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: EFFECTS OF STARCH SUGAR BY-PRODUCT ON RUMEN IN VITRO DIGESTIBILITY, IN SITU DISAPPEARANCE RATE, AND MILKING PRODUCTIVITY OF THE LACTATING DAIRY COW
Review round: 1
Reviewer: 1

Output:
Basic reporting: The presented manuscript “Eﬀects of starch sugar by-product on rumen in vitro digestibility, in situ disappearance rate, and milking productivity of the lactating dairy cow” is directly related to several contemporary issues and thus provides a great value to the community. However, in its current form, the presented manuscript suffers from several shortcomings.

The English language and readability of the presented manuscript should be improved. Although being a non-native English speaker, this reviewer has identified several parts that are grammatically incorrect and also the overall fluency of the text could be improved. This reviewer suggests to ask an English-speaking colleague to proof-read the manuscript or pay for a professional service. A couple of examples with unclear phrasing or mistakes can be found in lines: 37, 46-48, 56, 62-64, 75-77, 84, 88, 94, 102, 208, 219-220, 241, 246-250, 256, 261-272 (but please, focus on the entire text).

Overall, the structure of the manuscript is appropriate. This reviewer would just suggest to extend the introductory and result sections and possibly reorganize the individual method sections as described below.

The introduction could be improved and extended to better justify the need for this study. In the introduction, only a handful of references were used. In lines 53-55, it is not clear whether this is the authors’ speculation or whether it is a known fact. Similarly, in the line 55, it is not clear where the assumption that silica contaminates SSB comes from. In line 58, more than just two studies could be cited. In line 59, the “rare” research studies should also be properly cited to better place the current study in the context of known literature. Line 62 was not researched so its importance in the introduction is not clear – or can the predisposition to urinary tract stones be implied from the blood urea nitrogen (if so, it should be specified in the text)? In general, it is not clear how the blood samples are relevant for the study so it could be introduced. Similarly, the impact of fatty acid synthesis on milk production and composition is not introduced.
Experimental design: The objectives of the study are within the scope of the journal. The research questions and aims of the study could be defined more clearly.

The end of the introduction (lines 61-65) implies that the goal of the study is to: a) investigate the impact of silica present in SSB on cows’ physiology; b) determine whether SSB-enriched feed can be used in practice. However, the effects resulting from SiO2 presence in SSB were not sufficiently studied. Firstly, the values for SiO2 are missing in the raw data table characterizing the chemical composition of SSB. A control sample indicating if there is SiO2 present in the standard feed is missing. It would be better to quantify SiO2 also in the Table 2 and Table 3 to indicate the differences between standard and SSB-enriched feed (this reviewer does not ask for more experiments at this stage of the project; however, clear discussion of these aspects should be provided). In lines 244-245, the authors suggest that the ash is made up of silica. In its current form, this is just a speculation so this reviewer suggests to tone it down. Moreover, the effect of SiO2 is not covered in the results section. Overall, it would probably be better to modify/remove the parts regarding SiO2 as it is neither well introduced nor well investigated. A possible suggestion is either to improve these parts or keep the SiO2 part only for the discussion.

The remaining parts investigating the in vitro digestibility and in vivo disappearance of SSB are performed and described well. Methods are described with sufficient detail and information to replicate (if the English is improved) and the methodology used is adequate to the study.
Validity of the findings: The authors provided processed as well as raw data and described well the statistical analyses. Several comments regarding the data provided are below.

One of the most important finding relies in the altered gas production, ammonia nitrogen and A/P ratio upon increased SSB levels. The authors attribute the decreased gas production to inhibited microbial fermentation due to increased lipid and silica content (lines 252-253 and 261-262). However, note that sugars and starches (which constitute the nature of SSB) are broken down easily and quickly. It seems that the gas production is directly linked to digestibility of the feed; where with good digestibility the energy produced is used to increase weight and production of milk, which is associated with decreased enteric CH4 production (doi.org/10.3390/ani11071870; doi:10.1017/S1751731113000876). In line with these observations, with increasing SSB levels the authors identified increased levels of propionate (and thus decreased A/P ratio) which is known to be an end-product of starch and sugar fermentation and whose presence is associated with decreased CH4 and CO2 levels (Moran, 2005, How the Rumen Works). Considering the fact that the milk production was slightly increased and there were no dramatic changes to the pH, couldn’t this hypothesis, the simple fact that the feed was enriched for starch, better explain the results of the study? How the authors can be sure that the SSB-enriched feed does not provide enough nutrients to grow cellulolytic bacteria (line 266)? This is quite an overstatement since the authors did not investigate the grow of bacteria.
Additional comments: Typical reader of scientific literature first reads the results section and only in the case when something needs to be validated he/she seeks the methods. In the presented manuscript, the results are written in a way that it is difficult to follow, partly due to a high number of shortcuts used. Please consider re-phrasing the results to make this section more readable. Additionally, results could be extended to cover all aspects of the manuscript and in greater detail. Additionally, there are shortcuts that should be explained earlier. For example, DM, TMR, NRC in the abstract are not explained.

The organization of tables is a bit confusing. First, the authors present a composition of feed used in the in vitro (Table 2) and in vivo assays (Table 3), respectively. Then, they present data in the reverse order, i.e. first the in situ disappearance rate (Table 4), followed by the in vitro gas production (Table 5) and fatty acid synthesis (Table 6). Wouldn’t it be better to organize the tables in a more logical way and place the tables summarizing composition and results of the underlying assays next to each other? This would also help to understand the correlations between the composition and results of the analysis. In fact, it would be very helpful to provide a correlation analysis between the composition and analysis results to indicate a potential relationship between the variables and observed physiological changes (such as between EE, ADF, Ash and gas production, pH, ammonia nitrogen, fatty acids). For example, ADF is a good indicator of digestibility (doi:10.3390/foods8090364 and references therein) so it would be nice to see it confirmed also by this study. Additionally, a graphical representation of the data (similar to those in the supplementary excel file) would be highly appreciated by readers (indicating also the distribution/variability of the data).

Table 1. There are multiple categories (CP, EE, NDF, ADF, Ash, SiO2, WSC) that are expressed as % DM. However, when these numbers are summed up, it gives > 177%. Similar discrepancy can also be found for the individual measurements in the raw data excel table “1-2. Chemical characteristscs” (check also spelling of the word “characteristics” in all the tabs of the raw data table). Is it because these categories overlap? It is basically explained by the lines 244-245 where the authors suggest that the ash is made up of silica. But maybe it could be briefly explained somewhere in the text not to confuse readers.

In raw data excel file tab “2-1. IN SITU disapperance - DM” (please, correct the typo), the time point 0 in the graph is based on value 32.63 even though the value should be 32.37 as demonstrated in the Table 4. Similar discrepancy is present for the time point 8.

The authors should indicate based on which criteria they decided to use 3% DM of SSB for their “feeding trial”.

The authors should discuss the short duration (only six weeks) of the study. It is reasonable to assume that life-long supplementation of SSB could uncover more dramatic effects on cows well-being/milk production/weight gain.


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: EFFECTS OF STARCH SUGAR BY-PRODUCT ON RUMEN IN VITRO DIGESTIBILITY, IN SITU DISAPPEARANCE RATE, AND MILKING PRODUCTIVITY OF THE LACTATING DAIRY COW
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Dear authors,
Overall work here, I believe the authors have done an exemplary job in preparing this manuscript. The level of scientific rigor is apparent, and the attention to detail with regard to every aspect of the replication is appreciated.

I have a few minor suggestions that the authors might consider, but all of them would move forward.
1. The manuscript the authors discuss the connection between abstract statements and those of passive voice. This discussion includes some clarification from real data numbers in the world (statistical data in present), but I wonder if additional clarification about the connection between the abstract characteristics and passive voice would be worth discussing here.

2. On line 79 in the Materials section, the authors mention that the statements were evaluated so that
“the abstract and concrete versions used equally common language.” Was that equality evaluated in any specific way?
3. Please mention the different time frame variability and accuracy.

4. Please clearly mention all the reagent and chemical sources in this format; Company name, Manufacture location, Country in the manuscript.

6. The values are showing in table 8 is very close to human is this same in the dairy cow?


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: EFFECTS OF STARCH SUGAR BY-PRODUCT ON RUMEN IN VITRO DIGESTIBILITY, IN SITU DISAPPEARANCE RATE, AND MILKING PRODUCTIVITY OF THE LACTATING DAIRY COW
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors significantly improved the text quality in the revised version of the manuscript. Some language issues remained unresolved, thus this reviewer suggests one more proofreading round by an English speaking colleague.
Experimental design: The authors handled this reviewer’s comments well and applied the requested changes.
Validity of the findings: This reviewer suggests to rephrase the modified part and correct the English to improve its readability.
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: EFFECTS OF STARCH SUGAR BY-PRODUCT ON RUMEN IN VITRO DIGESTIBILITY, IN SITU DISAPPEARANCE RATE, AND MILKING PRODUCTIVITY OF THE LACTATING DAIRY COW
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript has been modified and approved for no further comments.
Experimental design: Looks good
Validity of the findings: Excellent and much improved
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: FUNCTIONAL NETWORK MOTIFS DEFINED THROUGH INTEGRATION OF PROTEIN-PROTEIN AND GENETIC INTERACTIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have neatly described a method to integrate genetic interactions (GI) to define functional network motifs (FNMs) to identify biologically relevant networks. They have critically analyzed the yeast protein-protein interaction network to show the utility of the workflow in identifying known a novel regulatory networks. This workflow provides useful information that needs to be integrated with hub identification from huge and complex networks.

The authors have clearly described the aim and citied multiple relevant articles in the manuscript which needs to be appreciated.

The authors have also provided the codes as GitHub repository, which will be highly useful to other researchers in the field.
Experimental design: The authors have defined FNMs and analyzed the identified FNMs to show their regulatory role. It will be great if the authors can conduct sensitivity analysis, to shown that the defined method identifies biologically relevant networks greater than by chance (eg: AUC).
Validity of the findings: It will be great if the authors can compare their method with other similar methods in the field to show the importance of adding GI.
Additional comments: No 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: FUNCTIONAL NETWORK MOTIFS DEFINED THROUGH INTEGRATION OF PROTEIN-PROTEIN AND GENETIC INTERACTIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: The research questions are not well-defined. The authors present a new concept that they call Functional Network Motifs or FNMs, and they propose that FNMs reveal new insights into biological networks. However, there are several major shortcomings:

1.) The definition of FNMs appears quite arbitrary, with a threshold of >= 50% frequency of genetic interactions between the genes in a set that is connected by physical interactions. There is no clear justification for this threshold and no analysis of alternate thresholds.

2.) There are confusingly non-parallel definition of FNMs relative to "conventional" network motifs. "Conventional" network motifs are architectures or conceptual graphs that occur more often than expected within a network. In this case, the main significant entity is the motif or architecture itself, not the specific examples. In contrast, FNMs appear to refer to specific sets of genes that are connected by physical and genetic interactions. There is no analysis of whether the observed architectures of FNMs are over-represented other than Figure 1C, but it seems that FNMs are defined as specific example sets of genes, regardless of the prevalence of the architecture. This makes comparisons of conventional network motifs and FNMs not straigtforward.

3) Related to point 2 above, a critical feature of conventional network motifs is the directionality of edges. This is particularly important for interpreting feedback or feedforward wiring. This feature is absent from protein-protein interaction and genetic interaction networks. As such, it is difficult to identify motifs that can be interpreted as relating to feedback or feedforward connections. Identifying such connections appears to be a major goal of the authors, but it is not clear that FNMs are a good way to identify them, and the claims that FNMs are useful for identifying them are not tested appropriately (see below).

4) As a smaller point relating to point 2, the FNMs are defined in terms of a "source node" but the definition of the "source node" in terms of identifying FNMs is not clear (are all nodes in each connected subgraph of the network considered as potential source nodes?). It is also not clear if the term "source node" is necessary or meaningful for FNMs.

The major claim of the manuscript is that FNMs are useful for identifying new functional relationships between genes or protein complexes, especially feedback relationships. These claims are not tested directly, and it is not clear if FNMs actually provide added value over what can be inferred directly from the original PPI and GI data sets.

5) The subsection titled "FNMs identify strong candidates for feedback regulation" and the phrase in the abstract "suggesting a promising strategy towards the systematic identification of feedback regulation in biological networks" suggest that FNMs have identified new feedback relationships that have been validated, but this is not the case. Instead, this subsection describes several FNMs and the relationships between the included genes based on previously published results. Potential regulatory connections are suggested, but the predicted feedback relationships are not concretely defined and they are not tested. Therefore it is totally unclear whether they are indeed feedback relationships. Furthermore, there is no roadmap presented for how feedback relationships might be systematically predicted based on FNM definitions.

6.) In Line 237, the authors comment that genetic interactions between complexes are almost perfectly consistent across the members of the complex. This is an observation that has been made previously by others, but it appears to be presented as a new finding. I believe the earliest description of this feature (relating in this case to pathways rather than complexes) was in Segre et al Nat Genetics 2004. It was also described in Roguev et al Science 2009, and in others as well.
Validity of the findings: 1. The authors state that it is remarkable that FNMs are two orders of magnitude less frequent than "conventional" network motifs, but the FNMs are defined with a threshold for frequency of genetic interactions which makes them less frequent than the conventional motifs by definition. The frequency of FNMs is entirely dependent on this threshold, and lower thresholds or higher thresholds would result in FNM definitions with either greater or lower frequency.

2.  The statistical test for enrichment of suppressors (line 193) would need to account for the fact that FNMs are enriched in GIs by definition. The authors do not account for this, stating "a single GI alone would not bias a motif towards an FNM". While a single GI will not be sufficient to make a set of genes meet the requirement for being an FNM, the reverse is clearly true. If a set of genes is an FNM, then it is by definition enriched for GIs. It then follows that they should be enriched for suppressor GIs as well. To demonstrate a true enrichment of suppressor GIs beyond what would be expected just from the definition of FNMs, the authors would need to perform a statistical analysis that corrects for the elevated frequency of GIs in FNMs. Is the fraction of GIs that are suppressor GIs higher within FNMs?
Additional comments: In summary, it is not clear whether the definition of FNMs proposed by the authors reveals new biological connections or facilitates the identification of feedback connections.


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: FUNCTIONAL NETWORK MOTIFS DEFINED THROUGH INTEGRATION OF PROTEIN-PROTEIN AND GENETIC INTERACTIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors propose an approach for selecting graphlets from PPI networks that also have genetic interactions.  In this case, two genes are defined to have a genetic interaction when the alleles of variations in one of the genes are correlated with the alleles with the variations in the other gene.  It is assumed that the correlated variations indicate relationships such as one variation compensating for another to maintain function.

- Language is clear and unambiguous
- Literature references are comprehensive, relevant, and appropriate
- Article structure, figures, and tables are all professional and high quality

On lines 56-57, the authors use the term "deletion mutants."  Do they mean "deleterious mutations"?

On line 195, did you mean "FNMs" instead of "FNM"?
Experimental design: - Research question is well defined, relevant, and meaningful.  The contribution and its relationship to previous work is clear
- The analyses were rigorous and conducted to a high technical standard
- Methods are described in sufficient detail
Validity of the findings: - The conclusions are well supported.
- The statistical analyses are clear.
- Conclusions are well stated.
Additional comments: Thank you for the opportunity to read about your work.  Your approach is interesting and compelling.

I think the clarity of manuscript could be improved for non-experts with a descriptive figure.  In particular, I would propose that the authors create an additional figure demonstrating a graphlet identified from the PPI and which edges also count as genetic interactions.  The figure could also illustrate why the genes encoding a pair of proteins are classified as having example a genetic interaction (e.g., show multiple sequence alignments for each gene showing the variations across samples).  This is completely optional, however -- I would not require this for acceptance.


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

Input:
Title: FUNCTIONAL NETWORK MOTIFS DEFINED THROUGH INTEGRATION OF PROTEIN-PROTEIN AND GENETIC INTERACTIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made great effort to address most of the concerning points, therefore would like to appreciate them.
Experimental design: The authors have addressed my comments efficiently.
Validity of the findings: No comments
Additional comments: No 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: FUNCTIONAL NETWORK MOTIFS DEFINED THROUGH INTEGRATION OF PROTEIN-PROTEIN AND GENETIC INTERACTIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: - There is a typo in the legend for Figure 1a.  ("recoreded")
- I appreciate the addition of figure 1a to clarify some of the terminology and concepts.

- I appreciate the additional explanation on lines 57-62.  That said, I think the language used by the authors is still confusing, however.

For example, your paper states: "Positive GIs, wherein the double deletion is less severe than expected, are often linked to dependencies within the same function while negative GIs,
derived from deletions of two genes whose combined effect is more severe than expected, are
frequently observed between redundant and compensatory processes (Beltrao et al., 2010)."

The source you cite explains the same concepts differently: "We have shown that pairs of yeast mutants that display positive genetic interactions often indicate two proteins that act in the same pathway or are physically associated (Figure 1B) (Roguev et al., 2008, Collins et al., 2007). A possible explanation is that if removal of a component of a complex disables that complex, then deleting a second component would have no additional effect, resulting in an epistatic (i.e., positive) interaction (Figure 1A). Alternatively, deletion of one component of a complex could result in partial dysfunction of that complex with a detrimental effect on cell viability. If the removal of an additional component completely disabled this detrimental function, then the result would be a suppressive relationship, another type of positive interaction."

I think the explanation provided by the source (e.g., the impact of deleting the pair is not much greater than deleting a single gene) is much clearer than the language used by authors.  Since the paper is focused on the functional nature of the interactions, the authors should expand their explanation.  Genetic interactions are very important concept to understanding the overall paper.

- Another reviewer commented on the confusion between the terms "conventional" network motifs and FNMs.  I think it would be clearer to label them as PPI network motifs and PPI+FNM network motifs.  All of the motifs you find are conventional PPI network motifs -- if I am understanding correctly, you are just selecting the subset for which a minimum fraction of the edges have GIs.
Experimental design: no comment
Validity of the findings: - It seems to me that you could have tried a few different approaches.  For example, you could generate a GI network and filter the motifs by a minimum fraction of PPIs.  Why is it advantageous to start with the PPI network and filter by GIs rather than the other way around?  PPIs capture direct interactions of adjacent components of a pathway, while GIs capture indirect relationships (such as two genes that serve in redundant pathways).  Please spend more time clarifying these differences and how the presence of a PPI but not GI, GI but not PPI, or both should be interpreted.
Additional comments: High level comment: I think this paper is well suited to an audience familiar with PPIs or GIs who are looking for new analysis methods.  Explanations of the fundamental concepts and how to interpret the results are insufficient for a generalist bioinformatician to decide whether FNMs would be a useful analysis to do for their given problem.


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: MOLECULAR IDENTIFICATION AND ANTIBIOTIC RESISTANCE PATTERNS OF DIVERSE BACTERIA ASSOCIATED WITH SHRIMP PL NURSERIES OF BANGLADESH: SUSPECTING ACINETOBACTER VENETIANUS AS FUTURE THREAT
Review round: 1
Reviewer: 1

Output:
Basic reporting: -The article title is overwhelmed. The study did not show the risk analysis on Acinetobacter venetianus properly. It is based on author's opinion.  Most of contents show the bacterial findings using molecular and microbiology methods. I suggest the authors adjust the title.
- The introduction part is redundant, for example a background on hatcheries was described on line 59 -85. It can be concluded within 2-3 sentences.  It should relate to the article topic.
-Result and discussion contents are repetitive in table, figure, and descriptive contents. Contents (bacterial isolation, molecular identification, and antibiotics susceptibility) are redundant which can summarize in tables. Because this study was local finding, discussion part should more precisely on comparison to other regions and reveal readers gain advantage how necessary to read and understand a situation in Bangladesh.
-Overall, of this article, lots of contents are unnecessary. The authors should analyze and summarize which readers can take advantage and do not waste a time to read.
Experimental design: - The sampling method does not show statistical methods. How to make readers confidently understand that samples were representative of PL-hatcheries in this study.
- To give reasonably for risk analysis, statistical analysis on Acinetobacter venetianus should be provided, not based on opinion.
Validity of the findings: - This study is local information, but it could be fundamental data comparing to other geographical regions or industries. Authors can provide more comparative information that be useful for readers.
Additional comments: no comment.


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

Input:
Title: MOLECULAR IDENTIFICATION AND ANTIBIOTIC RESISTANCE PATTERNS OF DIVERSE BACTERIA ASSOCIATED WITH SHRIMP PL NURSERIES OF BANGLADESH: SUSPECTING ACINETOBACTER VENETIANUS AS FUTURE THREAT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English writing needs substantial improvement. Standard wording and international numbering units should be used. Refrain from using 'lakh', 'crore' and 'ghers' on their own. Some sentences are unclear and needs to be rephrased. Please observe the consistency, eg., Gram positive/ negative vs gram positive/ negative; rod-shaped vs rod shaped

Additional literature refences are needed as to whether Vibrio should still be regarded as opportunistic pathogens after the emergence of acute hepatopancreatic necrosis disease (AHPND) involving a number of Vibrio spp besides V. parahaemolyticus.

Table 1 needs revision. The colony size should be in mm (diameter) instead of small, medium or large. The gel lanes in Fig 2A need proper labeling.
Experimental design: L146: Live PL?
L150: What tool/ equipment was used for the homogenization?
L159-160: Unclear description...'Only from treated water, 100 μl of processed raw sample and 10-2 dilution was inoculated on different agar plates'. Please revise for better clarity.

There should be negative control for the 16S PCR.

Please add a few more (instead of one or two) homologous 16S sequences from GenBank for each inferred species for multiple seq alignment and construction of phylo tree.
Validity of the findings: Conclusion
It should not be generalized based on the previous studies that all strains of certain bacterial species are pathogenic or non-pathogenic. To conclude if the isolates are pathogenic or otherwise, it should be proven by experimental infection on case by case basis.
Additional comments: Shrimp aquaculture does not actually protect the wild shrimp resources from depletion. While farming produces much more shrimp than catching from the seas, shrimp-fishing industry still continues. Also, some shrimp broodstocks are still being caught from the seas for PL production in hatcheries.

The B.licheniformis and B. pumilus isolates might be associated with probiotic use in the nurseries.

Some of the isolates could be the same strain.


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: MOLECULAR IDENTIFICATION AND ANTIBIOTIC RESISTANCE PATTERNS OF DIVERSE BACTERIA ASSOCIATED WITH SHRIMP PL NURSERIES OF BANGLADESH: SUSPECTING ACINETOBACTER VENETIANUS AS FUTURE THREAT
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall the manuscript is interesting and can be published after the revisions.
Experimental design: Good.

Please explain more on how many replicates of samples (PL, 3 kinds of water) were collected.
Validity of the findings: yes
Additional comments: It will be good to add more discussion on bacteria association with shrimps that were studied in other countries particular in SE Asia/Asia.

English grammar needs to be check thoroughly.


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: MOLECULAR IDENTIFICATION AND ANTIBIOTIC RESISTANCE PATTERNS OF DIVERSE BACTERIA ASSOCIATED WITH SHRIMP PL NURSERIES OF BANGLADESH: SUSPECTING ACINETOBACTER VENETIANUS AS FUTURE THREAT
Review round: 2
Reviewer: 1

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


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

Input:
Title: MOLECULAR IDENTIFICATION AND ANTIBIOTIC RESISTANCE PATTERNS OF DIVERSE BACTERIA ASSOCIATED WITH SHRIMP PL NURSERIES OF BANGLADESH: SUSPECTING ACINETOBACTER VENETIANUS AS FUTURE THREAT
Review round: 2
Reviewer: 2

Output:
Basic reporting: The writing still needs improvement to be clear and unambiguous, and also to get rid of typo and spelling errors, as well as the inconsistent format.

L90-91: Please revise the sentence to be more informative by mentioning the species reported (V. owensii and V. harveyi). Please also refer to this reference:
Vibrio parahaemolyticus and Vibrio harveyi causing Acute Hepatopancreatic Necrosis Disease (AHPND) in Penaeus vannamei (Boone, 1931) isolated from Malaysian shrimp ponds (https://doi.org/10.1016/j.aquaculture.2019.734227), which provides more info than Kondo et al (2015).

L177: Provide the reference for interpretation of susceptibility/ resistance.

L380-381: Please also refer to "Virulence properties and pathogenicity of multidrug-resistant Vibrio harveyi associated with luminescent vibriosis in Pacific white shrimp, Penaeus vannamei" (https://doi.org/10.1016/j.jip.2021.107594) for more relevant interpretation of MAR index value.

Please refer to the enclosed PDF for highlight and annotation.
Experimental design: Method description needs improvement.

Please refer to the enclosed PDF for highlight and annotation.
Validity of the findings: Conclusion:
The bacterial isolates in the present study have not been tested to be pathogenic. The pathogenicity status should not be based solely on the findings of previous studies by other researchers. “Potentially pathogenic” is more appropriate.

Please refer to the enclosed PDF for highlight and annotation.
Additional comments: L41-42: more than 0.2 means prior high exposure to the antibiotics
L61: “theshrimp” typo
L65: nauplii
L82: cause
L116: (after filtration and disinfection)
L120: used as pooled samples
L129: Vibrio spp.
L132: diverse
L133: μl inconsistent with μL
L135: "first processed water sample before dilution" - do you mean "undiluted". Please rephrase for clarity.
L152: Wizard SV Gel and PCR Clean-Up System
L159-160: “The reported sequences in this study have been deposited in the NCBI GenBank database under accession numbers MT368010 to MT368038.” should be in the results

L169: the bacterial isolates have not been tested to be pathogenic yet at this stage, so "pathogenic bacteria" is inappropriate. It should be "the isolates"

L175: "broth culture" instead of "bacterial suspension"
L174: Mueller
L194: estimated 1500 bp
Comment: DNA ladder only estimates the size of DNA band
L212: "100% identity" is more appropriate versus "were confirmed"
Comment:  Further in-depth analysis by multilocus sequence analysis (MLSA) may sometimes dispute the identity inferred from 100% similarity shown by BLAST pairwise alignment

L217-218:  “The identified bacterial strains have been summarized in the Table 3 according to the water and shrimp PL samples source collected from eight different shrimp PL nurseries.”  -  this should be placed after the phylogenetic results.

L221: “relatively short sequence” instead of “partial sequence”
Comment: Even the estimated 1500 bp sequences are not considered complete sequences.

L235: Be consistent with the term: antibiotic vs antimicrobial vs antibacterial

L239-240: Rephrase “Tetracycline showed high resistance since there was no sensitive isolates against tetracycline except V. xuii ”
No isolates were sensitive to tetracycline except V. xuii

L246: means prior high exposure to the antibiotics
L247: including
L253: The present study found Vibrio to be
L255: Revise “can turns into opportunistic pathogens” to “can turn pathogenic”
L262: revealed
L264: “caused” instead of “formed”

L266-267: “V. parahaemolyticus is responsible for shrimp disease known as early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) in cultured shrimp”…this is not generally true. Please revise.
AHPND is caused by Vp strains and other Vibrio spp that carry pir toxin genes (in plasmid). It should not be generalized that Vp causes AHPND.

L271: revealed
L293: incidents
L307: Revise “which was isolated from seawater” -  which has been isolated from seawater in previous study
L312: found
L313: . Z. denitrificans has been previously isolated from...
L314: both have been identified as novel species

L328: observed
L329: were reported to be present
L332: Danube River
L357-358: Revise “This is an indication that how discriminately antibiotics are being used”
This is an indication how antibiotics are being indiscriminately used

L358-359: Revise “and imposing health risk to shrimp and as well as to human by making multidrug-resistant bacterial strains.”
- and pose health risk to shrimp and humans by promoting emergence of multidrug-resistant bacterial strains

L365: suggested
L366: Revise “this species identified”  - the strain identified
L369: Most of the Shewanella spp. were
L371-372: found three S. algae strains that were resistant
L373: revealed
L376: showed
L377: showed
L378: high resistance prevalence against
L382: resistant to
L385: Vibrio species not identified?
L389: showed
L390: Revise "has been come to the spotlight" -  has come under the spotlight
L390-391: Please clarify how the antimicrobial resistance has come under the spotlight as the COVID-19 crisis is badly affecting almost the whole world

Revise “This study concludes the possible presence of both pathogenic and nonpathogenic bacteria”

Consider:  This study concludes the presence of both nonpathogenic and potentially pathogenic bacteria…

L407: potentially pathogenic


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: MOLECULAR IDENTIFICATION AND ANTIBIOTIC RESISTANCE PATTERNS OF DIVERSE BACTERIA ASSOCIATED WITH SHRIMP PL NURSERIES OF BANGLADESH: SUSPECTING ACINETOBACTER VENETIANUS AS FUTURE THREAT
Review round: 3
Reviewer: 1

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


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

Input:
Title: REMOVAL OF MICROCYSTIS AERUGINOSA CELLS USING THE DEAD CELLS OF A MARINE FILAMENTOUS BACTERIUM, AUREISPIRA SP. CCB-QB1
Review round: 1
Reviewer: 1

Output:
Basic reporting: Long and listing up sentences are sometimes not so fluent (red tread) from sentence to sentence. Example:
As the plant-based natural coagulants, some seeds and fruit/ peels are reported such
90 as Cicer aretinum, Moringa oleifera, cactus, etc. (Kumar, Othman & Asharuddin, 2017). Several
91 bioflocculant-producing bacteria belonging to genera Bacillus (Deng et al., 2003; Zheng et al.,
92 2008), Corynebacterium (He, Li & Chen, 2004), Klebsiella (Wang et al., 2007), Paenibacillus (Li
93 et al., 2013), Proteus (Xia et al., 2008) and Pseudomonas (Azzam & Tawfik, 2015; Liu et al.,
94 2016) were found during past decades. These bioflocculants were exhibited high removal
95 efficiency (80 - 99.6 %) on negatively charged clay particles, kaolin. Several investigations
96 demonstrated that bioflocculants were useful for removing M. aeruginosa.
Check grammar, example:
100 … The investigation conducted by
101 Zhang et al. demonstrated that the bioflocculant produced by Halobacillus sp. strain H9 was also
102 successfully removed M. aeruginosa cells (Zhang et al., 2019).
This should be moved to discussion section:
Our previous investigations demonstrated that CCB-QB1 cells were aggregate in the
210 presence of 7 mM CaCl2 (Furusawa, Hartzell & Navaratnam, 2015), and the dead cells of CCB-
211 QB1 formed aggregates with kaolin clay particles in the same condition (Hasyimah, Furusawa &
212 Amirul, 2020). In keeping with these data, we expected that the dead cells of CCB-QB1 could also
213 form aggregates and precipitate
This should be moved to discussion or method section:
FeCl3 was used as a flocculant for removing M. aeruginosa cells from aqueous samples (Li
250 et al., 2015; Hao et al., 2016)(GAO, SHIMIZU & XUE, 2012). In this study, we also performed
251 to use FeCl3 as a coagulant.
Please avoid leading regarding result, this is an open question still as the result has not yet been shown and the method should be neutral
176 First, to confirm the removal
Avoid expectations
213 form aggregates and precipitate with M. aeruginosa cells. To confirm the expectation, the dead
Result, discussion and conclusion:
278 the presence of 7 mM CaCl2. In addition, the removal efficiency of NISE 102 cells decreased under
279 low CaCl2 concentration (1 mM) (Figure 3), indicating that CaCl2 is crucial for removing NISE
280 102 cells.
Why is the removal efficiency lower in figure 3: 5 mg/ml than all the samples in the figure without CaCl2 (Fig 1) 5 mM should be comparable? Isn’t figure 1 without CaCl2? Then there is a higher removal efficiency without CaCl2, or? So why does removal efficiency decrease with addition of CaCl2. Same effect is seen in 10 mg/L in figure 4a. Please provide a negative control in all figures.
Experimental design: Indicate replicates in the figure text also provide controls as described
Related to result, discussion and conclusion:
Aggregate formation of the dead cells with NISE 102 cells: please describe figure 1 in more detail
Figure 1:
It is not so clear that this is a binding of the two cells from this image. Provide positive and negative control
Also show that this could be repeated, indicate how may replicates were used
Figure 2:
3 min incubation. What is the indication that 1 mg/ml and 2.5 mg/ml is not significantly different, and therefor the choice of 5 mg/l to continue?
Figure 3
Provide negative control
Figure 4
Provide negative control. Better describe the difference of a and b in the figure text. Especially b, what is the condition compared to a, is there anything else in there and how is b related to a. Not clear in the figure text. Is 4a without dead cells? If so be more clear reg. difference.
Figure 5
Provide positive and negative control
Figure text,: what is d? “(a), (b), (c), (d) Samples without FeCl3. (d), (e), and (f) Samples with FeCl3”. It makes it not possible to know what the figure tells.
Validity of the findings: Please see points in section 2 for next check the validity of the findings. Elaborated further;
As mentioned in the introduction it should be tested also absorption of e.g heavy metals with this method
“116 also absorbed heavy metals, such as Fe3+ and Cu2+, and removed it from the solutions (Hasyimah,
117 Furusawa & Amirul, 2020).”
The efficiency vs incubation time measured after finding the correct combination of cells and chemical. This seem relevant to avoid adding high concentrations of CaCl2 and FeCl3. e.g. concentration vs time. Adding longer time might be more economically beneficial v.s adding chemicals.
Additional comments: The results seem promising for a method of increased sustainability in removal of M. aeruginosa cells that might be also extended in further studies.
However there are some essential parts, as mentioned that should be changed.
Especially in the results, see points and that should be provided e.g in order to analyze and validate the results.
Figures/result/discussion conclusion: There are no positive or negative controls. Please provide these data and other points made here, in order to evaluate the data, and change the description of result, discussion and conclusion accordingly.
Check language, flow between sentences, and some grammar (see examples in section 1),
use of italic
Abbreviations can be useful but sometimes confusing if used in other known concepts such as PAM.


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: REMOVAL OF MICROCYSTIS AERUGINOSA CELLS USING THE DEAD CELLS OF A MARINE FILAMENTOUS BACTERIUM, AUREISPIRA SP. CCB-QB1
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The content and data of the article are insufficient and lack of scientific value. This work just evaluates the flocculation effect of M. aeruginosa by dead cells of Aureispira sp., did not explain why it can be flocculated, and what are the influencing factors of flocculation effect? In addition, the application area of this technology is water column. What is its impact on water quality? Do these dead cells have inhibitory effect on flocculated cyanobacteria?
2. There are too many clerical errors in the text. For example, (Hasyimah, Furusawa & A) in line 132; “5 gm/ml” in line 180 and 186; “5.0 mg/m” in line 233; “(Li et al., 2015; Hao et al., 2016)(GAO, SHIMIZU & XUE, 2012)” in line 250; “of FeCl3as” in line 257; “(Corpe, 1970; (Li et al., 2019; Pajerski et al., 2019)” in line 286, “flocculant, FeCl3, was improved by addition of CaCl2” in line 351. Please check the manuscript carefully before submitting it
Experimental design: The experimental design is puzzling. Only 10ml of algal liquid is used for flocculation test. What is the liquid depth in the flask, and can the supernatant be effectively separated? Please provide some photos of flocculation test. In addition, as the first experiment, how to determine the concentration of CaCl2 and dead cells in the section "observation of aggregation formation of the dead cells on NISE 102 cells". Please describe the purpose of the test in detail.
Validity of the findings: 3.	Two data graphs seem to be contradictory. Is the experiment in Figure 2 without CaCl2? The 5 mg/ml dead cells can remove more than 70% of cyanobacteria. However, the removal rate of cyanobacteria by 1 mM CaCl2 and 5 mg/mL dead cells in Figure 3 is only about 30%, which is puzzling.
Additional comments: Abstract:
1.	The abstract is a summary of this study. Generally, "our previous report demonstrated" cannot appear in abstract. Also, in the conclusion
2.	The abstract needs to be simplified. Generally, it is not necessary to list the “background”, “methods” and “results”, and the methods should be as brief as possible.
Methods and materials
1.	It is not rigorous to use the mass of wet cells weighed after centrifugation as the addition amount, which will have a great error.
2.	Line 142. Is H-ASWM the “high nutrient artificial seawater medium” mentioned above. Abbreviation should be marked when it first appears
3.	Line 152. “5 mg/ml of the dead cells” should be replaced by “50 mg of the dead cells”.
4.	Line 157-158. Is there BG11 medium or CaCl2 solution in the NISE 102 cell suspensions? In other words, what else is in the cell suspensions besides NISE 102 cells? This needs to be explained clearly, as well as in the following experiments
Discussion:
The discussion part needs to pay more attention on why dead cells can be used as flocculants to remove cyanobacteria, instead of why the addition of CaCl2 and FeCl3 can improve the removal efficiency.
Figures:
1.	A control group without CaCl2 should be added in Figure 3.
2.	The title of figure 4a should indicate whether there were dead cells, and what was the concentration of CaCl2. The title of figure 4b should indicate whether there was CaCl2 and what the concentration was.
3.	“(a), (b), (c), (d) Samples without FeCl3” in the title of Figure 5 should be replaced by “(a), (b), (c) Samples without FeCl3”. In addition, no figure 5g.
Reference:
The order of references is not correct. If you want to follow the order in the text, you should replace “(… et al, ….)” with numbers, otherwise, you should order it alphabetically.


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: REMOVAL OF MICROCYSTIS AERUGINOSA CELLS USING THE DEAD CELLS OF A MARINE FILAMENTOUS BACTERIUM, AUREISPIRA SP. CCB-QB1
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.	Language of the manuscript should be improved. Poor sentence formation with lot of grammatical mistakes are present in the entire manuscript.

2.	Punctuation errors are present at various places in the manuscript, for e.g line 149, 151-152, 189, 192

3.	Line 142- explain what is “modified H-ASWM”

4.	Avoid starting a sentence with a number, e.g line 152,157, 186. Either frame your sentence in such a way so that it does not starts with a number or write the number in words.

5.	Formatting errors are present, e.g line 282

6.	All the references should be in uniform pattern. There are variations at various places.
Experimental design: Data indicating cell surface chemistry should also be included
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: REMOVAL OF MICROCYSTIS AERUGINOSA CELLS USING THE DEAD CELLS OF A MARINE FILAMENTOUS BACTERIUM, AUREISPIRA SP. CCB-QB1
Review round: 2
Reviewer: 1

Output:
Basic reporting: The questions were answered according to the last review.
Experimental design: The questions were answered according to my last review and what was possible and relevant.
Validity of the findings: The questions were answered according to my last review and what was possible and relevant, I have no further comments.
Additional comments: Thanks for carefully going trough the comments, I have no further questions.


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

Input:
Title: COMPARATIVE ANALYSES OF CHLOROPLAST GENOMES IN ‘RED FUJI’ APPLES: LOW RATE OF CHLOROPLAST GENOME MUTATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript by Miao and colleagues report on the determination of chloroplast genomes from 24 Red fuji with detailed analysis of genetic variants study from comparative analysis of chloroplast genomes. In fact, chloroplast genomes at the same species level are generally not different. Although the findings of the authors are very clear, the purpose of the comparative analysis with the chloroplast genome analysis of 24 samples of the same species should be clarified. In a way, this result can be a natural result. Considering the result of producing 50 Gb of data, I thought that it may have been carried out as a previous study for population genetic research such as GBS, GWAS for Red fuji. Please state clearly the purpose of using the 24 samples of Red fuji.
Experimental design: It is very clear except for the material part.
Validity of the findings: It should be noted that the 24 chloroplast genomes are highly conserved.
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: COMPARATIVE ANALYSES OF CHLOROPLAST GENOMES IN ‘RED FUJI’ APPLES: LOW RATE OF CHLOROPLAST GENOME MUTATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are numerous language error in the manuscript. I would suggest that the author send the manuscript for a proofread by a native speaker.

Some of the figure were not clear, in particular Figure 5 the annotation for tRNA was not clear.

The author should indicate the published apple chloroplast genome by Yan et al. (2019) in the introduction.
Experimental design: Author showed lack of analysis for the chloroplast that were sequenced. There were also lack of description for de novo assembly of the chloroplast and information about the reads obtained from sequencing. Details on the comment for methods can be find in attachment. Author also did not mention that the reads or the sequence assembled were deposited to the public database. Method for DNA extraction is also lacking and the sequencing platform used is also not mentioned.
Author should also include a comparison with the published apple chloroplast as reference as it is already available.
Research question is well defined but the analysis done is not enough to answer the research question. Phylogenetic analysis was lacking and the description to the variation seen across the 24 chloroplast sequenced were also not detailed in manuscript.

I also have a concern over the correction done at the start and stop codon as indicated by the author. What is the exact correction that was done to determine the start and stop codon?
Validity of the findings: The author certainly has develop a good research question but the analysis done was not enough to answer the question.
The author has sequenced 24 chloroplast but in analysis only include 9 chloroplast for comparison by localities, so how to justify variation that may exist for sample from same localities. Some of the literature used to support argument are also very old (eg line 230).
Additional comments: The research has a merit but the analysis done is lacking and still has a room for improvement. Author can further improved in term of phylogenetic analysis and to describe in detail the variation exist among the 24 chloroplast sequenced.


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

Input:
Title: COMPARATIVE ANALYSES OF CHLOROPLAST GENOMES IN ‘RED FUJI’ APPLES: LOW RATE OF CHLOROPLAST GENOME MUTATIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Miao et al. sequenced and characterized the entire chloroplast genomes of 24 Fuji apple cultivar 'accessions.' The article is written in a standard format similar to other published articles, and the science is sound and easily understandable, though it could be improved further, as indicated in the comments below.

For a strong basis for the rationale of this study, consider introducing what has been done in terms of chloroplast genome sequencing in Malus, with a special emphasis on cultivated apples such as M. domestica and M. pumila. Although the topic of this article appears to be confined to within cultivar characterization and comparison (identification of mutation hotspot areas), you can use previously reported cp genomes to provide insights into the evolution of the cp genome in cultivated species/cultivars. Also, it is not clear why you used the cp genomes of M. sierversii and M. sylvestri in the various analyses for comparison, and why you excluded the cultivated species. The language requires thorough polishing to enhance clarity; see a few examples below.

Specific comments:
Language.
Line 20: ‘…, which belongs to…’
Line 24: what materials? You may consider using terms like varieties/accessions/cultivar…
Line 39: “…fruits (Jung, S & D, Main, 2014). In 2018, China, which is the largest producer of apples in the world (Li et al., 2019), produced more than half (43.88 million tons) of the global production (80.82 million tons).
Line 45: such as
Line 48: how is ‘colorfulness’ an advantage?
Line 50: ‘…longer shelf-life…’
Line 51 delete ‘by consumers’
Line 52: …on genetic…
Line 56 …organelle…
Line 57… commonly used
Line 71: In recent years, the number of…
Line 77: …not only deal with…
Line 81: ‘climate…’, ‘…not yet known…
Line86: ‘…to investigate mutations in the chloroplast DNA of the Red Fuji apple.’
Line 91: ‘…was collected from nine regions in China.’
Line 93: ‘…taken to the laboratory.’
Line 96’…using second-…
Line 100: ‘…to correct…’
Line 218: ‘…was rarely low’ this is somewhat ambiguous as it can be interpreted to mean that the variation rate was always high?

The following corrections can significantly improve
Consider stating the results without repeating details that are already presented in the methodology section. For example, you can delete the first sentence in line 194; ‘Using the MISA software… in line 199; line 207-208; line 214-215 restructure the sentences to remove the software used.
Line 139: delete ‘collected’
Line 139-140: what about the structure of the cp genome?
Line 157: Give details in the materials and method section on how the nine samples were selected. Was it random?
Discussion
Lines 270-273: which study is relevant here Li et al’s or Fang et al’s, both please rewrite the sentence and consider adding the year for Li et al’s study.
Experimental design: The study's objectives are in line with the journal's scope.
The methods require further input to allow for reproducibility. See comments below.

Give details on how the DNA was extracted, how quality was checked
How data cleaning was attained, and using which programs/parameters?
What was the amount of clean data for each sample?
Line 98: provide the version and reference for each of the software/program/website-based tools used.
Line 104: please consider writing all abbreviations in full at first mention…
Line 126: expound on the motif settings to allow for reproducibility
Validity of the findings: There is no information on whether and where the data was deposited. The data could not be reviewed and verified.

Conclusion:
Lines 280-281: This conclusion is not supported in any way by the performed analyses. How can you explain that timeline?
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: COMPARATIVE ANALYSES OF CHLOROPLAST GENOMES IN ‘RED FUJI’ APPLES: LOW RATE OF CHLOROPLAST GENOME MUTATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript has been properly revised for publication.
Experimental design: It has been properly prepared for publication in this journal.
Validity of the findings: It has been properly prepared for publication in this journal.
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: CLIMATE IN AFRICA SEQUENTIALLY SHAPES SPRING PASSAGE OF WILLOW WARBLER PHYLLOSCOPUS TROCHILUS ACROSS THE BALTIC COAST
Review round: 1
Reviewer: 1

Output:
Basic reporting: Comments attached!
Experimental design: Comments attached!
Validity of the findings: Comments attached!
Additional comments: Comments attached!


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: CLIMATE IN AFRICA SEQUENTIALLY SHAPES SPRING PASSAGE OF WILLOW WARBLER PHYLLOSCOPUS TROCHILUS ACROSS THE BALTIC COAST
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, I found the work to be well cited, putting this work in the context of previous work to come before it. I do, however, think that there are areas that could be more concise, which would improve the readability (and hence, accessibility) of the work. I found some sections to be somewhat repetitive.
Experimental design: The general questions and approach used to answer these questions is sound. More detail is needed in areas to more clearly describe what was done. See detailed comments in 'General comments to the author' section.
Validity of the findings: Some additional work is needed to clarify what was done and some additional tweaks to analyses are needed. For instance, there are areas where p-values are presented that probably should not be. There are other areas where multiple comparison need to be controlled for if presenting p-values. Other times I was unsure what was done, making it difficult to assess the appropriateness of the approach used. Those areas are noted in the detailed comments below.
Additional comments: General comments:

This study makes use of a long-term dataset to attempt to better understand what drives the migratory phenology of a long-distance migrant. The approach is interesting and does go beyond what was previously done. The suggestion that these various indices influence arrival at different periods of time is interesting, as is the notion that one sub-species arrives before another on the Baltic coast (the opposite of what is seen on the breeding grounds). Overall, however, I think there are a number of points that need to be addressed before this work is ready for publication. See below for more detailed comments on the manuscript:

INTRO

L 74: I don’t think “Previous life stages” is the best term here. Something like ‘at overwintering grounds’ would be more appropriate.

METHODS

Fig. 1: The x-axis in B-J is difficult to interpret. I think this caption could also be more succinct. Also, I think all subplots should have the same y-axis range.

L 179: So number of juvenile captured for a given day was actually number of juveniles captured for a given day / total number of birds captured throughout the entire season?

L 193: Again, I’m not sure that ‘life stage’ is the best language here and overwinter period would be better. How were these months selected (the reader should not have to refer back to the previous paper for this)? From Fig. 1, it looks like the NAO has an effect ranging from Europe down to western Africa, above the Sahara. Are these birds only arriving in this region of Africa in April? It also isn’t clear which of these are ‘lagged’ (from the previous year) and which are not. This needs to be presented in Table 1. At the moment is looks like NAO in the Jun-Jul was used as a predictor of arrival in May of that same year (I don’t think this was what was done, but it is presented this way in the table).

L 197: The reader should not have to refer back to the previous publication for this information.

Fig. 2: The labeling of these period is not very intuitive, making it difficult to follow and interpret the figure as a reader. The larger windows represent 1/3 of the total area under the cross-year average curve. How were the smaller windows selected?

L 229: What does this mean? It was stated previously that they were standardized. Is it that they were modeled as a linear function of AA rather than as a polynomial, etc.?

General: If the question of interest is what drives migration, I’m not sure including year in these models is appropriate. There could be one models looking at trends over time and a set of models exploring the influence of climatic factors on phenology. In reading further down, it seemed like year was not included, but then in the Discussion it’s clear it was. I think what needs to be done needs to conveyed more clearly.

L 234: Please explain what this is. I assume this means that AICc was calculated for every subset of the full model (all covariates).

L 245: Somewhat odd to refer to ‘successes’ here. Suggest removing.

L 254: For which model were these pR calculated from? The full model? The “best” model?

L 265: How large? Is the possibly due to changing migration corridors from year to year?

RESULTS

Table 2: The best models are presented here. Typically models within 2 or 3 AIC of one another are considered equivalent. I see that more results are presented in the supplement which is helpful. I wonder if presenting something like the weights of each predictor (climate index) would be helpful here (basically, a metric how often each variable shows up weighted by the AIC of the model). Also, I find it somewhat odd to present p-values with these many models. Better just to present the r squared values, in my opinion (given that many models are fit in this type of analysis and multiple comparisons are not controlled for).

Fig. 3: The text is very small in the figures. It would be helpful to increase the font size.

Table S11: If anything about statistical significance (i.e., p-values) is going to be presented here, multiple comparison need to be controlled for (using something like a Benjamini-Hochberg correction), as multiple statistical tests inflate the possibility of false positive (incorrectly saying there is an effect with there is no effect).

S12: Multiple comparisons should be accounted for here as well.

L 345: What does SCAND having an effect have to do with the relationship between breeding success and timing? I think it is interesting to look at the effect of (a proxy of) breeding success, but I don’t understand the logic here.

DISCUSSION

General: I found the Discussion to be somewhat repetitive, with statements made multiple times.

L 362: Which indices were related to what areas? Please be explicit. It seems that SOI is representative of conditions in both southern and western Africa. How do you disentangle the effect of SOI on western vs. eastern migrants? How was it determined which geographic areas each index affected most strongly?

L 391: Temperatures on overwintering grounds? Breeding grounds?

L 407: I don’t think it makes sense for year to be included in the ‘full model’ used for model selection, as noted above.

L 412: Earlier in the paragraph, it’s stated that some indices showed trends over time and not others (L 401). Now it’s mentioned which indices these are. I think the writing could be more concise here, simply stating which ones changed. I think this could also improve the readability.

L 432: Are they actually under weaker pressure to arrive early? Is there evidence to support this? Females still need to breed at a time that matches favorable environmental conditions, even if they aren’t establishing territories.

L 592: This is SCAND the previous year, is that correct (given that birds have already migrated through the area by the time June comes around)? The Discussion makes it seem otherwise. I’m unclear on what was done here.

L 628: How was effort controlled for? What about pop size (number of adults, since more adults will = more juveniles without having higher breeding success)?

L 637: Wouldn’t they be second year birds (i.e., one year old) if they are arriving the spring after they were hatched?

L 651: I’m not sure this paragraph is necessary

General: Somewhat surprised that no mention of the potential role that future tracking efforts might play in confirming the proposed ecological explanation for the varying temporal effects of these indices (i.e., that one sub species arrives earlier than the other along the Baltic Coast).


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: CLIMATE IN AFRICA SEQUENTIALLY SHAPES SPRING PASSAGE OF WILLOW WARBLER PHYLLOSCOPUS TROCHILUS ACROSS THE BALTIC COAST
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: In this manuscript, the authors use the same dataset and the same methods used in a previous paper (Remisiewicz & Underhill 2020; DOI: 10.7717/peerj.8770) to address a similar question. The main difference to the previous study is here the authors analyze the effect of climate indices on three main sub-periods of passage (early, middle and late) and another 8 “minor” sub-periods. Furthermore, in this study, they consider the effects of juvenile counts in autumn, as a proxy for breeding success, on spring passage.

I find the manuscript interesting, since it presents a new approach using the effects of different meteorological conditions on the trapping patterns of willow warbles during different periods of passage to identify different populations of birds wintering in different areas in Africa. Although there is some overlap with the previous manuscript, this new approach and the novelty of results would justify the publication of this manuscript. Although I find the manuscript well and clearly written, I find it too speculative for the reasons outlined below.
Validity of the findings: One problem with the study system used, is that neither birds from different subspecies can be identified, nor can first-year birds be separates from adults, or males from females. Differential migration is found in most European passerine migrants, whereby adults migrate earlier than juveniles and males earlier than females. Differential effects of climate change on the timing of spring arrival and breeding in male and female, and juvenile and adult willow warblers were found by Hedlund et al (2015). Since there is evidence for differential migration in this species, the effects of age, sex and population are potentially confounded. Because of this, you should interpret your result with caution. How could changes in the age and sex composition of the willow warbler population on passage have influenced your results? You should discuss these effects as a potential source of error.

Although, I find the explanations for some of your different results plausible (e.g. for why P. t. trochilus migrates later than P. t. acredula, lines 465-474, or why SCAND and the number of juveniles in autumn delayed migration in late spring, 588-649), they are speculative since they lack empirical evidence. I would ask you to provide some empirical evidence for your explanation: e.g. ringing recoveries from your station that indicate that birds recovered in, or from, southern Sweden (or Norway) migrate through your station later in spring than birds recovered in northern Sweden (or Norway), Finland or Russia. If you do not have such data, you should explicitly state on which assumptions your explanations are based, and which data would be needed to test them.

The reason given for subdividing the migration period is that you assume that birds migrating though Bukowo at different periods in spring have wintered in different areas. Thus, they should experience different wintering conditions and different conditions during migration.  This, however is an untested assumption. In principle, the effects of different climatic indices could be indirect, e.g. through correlation with other indices or meteorological variables in other areas, or due to interactions with species, which are affected directly by these indices, during migration. The main problem of the subdivision of the migration period used is that it is somehow arbitrary, and that it is not based on actual knowledge of differences in the timing of passage between willow warblers originating from different breeding populations. Although you justify the subdivision into the 3 periods by practical criteria (lines 651-673), and the results obtained further justify your choice (667-671), a validation of results would be needed.

To demonstrate that your approach yields reliable results and that your conclusions on willow warbler migration are correct you would need to test your main assumptions, i.e. that differential effects of different climatic indices on passage reflect different wintering areas. A conclusive test would be repeating your analyses with a species exclusively wintering in western sub-Saharan Africa - the wintering area of Ph. t. trochilus – (e.g. Acrocephalus scirpaceus), and a species wintering exclusively in southeast Africa, the wintering area of Ph. t. acredula (e.g. Sylvia curruca). If you found that in these species the same climatic indices, which had an effect on the presumptive spring migration passage of the two willow warbler subspecies, then you would very convincingly demonstrate that (1) with your method you can identify different wintering areas and (2) that the climatic indices affecting these areas have an effect on spring migration of trans-Saharan migrants.

Specific comments:

Line 305: In table 2 the variance explained is 30.7% not 24.9%.

Lines 450-453, 465-473: According to Hedenström & Pettersson (1984) P. t. trochilus arrives about 2 weeks earlier that Ph. t. acredula in central Sweden. Is it plausible that willow warblers would need more than a month longer to fly to central Sweden via the Baltic States, Russia and Finland than directly via Denmark or crossing the Baltic Sea? You may estimate the time it takes if you know how much longer this route is and you know the approximate migration speed of willow warblers in spring.

Line 631: Can you exclude that populations change migration routes from year to year (e.g. because of winds)? If they did, this could explain the unexplained peak of juveniles in 1982.
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: CLIMATE IN AFRICA SEQUENTIALLY SHAPES SPRING PASSAGE OF WILLOW WARBLER PHYLLOSCOPUS TROCHILUS ACROSS THE BALTIC COAST
Review round: 2
Reviewer: 1

Output:
Basic reporting: Comments attached!
Experimental design: Comments attached!
Validity of the findings: Comments attached!
Additional comments: Comments attached!


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: CLIMATE IN AFRICA SEQUENTIALLY SHAPES SPRING PASSAGE OF WILLOW WARBLER PHYLLOSCOPUS TROCHILUS ACROSS THE BALTIC COAST
Review round: 2
Reviewer: 2

Output:
Basic reporting: Overall, I find both the text and figures to be much clearer, compared to this initial submission. I applaud the authors on their efforts here and in addressing concerns (from myself and the other reviewers) with the previous version of the manuscript. I found the _1yr notation and renaming of the ‘periods’ (early, mid, late) to be particularly helpful. While this paper is quite long, a substantial amount of information is presented here (both in the main text as well as in the supplement). Detailed explanations of the effect of each predictor are given, which may be useful for some readers. Some points could be conveyed more succinctly throughout the text, though I don’t think this is a major point of concern.

My remaining comments focus primarily on the readability of the manuscript. After addressing these issues, I feel this manuscript would be suitable for publication and make a nice contribution to the existing literature in this area.
Experimental design: The general questions and approach used to answer these questions is sound.
Validity of the findings: After addressing previous concerns from myself and other reviewers, I found assertions to be support by the findings of the authors' analyses.
Additional comments: This is a resubmission. I reviewed the initial submission of this manuscript as well.

More detailed comments on the manuscript:

INTRO

Fig. 1: I find the ‘migratory divide’ to be somewhat confusing – this is seemingly used to separate the two subspecies breeding ranges, but also separate Ph. t. tochilus pre- vs post-Bukowo migration. Suggest removing dotted lines associated with ‘migratory divide’ (particularly since there are already two types of dashed lines in the figure).

Fig. 1: I’m confused as to what the months indicate. The caption states those months are when birds occur in various regions. But if that were the case, why is there any _1y?

METHODS

General: Best not to refer to the ‘nominate subspecies’ for those not familiar with which subspecies is, in fact, the nominate subspecies. This term is used throughout the manuscript.

L 320: Just as a note, normality of the predictors isn’t a necessary condition for linear regression. The focus should be on the normality of the residuals (deviations between the linear predictor [e.g., regression line] and data points). Transformations are typically applied to the response variable. This transformation of the predictor may serve to curb the influence of ‘outliers’ though and I don’t think the analysis necessarily needs to be rerun.

RESULTS

Fig. 3: Lettering for figure panels needs to be fixed. B and C should be swapped, based on how they are referred to in the text (e.g., L331-334).

Table 3: It seems there is something missing after p < 0.0 in the caption.

Table 3: It seems the _1y is missing from the variable names in the table.

Fig. 4: While not a formal analysis, I like the exploratory nature of this figure and think it conveys valuable information to the reader.

Fig. 5: The x-axis appears to be the standardized square roots of number of juvs rather than the
square-root of the standardized number of juvs (as noted in the figure).

DISCUSSION

L 499-509: The addition of these points strengthens the argument here.


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

Input:
Title: CLIMATE IN AFRICA SEQUENTIALLY SHAPES SPRING PASSAGE OF WILLOW WARBLER PHYLLOSCOPUS TROCHILUS ACROSS THE BALTIC COAST
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: The authors have thoroughly revised the manuscript and replied to all of my comments. They have followed my suggestions to test some of their central assumptions. Despite this effort, however, they could not concinigly demonstrate differential passage of the two warbler subspecies, which winter in different areas in Africa. Although this conclusion is plausible, and their analyses and data do not contradict them, it is not supported by the results of the additional tests they have conducted:

Neither do their ringing recoveries show that P. t. acredula migrates earlier than P. t. trochilus at their ringing site (see, below), nor does the analysis of species wintering exclusively (or predominantly) in the wintering areas of the two willow warbler subspecies, i.e. western and eastern Africa, give clear results. While the results for Sylvia curruca are in line with the results predicted, the analyses of Ficedula hypoleuca and Sylvia communis are not (see, table R1). The authors explain the discrepacy (i.e the influence of IOD on migration of birds wintering in western Africa) by the observation that individuals of these species breeding in northern and eastern Europe may winter in eastern Africa. However, to my knowledge, most pied flycatchers winter in western Africa. Thus, if the author’s interpretation of results is true, the IOD should not significantly influence migration of this species. How could we then interpret the strong effect of IOD on the migration phenology of this species?

Furthermore, the author’s conclusion that the fact that wing-length decreases over the season supports the assumption that P.t. acredula migrates earlier than P.t. trochilus (lines 515-517) is not convincing. As they discuss in the paper (517-520), this decrease in wing-legth could be due to differential migration among sexes, females, which have shorter wings than males, migrating later. This could also be due to juveniles, which also have shorter wings, migrate later than adults. Can you exclude that during spring passage you catch a significant number of juveniles on passage? The fact that juvenile numbers in autmn significantly explains annual annomaly in spring passage (figure 5), could be an indicator that the proportion of juveniles in spring strongly affects spring phenology. To control for this pontential source of error you should include this variable in all the models, even if you need to eliminate some 1-2 years from the analyses. Would this change your results?

The discussion of the effects of different climatic indices (lines 536-731) is way too long and speculative, particularly when it refers to the potential effect of climatic effects on different populations, which presumably winter in different areas in Africa (as deduced from the differential effects of different climatic indices). I would suggest shortening this part and omitting speculative interpretations of the results.

Specific comments

Lines 499-509: Is there any evidence that Ph. T. acredula migrate along the coastline instead of crossing te Baltic Sea? Please, give reference.

Lines 510-517: The ringing dates do not support the assumption that Ph. t. acredula migrates earlier than Ph. t. trochilus. The only presumptive record of the acredula subspecies was ringed later than 8 of the 9 presumptive Ph. t. trochilus individuals you caught. Therefore, these data, if considered informative, rather suggest the opposite of what you expect:  Ph. t. trochilus migrating earlier than Ph. t. acredula. Of course, the value of these few records is limited, since you do not have data on sex are age, which could confound any result. Since these data are not conclusive, I would suggest that you either delete this paragraph or revise the interpretation of results.

Lines 515-534: The overlap in wing-length between the two subspecies is considerable (Fig. S6). Therefore, I don’t think that you can use wing length to separate them. Moreover, Fig. S6  shows that the largest wing lengths are not found in individuals that are trapped very early in the season, but in the middle (between, April 20 and May 7). The same hold true, for the willow warblers with the shortest wings. Considering that males have longer wings than females and adults longer wings than juveniles, any change in the composition of subspecies, sexes or age-groups will cange the distribution of wing lengths and of arrival dates. You should explicitly state this limiation in your dataset and the interpretation of results.
Additional comments: Im conclusion, I think that this is a interesting study with new and relevant results. However, considering the limitations of the data and the ambiguity of the results of the tests I suggested, the authors should be more cautious with the interpretation of results and discuss in more detail the shortcomings of the study and alternative interpretations of the results.


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

Input:
Title: CLIMATE IN AFRICA SEQUENTIALLY SHAPES SPRING PASSAGE OF WILLOW WARBLER PHYLLOSCOPUS TROCHILUS ACROSS THE BALTIC COAST
Review round: 3
Reviewer: 1

Output:
Basic reporting: I reported this in the earlier version.
Experimental design: I reported this in the earlier version
Validity of the findings: I reported this in the earlier version
Additional comments: I am satisfied with the author's revision. They have addressed most of my concerns. I recommend acceptance of this manuscript but with minor revision. In lines 232-238, the author still needs to include some sentences on why it is acceptable (or justify) to have differences in the time period of averaging for the Sahel and Willow Warbler.


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: CLIMATE IN AFRICA SEQUENTIALLY SHAPES SPRING PASSAGE OF WILLOW WARBLER PHYLLOSCOPUS TROCHILUS ACROSS THE BALTIC COAST
Review round: 3
Reviewer: 2

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: Overall, I am satisfied with the authors additions to this manuscript. My concerns have been address. I have only a few minor suggestions below.

General: I believe the terms used for the different AA periods in the supplement (which uses AA1.4_15.5, etc.) were not changed to reflect changes to those terms in the main manuscript (which uses MP1, etc.). I suggest making sure these are coherent.

L 125: Note lat/lon Bukowo and note that it is in Poland


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: CLIMATE IN AFRICA SEQUENTIALLY SHAPES SPRING PASSAGE OF WILLOW WARBLER PHYLLOSCOPUS TROCHILUS ACROSS THE BALTIC COAST
Review round: 3
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have made a great effort to revise thier manuscript considering the reviewer's comments and suggestions. They have satisfactorily and in great detail replied to all points raised by the reviewers, and have made changes accordingly. I particularly appreciate that in this new version of the manuscript the authors are more cautious with their interpretation of results, presenting alternative explanations. Moreover, they have tested and taken into account possible confounding effects, and have excluded those analyses and parts of the discussion that were inconclusive (e.g. the analysis of wing length). Thank you to the authors for giving background information on pied flycatchers and common whitethroats migrating through Bukowo, which allowed me to better understand their reasoning.


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: DECIPHERING THE HETEROPTERYS PANNOSA SPECIES COMPLEX (MALPIGHIACEAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The Heteropterys pannosa complex has perplexed botanists for generations, and the resolution presented here is a valuable contribution to the understanding of the Brazilian flora. It was a pleasure to read this exceptionally well-prepared manuscript. The illustrations are of fine quality (but see notes below). I have made a few suggestions for the text with the “comments” tool on the pdf. Below are some detailed notes.

Materials and Methods. The authors cite eleven herbaria, but many more are listed in the sections of specimens examined. It is customary to list under “Acknowledgments” all the herbaria that contributed to the study. Perhaps a note of explanation would clarify why only the eleven herbaria are listed.

H. tocantinensis. Throughout the treatment of this novelty the authors emphasize that the pedicel is greatly reduced and only about half as long as the pedicel. Yet, in Fig. 5 both in the habit sketches (A, B) and the detail (F) the peduncle and pedicel are roughly equally long.

Illustrations and captions. The drawings of the androecia confused me. They appear not to be bilaterally symmetrical; is that correct? It would be helpful to note in the caption the position of the stamens relative to the perianth, at least by noting which stamen opposes the anterior sepal (the longest one?).— Throughout the captions the word “nut” should be changed to “mericarp.”


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: DECIPHERING THE HETEROPTERYS PANNOSA SPECIES COMPLEX (MALPIGHIACEAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1- Organize the description of the genus by parts: 1) morphology, 2) systematic and 3) distribution (line 41, move the habit to morphological description).
2. Line 51, change word work.
3. Line 52, different habitats? Change word, too ambiguous, preferably delete.
4. I suggest that in the sentences that go from line 59 to 69, the examples are reduced and only the consulted investigations remain.
5. Appointment for the sentences of line 73 and 75?
6.Capitalize the letters of the figures that appear in the text. They appear in capital letters in the figure captions, as in the image.
7. Line 114 and 120 change work for studie.
8. Line 121 add (“).
9. Line 147 Goyas or Goiás?
10. I suggest a brief description of the genus Heteropterys
11. Line 158 margin in singular without the the
12. The description of the indumentum of lamina is missing, only that of the margin appears. It's confusing.
13. Description of bract indumentum is lacking. In H. pannosa.
14. In materials and methods it is not mentioned that the HDJF herbarium was reviewed, in line 199 it is cited. Like NY, IBGE, HEPH and MBM.
15. line 240 put first “after.
16. Is the elevation and biome known where H. pannosa is distributed? Add.
17. In all descriptions it appears as stems erect and cylindrical, I suggest removing it.
18. In line 279 put margin in singular. The description of the outfit seems confusing, you only have outfit in the margin?
18. Why is Francener listed as the author of all new species and is not in the article? I suggest adding it.
19. In H. tocatins, lines 367 and 368, the arrangement of the description of the characters is different from the previous ones, homogenize: size, shape, margins, eglandular or not, persistent or not. Indument is lacking for bract descriptions of H. rosmarinifolia. Bract indumentum is also lacking.
20. add the indumentum of the stipulates if they are present.
21. In the descriptions of all species, accommodate the description of characters for stamens and styles, size, shape, clothing. Homogenize.
22. All the collectors have their abbreviated names, homogenize in line 404.
23. Homogenize with the previous descriptions the characters of the bracts and bracts in H. walteri.
24. Figure 1, manifolds in italics.
25. figure 3, add space between H. pannosa and complex
26. Figure 3 I suggest adding caption for each image, it would be much better to interpret.
27. Figure 3, remove the citation from the text, it already appears in materials and methods.
28. Figure 4, manifolds in italics.
29. Homogenize the bibliographic references, in some citations the volume is added and in others not.


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: CHARACTERIZING THE SUCKLING BEHAVIOR BY VIDEO AND 3D-ACCELEROMETRY IN HUMPBACK WHALE CALVES ON A BREEDING GROUND
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript has been carefully and fluently written. The aims and results are clearly articulated. It follows a logical structure and is well referenced. There is room for some minor shortening, as I have indicated with tracked changes and there are couple of very minor typos. It was a pleasure to read.
Experimental design: This work fits the scope and aims of the journal and it fills some important knowledge gaps. There is room to briefly expand the relevance of the findings in the discussion (e.g. regarding threats from ship-strikes), as I have indicated using tracked changes. The ethical standards are difficult to assess from the minimal details presented. As I have mentioned, in the tracked comments, more information should to be provided about ethical approval (in line with the standards for this journal). Furthermore, information about the response of the animals to the tagging procedure should be reported.
Validity of the findings: The work appears to be of a high technical standard and I admit that I am not sufficiently familiar with machine-learning to critically review the statistical methods. The conclusions are concise and well reasoned from the results.
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: CHARACTERIZING THE SUCKLING BEHAVIOR BY VIDEO AND 3D-ACCELEROMETRY IN HUMPBACK WHALE CALVES ON A BREEDING GROUND
Review round: 1
Reviewer: 2

Output:
Basic reporting: Line 367: The meaning of “N” here is inconsistent with that in Line 281 and Line 332. Please use different symbols to represent different meanings or you could describe them using text directly.

Line 370: In Table 1, it’s better to explicit the detailed phases of dives, i.e., changing “during dive” into “during dive (descent)” or “during dive (bottom)”.

Line 394: I believe you want to refer to “Table 3” not “Table 2”. Please check it in the subsequent content.

Line 437-441: The contents should be placed in the methods section.

Line 441: In Figure 5, please unify the expression of runs and iterations, i.e., using either runs or iterations.

Line 465: “training set was small” not “test set was small”.

Line 523-525: Could you first explain what’s meaning of the depth-derived data? What does it include? It seems that the currently used features for machine learning contain all the features (Line 226-228), not limited to the accelerometer and depth-derived data.

The text inside figures is not consistent, and it should be consistent with the manuscript text size and style.
Experimental design: Line 263-265: Preliminary data processing has been conducted, i.e., removing some unwanted blocks. But, as we all know, the collected data might contain some noise resulting from some device or environmental factors during the data collection process. I suggest that you conduct some denoising operations such as signal filtering before feature extraction. It would be beneficial for performance improvement.

Line 277-278: The authors only trained three types of supervised machine learning methods and did not explain why to use them. I suggest that you try a support vector machine and random forest algorithm, which might obtain higher performance.
Validity of the findings: Line 444-446: Please add the detailed results of global accuracy and False Positive Rate for all of the pre-selected models. You could add an external table or figure to show them.

Line 506-514: In Figure 6, it looks that it doesn’t work well for the second combination when using the restricted list of features. I suggest the authors add some explanations. In addition, the sensitivity is a little low and it may be improved if you try some pre-processing method or try other classifiers like SVM. You can also elaborate more about the reason why it’s relatively low (Line 686-687).

Line 686-689: The reason why the priority goal is to maximize precision instead of sensitivity is not clearly explained. It needs further explanation based on the practical significance.

Line 710-712: I have concerns about the main difficulty when applying the automatic identification method in practice? The authors should clearly state the limitations of the proposed method in practical applications and should mention them in the discussion section. In addition, it would be better to add some future works.
Additional comments: Overall, the manuscript is well and detailly written. Looking forward to seeing you with the revised version.