Mitochondrial Genome Uncovered Hidden Genetic Diversity in Microdous chalmersi (Teleostei: Odontobutidae)

Round 1

Reviewer 1 Report

Journal: Fishes (ISSN 2410-3888)
Manuscript ID: fishes-2332355
Type: Communication
Title: Mitochondrial genome uncovered hidden diversity in Microdous chalmersi, (Teleostei: Odontobutidae)
Section: Biology and Ecology
Special Issue: Genetics and Evolution of Fishes

In this research, the authors have conducted the mitochondrial genome sequencing of Microdous chalmersi, a freshwater fish species with limited distribution in China. Phylogenetic and genetic differentiation analyses were performed in this study to evaluate if geographically isolated populations could be considered cryptic species. This is an interesting communication. Nevertheless, I have some questions about the methodology, and more information is required from the authors. I have found some typos that must be corrected. The pending issues that must be addressed before the manuscript can be considered for publication are listed in the attached file in two sections: (1) Content issues and (2) Formatting issues.

Comments for author File: Comments.pdf

Author Response

Dear Editor,

Thank you and the reviewers for your comments and suggestions. We have reconstructed the phylogeny using all protein coding genes and rRNA genes of the target species and outgroups according to reviewers suggestion, and uploaded all mitochondrial genomes. We still waiting for the AC number from NCBI for the submission and we will update the numbers once we received them. We addressed all concerns brought by the reviewers. Please find the following responses to reviewers point-by-point.We also checked the format of references carefully.

Reviewer 1:

In this research, the authors have conducted the mitochondrial genome sequencing of Microdous chalmersi, a freshwater fish species with limited distribution in China. Phylogenetic and genetic differentiation analyses were performed in this study to evaluate if geographically isolated populations could be considered cryptic species. This is an interesting communication. Nevertheless, I have some questions about the methodology, and more information is required from the authors. I have found some typos that must be corrected. The pending issues that must be addressed before the manuscript can be considered for publication are listed below in two sections: (1) Content issues and (2) Formatting issues.

CONTENT ISSUES

Keywords

—Line 22: I would change “The Hainan small-toothed sleeper” to “Microdous chalmersi”.

Changed as suggested.

Abstract

—Line 12: I would add before “disjunctly”: “is an endemic fish”. If this species is unique

in China, this should be highlighted. Furthermore, I consider it important to indicate what

type of organisms is at the beginning of the manuscript to aid future readers.

Changed as suggested.

—Line 21: Please change “belong” to “could belong”. Please, soften this statement.

Changed as suggested.

Introduction

—Lines 26-28: If this species is an endemism, please add it somewhere.

Changed as suggested.

Material and Methods

I have missed a map as Supplementary Material with the known species distribution range

and the two sampling locations used in this study. I strongly recommend doing it. In how

many river basins is this species found?

Now we added a map  as suggested. The species is only found in limited areas in upper reaches of the Pearl River of Guangxi Province and the Changhua River and the Wanquan River of Hainan Island

—Line 72: Please add “GenBank accession:” before “MH644035”. The first time one of

these is cited in the main text.

Changed as suggested.

—Line 83: What was the quality base criterium used? Please add it to the main text.

Q-score < 20

—Line 85: What BWA alignment algorithm was used? BWA has three alignment

algorithms: 'mem', 'bwasw', and 'aln/samse/sampe'. Please add it to the main text.

We used mem algorithm, added as suggested.

—Lines 93-94: This link does not work. The link is correct, but the hyperlink may be cut

in two. Please, check it (https://chlorobox.mpimp-golm.mpg.de/geseq.html).

Now it works.

—Line 98: Please change “NC_021763” to “RefSeq accession: NC_021763”.

Added as suggested, as well as to other species.

Results

—Line 126: How were detected the PCR duplicates? Please explain it in the Material and

Methods section.

PCR duplicates were excluded by using “-bfastx_uniques” command in USEARCH (v10.0.240). Added at lines 84-85.

—Line 130:

According to the information available at NCBI

(https://www.ncbi.nlm.nih.gov/nuccore/ON312089.1/), the number of bp would be

16,485 (Figure 1). Please review it.

Thanks for pointing it out. It was our mistake for not updating it for the final version. 

Discussion

In a general way, I lack more information and context. Is this species endangered? What

threats are there over it? In how many water basins/rivers are present? This manuscript

has interesting results, but what would be the next step? This information could be used  

to define management units (MUs), improve conservation management, etcetera. All of

this should be in the Discussion section in some sentences.

Great suggestions, we add those discussion in the main text.

I have another question: Why do you define two species and not two subspecies due to

geographic isolation and this genetic differentiation?

These questions are mandatory for me.

Actually, we can not make a conclusion on whether they are two species or subspecies without collecting more fish and check their morphological characters. We now soften those conclusion in discussion.

Conclusion

—Line 214: Please change “formed” to “could be”. Please, soften this statement. This is

mandatory for me. You would need more sampling size and data to do this affirmation.

Totally agree, edited as suggested.

If I understood well, these samples are from two sampling locations: Guangxi (1) +

Hainan (1). For me, it would be recommendable to take different samples from both

regions, for instance: Guangxi (3) + Hainan (2), to compare the genetic differentiation

between populations in the same water basin/island.

We have extensively sampled both Guangxi and Hainan from previously known distribution sites for two years, but failed to collect more samples from different locations.

Reviewer 2 Report

Dear author,

 

your paper is interesting in a term of special research of deviant forms of Microdous occurs in remote and limited water reservoir location. Data revealed with genetic distance analysis based on 212 mitochondrial genome may show differences of isolated population, but not different taxon. In fishes spawn may be transferred by flying animals for a long distance remote water reservoirs. Such phoresy transmission is usually provides genetic drift necessary for species population stability. 

 

Phylogenetic analysis used in your paper is only concerned the genetic research and can not be considered as real phylogeny. Ichthyology systematics operates number of morphological characters and their ratio, but this subject is absolutely absent in your work. That is why the results presented in your paper is only supplementary data to pay attention for biodiversity of Microdous, but not uncovering hidden diversity of this genus. 

Well, I believe, the main question  of the research is: “What character may be used for explanation of differences in locally distributed population of Microdous from more widely spread populations?” And the authors tried to use genetic data for this purpose.

 

In case when traditional taxonomic methods cannot be used for characterization of rare and divergent from, other methods are welcomed. The present study gives us understanding of genetic difference occur in local population of fishes. It is the subject that should have to be explained in future. That is why the paper presented is interesting and actual for poorly studied territories fauna revision.

 

Revealed differences in mitochondrial genome of the locally distributed population of the species is presented for the first time. We can see, that this population genome is clustering independently with genome of M. chalmersi, and this fact have to be explained.

 

At first, the authors should renounce their point of view on taxonomic decision in this paper. They wrote in abstract: “We concluded that Microdous spp. from Guangxi and Hainan belong to two different species», but did not propose any necessary nomeclatural acts below. In this case, “taxonomic rhetoric” is irrelevant and can only confuse readers waiting for systematic changes in the present article.

Secondary, it is strongly recommended to the authors provide supplementary explanation why mitochondrial genome may be used ищер for generic, specific or population division. Such implementation of genome method confuse systematic hierarchy when each level of taxon demands individual comparison, not mixture of different taxa or specimens.

 

 

In whole the conclusion is coincides with the topic of the article and discoveries the main idea of the researched problem. I am not happy with the fact, tat it is sounds “taxonomically”: “… Microdous spp. … may have been differentiated and formed different species due to long-term geographical isolation…” May or not, we do not know. Also, we can not consider the data gained in the research as absolutely reasonable for taxonomic revision. It would be better tu change “different species” into “different populations” to fix the gap that have been studied and explained by the present investigation.

 

In my point of view, all the references appropriate.

 

 

I’d like to point out that this research can not be considered as taxonomic study, and can not be evaluated methodologically by systematics approach. A new data set revealed by the authors does not need comparison with “control group” as it is not en experimental research. Basing on this fact I believe the present paper can be recommended for publication, may be with minor revision of introduction and Methods in term of explanation of non-taxonomic concept of the main idea of the study.

Wish you all the best in future investigation of fish biodiversity using all necessary methods and not limiting with the mitochondrial genome data.

 

 

Author Response

Dear Editor,

Thank you and the reviewers for your comments and suggestions. We have reconstructed the phylogeny using all protein coding genes and rRNA genes of the target species and outgroups according to reviewers suggestion, and uploaded all mitochondrial genomes. We still waiting for the AC number from NCBI for the submission and we will update the numbers once we received them. We addressed all concerns brought by the reviewers. Please find the following responses to reviewers point-by-point.We also checked the format of references carefully.

Reviewer 2: 

Your paper is interesting in a term of special research of deviant forms of Microdous occurs in remote and limited water reservoir location. Data revealed with genetic distance analysis based on 2 mitochondrial genome may show differences of isolated population, but not different taxon. In fishes spawn may be transferred by flying animals for a long distance remote water reservoirs. Such prophesy transmission is usually provides genetic drift necessary for species population stability.

Phylogenetic analysis used in your paper is only concerned the genetic research and cannot be considered as real phylogeny. Ichthyology systematics operates number of morphological characters and their ratio, but this subject is absolutely absent in your work. That is why the results presented in your paper is only supplementary data to pay attention for biodiversity of Microdous, but not uncovering hidden diversity of this genus.

Well, I believe, the main question of the research is: “What character may be used for explanation of differences in locally distributed population of Microdous from more widely spread populations?” And the authors tried to use genetic data for this purpose.

In case when traditional taxonomic methods cannot be used for characterization of rare and divergent from, other methods are welcomed. The present study gives us understanding of genetic difference occur in local population of fishes. It is the subject that should have to be explained in future. That is why the paper presented is interesting and actual for poorly studied territories fauna revision.

We totally agree with the reviewer’s comments. It was a surprise from routine work for collecting mitochondrial data of fish collection in our lab. We found large genetic difference between samples of different populations. The specimens collected was too deformed to do further morphological work. We tried going back and collecting more specimens but yet succeeded. The genetic difference between populations, however, is equal or higher than that between species of sister genus. We think it is important to report the results to call attention to researchers to this understudied, but probably highly threatened species. We have softened the wording toward taxonomy in revised manuscript, but emphasized more on population difference as suggested. 

We changed the title to “Mitochondrial genome uncovered hidden genetic diversity in Microdous chalmersi, (Teleostei: Odontobutidae)” by adding genetic in front of diversity.

Revealed differences in mitochondrial genome of the locally distributed population of the species is presented for the first time. We can see, that this population genome is clustering independently with genome of M. chalmersi, and this fact have to be explained.

At first, the authors should renounce their point of view on taxonomic decision in this paper. They wrote in abstract: “We concluded that Microdous spp. from Guangxi and Hainan belong to two different species», but did not propose any necessary nomeclatural acts below. In this case, “taxonomic rhetoric” is irrelevant and can only confuse readers waiting for systematic changes in the present article.

We have revised abstract as “We propose that M. chalmersi from Guangxi and Hainan could belong to two different species, but further morphological studies should be carried our to test this hypothesis.”

Secondary, it is strongly recommended to the authors provide supplementary explanation why mitochondrial genome may be used for generic, specific or population division. Such implementation of genome method confuse systematic hierarchy when each level of taxon demands individual comparison, not mixture of different taxa or specimens.

We used mitochondrial genome data for two reasons: mitochondrial loci have been used in studies of other species of Odontobutidae, which can be used as references; with mitochondrial genome, we can have more loci and data to compare. I agree with the reviewer that different level of systematic investigation should use different loci. Our comparison was focus on interspecific level, when COI, Cytb are often used.

In whole the conclusion is coincides with the topic of the article and discoveries the main idea of the researched problem. I am not happy with the fact, tat it is sounds “taxonomically”: “… Microdous spp. … may have been differentiated and formed different species due to long-term geographical isolation…” May or not, we do not know. Also, we can not consider the data gained in the research as absolutely reasonable for taxonomic revision. It would be better tu change “different species” into “different populations” to fix the gap that have been studied and explained by the present investigation.

We tuned down the taxonomic wording, changed  “different species” to “different populations” as suggested.

In my point of view, all the references appropriate.

I’d like to point out that this research cannot be considered as taxonomic study, and can not be evaluated methodologically by systematics approach. A new data set revealed by the authors does not need comparison with “control group” as it is not an experimental research. Basing on this fact I believe the present paper can be recommended for publication, may be with minor revision of introduction and Methods in term of explanation of non-taxonomic concept of the main idea of the study.

Wish you all the best in future investigation of fish biodiversity using all necessary methods and not limiting with the mitochondrial genome data.

Thanks for the advice. We will keep searching for more specimens of the suspect species of Microdous chalmersi, and hopefully can solve the mystery with a thorough morphological study.

 

Reviewer 3 Report

In this paper, Jiang et al. attempt to resolve the issue of whether the morphological differences observed between populations of the Hainan small-toothed sleeper in the Hainan and Guangxi provinces of China are indeed the evidence of speciation. By analyzing five mtDNA loci from five individuals from each population, the authors concluded that the genetic distance between populations was greater than that within populations. Since the genetic distance between populations was comparable to that between the species of the closely related genus Odontobutis, the authors concluded that Hainan and Guangxi populations represent two different subspecies.

The study is technically straightforward and uses established NSG techniques. It is unclear, though, why the authors did not use a more straightforward and cost-effective approach of amplicon sequencing. This fact is even more puzzling, considering that only five loci rather than full mtDNA sequences were used for analysis. Why not just amplify these loci and sequence them if the rest of mtDNA sequence data are discarded, anyway? 

Thee conclusion may be potentially skewed by the small number of specimens collected and analyzed at each location and by the fact that sample collection occurred at only one site at each location. The study should be amended by additional specimens collected at different sites within each areal.

Also, the authors would need to justify: 1) using only mitochondrial loci in their analysis and excluding the nuclear ones to make conclusions about the speciation. This approach appears unconventional. 2) Using five mitochondrial loci rather than full mtDNA sequences for analysis. Such an approach reduces the credibility of the conclusions.

 

Finally, of the ten sequenced mitochondrial genomes, only two (one from each location) were deposited in public collections and therefore are available for re-analysis and re-validation of the authors’ conclusions. This deficiency has to be rectified.

 

Author Response

Dear Editor,

Thank you and the reviewers for your comments and suggestions. We have reconstructed the phylogeny using all protein coding genes and rRNA genes of the target species and outgroups according to reviewers suggestion, and uploaded all mitochondrial genomes. We still waiting for the AC number from NCBI for the submission and we will update the numbers once we received them. We addressed all concerns brought by the reviewers. Please find the following responses to reviewers point-by-point.We also checked the format of references carefully.

Reviewer 3: 

In this paper, Jiang et al. attempt to resolve the issue of whether the morphological differences observed between populations of the Hainan small-toothed sleeper in the Hainan and Guangxi provinces of China are indeed the evidence of speciation. By analyzing five mtDNA loci from five individuals from each population, the authors concluded that the genetic distance between populations was greater than that within populations. Since the genetic distance between populations was comparable to that between the species of the closely related genus Odontobutis, the authors concluded that Hainan and Guangxi populations represent two different subspecies.

The study is technically straightforward and uses established NSG techniques. It is unclear, though, why the authors did not use a more straightforward and cost-effective approach of amplicon sequencing. This fact is even more puzzling, considering that only five loci rather than full mtDNA sequences were used for analysis. Why not just amplify these loci and sequence them if the rest of mtDNA sequence data are discarded, anyway? 

Frankly, collecting mitochondrial genome data is a routine practice in our lab. We are using a gene-capture method, which is quite cheap comparable to sequencing five loci. We now used all 13 coding genes of the mitochondrial genome and rerun the phylogenetic analyses as the reviewer suggested.

Thee conclusion may be potentially skewed by the small number of specimens collected and analyzed at each location and by the fact that sample collection occurred at only one site at each location. The study should be amended by additional specimens collected at different sites within each areal.

Totally agreed. We tried collecting more specimens from its know distribution sites for two years, but failed, probably due to the decline status of the species. We added comments on limitation of our study and proposed to collect more samples and do a thorough morphological study in discussion (lines 213-230).

Also, the authors would need to justify: 1) using only mitochondrial loci in their analysis and excluding the nuclear ones to make conclusions about the speciation. This approach appears unconventional. 2) Using five mitochondrial loci rather than full mtDNA sequences for analysis. Such an approach reduces the credibility of the conclusions.

Now we refrained from making conclusions about taxonomy revision, but focus on genetic difference of the two populations and comparing it with other odontobutids using mitochondrial loci. We now used all coding sequence of the mitochondrial genome.

Finally, of the ten sequenced mitochondrial genomes, only two (one from each location) were deposited in public collections and therefore are available for re-analysis and re-validation of the authors’ conclusions. This deficiency has to be rectified.

We now uploaded all 10 mitochondrial genomes to NCBI site as suggested. The AC numbers for the 8 additional samples are: xxx-xxx

Hopefully, we have addressed all concerns of the editor and reviewers and the revised version could meet the standard of Fishes.

Round 2

Reviewer 1 Report

Journal: Fishes (ISSN 2410-3888)
Manuscript ID: fishes-2332355
Type: Communication
Title: Mitochondrial genome uncovered hidden diversity in Microdous chalmersi, (Teleostei: Odontobutidae)
Section: Biology and Ecology
Special Issue: Genetics and Evolution of Fishes

The authors have answered all the questions and issues in this new version research. The quality of the manuscript has improved adequately for its publication. Nevertheless, I have found some typos that must be corrected (e.g., lack of bibliographical references). The pending issues that must be addressed before the manuscript can be finally published are listed in the attached pdf in two sections: (1) Content issues and (2) Formatting issues.

Comments for author File: Comments.pdf

Author Response

We followed all advice from the reviewer 1 and revised the manuscript accordingly.

Reviewer 3 Report

My concerns were addressed

Author Response

Thanks for your comments