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

Input:
Title: THE INTERPLAY BETWEEN COGNITIVE TASKS AND VISION FOR UPRIGHT POSTURE BALANCE IN ADOLESCENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.1. The authors used a clear English language.
1.2. Raw data was supplied.
1.3. Figures have a good quality and are relevant.
1.4. The authors presented the context related to balance maintenance and the role of cognition during balance tasks. The study was focused on adolescents individuals, however the context described above applied to this population and the gap existent in the literature are not well addressed and need to be improved.
Experimental design: 2.1. The research question is well defined and relevant.
2.2. Some aspects should be improved and clarified in the Methods section:
- Did the adolescents included in the study present a similar cognition condition?
- What was the range of age of the included subjects?
- Was the number of errors controlled during the cognitive tasks? If yes, they need to be reported.
- It is not clear in the Methods section which was the total duration of each trial for the balance tasks, nor if the participants needed to do more than one subtraction for the same trial. Please, describe the total duration of each trial, the number of trials performed for each task and if the random 3-digit number was other for each trial.
- Please, describe the Romberg Quotient in the Methods section and for which direction it was determined. Maybe the information presented in the 3.3 section could be replaced in the Methods section.
- Another aspect that can influence the individuals’ balance is the base of support. Due to this, it is necessary to clarify what did the authors mean with the term “feet together” for the balance tests and how this position was controlled for all subjects (e.g. was there a distance between the feet? Or was one foot touching the other?).
- Please, specify in the Methods section and in the Abstract that the RMS will be reported for both ML and AP directions.
Validity of the findings: 3.1. The main objective of the study was to investigate the effects of varying the difficulty of a cognitive task on the visual control of upright posture among adolescents. However, from the results presented, it is not clear if the difficult cognitive task in comparison to the simple cognitive task lead to alterations in the balance. It was only demonstrated that both were different from the no cognitive task, but the results for the comparison between them was not presented. Please, clarify this for all variables.
3.2. For the eyes closed tasks it is also necessary to demonstrate if there were differences between the tasks or if they were similar. From the figures presented, one can suppose that they were similar. Anyway, this need to be clearly stated for the readers.
Additional comments: 1. The results presented in the Abstract are a little bit confused. Please, make clear for the readers what were the results according to the aim of the study.
2. In the Abstract section the term “difficultly” seems not appropriate. Did you mean “difficulty”?
3. The statistical analysis presented in the Results subsection in the Abstract should be replaced to the Methods subsection in the Abstract.
4. From the discussion and the title of the manuscript, one could think that when the difficulty of the task increase (no cognitive task, simple cognitive task and difficult cognitive task), the balance alters. However, from the results presented (as mentioned in the above comments) it is not clear if the difficult task was different from the simple cognitive task.


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

Input:
Title: THE INTERPLAY BETWEEN COGNITIVE TASKS AND VISION FOR UPRIGHT POSTURE BALANCE IN ADOLESCENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors changed significantly the manuscript, and improved its quality, especially regarding the language review.
Experimental design: Line 134: include that guardians signed the consent, when testing subjects younger than 18.
Validity of the findings: Please check the general comments about the interpretation of the data.
Additional comments: First, I want to congratulate the authors for the revision. I have still some concern about the interpretation of your result and, consequently, about the title of your paper. The generalization of the results is also an issue to revise.

- Line: 282-284: “The main effect of cognitive factor was found to be significant in the analysis of RMS values for the ML and AP directions and for the MV of total body sway of the upright posture in adolescents.” Please, specify that the results were found for open eyes.
- Line: 300-304: “Donker et al. (2007) found that processing cognitive tasks while standing with closed eyes increases body sway. ….. Our findings are consistent with those of both of these studies”
Please, check this again, because your results did not show significant increases in balance parameters for dual task conditions when eyes were closed.

- Line 322-327: especially “will lead to a reduction in the visual processing channels”. I would not affirm this/ or I would write this carefully, because you did not directly measure the flow of signals.
Another point about this affirmation is that: If the instability happened during dual tasks is explained by the fact that a “reduction of visual processing channels” occurs, it raised the question:
Why did you not find an effect of dual tasks in a situation in which the visual processing channels are naturally reduced, such as your eyes closed condition? I mean, your data showed no differences between both cognitive task conditions for any parameters when participant´s eyes were closed (EC).
Your data indeed demonstrates that the dual task has an effect on postural control during eyes open. However, you cannot affirm that the reason of this is based on the reduction of visual processing channels. As you also mentioned in your paper, postural control is a complex process, involving the integration of afferent information provided by various systems. The integration and the priority of afferent information from one or more systems seems to change constantly according to necessity, thus recruiting more information and reweighting the importance of each available input. Therefore, I think that it can be a speculation to state that a reduction of visual information occurs during dual tasks conditions and that it is the explanation for the instability in general.  This is also because when the visual information was inhibited (eyes closed) in your study, you did not present effects of dual task in balance. Hence, I would suggest more caution in the interpretation/extrapolation of the data, since your affirmation is in the abstract, in the discussion, in the conclusion and also in the title of the manuscript.


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

Input:
Title: THE INTERPLAY BETWEEN COGNITIVE TASKS AND VISION FOR UPRIGHT POSTURE BALANCE IN ADOLESCENTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comments.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: I would like to thank the authors for the paper revision. However, I have still some minor suggestions.
- It is necessary to clarify and contextualize in the Introduction section why this project is important to be performed with adolescents individuals. By this way, the importance of your study will be strengthened for the readers.
- Please, include in the Methods section how many balance trials were performed for each of the six conditions.
- It would be more appropriated to replace the second paragraph of the “Experimental procedures” subsection (Methods section) close to the first paragraph of the “Experimental tasks” subsection (Methods section), once that one completes the other.


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

Input:
Title: THE INTERPLAY BETWEEN COGNITIVE TASKS AND VISION FOR UPRIGHT POSTURE BALANCE IN ADOLESCENTS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The requested changes have been submitted.
Experimental design: The requested changes have been made.
Validity of the findings: The authors changed the text in the discussion “Owing to limited information processing resources…”. However, the interpretation/extrapolation of the data is still present in the abstract, in the conclusion, and in the title of the paper. Since you cannot affirm that a reduction of visual control happened, you should change this affirmation in the abstract, in the conclusion, and also in the title of the manuscript.
Additional comments: The authors answered the questions and made most of the requested changes. However, there are still some essencial changes to be made.


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

Input:
Title: THE INTERPLAY BETWEEN COGNITIVE TASKS AND VISION FOR UPRIGHT POSTURE BALANCE IN ADOLESCENTS
Review round: 3
Reviewer: 2

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


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

Input:
Title: GENETIC DIVERSITY, FUNCTIONAL PROPERTIES AND EXPRESSION ANALYSIS OF NNSBE GENES INVOLVED IN STARCH SYNTHESIS OF LOTUS (NELUMBO NUCIFERA GAERTN.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Good in all indicated aspects
Experimental design: Acceptable
Validity of the findings: Findings are valid
Additional comments: Scientific design and writing is within the standard frame


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

Input:
Title: GENETIC DIVERSITY, FUNCTIONAL PROPERTIES AND EXPRESSION ANALYSIS OF NNSBE GENES INVOLVED IN STARCH SYNTHESIS OF LOTUS (NELUMBO NUCIFERA GAERTN.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors identified two isoforms of NnSBEs, analyzed their genetic polymorphisms and variation, determined their spatial and temporal expression, and investigated their effects on amylopectin structure. This study can provide some fundamental information for regulating the starch content and structure in lotus. I recommend accepting it for publication after minor revision.
1. It is better to concise the Section of Introduction.
2. In the Section of Conclusion, it is better to give your main conclusion and the significance of the study, and avoid citing references and discussing them.


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

Input:
Title: GENETIC DIVERSITY, FUNCTIONAL PROPERTIES AND EXPRESSION ANALYSIS OF NNSBE GENES INVOLVED IN STARCH SYNTHESIS OF LOTUS (NELUMBO NUCIFERA GAERTN.)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript reads well, contains useful information mainly for those interested in starch accumulation in scared lotus.
Experimental design: no comment
Validity of the findings: The connections between the results and conclusion should be enhanced.
Additional comments: Zhu et al. identified two SBE family genes, NnSBEI and NnSBEIII in sacred lotus (Nelumbo nucifera), analyzed the phylogenetic tree, enzyme activity and the expression profiles. The experiments were well designed. The study will provide some information for the research of starch in sacred lotus. However, the connections between the results and conclusion should be enhanced.

1.	In the “Abstract”, the authors concluded that “NnSBE genes received artificial selection during the process of cultivation and domestication in lotus seeds.” However, in the present study, only 6 SNPs and 45 lotus individuals were performed. More results and statistical analysis should be provided to validate the artificial selection of NnSBE in sacred lotus (Nelumbo nucifera), such as the SNPs from upstream and downstream of the NnSBE and more lotus individuals or whole genome resequencing data and so on.
2.	In the study, the authors identified the two isoforms, NnSBEI and NnSBEIII in sacred lotus to provide some information for the breeding lotus with high starch content. However, the expression profiles of NnSBE showed that the highest transcript levels of NnSBEI and NnSBEIII were observed in leaves (Line 220). As we know, rhizome and seeds of scared lotus play the key role in a daily diet (Line 37~38), how these two genes (NnSBEI and NnSBEIII) will be used for the edible starch content breeding of sacred lotus?
3.	In “Results” section, Line 182 “two SBE genes were obtained using RT-PCR and RACE technique”, but there is no RACE used in the “Material and methods”. And the correct amplification of cDNA fragments by electrophoresis should be provided.
4.	The authors descripted that “SBEI and SBEIII subunits showed homology in their α-amylase catalytic domain, but highly dissimilar sequences in N-terminus and C-terminus” (Line 288~290). However, no relative results shown that difference. The structures of NnSBEI and NnSBEIII, should be further analysis, especially the difference between them.
5.	The authors analyzed genetic polymorphism of NnSBE in 45 individuals of lotus using Sanger sequencing. How many clones were sequenced for each sample? Although the author used high fidelity KOD DNA polymerase, enough clones should be ensure the sequences correct. While, whole genome resequencing data for some of the lotus accessions have been reported (Huang et al., 2018). Using the whole genome data will be useful for the genetic variations and the evolution of the two isoforms of NnSBE.
6.	The different haplotypes of NnSBE in different types of scared lotus is useful for breeding (Line 213~214). More results and discussion can be included in the manuscript.
7.	Figures should be high quality and well labelled, some examples:

Figure 1  The labels “NnSBE1” and “NnSBE3” should be “NnSBEI ” and “NnSBEIII”.
Figure 6b  The horizontal ordinate had a unexpected “A”.
Figure 7b  The line chart represents “NnSBEI ” or “NnSBEIII”? should be clear. And the legend was not include (b).

8.  There are many grammatical errors and typos throughout the manuscript. The manuscript should be more carefully edited by native English speakers or professional language services to fix the language issue. Not only include the following examples:

Line 66~67  “while SBE B family showa higher affinity for amylose.”
Line 77   “To study SBEs further”
Line 86   “Nelumbo nucifera. cv. Taikong lotus 36” should be italics. There are many other similar cases.
Line 151~152  “Cheng et al.’s protocol of measuring peak period of SBE activity during the rice grain filling stage was used in this study” here the “in this study” is easy to misunderstand.
Line 261~263   “finding that rhizome lotus had the lowest genomic diversity and a closer relationship to wild lotus, whereas the genomes of lotus seed and flower were admixed”, here the “rhizome lotus” or “lotus seed and flower” is unambiguous? What they are mean?
Line 274  “The results of q-PCR showed that the two NnSBEs were expressed throughout plant tissues. ”, here the “q-PCR” should be “qRT-PCR”.


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

Input:
Title: GENETIC DIVERSITY, FUNCTIONAL PROPERTIES AND EXPRESSION ANALYSIS OF NNSBE GENES INVOLVED IN STARCH SYNTHESIS OF LOTUS (NELUMBO NUCIFERA GAERTN.)
Review round: 1
Reviewer: 4

Output:
Basic reporting: Lotus is now a very popular vegetable in Southeast Asia. Starch is one of the major nutrients in lotus seed and rhizome, the two major organs that are consumed as vegetables. To know the mechanism of stach biosynthesis in these tissue might be very helpful in lotus breeding and cultivation. This manuscript focusing on one of the critical enzymes SBE that contribute greatly for the biosynthesis of amylopectin. Valuable information could be obtained. However, before it could be accepted, the authors may have to concern about the following questions and comments.
1, The writing is not good in English. It should be polished by some native speakers.
2, Line 46: in the synthesis of amylose and amylopectin.
3, Line 79: “using genes and protein sequences of the two NnSBEs”. In this sentence, it might be better to use DNA rather than genes.
4, The Latin name of lotus “Nelumbo nucifera” should be italic. Furthermore, when the “SEB” was used to stand for gene, it should also be italic
5, There should be a space between the digital number and the unit, eg. 5 μl.
6, In table 1, it might be better to mark the forward and reverse primers. Table 2, amino acid (Aa) should be number of amino acids.
7, Figure legends for figures 2-7 should be revised, to provide more suitable and detail information.
Experimental design: It is ok.
Validity of the findings: Acceptable with some new information.
Additional comments: As stated above.


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

Input:
Title: GENETIC DIVERSITY, FUNCTIONAL PROPERTIES AND EXPRESSION ANALYSIS OF NNSBE GENES INVOLVED IN STARCH SYNTHESIS OF LOTUS (NELUMBO NUCIFERA GAERTN.)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have revised their manuscript according to my comments. I recommend accepting it.


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

Input:
Title: GENETIC DIVERSITY, FUNCTIONAL PROPERTIES AND EXPRESSION ANALYSIS OF NNSBE GENES INVOLVED IN STARCH SYNTHESIS OF LOTUS (NELUMBO NUCIFERA GAERTN.)
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The problem have been well revised.


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

Input:
Title: GENETIC DIVERSITY, FUNCTIONAL PROPERTIES AND EXPRESSION ANALYSIS OF NNSBE GENES INVOLVED IN STARCH SYNTHESIS OF LOTUS (NELUMBO NUCIFERA GAERTN.)
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have answerred nearly all my concerns, but the following two aspects:
1) They still did not spell the gene name in an italic way (eg. SEB A and SEB B, when are used to refer to gene, they should be italic).

2) the legends for figure 5, 6 and 7 should contain some information about statistics. eg. how many repeats, and what is the p-value?
Experimental design: Good
Validity of the findings: Valuable
Additional comments: See the basic report


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

Input:
Title: GENETIC DIVERSITY, FUNCTIONAL PROPERTIES AND EXPRESSION ANALYSIS OF NNSBE GENES INVOLVED IN STARCH SYNTHESIS OF LOTUS (NELUMBO NUCIFERA GAERTN.)
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: The authors have revised all my concerns.


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

Input:
Title: GENETIC DIVERSITY, FUNCTIONAL PROPERTIES AND EXPRESSION ANALYSIS OF NNSBE GENES INVOLVED IN STARCH SYNTHESIS OF LOTUS (NELUMBO NUCIFERA GAERTN.)
Review round: 3
Reviewer: 2

Output:
Basic reporting: It is now acceptable for me.
Experimental design: Good
Validity of the findings: ok
Additional comments: No additional comments


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

Input:
Title: GENETIC DIVERSITY, FUNCTIONAL PROPERTIES AND EXPRESSION ANALYSIS OF NNSBE GENES INVOLVED IN STARCH SYNTHESIS OF LOTUS (NELUMBO NUCIFERA GAERTN.)
Review round: 4
Reviewer: 1

Output:
Basic reporting: It is now ok for me.
Experimental design: ok
Validity of the findings: good
Additional comments: All my concerns have been addressed.


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: CLONING EXPRESSION AND IMMUNOGENICITY ANALYSIS OF INHIBIN GENE IN YE MULE ARIES SHEEP
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled "Cloning expression and immunogenicity analysis of Inhibin Gene in Ye Mule Aries sheep" by Zengwen Huang et al. characterized the inhibin-a subunit gene (INHa) of Ye Mule Aries sheep, which is found locally in Emin County, Tacheng District, Xinjiang, China. Homologues of INHa gene have also been investigated in many other mammals in correlation to the breeding performance.

I have found serious issues with this manuscript thus I do not recommend it for publication at its present form unless the manuscript is extensively revised.

The English of the manuscript is rather poor. Not only dozens (!) of spaces are missing and plenty of typos show up everywhere, but also many times it is not easy to find out the meaning of the sentences. Most of these mistakes can easily be corrected using a spell check of text editor software, but help of an academic expert with good language skills is inevitable.
Experimental design: Experimental design is satisfactory, however the manuscript is poorly written, due to this the major impact of this manuscript is hidden.
I found many formatting mistakes like space between two words, font style and font size, subheadings of the different section. Introduction, results, and discussion sections are poorly written.
Validity of the findings: Manuscript by Zengwen Huang et al. characterized the inhibin-a subunit gene (INHa) of Ye Mule Aries sheep, which is found locally in Emin County, Tacheng District, Xinjiang, China. The study is very specific to animal of particular region and has limited global commercial values.
Additional comments: Line 11-13: Long and incomplete sentence.
Line 13-15: It is advisable to write the background, result and discussion section mostly in past tense. Sentence is ambiguous, unclear and incomplete.

Line 17-18: “The coding region sequence (GenBank accession number: XM-004004955.1) was designed to synthesize primers, and the target sequence was obtained by reverse transcription amplification.”
The sentence is technically incorrect. Coding region sequence can’t be designed. It was the primer, which was designed using coding sequence as template.

Line 18: “transcription amplification” both is verb and can’t be used in tandem at the end of the sentence.

Line 19: “cleavage sites” it should be ‘restriction sites’.

Line 22: In the sentence “The plasmid (pEGFP-INHα) was used as antigen”. Is it the plasmid that has been used as an antigen??

Line 25: “was also 1109 bp long” what else was also 1109bp??
“Protein size was about 40 KD” it is not size, proteins are depicted in molecular weight. KD can be written as KDa.

Line 29: “This study successfully constructed eukaryotic expression plasmid PEGFP-INH α and verify the plasmid PEGFP-INHα immunogenicity, through the biological software analysis and PEGFP-INHα cells and immune experiment, we get the conclusion is highly conservative INHα gene sequences, INHα gene carrier by high expression in cells, INH alpha gene within the animal body on the secretion of FSH has important influence”
Change “PEGFP-INHα” to “pEGFP-INHα”.
The biological software can predict only, and not verify the immunogenicity.
The sentence is long, not clear and abruptly ended.

Line 38-42: Since 1932, McCullagh has found a water-containing factor in the aqueous extract of bovine testes, which has a feedback-inhibiting activity on FSH secretion, named "inhibin" (Inhibin INH or IB), but scientists A large number of studies have begun on statins, but it has not been possible to obtain breakthroughs in the study of statins until Robertson et al. and Ling et al. obtained isolated and purified statins from follicular fluids of bovine and porcine, respectively [1].
Authors are advised to avoid long sentences. This sentence is very long, poorly written and full of grammatical errors.

Line 50: change “Prove” to prove.
Line 68: In to in.
Line 67 to 70 is not clear:
It is from the middle of the 19th century that the majority of Kazakh herders In the unique geographical and climatic conditions of the locality, the mutton for obtaining a large amount of Ye Mule Aries sheep and small fat hip fat body was formed from Kazakh sheep through long-term breeding.
Line 70: “It should be paid attention to in the” use of ‘to’, ‘in’, ‘the’ is grammatically incorrect.

Line 74-77: “In order to implement the policy of protecting the valuable resources of local resources and improve the productivity of the Ye Mule Aries, this experiment used Xinjiang Ye Mule Aries as the research object, cloned its inhibin a subunit gene (INHa), and constructed the gene.”
What authors want to say in the above sentence? What is the meaning of “valuable resources of local resources” in line 74-75??
“Ye Mule Aries” should be italicized throughout the manuscript.

Line 94-95: The liquid nitrogen was quickly frozen, and then placed in -80℃ refrigerator for use.
What do you mean by “liquid nitrogen was quickly frozen??

Line 100: “The amplified product fragment size is expected to be approximately 1100 bp”. “size was”.

Line 105-107: “PCR amplification of the cDNA sequence obtained by reverse transcription using the designed primers, the amplification system is 25μL, of which 2xMix 12.5μL, the upstream and downstream primers (100μmol/L) are each 0.4μL, the cDNA is 2μL, and the deionized water is added.”
Sentence should be in past tense, poorly written.

Paragraph from Line 121-130 should be merged together.

Line 132: “The cloned bacteria that were initially identified as positive were DNA-sequenced by Shanghai”
Is it the bacteria were sequenced??

Line 135: Sequence alignment analysis of the INHα subunit gene.
What do you want to say in this sentence??

Rewrite ‘Materials and Methods’ again as I give up in reviewing the section. The whole section is full of grammatical errors and so it is difficult to understand the sentences and message. I would request the authors to take the help of native English speaker.

Line181: “visible (Figure 1), indicating that no degradation occurred in the extraction process of.”
Extraction process of what??? Sentence is incomplete.

Result and Discussion section are also full of errors and authors are advised to rewrite the whole section.

Figures and figure legends needs an extensive rearrangement. Protein domain architecture in figure 4 is not acceptable! This is the screenshot of NCBI CD search. Authors are required to prepare this figure in ‘line and graph’ format of the same in any image tool like Powerpoint or Illustrator.
All figures should be grouped in 2-3 figures.


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: CLONING EXPRESSION AND IMMUNOGENICITY ANALYSIS OF INHIBIN GENE IN YE MULE ARIES SHEEP
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript titled “Cloning expression and immunogenicity analysis of 1 Inhibin Gene in Ye Mule Aries sheep (#32706)” is publishable In Peer J, but I have few concerns mentioned below:
Overall the english language should be thoroughly checked throughout the manuscript to make it more understandable.
Abstract:
In abstract, line 6, modify this sentence
“which is used as a template and according to the Aries INHα gene “
Modify this sentence
“The plasmid (pEGFP-INHα) was used as antigen and immunoassay”
Please modify english language in abstract.
Background
I will suggest to reorganize the introduction part and fix grammatical errors.
Line 39: Elaborate FSH here.
Line 39-40: Modify the sentence below
“but scientists A large number of studies have begun on statins “
Line 50: replace Prove by prove
Line 57: replave Vaccine by vaccine.
Line 68: replace In by in
Line 92: How many Ye Mule Aries sheep ovarian tissues were collected? Please define.
Line 138: What ratio?
“recovered, mixed with a transfection reagent (Lipofectamine 2000) in a certain ratio”
Line 102: Remove the primer
Line 142: Correct this sentence
“divided into two, one for RT-PCR amplification, and the other for RT-PCR amplification”
Experimental design: The experimental design is satisfactory.
Validity of the findings: No Comments
Additional comments: No additional comments


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

Input:
Title: CLONING EXPRESSION AND IMMUNOGENICITY ANALYSIS OF INHIBIN GENE IN YE MULE ARIES SHEEP
Review round: 2
Reviewer: 1

Output:
Basic reporting: There are still serious issues with this revised manuscript, thus I do not recommend it for publication.

First, the study of the Ye Mule Aries sheep, which is a local breed and found in very specific geographical location, so this study doesn’t have broad significance.

Starting from the abstract, the tense of the sentences are not consistence throughout. Abstract should present the broad picture of the study and not the facsimile of the manuscript.

In Background section of the abstract, last sentence in line 17 is ending abruptly and like that there are many more grammatical and language error throughout the manuscript that I had given up.

In the method section of abstract, rather than protocol detail, author should present the broad methodology of the work. Sentences are still abrupt and transition between sentences and paragraph is still missing. It is very hard for reader to understand the manuscript.

Line 85: “INHα is also 1109bp long” author should specify what else is 1109 bp?? Why use “also” ?? This point was also raised in earlier review but not addressed by author! Like this, many more issues hadn't been addressed by the author.

Subheadings, as it was also pointed in earlier review, in methods and result section still didn’t improved. e.g. in result section the subheading “Total RNA isolation” doesn’t make sense?? What result you got from RNA isolation?? ‘RNA isolation’ is no longer mentioned as a separate subheading even in methods section and here discussed in result. All the subheadings in the result section are poorly constructed and no significant improvement is found in revised manuscript. Thus I do not recommend for publication.
Experimental design: Has been reviewed and mentioned earlier.
Validity of the findings: Not significant for broad audience.
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: CLONING EXPRESSION AND IMMUNOGENICITY ANALYSIS OF INHIBIN GENE IN YE MULE ARIES SHEEP
Review round: 3
Reviewer: 1

Output:
Basic reporting: Revised version of the manuscript entitled "Cloning expression and immunogenicity analysis of Inhibin Gene in Ye Mule Aries sheep" by Zengwen Huang et al. characterized the inhibin-a subunit gene (INHa) of Ye Mule Aries sheep.

Manuscript has significantly improved after revision but still the manuscript is full of typos,  which cannot be overlooked and hence needed a major revision.

Line 12: ‘Background: Ye mule aries is one of the most important sheep breeds in Xinjiang, China, which as it is’ The use of ‘which as it is’ is incorrect, either use “as it is” or “which is”.

Line 14-16: ‘In order to analyze the clonal expression and immunogenicity of the Ye mule aries sheep inhibin follistatin gene, we extracted total RNA from sheep ovarian tissue which was used as a template to generate an eukaryotic expression vector and study its immunogenicity.’

This sentence is not clear, and it should be the part of method section of abstract.
Probably author want to state in this sentence is that ‘isolated total RNA was used as to synthesize cDNA, then this cDNA was used as a template to PCR amplify the gene which was cloned in eukaryotic expression vector.’

Line 38: (INH or IB) [1].INH: space missing after sentence.

Line94: -80℃for reserve: space missing. Please check throughout the manuscript for this type of error.

Line 84: China,Lipofectamine: space missing
company,T4: Space missing.

Generally space is provided between numerals and unit for e.g. 100μmol/L should be 100 μmol/L .

Line115: Five positive clones werer and omly picked: Please check for typos.

Line 121:  “Construction and identification of pEGFP-INHα plasmid”: change identification to characterization

Line 174: Total RNA isolation: Subheading of results section is not appropriate. I had already mentioned in earlier comments. Please update accordingly.

Either bioinformatic analysis should be presented first or in the last.

Combine figure 3 to 6 into one figure.

Please combine all molecular biology work in one section and one figure.

Rewrite Acknowledgement in formal language.
Experimental design: Already provided in earlier comments.
Validity of the findings: Already provided in earlier comments.
Additional comments: Typos are present throughout the manuscript, so it is requested to re-analyze the manuscript again.


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: CLONING EXPRESSION AND IMMUNOGENICITY ANALYSIS OF INHIBIN GENE IN YE MULE ARIES SHEEP
Review round: 4
Reviewer: 1

Output:
Basic reporting: Manuscript has significantly improved, and authors did satisfactory job in improving the sentences and paragraph structure, however the result section is still heavily unorganized. From the middle of nowhere, bioinformatic analysis pops up in the molecular biology section. And again RT-PCR analysis is mentioned in between protein localization and western blot data. I already commented earlier and again mention below the steps to be taken to improve the structure of result section. Hence, author need to structure the result section again and a major revision is recommended for the manuscript.
Experimental design: NA
Validity of the findings: NA
Additional comments: It will be nice to present bioinformatic analysis at the start of result section as it paves the way for further gene/protein experimental analysis. As, I also suggested in my last remark, that please move all the bioinformatic analysis of INHα gene and protein sequence as Figure 1 and accordingly move the result subheading “178 Bioinformatics analysis of Ye Mule Aries sheep INHα” as the first subheading in result section and accordingly update the text and figure number in revised result section.

As I already suggested in my last comment, please combine all DNA and RNA molecular biology work in one section. It is no longer in trend that one RNA isolation gel image or PCR gel image etc. qualifies for one single figure. Please combine figure 1, 2, 4 and 6 as figure 2 and accordingly update the manuscript as suggested above for earlier section. Please check the text and labelling in figures as text in figure 2 is not readable.

It will be nice if the protein expression and analysis work is presented as third section and figure 5 and 7 can be combined together as single figure 3.
Please improve the image quality of western blot in figure 7. Resize the image in figure 5.

Text in table 1 also needed to be improved.

Change the last sentence of ‘Acknowledgement’ as it is not required to acknowledge editor and reviewer.


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: CLONING EXPRESSION AND IMMUNOGENICITY ANALYSIS OF INHIBIN GENE IN YE MULE ARIES SHEEP
Review round: 5
Reviewer: 1

Output:
Basic reporting: Line 168: Cloning of the Ye Mule Aries sheep INHα gene coding region and Construction of Recombinant
Plasmid
Author should use uppercase/lowercase uniformly throughout the headings. Here no need to capitalize “Construction of Recombinant Plasmid”.

Line 174: The obtained produces a specific target band’
What obtained???

Line 175-176: Subsequently, the was cloned into the PMD18-INHαvector and transformed into E. coli DH5a competent cells.
What ‘the’ was cloned??

Line 186:  The cloned expression vector pMD18-INHα was sequenced and detected by Shanghai Shenggong Bioengineering Co., Ltd.
What do you mean by ‘and detected by Shanghai Shenggong Bioengineering Co., Ltd.”???
The construct was sequenced by Shanghai Shenggong Bioengineering Co., Ltd. But why it is detected here???

Line 191: nucleotide was 13.6%
The sentence is plural. Use ‘were’ instead of ‘was’.
Experimental design: Alreadry stated.
Validity of the findings: Valid findings
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: CLONING EXPRESSION AND IMMUNOGENICITY ANALYSIS OF INHIBIN GENE IN YE MULE ARIES SHEEP
Review round: 6
Reviewer: 1

Output:
Basic reporting: Still language is not great but acceptable. Authors fail to make recommended correction again. It is not possible to accept the manuscript without correcting the errors as mentioned in the last report and again in this report.

Please go through these lines carefully again and make the necessary corrections.

 Line 174 The obtained produces a specific target band with the length of 1109 bp (Figure1B).

175Subsequently, the was connected to the PMD18-Tvector and transformed into E. coli DH5a competent cells
Experimental design: NA
Validity of the findings: NA
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: CLONING EXPRESSION AND IMMUNOGENICITY ANALYSIS OF INHIBIN GENE IN YE MULE ARIES SHEEP
Review round: 7
Reviewer: 1

Output:
Basic reporting: Manuscript has significantly improved after revision.
Experimental design: Experiments were well defined and adhered to the objective of the paper.
Validity of the findings: The findings validate the major questions raised in the manuscript.
Additional comments: Manuscript accepted.


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

Input:
Title: HUMAN DISTURBANCE CAUSED STRONGER INFLUENCES ON GLOBAL VEGETATION CHANGE THAN CLIMATE CHANGE
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, the paper is well written and organized. The English has few grammatical errors and I have provided comments on needed corrections directly in the manuscript.
The authors present argument as to why the study is important set up the reasoning behind the experimental design. While the authors do provide some references regarding changes brought about by “human activities”, I think the discussion could be improved by reviewing the Key Findings of the IPBES assessment on land degradation and restoration (IPBES (2018): The IPBES assessment report on and degradation and restoration. Montanarella, L., Scholes, R., and Brainich, A. (eds.). Secretariat of the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services, Bonn, Germany. 744 pages.
Experimental design: Lines 61-63. The goals are clearly stated and general approach is clearly described.

Lines 72-79, the authors should acknowledge that differences NDVI reflect differences in “greenness”, but as a coarse measure, this may mask changes to vegetation species composition

Lines 93-96. This sentence appears to be contradictory, or at the least confusing, in relation to the design premise. While it is clear that changes to “potential” vegetation could reflect influences of climate change, stating that “climatic vegetation types include effects of human influences” is confusing. I would suggest changing terminology from “climatic vegetation” to “existing vegetation”, as that is what I think is meant.

Line 118. Again, the authors state that climate and NDVI data were used to classify “climatic vegetation types”, yet, earlier they state that climatic vegetation reflects human influences.
Validity of the findings: Lines 147-152, especially lines 149-150. A shifting of ecotones seems more likely the result of climate change influencing areas that are, literally, borderline based on climate...thus, a small shift in climate is likely to result in a change in vegetation in ecotones rather than in central veg areas. Thus, contrary to the idea that changes due to climate change could not be detected in the short time frame, what the authors report is, in fact, observation of changes resulting from climate change. The authors statement of requiring a longer time period should be applied only to the central portion of the biome.

Lines 159-160. This sentence employs circular reasoning…changes in NDVI is how changes in vegetation were defined.

Lines 168-169. I am not convinced that the authors did, indeed, completely separate human activity effects from climate change; I think the authors presented data that is consistent with hypothesis that changes at borders of biomes is actually due to climate change.

Lines 196-201. Contrary to what the authors argue in these lines, I think they have in fact documented changes in vegetation due to climate change. While their general conclusion that human activities (primarily land use change) is a larger factor in vegetation changes is demonstrated, I think it would make for a more nuanced discussion if the authors embrace the fact that they have demonstrated vegetation changes due to climate change.
Additional comments: I find this a very interesting study and I would recommend it for publication, provided that the authors better acknowledge that they have documented changes due to climate change.


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: HUMAN DISTURBANCE CAUSED STRONGER INFLUENCES ON GLOBAL VEGETATION CHANGE THAN CLIMATE CHANGE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article aims to separate the influences of human activity and climate changes on the vegetation changes. This is an interesting topic. The writing is not clear enough. In addition, there are some grammatical and structural mistakes in the manuscript and I am not able to highlight/revise all of them. So, please revise the manuscript and improve it considerably in terms of grammatical and structural mistakes. There are important assumptions here about whether vegetation changes are affected by human activity and climate changes— and they should always be stated. There are few empirical studies and many do not confirm that assumption.
The literature is suitable for the purpose of the article.
Experimental design: 1) Within Scope of the PeerJ journal.
2) Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap.
3) Structure conforms to technical & ethical standard.
4) Although the experimentation is appropriate, the paper would gain in interest if it would provide a more in-depth discussion about the selected topic.
Validity of the findings: 1) Data is robust, statistically sound, & controlled.
2) Conclusions are well stated, linked to original research question & limited to supporting results.
3) Speculation is welcome.
Additional comments: 1.	The Introduction & Data and methods need to be re-organized and written in a clear way. These severely limits any reader to understand what (and how) this study was made.
2.	A sketch map of this study should be added in the revision.
3.	The authors should thoroughly address the novelty and importance because so many works have focused on this point.
4.	The abstract should be redone, it is hard to follow at present.
5.	The section of introduction did not well review and cite existed works.
6.	In fact, the author should examine the applicability and generality of normalized difference vegetation index (NDVI). Sometimes, the specific thresholds are necessary. Some other vegetation indices (EVI, DVI…) may be useful.
7.	The authors conducted the resampling processing to all input ecogeographical factors (0.5°×0.5°). Why the finer resolution (0.0833°×0.0833°) was not considered in this study? In addition, the author should illustrate why the window size of 6×6 (which method) was used.
8.	It is true that the vegetation changes are influences by both human activities and climate changes in the real world. The author used the specific method which mentioned in your study is not clear enough. It is noted that this section is critical for this study. I think it should be described in detail.
9.	Why CRU TS 3.40 dataset were applied in your study specifically? There are some other datasets available.
10.	The matrix X should be listed in order.
11.	The results and discussions are weak and unsatisfied, the author should make the best effort to modify these sections. I want to see a considerable improvement in this regard.
12.	The section of conclusion is missing, please address this problem.
13.	The conclusion I not well stated and is written more as an abstract. A clear conclusion needs to be written.
14.	The section of validation is not well described.
15.	All tables/figures: Caption needs to completely explain the table, including what the abbreviations mean. Work on text clarification and formatting, improve figures, list of the abbreviations would be useful in this case to easily understand the text.


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: HUMAN DISTURBANCE CAUSED STRONGER INFLUENCES ON GLOBAL VEGETATION CHANGE THAN CLIMATE CHANGE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Line 32, it is a little odd to see "human activity" and "climate change" as two separate things because we usually assume the changing climate is mainly due to human activities. Probably authors can use "perturbation", "land use change" or "human disturbance" instead of a general "human activity".

Line 33, maybe use "construction" instead of "building", and in line 178 too

Line 34, the two parts of this sentences seem to be the same. "modified" meaning "changing" right?

Figure 2. Did authors see a movement of boundary lines? instead of reporting changes.

Figure 3. Did not authors separate the study period to two? how did authors calculate the impacts from 1982-2013? was that mentioned in the Method section?

Figure 4, it might be too much, but maybe can replace the blank background map of China to contour background or label some big cities to show near which location has the large changes.
Experimental design: Line 111, so this equation does not have parameters that account for interannual or long-term variation changes but only seasonal changes? and the monthly mean temperature or precipitation is the average of 15 years period?

Line 128-129, if an interval of at least 15 years is needed, why having more years that less than 15 years for those two periods? why not use 1982-1990 vs. 2006-2013 so those two periods have at least 15 years interval?
Validity of the findings: Line 144, how much is the largest change? I compared the Figure 1a and 1b, very similar. maybe because of the large scale. The 1c graph of difference is very helpful but I suggested to add the percent change scale on it or even color it to show difference levels of changes.

Line 186, the starting data of this study is in 1982, so this sentence "Transformations in the distribution of vegetation accelerated after the industrial revolution..." can not be written or proved in this study. The Industrial Revolution took place from the 18th to 19th centuries.

Line 208, yes but no. human activity already disturb the environments for the growth of potential vegetation type. If we return the farmland to forest, it might be a different forest type comparing with the potential vegetation type. For example, under the warmed environment already, we can only return abandoned lands to deciduous forest but not conifers.

Line 212, how to define this "low intensity of human interference"?

Adding a Conclusion section would be better, and is common for articles published in PeerJ.
Additional comments: This study reports and compares the changes in vegetation types due to changing climate only vs. land use change only. It concludes that land cover change causes more severer damage to global vegetation change than climate change. I find this paper has important values to the society.


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: HUMAN DISTURBANCE CAUSED STRONGER INFLUENCES ON GLOBAL VEGETATION CHANGE THAN CLIMATE CHANGE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: With clarification of some of the terminology, as I suggested in my original review, I feel that the experimental design appears stronger than in original manuscript.
Validity of the findings: Again, I originally questioned the validity of some of the conclusions, but editing of the manuscript and clarification of methods, I find the conclusions much more convincing.
Additional comments: The authors did an excellent job of responding to my original comments. I have just a few, non-critical comments that I have edited into the attached pdf.


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

Input:
Title: HUMAN DISTURBANCE CAUSED STRONGER INFLUENCES ON GLOBAL VEGETATION CHANGE THAN CLIMATE CHANGE
Review round: 2
Reviewer: 2

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: None
Additional comments: I find this paper can be accepted as it is.


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

Input:
Title: THE SUGARCANE MITOCHONDRIAL GENOME: ASSEMBLY, PHYLOGENETICS AND TRANSCRIPTOMICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper describes the mitochondrial genome assembly, phylogenetics and transcriptomics analysis of a variety of sugarcane cultivars.
The work adds to an understanding of the plant mitochondrial genomics, and although the most of the results are interesting, including the description of the mitochondrial CMS factor and the three putative genomic restoration factors, I have to recommend significant revision of this paper for several reasons I will briefly explain.
One reason is that the manuscript in an intelligible fashion. It seems that the authors submitted a draft version that was not rigorously corrected. Many sentences and paragraphs throughout the manuscript are poorly structured so that they become hard/impossible to understand.
The introduction does not inform the reader adequately about the plants and sugarcane cultivars used, and some of the results are very poorly introduced, insufficiently described and often incomplete, sometimes shuffled with the discussion section
Experimental design: please see general comments for the author
Validity of the findings: please see general comments for the author
Additional comments: Major issues:

Different sugarcane cultivar and other plant genera were used in this work. The abstract and background section was difficult to follow due to the number of sugarcane cultivars and samples used, which reflect in challenging to track the results obtained. I strongly suggest the authors clarify the abstract and introduction sections to better describe and summarize the sugarcane cultivars and other plant species explored in this work. A table in the material and methods section showing this information will be precious.
For instance in the abstract L24: “We attempted to assemble a reference sugarcane mitochondrion from cultivar SP80-3280...”
L33: “...the mitogenomes of three additional cultivars (LCP85-384, RB72343, and IJ76-514) were assembled...”
Introduction from L149 to L154:
The three sugarcane cultivars were formally presented in the manuscript (LCP85-384, RB72343, and IJ76-514). However, the authors also state that Saccharum spontaneum SES234B and Miscanthus sinensis cv Andante were also assembled.
In the results section (L393, L412, L442, L450) its also presented four additional cultivar (SP70-1143, SES234B, Btx623, Khon Kaen3 ).
Several other sugarcane cultivars were also used for chloroplast assembly and analyses (see L504 to L508).
These are some examples that make the manuscript difficult to follow.

M&M and Result Sections
Transposable Element Analyses
Since TEs are very diverse among even closely related species, I don’t think that using the Poaceae database alone as a query for potential TEs insertion integration identification is the proper approach for this work. Nowadays, several publications contains well known and annotated sugarcane TEs (e.g., doi: 10.1186/1471-2164-13-137 (Additional file 6) , doi: 10.1186/1471-2164-15-540 (Additional file 3), and doi:10.1038/s41467-018-05051-5), and I strongly suggest the authors refine this analysis adding the data provided from these previous work, and update the presented results.

Results and Discussion Section
L389 to L291 and L539 to L544
Even few and small repeats shared between both chromosomes can be enough for site-specific and recombination events.  Besides nonhomologous recombination can also occur.
Also, the authors conclude by comparative and read mapping analysis that the Saccharum spontaneum SES234B has multiple reads joining chromosome 1 and chromosome 2 (L412 to L421). This may also suggest that a master-circle molecule can occur in vivo?
Moreover, the splicing event joined the start of chromosome 1 with the beginning of chromosome 2 of the SP80-3280 (L474 to L483), which may suggest for a master circle structure occurring in vivo.
Therefore, except showing a reliable piece of evidence, I don’t think that the authors can state that both chromosomes from SP80-3280 unlikely recombine, thereby this conclusion is too speculative, and I recommend to be toned down along the manuscript (e.g., see L32 - L735-L744).
Also, in L740 the authors indicate that “there are no signs of repeat or shared sequence between the two mitochondrial chromosomes that would allow recombination into a master circle.”
This is not following the results, where is stated at L542 “...few repeats were found to be common between both chromosomes...”.

L501 to L582 (only eight substitutions and a single insertion as differences)
The authors did not show these results.
Are these differences are typical or unusual? These differences were a product of the error correction procedures (Spades and Pilon), or can be considered artifacts?
After reading the ms, I’d noted that these results are appropriately presented in the discussion section.
Therefore, the results and discussion need to be re-written and re-organized.

L521 to L537 (as well in the introduction L157 and L158)
Don’t understand the relevance to show the rbcL molecular modeling analysis. Gene transfer between the organelles is well documented over the past years.
Also, these results *do not* indicate that sugarcane mitochondrial rbcL could be active and functional (L534 and L535), but indeed, it suggests for an only small piece of evidence. What about the transcriptome data, and what it reveals concerning mt rbcL expression?
Figure 6 and 8 are useless to answer whether rbcL is active or not.

L545 and L546:
“Subsequent our initial assembly of the SP80-3280 mitochondrion, the paper of Shearman et al., (2016) was published”
Don’t understand this statement. Since SP80-3280 mt genome is presented in this work (2019), how Shearman et al which presented an mt assembly subsequent from this work in 2016?
Rephrase the sentence for clarification

L559 to L563:
This is paragraph is entirely not clear. Please clarify. What is “ACR”?

L620 to L671
The authors presented merged results and discussion version of the identification CMS factors in Sugarcane.
The identification of the CMS factors was not presented, including the figure 7,  in the results section.
The results and discussion need to be re-written and re-organized.

L672 to L676: This is material and methods, not discussion.

L752 to L754:
sugarcane hybrids have diverged significantly from all these species, or there were not enough reads for a proper assembly?

L767-L769:
The GC content analysis and results is firstly presented in the discussion section. The results and discussion need to be re-written and re-organized.

There is no reference for Figure 7 and Figure 8 along the text.

Minor Comments:

Abstract:
“The sugarcane mitochondrion is comprised of independent chromosomes...”
to
“The sugarcane mitochondrion is comprised of two chromosomes...”

There are several results that point out that both chromosomes are not indeed independent (e.g., see L476 and L477, and L599 and L600).

L153 and L154
“For phylogenetic analyses, Illumina reads from Saccharum spontaneum SES234B and Miscanthus sinensis cv Andante were partially assembled against the sugarcane template.”
to:
“For phylogenetic analyses, Illumina mitochondrial-related reads from Saccharum spontaneum SES234B and Miscanthus sinensis cv Andante were partially assembled against the sugarcane SP80-3280 template.”

L201: “single-end”

L258: typo
“Potential Rf Transcirpt Assembly and Sequencing”
to
“Potential Rf Transcript Assembly and Sequencing”

L259:
“Restorer of Function (Rf) transcripts were identified from the Oryza literature.”
Please cite these references

L298: missing period
“IGV viewer (Thorvaldsdóttir et al., 2013) for further analyses”
to
“IGV viewer (Thorvaldsdóttir et al., 2013) for further analyses.”

L389:
“maxicircle”
to
“master circle”

L596: “For the first time we have mapped...”
to
“We mapped ...”

L632, L633: nad9 (italic)

L643: “S. officinarum” (italic)

L715: “(on s small sample)”

L732: gnome

L739: my

L781: nad9 (italic). Please verify for the correct spelling of scientific and genes names along the manuscript.

General minor comment
The text font and size does not present standardization. Different font type and sizes are displayed along with the manuscript; please correct it accordingly.


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

Input:
Title: THE SUGARCANE MITOCHONDRIAL GENOME: ASSEMBLY, PHYLOGENETICS AND TRANSCRIPTOMICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Language is clear and unambiguous mostly however there are typographical errors throughout the text. This needs attention and corrected accordingly for an easy flow
Experimental design: Well planned experimental procedures
Validity of the findings: As the authors claim, mitochondrial genomes will bring new insights into the evolutionary processes and also help in the widening of the genetic background of sugarcane hybrids.
Additional comments: The following edits are necessary in the manuscript

Line numbers
3- remove full stop in the title
21-insifficient is insufficient in explaining, can be expanded with one or two reasons
26-improve the annotation, is the annotation already available?
28-blast, Blast, BLAST, please use uniformly
32-cannot recombine, why?other than the reason cited, just sequence difference alone prevents recombination?
35-assembled the chloroplast of-chloroplast genome of
38-S.officinarum and S.cultum-usage and claim have to be substantiated by supporting literature other than your own publications.
42-without means without coding sequences or without splice sites?
63-belong to
80-reference has to be checked
90-uniformity in usage bp, Kbp etc
123, 124-these phenomena, these effects
187- SP80-3120?or is it SP80-3280?
205-novel pipeline, Does  the following paragraph explains the pipeline?
212-SP80-3180-same as 187
19-blast
220-mitochondrial genome annotation
234-blast
242-sugarcane chloroplast genome assembly
258-transcript
261-Blast
269-sequences for three transcripts sequenced-rephrasing required
287-biocolor
293,297-Samtools, samtools-uniform usage
298-fullstop at the end of the sentence
318,324-reference format
331- the tree prior, what does that mean?
392,393-mitochondrial genomes, mitogenomes, both terms are implying same meaning, why use differently in different places?
446-Sorghum, sorghum-uniform usage, italicize
510- 18
529-rubisco or RuBISco
514-544-can be elaborated
551-delete however
553-so that can be added
561-plastid, what is ACR?
622-ORF113
623-open reading frame
628-orf
632-nad9-italicize
639-ORF
643-646-long sentence, need to be rephrased
652-reveal
665-assembled
665 and 66-CDSes, no e in case of CDSs
734-only two, why use multiple?
735-sites
739-may


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

Input:
Title: THE SUGARCANE MITOCHONDRIAL GENOME: ASSEMBLY, PHYLOGENETICS AND TRANSCRIPTOMICS
Review round: 2
Reviewer: 1

Output:
Basic reporting: All the issues pointed by the reviewers were fully addressed
Experimental design: All the issues pointed by the reviewers were fully addressed
Validity of the findings: All the issues pointed by the reviewers were fully addressed
Additional comments: I would like to thanks the authors for all clarifications made in the rebuttal letter. The revised manuscript improved a lot and will add valuable knowledge regarding sugarcane genomics, and plant  mitochondrial genomics, including bioinformatics techniques related to elucidating the master-circle structure and assembly.
I'm happy to see this manuscript published in its current format.


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: IN SITU GROWTH AND BIOEROSION RATES OF LOPHELIA PERTUSA IN A NORWEGIAN FJORD AND OPEN SHELF COLD-WATER CORAL HABITAT
Review round: 1
Reviewer: 1

Output:
Basic reporting: -	The paper is clearly written but there are a few areas where the English language and grammar could be improved. Below are a few suggestions:
o	Line 127 – “measured under in situ conditions” should be something like “ current conditions measured in situ”
o	Line 144 – “found to being” should be “found to be”
o	Line 151 – “Besides” could be deleted or changed to “However”
o	Line 348 – “Massy” is not a word. The authors could say “coral fragments with less mass” or “heavy”

-	I believe there are too many instances of “i.e.” and “e.g.” throughout the manuscript (specifically the introduction). The writing will flow more smoothly if the authors can eliminate a few of them and write the example(s) as a sentence.

-	The introduction is well written and includes relevant information and references. I do think the authors could add a bit more about projected ocean acidification and potential changes in the aragonite saturation state in the context of cold-water corals, since they are potentially the first ones to be impacted by low saturation states.
o	Refs: Orr et al., 2005; Guinotte et al., 2006; Georgian et al., 2016a
Experimental design: -	This study aims to address interesting and not fully explored questions around CWC calcification and bioerosion in perspective of current environmental conditions and looking to the future under climate change.

-	The authors applied a variety of different methods to measuring calcification and impacts of bioerosion. I think these multiple methods strengthen the overall experimental design and impact of this study. Specifically, the CT scanning was a very innovative approach I hope to continue to see used in similar studies.

-	I am hesitant about the mixing of the Sula and Nord-Leska dead erect coral framework. The author states that the structural framework is quite different between the two sites so it would have been interesting to compare the effects of bioerosion on the different skeletal “types”.

-	The authors do acknowledge the missing treatment, in that there was not an experiment run on the reef of Sula. I am curious, why did the authors choose to run the samples they did off of the reef? Vs. having two on the reef treatments (one at Sula and one at Leska)?

-	I would appreciate it if the authors could discuss why they chose to use Alizarin Red S instead of another stain like Calcein. Also, do the authors think the long exposure to Alizarin Red could have contributed to some of the mortality observed?
Validity of the findings: -	No need to state “We provide the first” in line 485

-	While the findings of this study are very interesting, I do feel like the authors engaged in a lot of speculation throughout the discussion.
o	Lines 501-504
o	Lines 518-519
o	Lines 651-668
o	Lines 668-671
o	Lines 694-696
o	Lines 758-760

-	I think the authors should be cautions in the section at the beginning of the discussion from lines 491-501 where they directly combine the calcification and bioerosion estimates. They mention that the “they are to be regarded as rough approximation of relative rates, assuming similar amounts of live and dead coral framework and do not reflect the actual reef carbonate budget.” However, even with this statement it is taking the data too far to say, “overall net production rates remained still positive.” I think to make a statement about net productivity of the reefs the authors would need to create or model estimates of the amount of live versus dead framework for these reefs.

-	The paragraphs in the discussion (lines 551-631) about the different methods for measuring linear extension and results from previous studies could be greatly reduced.
Additional comments: -	I think this is a very interesting paper and much needed in the realm of CWC experimental biology. While I agree with the authors about the need for more in situ experimentation, I wish they had focused a bit more on the importance of recording and understanding the environmental and oceanographic conditions in the regions where experiments are occurring. Yes, we need more experiments in natural settings, but those experiments won’t hold a lot of meaning if we can’t compare the findings across different geographic areas through the environmental conditions they are exposed to.

-	If the data is available, I would suggest adding a table of environmental conditions (depth, temperature, dissolved oxygen, salinity, pH, total alkalinity, aragonite saturation, etc.) for the two different sites. I think this will provide some needed context for the similarities/differences between the three study sites.

-	I commend the authors on including the process of dissolution (as well as calcification) in the discussion of cold-water coral reef formation. I believe this is a very important component to understanding this system that has be overlooked for far too long. While they do make a compelling case about the significant of bioerosion specifically, I do wish they had considered how to include chemical dissolution as well.


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

Input:
Title: IN SITU GROWTH AND BIOEROSION RATES OF LOPHELIA PERTUSA IN A NORWEGIAN FJORD AND OPEN SHELF COLD-WATER CORAL HABITAT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is comprehensive, structured according to PeerJ standards and well-referenced with relevant literature. The figures are relevant, well labeled and described, and the raw data is supplied. The paper is generally well written, but the authors are not native English speakers and there are a few quirks throughout. These can be easily remedied through a review by a native English speaker. The sentence structure often tends towards the verbose, and as the paper is rather long, I recommend the authors work to make the language more concise.
Experimental design: The paper is based on original research within the scope of the journal; it has a well-defined question and clearing states the knowledge gap addressed by the research topic. The study was performed to a high technical standard and the methods are scientifically rigorous and described in sufficient detail to be replicated.
Validity of the findings: There is no control per se, as the experiment was focused on in situ observations rather than laboratory experimentation; however, the data is robust and statistically sound. Conclusions are well stated and linked to results. There is some speculation, which is generally noted as such.
Additional comments: This is a good and important paper that deserves to be published. I have made some minor comments and suggestions in the PDF document (attached). My major criticism is that the language is wordy, sometimes with very long and confusing sentences, and there are minor grammatical errors scattered throughout the document.. I recommend a thorough critique by the authors to make the language more concise and reduce some redundancies, and a review by a native English speaker to resolve some of the grammatical quirks.


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: IN SITU GROWTH AND BIOEROSION RATES OF LOPHELIA PERTUSA IN A NORWEGIAN FJORD AND OPEN SHELF COLD-WATER CORAL HABITAT
Review round: 1
Reviewer: 3

Output:
Basic reporting: This study measured in-situ growth and erosion rates (1 year) of an important cold-water coral, which provides a critical piece of information of these processes in the natural habitat. It is clear, and professional English was used throughout. Thereby, this study has the potential to improve the understanding of the calcification and dissolution processes in cold-water ecosystems. In general, the paper meets the standard, However, there are some concerns that need to be addressed.

Specific comments

intro
The Introduction provides a thorough literature background on the subject and generally is well written. However, it is to some context too extent and can be reduced.

Line 103: delete “suggest”

Line 112-121: this paragraph can be linked to the previous one and can be more synthesized.

As stated before, authors should provide some hypothesis regarding the experimental design. For example, in the results and discussion there is a good deal of text comparing coral growth and bioerosion between on-reef vs. off-reef, however it is not mentioned the reason for choosing this set-up, in the intro or in the methods.

Authors provided access to raw data
Experimental design: This study is an original primary research, which has not been done in cold-water ecosystems, thus is within the scope of PeerJ and will benefit the cold/deep-water community interested on these ecological processes.
The research questions are well defined based on the objectives and the methodological approach, however, there is no any hypothesis that can guide the reader about why or what was the main purpose of some of the experimental design. For example, what was the criteria to select the places? Why some experimental fragments were set on-reef vs. off-reef? Why Sula reef had only fragments set-up off-reef ad not on-reef?

The investigation was performed with high technical and ethical standard.

Methods are describe in most of the paper with sufficient detail, however, see specific comments in the attached document.

Those basic questions should be address somewhere in the intro as well as in the methods.

Specific comments:

Methods
The methods are clear and well presented. Clearly explain in detail all the experimental design, manipulation that were performed and data analysis for other study to replicate it.

Line 209-214: “This offshore location comprises a relatively constant habitat in terms of environmental parameters such as temperature, salinity, pH, and currents, while the selected inshore location, a reef near the island Nord-Leksa in the outer Trondheimsfjord (henceforth referred to as Leksa Reef), is exposed to a highly variable environment due to strong tidal and compensatory currents (Form et al., 2015 Cruise Report POS473)”
It would be great if authors include a table with the environmental variables from both sites, due to the importance of this in the whole context of the paper. I tried to find the values in the cruise report Form et al. 2015 (is in the citation) but did not find any table with the environmental conditions of the selected places.

Line 214: What is the reason for the two inshore locations? Why this design was not applied to off-shore locations?

Line 224-226: I would suggest this: “All samples were collected by means of the manned submersible JAGO (GEOMAR, 2017)”

Line 228: there is a typo before 500 L

Line 234: Could the authors be more specific about the size of the coral fragments?

Line 238: Based on what methodology did the authors choose the dye concentration? Is there any empirical evidence?

Line 252: I would suggest start the sentence with “Several fist-sized…”, and again, it would be better if the authors provide a value of the size.

Moreover, in the discussion section (lines 753-754), the authors refer to a different bioerosion stages (3 to 6), however it is never explained in the method section. Authors should add one or two sentences explaining this.

Line 262-265: Avoid repetitive use of JAGO. I think it only needs JAGO in the first sentence (line 262).

Lines 331-339: It would be great if the authors provide more description about the statistical analysis performed, i.e. type of routines run, type of variables used for the analysis (dependent/independent), number of effective replicates, more explanation about the pooling procedures used for the analysis, and assumptions of the data.
Validity of the findings: In general, the experiments were conducted in a sound manner.
The data is robust, especially when the analysis is done with pooling. However, need to provide statistical validation specially to support the decision of pooling (p-value not shown, see Underwood, 1997). There are parts in the methods that are not well explained, especially the type of statistical analysis performed, and why.
Moreover, need to unify the way to present the statistical support, especially in the results. The analysis would be greatly improved if the authors include environmental data, especially because the differences in response variables are speculated to be because of differences in environmental conditions.

Specific comments:
Results

Need to provide tables with the statistical analysis, i.e. ANOVA table.

I would suggest the authors to incorporate sections 3.1.1 and 3.1.2 into one section, and more synthesized. Specially section 3.1.1 that looks detached from the paper. Moreover, this part is not taken into consideration in the discussion. I would suggest report an average size of the fragments per colormorph and per site. In this way it will look more integrated into the whole paper.

Line 347: It is not clear to me why to perform post-hoc analysis for the ANOVA if only two location are compared?

Line 350: Why two different analysis for the same comparison? It is t-test? Or ANOVA? Also please unify the way you want to present the statistical support.

Line 363: This is interesting. Any hypothesis why this can be?

Line 379: The figure 4 shows P=0.003. Moreover, please unify the way you want to present the statistical support throughout the manuscript

Line 399: Please change the N value in the Fig. 7 to match the results. I am curious if the authors perform a correction for the uneven sample size, N=14 vs. N=4.
Line 439: As mentioned in the methods, I would encourage the authors to provide environmental data since it is an important part of the hypothesis and the whole analysis of the paper.

Line 449-450: I would replace the term “statistically highly significantly different” to “statistically different” and again, it is not clear to me why to use post-hoc with only two group comparisons. Please check.

Line 472-481: This is not a part of results. Suggest move to methods section.

Discussion

Line 514: It is a little bit counter intuitive to say, “cold-water coral degradation may be particularly affected”. I would say that coral degradation is enhanced by….

Line 587-591: I would suggest link the two sentences.
“Highest extensions in aquarium cultivations were obtained by Orejas et al. (2008) in L. pertusa from the Mediterranean Sea, with measured linear extensions of 15 – 17 mm yr-1 at temperatures around 12 °C”

Line 591: Reference citation from line 592 should be changed to line 591, “Orejas et al (2008)”

Line 626: Please provide citations related to extension rates.

Lune 629: change “elevated extension” to “higher extension rates”

Line 628-631: Overuse of the words “extension rates”

 Moreover, is there any explanation why corals grew more inshore sites of Nord-Leksa? This should also be addressed in the discussion.

Line 751-755: I would suggest moving to methods. It is not explained the different bioerosion stages.

Lines 808-815: I would suggest moving to intro

References:
Sone references are cited but missing from the list.

Line 76: Freiwald 2009

Line 86: should read as “Reported estimates of growth rates from in situ and laboratory conditions range from 2.4 to 35 mm per year……..” not all references provided come from in-situ studies.

Line 148: Include Provan et al (2016) in the reference list

Line 161: You should include in the reference:
Neumann, A. 1966. Observation on coastal erosion in Bermuda and mesurement of the boring rate of sponge Cliona lampa. Limnol. Oceanogr. Vol. 11(1): 92-108.
This is the first paper that coined the term Bioerosion

Conclusions are well stated and summarize the findings.
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: IN SITU GROWTH AND BIOEROSION RATES OF LOPHELIA PERTUSA IN A NORWEGIAN FJORD AND OPEN SHELF COLD-WATER CORAL HABITAT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors did a sufficient job of addressing the "basic reporting" comments from the first review - no further comments.
Experimental design: The authors did a sufficient job of addressing the "experimental design" comments from the first review - no further comments.
Validity of the findings: The authors did a sufficient job of addressing the "validity of the findings" comments from the first review - no further comments.
Additional comments: Thank you for spending the time to thoroughly address the comments and concerns of the first review. Congrats on a great paper!


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

Input:
Title: IN SITU GROWTH AND BIOEROSION RATES OF LOPHELIA PERTUSA IN A NORWEGIAN FJORD AND OPEN SHELF COLD-WATER CORAL HABITAT
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have addressed my comments and those of the other reviewers adequately for publication. I look forward to seeing this in print.


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: ESTIMATING FLOWERING TRANSITION DATES FROM STATUS-BASED PHENOLOGICAL OBSERVATIONS: A TEST OF METHODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: General Comments: This is a well thought out, useful paper that that is generally well-written, and will be of interest and practical utility to phenological researchers in the future.
Experimental design: This experiment seems well designed, although there are a few places where aspects of it could be better communicated (see comments to the author)
Validity of the findings: These findings seem legitimate.
Additional comments: Lines 15-17 since you bring up herbarium data earlier, it might be helpful to mention that this is an in-situ dataset for purposes of clarity.

Lines 19-21 It seems a little odd to just say you tested estimates, when the paper is testing the accuracy of estimates produced through multiple different methodologies. This absence may be a reflection of the word limit associated with the abstract, but some mention that you are testing estimates constructed using multiple methodologies would be appropriate here.

Line 35 would ‘phenological’ be more appropriate than ‘phenology’ here, since it’s being used as an adjective?

Line 66 This is a minor point, but if you’re referring exclusively to spatial scales, it might be best to say that explicitly – phenological studies can also exist across scales not only spatially, but also temporally and at individual – population levels.

Line 84: what did the phenological observations consist of? Clearly they record more than simple flowering/nonflowering, but a little more explicit explanation would be helpful. (i.e., are the number of disk flowers within a head recorded, or the proportion? How many flowering heads were recorded per individual, and what was the protocol for selecting them, or handling cases where the proportion of open disk flowers differed among heads?)  Also, I think ‘phenological’ might be more appropriate than ‘phenology’ since it’s a descriptor.

Line 91-92 – to what accuracy can the date of first-flower be back-estimated? How was this tested? If the accuracy wasn’t explicitly tested I don’t think that’s an insurmountable problem, but if you’re claiming total precision (as seems to be the case here) then some explanation of how you can be confident that’s the case seems appropriate.

.	Lines 101-103 - This statement is a little ambiguous to me.  Did you randomly select a subset of the actual observations of each individual (which are not made every day, and are unlikely to be made during parts of the year when the plant is unlikely to be in flower), or did you, knowing the actual dates of bloom onset and termination, randomly select a number of dates throughout the year (on which an actual observation may or may not have occurred) and then populate them with what you can infer the phenological status was using the actual observations? Based on the following text I believe it’s the latter. If so, it needs to be more explicit, as my initial reading from this statement was that it was the former.
.	Lines 106 “observations”
.	Line 142 some brief description of the Weibull distribution might be helpful here, as the reader may not be familiar with it.  For example, this particular distribution has a number of advantages over several other probablility distributions in that it can be quite flexible in its shape, or handle skewed distributions.
.	Lines 146 and 147 – it’s probably appropriate to note what language system the code is for in the main text (i.e., python, R, etc). Also, it might be best to include the web address or DOI for the code repository here.
Lines 150, 163 - it seems odd to have these orphaned lines of R code inserted into the main text.  I would either simply state that you used this method in R using XYZ package, or include the full code in the supplement and then refer to that in the main text.
Lines 178-182 – For form’s sake, it’s probably best to include the version of R (and of the relevant packages) that were used in this study.
.	Line 290 – It may also be worth noting that many herbarium specimens do not explicitly document phenological status, even within herbaria that typically do record phenology – thus, while specimens recorded as in flower can be assumed to be in flower, specimens not recorded as being in flower (which are often the majority of specimens) may potentially be either in flower or not, and cannot therefore be reliably used as observations of flowering absence unless accompanied by imagery or a manual assessment of flowering status
.	Line 322 replace midway with midiway
.	Discussion – I think some discussion of the applicability of each method to different kinds of existing phenology datasets would improve the utility of this manuscript.  For example, while the midway-7 day method may perform best in some cases, it may not be usable with datasets such as herbarium specimens, in which absence observations are not present or are unreliable. Conversely, this would further underscore the utility of the Weibull method, as the fact that it does not utilize observations of absence makes it a better fit for studies using those types of phenological data that do not provide them.  Emphasizing that it not only performs best in many cases, but also is a better fit to the nature of many phenological datasets, would help communicate the importance of this finding.


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: ESTIMATING FLOWERING TRANSITION DATES FROM STATUS-BASED PHENOLOGICAL OBSERVATIONS: A TEST OF METHODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I suggest editing text in the last paragraph of the introduction where you introduce what you did and why and set up the methods - I think you need a sentence linking the sentences on line 76 and 77, and a statement of the overall objective of the study. I also suggest adding subheadings in the discussion section for readability and flow.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Overall, this is a great contribution to the phenological literature, as there is an abundance of methodologies used to generate the unit of measurement across many disparate datasets and this provides some “best practices”.  The synthesis presented in a very useful and straightforward manner, and the methodology is innovative and robust.  Would be interesting to follow up with a comparison of peak flowering at the individual level.  I’d also suggest including context in the discussion of when someone might choose to use individual estimators in comparison to using peak estimators – for decision making in data analyses and applications.

A few small thoughts: I suggest not using the term “Julian day” for the day of year, as Julian day technically refers to the number of days since Jan 1, 4713 BC. [Or clarify that you are using a specific definition of this term that restricts DOY values between 1-365].  I would also clarify that the “mean occurrence day” is not the mean of all the first observed DOYs but rather the mean of all the positive records for a year.  I’d suggest citing Renzi et al. 2019 American Journal of Botany for a similar (but less detailed) look at population vs individual level metrics.  I’d suggest using the term “metrics” rather than “estimators”; as it was somewhat confusing to go back and forth between the term estimates (of onset and end dates) and estimators.


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: ESTIMATING FLOWERING TRANSITION DATES FROM STATUS-BASED PHENOLOGICAL OBSERVATIONS: A TEST OF METHODS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The reporting in the article is unambigous and professional English throughout. However, I do suggest to make sure that all figures have a full caption without cross references.
Experimental design: The research is well defined, and full access to the code and data is provided. The code runs. I did not check for bugs or other calculation errors as I consider code review beyond the scope of a manuscript review. I assume good faith and relative stability of the code provided.
Validity of the findings: The study is a meaningful addition to previous work, in particular Moussus et al. 2010 / Pearse et al. 2017, while adding transparency in code and data used. I would include some stronger (broader) statements providing the context provided by these previous studies (see general comments).
Additional comments: The manuscript titled "Estimating transition dates from status-based phenology observations: a test of methods" compares various approaches to characterize and estimate true transition dates from status based observations.

Status based observations dominate various phenology records not in the least those derived from herbarium specimen, but also and certainly not less important in a contemporary setting, crowd-sourced data. All these data sources are important in understanding past and current phenology responses (and their interactions). A better understanding of the influence of how data is processed and its influence on error distributions is a valuable addition to ongoing work in the field of (vegetation) phenology.

I think this study certainly merits publication. However, I feel that the scope has been left too narrow in either defining the question and the final conclusion. This is more a matter of how the text is structured rather than any flaws in referenced literature. I would encourage the author to look for ways to improve the narrative, and in generally make the manuscript more concise.

Reading through the discussion I feel that there was some repetition involved and this section might benefit from some tightening up and more concise writing. While references to common data sources such as the PEP725 dataset, USA-NPN data or other citizen science based data is sparse. Given the scope of the analysis I think some more context in this respect would benefit readers to have an overview of some of these (and other data sources), if not in a small overview table.

I would rephrase the language in the final concluding paragraph, and as a lead up in your introduction, to be a bit stronger (especially when combining the current results with those of other studies, i.e. Moussus et al.). I think it is fair to state that in large but sparse datasets (low presence large absence data) either Weibull or GAM methods merit consideration. Their should be caution when dealing with smaller datasets (which might be common if not the norm in certain contexts, it might be beneficial to highlight those). Absence values are of importance, and hence should be gathered where and if possible. This is mentioned in passing in the text, but I would argue that repeating this message again in a conclusion as it would benefit the design of future studies. The presence of the analysis code also ensures that users can evaluate their methodology up front before preceding with additional analysis which should limit future ad-hoc decisions. I think this is one of the main advantages of open science, and therefore should not be undervalued! However, some work might be needed to make the coding framework a bit more transparent (see below technical comments).

I recognize that the limitation of using one species, which can't be used to generalize, the availability of code and data ensures scalability of this analysis to other data sources. In this context I'm missing an explicit reference to a code and data repository in the methods or a data availability statement. This would help people find code and data quicker, and repeat the analysis on their own data if so desired (again more on this in the technical comments).

Given the above comments, and some minor details below I think this should make a nice reproducible paper. I can recommend a minor revision and wish the author all the best with implementing these changes.

Technical comments:

I've ran the code, although it did not complete (longer computational time, and my impatience). It might be useful to include a README with details on:

1. the system requirements (R packages mainly)
2. how to use the code (what to run first)
3. what to expect in terms of processing time.

My suggestion would also be to create a formal R package as there is no file outlining package requirements. A formal R package DESCRIPTION file (analogues to a python requirements.txt file) would limit issues regarding missing packages before running the code (and frustration that might come with it for novel users). Alternatively, I encourage the author to include an R script which checks if all requirements are met.

As mentioned above, the strength of an open publication in all its aspects (manuscript + code + data) lies in the fact that the analysis ideally can be run on different datasets with ease in order to limit future ad-hoc decisions. I think this idea should be kept in mind when providing code.

Minor comments:

L16 Be explicit about the species / dataset name, this seems to be ommitted but rather key information even for an abstract.

L21 Mentions 'recently' twice in short succession, might want to look for an alternative.

L22 Why is the Weibull curve method new and what are the advantages? Add a line with this info to the abstract as to get people up to speed. Repeat this line in the method section as well.

L232 Is the described pattern a form of overfitting?

L250 I suggest to combine the individual estimates (onset / end) into one section.

L281 Absence data might be as valuable as presence data, how is this currently implemented in observations schemes in particular citizen science components (USA-NPN etc.) (resolved in L293 - but might need consolidation)

L273 How does this observation reflect on the use of these statistics on herbarium data? I suspect they all might have low(er) observation numbers?