Towards a Comprehensive DNA Barcode Library of Stenochironomus Kieffer, 1919 (Diptera: Chironomidae) from China

Round 1

Reviewer 1 Report

I suggest to clarify better the aim of the paper; see attached files

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thanks for your valuable time and sincere suggestions. Chironomid species are not only an environmental indicator but also a good model for adaptive evolution. Our primary objective in this study was to construct a comprehensive DNA barcode library of Stenochiromus Kieffer in China. In fact, we have included the majority of Stenochironomus species that could been collected in China to make sure a good coverage of DNA barcode library. We fully acknowledge the importance of both morphological description and DNA barcoding in a DNA library. Thus, all species identification was implemented with both morphological and DNA barcode method. For those species named with Stenochironomus sp. N ,such as Stenochironomus sp. XL19, they actually were unpublished new species. Additionally, we have revised the format of species names and incorporated any missed reference based on comments recevied. We sincerely appreciate your time and input once again. Your contributions have undoubtedly enhanced the quality and accuracy of our study.

Reviewer 2 Report

In this manuscript, the authors assessed the genetic variability of chironomid species inhabiting regions in China, with particular regard to the genus Stenochironomus. This study is interesting as it broadens the knowledge of this dipteran family that is important not only on taxonomical viewpoints but also considering that these organisms are useful hydrobiological markers. In this sense, I have missed information on the quality of water reservoirs that were visited for chironomid collections.  I think this is of broad interest for the readers and also for the authors as the putative presence of potential mutagenic agents in those environments might affect the results on genetic variability. 

I think the authors could justify a little bit more the choice of the genus for this study. Sometimes the text give to the the impression that only Stenochiromus has been collected in China.

In Materials and Methods (lines 115-116), what exactly do the authors mean? Could you describe the "analytical method" you refer?

The authors obtained data suggesting low genetic variability. Could this be related to the nature of the gene sequenced? I mean, as it is a very conserved mitochondrial gene, would you expect the same if a nuclear gene was studied. I would see something on this issue discussed in the text.

Although the work is very well presented, I think the readers will like to see a larva figure added to the adult one already present the manuscript.

It is ok.

Author Response

Dear Reviewers,

Thanks for your valuable time and sincere suggestions. Chironomid species are not only an environmental indicator but also a good model for adaptive evolution. Our primary objective in this study was to construct a comprehensive DNA barcode library of Stenochiromus Kieffer in China. Therefore, we collected all the DNA barcode sequences with morphological information, and further checked the genetic distance and phylogenetic relationship of Stenochiromus in China. We totally agree with that putative presence of potential mutagenic agents in those environments might affect the results on genetic variability. Unfortunately, the majority of samples examined in this study were adult individuals, making it challenging to trace their exact habitat origins. Nonetheless, we appreciate the suggestion and acknowledge its value, especially when working with larvae. This will enable us to gain deeper insights into the environmental factors that shape genetic variability.

For the "analytical method", we were mentioned about species identification process combined morphological identification, DNA barcode and voucher specimen.

For the issue about "genetic variability", it's does related to the nature of the gene. Genetic markers exhibit varying genetic distances, even within the same type of gene marker. A study conducted on helminths using different genetic markers, including nuclear and mitochondrial genes, revealed distinct patterns. Researchers discovered that the nuclear ribosomal ITS regions exhibited the largest genetic distance, whereas the nuclear rRNA genes displayed the smallest genetic distance (https://doi.org/10.1186/s13071-021-04737-y). The genetic distance observed in mitochondrial rRNA genes was comparable to that of mitochondrial protein-coding genes, but significantly higher than that of nuclear rRNA markers. Following your suggestions, we discussed about this issue in this study. We have also included a picture of the larvae species in manuscript.

We sincerely appreciate your time and input once again. Your contributions have undoubtedly enhanced the quality and accuracy of our study.

Round 2

Reviewer 1 Report

You have correctly updated the  manuscript following our suggestions