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: TROPHIC DEGRADATION PREDISPOSITIONS AND INTENSITY IN A HIGH-FLOW, SILTED RESERVOIR
Review round: 2
Reviewer: 2

Output:
Basic reporting: Review of Manuscript Number:  #44688
The revised version of the manuscript "Trophic degradation predispositions and intensity in a highflow, silted reservoir" (earlier "Predispositions and intensity of trophic degradation of the highly flow, silted reservoir") by L. Bartoszek et al. is improved. The overall coherence of the paper and clarity has visible increased. The simplifications used in the manuscript have been developed. References was supplemented. Methods used by Authors was detailed clarified in manuscript and supplements. On this basis, I recommend the manuscript for publication.
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: BEHAVIORAL, MORPHOLOGICAL, AND ECOLOGICAL TRAIT EVOLUTION IN TWO CLADES OF NEW WORLD SPARROWS (AIMOPHILA AND PEUCAEA, PASSERELLIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is written clearly, the tables and figures relevant and well done, and background information and context are sound.
Experimental design: The phylogenetic methods used are “industry standard” and thus ought not to draw any undue scrutiny.
Validity of the findings: To me, the results are what they are. In some instances the authors presented a priori expectations drawn from Wolf’s monograph. In other instances no a priori expectation was stated, yet results are of interest nonetheless. Some may quibble with coding of traits, but choices are transparent and, hence, the study repeatable.
Additional comments: Near the end of the Introduction, the authors remark that, with respect to traits and habitats, “If we find a strong association, then species in the same habitat type (i.e., same ecological group) may have been subjected to similar selective pressures from the local environment and shared trajectories of trait evolution within their lineage.” Another possibility is the process (not just pattern) of phylogenetic niche conservatism.

Results for the lability index confuse me. Probabilities shown in Table 1 are for D ≠ 1 and D ≠ 0, with certain values in boldface type that, if used per custom, are meant to draw attention to those considered to be statistically significant. But how can that be? As reported, ff the probability that D ≠ 1 is 0 for, say, nest position, then there is no chance D is not 1, which is another way to say D = 1. But that does not make sense, either, given that tabled values of D nearer to 1.0 have P higher (i.e., a bigger chance D ≠ 1) than values further from 1.0. And there is the matter of the plumage brightness result, which implies Schrödinger's D, in that there D = 1 and D = 0 at the same time. I take it, then, that the header really ought to be the probability D = 1 and D = 0? If that is true, then at least the plumage brightness result makes some sense: D definitely is neither 1 nor 0, which seems reasonable given the estimate is roughly midway between the two.

One broader question is whether the authors have any data on territoriality across the various species? They mention territoriality as a prospective predictor of duetting, which makes sense, but it could be particularly interesting to explore, perhaps in a future paper, whether tendency toward or type of duet is associated with pair bond duration and associated aspects of whether territories are defended by the male or by the pair.

Specific comments:

l. 104: It may reflect nothing more than personal preference, but when I see a genus or species name in quotes I assume it is meant ironically, to signal a dubious name. It would be clearer to say Aimophila sensu lato.

l. 240: breeds

l. 299: Pearson product-moment correlation?

l. 340: By definition, negative values are less than zero. Is something else meant here?

l. 470: None of the species in Aimophila (sensu lato) or Melozone or Pipilo is a long distance migrant, so this statement puzzles me.

l. 482: Not to nitpick, but the “Brown Towhee complex” is only part of Melozone and could be viewed as a single species pair (M. fusca and M. crissalis). As written, it is implied that the whole genus is in the complex.

ll. 493–494: Regarding “. . . reinforcing the notion that evolution does not always favor more elaborate plumage, but can also create drabness,” has there been a suggestion somewhere that elaborate plumage is a directed trait? (If so, holy crap.) I imagine virtually any clear-headed evolutionary biologist would respond “it depends”: strong sexual selection could favor elaborate plumage regardless of, say, increased parasitism risk, whereas strong natural selection for crypsis (which the authors mention later) in the face of strong predation pressure could favor drab plumage regardless or, say, female choice (or the choice could select to elaborate song instead, as perhaps hinted at by negative correlations reported herein).

Fig. 1: Why Ammodramus rather than Ammospiza for A. nelsoni and A. lecontei?


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: BEHAVIORAL, MORPHOLOGICAL, AND ECOLOGICAL TRAIT EVOLUTION IN TWO CLADES OF NEW WORLD SPARROWS (AIMOPHILA AND PEUCAEA, PASSERELLIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors of this manuscript analyzed the evolution of behavioral, morphological and ecological traits among the species of two clades within the New World Sparrows. They focused on the same species and traits considered by a paper from the 70s (Wolf 1977), which used such traits to infer phylogenetic relationships. Now, the authors use a molecular phylogeny to analyze evolution of the same traits in a more modern framework and adding some species.
Previous references and context provided are adequate. The general structure could be improved, as I found it hard to follow which species where considered for each analysis, and more importantly, inclusion/exclusions of species are not always properly justified.
The raw data shared could allow replication of the study, but how some of that data was produced is not completely clear (see comments below on how song was classified into "simple" or complex")
Experimental design: Some parts of the introduction still lead to the idea that one of the objectives of the paper is to use phenotypic/behavioral traits to infer phylogenetic relationships between the species, which is not what the authors did (see lines 47-49 and 128-129 in the Introduction).
I see the value in re-visiting classic studies, specially when they have produced a dataset still informative nowadays if re-analyze with modern statistical techniques (such as those that take into account phylogenetic relationships). However, some of the very interesting traits considered here, such as plumage pattern (characterized as present or absent) and song structure (divided into "simple" or "complex" in an obscure way), should be analyzed in a more rigorous and informative way than it was done 40 years ago. Adding a phylogeny is certainly important but not the only thing necessary to provide a “modern framework” when reconsidering a classic paper.
In the case of song, if authors decide to keep this oversimplified, binary classification for song structure, they should be more explicit about how they classified the songs of the species they had to analyze themselves. They say they were based on the methods by Wolf (1977), who states that  “For this report the types of vocalizations will be divided primarily on the bases of their supposed function and internal complexity” but I could not find much information about how he did so, and therefore it is not clear how the authors of the present manuscript replicated this method for the recordings they analyzed themselves.
Validity of the findings: Previous reviewer raised concern about the validity of the conclusions considering that most analysis where based on a small subset of species. I do not think a “small” dataset in terms of number of species is a problem in itself, as long as traits are measured in a less simplistic way than it is usual for studies considering entire families or even orders. This is not case in this manuscript for some traits such as plumage pattern and song structure, which I suggest re-considering to better support the conclusions drawn from results obtained.
For example, analyzing song evolution by classifying it into either “simple” or complex” is an oversimplification. There is an ongoing debate of what makes a song more “complex”, so in its current way the relationship found between habitat and song structure seems uninformative. This level of simplification questions the validity of the conclusions made base on the obtained results. For example, many of the species in this family have trilled songs, and the separation between notes in a trill can be strongly influenced by the level of vegetation coverage in the habitat.  Here, apparently all trilled songs have been classified as “simple”.
Additional comments: I can’t help thinking that this seems like a missed opportunity to build a more comprehensive phylogenetic hypothesis for the New World Sparrows. It is noticeable that none of the many previous studies on New World Sparrows systematics could provide the necessary framework for the 2 clades of interest and species in-between (due to differences in taxon and character sampling). A previous reviewer raised a similar point about including more species for the phenotypic data and I agree with the authors that “add more species” is usually an unfair a criticism that could potentially be done to any study. I also understand that the main objective of the authors was studying trait evolution and the molecular phylogeny was just a tool to achieve this, not an objective in itself. But building a more comprehensive phylogenetic hypothesis (after so many previous papers on the topic that still not providing the necessary framework for studying trait evolution in the entire family) certainly would have made the work stronger.


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: BEHAVIORAL, MORPHOLOGICAL, AND ECOLOGICAL TRAIT EVOLUTION IN TWO CLADES OF NEW WORLD SPARROWS (AIMOPHILA AND PEUCAEA, PASSERELLIDAE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article has been well structured after much editing, due to observations of other reviewers and from the editor of PeerJ, so one can see a better structure and clarity.
Experimental design: The experimental design is appropriate, both for phylogenetic reconstruction and for the analysis of the traits mapped and infered in the phylogeny in the two clades chosen.
Validity of the findings: I consider that a more robust phylogeny is offered here, after previous studies and a more complete sampling of taxa, so, this part represents an advance. The analyzes of the behavioral, ecological and morphological traits are satisfactory, being in the majority of the comparisons between the two clades, contrasting situations.

This paper is interesting for a more general audience, because it explores evolutionary issues that are relevant not only for this group of birds, but for others that have similar habitat, behavior or morphological characteristics.
Additional comments: I only noted a few small spelling mistakes in the following lines:

138 … traits remain an open question, however, we do not have a priori [without “and”]
464 Trait evolution within the Aimophila and Peucaea clades [a missing a in Peucaea]
607 Adolfo Navarro


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: BEHAVIORAL, MORPHOLOGICAL, AND ECOLOGICAL TRAIT EVOLUTION IN TWO CLADES OF NEW WORLD SPARROWS (AIMOPHILA AND PEUCAEA, PASSERELLIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Title: Behavioral, morphological, and ecological trait evolution in two clades of New World Sparrows (Aimophila and Peucaea, Passerellidae)
	Authors: Carla Cicero, Nicholas A. Mason, Lauryn Benedict, and James D. Rising

I appreciate the authors’ responses to the few comments I offered on my initial review of this manuscript. I have little new to add, and I continue to feel this paper is scientifically sound and will be of general interest. Its importance lies, I believe, in its basic goal to quantify and test ideas presented in Wolf’s seminal monograph from nearly a half-century ago. Wolf’s monograph is a showpiece in how to wed thinking in evolutionary ecology to phylogeny. It was ahead of its time.

I have but a single comment on this revision. The expanded explanation in the Methods of the D statistic is good, especially in light of Fritz and Purvis’ less than clear explication when they first presented the statistic. I nonetheless cannot wrap my head around results presented in Table 1. In the original paper, Fritz and Purvis presented similar results with associated p-values. In their Table 3 one can see the two null hypothesis tests (for D > 0 and D < 1; i.e., tests of Brownian and of random) and can make sense of associated p-values relative to the value of D itself. For instance, if D is small, then the first test may be “n.s.” whereas the second may be significant (i.e., the null hypothesis of D = 1 was rejected). Results in Table 4 are less readily interpreted, unless negative values of D are, in effect, spurious, by which I mean that values < 0 are treated statistically as = 0 when the null hypothesis test is conducted. I imagine this is a function of how the statistic was implemented in the R package, and as such perhaps no further explanation is needed. Even so, a glance at Table 4 can be jarring if p-values were interpreted by the reader as, say, strength of evidence (i.e., in a Bayesian way).

–Michael A. Patten
  Oklahoma Biological Survey
  University of Oklahoma


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: PROFILING TEAR PROTEOMES OF PATIENTS WITH UNILATERAL RELAPSED BEHCET’S DISEASE-ASSOCIATED UVEITIS USING DATA-INDEPENDENT ACQUISITION PROTEOMICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: Line 162, "Other settings included the default parameters. All identified proteins had a false
162 discovery rate (FDR) of ≤1%, calculated at the peptide level." This is extremely confusing. All proteins had FDR of 1% at peptide level. So this is actually peptide FDR not protein FDR, correct? Please make sure to filter your proteins through protein FDR in addition to the current peptide FDR as the false positive problem can amplify exponentially with subsequent biomarker discovery.

Line 221, " fold change ≥ 1.5 between the two groups and a P-value of paired T-test<0.05." Did the authors perform correction for multiple hypothesis testing? Please clarify. If not, please perform the correction and use corrected p-values.
Validity of the findings: Line 201, "Of the 3718 tear proteins identified in total, only 39 proteins were detected in all three studies (Fig.2). The great inter-study variability of tear protein profiles suggests that only a small proportion of tear proteins are constitutively expressed and necessary for physiological tear functions." Incorrect claim. Not detecting a protein by proteomics does not mean it is not expressed. In fact, at least 2779 proteins were identified by the DDA method when constructing the library. There is absolutely more proteins not detected by the authors. Please rephrase. Also please comment on the technologies used by the previous studies and what potential effects that might have on the overlaps.

Line 216, "These results suggest that tear proteomes may reflect the status of other tissues/organs and thus can be a window for disease biomarker discovery" Like the author stated previously, a lot proteins are constitutively expressed, such as fibronectin etc.. Overlap with other tissues totally makes sense as these proteins are just essential to cell survival. It does not mean they can reflect status of other tissues. In fact with only 514 proteins identified, I am assuming most of these proteins are just super abundant proteins that most of cells make.  Please rephrase.

Line 230, " The trends in each protein abundance changes were inconsistent among the 15 pairs of tears, but 13 (0.7%) proteins showed the same trend in more than half of the sample pairs (Table 3)." It does not make much sense by doing such comparison. This is why you do statistical test. 13 proteins having the same trend in more than half samples do not mean anything statistically. They could just go to the other extreme in the other half of the samples and would not be counted as significantly regulated proteins. If the authors really want to show proteins with same trend, please only focus on proteins that are statistically significant.
Additional comments: This is a well-designed research and it has values for people who are studying the disease. Therefore it should be published. However, there are some flaws that need to be addressed first.


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: PROFILING TEAR PROTEOMES OF PATIENTS WITH UNILATERAL RELAPSED BEHCET’S DISEASE-ASSOCIATED UVEITIS USING DATA-INDEPENDENT ACQUISITION PROTEOMICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors present a study using LC-MS/MS analysis to identify tear proteins differentially deregulated in the disease eye versus quiescent eye in patients with unilateral relapse of Becher`disease associate with uveitis.
Experimental design: The work has been methodologically well conducted, and high technical and methodological detail is provided in Materials & methods section.
Validity of the findings: Although the results obtained and data presented are robust and consistent, in general the discussion of results is quite short. Further discussion is recommended.
Besides there are some inconsistences between results and discussion & conclusions. For example:

In paragraph 3.1, lines 212-.217, the authors indicate “These results suggest that tear proteomes may reflect the status of other tissues/organs and thus can be a window for disease biomarker discovery”. The tears as a source of biomarkers is obvious. It has been repeatedly reported in previous publications and this statement is not a direct result of the work here presented.

In paragraph 3.2, the authors indicate that of 62 differentially expressed proteins in (A) vs (Q), authors presented in table 2 only those related with immunity. Although other ocular diseases was an exclusion criteria in the study, most of the proteins listed in table 2 have been previously reported in tear proteomics studies of dry eye (DE). I recommend authors to review the paper “TFOS DEWS II Tear Film Report. Willcox,M et al. The Ocular Surface 15 (2017) for further discussion. For example in this study Annexin A1 (ANXA1), is reduced in active BDU in comparison with quiescent, but increased in ADDE and SSDE; lysozyme is decreased in both, active BDU and DE, etc...

The number of interindividual variations presented in Table 3 is not informative. I suggest to remove this table, and maybe replace it by a table / figure presenting the physiological processes in which the remaining 54 (or total 62) are involved.

A key point in this kind of studies is the immunological validation of candidate biomarkers. I recommend to perform a validation/verification step to confirm protein expression in novel tear samples.

Conclusions paragraph presents a summary of work more than conclusions. Which are the most relevant candidate biomarkers?
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: PROFILING TEAR PROTEOMES OF PATIENTS WITH UNILATERAL RELAPSED BEHCET’S DISEASE-ASSOCIATED UVEITIS USING DATA-INDEPENDENT ACQUISITION PROTEOMICS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript entitled “Profiling of tears proteome of patients with unilateral relapsed Behcet’s disease-associated uveitis via data-independent acquisition proteomics” investigates novel marker proteins on tear fluids of Behcet’s disease-associated uveitis using DIA based proteomics approach. Anyi Liang et al., carried out the comparative proteomic analysis of 15 uveitis patients resulting identification of 1,797 proteins including 62 DEPs which are significantly altered in active eye compared to the opposite quiescent eye of the same patient. This result suggests potential valuable biomarkers of Behcet’s disease-associated uveitis in tear fluids. There are several concerns, both in the structure/organization of the manuscript and their findings.
Experimental design: 1) The authors compared identified proteins from tear fluids with two previous studies (in results 3.1). It is hard to understand how authors compared protein lists. This is a very critical part of your paper and needs to be explained. It makes no sense that only 1% of proteins are expressed simultaneously in tears. In the current version, the detailed comparison method are not described. It seems to be unfair comparison. For a fair comparison, download the raw data of the two papers and search using same database and parameters.
You also need an explanation for the sentence below. As you explain, the differences of expressed tear proteins among individuals should be very large, but the difference among the 15 patient's tears used in your study is not significant. This can be very confusing to readers and may mean that tear fluid cannot be used as a biomarker source. You need to have a clear explanation for this.
“The great inter-study variability of tear protein profiles suggests that only a small proportion of tear proteins are constitutively expressed and necessary for physiological tear functions.” (in line 202)

2) Following question 1, why did you compare the proteins identified in DIA, not the proteins identified in DDA?

3) In results 3.1, identified proteins in tear fluid were compared with tissue proteome data. However, there is no explanation for the comparison criteria in your manuscript. You should explain detailed criteria such as the kind of tissue you compared. Furthermore, interpretation of your data is too weak so you should describe more detailed conclusion of this data.

4) LFQ data such as DIA, DDA must be confirmed for a subset of proteins with antibody-based assays for additional validation. LFQ will miss out on isoform abundance levels in the total proteome.

5) The reproducibility of the DIA analysis was confirmed by the DDA data of the pooled peptides in triplicates. Although this can confirm the inter-day variation of LC-MS sensitivity, it is difficult to confirm that there is no variation in the analysis of tear fluid samples. To confirm the reproducibility of the DIA assay, show the CV (%) of retention time and intensity of the spiked iRT peptide.

6) What is the difference between general glaucoma and Behcet's disease-related glaucoma? There is no interpretation of the results related to Behcet disease. Please describe in the discussion part.

7) The manuscript is rather poorly organized and each section (method and results) does not serve its purpose. Especially, 3 and 4 in methods part have to be cleaned up and combined to prevent confusion for readers. In addition, the results part should be explained in more detail.
Validity of the findings: 1) The amount of tear fluid sample collected from the active eye and quiescent eye in each patient and the protein per unit volume are not given. Is there any significant variation in the opposite eye?

2) Why did the authors use HLB for desalting?
“Oasis HLB cartridges (Waters, USA). The resulting peptides were dried and desalted in a SpeedVac (Thermo Fisher Scientific, Waltham, MA).” (in line 112)  should be changed.

3) “The eluent was transferred to a reversed-phase analytical column (50 μm × 500 mm, 2 μm, C18, 100 Å). The eluted gradient was 5–30% buffer B (0.1% formic acid in 99.9% acetonitrile; flow rate of 0.6 μl/min) for 60 min.” (in line 134)
 Although the used column was 50 μm (inner meter) x 50 cm (Length), you used flow rate of 600nl/min. Perhaps the maximum pressure seems to be exceeded on easy nano-LC. You did not even explain the column temperature. Please describe it exactly.

4) Revise the wrong number with four-digit in this manuscript. 1 797 should be 1,797 in line 29. Please check all typing errors in word and number.

5) Despite your sample is tear fluid not cell or tissue samples, why did the author use the FASP method for protein digestion instead of in-solution digestion? Please explain.

6) Line 128 : EASY-nLC 1000 HPLC System  should be UHPLC, not HPLC
7) “The most intense ions per survey scan (top speed mode) were selected for collision-induced dissociation fragmentation, and the resulting fragments were analyzed in the Orbitrap with the resolution set to 60 000. The normalized collision energy for HCD-MS2 experiments was set to 145 32%, the AGC target was set at 5e4 and the maximum injection time was set to 30 ms.” (in line 142)
 You should write an exact collision type to prevent confusion. CID : Collision-induced dissociation, HCD : Higher energy collision dissociation.

8) Why did the authors use only FDR of ≤1%, calculated at the peptide level? The protein level of 1% FDR should be applied in DDA analysis.

9) Table 3 is ambiguous to understand. Change the representation or format of Table 3 to make it easier to understand.
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: PROFILING TEAR PROTEOMES OF PATIENTS WITH UNILATERAL RELAPSED BEHCET’S DISEASE-ASSOCIATED UVEITIS USING DATA-INDEPENDENT ACQUISITION PROTEOMICS
Review round: 2
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: The authors addressed my comments sufficiently.


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: PROFILING TEAR PROTEOMES OF PATIENTS WITH UNILATERAL RELAPSED BEHCET’S DISEASE-ASSOCIATED UVEITIS USING DATA-INDEPENDENT ACQUISITION PROTEOMICS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: As a general consideration, authors should have clearly indicated the page/line of changes performed in the text in response to the questions raised each reviewer.  It has not been easy for me to locate the changes indicated in the Response to editor Letter.

The authors have made great efforts to address the reviewers’ concerns and have answered properly most of the questions and considerations indicated for the original version of the manuscript.

However, the main question that remains is related to validation of candidate biomarkers. Regardless of the technical used for validation, immunoassays (gold standard) or Mass Spectrometry (MS) based-methods (as argued by authors), the point is that the candidate biomarkers are not validated in the present study, and I must be clearly indicated in the discussion section as a limitation of the study. Maybe also indicate that the validation will be further presented in another manuscript.

A minor consideration:
Next references indicated in the Response to editor Letter are not included in References of manuscript.
[1]	Willcox M, Argüeso P, Georgiev GA, Holopainen JM, Laurie GW, Millar TJ, Papas EB, Rolland JP, Schmidt TA, Stahl U, Suarez T, Subbaraman LN, Uçakhan OÖ, Jones L. TFOS DEWS II Tear Film Report. Ocul Surf. 2017. 15(3): 366-403.
[6]	Aebersold R, Burlingame AL, Bradshaw RA. Western blots versus selected reaction monitoring assays: time to turn the tables. Mol Cell Proteomics. 2013. 12(9): 2381-2.
[7]	Mermelekas G, Vlahou A, Zoidakis J. SRM/MRM targeted proteomics as a tool for biomarker validation and absolute quantification in human urine. Expert Rev Mol Diagn. 2015. 15(11): 1441-54.


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: MITOCHONDRIAL GENOME OF THE NONPHOTOSYNTHETIC MYCOHETEROTROPHIC PLANT HYPOPITYS MONOTROPA, ITS STRUCTURE, GENE EXPRESSION AND RNA EDITING
Review round: 1
Reviewer: 1

Output:
Basic reporting: Raw data: The SRA accession number of the data is already accessible because it was mentioned in a previous publication.
The accession numbers of the mitochondrial genome of H. monotropa are included in Table 1 but are not yet accessible. Given the number of errors in Genbank data bases on angiosperm mitochondrial genomes because reviewers are normally not able to access the deposited data in advance, I suggest that the data be accessible to reviewers at this stage.

Even though the English language was generally clear and unambiguous, some sentences should be improved to clearly understand your text.
Some examples where the phrasing could be improved include lines 76-78, 128-131, 289-291.
Some examples where I identified grammatical mistakes include lines 50-51, 54-55, 293-294, 310-311.

The introduction includes appropriate context and the literature is up to date and relevant. A few comments regarding the Introduction:

Lines 40-41: in Bjorkman 1960 this species was named as Monotropa hypopitys, if the name was changed or if it is a case of synonymy it should be noted.

Lines 67-69: the authors mentioned the mitogenomes of Rafflesiaceae and Cynomoriaceae as examples of massive HGT. However, the mtDNA of Cynomorium does not show massive HGT, while the holoparasite Lophophytum mirabile does and was not mentioned (Sanchez-Puerta et al 2017 New Phytol, 2019 Mol. Phylogenet. Evol). Also, citations of these studies are missing.

Line 74: ccmC, ccmFc and ccmFn are annotated as functional (but very divergent) genes in Viscum species

Lines 86-87 and lines 224-225: the gene nad4L is not a pseudogene in Vaccinium because the ORF is intact and it only needs RNA editing in the first codon to go from ACG to ATG. Otherwise, it is well-conserved and of the expected length.
Experimental design: The research questions are well defined, relevant and meaningful. It is stated how the research fills an identified knowledge gap. The methods are described with sufficient detail in general. A few comments on the experimental design:

Lines 92-94: Relevant DNA and RNA library details should be included here along with the citation of the fully described protocol. For example, important details include the library insert size, read length, paired-end reads or not, size of sequence data.

Line 95: Given that plant mitochondria normally contains about 50% of its sequence with no homology to any sequence in the NCBI databases, what did you do to make sure that mitochondrial contigs with no hits to Vaccinium mtDNA were included in the final mitochondrial assembly?

Lines 101-102: I recommend doing BLASTN searches against all mitochondrial genome plants, not only Vaccinium macrocarpon for the first screening of mitochondrial contigs. Otherwise, you might be missing some mitochondrial contigs.
Lines 109-111: What are the coverage’s intervals that you considered as nuclear, mitochondrial or chloroplast?
What was the coverage of nuclear contigs?

Lines 120-138: The term chromosome should be replaced by contigs because there is not enough information to support the presence of two autonomous chromosomes. I suggest using contigs or molecules to refer to the circular and linear assemblies of the MT.
Line 129-130: The explanation for the decrease in coverage at the ends of the linear contig is not clear. In fact, I believe that it is normal to see a decrease at the ends due to the alignment of reads, as stated in the legend of Figure S1B “The drops on the ends of the larger chromosome follow from its linearity.”
Lines 147-148: if the read depth is used to suspect unnoticed protein-coding genes, then the alignment of the RNAseq reads should be more stringent (not relaxed) because lots of noisy reads could result in an artefactual high coverage.

Lines 163-164: dN/dS refers to the number of nonsynonymous and synonymous changes per nonsynonymous and synonymous sites, respectively. Therefore, the calculation done here is not really similar to dN/dS.

Lines 168-169 the mitochondrial plastid DNA are commonly known as MTPTs, I don’t see the need to introduce a new term (MIPT) for the same feature.
Lines 169-171: why only the genes of Cammellia and not the intergenic regions, that is, the whole cpDNA was aligned to the H. monotropa mtDNA? It is common to see MTPTs derived from plastid intergenic regions.

169-170: I suppose you are talking about Camelia plastid genes. Please clarify in the text.

Lines 181-183 and 262-263: based on the alignments produced here, it would be simple and interesting to run phylogenetic analyses and test whether any of these MTPTs were foreign. It is very common in plant MT to find foreign MTPTs (see Gandini et al 2015 Sci. Rep.). In particular, it would be relevant in this case that a few plastid genes are not present in the plastid genome of H. monotropa. Therefore, if these genes are native, it would indicate an ancestral plastid-to-mitochondrion intracellular gene transfer. If foreign, there could be more recent cases of horizontal gene transfer.

Lines 184-189: It would be better to run BLAST searches against nr instead of restricting the search to Russula brevipes. BTW, is Russula brevipes a fungus normally associated to H. monotropa? Do they have a specific relation or is H. monotropa generalist? Please clarify the rationale behind your limited search criteria.
In addition, searching against Russula brevipes gives unsufficient information for the goal state here.

Lines 191-194 and 303-305: were editing sites removed from the alignments before running the
 phylogenetic analyses of individual genes? To validate the statement that all the topologies were identical, please include the individual gene phylogenies as supplementary.
How many RAxML bootstrap replicates were done?
Validity of the findings: The results are meaningful, although a little disorganized and unclear at times. The conclusions are generally limited to supporting results. See the comments on the Results and Discussion sections below:

Lines 200-201 belong to the Methods section.

Supplementary Figure S1C legend: the authors stat:e “Coverage of the chromosomes by reads of all three sequencing libraries: the paired-end library and both mate-pair libraries” What is the difference between a paired-end library and a mate-pair library? I believe there are no differences and the three libraries should be distinguished by the insert size or any other variable feature.

Lines 236-237. Given that H. monotropa has the cox1 intron and Vaccinium does not, it indicates that it has been acquired by HGT. It would be worth including a tree of the cox1 intron to find out its phylogenetic affiliation.

Lines 239-240: nad5 has 5 exons. Please search for exon 3 which is only 22bp long.

Lines 240-245: interchromosome trans-splicing has been previously reported in multichromosomal mitochondrial genomes such as Silene and Lophophytum. In addition, I don’t consider it a rare event, as much as the rarity of the presence of multichromosomal mtDNAs.

Lines 270-272: Plastid genes that are missing in the plastid genome could be lost from the plant or could have been transferred to the nuclear genome. I believe this possibility or the opposite ( if the absence of these missing genes in the nuclear genome is confirmed) should be included in the discussion.

Lines 281-287: How was the differential expression of the mitochondrial genes evaluated? If the relaxed alignment method described in Methods was used, it could be artefactual. To avoid misleading conclusions, the alignments of RNAseq data on each gene should be visualized to detect the mismapping of reads. In addition, a figure showing the read depth of the RNAseq data over the mitochondrial genome should be included.

Line 291 “most of them are C-U”. Given that only C-U editing is known in plant mitochondria, any case of a different editing type should be clearly stated, verified and reported. If not, the mismatch should be explained by alternative hypothesis.

Lines 297-298: stop codons cannot be edited because none of the 3 stop codons lack any cytosine.

Suppl Table 1: it should include each editing site, including the change, if different from C-U, whether it is synonymous or nonsynonymous and the editing extent of each site. The bulk or average numbers provided in this table are not very informative.
Did you identify edition of RNAs and the sequences that surrounding protein-coding regions? Are intergenic regions edited?

329-337: the fact that the authors assembled a linear molecule does not mean that it exists in vivo. It could be the result of missing sequences in the assembly that would allow the assembly of one or more circular molecules (see comment above on the assembly methods). Please comment on this or provide a possible explanation for the replication and maintenance of this linear molecule.
The title mentions the unusual structure; however, this should be revised because most angiosperm mtDNAs are lineal in Genbank and published as such due to the complexity in the assembly process and not necessarily due to a real replicating-structure in vivo.

360-365: The fact that this mycoheterotrophic plant and also Gastrodia do not show any fungal sequences despite their intimate relationship could be explained by the “mitochondrial compatibility fusion model” of plant HGT described in Rice et al 2013 Science”. Fungal mitochondria cannot fuse with plant mitochondria and this is the hypothetical mean for plant to plant HGT.

Line 368: what is the meaning of MHT plants? It should be defined the first time this term is used.

373-386: the discussion of the editing sites is incomplete. For example, much has been discussed on the editing of synonymous sites and it correlation with low editing extent or efficiency. Also, the statistical dEN/dES= 1.23 is not well explained and thus, it is difficult to interpret (see comments above).
Also, the phrase in the title “extensive RNA editing” is somewhat misleading because knowing that plant mitochondria hace extensive RNA editing, it seems that this plant shows particularly extensive RNA editing, which is not the case.

395-396: the presence of repeats at the end of the linear molecule is also compatible with assembly problems or artifacts. Assemblers have trouble when repeated sequences are present and tend to produce contigs and stop at repeats.

Figures and tables are relevant, high quality, and appropriate. However, there are a few suggestions for improvement:
Table 1 indicates the number of genes in each mt chromosomes (should be replaced by contigs or molecules) but it is really the “number of protein-coding genes” as it is not considering tRNAs or rRNAs. Please modify. 5SrRNA is missing in the Table.

Figure 1: There are mistakes in gene color assignment (e.g. ccm genes are in violet instead of being in dark-green). I don’t see the rRNA 5S. Repeats and MTPTs should be included. Trans-spliced lines are not needed.

Figure 3: the phylogenetic tree is rooted in Liriodendron but it should have been rooted in Cycas because it is the only non-angiosperm present.
Additional comments: The results, that is, the report and analysis of the mtDNA of H. monotropa is original, interesting and worthy of publication. However, the assembly, the analyses and text should be severely improved. The data should be analyzed in more detailed, making better and more in-depth interpretations to reach solid conclusions and make statements that are clearly supported by the data and the bibliography. I hope that the detailed comments I provide are helpful to the authors.


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: MITOCHONDRIAL GENOME OF THE NONPHOTOSYNTHETIC MYCOHETEROTROPHIC PLANT HYPOPITYS MONOTROPA, ITS STRUCTURE, GENE EXPRESSION AND RNA EDITING
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study focuses on the assembly of mitochondrial genome of H. monotropa and analyses regarding mitochondrial RNA editing and horizontal gene transfer.However, this article has major problems in their experimental design and the following analyses. If done correctly, this study would be a good resource for the field. However, this manuscript is not ready to be published before the following corrections have been made and raw data deposited.
Experimental design: Assembly:
a.The authors used the mitogenome of V.macrocarpon as the reference for selecting mito sequences from H. monotropa because the mito genome of the two species are ‘phylogenetically closest’. However, the authors did not provide any evidence supporting their ‘phylogenetically closest’ relationship. Although in their phylogenetic analysis later, they are in the same clade, but this may be simply due to the fact that V.macrocarpon was used as the reference for assembly. In addition, V.macrocarpon is a photosynthetic plant whereas H.monotropa is not. In sum, it seems reckless to use only one species as the reference genome for assembly. The authors should use more species as the reference.
b. It seems that the authors excluded the cp sequences for further analysis in the assembly of mito genome (L108). However, this is not reasonable due to ‘the presence of inserts from the cp genome into the mito genome. It has been reported in many species that cp and mito genome share similar sequences. Furthermore, it is unclear how the authors mapped reads to nuclear contigs (which organism was used as reference?). Thus, it is hard to evaluate if they did it correctly in removing nuclear sequences.

HGT:
a. The authors attempted to identify the sequence that transferred from the plastome to the mitogenome. However, they excluded the cp sequences for further analysis in their sequence assembly. Therefore, this analysis does not make any sense. Second, they used Camelia sinensis’s sequence as the reference genome in this step. In the results, they said they intended to look for ‘intracellular’ horizontal transfers. For intracellular transfers, the H.monotropa’s cp genome should be used as the reference. In addition, their claim that C.sinesis is closest to H.monotropa does not have a reference.
b. For examining possible horizontal gene transfers from fungi, the authors used sequences from R. brevipes. However, the assembled mito sequence was based on a blast to the V. macrocarpon genome. Given that V.macrocarpon is a photosynthetic plant, it is less likely to contain sequences from R.brevipes. Therefore, sequences from fungi would be eliminated in a prior step, thus they did not find any horizontal gene transfer from fungi is expected.

RNA editing:
a. RNA-seq of at least two bio reps needs to be performed for identification of RNA editing sites for statistical significance.
b. How the expression level (L284) is derived is a mystery.
c. ‘Most of them are C-U. Was there other types of editing? Please clarify.
d. “Level of editing’ should refer to the editing extents (up to 100%), not how many sites are edited in 100 bp.
e. Please provide a full list of all editing sites and corresponding editing extents.
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: MITOCHONDRIAL GENOME OF THE NONPHOTOSYNTHETIC MYCOHETEROTROPHIC PLANT HYPOPITYS MONOTROPA, ITS STRUCTURE, GENE EXPRESSION AND RNA EDITING
Review round: 1
Reviewer: 3

Output:
Basic reporting: Basic Reporting:  Although this is an interesting plant to study, the introduction section would benefit from some reorganization, revisions and better review of background literature.

For instance, the concluding paragraph of the Introduction on lines 83-89 are spent introducing the Vaccinium mitogenome.  This would be better placed before the section of the introduction that outlines the objectives of the project.

On lines47-49 the authors refer to most attention focused on plastid genomes of mycoheteroptrophs but only refer to two studies on Monotropa (2016 but still “in press” in the Lit. cited?) and a second study of their own.  This is really a biased and insufficient treatment of a vast literature.

In lines 52-57 the authors need to improve the writing because in the first sentence they state that plant mitogenomes are highly variable in size and gene content, yet in line 57 they state that gene content is “rather stable”.  Since this is a topic of most relevance to the rest of the paper, this paragraph probably needs to be improved.

On line 62 the authors refer to non-photosynthetic plants as being either parasitic and mycoheterotrophic.  There are also photosynthetic parasites and mycoheterotrophs so I am not sure why these are excluded from the literature review here.
Experimental design: Experimental design:  The methods used are largely clear and seem robust.  It is really excellent that the authors used RNAseq to compare to the genomic sequences obtained.

It would be beneficial for the authors to justify why this particular mitogenome is important to study.  There is already one mycoheterotroph studied.  Is it a particularly good representative to estimate HGT and study RNA editing?  If so, why?

Lines 120-138:  It might have been useful to use PCR to attempt to experimentally verify contig assemblies and chromosome structure.

Lines 160-166:  It is entirely unclear to me why the authors are studying RNA edit sites.  I personally find RNA editing to be fascinating but no justification was given for why it is investigated in this study.

Line 184-189:  It is not clear that a rigorous investigation of HGT can be achieved if sequences are only compared to Russula.  Couldn’t there be other donors?
Validity of the findings: Validity of the findings:  Largely, I think that the results are justified and nicely presented.  However there are shortcomings.

Lines 220-223: The authors claim that “Unlike parasitic plants, which show a certain degree of reduction, from limited to high….the gene content in H. monotropa is not reduced.”  It is not clear to me how the situation for H. monotropa is unlike parasitic plants since some parasitic plants show gene content that is not reduced as well.

Lines 225-228:  The authors indicate that pseudogenes exist within the H. monotropa mitogenome but we are never told what the basis for that determination is.  This should either be explained here or in the methods section.

Lines 229-261:  This section needs help.  First, the authors discuss group I introns, cis and trans-spliced introns in a non-organized way that is hard to follow and then the rest of the paragraph is focused on ribosomal RNA and tRNA sequences.  Probably this would benefit by being separated into different paragraphs.

Lines 231-238:  The authors probably need dig more carefully into the coxI intron literature, especially as it relates to horizontal gene transfer and also parasitic plants.
First, the Cusimano 2008 paper has some valuable contributions regarding some degree of vertical inheritance of the intron, especially in Araceae and other have shown a case of limited vertical inheritance in orchids but this paper is flawed in other ways as shown convincingly by Sanchez-Puerta et al., 2008.
Second, the authors should cite their source for “The cox1 intron is highly overrepresented in the parasitic plants…”.  Presumably that is Barkman et al., 2010?
Third, the authors only compare Vaccinium and H. monotropa for coxI intron presence; yet, numerous Ericales species have the intron, perhaps of most relevance is Pyrola since it is also in Ericaceae.
Fourth, the authors claim there is no evidence for HGT in the H. monotropa mitogenome but never show a phylogenetic analysis for this coxI intron.  A phylogenetic analysis is needed to show what other species the intron is closely related to.  If Pyrola, perhaps it was vertically inherited.  If not, it is likely present due to HGT and therefore the claim on line 356 needs to be revised.

Line 241-243:  The authors report a new ORF but suggest that it has no significant similarity to other sequences.  It would be better to show the evidence for this.  Trying lower stringency would be important.  It is also not clear if it has no similarity to ANY sequence in GenBank or if it just doesn’t match sequences in mitogenomes.

Lines 265-267:  If these plastid sequences are due to IGT then they should be shown by phylogenetic analysis to be closely related to the same genes from the H. monotropa plastid genome.  A phylogenetic analysis needs to be performed to show this.

Lines 284-287:  It is not clear from the results or methods what the expression levels are based upon.  FPKM?

Lines 288-299:  The authors should compare their reported edit sites to those that are known from other plants.  Usually these show conservation across species.  Although atp1 is listed as having zero edit sites, it has been reported to show editing at a few sites.

Lines 301-314:  The authors suggest that all single genes and combined genes give the same topology.  There are no data shown to substantiate this claim.  I would be fairly surprised if this were to be the case.  RNA edit sites  can have a huge impact on phylogenetic reconstruction as demonstrated by Bowe and dePamphilis back in 1996.  There is no indication that these were removed prior to tree estimation.

Lines 365-372:  The authors are over-interpreting their results:  the suggestion that little HGT in H. monotropa means that mycoheterotrophs (MHT) are somehow indicative fundamentally different from parasitic plants in this regard should probably be tempered.

Lines 374-386:  It would help readers to understand why RNA editing studies in mycoheterotrophs is important.  There is no hypothesis presented in this manuscript for why it should be different from any other plant.  Again, without comparing the edit sites to those known from other plants we cannot even assess whether they are unique in this species.
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: MITOCHONDRIAL GENOME OF THE NONPHOTOSYNTHETIC MYCOHETEROTROPHIC PLANT HYPOPITYS MONOTROPA, ITS STRUCTURE, GENE EXPRESSION AND RNA EDITING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is largely clear and provides a nice data point from a well-known plant species but would benefit from minor editing for grammar.

The authors do a reasonable job summarizing the literature.

However, if they want to be more complete with regard to RNA-editing in parasitic plants on lines 457-458, where they state:

"By now the data on RNA-editing in mitochondria for non-photosynthetic plants are scarce. C-to-U RNA editing in seven genes (atp1, atp4, atp6, cox2, nad1, rps4, and rps12) was found in R. cantleyi (Xi et al., 2013)."

they could include the study by Barkman et al., 2007 which showed RNA editing in the putatively horizontally transferred atp1.
Experimental design: No problems for the most part.  Individual gene trees are provided in SI.
Validity of the findings: The authors should be careful of over-interpreting results.

1.  Lines 390-394:  "All available evidence suggests that H. monotropa mitogenome has a linear fragment. While linear plasmids are found in mitochondrial genomes of several plants (e.g., in the work of Handa at el., (2002)), the linear fragment of the H. monotropa mitogenome lacks characteristic features of these plasmids: a terminal inverted repeat, the small size (not over 11 Kbp) and genes of RNA and DNA polymerases (reviewed in (Handa, 2008)).

While this may be true, the authors should be more cautious.  The study would have benefited from use of more "direct" methods of analysis for genome structure.  For instance, PCR or pulsed field gel electrophoresis (PFGE) is useful to document mt subgenomic circles, etc as has been done in other plants.

2.  Lines 443-446: "MHT plants are fundamentally different in terms of the interaction with their hosts and, potentially, in many other features that stem from this. Thus the knowledge on MHT plant biology should be obtained using MHT models, not by the simple transfer of knowledge from the plant parasites."

I simply do not know of any scientists in the world that are trying to advance the notion that parasitic plants and mycoheterotrophs are some how identical and therefore whatever we know from parasitic plants must be true for MHT.  Instead, I think the authors of this paper may be over-interpreting the results of their study in the opposite regard!  Do the results from this study make the rule for all MHT?

3. Lines 470-473:   "In many cases RNA editing has clear functional role (e.g. restoration of typical start codon in plastid genes rpl2 and psbL (Kudla et al., 1992). We found one such case in H. monotropa (nad4L). However, the dEN/dES value in mitochondrial transcripts is 1.23 (does not significantly differ from 1, p-value of 0.060) which indicates that in general edited sites are unlikely to have functional significance."

While I appreciate that the authors want to somehow quantify the proportion of edit events that either change or do not change the encoded amino acid for an ORF, to make the claim that since their metric is not different from 1 it means editing is not functionally important is erroneous.  Even if only 1 out of 100 editing events changed a codon for an amino acid that altered a protein from non-functional to functional, wouldn't that be "functionally significant"?   It may be true in this study that there are a lot of silent editing sites but to state that "in general edited sites are unlikely to have functional significance" is preposterous.
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: RESPONSE OF ARBUSCULAR MYCORRHIZAL FUNGAL COMMUNITY IN SOIL AND ROOTS TO GRAZING DIFFERS IN A WETLAND ON THE QINGHAI-TIBET PLATEAU
Review round: 1
Reviewer: 1

Output:
Basic reporting: The hypotheses are not well developed (not clearly answered either). Listing what previous studies have reported about AM fungal changes in other systems doesn’t automatically give the basis and the logic development of your own hypotheses. I’d like to see why you hypothesize moderate grazing would increase AM fungal spore density and GRSP content, but not to change the hyphal density, and why moderate grazing would change the community composition of AM fungi but not richness in roots and soil in the Zoige wetland.

The language needs to be improved substantially. Quite many sentences are difficult to understand, for example Lines 37-38, Line 43 (the use of “On the contrary”), Line3 234-235, etc.
Experimental design: According to the description of your sampling strategy, root and soil have to be considered as paired samples in statistical analysis. The paired characteristic of root and soil sample requires root versus soil to be nested in grazing treatment, i.e. nested ANOVAs and nested PerMANOVA should be used.
Validity of the findings: Should be more explicit about whether the two hypotheses are supported or not.
Additional comments: This study examined whether and how grazing would change arbuscular mycorrhizal (AM) fungal spore density, hyphal density and GRSP content in soil and fungal richness and community composition in both roots and soil in a Qinghai-Tibet Plateau wetland. The authors reported that moderate grazing increased AM fungal spore density and GRSP content, but had no effect on hyphal density and fungal OUT richness. AM fungal community composition differed between roots and soil, and was significantly influenced by grazing in soil but not in roots. Overall, the methodology and findings are sound and the manuscript is easy to follow.

My concerns and comments are:
1, The hypotheses are not well developed (not clearly answered either). Listing what previous studies have reported about AM fungal changes in other systems doesn’t automatically give the basis and the logic development of your own hypotheses. I’d like to see why you hypothesize moderate grazing would increase AM fungal spore density and GRSP content, but not to change the hyphal density, and why moderate grazing would change the community composition of AM fungi but not richness in roots and soil in the Zoige wetland.
2, The language needs to be improved substantially. Quite many sentences are difficult to understand, for example Lines 37-38, Line 43 (the use of “On the contrary”), Line3 234-235, etc.
3, According to the description of your sampling strategy, root and soil have to be considered as paired samples in statistical analysis. The paired characteristic of root and soil sample requires root versus soil to be nested in grazing treatment, i.e. nested ANOVAs and nested PerMANOVA should be used.

Line 94: Sources of MAT and MAP?
Lines 102-103: Provide the criteria for defining moderate vs light grazing intensity.
Lines 109-110: but the soil was passed through 1-mm sieve.
Line 114: This is sufficient for the description of soil moisture measurement.
Lines 194-196: Results of soil analyses need to be reported in the section Results.
Line 310: what does “seasonal nature” mean? be explicit.
Lines 261-279: The IDs of these OTUs are meaningless, so it seems quite redundant to list them. What the point to report meaningless OUT IDs here?


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: RESPONSE OF ARBUSCULAR MYCORRHIZAL FUNGAL COMMUNITY IN SOIL AND ROOTS TO GRAZING DIFFERS IN A WETLAND ON THE QINGHAI-TIBET PLATEAU
Review round: 1
Reviewer: 2

Output:
Basic reporting: The draft is well written and it is a descriptive study of the effect of grazing to the arbuscular mycorrhizal fungi communities and activities in wetlands. The background context is good. The structure is clear and figures are self-explanatory. However, the hypotheses are not well introduced, I miss a bit more background on the meaning of the variables measured in relation to the effect of grazing.
Experimental design: The aim fits the journal and the questions/aims are relevant, although its meaning is not clear in the introduction. the design is fine the statistical analyses should be improve moving from ANOVA to GLM in particular when the data is lof or square-root transformed (see comment to authors for details).
Validity of the findings: I suggest repeating the statistical analyses, as the model assumption might not have met in the current ones. I lacks a deeper interpretation in the context of the paper, so a little speculation might help in the discussion and a more ecological conclusion is needed (instead of a description of results).
Additional comments: The manuscript of dr. Zhong-Feng Li and coworkers deals with an interesting topic, the effect of moderate grazing to the soil properties in relation to arbuscular mycorrhizal fungi (AMF), in terms of soil glomalin content, AMF spore density, extraradical hyphal density and AMF richness and composition in soils and roots. The text is clear and easy to follow and the results seem logical, however, there is relevant information missing about the context how was the grazing areas (density of herbivores and grazing period), and the logic of choosing all of these measurements related to AMF and the timing of sampling. Besides the introduction and discussion goes smoothly but maybe too superficially through the meaning of the measurements and the results, see my main concerns below.
*.- I feel the introduction and discussion are superficial and do not introduce the logic and scientific meaning of measuring soil glomalin, soil spores density and extraradical hyphal density in wetlands. Thus i recommend the authors to expand the reasoning and expectation of measuring these variables in this particular habitat, possibly separating each measure a bit more. This would lead to having a more clear hypothesis and a more deep discussion about them.
*.- Some of the analytical methods sound not completely appropriate. For example the use of ANOVA for variables that are not normally distributed or are discrete (richness) or non-negative (densities), force the use of data transformation. These transformations have been (in the last 10 years) highly unrecommended as they modify the data characteristics sometimes in unpredictable ways. See for example:
O’Hara, R.B. and Kotze, D.J. (2010), Do not log‐transform count data. Methods in Ecology and Evolution, 1: 118-122. doi:10.1111/j.2041-210X.2010.00021.x
Warton, D.I. and Hui, F.K.C. (2011), The arcsine is asinine: the analysis of proportions in ecology. Ecology, 92: 3-10. doi:10.1890/10-0340.1
  Besides the transformation step, also the ANOVA does not help in analyses validation as residuals are not estimated. Thus I strongly recommend the authors to move to generalized linear models (using Poisson or negative-binomial for counts and gamma for non-negative continuous and see whether the model is valid) and provide validation with the residuals of the model vs the fitted values and check heteroscedasticity in the residuals. For guidance I recommend:
Mixed E_ects Models and Extensions in Ecology with R
Alain F. Zuur, Elena N. Ieno, Neil J. Walker, Anatoly A. Saveliev, Graham M. Smith
Springer-Verlag, New York, 2009. ISBN 978-0-387-87457-9. 574 pp.
*.- The conclusion just describes the results and not the consequences of the results in the context of our knowledge and specifically in wetlands.


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: RESPONSE OF ARBUSCULAR MYCORRHIZAL FUNGAL COMMUNITY IN SOIL AND ROOTS TO GRAZING DIFFERS IN A WETLAND ON THE QINGHAI-TIBET PLATEAU
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well written, with enough information about the area of research, however I think that the authors should reduce the number of references by choosing the relevant, since there ae more than ten pages of references. This manuscript is well estructured, with figures and raw data shared. The results are clear, however the hypotheses can not be proved and supported with the experimental design choseen by the authors (see next section).
Experimental design: The research question is well defined, however the experimental design do not allowed to prove the hypotheses, since there are no previous knowledge about the arbuscular mycorrhizal fungi communities in the grazing treatment, so it is hard to know if the communities detected in this research are the result of grazing treatment.
I would suggest to change the research question, in order to improve the manuscript findings, consequently the data analysis and statistics analysis  must change.
Validity of the findings: Since the experimental desing do not allowed to prove the hypotheses, the conclusion the authors reach are not well supported with the results obtained.
Additional comments: No additional comments


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

Input:
Title: RESPONSE OF ARBUSCULAR MYCORRHIZAL FUNGAL COMMUNITY IN SOIL AND ROOTS TO GRAZING DIFFERS IN A WETLAND ON THE QINGHAI-TIBET PLATEAU
Review round: 2
Reviewer: 1

Output:
Basic reporting: The reviewed manuscript is clear, well written, with appropriate references, manuscript structure and with an improved interpretation of the results.
Experimental design: The question section, at the end of the introduction, can be further improved by adding a line explaining the meaning of the selection of the response variables in the text. I think the authors integrate better this information in the discussion, but in the introduction, readers could not get the aim of the paper, and why all these variables are chosen and not others. I tried to be more explicit and suggest a line structure to add, see my comments in the general comments to the authors' section.
Validity of the findings: The findings of this paper are interesting, as this is the first study to my knowledge of grazing effect on AM fungal properties in wetlands. The data and analyses have been fixed and seem to be appropriate. Conclusions are logical and reasonable. Authors could speculate a bit more given all the information provided in the literature and with their own results, identifying it as the potential mechanisms ruling in the conditions given. This speculation is key to the development of further hypothesis in future studies.
Additional comments: The manuscript has improved significantly and most of my previous concerns have been successfully fixed.  However, I found still some issues I recommend the authors to deal with before further considerations:
The questions/hypotheses. All this information is more clear and integrated into the discussion. The authors should consider adding a line integrating the meaning of the measurements selected. It is still not completely clear in the introduction why authors have chosen these variables and only readers can understand it when reading the discussion. In other words, some bits of this information needs to go to the introduction. I suggest the authors an integrating line such as:
„Overall, the effects of intense grazing on mycorrhizal symbiosis may lead to a shortage on C from the plant to the fungi, what has been related to fungal response with a different production of glomalin, spores and extraradical fungal biomass (add refs): Besides grazing may cause changes in AM fungal richness and community composition in plant roots and surrounding soil (refs). However, these responses are environmentally dependent and habitat-sensitive and may be influenced by soil characteristics and dynamics (refs). This study represents the first one to analyze AM fungal responses to grazing in Wetland ecosystems.“
The abstract needs a more sound final line, perhaps rephrasing and bringing to the abstract the last new a nice line of the conclusion. Also, I recommend combining a line of the results in the abstract for better information flow. For example replace „AM fungal richness was significantly lower in roots than in soil, but not significantly influenced by grazing. AM fungal community composition was significantly different between roots and soil, and was significantly influenced by grazing in soil but not in roots. “ with something like „While AM fungal richness and community composition differed between roots and soils, grazing was found to influence only the community composition in soils“.  Then there is more room to interpret these results in the abstract.


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: RESPONSE OF ARBUSCULAR MYCORRHIZAL FUNGAL COMMUNITY IN SOIL AND ROOTS TO GRAZING DIFFERS IN A WETLAND ON THE QINGHAI-TIBET PLATEAU
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is well written and now the number of references is adequate, the results are clear, and the hypotheses has been  changed to questions by the authors, so it is better according to work they present.
Experimental design: The authors have now replaced the hypotheses by question to explore the effect of moderate grazing on AM fungal parameters and GRSP content, richness and community composition. The methods are sufficiently described.
Validity of the findings: Now the authors showed a well defined questions, and the conclusions are well structured and are supported by results.
Additional comments: The manuscript are now been improved, the question are answered appropriately, the methods and results are clear, the discussions and conclusion are referred to result reached.


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: TEMPERATURE ALTERS THE SHAPE OF PREDATOR–PREY CYCLES THROUGH EFFECTS ON UNDERLYING MECHANISMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: General:
This study addresses important issues in a useful and original way.  I am fully supportive of the questions asked, the approach used to answer them, and (mostly) the findings and their interpretation.  I start with my main (and really only) concern.  I then provide, following the Journal’s guidelines, some other comments, for which I have hopefully indicated possible changes.
Main concern

Paramecium is unlikely to ever be able to divide more than two time in a day (i.e. maximum growth rate is 1.4 d-1).  It’s just not built to do more.  Some ciliates, when faced with a nutritional shift down will undergo rapid divisions without an increase in size (e.g. see Lynn et al. 1987, Divider size and the cell cycle after prolonged starvation of Tetrahymena corlissi. Microb Ecol 13:115-127).  So, then let’s say Paramecium might have divided 3 times in a day, and growth rate would be 2 d-1, under transient conditions.  You, however, report apparent growth rates of up to 5 d-1 from your ODE (Fig. 3 a), and these differ substantially from the independently determined estimates (without predators) and from those presented in the literature (e.g. Salt et al 2017,which you cite, but also see Krenek et al. 2011, Thermal performance curves of Paramecium caudatum: a model selection approach. European Journal of Protistology. 2011 47:124-37.)

Where does that leave us?  I’m no expert on the method you used to obtain parameters from ODE, but it strikes me that if one of your parameters is way off, then others will be affected.  Is this so, and if so, how will it alter your other parameter estimates and conclusions?  Would it not be possible to constrain some of the parameter estimates (not just prey growth rate) using literature values and your own experimental values?  Would this not then provide more realistic estimates and trends?

Have I missed something here?  If I'm right, then the study might require a major overhaul,and if I'm wrong, then I only ask the Authors to:
1. explain why it's ok to have these high growth rates (I don't really accept the current explanation in the paper that Paramecium was growing at these rates)
2. think about the other changes/additions I've suggested.

Clear and unambiguous, professional English used throughout:
Generally, the manuscript was well written and clear.  There were a few very minor issues:
Line 118: is this “Paramecium” or “Paramecia”?
Line 132: missing a degree symbol before C.
Line 140: you might indicate why C and R are used (presumably Consumer and Resource?
Line 150: I don’t think this is actually “volume cleared of prey…”.  It is volume processed by a consumer per unit time x the efficiency of capturing a particular prey type (i.e. it is prey dependent).  Few people note this distinction, but it IS worth doing so, I feel.
Line 161: change “challenging” to “impossible”?  If it was only challenging, why did you not rise to it?
Line 162: “arose” not “arise”?
Line 172: delete “that”
Line 192 (and elsewhere): delete “in order”: “to” is sufficient.
Line 208: did it really “increase”; the data are not so strong here, I feel.

General: I also felt that the Discussion could be tightened up:
Lines 307-312; could this information be moved to the Methods?
Lines 324-331; I feel this could have been reduced and put into the Introduction by saying what this model can do, rather than ending with what it cannot do.  However, these are stylistic issues, that the Authors can ignore if they wish.  I just feel that these changes might make it a punchier paper.

Finally: I felt that in a few places in the Discussion, the obvious was stated.  These related to a unimodal or monotonic increase, rather than an exponential increase.  If the underlying experimental design is based on the full consumer thermal response (which is almost by definition unimodal (and right-skewed), then I think we might expect most underlying responses to be unimodal.  I think this could be explored in a more rigorous fashion, rather than just stating it (see literature below for some thoughts)

Literature references, sufficient field background/context provided:
I think that the use of the literature is sufficient, but I would like to give the Authors the opportunity to consider the studies below, as they may provide additional insights and useful background on temperature-food concentration interactions, model elaborations, and predator-prey dynamics of protists.  Clearly, the Authors need not cite any of these, and they may be aware of all of them, but I know I miss stuff, so here it is:

Montagnes DJS, Zhu X, Gu L, Sun Y, Wang J, Horner R, Yang Z (2019) False exclusion: a case to embed predator performance in classical population models. American Naturalist. 194:654-670
Wang Q, Lyu Z, Omar S, Cornell S, Yang Z, Montagnes DJ. (2018) Predicting temperature impacts on aquatic productivity: Questioning the metabolic theory of ecology's “canonical” activation energies. Limnology and Oceanography. 64: 1172-1185doi.org/10.1002/lno.11105
Yang Z, Zhang L, Zhu X, Wang JK, Montagnes DJS (2015) An evidence-based framework for predicting the impact of differing autotrophic-heterotroph thermal sensitivities on consumer-prey dynamics, ISME Journal 10:1767–1778 doi: 10.1038/ismej.2015.225
Yang Z, Lowe CD, Crowther W, Fenton A, Watts PC Montagnes DJS (2013) Strain-specific functional and numerical responses are required to evaluate impacts on predator-prey dynamics ISME Journal 7:405-416
Cooper JK, Li J, Montagnes DJS (2012) Intermediate fragmentation per se provides stable predator-prey metapopulation dynamics. Ecology Letters 15:856-863
Montagnes DJS, Roberts E, Lukeš, J, Lowe C (2012) The rise of model protozoa. Trends Microbiol. 20:184-91
Kimmance S, Atkinson D, Montagnes DJS (2006) Do temperature - food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina. Aquat Microb Ecol 42:63-73 66.
Atkinson D, Ciotti BJ, Montagnes DJS (2003) Protists decrease in size linear with temperature: ca. 2.5 ºC-1. Proc Royal Soc 270:2605-2611
Montagnes DJS, Kimmance SA, Atkinson D (2003) Using Q10: can growth rates increase linearly with temperature? Aquat Micob Ecol 32:307-313 71.
Weisse T, Stadler P, Lindström ES, Kimmance SA, Montagnes DJS (2002) Interactive effect of temperature and food concentration on growth rate: a test case using the small freshwater ciliate Urotricha farcta. Limnol Oceanogr 47:1447-1455 78.
Montagnes DJS, Weisse T (2000) Fluctuating temperatures affect growth and production rates of planktonic ciliates. Aquat Microb Ecol 21:97-102 83.
Weisse T, Montagnes DJS (1998) Effect of temperature on inter- and intraspecific isolates on Urotrichia (Prostomatida, Ciliophora). Aquat Microb Ecol 15:285-291

Professional article structure, figures, tables:
I wonder if Figs 1 and 2 could be combined?  Just a thought.


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: TEMPERATURE ALTERS THE SHAPE OF PREDATOR–PREY CYCLES THROUGH EFFECTS ON UNDERLYING MECHANISMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: It was my pleasure to read and review the article “Temperature alters the shape of predator-prey cycles through effects on all of underlying driving mechanisms”. It deals with a very timely subject – how temperature affects ecological dynamics through changes in underlying mechanisms that govern populations of interacting prey and predator. The authors used a novel method of direct parameter fitting to an ordinary differential equation model, a method that is very powerful and allows for a reliable characterization of mechanistic system behavior.

I do have however a number of comments that mostly focus on model formulation, model choice, and subsequently on parameter interpretation. Many of this issues are somewhat connected, but still I tried to list them one-by-one below.

1. My first concern is the model formulation. I think that it is rather complicated for the given system with its simple dynamical pattern (i.e. one abundance peak followed by extinction). An overly complex model could create problems with parameter redundancy, leading to a situation in which our confidence in fitted parameter values is low as many of them are strongly statistically correlated. The authors partly took care of that issue by performing a simple model selection procedure. However, this in turn created another issue – that estimates of each parameter, when compared across temperature gradient, are compared between different models, and each model formulation changes (in a statistical sense) the interpretation of parameters as they correlate.

First, using the parameter r_slope (=r/K) instead of just the carrying capacity K produces a very strong correlation between parameters r and K. Such situation should be avoided as this hinders us in identifying a single set of mechanistically interpretable parameter estimates. This could be, for instance, the reason why r and K behave very similarly across temperature (Fig. 3 A and B, full circles) – I believe they do that because there is no other choice when strongly correlated. Also, if r_slope is close or equal to 0, it is hard to know what it should mean for parameter r. Most likely, this means that there is not enough data in the time series in which the prey gets sufficiently abundant so that K can be identified. This could also be the real reason why estimates of r differ between predator-present and predator-absent treatments – when predator is present, the prey never gets very abundant therefore the model fitting “does not see” the carrying capacity, therefore the estimate of r gets high (Fig. 3 A).

Second, the formulation of prey-density-dependent predator mortality is somewhat awkward. Note that, in principle, such formulation means that there is an extra prey-density-dependent growth term for the predator “+(d*R)/(Cd+R)”. It feels not needed, as predator growth is already, through the functional response, a positive function of prey density. Then, if there is any mortality in the predator during experiments, it means that parameter d has to be positive. This in turn would force parameter Cd to be non-zero and identifiable as well, because otherwise the whole mortality term would cancel out. And this latter option is actually what seems to be happening here – the estimates of Cd are equal to 0 or very close to it (Fig. 3 H) so that the mortality cancels out – and this means there was virtually no Didinium mortality during the experiments! This might mean that the estimates of d are just statistical artifacts as anyway the whole term “-d+(d*R)/(Cd+R)” is equal to 0.

As the best way to deal with these issues, I would recommend to run a full model selection. That is, compare all possible model combinations, with the current Full Model on one end, and a simple Lotka-Voltera model (with type I functional response, no predator interference, and no predator mortality) on another end. In addition, it is important to select a model that gives the lowest AICc values at all temperatures combined. This will ensure that parameter estimates are compared within the same model across all temperatures, therefore within a single interpretation of the parameters. Only then your statement in the discussion section (lines 308-312) can be considered true, that is, the chosen model would truly contain only the essential parameters.

2. Another thing that got me worried is the fact that the authors used replicate averaging, and that they added extra interpolated time series points. I understand that this might be needed in order for the fitting procedure to work, however in principle such things should not be done. I wonder if this issue would remain if a simpler model is fitted. Then, as parameters would be more strictly defined and hopefully less correlated, averaging and extra data points might not be needed anymore. It is also possible that high uncertainty is an immanent property of protist predator-prey systems, and in that sense it is important to show it to the readers.
Either way, it can also mean that the authors are reaching the limit of what the ODE fitting method can achieve. Perhaps with that many time series data points, and for that many parameters to be estimated, it just does not work satisfactorily well. It might be a good idea to highlight this potential limitation in the discussion section.

3. I am slightly sceptic to the story about long-term warming effects. Obviously, acclimation, adaptation, selection etc. can be happening in the studied experimental system. However, with the current modeling approach, all such things are treated as implicit processes inside the black box of  population-level mechanisms, brought together by a small bunch of fixed, non-evolving parameters. In other words, model parameters are explicit functions of temperature only, so that other effects (long-term or not) can be only seen as the net effect. The authors mention that in the discussion, however when I read the abstract and the introduction, I almost expected to see an eco-evo model. It might be a good idea to tone down a bit this story early in the manuscript.

4. More of semantics and interpretation issue: If the predator-prey cycles shrink in state space with warming, it should mean that the cycle amplitude also has to shrink. In Fig. 1 B it seems like the Didinium cycle first goes up and then down with its amplitude, but it is not very clear, and Paramecium does not do that. I would probably not mention the unimodal response of the amplitude to warming, but just stick to the observation that it generally declines, and so the state space of the cycle shrinks. Additionally, as there is only one cycle peak, I am not sure how informative it is to talk about the cycle period defined as the distance between peaks.

5. What did Paramecium feed on during the experiments? Could the dynamics of this food item be in any way temperature-dependent?

6. The first paragraph in the discussion section (lines 235-249) can probably be completely removed from here, with some parts incorporated to the introduction instead.


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: TEMPERATURE ALTERS THE SHAPE OF PREDATOR–PREY CYCLES THROUGH EFFECTS ON UNDERLYING MECHANISMS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The Authors have fully addressed all my concerns except the first one, which I still would like them to consider a bit further in the manuscript.

If possible, I would like them to add the following to the Discussion:

1.	A clear recognition that for short periods, their estimates of Paramecium maximum growth rate are about 4 times higher than has ever been reported and much higher than would be expected for ciliates of this size.
2.	A range of explanations for these that could include:
i.                     Incomplete parameterisation of the model (i.e. something was missing such as shifts in cell size of either Paramecium or Didinium, which would affect conversion efficiency).
ii.                   That the high grow rate was real and could have been due to the presence of predators (a very novel and interesting speculation).
iii.                 The growth rates may have been short-term (transient) effects due to a burst of synchronized divisions.
iv.                 Anything else the Authors can think of.
3.	It might also be useful for the Authors to include a caveat about how estimates are model-dependent, and the cross-temperature pattern is the important part.
Experimental design: see abovesee
Validity of the findings: see above
Additional comments: The Authors have fully addressed all my concerns except the first one, which I still would like them to consider a bit further in the manuscript.

If possible, I would like them to add the following to the Discussion:

1.	A clear recognition that for short periods, their estimates of Paramecium maximum growth rate are about 4 times higher than has ever been reported and much higher than would be expected for ciliates of this size.
2.	A range of explanations for these that could include:
i.                     Incomplete parameterisation of the model (i.e. something was missing such as shifts in cell size of either Paramecium or Didinium, which would affect conversion efficiency).
ii.                   That the high grow rate was real and could have been due to the presence of predators (a very novel and interesting speculation).
iii.                 The growth rates may have been short-term (transient) effects due to a burst of synchronized divisions.
iv.                 Anything else the Authors can think of.
3.	It might also be useful for the Authors to include a caveat about how estimates are model-dependent, and the cross-temperature pattern is the important part.


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: TEMPERATURE ALTERS THE SHAPE OF PREDATOR–PREY CYCLES THROUGH EFFECTS ON UNDERLYING MECHANISMS
Review round: 2
Reviewer: 2

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: TEMPERATURE ALTERS THE SHAPE OF PREDATOR–PREY CYCLES THROUGH EFFECTS ON UNDERLYING MECHANISMS
Review round: 2
Reviewer: 3

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

In this paper the authors use combination of microcosm lab experiments and mechanistic ODE modelling to investigate how predator–prey cycles are affected by temperature. They find that both predators and prey cycle periods shorten with increasing temperatures, and that the amplitudes of cycles shifted with temperature as well.

## General ##

The questions around how predator–prey dynamics shift with temperature are interesting, and the approach taken by the authors to combine experiments with dynamical modelling is very refreshing. The authors'  findings are well put in the context of the literature, and their investigations are for the most part well explained. Barring some technical details I believe have to be further discussed by the authors, I believe this manuscript would make a good contribution to PeerJ.

Main comment 1: My main concern regarding this manuscript is around some of the assumptions going into the modelling framework, leading to some interrelated potential problems.

1a) The set of ODEs describing the predator–prey dynamics can, given positive initial conditions, never reach zero density in finite time, whereas virtually all the experiments conducted by the authors did. Furthermore, ODE models are usually approriate in situations with sufficiently high densities, but here the initial conditions for the predators are just 2 individuals. While I certainly don't think this sinks the authors' approach, I do think that these approximations should be mentioned and justified.

1b) Another problem relating to necessary positive solutions of the ODEs is the structure of variance. Typically in situations like these, one would assume that the solution can be captured by some deterministic, mechanistic part, plus some random variation around this mean, so that e.g., y(t) = μ(t) + N(0,σ(t)), where y is some quantity of interest, t is time, μ is the deterministic mean response, and N is some distribution of errors with SD σ (e.g. normal). Even if one does not make such an assumption explicitly, when performing optimization for some objective function, such an assumption is always implicit. For example, using a least-squares objective function a normal distribution of errors is implicitly assumed. This, however, presents a problem when modeling strictly non-negative quantities such as organism densities. Since negative densities cannot be observed, the distribution of errors cannot be normally distributed. For large densities this is not really a problem in practice, but for densities close to zero the estimates will be clearly biased. This problem can normally be circumventing by studying the positive quantity on a log scale, but since the authors have several data points that are identically zero, this approach cannot be taken. Once again, I do not believe that this problem merits changing the model, but it I do think it merits some discussion by the authors justifying their approach.

Major comment 2: My second major concern is that the description of how the fitting procedure was carried out is insufficient. I glanced at the PotterWheel Matlab toolbox the authors used to fit their model, and it appears to have many different options for fitting models. The authors should provide sufficient description of their process for replication to be possible. As I understand that such a description might be rather lengthy and technical I think it would be fine to put this information in an appendix or in the supplementary materials.

I also have a specific question related to the fitting process. As far as I could understand from the documentation, the PotterWheel toolbox optimizes the objective χ^2 = d1^2/σ1^2 + ... + dn^2/σn^2 for data points 1 through n, where d_i is the distance of the fit from the data point i. However, several of the standard errors (especially towards the end of many time series) are in fact zero, which would imply an infinite χ^2. Could the authors clarify this point?

## Minor and line-by-line comments ##

Line 90: The sentence starting on this line appears to be incomplete.

Line 147 (and others): When a subscript is not a mathematical symbol it should be set in upright font (here, 'slope').

Line 167: For the sake of replicability, version numbers for matlab and the toolbox should be reported.

Line 183: X^2 -> χ^2

Lines 205-207: I cannot understand this sentence. Consider rephrasing.

Line 217: Given the error bars, I am not sure the results necessarily supports the conclusion of unimodality for r and r_slope. Did the authors perform any statistical tests supporting this conclusion? If so, they are not reported.

Line 243: 'More complete depiction' -> 'A more complete depiction' or 'More complete depictions'

Line 283: '...time estimated as the inverse of the r' -> '...time estimated as 1/r'

Figure 2 figure text: What does "SE of the mean of each point mean" Is this referring to the sample SD among the replicates, or is it something else? Why is it interpolated between points?


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: TEMPERATURE ALTERS THE SHAPE OF PREDATOR–PREY CYCLES THROUGH EFFECTS ON UNDERLYING MECHANISMS
Review round: 2
Reviewer: 4

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This study uses a Didinium-Paramecium predator-prey system to infer how temperature affects its dynamics. While the experimental data shows a clear trend in shifting cycles with warming, the main focus is uncovering the mechanisms leading to this pattern. This is an import and timely issue since, beyond individual increase of biological rates, these processes are not fully understood yet. An ODE-based predator-prey model is fitted to the observed time-series, allowing the direct estimation of all parameters involved. The presentation of the methods and the results is clear and straightforward. I really appreciate the effort and the complexity of confronting a continuous-time dynamical model with experimental time-series data. I have one concern about the methodology and hope that the authors carefully take it under consideration.

My main concern with their approach is the identifiability of the model. It includes 8 parameters describing a variety of processes: growth + density dependence, feeding interaction, interference, mortality + density dependence. All of them have to be informed by just 1 cycle in the dynamics. I understand that a flexible model is required to fit the data well, but that does not automatically imply identifiability of all parameters. Due to (1) process or observation stochasticity, (2) parameter redundancy in this highly nonlinear context, or (3) a general mismatch between model and reality, parameter estimates could be biased. Even if (3) could be ruled out by theory, with (1) and (2) the question remains, how can we trust the estimates.

The authors cite a previous simulation study (DeLong et al 2018), that tested model identifiability by fitting data which was generated with known parametrisation. However, that study used a simpler model with 4 parameters r,a,e,d (or 5, if carrying capacity K was included in the validation, I could not figure that out at first glance). I recommend that the authors include such a simulation study with their full 8 parameter model. Thus, doubts about biased parameters would be either ruled out, or, found bias or uncertainty in estimates could be included in the interpretation of their results. I believe that the paper would greatly benefit from such a validation.

other comments:

l. 190: “but these populations did not achieve and identifiable carrying capacity at all temperatures”. Are the observed densities in the range of these with predator treatments? If it is already difficult to fit carrying capacity in control treatments, I guess this holds for the full model under predation, too. Again, this can be tested in the proposed simulation study.

l. 216: “all of the parameters governing these equations changed with temperature”. Please be careful when comparing parameter estimates without a statistical test for difference. E.g., the variation in parameter e in Fig. 3F could be just random. Especially when the quantified uncertainty is just 1 sigma (68.5% CI).

Fig. 4: I find this figure hard to interpret. Maybe separate plots for pred and prey densities would make it more readable (as in Fig 2).