Comparative Mitogenomics of True Frogs (Ranidae, Anura), and Its Implications for the Phylogeny and Evolutionary History of Rana

Round 1

Reviewer 1 Report

This study mapped the mitogenome of 2 species in the genus Rana and compared results to 18 other Rana species and 2 outgroups.

They found that Rana showed 2 clades and four gene arrangement patterns. I found the manuscript complete, detailed and understandable.

Minor sentence structure fixes are necessary. For example, the sentence in the abstract "However, the evolutionary history and population genetics in Rana were not well understand, which may be limited by the absence of mitogenome information." could be corrected to However, the evolutionary history of the genus Rana is not well understood and may be limited by the absence of mitogenome information.

I recommend removing the phrase population genetics because I did not see reference to the topic in the main text.

Author Response

Reviewer 1

Comments and Suggestions for Authors

This study mapped the mitogenome of 2 species in the genus Rana and compared results to 18 other Rana species and 2 outgroups. They found that Rana showed 2 clades and four gene arrangement patterns. I found the manuscript complete, detailed and understandable.

>> Thank you for your constructive comments which are very helpful in preparing a revised manuscript. And many thanks for your encouragement.

Minor sentence structure fixes are necessary. For example, the sentence in the abstract "However, the evolutionary history and population genetics in Rana were not well understand, which may be limited by the absence of mitogenome information." could be corrected to However, the evolutionary history of the genus Rana is not well understood and may be limited by the absence of mitogenome information.

>>Revised. See in Line 32.

I recommend removing the phrase population genetics because I did not see reference to the topic in the main text.

>>Revised. See in Line 20 and Line 32.

Reviewer 2 Report

This manuscript reports sequencing of mitochondrial genomes of two frog species with comparative analysis from 18 species of genus Rana. Authors have analyzed different aspects such as mitochondrial genes features, phylogenetic relationships, and gene rearrangement mechanisms. The main findings of this study include the four kinds of gene arrangement patterns and the purify selection of mitochondrial genes in frog species. The mitogenomic analysis is adequate to support the conclusion of this work and overall manuscript is well written. However, I have the following two major concerns to assess the novelty of this work.

1. The complete mitochondrial genome of frog Rana zhenhaiensis was already published elsewhere (see et al. Huang et al. 2017; GenBank ID: MN218687.1). However, in this study, it is claimed as newly sequenced mitogenome.
2. After checking the given accession IDs from the “Data Availability Statement”, there is no record available in NCBI for any of these accession in both SRA and Genebank databases. The availability of this data is very important to evaluate the quality of mitogenome assemblies and annotations.

Many readers will also be very interested in this data for future work related to mitochondrial phylogenetics.

Minor comments:

1. In the Line. 102. “A total of 6.0 Gb raw reads were generated by next-generation sequencing on the 102 Illumina platform”. It is not clear whether how many of raw reads correspond to each species? Further the specific Illumina platform (Miseq, Hiseq or other) used should be informed. Also, the sequence coverage/depth of this generated data should be informed.
2. In the Line 133. “The tandem repeats were searched in the CR using the Tandem Repeats Finder program (https://tandem.bu.edu/trf/trf.html)”. This information is missing in the results sections how many and what type of repeats were found?
3. It is recommended to change the term “Genomic Rearrangements” (Line. 225) by “Mitogenomic Rearrangements”. Genomic Rearrangements is actually used for whole genome (or chromosomes).
4. The Figure 4, 5 and 6 represent and describe the data for the related topic of Genes organizations. It would be better to merge these as Panels (a), (b) and (c) into a single figure.

Author Response

Reviewer 2

Comments and Suggestions for Authors

This manuscript reports sequencing of mitochondrial genomes of two frog species with comparative analysis from 18 species of genus Rana. Authors have analyzed different aspects such as mitochondrial genes features, phylogenetic relationships, and gene rearrangement mechanisms. The main findings of this study include the four kinds of gene arrangement patterns and the purify selection of mitochondrial genes in frog species. The mitogenomic analysis is adequate to support the conclusion of this work and overall manuscript is well written. However, I have the following two major concerns to assess the novelty of this work.

>> Thank you for your constructive comments which are very helpful in preparing a revised manuscript. 

1. The complete mitochondrial genome of frog Rana zhenhaiensis was already publishedelsewhere (see et al. Huang et al. 2017; GenBank ID: MN218687.1). However, in this study, it is claimed as newly sequenced mitogenome.

>> Thanks a lot. We have revised. Please see Line 77.

2. After checking the given accession IDs from the “Data Availability Statement”, there is no record available in NCBI for any of these accession in both SRA and Genebank databases. The availability of this data is very important to evaluate the quality of mitogenome assemblies and annotations. Many readers will also be very interested in this data for future work related to mitochondrial phylogenetics.

>> Thank you for your careful reading. The data have been uploaded in GenBank, and it will be automatically released on December 1, 2022, or when the article is published. The details are shown in the attached file

 

Minor comments:

1. In the Line. 102. “A total of 6.0 Gb raw reads were generated by next-generation sequencing on the 102 Illumina platform”. It is not clear whether how many of raw reads correspond to each species? Further the specific Illumina platform (Miseq, Hiseq or other) used should be informed. Also, the sequence coverage/depth of this generated data should be informed.

>> Revised. See in line 102, 113-114.

2. In the Line 133. “The tandem repeats were searched in the CR using the Tandem Repeats Finder program (https://tandem.bu.edu/trf/trf.html)”. This information is missing in the results sections how many and what type of repeats were found?

>> Revised. See in Line 227-231.

3. It is recommended to change the term “Genomic Rearrangements” (Line. 225) by “Mitogenomic Rearrangements”. Genomic Rearrangements is actually used for whole genome (or chromosomes).

>> Revised. See in Line 233.

4. The Figure 4, 5 and 6 represent and describe the data for the related topic of Genes organizations. It would be better to merge these as Panels (a), (b) and (c) into a single figure.

>> Revised. See in Line 272-285.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript entitled “The evolution of mitochondrial genomes in true frogs (Ranidae, Anura) revealed diverse gene arrangement in Rana species” address the mitogenome of two Rana sp. species of China. Although the data obtained is always useful for further studies, the novelty of the work is low, as the main contribution to the science is the data generated and not the conclusions we can extract from the work. However the work is scientifically correct, well written and in my opinion rigorous.   I think it could be interesting for a local audience, and as a basis for further evolutionary or ecological works. However, this work would benefit from an expansion, including echological analyses for both species, and relating it with mitogenome results per example. This would allow to reach more conclusions with a wider interest.

 

For this reason I propose the work for major revisions, recognizing that with piece of additional work it is publishable in Animals.

Author Response

Reviewer 3

Comments and Suggestions for Authors

The manuscript entitled “The evolution of mitochondrial genomes in true frogs (Ranidae, Anura) revealed diverse gene arrangement in Rana species” address the mitogenome of two Rana sp. species of China. Although the data obtained is always useful for further studies, the novelty of the work is low, as the main contribution to the science is the data generated and not the conclusions we can extract from the work. However the work is scientifically correct, well written and in my opinion rigorous.  I think it could be interesting for a local audience, and as a basis for further evolutionary or ecological works. However, this work would benefit from an expansion, including echological analyses for both species, and relating it with mitogenome results per example. This would allow to reach more conclusions with a wider interest. 

For this reason I propose the work for major revisions, recognizing that with piece of additional work it is publishable in Animals.

>> Thank you for your constructive comments. We read and study the recent mitogenome research papers published in Animals, then revised this manuscript. Two papers were cited in the references. See in Line 65-66.

Round 2

Reviewer 2 Report

I appreciate authors revisions. This revised manuscript is improved and I have one recommendation regarding figures. It is recommended to increase the text size in figure 1 and figure 4. I do not have any further comments.

Author Response

Reviewer 2

I appreciate authors revisions. This revised manuscript is improved and I have one recommendation regarding figures. It is recommended to increase the text size in figure 1 and figure 4. I do not have any further comments.

>> Done. Thank you for your comments.

Reviewer 3 Report

I still think this work needs additional work, with an extensive review of Rana sp. mithogenome. Authors have limited their review to only to references in Annimals and it is not enough.  They shoud inclede all knowledge available in all scientific literature. Thus, I can't recommend this work for publication in the present form. 

Author Response

Reviewer 3

I still think this work needs additional work, with an extensive review of Rana sp. mitogenome. Authors have limited their review to only to references in Animals and it is not enough.  They shoud inclede all knowledge available in all scientific literature. Thus, I can't recommend this work for publication in the present form.

>> Thank you for your advice. We have read, study, and cite the recent mitogenome research in Rana from the journal of Molecular Phylogenetics and Evolution, PLoS ONE, ZooKeys, Asian Herpetological Research, and so on. Then revised this manuscript.  This study was only invested effort in generating new genomic resources, which is always a welcome thing to see and crucial to facilitate future research in various fields of Rana research such as phylogenetics, population, conservation, functional genetics etc. And we want to reveal the spatiotemporal diversification of the genus Rana by using three genes (Table 1). But we think that these contents are of little relevance to this study.

>> Thanks again for your constructive comments.

 

Table 1. GenBank numbers for samples of the genus Rana used.

ID	Species	16S	Cyt b	ND2
1	Rana wuyiensis sp. nov.	MZ337980	MZ355497	MZ355540
2	Rana zhengi	MZ337992	MZ355509	MZ355552
3	Rana sangzhiensis	MZ338002	MZ355517	MZ355559
4	Rana jonhsi	MZ338010	MZ355525	MZ355566
5	Rana weiningensis	MZ338023	MZ355538	MZ355576
6	Rana amurensis	KX269203	KX269349	KX269418
7	Rana areolata	AY779229	KX269300	KX269369
8	Rana arvalis	KX269197	KX269344	KX269413
9	Rana asiatica	KX269200	KX269346	KX269415
10	Rana berlandieri	AY779235	KX269301	KX269370
11	Rana boylii	KX269178	KX269299	KX269368
12	Rana cascadae	KX269176	KX269302	KX269371
13	Rana catesbeiana	KX269208	KX269354	KX269423
14	Rana chaochiaoensis	KX269192	KX269339	KX269408
15	Rana chensinensis	KX269186	KX269333	KX269402
16	Rana chiricahuensis	AY779225	KX269303	KX269372
17	Rana clamitans	AY779204	KX269304	KX269373
18	Rana coreana	KX269202	KX269348	KX269417
19	Rana culaiensis	KX269190	KX269337	KX269406
20	Rana dunni	AY779222	KX269305	KX269374
21	Rana dybowski	KX269188	KX269335	KX269404
22	Rana forreri	AY779233	GU184219	GU184250
23	Rana graeca	KX269199	KX269345	KX269414
24	Rana hanluica	KX269191	KX269338	KX269407
25	Rana huanrensis	KX269183	KX269330	KX269400
26	Rana iberica	KX269195	KX269342	KX269411
27	Rana japonica	KX269220	KX269364	KX269434
28	Rana kukunoris	KX269185	KX269332	KX269401
29	Rana kunyuensis	KX269201	KX269347	KX269416
30	Rana longicrus	KX269189	KX269336	KX269405
31	Rana luteiventris	KX269213	KX269358	KX269428
32	Rana macrocnemis	KX269194	KX269341	KX269410
33	Rana macroglossa	AY779243	KX269306	KX269376
34	Rana maculata	AY779207	KX269307	KX269377
35	Rana magnaocularis	AY779239	KX269308	KX269378
36	Rana montezumae	AY779223	KX269309	KX269379
37	Rana neovolcanica	AY779236	KX269310	KX269380
38	Rana omeimontis	KX269193	KX269340	KX269409
39	Rana omiltemana	AY779238	KX269311	KX269381
40	Rana ornativentris	KX269187	KX269334	KX269403
41	Rana palustris	KX269207	KX269353	KX269422
42	Rana psilonota	AY779217	KX269312	KX269382
43	Rana pustulosa	AY779220	KX269313	KX269383
44	Rana sakuraii	KX269205	KX269351	KX269420
45	Rana sauteri	KX269204	KX269350	KX269419
46	Rana septentrionalis	KX269179	KX269314	KX269384
47	Rana shuchinae	KX269210	KX269356	KX269425
48	Rana sierrae	KX269211	KX269357	KX269426
49	Rana sierramadrensis	AY779216	KX269315	KX269385
50	Rana spectabilis	AY779227	KX269320	KX269390
51	Rana sphenocephala	AY779251	KX269321	KX269391
……
……

 

Round 3

Reviewer 3 Report

Although I disagree with the authors (the contextualization of Rana sp. mithogenome is necessary in order to provide clear conclusions and not only data), I appreciate the effort to review additional references.

Author Response

>> Thank you for your encouragement and constructive comments. We have learned a lot from your advice, which will be of great benefit in our future study. And we have modified the grammar a in the manuscript to make the article more readable. Thanks again.