Karyotypes and COI Gene Sequences of Chironomus agilis2, Ch. balatonicus, and Camptochironomus tentans (Diptera, Chironomidae) from Kurchatskoe Lake, Tyumen Region, Russia

Round 1

Reviewer 1 Report

Very interesting paper.

The paper is very rich in information and of very large interest. I have made only some language adjustment and I note that Camptochironomus was considered a subgenus of Chironomus see: Martin, J., Blinov, A., Alieva, K., Hirabayashi, K.   2007       A molecular phylogenetic investigation of the genera closely related to Chironomus Meigen (Diptera: Chironomidae). In: Andersen, T. (ed.): Contributions to the systematics and ecology of aquatic Diptera. A tribute to Ole A. Sæther. Caddis Press, Columbus (OH); pp. 193-203.

 

 If you have more recent literature or if you are of another advice, it is necessary you specify this, for example restoring the subgenus or the genus status; you can consult J. Martin, who is the most competent in this matter.

 

You should be cautious to call your Bayesian tree a phylogenetic tree;  your Bayesian tree emphasizes  a separation of Camptochironomus from other groups, but only a comparison of cytogenetic, morphological (including adults and pupal exuviae !) and molecular analysis (including other markers ?) should allow a sustainable phylogenesis.

I am not expert of the genus, J. Martin is surely the most one I know, but you have also Russian colleagues (e.g. from the Novosibirsk group) very competent; so I suggest you contact them, if you have not yet done, to add some general conclusion about your results 

Comments for author File: Comments.pdf

Author Response

Thank you very much for your recommendations!

Yes, indeed, you are right, Camptochironomus is a subgenus. First of all, it is distinguished by the cytocomplex «camptochironomus» - AB CF DE G (arm combination), in Ch. plumosus is «thummi» cytocomplex – AB CD EF and G. This is beyond question. In this work, we do not set ourselves the goal of such significant interventions in the systematic position of this species. You are right again, according to many scientists, phylogeny requires all the parameters listed by you (including the COI gene, CytB, gb2b …). Only one sequence of the COI gene is not enough to study phylogeny. There is an excellent study dedicated to this question (Proulx et al. 2013). Jon Martin is really a great man; he reviewed my previous works. For example, this study (Bolshakov and Movergoz 2022). There is a similar tree there, and he had no questions. He has given us a lot of good advice.

Bolshakov V V, Movergoz EA (2022) Karyotype and COI gene sequences of Chironomus melanotus Keyl, 1961 from the Yaroslavl region, Russia, and the difficulties with its identification using GenBank and BOLD systems. Comparative Cytogenetics 16: 161–172. https://doi.org/10.3897/compcytogen.v16.i3.90336

Proulx I, Martin J, Carew M, Hare L (2013) Using various lines of evidence to identify Chironomus species (Diptera: Chironomidae) in eastern Canadian lakes. Zootaxa 3741: 401–458. https://doi.org/10.11646/zootaxa.3741.4.1

 

 

The paper is very rich in information and of very large interest.

I have made only some language adjustment and I note that

Camptochironomus was considered a subgenus of Chironomus see:

Martin, J., Blinov, A., Alieva, K., Hirabayashi, K.  2007  A molecular phylogenetic investigation of the

genera closely related to Chironomus Meigen (Diptera: Chironomidae).  

In: Andersen, T. (ed.): Contributions to the systematics and ecology of aquatic Diptera. 

A tribute to Ole A. Sæ ther. Caddis Press, Columbus (OH); pp. 193-203.

 

If you have more recent literature or if you are of another advice, it is necessary you specify this, 

for example restoring the subgenus or the genus status; you can consult J. Martin, who is the most

competent in this matter.

You should be cautious to call your Bayesian tree a phylogenetic tree;  your Bayesian tree emphasizes 

a separation of Camptochironomus from other groups, but only a comparison of cytogenetic,

morphological (including adults and pupal exuviae !) and molecular analysis (including other markers ?)

should allow a sustainable phylogenesis.

I am not expert of the genus, J. Martin is surely the most one I know, but you have also Russian

colleagues (e.g. from the Novosibirsk group) very competent; so I suggest you contact them, if you

have not yet done, to add some general conclusion about your results  

Author Response File: Author Response.docx

Reviewer 2 Report

This is an interesting contribution on a poorly studied group, based on an integrative approach using different sources of characters (i.e., morphology, karyotypes, and COI sequences).  It was not clear to me what was the sampling design, so that in the discussion original data from the study was somehow mixed with data from the literature, which is fine to do, but should be clear from according to the methods in the paper.  Often Chironomus was mentioned, but probably chironomids were meant, as not all larvae belonged to this genus.  It is not clear why only one species was morphologically diagnosed.  Other minor writing observations are marked to the text.  A general English style revision would be appropriate.

Comments for author File: Comments.pdf

Author Response

We would like to express our appreciation for the Reviewers comments to our manuscript! We thank you for the language correction! With your permission, we will not indicate small errors here (language corrections), they are highlighted in yellow in the text of the manuscript.

 

The changes are highlighted in yellow.

 

Lines 32, 39 etc. Chironomus - here we mean the species of the genus Chironomus. in order not to write it every time, we write only Chironomus. This is written in many articles, and this is the first time we receive such a comment.

Line 83-85. Thank you! New version.

The current study aims to precisely identify the cryptic species of Chironomus larvae from Kurchatskoe lake with mineralized water using a variety of approaches, including morphology, cytogenetics, and molecular genetics, as well as a study of the level of chromosomal polymorphism in the identified species.

Line 120. perhaps this could go to a table in an appendix.

We'll wait to see what the editors say.

Line 147. why only this species is morphologically diagnosed?  Could this be explained or justified?

“Photos of the morphology of the larva are presented for the first time in this study (Fig. 1)”.

Line 295. not all species are Chironomus…

Genus Chironomus, subgenus Camptochironomus.

“Figure 5. Bayesian tree of the analyzed samples of Chironomus (Camptochironomus) spp. inferred from COI gene sequences. Species name, GenBank accession numbers and group name are shown to the right of the branches. Support p-values are given if they exceed 0.3.”

Line 377. …with respect to ___ populations…

“Only in C. tentans from China the distance was 4.5%, we suggest that additional research with the use of cytogenetic analysis is required.”

 

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript by Bolshakov et al. describes the identification of some chironomid species inhabiting the Kurchatskoe lake (Russia). Species were identified by means of morphology, cytogenetics and  molecular biology as well. The study was justified by (1) lakes undergoing increased mineralization may present unusual organisms for that area and (2) species discrimination is difficult solely on the basis of morphology. 

In the Introduction, I have missed more information on the mineralization process regarding the lake. Particularly nowadays, readers are interested in diversity connected to changes in the flora/fauna as a function of climate, pollution, mineralization, etc. For this reason, I think further information on the origin of increasing mineralization could attract the attention of more readers.

It is true that a reliable assessment on chironomid fauna of a given area is necessary for water bodies I have a doubt in relation to chironomids participating in "water purification" as stated by the authors in the Abstract and Discussion as well. To my knowledge, chironomids are important biological markers for water body contamination from several sources. However, the author information gives the impression that biological activity from chironomid species results in water purification. If I am not mistaken, the two sentences must be rephrased. Otherwise, the authors must include references where "water purification" from chironomids are clearly stated.

In Materials and Methods, that are well described, there is  two citation forms, numbering and author names. I do not know whether Diversity accepts both. Otherwise, please homogenize citations using just numbering.

In Results, karyotype descriptions, including figure legends, the authors wrote "nucleous" (N) instead  nucleolus. Please, correct the conceptual mistake using either nucleolus (singular) or nucleoli (plural) when it is necessary.

Finally, Discussion and Conclusion are too descriptive and could be enriched as I suggested for the Introduction. For instance I have also missed comments on how the data obtained in the study (with particular regard to the low heterozygosity and genetic distances) establish (or not) some dialog with the mineralization process in course. This will be welcome not only to the readers but also to the authors.

 

Author Response

The manuscript by Bolshakov et al. describes the identification of some chironomid species inhabiting the Kurchatskoe lake (Russia). Species were identified by means of morphology, cytogenetics and molecular biology as well. The study was justified by (1) lakes undergoing increased mineralization may present unusual organisms for that area and (2) species discrimination is difficult solely on the basis of morphology.

 

In the Introduction, I have missed more information on the mineralization process regarding the lake. Particularly nowadays, readers are interested in diversity connected to changes in the flora/fauna as a function of climate, pollution, mineralization, etc. For this reason, I think further information on the origin of increasing mineralization could attract the attention of more readers.

--Thank you for the recommendation. These lakes have not been studied much yet. I added a line. The fluctuation of salinity in these lakes is a natural process.

“It is noted that most of the lakes are non-flowing and shallow, growing over with vegetation; the shores are often swampy. Intensive evaporation of water and a low rainfall contributes to increased mineralization [2]. Lakes with a mineralization of 1–10‰ make up about 28% of all Ishim Plain lakes [3], at the moment of sampling, the water mineralization of Kurchatskoe Lake was 7‰.”

It is true that a reliable assessment on chironomid fauna of a given area is necessary for water bodies I have a doubt in relation to chironomids participating in "water purification" as stated by the authors in the Abstract and Discussion as well. To my knowledge, chironomids are important biological markers for water body contamination from several sources. However, the author information gives the impression that biological activity from chironomid species results in water purification. If I am not mistaken, the two sentences must be rephrased. Otherwise, the authors must include references where "water purification" from chironomids are clearly stated.

-- about the significance of chironomids in the biological purification of water is in the work of Shobanov.

«by building tube houses in bottom sediments, they can increase the water-soil phase interface by several times, so significantly intensifying microbiological processes; in addition, surpassing the zebra mussel (Dreissena polymorpha) in relative sedimentation activity, the Chironomus performs the function of biological purification of waterbodies».

In- Full Doctor Thesis of dissertation (in Russian).

«Genus Chironomus Meigen (Diptera, Chironomidae): Systematics, biology and evolution».

https://www.dissercat.com/content/rod-chironomus-meigen-diptera-chironomidae-sistematika-biologiya-evolyutsiya  

(Shobanov, 2000).

 

In Materials and Methods, that are well described, there is  two citation forms, numbering and author names. I do not know whether Diversity accepts both. Otherwise, please homogenize citations using just numbering.

--corrected. Thank you.

In Results, karyotype descriptions, including figure legends, the authors wrote "nucleous" (N) instead nucleolus. Please, correct the conceptual mistake using either nucleolus (singular) or nucleoli (plural) when it is necessary.

--corrected. Thank you.

Finally, Discussion and Conclusion are too descriptive and could be enriched as I suggested for the Introduction. For instance, I have also missed comments on how the data obtained in the study (with particular regard to the low heterozygosity and genetic distances) establish (or not) some dialog with the mineralization process in course. This will be welcome not only to the readers but also to the authors.

-- I think I don't fully understand your comment, perhaps this is a topic for another publication devoted specifically to this theme. Reasoning on this theme will significantly increase the scope of this paper.

“We know that the quantity of individuals is not enough for statistical analysis, but we can assume that there is an excess of heterozygotes for balD1.2 in Kurchatskoe Lake. It was shown, that in the distribution of banding sequences and the compositions of zygotic combinations on range of habitat depend more on conditions in water bodies, than on geographical localization [13,24,46]. In addition, in experiments with infection by the entomopathogenic bacterium Bacillus thuringiensis israelensis, it was found that the presence of balD1.1 and balD2.2. reduces the possibility of infection [72]. Based on these data, we suggest the presence balD1.2 combination has adaptive significance for larvae to mineralized water in Kurchatskoe Lake.”

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

According to my comments, the authors made changes to my satisfaction except that concerning chironomids participating in water purification process. References for this process were in fact provided in their comments but they were not added to the Discussion. I still think that references are necessary in this case particularly to the readers. The authors may also want to consider adding to the Discussion the explanation for the process given in their responses

Author Response

We would like to express our appreciation for the Reviewers comments to our manuscript!

Thank you for your time!

The changes are highlighted in yellow.

According to my comments, the authors made changes to my satisfaction except that concerning chironomids participating in water purification process. References for this process were in fact provided in their comments but they were not added to the Discussion. I still think that references are necessary in this case particularly to the readers. The authors may also want to consider adding to the Discussion the explanation for the process given in their responses

--sorry, I just forgot to add references to the work.

They build tube-shaped houses; as a result, the area of interaction between bottom sediments and water increases, and microbiological processes intensify; they are also surpassing the Dreissena polymorpha (Pallas, 1771) in relative sedimentation activity, all of which contribute to the biological purification of water [72].

 

 

Author Response File: Author Response.docx