Evaluation of Gut Microbiota Stability and Flexibility as a Response to Seasonal Variation in the Wild François’ Langurs (Trachypithecus francoisi) in Limestone Forest

ABSTRACT The coevolution between gut microbiota and the host markedly influences the digestive strategies of animals to cope with changes in food sources. We have explored the compositional structure and seasonal variation in the gut microbiota of François’ langur in a limestone forest in Guangxi, southwest China, using 16S rRNA sequencing. Our results demonstrated that Firmicutes and Bacteroidetes were the dominant phyla in langurs, followed by Oscillospiraceae, Christensenellaceae, and Lachnospiraceae at the family level. The top five dominant phyla did not show significant seasonal variations, and only 21 bacterial taxa differed at the family level, indicating stability in gut the microbiota possibly with respect to foraging for several dominant plants and high-leaf feeding by the langurs. Moreover, rainfall and minimum humidity are important factors affecting the gut microbiota of the langurs, but they explain few changes in bacterial taxa. The activity budget and thyroid hormone levels of the langurs did not differ significantly between seasons, indicating that these langurs did not respond to seasonal changes in food by regulating behavior or reducing metabolism. The present study indicates that the gut microbiota’s structure is related to digestion and energy absorption of these langurs, providing new perspectives on their adaptation to limestone forests. IMPORTANCE François’ langur is a primate that particularly lives in karst regions. The adaptation of wild animals to karst habitats has been a hot topic in behavioral ecology and conservation biology. In this study, gut microbiota, behavior, and thyroid hormone data were integrated to understand the interaction of the langurs and limestone forests from the physiological response, providing basic data for assessing the adaptation of the langurs to the habitats. The responses of the langurs to environmental changes were explored from the seasonal variations in gut microbiota, which would help to further understand the adaptive strategies of species to environmental changes.

bacteria that had significant effects on T3 and T4, when in reality they only identified taxa whose abundance is correlated with T3 and T4.
Language and interpretations: There are some issues with interpretations. For example, at L279-90, 283-4, 287-8, 292-6, 298, 299-302, 302-3, 303-4, 305-6, 311 and 312-4 (I don't list beyond here, but these represent many issues in a short span), the interpretations are overstated. In some instances I think this is because of language -for example, at L283-4 the authors are likely citing a previous finding, but it is phrased as a conclusion. Others, maybe also be languages, such as L 311 "gut microbiota varied significantly on smaller timescales [similar to humans]" the structure of the sentence implies that human microbiomes vary across small time scales but not large ones and cites papers that only support stability. I think the authors only intend those references to apply to the statement about long stability, but the sentence needs to be restructured to reflect this. Finally, for this issue about long-term stability and short-term variation I think this needs to be better explained in the text, as any short term variability must by definition mean there is long-term variability (but the cited studies are correct, the difference between the results just needs to be reconciled better in the text). Generally, I think the authors need to pay very close attention to the intro and discussion to make sure findings are not over interpreted.
Use of supporting information: I think some the information in the supporting information could be moved to the main text to keep the reading clearer. Certainly there need to be more details in the methods (L121 -what are the kits? what are the per parameters? L123 -what software packages were sued on the MajorBio Cloud platform, etc). Also, the authors' selection of figures adds some confusion to the manuscript. For example, there are no main text figures supporting measuring T3 or T4 levels, animal activity (these are in supplemental), and there is no taxon plot showing the overall microbiomes of the samples. These changes may not be required, but I recommend the authors pay attention to them to ensure the manuscript is clear and direct.
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Responses to Reviewers' Comments
NOTE: The reviewers' comments are in black, and our responses are in red. The line numbers in our responses are those in the clean version of current manuscript.
Associate Editor Comments to Author: Thank you for submitting your manuscript to Microbiology Spectrum. It has now been reviewed by three experts in the field, and they have raised issues that must be addressed before I can consider the manuscript appropriate for acceptance to Microbiology Spectrum. I encourage you to consider submitting a revised version of the manuscript, and, if you do so, to pay attention to themes raised by multiple reviewers. Additionally, one reviewer raised concerns about the differences in this study relative to one of your previous studies, and I encourage you to be sure to document these differences clearly in your response. Evaluating if this is a replication finding or not is an important part of the review process (some replication findings are useful whereas others are not).
Thanks for your comments. In current version, we revised manuscript according to the comments from editors and reviewers.
Our study is different from the previous study. Current study aims to examine the seasonal shift pattern of the gut microbiota of François's langurs, whereas the previous one attempts to detect the effect of captivity on the gut microbiota of the wild Francois's langurs. Moreover, current works cover a completed year circle and provide data based on 152 fecal samples from the wild langurs. The previous paper is based on 32 fecal samples (15 from the provisioned members and 17 from the wild individuals), which provides limited descriptive information without any data on seasonality. In addition, we combined behavioral and fecal hormone data into current study. Actually, the fecal samples used in the two studies were different because of the different sampling times and number of samples.

Reviewer 1:
The authors studied the compositional structure and seasonal variation in the gut microbiota of François's langur by 16S rRNA sequencing and fecal hormone assay. The design of this study is good, and the results can provide data basis for wildlife protection. The writing of the article is relatively rough, and English writing needs professional modification and improvement.
The main questions of this manuscript are: 1. The logic of content writing and key content are not highlighted: for example, the title of the manuscript indicates that it is an assessment of the gut microbiota of langurs under seasonal variation, while the abstract does not summarize the flora related to seasonal changes; The results did not show the information of genus level flora. The accuracy and recognition of 16S rRNA amplicon sequencing technology in the analysis of flora at the genus level are relatively high now. It is suggested that the author can summarize and display the flora at the genus level under the key flora at family level, so that the analysis will be more in-depth and enhance the readability.
We deeply appreciate for your comments, and we have summarized the seasonal changes of the gut microbiota in the langurs in the abstract, please see L28-31.
Indeed, we tried to analyze the compositional structure of the gut microbiota of the langurs at the genus level, but more than 250 bacterial taxa out of 664 were undefined, as were some taxa in the top 5 dominant families (Table 1), likely due to the abundant novel species in the gut of study langurs. And about 50% of the top 15 bacterial taxa in the genus level with high relative abundances were undefined ( Figure 1). This is why the subsequent classification of bacterial taxa only reached the family level.  2. The description of the contents and statements in many parts of this article is vague: For example, some contents of the method steps are not clearly written, the sentence "We studied two cohorts of wild Francois' langurs with stable numbers over the periods" led me to believe that this study studied two cohorts of wild Francois' langurs with stable numbers over the periods. In addition, why does the determination of fecal hormones in the results not use the samples in Table s1, but use the fecal samples in Table s2 collected at different times? This makes me very confused when reading this manuscript! We thank you for pointing out this problem. The sentence " we studied two cohorts of wild Francois' languages with stable numbers over the periods " is poorly formulated and we revised, please see L108-112.
It should be mentioned that we supplemented the second period experiment by discovering in the first stage that there is no significant seasonal variation in the relative abundance of most bacterial taxa of the langurs, trying to probe the seasonal regulation of energy metabolism of the langurs from physiological hormone studies, and further understand the adaptation by the langurs to limestone forest. This may have reduced the support for the results of gut microbiota during the first stage, and we acknowledge that the combined data on gut microbiota, physiological hormones, and activity time in the same period are more convincing. However, our study is the first report to combine these data to provide a comprehensive analysis of the adaptation by Francois' langurs to limestone habitat. The current study should properly reflect the characteristics and seasonal variations in the gut microbiota of the langurs living in the limestone forest.
3. Many of the words used in the full text are inaccurate: for example, the title in Table s16 uses " several primates", but this table clearly listed 11 animals, so it can be written directly as "11 primates". In addition, the remarks under this table are not properly written as "1-Firmicutes"; the "invsimpson" in Table s17 does not need to be italicized; The notional words in the title of Figure 2 need not be capitalized, and the four figures in the figure 2 need to be written with a, b, c and d respectively; What do a and b in Figure 3 represent? Figures 2 and 5 also need to refer to a, b, c and d.
Thanks for your valuable comments and help, we modified. Please see Table S16, Fig.2, Fig.3 and Fig.5. In addition, based on the comments of other reviewers, we deleted the original Table   S16 and changed the original Table S17 to Table S16.
4. Some writing of the full text are inconsistent: for example, the letter P should be capitalized; "triiodothyronine (T3) and tetraiodothyronine (T4)" are unified if the initial letters are all capitalized.
Thank you for your tips, we have modified it. Please see L251-252.

Reviewer 2:
The manuscript described microbiome characterization by seasonal variation in the wild Francois's langurs living in limestone forest. This paper revealed that microbiome represented relatively stable during the seasons. Only the lower bacteria taxa at the family level showed variable depending on seasons.

Major revision
1. Significant different microbiota composition at phylum and family level was identified in wild Francois langurs reared in the same place between this paper and a previous paper Thanks for your interest in this problem. Our study is different from the previous paper.
Current study aims to examine the seasonal shift pattern of the gut microbiota of François's langurs, whereas the previous one attempts to detect the effect of captivity on the gut microbiota of the wild François's langurs. Moreover, current works cover a completed year circle and provide data based on 152 fecal samples from the wild langurs. The previous paper is based on 32 fecal samples (15 from the provisioned langurs and 17 from the wild individuals), which provides limited information without any data on seasonality. In addition, we combined behavioral and fecal hormone data into current study. Actually, the fecal samples used in the two studies were different because of the different sampling times and number of samples.

Direct proof should be added regarding increased level of Christensenellaceae is associated
with dealing with foods such as young leaves and fruits.
Thanks for your suggestion. We had data on feeding activities of the langurs and could not https://doi.org/10.1186/s40168-020-00977-9 3. The energy metabolism have been known for association with various factors including other hormones.
Thank you for your information. Actually, after the first period found that there was no significant seasonal variation in the relative abundance of most bacterial taxa in the gut microbiota of the François's langurs, we examined the seasonal difference of thyroid hormones in their feces during the second period to explore their energy status, which may help us better understand the adaptations of the langurs.

Reviewer 3:
The manuscript reflects a broad and robust dataset that reports on the fecal (not the gut) microbiota of wild langurs, and also reports on the collection of some other animal data and the correlation between these values and microbiota abundance. The authors use numerous statistical tests and they are generally appropriately applied; however there are superior tests that the authors could and should use in their analyses. Finally, I am very concerned that the paper is not very easy to read, and the authors could do some work to improve the structure of the results and discussion.
Tests: The authors use ANOVA and adonis to discriminate differences in overall microbiota composition between treatments, and also use Wilcoxon tests to discriminate changes in abundance of individual microbes. I recommend the authors replace these analyses with PERMANOVA (adonis is PERMANOVA, but when run through some applications such as the qiime2 software, it may not be possible to use multiple covariates. If the authors move to R, they should be able to perform a PERMANOVA using the vegan package (function is named adonis). Also, a method such as ANCOM or LefSe could be used to identify individual microbes that vary in abundance between treatments.
Thanks for your suggestion. We used the "vegan" package in R (4.2.2) for PERMANOVA (adonis function) to evaluate the differences in gut microbiota communities between seasonal groupings, please see Fig S2. In addition, LefSe can display bacterial taxa with inter-group differences, but some studies also use Wilcoxon tests (Holmes et al., 2022, Huang et al., 2022, Rocha et al., 2022, which is also a method that effectively tests for difference in the relative abundance of bacterial taxa, and FDR is used to correct the P value of Wilcoxon tests, removing false positives. We believe that this method can effectively display whether there are seasonal differences in bacterial taxa in our study.
References mentioned above: the authors report that there is no difference in T3 or T4 levels with season; yet then they go on to report microbial abundances that covaried with T3 or T4 levels. This does not make sense, since there is no variation to calculate correlations for. Note that L251 says the analysis revealed bacteria that had significant effects on T3 and T4, when in reality they only identified taxa whose abundance is correlated with T3 and T4.
We have carefully considered your suggestion. These statistical results show no conflict with our theme. There were no significant seasonal differences in fecal T3 and T4 concentrations, and the relative abundances of most bacterial taxa did not vary significantly on the seasonal scale, and the Random Forest model indicated that only two low abundant taxa were associated with T3. Surely, we only identified the relative abundances of bacterial taxa correlated with T3 and T4, which should be mentioned without misinterpretation.
In some instances I think this is because of language -for example, at L283-4 the authors are likely citing a previous finding, but it is phrased as a conclusion. Others, maybe also be languages, such as L 311 "gut microbiota varied significantly on smaller timescales [similar to humans]" the structure of the sentence implies that human microbiomes vary across small time scales but not large ones and cites papers that only support stability. I think the authors only intend those references to apply to the statement about long stability, but the sentence needs to be restructured to reflect this. Finally, for this issue about long-term stability and short-term variation I think this needs to be better explained in the text, as any short term variability must by definition mean there is long-term variability (but the cited studies are correct, the difference between the results just needs to be reconciled better in the text). Generally, I think the authors need to pay very close attention to the intro and discussion to make sure findings are not over interpreted.
We deeply appreciate for your comments. First, we have rewritten the original sentences of L279-L281, please see L280. Second, we are aware that the difference in physiological structure and diet between western lowland gorillas (Gorilla gorilla) and François's langurs mentioned by L281-L282, which may not be a strong support for our results, and we chose to delete the sentence.
It was previously suggested that the ratio of Firmicutes to Bacteroidetes (F/B) may be involved in energy absorption (Sun et al., 2019, Xia et al., 2021, Lai et al., 2023, but we reviewed more literatures and found that F/B presented different states in different species without a uniform presentation (Ley. et al., 2006, Turnbaugh et al., 2006, Filippo et al., 2010, Barelli et al., 2020, so we decided to delete L283-L288 in the discussion. At the same time, we reworded these sentences because of possible misrepresentation problems in the L292-296, L298-302, L302-303, L303-304, L305-306, Please see L284-294. And we have modified some sentences on the introduction and the discussion that may be poorly formulated.
Finally, we supplement the references with the L311-314, illustrating the elastic changes of the gut microbiota. For the long-term stability and short-term flexibility mentioned in this paper are relative, the existing reports mostly also set long-term and short-term groups in their studies.
Our study demonstrates that the gut microbiota of François' langurs exhibits significant shifts in the relative abundances of bacterial taxa, community structures, and alpha diversity during relatively short months, whereas those did not exhibit significant differences in relative abundances, community structures and alpha diversity over the relatively long seasonal scale.
References mentioned above: We have carefully considered your suggestion. Firstly, we supplement the method description in the L121. Secondly, the DNA extraction, RCR amplification and sequencing of this study are provided by MajorBio Cloud platform, and the detailed steps are only listed in the supplementary information. Then, we put the monthly changes of T3 levels, T4 levels and activity budgets into the text, as shown in Fig.6 and Fig.9. Finally, Fig. 2 shows the main community structure of the gut microbiota of the langurs, and Table S4 and S5 show overall bacterial taxa at the phylum and family level.
which were distributed within a certain range. At family level, Oscillospiraceae were significant different (approximately 10% to 25% vs 30.21% {plus minus} 4.87%). Moreover, Eggerthellaceae was not identified in all months in this report but was fourth bacteria (8.24% {plus minus} 8.37%) in wild langur in the same place. You should explain for this difference especially Eggerthelaceae. 2. Energy metabolism is regulated by a complex interplay hormones and other signaling molecules. Hormones play important roles in energy metabolism, including insulin, glucagon, cortisol, growth hormone as well as thyroid. Furthermore, fecal thyroid is not commonly used in clinical practice for evaluating metabolism as blood tests are the standard method for measuring thyroid hormones. Therefore, I can't trust the result that there was no metabolism variation according to seasonality based on only fecal thyroid hormone level. Also, sample source should be added for thyroid hormone evaluation. You should add the proof that only evaluation of fecal thyroid can reflect for metabolism in NHPs.
Reviewer #3 (Comments for the Author): The authors' response has addressed some of my concerns in part, but I am concerned that the foundational concerns have not fully been addressed. My remaining concerns are below.Line numbers are based on the track-changes version. I apologize if my brief writing is terse -I am simply trying to be brief, and have not recommended exactly how the authors should make each change.
Language and interpretations. The authors continue to overstate their findings, including at L26-7, 29-30, 30-31, 35, and 35-7, 277 (need to rephrase as being correlated with, not affecting), 311-2, 313 (Bacteroides can play), 324-5, 327 (can efficiently), 330 (can help), 331 (may respond), 411 (no evidence), 419 ( no evidence), 421 (may effectively), 421-2 (they did not increase to cope with the change, their response was correlated with dry season onset and their increase may have provided a coping mechanism to the animals), 425-6, 483-4, 499 (may assist), 530, and 532-7. All of these lines of text are over interpreted and must be rephrased. Two common issues are: 1) the authors conclude that microbes in the langurs are performing a function because some strains of the microbial group that strain belongs to have the function; this is inappropriate because of strain-level differences. 2) inferring that correlation is causation (because the microbes vary they cause the change or result from the change, for example, in season).
Additionally, I remain concerned about the authors' comparison between a dry and wet season. There is no replication for season, so it is impossible to discriminate if season versus any number of other possible covariates may underlie the differences in a few sampling groups versus a few other sampling groups. My main concern remains: the authors say there is no long term change but there is short term change. This is impossible. The human literature reconcile this in terms of a discussion of steady states (David 2014 Genome Biology comes to mind first), despite the idea that major perturbations, including changes in diets, medicine, or lifestyle may cause shifts between states. The authors do appear to hint at this, but none of it is explicit and it should be. Additionally, the interpretations are not consistent. At 224-9 and 425 there are seasonal differences, but at 337-8 there are no significant differences. The authors should be consistent and frame accordingly. I believe they are leveraging that there are no major differences in the five most abundant taxa, but this is not the same thing as saying there are no differences. Additionally, because season is not replicated, it is not clear to me that the authors have any power to conclude anything about season.
I continue to recommend that methods details be placed in the main text. Spectrum does not have space limitations, and there is insufficient detail in the methods section for readers to evaluate if the methods are appropriate.
Staff Comments:

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Thank you for submitting your paper to Microbiology Spectrum. (may respond), 411 (no evidence), 419 ( no evidence), 421 (may effectively), 421-2 (they did not increase to cope with the change, their response was correlated with dry season onset and their increase may have provided a coping mechanism to the animals), 425-6, 483-4, 499 (may assist), 530, and 532-7. All of these lines of text are over interpreted and must be rephrased.

Responses to Reviewers' Comments
Two common issues are: 1) the authors conclude that microbes in the langurs are performing a function because some strains of the microbial group that strain belongs to have the function; this is inappropriate because of strain-level differences. 2) inferring that correlation is causation (because the microbes vary they cause the change or result from the change, for example, in season).
Many thanks for your comments. 1) We have revised or rewritten these sentences. 2) In addition, we also attempted to explore the function of gut microbiota after isolating strains, but the realistic sampling difficulties did not allow us to do such analysis, requiring higher precision sequencing technology. 3) Actually, some articles describe that diet "affects " the gut microbiota of host (Leshem et al., 2020, Huang et al., 2022, Bourdeau-Julien et al., 2023, but your suggestion is a more rigorous expression that we accept and modify. Thank you for your comments. We admitted that there should be weaknesses in current study (as you pointed), however, this is the first reports on the seasonal variations in the gut microbiota of these karst-dwelling langurs and should provide general pattern in the seasonality in their gut microbiota. We added these into the discussion section, please see line L414-7. 1) We acknowledge the limitations of this study, with incomplete sample data spanning 16 months. Current study could reflect the compositional structure and basic seasonal regularity of the gut microbiota in the François' langurs, which has a similar setting in other studies (Smits et al., 2017, Wu et al., 2017, Sun et al., 2018, Orkin et al., 2019, Xia et al., 2021. 2). We describe nonsignificant seasonal changes in relative abundance of bacterial taxa (relative stability) and significant changes between months (relative short-term resilience), which we speculate is related to the high leaf feeding of these langurs in previous behavioral ecology studies. Such features are exhibited in the gut microbiota of humans. 3) Finally, we corrected for the previously mentioned significant seasonal variations in L224-9 and L425, and Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines reviewer 2 has recommend. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
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Editor, Microbiology Spectrum Reviewer comments: Reviewer #2 (Comments for the Author): The authors have addressed the comments and I recommend acceptance of the manuscript Reviewer #3 (Comments for the Author): All line numbers are from the tracked changes version. the authors have partially addressed my concerns, but I think there is some room for further revision.
Language and interpretations. I think the authors have generally addressed this concern, although some issues remain, for example L 288 it says relative humidity has the largest effect, but it should be rephrased as 'explains the most variation'.Additionally, there are no experiments to support conjecture at L 29-32 and while such statements could be included in the discussion they are inappropriate for the abstract.
Season. The text at 375-84 remains inappropriate and needs to be revised. There cannot be variation on short periods but not on long periods since the long periods include the short periods. The text does an insufficient job of explaining the subtleties here and needs to be made very explicit and clear. I do wonder if using an ASV-based approach might resolve some of the discrepancy (see comment below), and I would recommend that the authors re-analyze their data using an ASV-rather than OTU-based approach; however, this may not be necessary based on how the authors can respond to this concern below. Methods. The additional methods are helpful in some cases but remain incomplete. For example: L146 -the authors should state how the PCR was performed, which primers were used, and how barcodes and Illumina adapters were added L146 -please explained what 'qualified' PCR products are. Also, there are usually steps between PCR and submitting for sequencing (sometimes not if the company did the work for you) L147 -it is not clear which software packages the authors used to filter the data. maybe the microbiome package on major bio? which executables? I'm unfamiliar with this platform, but there must be some level of detail they can explain. L158 -please rephrase what are "non-repetitive sequences in repetitive sequences" L163-4 -I don't understand this line. please rephrease "equaling all sequences according to the minimum sequence", l180 -train > training? L165 -you say you couldn't refine most OTUs to the genus level, but then you report on genus and species levels at L246; this seems inconsistent but I may be misunderstanding, please add some text to help explain the distinction The authors' additional details show that they are using an OTU instead of an ASV clustering method. The authors may find differences in their results if they used an ASV method, which I think is generally more common and widely accepted in recent years. This is not an objective requirement, but if they wish to do so, it may help provide insight into the short-vs. long timescales issue.

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Responses to Reviewers' Comments
NOTE: The reviewers' comments are in black, and our responses are in red. The line numbers in our responses are those in the clean version of current manuscript.
Associate Editor Comments to Author: Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines reviewer 2 has recommend. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
Thanks for your comments. In the clean manuscript, we have made modifications based on the reviewer's suggestions.

Reviewer #3 (Comments for the Author):
All line numbers are from the tracked changes version. the authors have partially addressed my concerns, but I think there is some room for further revision.
Language and interpretations. I think the authors have generally addressed this concern, although some issues remain, for example L 288 it says relative humidity has the largest effect, but it should be rephrased as 'explains the most variation'.
Additionally, there are no experiments to support conjecture at L 29-32 and while such statements could be included in the discussion they are inappropriate for the abstract.
Thank you very much, we have made the corresponding modification, please see L172.
Season. The text at 375-84 remains inappropriate and needs to be revised. There cannot be variation on short periods but not on long periods since the long periods include the short periods. The text does an insufficient job of explaining the subtleties here and needs to be made very explicit and clear. I do wonder if using an ASV-based approach might resolve some of the discrepancy (see comment below), and I would recommend that the authors re-analyze their data using an ASV-rather than OTU-based approach; however, this may not be necessary based on how the authors can respond to this concern below. Thanks a lot. We have decided to retain the analysis on the seasonal scale and remove the analysis between months, which is more in line with the theme of exploring the seasonal patterns of the gut microbiota of these langurs. In fact, there are still studies using OTU clustering (Huang et al., 2022, Chen et al., 2023, Gan et al., 2023, Zhou et al., 2023, and ASV clustering sequencing is more accurate, but it may also mistakenly remove sequences with low abundance. Both clustering methods have their own advantages and disadvantages. We are considering combining ASV clustering, OTU clustering, metagenomics and other technologies to explore the structure and function of the gut microbiota of François's langurs.
L147 -it is not clear which software packages the authors used to filter the data.
maybe the microbiome package on major bio? which executables? I'm unfamiliar with this platform, but there must be some level of detail they can explain.
We used Fastp and FLASH to filter the original sequence and splicing sequence, please see L141-143 L158 -please rephrase what are "non-repetitive sequences in repetitive sequences" We have corrected it, please see L148 L163-4 -I don't understand this line. please rephrease "equaling all sequences according to the minimum sequence", We used the minimum sequence number to unify the sequence number of all samples, and then carried out subsequent analysis. Please see L152-153 l180 -train > training?
We corrected it, please see L168.
L165 -you say you couldn't refine most OTUs to the genus level, but then you report on genus and species levels at L246; this seems inconsistent but I may be misunderstanding, please add some text to help explain the distinction The sequencing results can determine the number of taxa at the species level and genus level, but there are also many unranked or unclassified bacterial taxa, so, we analyzed the bacterial taxa at the phylum level and family level. Let's put it another way, see L154-155 The authors' additional details show that they are using an OTU instead of an ASV clustering method. The authors may find differences in their results if they used an ASV method, which I think is generally more common and widely accepted in recent years. This is not an objective requirement, but if they wish to do so, it may help provide insight into the short-vs. long timescales issue.
Thank you very much. Both OTU clustering and ASV clustering have their own advantages and disadvantages, and both can reflect the composition characteristics of the microbial community of the research objects to a certain extent. In the subsequent analysis, we can combine ASV clustering, metagenomics and metabolomics to explore the structure and function of the gut microbiota of François's langurs.