Genetic Variation and Phylogeography of Lumbriculus variegatus (Annelida: Clitellata: Lumbriculidae) Based on Mitochondrial Genes

Round 1

Reviewer 1 Report (Previous Reviewer 2)

I am glad to see that the manuspcript „Genetic variation and phylogeography of Lumbriculus variegatus (Annelida: Clitellata: Lumbriculidae) based on mitochondrial genes“ has been improved since our last review, although I still have some major issues with the paper that I mention here and in the attached pdf. I think these major issues cannot be adressed unless a lot of rework is done, which is probably not possible for the authors. A bit more emphasis should be given on them.

 

One of the major limitations I see, is still the age of the species which is very old (and the low number of potential cryptic species). I think this should be stated clearly from the abstract and through the text that it is surprising. It may have been addressed in between but I did not find it clearly stressed to notice it at least.

I am also wondering why the data from Gustafsson et al. 2009 are not all included. I think it has to be. If it is not possible to include in the study because it would necessitate to start the study from scratches, then it has to be stressed in the text. This is a very important flaw of this study which make it a bit disarticulated from the rest of science and specifically works on this species. A new paragraph has been added discussion this study but it has to go further.

After WoRMS there are 13 accepted species of Lumbriculus, this should be discussed somewhere at least in the discussion. What about their repartition ?

Also since you discuss the glacial refugia, if ever you have some informations about the altitude of the samples that would really give an interesting information to add in the supplementary material as well as a dissussion in general on this information (if you have it, and I hope you have it). This would be crucial on a phylogeographical point of view.

All in all I see a great improvement from the previous version but think that there is still a lot of room for further improvements.

Sincerely.

Comments for author File: Comments.pdf

Author Response

We would like to thank you for giving us the opportunity to revise our manuscript. Those comments were all valuable and very helpful for revising and improving our paper. In the revised version, we have carefully taken your comments into consideration in preparing our revision. The specimen sequences with both COI and 16S genes from Gustafsson et al. 2009 was used for analysis. For example, CE1103, CE1105 and CE1109 and so on. For the glacial refugia, I added the information of altitude of the specimens in the supplementary material. The altitude of lineage I was higher than lineage II. In addition, we provide our point-by-point responses to each of the comments in the following pages.

Author Response File: Author Response.docx

Reviewer 2 Report (Previous Reviewer 3)

I congratulate the authors for the significant improvement of their manuscript. There are still some minor editing issues and after their revision I consider the manuscript can be published.

Author Response

We would like to thank you for giving us the opportunity to revise our manuscript. Those comments were all valuable and very helpful for revising and improving our paper.

Reviewer 3 Report (Previous Reviewer 1)

The authors revised the manuscript by analyzing sequences from mitochondrial COI and 16S rRNA. However, sampling methods and data analysis is still poorly written. The authors claim two lineages of L. variegatus, however, I could not locate the possible justification for samples from Tibetan Plateau forming a clade with samples from Sweden and US.

I have highlighted the areas that are to be improved in the manuscript in annotated PDF.  

 

Comments for author File: Comments.pdf

Author Response

We would like to thank you for giving us the opportunity to revise our manuscript. Those comments were all valuable and very helpful for revising and improving our paper. In the revised version, we have carefully taken your comments into consideration in preparing our revision. In addition, we provide our point-by-point responses to each of the comments in the following pages.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report (Previous Reviewer 2)

I am fine with the answers of the authors and think that now the paper has been greatly improved, with a way better scientific framework. I have very few minor comments now.

Nice to see the altitude in the supplementary material, it really shows that most of the specimens come from a high altitude and strenghtenth some conclusions of the paper. On thing that could be mentioned, discussed may be about a biais in sampling ? Where the collecting campaigns mostly in high altitude or have they been also researches of Lumbriculus variegatus without success in lower altitude. This can be answered in one sentence :)

 

Line 265 : please remove the dash in „Pangean“

My last kind of important remark is this one : if I am not wrong, you found only two species of Lumbriculus in WoRMS because you may have forgotten to uncheck the „marine only“ cursor on the top right of the page. Here is the list of species I found :

Lumbriculus alexandrovi Popchenko, 1976

Lumbriculus ambiguus (Holmquist, 1976)

Lumbriculus genitosetosus (Holmquist, 1976)

Lumbriculus illex Timm & Rodriguez, 1994

Lumbriculus inconstans (Smith, 1895)

Lumbriculus japonicus Yamaguchi, 1936

Lumbriculus kolymensis Morev, 1982

Lumbriculus mukoensis Yamaguchi, 1953

Lumbriculus multiatriatus Yamaguchi, 1937

Lumbriculus olgae Sokolskaya, 1976

Lumbriculus sachalinicus Sokolskaya, 1967

Lumbriculus tetraporophorus Popčhenko, 1976

Lumbriculus variegatus (Müller, 1774)

If I am right then this should be discussed in regard to repartition, characters etc. In the discussion at least briefly, at best as a new paragraph. It would open research programs on the species and show that you considered the putatively closely related species (eventhough it seems that there is no morphological or molecular phylogeny with all these species in my knowledge).

Anyway I am looking forward to see the article published.

Sincerely

Author Response

Dear reviewer,

 

I appreciated all of your comments, thank you very much.

 

For the biais in sampling, there were also collections at lower altitude, but the number was small. Most specimens were collected at high altitudes, but less at low altitudes. It just goes to show that the L. variegatus is distributed mainly at a high altitude.

 

The dash in Pangean has been removed. Thank you for your advice.

 

I am so sorry about the species of Lumbriculus, and you are right. There are indeed thirteen species. We value your feedback and suggestions. Below is the added discussion.

Thirteen species of the genus of Lumbriculus are recognized. These species are morphologically identical, with chaetae sigmoid, bifid and upper tooth reduced. The identification of species within the genus depends on the distribution and pattern of the genitalia. Lumbriculus ambiguus (Holmquist, 1976) shares with L. variegatus the character of the atria paired in segment VIII, yet the former species has two pair testes and the latter with only one pair. Lumbriculus tetraporophorus Popčhenko, 1976 is similar to Lumbriculus kolymensis Morev, 1982 based on the atria in segment VIII and IX. They differ in the number of the spermathecae with two and four pairs, respectively. There are two groups of four species whose atria are located at segment X. In the group with only one pair of ovaries, the spermathecae of Lumbriculus alexandrovi Popchenko, 1976 and Lumbriculus illex Timm & Rodriguez, 1994 open in segment IX and XII separately. Two pairs of ovaries were found in Lumbriculus genitosetosus (Holmquist, 1976) and Lumbriculus inconstans (Smith, 1895), while the genital chaetae were modified in the former. Both Lumbriculus japonicus Yamaguchi, 1936 and Lumbriculus sachalinicus Sokolskaya, 1967 have atria in the segment XI. They are different because of the number of spermathecae, three pairs and one pair, respectively. The atria of Lumbriculus mukoensis Yamaguchi, 1953 were paired in segment XII. Three pairs of atria are located from segments X to XIII in Lumbriculus multiatriatus Yamaguchi, 1937. Lumbriculus olgae Sokolskaya, 1976 differs from L. variegatus by the connective position between the vas deferens and the atrium. There is no gene sequence with all these species, except for L. variegatus. More molecular data are needed to support species classification and phylogenetic relationships construction in the future.

 

I hope you're all right.

Reviewer 3 Report (Previous Reviewer 1)

The authors revised the manuscript and made pointwise responses to the previous comments. The MS seems much improved than previous. One of the major problems which I am still feeling and the authors also could not justify is- samples from Sweden and US belonging to two lineages from the Tibetan Plateau. 

One of the basic prerequisites of the phylogeographic analysis is fine-scale sampling representing the entire study area. The authors have included about a dozen of samples from Sweden and the US together with the remaining samples from China. That has violated the basic assumption of phylogeographic study- samples could represent the entire study area and sample by almost equal sampling efforts. Sampling one locality from Europe and another one from the new world (US) does not represent those areas. What about other missing areas?

Therefore, I think it is wise to drop samples from Sweden and US and perform a phylogeographic analysis of the Chinese samples only. 

For the phylogenetic analysis, I suggest authors use 29 haplotype sequences and the outgroups. No need to use all samples. 

I have made some comments in the PDF. All the best!

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

Thank you very much for your suggestion. 

I performed a phylogeographic analysis of the Chinese samples only. And my sense is that the results have changed very little. 

Beat wishes.

Author Response File: Author Response.docx

Round 3

Reviewer 3 Report (Previous Reviewer 1)

The authors have revised and improved the manuscript. They have added the Figure 1 showing sampling localities. It would be good if they add layers of major rivers and DEM as they have discussed the roles of rivers and mountains in shaping the genetic structure of L. variegatus. 

Author Response

Dear reviewer,

Thanks for your advice. It is very good. We added layers of major rivers and DEM in the Figure 1. It looks clearer when explored the roles of rivers and mountains in shaping the distribution pattern of L. variegatus.

Thank you, sincerely grateful, I wish you all fine.

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

Zhou et al. explored genetic variation and phylogeography of Lumbricus variegatus using mitochondrial COI and 16S rRNA sequences. The research was conducted on an ecologically important but understudied species. However, it needs careful revision and redrafting, especially in the methods and results sections. There are noticeable errors in the interpretation of phylogenetic trees, neutrality tests, and haplotype networks. Please find attached annotated pdf with some of my comments. 

 

Comments for author File: Comments.pdf

Author Response

We would like to thank you for giving us the opportunity to revise our manuscript. Those comments were all valuable and very helpful for revising and improving our paper. In the revised version, we have carefully taken your comments into consideration in preparing our revision. We re-analysed phylogenetic trees, neutrality tests, and haplotype networks. All revised parts were marked in red. In addition, we provide our point-by-point responses to each of the comments in the following pages.

Author Response File: Author Response.docx

Reviewer 2 Report

The topic of the paper is Lumbriculus, an inconspicuous genus of annelids which needs more studies, and is often neglected in biodiversity studies due to a low variation of morphology. I am happy to see people working on this matter and from this point of view, the raw data (sequences) are important for science, and should be published in a way or another. However, the science of the paper is very limited to me and it is discussed more in details in the comments of the paper in the attached pdf.

First of all the time estimation of the divergence of Lumbriculus variegatus is way too old, considering that there is no time calibration as much as I saw, this MUST be discussed at least, at best this has to be all in all removed. If there is no time calibration (fossils or events) then discussing about the time divergence makes no sense, especially with only two markers.

Consequently a species delimitation analysis is necessary and is not found in the paper. This HAS to be included for this to make at least a bit of sense. If you still have the DNA in the lab you should consider amplify and sequence more loci for future studies. I know money and time are rare and this is a rather demanding task, but unfortunately, as it is, your results are too limited, and that is why most of your conclusions do not stand scientifically from my point of view (other reviewer may disagree). Otherwise you have to humble down the aims of the paper. The data you present cannot support most of the conclusions you make.

This is my main issues but you will see that I have several others like the weakness of the biogeographical discussion, problems with the phylogenetic analysis and interpretations, lack of explanation of the figures, lack of logical organization of the introduction, lack of conclusion, etc.

I am sorry Ito reject the paper, and I hope my comments will be constructive (as well as the comments of the other reviewers) for your resubmission here or somewhere else. I know oligochaetes are animals difficult to work with and as I said at the beginning, the raw data ought to be used in a study and valued in our field of annelid evolution!

Sincerely,

 

Comments for author File: Comments.pdf

Author Response

We would like to thank you for giving us the opportunity to revise our manuscript. Those comments were all valuable and very helpful for revising and improving our paper. In the revised version, we have carefully taken your comments into consideration in preparing our revision. The molecular clock correction was performed using the divergence time of Naididae and Lumbriculidae (354 MYA), and Lumbricidae and Megascolecidae (157 MYA). Lumbriculus variegatus is not a single species, and we added the part. I was so sorry that there were no DNA in the lab. We could not obtain more sequences. We rewrote the introduction and added the conclusion. All revised parts were marked in red. In addition, we provide our point-by-point responses to each of the comments in the following pages.

Author Response File: Author Response.docx

Reviewer 3 Report

Dear authors,

Congratulations for the work performed! The study design and results are clearly presented and relevant for the genetic and phylogeographic studies of this species. You can find in the manuscript attached a few comments related to the scientific part. I recommend you an extensive revision of English.

Comments for author File: Comments.pdf

Author Response

We would like to thank you for giving us the opportunity to revise our manuscript. Those comments were all valuable and very helpful for revising and improving our paper. In the revised version, we have carefully taken your comments into consideration in preparing our revision.