Characterization of Two New Apodemus Mitogenomes (Rodentia: Muridae) and Mitochondrial Phylogeny of Muridae
Round 1
Reviewer 1 Report
Data looks good, but there are no details on the data collection, how many samples? How were they collected? Also no information about animal ethics. How did you identified them as A. a. ningpoensis and A. d. draco? What do you mean that the molecular characteristics of these subspecies are limited? You mainly took some samples of those two subspecies (that are not even from the same species), and studied their mitogenomes. Then you created phylogenetic trees based on information already collected and available in NCBI. That is good information to know, and to add to literature, but I am missing the significance of this paper. The fact that Apodemus is a monophyletic genus is well known, there is no need to confirm that by looking at the mitogenome of two subspecies from different species. The aim of the paper is very descriptive, it is mainly adding up data on two subspecies to the current literature. Is that enough? I would like to see some more theoretical reasoning for publishing this paper.
Author Response
Dear reviewer, thank you for your comments on our manuscript. We have further revised it. The following are our revision contents.
Point 1: Data looks good, but there are no details on the data collection, how many samples? How were they collected? Also no information about animal ethics.
Response 1: We thank the reviewer for pointing out this issue. We indeed should mention the data collection information in the paper. Samples were collected by the clip-night method, using fresh peanut rice as bait and combining with habitat characteristics to select standard iron plate clips, and 100 clips were placed in each sample plot. The clips were placed at dusk each day and collected the next morning. The captured small animals were first weighed and numbered, and then the conventional data such as head body length, tail length, ear length and hind foot length were measured, and the sex was identified. After morphological identification, BLAST analysis in NCBI using DNA barcoding sequences verified their classification. One A. a. ningpoensis and one A. d. draco sample were taken for DNA sequencing respectively. The animal study protocol was approved by the Institutional Animal Care and Use Committee of Anhui Zoological Society (2022102001) on October 20, 2022. Your point is necessary, so I have added data collection information and animal ethics information to the paper. Revision positions are given at lines 87-93 and 345-347.
Point 2: How did you identified them as A. a. ningpoensis and A. d. draco?
Response 2: The question you mentioned is very deep. The identification was carried out based on the geographical distribution and body shape of rats. A. a. ningpoensis is distributed in the south of Qinling Mountains and Huaihe River. Most of the black line of the central dorsal body of the mouse is not clear, some only have the central dorsal dark area, or the black line is visible, but the boundary between the dorsal hair is unclear, the dorsal hair and body side hair color is darker, brownish-yellow tone. A. d. draco is distributed is distributed in Anhui Province. The body color is bright, the hind foot ankle is dark. The tail is longer than the body length, with an average body length of 91.6 mm and an average tail length of 91.3 mm; the hindfoot length is shorter by 20-23 mm; the ear is shorter, with an average of 15.5 mm. After morphological identification, BLAST analysis in NCBI using DNA barcoding sequences verified their classification.
Point 3: What do you mean that the molecular characteristics of these subspecies are limited?
Response 3: Your statement is very scientific. At present, only scholars have sequenced the cytb gene of A. a. ningpoensis, and the mitochondrial genome contains information such as gene rearrangement, tRNA secondary structure, genetic code changes, replication and transcriptional regulation patterns, which are lacking in the fragment. The mitochondrial genomes of A. a. ningpoensis and A. d. draco have not been described, and their molecular characteristics have not been well documented.
Point 4: You mainly took some samples of those two subspecies (that are not even from the same species), and studied their mitogenomes. Then you created phylogenetic trees based on information already collected and available in NCBI. That is good information to know, and to add to literature, but I am missing the significance of this paper. The fact that Apodemus is a monophyletic genus is well known, there is no need to confirm that by looking at the mitogenome of two subspecies from different species. The aim of the paper is very descriptive, it is mainly adding up data on two subspecies to the current literature. Is that enough? I would like to see some more theoretical reasoning for publishing this paper.
Response 4: Thank you for the deeper guidance. I really should have noticed that. As one of the major vector organisms, Apodemus can transmit a variety of virulent infectious diseases such as plague and renal syndrome hemorrhagic fever. A. a. ningpoensis and A. d. draco are widely distributed an ideal species for biogeographical research and understanding of environmental changes. Mitochondria have a relatively independent genetic transcription system within them, and they have a long symbiotic evolutionary history with eukaryotes. Their genomes contain key information reflecting the evolution of species, which is an effective tool for analyzing the phylogeny and genetic evolution of organisms. Only the cytb gene of A. a. ningpoensis has been sequenced by scholars. In this study, the mitochondrial genomes of A. a. ningpoensis and A. d. draco were reported for the first time. Their genome sequences, nucleotide composition, genome size, codon usage, gene overlap, and tRNA secondary structure were analyzed to explore their phylogenetic position and the composition and evolution of the mitochondrial genomes. There is no doubt that the genus Apodemus is monophyletic, and the present study confirms exactly this. The results of this study will provide a reference for future research on the evolution of Apodemus mitochondrial genome, lay a foundation for the identification and classification of species, better promote the study of rodent taxonomy, and provide a scientific basis for the conservation and utilization of germplasm resources of Apodemus. Revision positions are given at lines 76-83 and 325-330.
Thank you again for your comments and instructions, which have greatly helped in the improvement of this manuscript.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript "Characterization of Two New Apodemus Mitogenomes (Rodentia: Muridae) and Mitochondrial Phylogeny of Muridae" provided new important information related to the mitochondrial genome of Apodemus agrarius ningpoensis and Apodemus draco draco using whole mitogenome sequencing. Furthermore, new data have been used in the phylogeny analyses of the Muridae family. The description of mitogenomes divergence is presented adequately.I found this paper interesting and clearly written. However, it contains some points which deserve clarification.
Lines 45-46 "However, single mitochondrial gene sequences often have limited phylogenetic information [6, 16-18]. "This is why two, three, or more mitochondrial markers have been used in some phylogenetic studies (e.g., Bellinvia, 2004; Yalkovskaya et al., 2022). Since you have given a historical overview of the progress of phylogenetic research, it would be good to mention this before pointing out the advantage of the method you used.
Lines 68-69 "Of note, A. agrarius is morphologically similar to A. draco, but morphological studies are somewhat subjective." Morphological studies have limitations, but they are not subjective in all segments. Measurements and new methods in morphological research offer more and more opportunities, so they cannot be considered subjective. I suggest you remove this statement.
Lines 66-67 – "Based on complete mtDNA cytochrome b sequences approximately 8.1 Mya ago, Apodemus underwent two stages of radiation evolution to produce the agrarius and draco subgroups [27]." It is more informative if it states "about 7.17–9.95 Mya," as the authors point out, since the deviation from the value you give is quite significant.
Discussion
Parallel analyses in the nuclear genome and mitogenome will probably influence phylogenetic studies in the future. The cytogenetic characteristics of the analysed species are interesting to discuss (e.g., Motokawa et al., 2018). Chromosomal divergence is thought to play a role in reproductive isolation, so diversity among sister species at the chromosome level is significant to mention herein.
In conclusion, this manuscript is interesting and provides new information about mitogenome diversity in the Apodemus genus. I would recommend taking into account the comments given above before final acceptance.
Author Response
Dear reviewer, thank you for your comments on our manuscript. We have further revised it. The following are our revision contents.
Point 1: Lines 45-46 "However, single mitochondrial gene sequences often have limited phylogenetic information [6, 16-18]. "This is why two, three, or more mitochondrial markers have been used in some phylogenetic studies (e.g., Bellinvia, 2004; Yalkovskaya et al., 2022). Since you have given a historical overview of the progress of phylogenetic research, it would be good to mention this before pointing out the advantage of the method you used.
Response 1: Your suggestions are great! I will add it before pointing out the advantage of the method I used. Revision positions are given at lines 47-48.
Point 2: Lines 68-69 "Of note, A. agrarius is morphologically similar to A. draco, but morphological studies are somewhat subjective." Morphological studies have limitations, but they are not subjective in all segments. Measurements and new methods in morphological research offer more and more opportunities, so they cannot be considered subjective. I suggest you remove this statement.
Response 2: I'm sorry I didn't take that into account. I will remove this statement. Revision positions are given at lines 67.
Point 3: "Based on complete mtDNA cytochrome b sequences approximately 8.1 Mya ago, Apodemus underwent two stages of radiation evolution to produce the agrarius and draco subgroups [27]." It is more informative if it states "about 7.17–9.95 Mya," as the authors point out, since the deviation from the value you give is quite significant.
Response 3: The comments you give are very pertinent, and the deviations from the values I gave are quite large, as the author points out. I will change 8.1 Mya to 7.17-9.95 Mya. Revision positions are given at lines 65-66.
Point 4: Discussion Parallel analyses in the nuclear genome and mitogenome will probably influence phylogenetic studies in the future. The cytogenetic characteristics of the analysed species are interesting to discuss (e.g., Motokawa et al., 2018). Chromosomal divergence is thought to play a role in reproductive isolation, so diversity among sister species at the chromosome level is significant to mention herein.
Response 4: Thank you for the deeper guidance. I will add the following to the paper. Parallel analyses in the nuclear genome and mitogenome will probably influence phylogenetic studies in the future. The cytogenetic characteristics of the analysed species are interesting to discuss. A. agrarius and A. draco belong to the Apodemus Group and Draco Group, respectively, based on morphological characteristics. The karyotype of A. agrarius had 3–5 biarmed metacentric autosome pairs (2n=48, FNa=54), whereas that of A. draco had one small metacentric pair (2n=48, FNa=48). The karyotype of Apodemus chevrieri, a sister species of A. agrarius possesses four small metacentric pairs (2n=48, FNa=54) which was consistent with the standard karyotype of A. agrarius. The sister species of A. draco, Apodemus latronum, has a similar karyotype to A. draco. However, they have different chromosome histotypes and central heterochromatin is present in many distal center pairs. Two DNA phylogenetic species groups were distinc. Chromosomal rearrangements at the cytological level have contributed to the formation of post-mating reproductive isolation. I will add that content to the paper. Revision positions are given at lines 309-320.
Thank you again for your comments and instructions, which have greatly helped in the improvement of this manuscript.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
I appreciate the authors attempted to integrate my comments. And I think they did a good job. I still feel, however, that the rationale for the paper is a bit weak. You now state the following:
The results of this study will provide a reference for future research on the evolution of Apodemus mitochondrial genome, lay a foundation for the identification and classification of species, better promote the study of rodent taxonomy, and provide a scientific basis for the conservation and utilization of germplasm resources of Apodemus
But those claims are not fully discussed/integrated in the conclusion. I suggest putting some more information, in the discussion/conclusion, on future application of your dataset. Also, I was not against you including a statement about the monophyly of Apodemus (so I would leave it somewhere in the discussion). I was just arguing that was not enough to justify the originality of your paper.
Author Response
Dear reviewer, thank you for your comments on our manuscript. We have further revised it. The following are our revision contents.
Point 1: The results of this study will provide a reference for future research on the evolution of Apodemus mitochondrial genome, lay a foundation for the identification and classification of species, better promote the study of rodent taxonomy, and provide a scientific basis for the conservation and utilization of germplasm resources of Apodemus. But those claims are not fully discussed/integrated in the conclusion. I suggest putting some more information, in the discussion/conclusion, on future application of your dataset. Also, I was not against you including a statement about the monophyly of Apodemus (so I would leave it somewhere in the discussion). I was just arguing that was not enough to justify the originality of your paper.
Response 1: Thank you very much for the reviewers' comments. Your comments are indeed the shortcomings of this paper. I will add more information about the future applications of the dataset in the conclusion. In the future, primers can be developed by combining the mitochondrial genome sequence to identify the most abundant loci for rodent polymorphic types, and construct DNA barcode candidate markers to distinguish rodent populations, so that a large enough sample size of rodents can be distinguished with a minimum number of markers to effectively make definitive identification and diagnosis of confusing species. Continuous expansion of the mitochondrial genome database of the Apodemus allows for the development of SSR markers for germplasm surveys. The mitochondrial coding region sequence can be used to understand the disease information related to the source of the sample, and provide an important reference basis for the epidemiological investigation and control of Apodemus. However, because of their wide distribution, there are particularly high levels of morphological and genetic variations among representative species. The conclusions obtained in this study are preliminary and tentative. In future studies, the use of genetic diversity of mtDNA, combined with geographic distribution and other information to expand the sample size of the geographic population of the Apodemus, will help to better understand the genetic differentiation and dispersal of the Apodemus, and understand the mechanism of environmental influence on its evolution. The monophyly of Apodemus is well known and is indeed not sufficient to prove the originality of this paper. Therefore, Only the discussion section has relevant statements. Revision positions are given at lines 338-349.
Author Response File: Author Response.docx
