Characterization of the Complete Mitochondrial Genome of the Central Highland Grey-Shanked Douc Langur (Pygathrix cinerea), a Critically Endangered Species Endemic to Vietnam (Mammalia: Primates)

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

 

The authors describe a new mt genome sequence from a Vietnamese langur.  The paper is largely descriptive, with comparisons to already published data.  The number of authors is extensive given the amount of new data collected and the number the analyses done (which do not test any scientific hypotheses really).  More discussion is necessary for interpreting the phylogeny of P. cinerea, which is paraphyletic in the mt genome tree that is presented.  The authors also might consider the following in revising their manuscript:

1)   line 86.  This line does not read correctly.  Adjust the English a bit?

2)   Line 127.  There seem to be several P. cinerea mt genome sequences in the authors tree figure (Figure 6), but the authors note that the mt genome of this species has not been sequenced before, so this was a bit confusing to me.  Are the previously published P. cinerea mt DNA not complete genome sequences?  Or, were these previous genomes just not from this area of Vietnam?  In part, this confusion is caused by the authors using ‘species’ and ‘specimen’ interchangeably throughout the text, which needs to be fixed (also see comment #4 below).

3)   Line 112.  Is ‘Passeriformes’, a bird clade (?), a typo here?  Should this be ‘Pygathrix’?

4)   Lines 160 and 168 and line 208 and line 223 (among many other places).  Change ‘other Pygathrix cinerea species’ to ‘other Pygathrix cinerea specimens’ because these are difference individuals of P. cinerea and not different species?  Also, in lines 175-176, refer to two individuals of P. cinerea as different ‘species’.  All of these sequences have the exact same species name so should not be referred to as different species.

5)   In Figures 2-3 and Tables 2-3, why is the new mt genome for P. cinerea (highland Vietnamese) compared to just P. cinerea NC018063 when there seem to be multiple mt genomes available for P. cinerea (see tree in Figure 6)?  Also, I do not see P. cinerea NC018063 in this Figure 6 tree.

6)   Figure 6.  The tree shows that P. cinerea mt sequences are not monophyletic because P. nemaeus is nested within the set of P. cinerea sequences.  As mentioned above, the mt genome NC018063 for P. cinerea does not seem to be in this tree but is utilized in earlier analyses in the paper?  The new P. cinerea mt genome (PP623106) seems to be almost indistinguishable from P. cinerea mt genome JG821842 based on the very short terminal branch lengths that separate these individuals in the tree.  It would be profitable for the authors to show an ML tree analysis of just the Pygathrix sequences and the closest outgroups (Nasalis and Simias).  A tree with just these few sequences would allow the reader to better see actual branch lengths and relationships that are most relevant to the present study.  Such a tree should include where the various mt genomes are from (geographically).  This is especially critical as P. cinerea is not monophyletic in the tree, with JQ821840 very divergent from the other included P. cinerea specimens for mt sequence.  So, the authors should comment on this part of the tree given this situation and try to explain why P. cinerea is paraphyletic with P. nemaeus nested within.  There might be several possible explanations for this pattern, including introgression, mis-identification of specimens, cryptic speciation, broad geographic varation, etc., etc.

7)   Line 355.  Why were there so many nucleotide positions (25,578 sites) in an analysis of mt genomes that average about 16 kb in length each?  Are there really almost 10kb of indels (insertions + deletions) in the alignment that was analyzed?  This does not seem possible to me, and the authors should check their alignment by eye to see if there is a problem, or perhaps this number is a typo?

8)   Line 360.  This sentence is wrong in many ways and has multiple errors that the authors need to sort out.  All of the taxa are Primates contrary to what the authors state, and there are several other mistakes.

9)   Line 382.  Where the authors state that, “While the Vietnamese Highland Pygathrix cinerea's whole mitogenome displayed similar characteristics to those of other Pygathrix cinerea species, it was unique from the others in terms of mitogenomic characteristics”,  it is challenging to see this uniqueness of the new Vietnamese P. cinerea mt genome sequence in the tree figure shown.  The terminal branch length that leads to the new mt sequence is extremely short according to the tree figure.  It would be good to note how many inferred nucleotide changes there are on this tiny terminal branch which are the substitutions unique to the new specimen.

10)                   Line 394.  Is just 2 bp more really a ‘significantly larger size’ for this D-Loop?  I mean, 1 bp is the minimal difference in length between two sequences, and 2 bp is just not much more than that.  Do the authors think this tiny difference actually has any significance in terms of function?  Also, any speculation here on why there is a 2 bp difference in length (‘selection’, ‘drift’) is not particularly useful.

11)                   Line 412.  This text did not make sense to me as the authors are discussing translations and amino acid positions in mt rRNA genes that do not encode proteins, or I am not understanding their text here.

12)                   Line 423.  The various statements in these descriptive statements on various genes about possible functional implications are more or less throwaway speculative statements that I think are not justified to include in the text of a paper like this one that characterizes a single mt genome.  The authors really have no information on functional implications of the various DNA changes they describe?  There are many speculative comments like this in the discussion section that do not enlighten.

13)                   Line 452.  A minor grammatical error to fix here but also in various other places in the text.  Actually, there are quite a few grammatical errors in this final paragraph which should be proofread again by the authors and/or the editorial staff.

14)                   Line 453.  Sometimes ‘highland’ has been in capitialized in the text and other times not.  Should probably not be capitalized throughout?

15)                   Line 465.  I think ‘fascinating’ here is not the correct adjective as the paper is a simple description of one new sequence and there are a few variable sites relative to other individuals in the species.  It is impossible to know the implications of really any of these changes for the different individuals of P. cinerea analyzed here.

Comments on the Quality of English Language

The English is generally fine.  There are a few things to clean up.

Author Response

Dear reviewer!

Thank you so much for graciously taking the time to read through our manuscript and provide us with such genuinely valuable feedback. We greatly appreciate you sharing your insightful thoughts and suggestions with us. We would now like to respond as follows

• line 86.  This line does not read correctly.  Adjust the English a bit?

Respond: Thank you for your detail suggesting. I rewrite it by: “The raw sequencing data, after undergoing optimization, de novo assembly, and genome annotation, were stored in the FASTQ file format (S2).’’

• Line 127.  There seem to be several P. cinerea mt genome sequences in the authors tree figure (Figure 6), but the authors note that the mt genome of this species has not been sequenced before, so this was a bit confusing to me.  Are the previously published P. cinerea mt DNA not complete genome sequences?  Or, were these previous genomes just not from this area of Vietnam?  In part, this confusion is caused by the authors using ‘species’ and ‘specimen’ interchangeably throughout the text, which needs to be fixed (also see comment #4 below).

Respond: Thank you for the comprehensive review of my document. The detailed notes and recommendations will be extremely helpful as I move forward with revising the text.

The mitochondrial genome of Pygathrix cinerea has been sequenced, and we have provided the link for further clarification. https://www.ncbi.nlm.nih.gov/nuccore/JQ821842.1/ , however, they didn’t indicate that sequence is Vietnamese Pygathrix cinerea mitochondrial genome.

About the ‘species’ and ‘specimen’, I had changed ‘other Pygathrix cinerea species’ to ‘other Pygathrix cinerea specimens’ through all manuscript.

• Line 112.  Is ‘Passeriformes’, a bird clade (?), a typo here?  Should this be ‘Pygathrix’?
• Lines 160 and 168 and line 208 and line 223 (among many other places).  Change ‘other Pygathrix cinerea species’ to ‘other Pygathrix cinerea specimens’ because these are difference individuals of P. cinerea and not different species?  Also, in lines 175-176, refer to two individuals of P. cinerea as different ‘species’.  All of these sequences have the exact same species name so should not be referred to as different species.

Respond: Thank you so much for the detailed feedback on my draft. I really appreciate you taking the time to provide these helpful suggestions. I changed ‘other Pygathrix cinerea species’ to ‘other Pygathrix cinerea specimens’ through all manuscript.

• In Figures 2-3 and Tables 2-3, why is the new mt genome for P. cinerea (highland Vietnamese) compared to just P. cinerea NC018063 when there seem to be multiple mt genomes available for P. cinerea (see tree in Figure 6)?  Also, I do not see P. cinerea NC018063 in this Figure 6 tree.

Respond: We have identified that the sequences NC018063 and JQ821842 represent the same genetic data.

https://www.ncbi.nlm.nih.gov/nuccore/NC_018063.1/

https://www.ncbi.nlm.nih.gov/nuccore/JQ821842.1/

In our initial analysis, we inadvertently processed these two accession numbers concurrently, rather than recognizing they referred to the identical sequence. We regret the inconvenience this may have caused in our earlier reporting of these results.

We have corrected this error and are now using only the JQ821842 accession number going forward, as this appears to be the appropriate and consistent identifier for this sequence.

• Figure 6.  The tree shows that P. cinerea mt sequences are not monophyletic because P. nemaeus is nested within the set of P. cinerea sequences.  As mentioned above, the mt genome NC018063 for P. cinerea does not seem to be in this tree but is utilized in earlier analyses in the paper?  The new P. cinerea mt genome (PP623106) seems to be almost indistinguishable from P. cinerea mt genome JG821842 based on the very short terminal branch lengths that separate these individuals in the tree.  It would be profitable for the authors to show an ML tree analysis of just the Pygathrix sequences and the closest outgroups (Nasalis and Simias).  A tree with just these few sequences would allow the reader to better see actual branch lengths and relationships that are most relevant to the present study.  Such a tree should include where the various mt genomes are from (geographically).  This is especially critical as P. cinerea is not monophyletic in the tree, with JQ821840 very divergent from the other included P. cinerea specimens for mt sequence.  So, the authors should comment on this part of the tree given this situation and try to explain why P. cinerea is paraphyletic with P. nemaeus nested within.  There might be several possible explanations for this pattern, including introgression, mis-identification of specimens, cryptic speciation, broad geographic varation, etc,.

Respond: We wanted to express our sincere appreciation for the detailed advice you shared on improving our draft. Your guidance is greatly valued and will make a significant difference as we continue editing. We are happy to rewrite this paragraph by:

The phylogeny of primates is shown in Fig. 6. The tree topology of the primates was consistent according to the Maximum Likelihood method and analysis with bootstrap support (>70). The closest living relatives of primates, Galagidae (Galago senegalensis) and Lorisidae (Otolemur crassicaudatus), were used to root the ingroup of Galagidae and Lorisidae. The tree was rooted with the outgroup Cynocephalus variegatus (Dermoptera) based on Mason et al. (2016) [35]. The entire Primates tree has its roots stemming from the species Dermoptera, specifically Cynocephalus variegatus, and the resulting structure is consistent with the structure obtained from previous studies [36–40]. The phylogenetic tree constructed using the complete mitochondrial genomes of over 50 primate species clearly shows that the three Pygathrix species (Pygathrix cinerea, Pygathrix nemaeus, and Pygathrix nigripes) form a well-supported monophyletic clade. In previous studies, the relative position of Pygathrix and Nasalis was closer than Rhinopithecus, the present study revealed the same thing [12,14]. A common origin of the group is now widely accepted, although the phylogenetic relationships among its genera and species are largely unknown [14]. Our analysis strongly supports this relationship among primates and confirms that the douc langurs (genus Pygathrix) are a distinct evolutionary lineage within the odd-nosed monkey group. The Pygathrix clade is most closely related to the genera Nasalis (proboscis monkey) and Simias (Mentawi langur). Together, these three genera form a larger monophyletic group of odd-nosed monkeys, which is consistent with previous morphological and molecular studies.

The mitochondrial genome sequence of Pygathrix cinerea generated in this study (GenBank accession PP623106) is significantly different from a previously published Pygathrix cinerea mitogenome (GenBank accession JQ821842) which 43 nucleotide differences, suggesting the presence of distinct genetic lineages within this species. This divergence between the mitogenomes indicates the previously published sequence (JQ821842) may have come from a different geographic population or subspecies of Pygathrix cinerea, which could account for the observed genetic differences or the possibility of cryptic subspecies or evolutionarily significant units within the grey-shanked douc langur population. The mitogenome sequence generated in this study is firmly placed within the Pygathrix clade, confirming the species assignment of the sample as Pygathrix cinerea. However, the distinct genetic differences from the previously published Pygathrix cinerea mitogenome suggest the need for more population investigation into the evolutionary history and population structure of this critically endangered primate species.

• Line 355.  Why were there so many nucleotide positions (25,578 sites) in an analysis of mt genomes that average about 16 kb in length each?  Are there really almost 10kb of indels (insertions + deletions) in the alignment that was analyzed?  This does not seem possible to me, and the authors should check their alignment by eye to see if there is a problem, or perhaps this number is a typo?

Respond: We are really thankful for the thorough feedback you provided on our work. The reported number of 25,578 sites was a typo. This sentence was be deleted.

• Line 360.  This sentence is wrong in many ways and has multiple errors that the authors need to sort out.  All of the taxa are Primates contrary to what the authors state, and there are several other mistakes.

Respond: Thank you so much for you detail feedback. We ran the phylogeny for the fourth version, so in the first analyze we got problem with the result, therefor that shown the closet with the closest living relatives of primates, Galagidae (Galago senegalensis) and Lorisidae (Otolemur crassicaudatus). We are so happy for your detail feedback to help us improve our manuscript by this paragraph: The phylogeny of primates is shown in Fig. 6. The tree topology of the primates was consistent according to the Maximum Likelihood method and analysis with bootstrap support (>70). The closest living relatives of primates are Hominidae. The tree was rooted with the outgroup Cynocephalus variegatus based on Mason et al. (2016) [35]. The entire Primates tree has its roots stemming from the species Dermoptera, specifically Cynocephalus variegatus, and the resulting structure is consistent with the structure obtained from previous studies [36–40]. The phylogenetic tree constructed using the complete mitochondrial genomes of over 50 primate species clearly shows that the three Pygathrix species (Pygathrix cinerea, Pygathrix nemaeus, and Pygathrix nigripes) form a well-supported monophyletic clade. In previous studies, the relative position of Pygathrix and Nasalis was closer than Rhinopithecus, the present study revealed the same thing [12,14]. A common origin of the group is now widely accepted, although the phylogenetic relationships among its genera and species are largely unknown [14]. Our analysis strongly supports this relationship among primates and confirms that the douc langurs (genus Pygathrix) are a distinct evolutionary lineage within the odd-nosed monkey group. The Pygathrix clade is most closely related to the genera Nasalis (proboscis monkey) and Simias (Mentawi langur). Together, these three genera form a larger monophyletic group of odd-nosed monkeys, which is consistent with previous morphological and molecular studies.

• Line 382.  Where the authors state that, “While the Vietnamese Highland Pygathrix cinerea's whole mitogenome displayed similar characteristics to those of other Pygathrix cinerea species, it was unique from the others in terms of mitogenomic characteristics”,  it is challenging to see this uniqueness of the new Vietnamese P. cinerea mt genome sequence in the tree figure shown.  The terminal branch length that leads to the new mt sequence is extremely short according to the tree figure.  It would be good to note how many inferred nucleotide changes there are on this tiny terminal branch which are the substitutions unique to the new specimen.

Respond: We rewrite it by: The mitochondrial genome sequence of Pygathrix cinerea generated in this study (GenBank accession PP623106) is significantly different from a previously published Pygathrix cinerea mitogenome (GenBank accession JQ821842) which 43 nucleotide differences, suggesting the presence of distinct genetic lineages within this species. This divergence between the mitogenomes indicates the previously published sequence (JQ821842) may have come from a different geographic population or subspecies of Pygathrix cinerea, which could account for the observed genetic differences or the possibility of cryptic subspecies or evolutionarily significant units within the grey-shanked douc langur population. The mitogenome sequence generated in this study is firmly placed within the Pygathrix clade, confirming the species assignment of the sample as Pygathrix cinerea. However, the distinct genetic differences from the previously published Pygathrix cinerea mitogenome suggest the need for more population investigation into the evolutionary history and population structure of this critically endangered primate species.

• Line 394.  Is just 2 bp more really a ‘significantly larger size’ for this D-Loop?  I mean, 1 bp is the minimal difference in length between two sequences, and 2 bp is just not much more than that.  Do the authors think this tiny difference actually has any significance in terms of function?  Also, any speculation here on why there is a 2 bp difference in length (‘selection’, ‘drift’) is not particularly useful.

Respond: We rewrite by this: The control region of Vietnamese Central Highland Pygathrix cinerea exhibits a significantly larger size (1,094 bp) compared to other Pygathrix cinerea specimens (1,092 bp), however, the two base pair difference is in a region of repetitive sequences and is therefore biologically insignificant, as this pattern is observed in other primate species that do not experience population bottlenecks or rapid evolutionary divergence [42]. Therefore, the minor size difference observed here does not warrant conclusions about distinct evolutionary lineages or population-level diversity.

• Line 412.  This text did not make sense to me as the authors are discussing translations and amino acid positions in mt rRNA genes that do not encode proteins, or I am not understanding their text here.

Respond: The remarkable conservation of rRNA gene size and A+T content across Pygathrix species underscores their fundamental role in mitochondrial protein synthesis [46]. This consistency highlights the value of these genes as reliable markers for phylogenetic analysis, particularly for resolving relationships within closely related taxa [47,48].   While the rRNA genes are generally highly conserved, there are four nucleotide insertion positions were found in rrnS and rrnL that may lead to regulate gene expression at the translational level and this rRNA modification patterns may occur in response to environmental shifts, developmental stages, and disease states [49] or indicate a unique evolutionary trajectory of Vietnamese Central Highland Pygathrix cinerea.

• Line 423.  The various statements in these descriptive statements on various genes about possible functional implications are more or less throwaway speculative statements that I think are not justified to include in the text of a paper like this one that characterizes a single mt genome.  The authors really have no information on functional implications of the various DNA changes they describe?  There are many speculative comments like this in the discussion section that do not enlighten.

Respond: As we have described, there is a published study that characterizes the complete mitochondrial genome of the Pygathrix cinerea species. However, it is unclear whether the sample used in that previous study was obtained from the Vietnamese population of this primate. Consequently, we currently lack sufficient comparative mitogenomic data from the Vietnamese grey-shanked douc langur to fully contextualize the findings of our own analysis. Without access to mitogenomes from other well-characterized Pygathrix cinerea populations, our ability to assess the significance of the nucleotide differences we have identified is limited. Further comparative genomic studies will be needed to better understand the evolutionary relationships and taxonomic status of the Vietnamese I. The lack of available reference data from other geographical regions represents an important constraint in interpreting the implications of the mitochondrial genome we have sequenced in this study.

• Line 452.  A minor grammatical error to fix here but also in various other places in the text.  Actually, there are quite a few grammatical errors in this final paragraph which should be proofread again by the authors and/or the editorial staff.

Respond: This study presents the first comprehensive characterization of the complete mitochondrial genome of the Central Highland grey-shanked douc langur, Pygathrix cinerea, a critically endangered primate endemic to Vietnam. The mitogenome sequence obtained provides valuable molecular data on this poorly studied species. Pygathrix cinerea mitogenome have shown that it retains most of the conserved genes of ancestral features. The mtDNA of this Vietnamese Central Highland Pygathrix cinerea consists of 37 genes that are typically similar with most vertebrates, arranged in a specific pattern. It is a circular molecule with a length of 16,541 base pairs (bp). These genes include 13 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs. This Vietnamese Central Highland Pygathrix cinerea is most closely related to Pygathrix nemaeus and the second closest relative is Pygathrix nigripes. The special thing is that within the population of Pygathrix cinerea, there are significant differences in the gene structure with 43 distinct variations in the DNA that alter 23 amino acids in proteins, highlighting the differences between this Vietnamese Central Highland Pygathrix cinerea and other Pygathrix cinerea.

To better understand the genetic diversity, population dynamics, and conservation status of Pygathrix cinerea, future studies will need to incorporate a much larger sample size representing the major subpopulations across the species' distribution. Comprehensive population genomic analyses incorporating nuclear as well as mitochondrial markers will be crucial to elucidate the evolutionary history and guide evidence-based conservation efforts for this threatened primate.

The mitogenome sequence reported here serves as an initial molecular resource, but additional research is needed to fully characterize the genetic diversity and evolutionary patterns within the grey-shanked douc langur. Expanding the genomic sampling and integrating it with ecological and demographic data will be necessary to inform effective conservation strategies for protecting this endangered species.

• Line 453.  Sometimes ‘highland’ has been in capitialized in the text and other times not.  Should probably not be capitalized throughout?

Respond: "We have changed 'highland' to Vietnamese Central Highland' and capitalize Highland to indicate the geographic location throughout the text."

• Line 465.  I think ‘fascinating’ here is not the correct adjective as the paper is a simple description of one new sequence and there are a few variable sites relative to other individuals in the species.  It is impossible to know the implications of really any of these changes for the different individuals of P. cinerea analyzed here.

Respond: Thank you for the review and suggestions. We appreciate you taking the time to provide feedback. We fixed it by delete this sentence.

Once again, thank you for your response and for the opportunity to provide additional feedback. Receiving constructive feedback is an important part of the revision process.

We hope that our corrections could meet your requirements.

Long Le, PhD.

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

This study reports in detail the results of molecular analyses of the structure and composition of the mitogenome of P. cinerea. A single individual was sequenced whose location and provenance within the geographical distribution of the species are unknown and have not been described. This is a preliminary study, of a molecular kind, not of a genetic or population genomic study. Population studies require the analysis of numerous samples from the main distribution areas of the species. These results are therefore difficult to interpret from a population genetics and evolutionary biology point of view. Unfortunately, these results cannot provide useful information for conserving P. cinerea.

The authors are advised to write a very abbreviated text of a purely molecular nature, moving many Tables and Figures to the Supplementary. When sequences of a significant number of samples are available, then it will be possible to evaluate their population significance and implications for conservation.

Title

Why “Highland”? It seems that primate taxonomists use simply the name: grey-shanked douc langur. I suggest changing the Title in: Characterization of the complete mitochondrial genome of the grey-shanked douc langur Pygathrix cinerea, a critically endangered primate endemic to Vietnam

Abstract

Line 30 - I suggest writing: In this study, we describe the Highland species’ complete mitochondrial genome (mitogenome - mtDNA)

L. 34 - … Vietnamese Highland Pygathrix cinerea … . Delete “Highland” throughout the text

L. 35 – 38 - We identified 43 nucleotide differences when comparing this genome with a previously published mitogenome of Pygathrix cinerea. There are distinct differences between the Pygathrix cinerea we are currently studying and the other Pygathrix cinerea.

This sentence is interesting and important. Authors should complete the Abstract by briefly indicating whether: - these 43 differences can be at least partly explained by sequencing errors; - the two sequenced divergent mitogenomes come from specimens sampled in distant areas; - and whether this suggests that populations of this species belong to distinct evolutionary lineages.

 

 Introduction

The Introduction should be rewritten, because: - it does not provide sufficient information on the distribution, status, and risks of the species, essential premises to understand if and how mitogenome sequencing is useful for conservation; - it goes into unnecessary details describing the basic information on the structure of mtDNA, information that has been known for decades and that is substantially identical for every mammal species. Therefore, I suggest that:

-          The first paragraph is changed, as follows:

 

The grey-shanked douc langur Pygathrix cinerea was described as recently as 1997, as a subspecies of Pygathrix nemaeus [11]. However, the subsequent evaluation of morphological differences and molecular divergence indicated that it is one of three species in the genus Pygathrix present [12–14]. The grey-shanked douc langur is one of the most critically endangered primates in the world [1–3], with fewer than 1,000 individuals remaining in the wild [4]. … here add details on the species’ geographical distribution and on morphological variability, if known … Its population is seriously threatened by a significant reduction in its projected distribution range between 2050 and 2070 … why population decline is expected … ? [5]. Conservation efforts for endangered species require a thorough understanding of their genetic diversity and evolutionary history [6–10]. …. Why conservation efforts for this endangered species is required … ?  … low genetic diversity … inbreeding ... admixture with other species … population structuring … ? lack of gene flow among population fragments … ? … others …? Please, try to be specific, explaining why mtDNA information is important for conservation.

 

-          Delete this paragraph and just add some references at the end of line 52. i.e., [13,19–22]:

Mitogenomes have high abundance in all cells, rapid rates of evolution, a small genome size, preserved gene content, maternal inheritance, and an absence of substantial recombination [15–18]. The mitogenomes of most animals are circular and exhibit conserved gene content. Most mitogenomes are estimated to be 16 kb in size and encode 37 genes, including one control region (A+T rich area), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and 13 protein-coding genes (PCGs) [13,19–22].

-          L. 62- 63: Phylogenetic analysis was conducted to assess the evolutionary relationships among these species.

 

This sentence is fine, but you already stated (L. 49 – 50) that: … the subsequent evaluation of morphological differences and molecular divergence indicated that it is one of three species in the genus Pygathrix.

Thus: the evolutionary relationships among these species … should have been clarified. If not, please, let us know why.

 

-          L. 90 - 96: this paragraph:

2.2. Evaluation Of Interpretation Data Quality  

The raw sequencing data after underwent optimisation, de novo assembly, and genome annotation were stored in the fastq file format (S2), containing read and sequence information and the corresponding quality score. The reading quality results are presented in Table S3.

is not in the right place and is redundant. I suggest including it in the following paragraph, after line 101.

3. Results and discussion

Also, the paragraph (L  127 – 136) is not in the right place. I suggest moving it in the Introduction, after line 62 (but please, after the moves, check carefully that the Introduction is OK, and fix everything that needs fixing):

 

This study presents the first complete mitochondrial genome sequence of Pygathrix  cinerea, a critically endangered primate endemic to Vietnam. While partial mitogenome sequences of this species were included in Liedigk et al., (2012) [14], a detailed analysis of its mitochondrial characteristics was lacking. We compared the complete mitogenome of Pygathrix cinerea to publicly available data from other primates, focusing on the precise location and orientation of genes within the mitochondrion. Our analysis revealed that the gene order and orientation in the Pygathrix cinerea mitogenome are generally conserved among douc langur species. However, we identified significant differences in the positions and lengths of a few genes, highlighting potential variations in mitochondrial evolution within this group.

The Results are described with too many details. Some Tables and Figures could be moved to the Supplementary, for instance: Table 1, 2, 3, 5; Figure 2, 3, 4, 5

3.9. Phylogeny of 55 primate mitogenomes

L. 370: delete

Paragraph L. 371 – 386 is redundant and should be deleted.

Figure 6: the position of Homo neanderthalensis makes no sense in this tree. Please add the Homininae sequences, or delete the neanderthal, and recompute the phylogeny

In this chapter, you should highlight the phylogenetic results of mtDNA analyses of P. cinerea. For instance: - genus Pygathrix is monophyletic, closely related to sister Nasalis and Simias … … . Why P. cinerea sequence JQ821841 is different … ? and so on.

 

4. Discussion

Sentences in paragraph L. 394 – 405 are inconsistent and wrong.

The control region of Vietnamese Highland Pygathrix cinerea exhibits a significantly larger size (1,094 bp) compared to other Pygathrix cinerea species (1,092 bp) … they are just two base pairs of difference in a region of repetitive sequences, exposed to the risk of sequencing errors and in any case biologically insignificant … and the species is the same!

L. 395 – 396: a pattern observed in other primate species experiencing population bottlenecks or rapid evolutionary divergence [42]. This sentence makes no sense. The CR is usually very variable within species independent of… bottlenecks or rapid evolutionary divergence.

L. 397 – 399: this sentence is confusing. Do you mean unique mutations and distinct lineage in this POPULATION or SPECIES? Anyway, the phylogenetic tree does not support any clear evolutionary distinction among cinerea sequences

5. Conclusion

 

The conclusions are not supported by the data. This study reports in detail the results of molecular analyses of the structure and composition of the mitogenome of P. cinerea. A single individual was sequenced whose location and provenance within the geographical distribution of the species are unknown and have not been described. This is a preliminary study, of a molecular kind, not of a genetic or population genomic study. Population studies require the analysis of numerous samples from the main distribution areas of the species. These results are therefore difficult to interpret from a population genetics and evolutionary biology point of view. Unfortunately, these results cannot provide useful information for conserving P. cinerea.

Comments on the Quality of English Language

English should be revised throughout the text.

Author Response

Dear reviewer!

We are deeply grateful for you taking the time to carefully read over our work. It is a true privilege for us to have had the chance to hear from someone with your caliber of experience and expertise. After giving the reviewer's insightful ideas considerable thought, we made a few adjustments that we believe have strengthened the manuscript. Specifically, we have:

Title

Why “Highland”? It seems that primate taxonomists use simply the name: grey-shanked douc langur. I suggest changing the Title in: Characterization of complete mitochondrial genome of Central Highland grey-shanked douc langur Pygathrix cinerea, a critically endangered primate endemic to Vietnam.

Respond: Thank you so much for the suggestion to revise the tittle of my research.

The reason why I included "Highlands" in the title is that there was a single complete mitochondrial DNA (mtDNA) sequence for the grey-shanked douc langur that has been deposited in the NCBI database, but the specific geographic location within Vietnam where this sample was collected was not provided. This research aims to clearly map the locations where grey-shanked douc langur mtDNA samples have been collected, as this endangered primate is found exclusively in Vietnam's Central Highlands region, which is made up of the provinces of Đắk Lắk, Đắk Nông, Gia Lai, Kon Tum, and Lâm Đồng. Therefore, would you mind if we revise the title to:

Characterization of the complete mitochondrial genome of the Central Highland grey-shanked douc langur Pygathrix cinerea, a critically endangered primate endemic to Vietnam.

Abstract

Line 30 - I suggest writing: In this study, we describe the Highland species’ complete mitochondrial genome (mitogenome - mtDNA).

Respond: Thank you so much for your suggestion. We have rewritten this sentence as: In this study, we describe the Central Highland species’ complete mitochondrial genome (mitogenome - mtDNA).

1. 34 -… Vietnamese Highland Pygathrix cinerea… . Delete “Highland” throughout the text

Respond: Like the description above about the name Central Highland, we have rewritten this sentence as:

'In this study, we describe the Central Highlands species' complete mitochondrial genome (mitogenome - mtDNA).'

L.34: We would like to replace the ‘’Highland’’ by ‘’Central Highland’’.

1. 35 – 38 -We identified 43 nucleotide differences when comparing this genome with a previously published mitogenome of Pygathrix cinerea. There are distinct differences between the Pygathrix cinereawe are currently studying and the other Pygathrix cinerea.

This sentence is interesting and important. Authors should complete the Abstract by briefly indicating whether: - these 43 differences can be at least partly explained by sequencing errors; - the two sequenced divergent mitogenomes come from specimens sampled in distant areas; - and whether this suggests that populations of this species belong to distinct evolutionary lineages.

Respond: We rewrite it by: “The grey-shanked douc langur Pygathrix cinerea is a recently described, critically endangered primate, endemic to Vietnam. In this study, we describe the Central Highland species’ complete mitochondrial genome (mitogenome - mtDNA). It is a circular molecule with a length of 16,541 base pairs (bp). The genome consists of 37 genes, consistent with those found in most other vertebrates, including 13 protein coding genes, 22 transfer RNAs, and 2 ribosomal RNAs. A comparison with the mitogenomes of more than 50 primates showed that the mitogenome of Vietnamese Central Highland Pygathrix cinerea has a conservative gene order. We identified 43 nucleotide differences when comparing this genome with a previously published mitogenome of Pygathrix cinerea. It is evident that there are distinct differences between the Pygathrix cinerea we are currently studying and other Pygathrix cinerea. These differences are unlikely to be solely the result of sequencing errors, as mitogenomes were generated using high-quality methods. The genetic divergence observed between the two Pygathrix cinerea mitogenomes implies the potential existence of at least two distinct lineages or forms of this primate species within its native range in Vietnam.

1. Introduction

The Introduction should be rewritten, because: - it does not provide sufficient information on the distribution, status, and risks of the species, essential premises to understand if and how mitogenome sequencing is useful for conservation; - it goes into unnecessary details describing the basic information on the structure of mtDNA, information that has been known for decades and that is substantially identical for every mammal species. Therefore, I suggest that:

-          The first paragraph is changed, as follows:

The grey-shanked douc langur Pygathrix cinerea was described as recently as 1997, as a subspecies of Pygathrix nemaeus [11]. However, the subsequent evaluation of morphological differences and molecular divergence indicated that it is one of three species in the genus Pygathrix present [12–14]. The grey-shanked douc langur is one of the most critically endangered primates in the world [1–3], with fewer than 1,000 individuals remaining in the wild [4]. … here add details on the species’ geographical distribution and on morphological variability, if known … Its population is seriously threatened by a significant reduction in its projected distribution range between 2050 and 2070 … why population decline is expected … ? [5]. Conservation efforts for endangered species require a thorough understanding of their genetic diversity and evolutionary history [6–10]. …. Why conservation efforts for this endangered species is required … ?  … low genetic diversity … inbreeding ... admixture with other species … population structuring … ? lack of gene flow among population fragments … ? … others …? Please, try to be specific, explaining why mtDNA information is important for conservation.

 Revised Introduction:

The grey-shanked douc langur Pygathrix cinerea is a critically endangered primate species endemic to Vietnam. It was initially described as a subspecies of the Indochinese douc langur (Pygathrix nemaeus) in 1997, but subsequent morphological and molecular studies have confirmed it as a distinct species within the genus Pygathrix [11-14]. The grey-shanked douc langur is one of the rarest primates in the world, with a highly fragmented population estimated at fewer than 1,000 individuals [1-4]. Its distribution is primarily within the provinces of Quang Nam, Quang Ngai, Kon Tum and Gia Lai of Vietnam, inhabiting a limited range of tropical forests [4]. Morphological variability within the species has been observed, with some populations exhibiting distinct phenotypic characteristics which coat coloration can range from light grey to dark charcoal, with some individuals exhibiting reddish-brown hues on the limbs and head [12,13]. The grey-shanked douc langur faces significant threats to its long-term survival. Its population is projected to decline substantially between 2050 and 2070, primarily due to habitat loss and fragmentation driven by deforestation, infrastructure development, and agriculture expansion [5]. Conservation efforts are critical to prevent the extinction of this endangered primate. Understanding the genetic diversity and evolutionary history of the grey-shanked douc langur is essential for effective conservation planning and management [6-10]. Analyzing the mitochondrial genome can determine the origin and genetic relationships between populations of a species. Populations with low genetic diversity will need to be prioritized for conservation to maintain the vitality of the species. This helps better understand the genetic structure and evolutionary history of the species, allowing for more effective conservation strategies.

-          Delete this paragraph and just add some references at the end of line 52. i.e., [13,19–22]:

Mitogenomes have high abundance in all cells, rapid rates of evolution, a small genome size, preserved gene content, maternal inheritance, and an absence of substantial recombination [15–18]. The mitogenomes of most animals are circular and exhibit conserved gene content. Most mitogenomes are estimated to be 16 kb in size and encode 37 genes, including one control region (A+T rich area), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and 13 protein-coding genes (PCGs) [13,19–22].

Respond: We deleted it add the references at the end of line 66 in new version of manuscript.

1. 62- 63: Phylogenetic analysis was conducted to assess the evolutionary relationships among these species.

This sentence is fine, but you already stated (L. 49 – 50) that: … the subsequent evaluation of morphological differences and molecular divergence indicated that it is one of three species in the genus Pygathrix.

Thus: the evolutionary relationships among these species … should have been clarified. If not, please, let us know why.

Respond: Previous research had established that the genus Pygathrix comprises three recognized species: Pygathrix nemaeus, Pygathrix nigripes and Pygathrix cinerea. The subsequent evaluation of morphological differences and molecular divergence between these three Pygathrix species indicated that they represent distinct evolutionary lineages. A comparison of the Pygathrix cinerea mitogenome with those of more than 50 other primates showed that the mitogenome of the Vietnamese Central Highland population has a conserved gene order. We identified 43 nucleotide differences when comparing this genome with a previously published mitogenome also attributed to Pygathrix cinerea. This genetic divergence implies the potential existence of at least two distinct lineages or forms of this primate species within its native range in Vietnam.

The key addition is the paragraph providing the necessary context about the three recognized Pygathrix species and how the present study relates to the broader evolutionary relationships within this primate genus. This helps establish a clearer foundation for interpreting the findings on the genetic divergence observed between the two Pygathrix cinerea mitogenomes.

2.2. Evaluation Of Interpretation Data Quality 

The raw sequencing data after underwent optimisation, de novo assembly, and genome annotation were stored in the fastq file format (S2), containing read and sequence information and the corResponding quality score. The reading quality results are presented in Table S3 is not in the right place and is redundant. I suggest including it in the following paragraph, after line 101.

Respond: We included it after line 101

3. Results and discussion

Also, the paragraph (L  127 – 136) is not in the right place. I suggest moving it in the Introduction, after line 62 (but please, after the moves, check carefully that the Introduction is OK, and fix everything that needs fixing):

This study presents the first complete mitochondrial genome sequence of Pygathrix  cinerea, a critically endangered primate endemic to Vietnam. While partial mitogenome sequences of this species were included in Liedigk et al., (2012) [14], a detailed analysis of its mitochondrial characteristics was lacking. We compared the complete mitogenome of Pygathrix cinerea to publicly available data from other primates, focusing on the precise location and orientation of genes within the mitochondrion. Our analysis revealed that the gene order and orientation in the Pygathrix cinerea mitogenome are generally conserved among douc langur species. However, we identified significant differences in the positions and lengths of a few genes, highlighting potential variations in mitochondrial evolution within this group.

Respond: We moved it the introduction.

The Results are described with too many details. Some Tables and Figures could be moved to the Supplementary, for instance: Table 1, 2, 3, 5; Figure 2, 3, 4, 5

Respond: We appreciate the suggestion to move some of the tables and figures to the Supplementary Materials. We are happy to explore alternative suggestions that would maintain the clarity and comprehensiveness of the Results as currently written. Our goal is to ensure the final manuscript is as clear, compelling and comprehensive as possible. However, after careful consideration, removing them to the Supplementary could risk the main text feeling incomplete or lacking important context. We feel it's important to keep this supporting data readily available to the reader within the main body of the manuscript. The information contained in those specific tables and figures is integral to fully supporting the key findings presented in the Results section.

3.9. Phylogeny of 55 primate mitogenomes

1. 370: delete

Respond: We deleted the sentence: ‘We found Cercopithecidae to be the sister group of Hominidae’

Paragraph L. 371 – 386 is redundant and should be deleted.

Respond: We deleted the paragraph line 371- 386.

Figure 6: the position of Homo neanderthalensis makes no sense in this tree. Please add the Homininae sequences, or delete the neanderthal, and recompute the phylogeny

Respond: I'm grateful for the feedback, as it allows me to strengthen the rigor and validity of the phylogenetic reconstruction presented in the Results. Homo neanderthalensis is indeed a species within the Homininae subfamily, so its placement in the phylogenetic tree makes sense. https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Undef&id=207598&lvl=3&keep=1&srchmode=1&unlock

Please let me know if there are any other aspects of the tree topology or analysis that you would like me to revisit or clarify.

In this chapter, you should highlight the phylogenetic results of mtDNA analyses of Pygathrix cinerea. For instance: - genus Pygathrix is monophyletic, closely related to sister Nasalis and Simias … … . Why Pygathrix cinerea sequence JQ821841 is different … ? and so on.

Respond: Thank you for the detailed feedback. Here are the revisions:

The phylogeny of primates is shown in Fig. 6. The tree topology of the primates was consistent according to the Maximum Likelihood method and analysis with bootstrap support (>70). The closest living relatives of primates, Galagidae (Galago senegalensis) and Lorisidae (Otolemur crassicaudatus), were used to root the ingroup of Galagidae and Lorisidae. The tree was rooted with the outgroup Cynocephalus variegatus (Dermoptera) based on Mason et al. (2016) [35]. The entire Primates tree has its roots stemming from the species Dermoptera, specifically Cynocephalus variegatus, and the resulting structure is consistent with the structure obtained from previous studies [36–40]. The phylogenetic tree constructed using the complete mitochondrial genomes of over 50 primate species clearly shows that the three Pygathrix species (Pygathrix cinerea, Pygathrix nemaeus, and Pygathrix nigripes) form a well-supported monophyletic clade. In previous studies, the relative position of Pygathrix and Nasalis was closer than Rhinopithecus, the present study revealed the same thing [12,14]. A common origin of the group is now widely accepted, although the phylogenetic relationships among its genera and species are largely unknown [14]. Our analysis strongly supports this relationship among primates and confirms that the douc langurs (genus Pygathrix) are a distinct evolutionary lineage within the odd-nosed monkey group. The Pygathrix clade is most closely related to the genera Nasalis (proboscis monkey) and Simias (Mentawi langur). Together, these three genera form a larger monophyletic group of odd-nosed monkeys, which is consistent with previous morphological and molecular studies.

The mitochondrial genome sequence of Pygathrix cinerea generated in this study (GenBank accession PP623106) is significantly different from a previously published Pygathrix cinerea mitogenome (GenBank accession JQ821842) which 43 nucleotide differences, suggesting the presence of distinct genetic lineages within this species. This divergence between the mitogenomes indicates the previously published sequence (JQ821842) may have come from a different geographic population or subspecies of Pygathrix cinerea, which could account for the observed genetic differences or the possibility of cryptic subspecies or evolutionarily significant units within the grey-shanked douc langur population. The mitogenome sequence generated in this study is firmly placed within the Pygathrix clade, confirming the species assignment of the sample as Pygathrix cinerea. However, the distinct genetic differences from the previously published Pygathrix cinerea mitogenome suggest the need for more population investigation into the evolutionary history and population structure of this critically endangered primate species.

4. Discussion

Sentences in paragraph L. 394 – 405 are inconsistent and wrong.

The control region of Vietnamese Highland Pygathrix cinerea exhibits a significantly larger size (1,094 bp) compared to other Pygathrix cinerea species (1,092 bp) … they are just two base pairs of difference in a region of repetitive sequences, exposed to the risk of sequencing errors and in any case biologically insignificant … and the species is the same!

1. 395 – 396: a pattern observed in other primate species experiencing population bottlenecks or rapid evolutionary divergence [42]. This sentence makes no sense. The CR is usually very variable within species independent of… bottlenecks or rapid evolutionary divergence.

Respond: Thank you for the detailed feedback on the issues with the discussion section of the paper. I appreciate you highlighting these specific concerns. Here is a suggested revision to address the problems you identified:

The control region of Vietnamese Central Highland Pygathrix cinerea exhibits a significantly larger size (1,094 bp) compared to other Pygathrix cinerea species (1,092 bp), however, the two base pair difference is in a region of repetitive sequences and is therefore biologically insignificant, as this pattern is observed in other primate species that do not experience population bottlenecks or rapid evolutionary divergence.

1. 397 – 399: this sentence is confusing. Do you mean unique mutations and distinct lineage in this POPULATION or SPECIES? Anyway, the phylogenetic tree does not support any clear evolutionary distinction among cinerea sequences

Respond: Thank you for the detailed feedback on the issues. I rewrite it by the following sentence:

This size variation, coupled with the presence of unique mutations, suggested a potentially distinct evolutionary lineage for this population, possibly reflecting a period of isolation or adaptation to specific environmental pressures [43]. These mutations might be associated with the effect of selection and genetic drift and supports the notion of a distinct evolutionary lineage [44]. However, the limited of the number of samples preclude any robust conclusions about the evolutionary history and population structure of this endangered primate.

5. Conclusion.

The conclusions are not supported by the data. This study reports in detail the results of molecular analyses of the structure and composition of the mitogenome of Pygathrix cinerea. A single individual was sequenced whose location and provenance within the geographical distribution of the species are unknown and have not been described. This is a preliminary study, of a molecular kind, not of a genetic or population genomic study. Population studies require the analysis of numerous samples from the main distribution areas of the species. These results are therefore difficult to interpret from a population genetics and evolutionary biology point of view. Unfortunately, these results cannot provide useful information for conserving Pygathrix cinerea.

Respond: Thank you so much for your comment which make me realize that we got problem in the conclusion. We rewrite it by:

This study presents the first comprehensive characterization of the complete mitochondrial genome of the Central Highland grey-shanked douc langur, Pygathrix cinerea, a critically endangered primate endemic to Vietnam. The mitogenome sequence obtained provides valuable molecular data on this poorly studied species. Pygathrix cinerea mitogenome have shown that it retains most of the conserved genes of ancestral features. The mtDNA of this Vietnamese Central Highland Pygathrix cinerea consists of 37 genes that are typically similar with most vertebrates, arranged in a specific pattern. It is a circular molecule with a length of 16,541 base pairs (bp). These genes include 13 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs. This Vietnamese Central Highland Pygathrix cinerea is most closely related to Pygathrix nemaeus and the second closest relative is Pygathrix nigripes. The special thing is that within the population of Pygathrix cinerea, there are significant differences in the gene structure with 43 distinct variations in the DNA that alter 23 amino acids in proteins, highlighting the differences between this Vietnamese Central Highland Pygathrix cinerea and other Pygathrix cinerea. Our analysis of the mitochondrial genome of this Vietnamese Central Highland Pygathrix cinerea reveals a fascinating blend of conserved features characteristic of primates and unique variations that offer insights into its evolution and conservation. It is evident that Vietnamese Central Highland Pygathrix cinerea is distinct an individual we are currently studying and other Pygathrix cinerea populations.

To better understand the genetic diversity, population dynamics, and conservation status of Pygathrix cinerea, future studies will need to incorporate a much larger sample size representing the major subpopulations across the species' distribution. Comprehensive population genomic analyses incorporating nuclear as well as mitochondrial markers will be crucial to elucidate the evolutionary history and guide evidence-based conservation efforts for this threatened primate.

The mitogenome sequence reported here serves as an initial molecular resource, but additional research is needed to fully characterize the genetic diversity and evolutionary patterns within the grey-shanked douc langur. Expanding the genomic sampling and integrating it with ecological and demographic data will be necessary to inform effective conservation strategies for protecting this endangered species.

We value your thoughtful guidance tremendously and believe these revisions have helped elevate the overall quality and impact of our work. Please let us know if you have any other feedback or suggestions - we remain open to continuing this collaborative process to ensure we achieve the full potential of our manuscript. Your input has been instrumental, and we are sincerely appreciative.

Thank you, so much and best regards,

Long Le, PhD.

Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Overall the authors do a good job at reporting the mitochondrial genome of Pygathrix cinerea. There are a few smaller things, and I have left comments on a few grammatical things and comments on some particular parts on an attached annotated PDF.

There are a few smaller things, and one thing that needs to be fixed throughout is the constant use of "Pygathis cinerea species." As this is only a single species, it is either "Pygathrix species" or, more likely, it refers to multiple specimens or samples of the species Pygathrix cinerea. If this is the case, this must be redone throughout the manuscript to clarify what it is they are referring to.

A somewhat larger aspect that should be clarified is how the different P. cinerea specimens and samples relate to each other. The authors mention the possibility of different populations potentially resulting in the differences in the data between Pygathrix cinerea specimens. If this is the result of different populations that could, conceivably, potentially be from different subspecies, it would be more useful to know if these P. cinerea specimens are from different populations within their range and how these populations relate geographically. This would be useful for helping clarify potential taxonomic issues within the species. This is something that should be further explored or explained in the paper that would help clarify the importance of the data and this paper.

The references section needs to be made more consistent as there is a variety of way that references are listed in this section.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

There were just a few minor issues and those I came across are marked in the annotated PDF.

Author Response

Dear Reviewer,

We appreciate you taking the time to provide such thorough and thoughtful feedback on our manuscript detailing the mitochondrial genome of Pygathrix cinerea. Your insights and suggestions are invaluable, and we are grateful for the opportunity to address the points you have raised.

Comments and Suggestions for Authors

Overall the authors do a good job at reporting the mitochondrial genome of Pygathrix cinerea. There are a few smaller things, and I have left comments on a few grammatical things and comments on some particular parts on an attached annotated PDF. There are a few smaller things, and one thing that needs to be fixed throughout is the constant use of "Pygathis cinerea species." As this is only a single species, it is either "Pygathrix species" or, more likely, it refers to multiple specimens or samples of the species Pygathrix cinerea. If this is the case, this must be redone throughout the manuscript to clarify what it is they are referring to.

Respond:

Regarding the consistent use of "Pygathrix cinerea species" - you are absolutely right that this is incorrect, as we are describing a single species, Pygathrix cinerea. We have carefully reviewed the manuscript and made the necessary corrections throughout to refer to "Pygathrix cinerea" or "P. cinerea" as appropriate.

A somewhat larger aspect that should be clarified is how the different P. cinerea specimens and samples relate to each other. The authors mention the possibility of different populations potentially resulting in the differences in the data between Pygathrix cinerea specimens. If this is the result of different populations that could, conceivably, potentially be from different subspecies, it would be more useful to know if these P. cinerea specimens are from different populations within their range and how these populations relate geographically. This would be useful for helping clarify potential taxonomic issues within the species. This is something that should be further explored or explained in the paper that would help clarify the importance of the data and this paper.

Respond:

Your comments about clarifying the relationship between the different P. cinerea specimens and samples are well-taken. We agree that providing more context around the geographic origin and potential population differences between the samples would greatly strengthen the paper. This will help shed light on any potential taxonomic considerations within the species.

The references section needs to be made more consistent as there is a variety of way that references are listed in this section.

Respond:

Finally, we have reviewed the references section and standardized the formatting to ensure consistency across all citations. Thank you for catching this - we appreciate you taking the time to provide this level of detailed feedback.

We are grateful for your expertise and guidance throughout this review process. Your comments have helped us identify areas for improvement that will undoubtedly enhance the quality and impact of our work. Please let us know if you have any other feedback or if there is anything else we can clarify.

Sincerely,

Long Le, PhD

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

We thank the authors for the careful revision of the original ms. Now this paper is in better shape for publication.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript describes a new mitogenome of an extremely rare primate: the grey-shanked douc langur (Pygathrix cinerea). The characterization and analysis of the mitogenome is adequate and up-to-date. I have a couple of concerns and comments, but these are easily addressed.

First, the nucleotide sequence of the new mitogenome is given in Supplementary data 2. Current standards dictate that any new sequence described or used in a publication must have been deposited at GenBank before publication, with the GenBank accession number cited in the paper.

Second, the authors compare the new mitogenome with another mitogenome of this species (JQ821842/NC_018063). This mitogenome was obtained by Liedigk et al. (2012), but curiously this paper is not cited in the manuscript. It would be good to cite this paper, and state that Liedigk et al. (2012) did not fully describe this species’ mitogenome, so that this manuscript is the first to fully describe it.

Liedigk, R., Yang, M., Jablonski, N. G., Momberg, F., Geissmann, T., Lwin, N., et al. 2012. Evolutionary history of the odd-nosed monkeys and the phylogenetic position of the newly described Myanmar snub-nosed monkey Rhinopithecus strykeri. PloS one, 7(5), e37418.

Third, mitogenome NC_018063 is not from Myanmar (as stated in the manuscript) but was obtained by Liebigk from the Endangered Primate Rescue Center, Vietnam.

Fourth, the phylogeny is presented as a cladogram (which has no information on branch lengths). However, it has been recommended that new mitogenome descriptions include a phylogram (which presents actual branch lengths). See:

Botero-Castro, F, Delsuc, F & Douzery, EJ 2016. Thrice better than once: quality control guidelines to validate new mitogenomes. Mitochondrial DNA Part A 27: 449-454.

Sangster, G & Luksenburg, JA 2021. Sharp increase of problematic mitogenomes of birds: causes, effects and remedies. Genome Biol. Evol. 13(9), evab210.

Fifth, because Pygathrix cinerea  is paraphyletic (with respect to Pygathrix nemaeus, see Liedigk et al. 2012) in mitochondrial DNA, it would be good to reconstruct a new maximum-likelihood phylogeny (phylogram) that also includes the following two mitogenomes. This can shed further light on the relationships between the two species.
Pygathrix cinerea  JQ821841
Pygathrix nemaeus JF293096

In addition, I recommend that the authors include:
Simias concolorJF293094
Because this may affect inference of the relationships of Pygathrix, Rhinopithecus and Nasalis.

Sixth, please state (i) where this sample was obtained, or if it is from a captive animal, where that animal was captured in the wild, if known, (ii) what the tissue number is and where the tissue has been deposited, (iii) what kind of tissue was used (liver, skin etc), (iv) how the animal was identified as Pygathrix cinerea (please describe the diagnostic character states of the animal).

Minor comments / suggestions:

Line 2. I suggest the authors rename the title as “Characterization of the complete mitochondrial genome of the critically endangered grey-shanked douc langur Pygathrix cinerea (Mammalia: Primates)”.

Lines 27-40: please rephrase the abstract as: “The grey-shanked douc langur Pygathrix cinerea is a recently described, critically endangered primate, which is endemic to central Vietnam. In this study, we describe the complete mitochondrial genome (mitogenome). It is a circular molecule with a length of 16,541 base pairs (bp). The genome consists of 37 genes, consistent with that found in most other vertebrates, including 13 protein coding genes, 22 transfer RNAs, and 2 ribosomal RNAs. Comparison with the mitogenomes of more than 50 primates showed that the mitogenome of Pygathrix cinerea has a conservative gene order. We identified 43 nucleatide differences with a previously published mitogenome of Pygathrix cinerea. Although in previous studies the Pygathrix was more closely related to Nasalis than to Rhinopithecus, the present study supports a closer relationship of Pygathrix to Rhinopithecus than to Nasalis.”

Line 45: the most severely endangered = critically endangered

Line 47: delete “most recently”

Line 48: significantly decreasing = a great reduction in the

Lines 53-57. Delete the sentences “According to …..many old ones vanished.”

Line 60: start a new paragraph with Pygathrix ….

Line 61: have been found = occurring

Lines 61-63: replace the sentence starting with “in the beginning …” with “It was described as recently as 1997 , as a subspecies of P. nemaeus [11]. However subsequent evaluation of morphological differences and molecular divergence indicated that it was better treated as a full species [12].”

Line 69: the absence = an absence

Lines 69-70: delete are the characteristics of a typical mitogenome

Line 71: Most mitogenomes are estimated ….

Lines 74-75: delete “However, for the purpose of clarifying the distribution of species requires a wide variety of complex data and detailed studies of species [23].”

Lines 84-85: “… sequence information and the corresponding quality scores.”

Line 95: will be = were

Line 97: would be = were

Line 101: will then be = was

Lines 116-117: The GenBank accession numbers of all sequences used is shown the Supplementary data.

Line 119: delete “The”

Line 120: replace the sentence “We shown … first time” with “We describe and analyse the complete mitochondrial mitogenome of Pygathrix cinerea for the first time. Two previous mitogenomes of this species were included in Liedigk et al. (2012) but this paper did not describe the mitochondrial characteristics in any detail.”

Line 129: contained ….. (Fig. 1). Transfer “which almost all the 129 genes in the mitogenome of Vietnamese Pagathrix cinerea were located on the H strand” towards line 134.

Thus, at line 134, after “… (table 1).”, include the sentence: Almost all the genes in the mitogenome of Vietnamese Pagathrix cinerea were located on the H strand, except ND6 and eight tRNAs tRNA Gln, tRNA Ala, tRNA Asn, tRNA Cys, tRNA Tyr, tRNA Ser, tRNA Glu which were found to be located on the L strand.”

Line 141, Figure 1. caption, rephrase the first sentence as “Figure 1. Genome structure of the complete mitochondrion of Pygathrix cinerea.

Please consider making figure 1 much larger. The text is almost illegible.

Line 144, Table 1.  caption, rephrase as “Location and length of 37 genes of Pygathrix cinerea.”

Figure 3. Please remove “Vietnam” and add GenBank accession number after the species name. Please remove “Myanma” and add GenBank accession number (NC018063) after the species name .

Table 2, page 7, please add the GenBank accession number for your mitogenome sequence of Pygathrix cinerea

Table 2. Please list the two (preferably three, see above) sequences of Pygathrix cinerea after each other so that they can be easily compared.

Line 169. The concatenated 13 PCGs of the mitogenome of Pygathrix cinerea were estimated ….

Line 171: delete “as seen in other primates”

Lines 176-177: rephrase this sentence as: “The AT- and GC-skew of the PCGs of Pygathrix cinerea and other primates are listed in Table 3.”

Line 182: “other” should not be italized

Lines 184-185:  Pygathrix cinerea should be italized

Line 187: Pygathrix nigripes should be italized

Table 3. Please list the two (preferably three, see above) sequences of Pygathrix cinerea after each other so that they can be easily compared. Please add the GenBank accession number for your mitogenome sequence of Pygathrix cinerea.

Lines 194-195. Rephrase as: “The D-loop was located between the trnP and trnF regions and was 1094 bp long (positions 15,584–16,541 and continuing at 1-136).” However, I strongly recommend that you cut positions 1-136 from your mitogenome and paste it after position 16,541, so that the D-loop is the last fragment of the linear mitogenome sequence, and it becomes aligned with the other mitogenomes of this species. In that case you should also modify the first sentence as follows “The D-loop was located between the trnP and trnF regions and was 1094 bp long (positions 15,448–16,541).”

Line 195: replace “common” with “typical”

Line 202 “the control region”; “the mitogenome”

Line 205: “… no discernible repeat.” (delete “were discovered”)

Line 215: replace “did not show” with “was not present”

Line 216: this region: which region do you mean?

Table 4: control area = control region. What do the grey bars mean?

Table 5: where is the T-C = pseudouridine in the table? What does “T-arm” mean?

Figure 5: the figure is not readable, please use a larger format in the original (e.g. 300%) and copy that before pasting it into the manuscript file.

Line 256: change “Besides,” to “In addition,”

Line 264: please add a reference after “gene expression”

Line 292: delete the word “position”

Table 6 and 7: It is not clear what the dots represent. Please list all upper sequence positions in both tables.

Figure 6. Please state that this is a maximum likelihood phylogeny. Please specify if you used PCGs only or the full mitogenome. If you make a new phylogeny (which I recommended above) plase use an outgroup.

Tarssidae = Tarsiidae

Line 333: has been = is

Line 342: change “have separated from”  with “”be the sister group of”

Line 364: delete “species of”

Line 365: with = to

Line 365: that to Nasalis

Please italize all Latin species names in the references (Lines 388, 397, 400, 408, 418)

Comments on the Quality of English Language

The quality of the English could be improved.

Author Response

Dear Reviewer.

We appreciate you taking the time to carefully read over our work. It is a privilege for us to have had the chance to hear from someone with your caliber of experience. After giving the reviewer's idea considerable thought, we made a few adjustments with the file attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Characterization of the complete mitogenome of Vietnamese Pygathrix cinerea” is well written and structured by the Authors to whom one can hardly object to anything or almost anything in terms of the methodological rigor adopted in the analyses and experimental plan.

Nevertheless, I believe that the introductory section could be expanded and made more interesting so as to serve as an invitation to the strictly technical part addressed in the experimental investigation.

The expression "de novo" should always be written in italics.

Figure 1 should be made more readable or, if this is incompatible with editorial requests, inserted in a more readable form within the supplementary material package.

On the contrary, the image inserted in the supplementary material, if, as I presume, is of one of the subjects part of the experiment, could also be inserted in the main text.

Figure 5 is extremely interesting but is even more illegible than figure 1 and this aspect absolutely needs to be resolved.

Author Response

Dear Reviewer.

We appreciate you taking the time to carefully read over our work. It is a privilege for us to have had the chance to hear from someone with your caliber of experience. After giving the reviewer's idea considerable thought, we made a few adjustments with the file attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscrirpt presents new mitochondrial data from a monkey species, but the manuscript needs a lot of work before it is of publishable quality.  Given the very limited new genetic data generated for the study, the long string of authors is surprising.  The paper should be much, much shorter given the that very little new information is given and there were not really any hypotheses that were tested.

Comments on the Quality of English Language

The English needs a lot of work in this manuscript.

Author Response

Dear Reviewer.

We appreciate you taking the time to carefully read over our work. It is a privilege for us to have had the chance to hear from someone with your caliber of experience. After giving the reviewer's idea considerable thought, we made a few adjustments with the file attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is substantially improved. I still have a couple of issues with the phylogeny that is presented.

1.    The phylogeny is presented as a cladogram (which has no information on branch lengths). However, it has been recommended that new mitogenome descriptions include a phylogram (which presents actual branch lengths). See:

Botero-Castro, F, Delsuc, F & Douzery, EJ 2016. Thrice better than once: quality control guidelines to validate new mitogenomes. Mitochondrial DNA Part A 27: 449-454.

Sangster, G & Luksenburg, JA 2021. Sharp increase of problematic mitogenomes of birds: causes, effects and remedies. Genome Biol. Evol. 13(9), evab210.

2.    In addition, because Pygathrix cinerea is paraphyletic (with respect to Pygathrix nemaeus, see Liedigk et al. 2012) in mitochondrial DNA, it would be good to reconstruct a new maximum-likelihood phylogeny (phylogram) that also includes Pygathrix cinerea JQ821841. This can shed further light on the relationships between the two species.

3.    Furthermore, I recommend that the authors include Simias concolor JF293094 because this may affect inference of the relationships of Pygathrix, Rhinopithecus and Nasalis.

4.    The phylogeny should be prepared with an outgroup. Cynocephalus variegatus is the closest relative of primates and is the ideal outgroup.

5.    The phylogeny should include bootstrap support values.

To help the authors, I have prepared a new phylogeny to replace the one in the paper, which includes branch length information, Pygathrix cinerea JQ821841, Simias concolor JF293094, an outgroup (Cynocephalus variegatus), and bootstrap support values. The authors may include the following text:

We constructed a Maximum Likelihood phylogeny of 55 primates using IQ-tree version 2.2.2.6 (Minh et al. 2020). The appropriate substitution model for the data set was selected using ModelFinder (Kalyaanamoorthy et al. 2017). Branch support was obtained using ultra-fast bootstrapping (Hoang et al. 2018). The tree was rooted with the outgroup Cynocephalus variegatus (Dermoptera) based on Mason et al (2016).

Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS 2018. UFBoot2: Improving the ultrafast bootstrap approximation. Mol Biol Evol. 35:518–522. https://doi.org/10.1093/molbev/msx281

Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS 2017. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Meth. 14:587–589. https://doi.org/10.1038/nmeth.4285

Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Von Haeseler A, Lanfear R. 2020. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 37(5):1530–1534. https://doi.org/10.1093/molbev/msaa015

Mason, V. C., Li, G., Minx, P., Schmitz, J., Churakov, G., Doronina, L. et al. 2016. Genomic analysis reveals hidden biodiversity within colugos, the sister group to primates. Science Advances, 2(8), e1600633.

Line 2. I suggest the authors rename the title as “Characterization of the complete mitochondrial genome of grey-shanked douc langur Pygathrix cinerea, a critically endangered endemic to Vietnam (Mammalia: Primates)”.

Please consider making figure 1 much larger. The text is almost illegible.

Good luck with the revision.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

I have no comments

Author Response

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