, CZECH REPUBLIC) SHED LIGHT ON THE EVOLUTIONARY HISTORY OF THE RARE CRICETID GENUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript titled “Early Miocene remains of Melissiodon from Mokrá-Quarry (Moravia, Czech Republic) shed light on the evolutionary history of the rare cricetid genus” by Bonilla-Salomón et al. presents a new population Melissiodon from the early Miocene of Mokrá-Quarry in Czech Republic. The manuscript includes a detailed description of the fossil teeth (focussing on quantifying the morphological variability) and is supported by precise measurements (including scatter diagrams of compared sizes) and good quality illustrations (SEM pictures). Following the description, the authors discuss the evolutionary trends and the possible ecological features of the genus during the early Miocene. Finally, the manuscript is overall well organised and the cited references are up to date.
The study is interesting and improves our knowledge of this, indeed, poorly known genus; it can be publish with minor revisions (see suggestions below):

-	Line 98: genera misspelled: Adelomyarion and Raricricetodon
-	Line 140: “referred” rather than “ascribed”
-	Line 151: italic missing for dominans
-	Line 161: “exceptional” not “exceptionally”
-	Line 170: “led” instead of “leaded”
-	Line 171: delete “The”
-	Line : 270: “is turned” or “turns”
-	Line 280: “Distinct” not “Disctinct”; additionally what do you mean by “No distinct mesostyle is discerned” ? Is the mesostyle absent or does something prevent from seeing it ?
-	From line 353 to 459: The discussion is long. I would suggest waiting until the end of the discussion to summarize the arguments and then naming the population M. aff. dominans. I would also explain at end of the discussion why not naming a new species and why not M. aff. schlosseri.
-	Line 367: who are “the authors”? previous authors or the authors of the present manuscript?
-	Line 369: Nevertheless
-	Line 387: those
-	Line 470: replace “is” by “being”
-	From line 571 to 598: all italics are missing for genera and species names
-	Line 581: not all sciurids are arboreal, please precise
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: EARLY MIOCENE REMAINS OF MELISSIODON FROM MOKRÁ-QUARRY (MORAVIA, CZECH REPUBLIC) SHED LIGHT ON THE EVOLUTIONARY HISTORY OF THE RARE CRICETID GENUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article describes new material from a very specialized murioidea species that is poorly known and therefore interesting for the scientific community.
The manuscript presents a classical structure. The language used throughout the article needs to be reviewed as it contains numerous mistakes that should be corrected and that, in some cases, make it difficult to understand the meaning of the sentences.
The framework of the study is, in general, well established, although a more detailed introduction of the evolutionary framework of the genus in periods prior to the Miocene might improve to understand better the main context. The references are adequate, although a citation, made in the text, is missing in the list of references. The figures are all relevant and of good quality and the data included are adequate.
Experimental design: The study has a classic design and addresses taxonomical questions in an adequate way.
The study, as indicated above, is relevant and presents new data from undescribed populations of Melissiodon, a rare genus from the Oligocene and Miocene of Eurasia. The description of the new material are very detailed but sometimes it is difficult to understand them since part of the terminology of the dental elements used in the study is not indicated in figure 2.
Validity of the findings: Regarding the structure of the discussion, it would be appreciated if, instead of comparing each of the morphological characters shown in Mokrá material with those seen on the other populations of the Miocene, a complete morphological comparison with the different Miocene populations were done. In this way it would be easier to understand what the differences between populations are and it would be easier to assess the similarity or difference between populations, being their taxonomic identity more evident.
Regarding the taxonomy of this work, I do not quite understand why the Mokrá material is described as M. aff. dominans, if throughout the text it is made explicit that it is clearly a form not related to this species. Strong resemblances to M. schlosseri are proposed, so if they think it could be this species it should be assigned to M. cf. schlosseri. If, on the contrary, they suspect that it may be a new species but do not want to establish it until a more in-depth study of that species is completed, they should name the population of Mokrá as M. aff. schlosseri. In any case, as I have indicated previously, the discussion should be restructured so that the similarities and differences with the type populations of these species are better established in order to better assess their affinities.
Additional comments: Figure 5 must be corrected since the ages of the localities and their correlations are erroneous in light of recent works (Van der Meulen et al 2011, Reichenbacher et al 2013, Prieto and Rummel 2016, etc...). If you do not agree with these correlations, you should discuss why and what this new proposal is based on.
In the attached file several corrections and extra comments have been proposed.