Comparative Analysis of Microtendipes Mitogenomes (Diptera: Chironomidae) and Their Phylogenetic Implications
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors present a valuable study that contributes new insights into the taxonomy of Microtendipes through mitochondrial genome analysis, making it a strong candidate for publication. A few revisions, as detailed below, would further enhance the clarity and accessibility of the manuscript for readers.
- In Figure 1, the representation of the eight mitochondrial genomes is quite small, which may make it challenging for readers to distinguish details, particularly in a printed version. Considering the reported consistency in the basic gene arrangement across these eight samples, the authors might consider presenting a single, representative genome map or a generalized diagram illustrating the basic gene arrangement. This would likely improve visual clarity.
- For the phylogenetic tree(s) presented, the authors mention analyzing multiple datasets. To enhance clarity and reproducibility, it is recommended that the figure caption for each phylogenetic tree explicitly states the specific dataset and the analytical method (e.g., software, algorithm) used for its construction.
- Additionally, concerning the phylogenetic analyses, including the results of model selection and partitioning (e.g., from PartitionFinder2) in the supplementary materials would be beneficial for transparency. The authors report using RAxML (version 8 series). It is worth noting that this version has limitations regarding the range of available substitution models. If the model selection process indicated that models other than GTR were optimal for certain partitions, the authors should consider, or discuss the implications of, using software that supports a broader array of models, such as RAxML-NG or IQ-TREE, to ensure the robustness of the phylogenetic inference.
- Figure 3, illustrating codon usage, could benefit from a more detailed explanation in its legend. While it is presumed that the different colors within the bar graph correspond to specific codons, the stacking order is not immediately intuitive (e.g., it appears potentially reversed). Furthermore, distinguishing between codons represented by similar colors, or those with very low relative frequencies, is challenging in the current format. The authors might consider if presenting this data in a tabular format, with precise numerical values for codon usage, would offer greater clarity and ease of interpretation for readers.
- The figure that compares genetic distances across different genes also requires further clarification. Specifically, the meaning or significance of the lowercase letters (e.g., a, b, c) associated with each gene should be clearly explained in the figure legend or the main text.
- A minor point: it appears there are two distinct figures labeled as 'Figure 4' in the manuscript. This should be corrected to ensure consistent and unambiguous referencing throughout the text.
Author Response
Q: In Figure 1, the representation of the eight mitochondrial genomes is quite small, which may make it challenging for readers to distinguish details, particularly in a printed version. Considering the reported consistency in the basic gene arrangement across these eight samples, the authors might consider presenting a single, representative genome map or a generalized diagram illustrating the basic gene arrangement. This would likely improve visual clarity.
Response: We have revised Figure 1 accordingly, as suggested, only presenting Microtendipes tuberusous， others provide in supplementary files.
Q: For the phylogenetic tree(s) presented, the authors mention analyzing multiple datasets. To enhance clarity and reproducibility, it is recommended that the figure caption for each phylogenetic tree explicitly states the specific dataset and the analytical method (e.g., software, algorithm) used for its construction.
Response: We have updated the figure caption.
Q: Additionally, concerning the phylogenetic analyses, including the results of model selection and partitioning (e.g., from PartitionFinder2) in the supplementary materials would be beneficial for transparency. The authors report using RAxML (version 8 series). It is worth noting that this version has limitations regarding the range of available substitution models. If the model selection process indicated that models other than GTR were optimal for certain partitions, the authors should consider, or discuss the implications of, using software that supports a broader array of models, such as RAxML-NG or IQ-TREE, to ensure the robustness of the phylogenetic inference.
Response: We fully agree that transparency in model selection is essential. As suggested, we have now included the detailed results from PartitionFinder2 (including model selections and partitioning schemes) in Supplementary File 1. And we used RAxML-NG to reconstruct the phylogeny and updated in supplementary file.
Q: Figure 3, illustrating codon usage, could benefit from a more detailed explanation in its legend. While it is presumed that the different colors within the bar graph correspond to specific codons, the stacking order is not immediately intuitive (e.g., it appears potentially reversed). Furthermore, distinguishing between codons represented by similar colors, or those with very low relative frequencies, is challenging in the current format. The authors might consider if presenting this data in a tabular format, with precise numerical values for codon usage, would offer greater clarity and ease of interpretation for readers.
Response: We try to present the data in a tabular format, but it was too large to present. The figure format may be better showing the results as similar research use such format in hassan et al. (2023) in BMC Genomics. https://doi.org/10.1186/s12864-023-09809-0.
Q: The figure that compares genetic distances across different genes also requires further clarification. Specifically, the meaning or significance of the lowercase letters (e.g., a, b, c) associated with each gene should be clearly explained in the figure legend or the main text.
Response: We have updated the figure.
Q: A minor point: it appears there are two distinct figures labeled as 'Figure 4' in the manuscript. This should be corrected to ensure consistent and unambiguous referencing throughout the text.
Response: We have updated the figure.
Reviewer 2 Report
Comments and Suggestions for AuthorsPlease see attachment.
Comments for author File: 
Comments.pdf
Author Response
Q: Reproducibility: Authors in their supplemental provide data, please provide (or link to) script that analyze the data to generate the output(Figures/Tables)。
Response: All script used have uploaded to the website, https://github.com/geduo42/mitogenome.
Q: Figure captions Figures 2–4 could benefit from brief explanations of what each color/track represents (e.g., codon-usage heatmap color scale, Manhattan plot axis labels).
Response: We have updated the captions of the figures.
Q: 3. Minor formatting A few typographical errors remain (e.g.,
- “optical codons” → “optimal codons” in text line 329;
- “Condon” → “Codon” in text line 345).
RESPONSE: updated.
Q: A final proofread will fix these.
Optional enhancements
- Provide branch-length units (substitutions per site) on the main phylogeny for readers interested in relative divergence times.
RESPONSE: updated.
Q: Offer a summarized table of support values comparing the four data-set configurations at a glance (PCG vs PCGrRNA etc.).
RESPONSE: Here are summarized table of four dataset support value.
Table 1 ML tree based on non-partitioned data supports of main stems.
|
|
PCG |
PCG rRNA |
PCG12 |
PCG12RNA |
|
Polypedilum complex |
0.99 |
1 |
0.99 |
1 |
|
Chironomus complex |
1 |
0.98 |
1 |
1 |
Table 2 ML tree based on partitioned data supports of main stems.
|
|
PCG |
PCG rRNA |
PCG12 |
PCG12RNA |
|
Polypedilum complex |
0.78 |
0.72 |
1 |
0.78 |
|
Chironomus complex |
1 |
0.72 |
1 |
0.60 |
Q: • If feasible, include Pseudochironomini mitogenomes in a supplementary analysis to firm up the tribal discussion.
RESPONSE: Pseudochironomini mitogenomes are not accessible in the nowadays phylogeny.
Reviewer 3 Report
Comments and Suggestions for AuthorsIn the reviewed MS a group of six scientists from China give their results on comparative mitogenomic study of the phylogeny of tiny dipterans of the genus Microtendipes (family Chironomidae). They sequences eight new mitogenomes of Microtendipes, provided their basic comparison and gene annotations, and performed e series of ML and BI phylogenetic analyses using differently organized alignments. The MS in general is well-written, however it needs substantial revision in terms of restructuring and reformatting. The MS is too long, the authors are requested considering to make it shorter 2 times. The section 4.1. is written in suboptimal way and needs major revision. The authors are requested to provide morphological data (photographs, drawings, measurements) supporting their identification of the sequenced dipteran samples. This data could be given as a separate figure plate embedded in the MS or as a supplementary figure. Additional remarks are given below.
Please, check the Supplement: one of the two folders in the archive is empty.
16 “Comparative analyses revealed significant intraspecific genetic distances (5.3–13.8% across 13 protein-coding genes), challenging COI-based barcoding for species delimitation.” -please revise this sentence: it is unclear if you speak only about Cox1 or about all mitochondrial protein coding genes, and what is the real connection between the percentages and the “barcode” region.
18 Microtendipes as a distinct clade - can you say anything about the structure of this clade? It seems there are some subclades?
19 “Notably, larval morphology-based species groupings conflicted with molecular data, suggesting cryptic diversity.” – This is unclear. The conflict does not immediately implies cryptic species, it only indicates different evolution of different characters. Please, revise. Note, that in the subsection 4.3. you do not interpret this conflict as cryptic speciation. Be consistent.
40 greatest – please, make the statement more moderate.
79 we have presented eight specimens – revise
102 mito genomes - revise
113 Rheocricotopus villiculus – why this species is absent in the Table 1?
Table 1: please, explain taxonomy of the insects from this Table. Insert data on all outgroups in the subsection 2.3., explain the out-group choice and overview all mitogenomic data on Chironomidae from GBank. There are many circular mitogenomes of Chironomidae (about 500 circular DNA sequences of 15000-16500 bp lengh), why they were not used in the analyses?
188 cox1 gen – please, check the format for gene names and follow in the entire MS
209 Stenochironomus – please, report on the topography when this rogue taxon is excluded
238: Microtendipes bimaculatus-clade 1 (MZ981734); Microtendipes bimaculatus-clade 2 (PP966948); Microtendipes bimaculatus-clade 3 (PP966952 and PP966953) – please, provide data confirming morphological identity of these speciemens. The sequences differences may be because of wrong morphological ID. However no data on this issue is provided.
section 4.1. This section reports Results and their interpretation. It is better (a) to specify in the Introduction that you have an additional goal, concerning Cox1 barcode region, (b) give all results on 658bp region comparison in Results, and (c) present a revised discussion on this in Discussion. Currently all these is given in suboptimal way.
Fig.4. This figure needs a better explanation including the number of species, number of conspecific samples, number of dots in the diagram and their origin, and letters abcdefg…. Please, join this with Supplement and the figure caption.
subsection 4.2. – no need to repeat the data from subsection 3.4. Please, profide only brief discussion and interpretations
Comments on the Quality of English Languageminor to moderate
Author Response
Q: The MS is too long, the authors are requested considering to make it shorter 2 times. The section 4.1. is written in suboptimal way and needs major revision.
A: Regarding manuscript length: In direct response to your request to significantly shorten the manuscript, we have fundamentally restructured the presentation of our findings. We have consolidated the Results and Discussion sections into a single integrated "Results and Discussion" section. Regarding Section 4.1 (now within the integrated Results and Discussion): We acknowledge your assessment that the original Section 4.1 was suboptimal and required major revision. We have undertaken a comprehensive restructuring and rewriting of this specific subsection.
Q: The authors are requested to provide morphological data (photographs, drawings, measurements) supporting their identification of the sequenced dipteran samples. This data could be given as a separate figure plate embedded in the MS or as a supplementary figure.
A: The genetic distance of PCGs between 9 specimens (contains 1 each of Microtendipes bimaculatus-Clade1, Microtendipes bimaculatus-Clade2, Microtendipes baishanzuensis, Microtendipes tuberosus, Microtendipes wuyiensis, and 2 each of Microtendipes bimaculatus-Clade3, Microtendipes robustus). The dots represent the pairwise genetic distances between the 9 specimens for each gene, while the box represents the first quartile, median, and the third quartile. The letters above each group indicate the significance level of genetic distance differences between genes. The presence of the same letter indicates that there is no significant difference in genetic distance between the two genes, while the absence of any common letters indicates that there is a significant difference in genetic distance between the two genes.
Q: Additional remarks are given below. Please, check the Supplement: one of the two folders in the archive is empty.
A: We have checked the supplementary files, and update the files.
Q: 16 “Comparative analyses revealed significant intraspecific genetic distances (5.3–13.8% across 13 protein-coding genes), challenging COI-based barcoding for species delimitation.” -please revise this sentence: it is unclear if you speak only about Cox1 or about all mitochondrial protein coding genes, and what is the real connection between the percentages and the “barcode” region.
A: Agree with the comment, and the sentence is deleted in the ms.
Q: 18 Microtendipes as a distinct clade - can you say anything about the structure of this clade? It seems there are some subclades?
A: We have rewrite the sentence in abstract.
Q: 19 “Notably, larval morphology-based species groupings conflicted with molecular data, suggesting cryptic diversity.” – This is unclear. The conflict does not immediately implies cryptic species, it only indicates different evolution of different characters. Please, revise. Note, that in the subsection 4.3. you do not interpret this conflict as cryptic speciation. Be consistent.
A: We have rewrite the sentence in abstract. Notably, larval morphology-based species groupings conflicted with molecular data, suggesting that classifications derived from larval morphological traits may be unreliable.
Q: 40 greatest – please, make the statement more moderate.
A: Chironomidae, normally known as non-biting midges, exhibit the remarkable diversity of species that have evolved to thrive under extreme abiotic environments.
Q: 79 we have presented eight specimens – revise
A: we have presented eight mitogenomes.
Q: 102 mito genomes - revise
A: revised to mitogenomes.
Q: 113 Rheocricotopus villiculus – why this species is absent in the Table 1?
A: We have updated the Table 1.
Q: Table 1: please, explain taxonomy of the insects from this Table. Insert data on all outgroups in the subsection 2.3., explain the out-group choice and overview all mitogenomic data on Chironomidae from GBank. There are many circular mitogenomes of Chironomidae (about 500 circular DNA sequences of 15000-16500 bp lengh), why they were not used in the analyses?
A: For phylogenetic reconstruction, we analyzed mitochondrial genomes from 22 Chironominae genera (Table 1), including eight newly sequenced mitogenomes. Rheocricotopus villiculus (Orthocladiinae: MW373526) was designated as the outgroup, reflecting its established position as the sister subfamily to Chironominae. This phylogenetically proximate outgroup minimizes long-branch attraction artifacts while ensuring robust tree rooting. Taxon selection prioritized taxonomic diversity, data completeness, and relevance to resolving the systematic placement of Microtendipes within Chironominae.
Q: 188 cox1 gen – please, check the format for gene names and follow in the entire MS
A: we have revised the format for all the genes in entire ms.
Q: 209 Stenochironomus – please, report on the topography when this rogue taxon is excluded
A: Upon excluding Stenochironomus from the dataset, we re-performed phylogenetic analyses using both Maximum Likelihood (ML) and Bayesian Inference (BI) methods. The resulting topologies remained consistent with those generated prior to taxon removal, demonstrating no structural alterations in the phylogenetic relationships within Chironominae. This stability suggests that Stenochironomus does not exert significant influence on the overall framework of the subfamily’s phylogeny under the current analytical parameters.
Q: 238: Microtendipes bimaculatus-clade 1 (MZ981734); Microtendipes bimaculatus-clade 2 (PP966948); Microtendipes bimaculatus-clade 3 (PP966952 and PP966953) – please, provide data confirming morphological identity of these speciemens. The sequences differences may be because of wrong morphological ID. However no data on this issue is provided.
A: In our prior study (Song et al., 2023), all specimens corresponding to clades 1–3 were rigorously morphologically examined and confirmed to align with the diagnostic characteristics of M. bimaculatus.
Q: section 4.1. This section reports Results and their interpretation. It is better (a) to specify in the Introduction that you have an additional goal, concerning Cox1 barcode region, (b) give all results on 658bp region comparison in Results, and (c) present a revised discussion on this in Discussion. Currently all these is given in suboptimal way.
A: We have amended the Introduction (Section 1) to explicitly state our additional research goal concerning the analysis of the Cox1 barcode region (658bp). All comparative results pertaining to the 658bp region have been moved from the original Section 4.1 into the dedicated Results section within the integrated Results and Discussion. The interpretation and broader implications of the 658bp region results have been comprehensively revised and integrated into the(now part of the integrated Results and Discussion.
Fig.4. This figure needs a better explanation including the number of species, number of conspecific samples, number of dots in the diagram and their origin, and letters abcdefg…. Please, join this with Supplement and the figure caption.
A: Figure 5. The genetic distance of PCGs between 9 specimens (contains 1 each of Microtendipes bimaculatus-Clade1, Microtendipes bimaculatus-Clade2, Microtendipes baishanzuensis, Microtendipes tuberosus, Microtendipes wuyiensis, and 2 each of Microtendipes bimaculatus-Clade3, Microtendipes robustus). The dots represent the pairwise genetic distances between the 9 specimens for each gene, while the box represents the first quartile, median, and the third quartile. The letters above each group indicate the significance level of genetic distance differences between genes. The presence of the same letter indicates that there is no significant difference in genetic distance between the two genes, while the absence of any common letters indicates that there is a significant difference in genetic distance between the two genes. CO1_658 represents the 658 bp COI barcode.
subsection 4.2. – no need to repeat the data from subsection 3.4. Please, profide only brief discussion and interpretations
A: Yes, we have revised the Results and Discussion part. And make brief discussion and interpretations.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI thank the authors for thoroughly addressing all my comments.
In particular, I really appreciate the inclusion of the analysis scripts via the GitHub repository and the updates made to the figure captions and typographical issues. The addition of summarized tables comparing support values across datasets and the clarification regarding Pseudochironomini mitogenomes are also helpful.
The manuscript has clearly improved in clarity and completeness, and I have no further questions. I think the paper is ready to go.
Author Response
Thank you very much for your positive assessment of our revised manuscript and your constructive feedback. We are delighted to learn that you found the revisions to significantly improve the clarity and completeness of the paper and that you consider it ready for publication.
It is particularly heartening to know that you feel the manuscript has improved in clarity and completeness and that you have no further questions. We have carefully addressed all points raised during the review process based on your comments and those of the other reviewers.
We will ensure the final version is meticulously proofread before publication.
Once again, we sincerely appreciate your valuable time and insightful review, which has greatly strengthened the quality of our manuscript.
Author Response File: 
Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsWhile the authors have addressed several of the reviewers' comments, the manuscript still requires linguistic polishing to improve clarity and readability. Additionally, the text remains excessively long and should be condensed where possible. The authors have disregarded the reviewer's explicit request to provide morphological data supporting the identification of the investigated taxa (e.g., diagnostic characters, images) in the supplementary material. Currently, their identifications rely solely on genotyping, which is insufficient given the well-documented inaccuracies in GenBank sequences. Without morphological validation, the taxonomic conclusions lack robustness, and the manuscript's reliability is compromised. This issue must be resolved before further consideration.
Author Response
Comment 1: Language Polishing and Length
Author Response:
-
Language Polishing: We have now engaged a professional English editing service to thoroughly polish the entire manuscript. This focused on improving sentence structure, terminological accuracy, clarity of expression, and overall readability. We believe the revised text demonstrates significant improvement in language quality.
-
Length Reduction: We have carefully reviewed the manuscript and condensed the text wherever possible, without compromising the core scientific content or logical flow.
Comment 2: Lack of Morphological Data Supporting Species Identification
Author Response: We wish to clarify that the species identifications in this study were not based solely on genotypic analysis. All species identifications reported in this study are grounded in systematic morphological investigations conducted during our prior research in Song et al. (2023). Crucially, the first author of that paper (Dr. Song) is the primary identifier (the person who determined the species) for the taxa discussed in the present study. The present research builds upon morphologically confirmed identities established by that prior work. Therefore, our identifications represent an integrative taxonomy approach, combining morphological and molecular evidence, rather than relying on genotyping alone.
Author Response File: Author Response.pdf
