Abstract

The plateau vole, Neodon fuscus is endemic to China and is distributed mainly in Qinghai Province. It is of public health interest, as it is, a potential reservoir of Toxoplasma gondii and the intermediate host of Echinococcus multilocularis. However, genetic data of this species are lacking, and its name and taxonomy are still a controversy. In the present study, we determined the nucleotide sequence of the entire mitochondrial (mt) genome of N. fuscus and analyzed its evolutionary relationship. The mitogenome was 16328 bp in length and contained 13 protein-coding genes, 22 genes for transfer RNAs (tRNA), two ribosomal RNA genes and two major noncoding regions (OL region and D-loop region). Most genes were located on the heavy strand. All tRNA genes had typical cloverleaf structures except for tRNASer (GCU). The mt genome of N. fuscus was rich in A+T (58.45%). Maximum likelihood (ML) and Bayesian methods yielded phylogenetic trees from 33 mt genomes of Arvicolinae, in which N. fuscus formed a sister group with Neodon irene and Neodon sikimensis to the exclusion of species of Microtus and other members of the Arvicolinae. Further phylogenetic analyses (ML only) based on the cytb gene sequences also demonstrated that N. fuscus had a close relationship with N. irene. The complete mitochondrial genome was successfully assembled and annotated, providing the necessary information for the phylogenetic analyses. Although the name Lasiopodomys fuscus was used in the book ‘Wilson & Reeder’s Mammal Species of the World’, we have confirmed here that its appropriate name is N. fuscus through an analysis of the evolutionary relationships.

Background

Mitochondria (mt) are virtually ubiquitous in eukaryotic organisms, playing an important role in a range of cellular processes. With few exceptions, metazoan mt genomes are covalently closed-circular molecules. In the case of mammals, these mt genomes range from 15 to 17 kb. In addition to the D-loop, which is involved in initiation of DNA replication, mt genomes contain 13 protein-coding genes: cytochrome b (cytb), subunits 1-3 of cytochrome oxidase (cox1-3), ATPase subunits 6 and 8 (atp6 and atp8) and NADH dehydrogenase subunits 1-6 and 4L (nad1-6, nad4L). Furthermore, they have genes for two ribosomal RNAs (rRNA) (12S rRNA and 16S rRNA) and 22 transfer RNAs (tRNAs). Although mt genomes are rather uniform in their length and structure, the primary sequences of their genes vary substantially. Because of their small size, simple structure, fast evolutionary rate, maternal inheritance, limited recombination and lack of tissue specificity [1–4], mtDNA sequences are used widely for analyzing phylogenetic relationships among vertebrates and for population differentiation [5–7].

The plateau (or smoky) vole (a rodent in the family Cricetidae, subfamily Arvicolinae) was first described as Lasiopodomys fuscus by Büchner in 1889 [8]. Its taxonomy has been unstable since then, with the species being referred to as several different subgenera/genera in recent years, in particular Microtus Schrank 1798, Lasiopodomys Lataste 1887 and Neodon Horsfield 1841. The authoritative ‘Wilson & Reeder’s Mammal Species of the World’ (third Edition, 2005) placed it in Lasiopodomys (see http://www.vertebrates.si.edu/msw/mswcfapp/msw/taxon_browser. cfm?msw_id = 4367), and this classification is commonly accepted (e.g., Smith and Xie) [9]. Liu et al (2012) [10] considered, partly on the basis of mt cytochrome b sequences, that the species should be transferred from Lasiopodomys to Neodon. This was also demonstrated, but not explicitly stated, by Chen et al (2012) [11] in a study using mitochondrial and nuclear DNA sequences. In the Chinese animal scientific database, the name of L. fuscus, however is also used, the Chinese public health literature, related to plague and other pathogens (see below), almost invariably uses the name Microtus fuscus.

This plateau vole is the natural host of a variety of parasites. Research has shown that the rate of Toxoplasma gondii infection was 12.5% (6/48) and that this species could serve as a potential reservoir of T. gondii [12]. Qinghai-Tibet Plateau is one of the two major Alveolar echinococcosis (AE, caused by Echinococcus multilocularis larvae) endemic areas (the other is Xinjiang) in China, and the vole is an endemic species and one of the endemic small mammals, and it plays a significant role in transmitting E. multilocularis [13]. According to our survey, the rate of E. multilocularis infection was 29.41% (32/102) in Jiuzhi county, Qinghai province [14]. Xu et al [15] found that the rate of E. multilocularis infection was different as the seasons changed, and that that in summer, the infection rate (3.4%, 12/353) was lower than in winter (11.66%, 76/652). Moreover, its fleas have been confirmed as infected with the natural plague (Yersinia pestis) [16,17]. A new study found that Neodon fuscus could also be the host of Taenia tianguangfui n. sp. [18]. It is, therefore, an actual or potential reservoir of animal and human zoonotic pathogens. Therefore, it is necessary to name this vole and to understand its taxonomy and phylogenetic relationships with other rodents to aid epidemiological investigations of zoonoses.

In the present study, we report the entire nucleotide sequence of the mt genome from the plateau vole, including information on the gene arrangement, nucleotide composition and codon usage. In addition, the phylogenetic and systematic position of this species is investigated. Based on the analysis of phylogenetic trees inferred from complete mt genomes and cytb genes, we strongly suggest that the vole examined in this study should be named N. fuscus.

Materials and methods

Samples and DNA extraction

The specimens of N. fuscus were collected from Wow Aryi Xiang, Jiuzhi County, Golog Autonomous Prefecture, Qinghai Province (33°19′N, 100°32′E) with an average elevation of 4100 meters above sea level. The collection and autopsy of the trapped voles was strictly conducted under national ethical guidelines (Regulations for Administration of Affairs Concerning Experimental Animals, China, 1988) for animal husbandry and humane treatment. Total genomic DNA was extracted from liver tissue using the TIANamp Genomic DNA Kit (TianGen).

PCR amplification, sequencing and identification

Primers were designed with reference to an alignment of sequences from published mt genomes of voles (Supplementary Table S1). Nine overlapping PCR products, ranging from 1829 to 2163 bp in length, were amplified from one specimen (specimen 1) to cover the entire mt genome. Primers used for the generation of PCR products and the sizes of the fragments are given in Table 1. PCR was carried out using a standard 3-step cycle: 94°C, 4 min, 35 cycles of: 94°C, 30 s, 52–56°C, 30 s, 72°C, 2 min 30 s followed by hold at 72°C, 10 min and a final hold at 16°C.

Table 1
Primers used to amplify the complete mitochondrial genome of N. fuscus
Primer name Primer sequence (5′→3′) Positions on the H-strand Size of PCR product (bp) 
F1 TGA AAA TGC TTA GAT GGA TGC 30–50 2123 
R1 CTC CAT AGG GTC TTC TCG TC 2133–2152  
F2 GGT AGC ATA ATC ACT TGT TC 2009–2028  
R2 GCT TTA TTA GCT GAC CTT AC 3819–3838 1829 
F3 CCC TAC ACC TAG AAA TAT GTC TG 3670–3692  
R3 CCC TGC TTC TAC TAT TGA TG 5642–5661 1992 
F4 CTC AGC CAT TTT ACC TAT GTT C 5283–5304  
R4 TGT TTT TAC TGT GAG GGC TG 7259–7278 1995 
F5 TCC AAC TAG GCT TAC AAG ATG 6995–7015  
R5 AGA YCC RTA AAT TCC GTC TG 9139–9157 2163 
F6 ACG RAA CAR YAT AAA CCA AGC 9038–9058  
R6 GGA TTA TRA TAG CGG TGA TGA C 11022–11043 2006 
F7 ATG RGG TAA CCA AAC AGA ACG 10538–10558  
R7 GTT GGC TTG ATG TTG AGA ATG 12612–12632 2095 
F8 AAC ARY ACY ATC YTR ACA GCC 12512–12532  
R8 TGA GGG TRG CTT TRT CTA CTG 14620–14640 2129 
F9 TGA GGA CAA ATA TCA TTC TG 14517–14536  
R9 ATG TAC TTG ATA CCC TCT CC 170–189 2001 
Primer name Primer sequence (5′→3′) Positions on the H-strand Size of PCR product (bp) 
F1 TGA AAA TGC TTA GAT GGA TGC 30–50 2123 
R1 CTC CAT AGG GTC TTC TCG TC 2133–2152  
F2 GGT AGC ATA ATC ACT TGT TC 2009–2028  
R2 GCT TTA TTA GCT GAC CTT AC 3819–3838 1829 
F3 CCC TAC ACC TAG AAA TAT GTC TG 3670–3692  
R3 CCC TGC TTC TAC TAT TGA TG 5642–5661 1992 
F4 CTC AGC CAT TTT ACC TAT GTT C 5283–5304  
R4 TGT TTT TAC TGT GAG GGC TG 7259–7278 1995 
F5 TCC AAC TAG GCT TAC AAG ATG 6995–7015  
R5 AGA YCC RTA AAT TCC GTC TG 9139–9157 2163 
F6 ACG RAA CAR YAT AAA CCA AGC 9038–9058  
R6 GGA TTA TRA TAG CGG TGA TGA C 11022–11043 2006 
F7 ATG RGG TAA CCA AAC AGA ACG 10538–10558  
R7 GTT GGC TTG ATG TTG AGA ATG 12612–12632 2095 
F8 AAC ARY ACY ATC YTR ACA GCC 12512–12532  
R8 TGA GGG TRG CTT TRT CTA CTG 14620–14640 2129 
F9 TGA GGA CAA ATA TCA TTC TG 14517–14536  
R9 ATG TAC TTG ATA CCC TCT CC 170–189 2001 

Each PCR product purified for sequencing was gel-cut and DNA was recovered through a column (AxyPrep DNA Gel Extraction Kit by AxyGen). The prepared products were sent to GENEWIZ to be sequenced using Sanger dideoxy chain termination in an ABI3730 DNA Analyzer. All the raw sequences were assembled, edited and aligned using the software package SeqMan (DNASTAR7.1) [19], Clustal W [20] and Bioedit v7.2.3 [21]. The boundaries of protein-coding genes and ribosomal genes were predicted by sequence homology with those of other voles. tRNAscan-SE1.2.1 [22] was used in a preliminary search for tRNA genes using the default search mode, the vertebrate mt genetic code (transl_table = 2) and ‘Mito/chloroplast’ source.

To examine the genetic variation of mtDNAs among additional individuals, primers were also designed for amplifying only the cytb gene (forward primer: 5′-CAAAGAAGTGCCTAAACAACCTA-3′; reverse primer: 5′-TAGAATATCAG CTTTGGGTGTTG-3′; expected length: 1402 bp) and D-loop region (forward primer: 5′-TCTCAGGGCATCAAGAAGGAAGG-3′; reverse primer: 5′-TGTGGCTAGGC AAGGTGTCTTTA-3′; expected length: 1231 bp) from 36 individuals from the area where specimen one was collected. PCR and sequencing of the cytb gene and D-loop region was performed according to the abovementioned description.

Phylogenetic analyses

To investigate the phylogenetic position of N. fuscus relative to other rodents, we used ClustalW to align the complete mt genomes of 32 rodent species which were downloaded from GenBank (Supplementary Table S2). Trees were constructed using maximum likelihood (ML) approaches (GTR+I+G4 model; 1000 bootstraps) in IQ-TREE [23] and Bayesian methods in MrBayes v3 [24]. The Bayesian analysis was run for 500000 generations and sampled every 1000 generations. The first 25% of trees were omitted as burn-in and the remaining trees were used to calculate Bayesian posterior probabilities. The mtDNA sequences of two species of pikas, Ochotona collaris and Ochotona curzoniae, were used as outgroups. Because the mt cytb is often used as a molecular marker for phylogenetic studies in the Arvicolinae, we also constructed trees from cytb genes of the same 34 (33 ingroup; 1 outgroup) species. The phylogenetic tree based on mt cytb genes was constructed using ML algorithms implemented in online IQ-TREE Web Server (http://iqtree.cibiv.univie.ac.at/) and TIM2+F+I+G4 model was chosen according to AIC implemented in IQ-TREE. Similarly, the tree based on mt cytb genes was also constructed by MrBayes v3.

Results

Genome structure and base composition

The complete mt genome of N. fuscus (specimen 1) obtained in the present study was a closed circle of 1328 bp (Accession number: MG833880/ NC_040138) (Table 2). The genome was comprised of two noncoding regions, 13 protein-coding genes, 22 tRNA genes and two rRNA genes, a structure similar to those of other rodents. The mtDNA base composition was 31.91% A, 14.36% G, 26.54% T and 27.20% C.

Table 2
Nucleotide composition data for the mitochondrial genomes of Neodon fuscus and other rodents
Family or Subfamily (Species) Size (bp) Nucleotide compositions of complete mt sequence Nucleotide compositions of 13 protein coding genes 
  %A %T %G %C %A %T %G %C 
Arvicolinae (Microtus fortis fortis16310 32.66 26.22 13.42 27.70 30.79 27.14 13.12 28.94 
Arvicolinae (Microtus kikuchii16312 32.71 25.99 13.59 27.71 30.72 27.02 13.42 28.84 
Arvicolinae (Microtus levis16283 32.95 27.29 13.59 26.17 30.93 28.50 13.41 27.15 
Arvicolinae (Neodon fuscus16328 31.91 26.54 14.36 27.20 30.63 26.47 13.41 29.48 
Arvicolinae (Neodon irene16367 32.33 26.78 13.89 26.99 30.32 27.84 13.59 28.25 
Arvicolinae (Proedromys liangshanesis16296 33.01 25.99 13.64 27.37 32.17 26.22 12.31 29.30 
Cricetinae (Mesocricetus auratus16264 32.63 31.07 13.07 23.22 31.26 32.85 12.51 23.38 
Murinae (Rattus rattus16305 34.01 27.89 12.57 25.53 31.72 29.32 12.37 26.59 
Murinae (Mus musculus musculus16300 34.61 28.55 12.31 24.53 32.66 29.83 12.19 25.33 
Sciurinae (Scinrus vulgaris16507 32.11 30.86 12.55 24.48 30.16 32.55 12.44 24.85 
Leporidae (Lepus capensis17722 31.64 29.53 13.19 25.63 29.62 31.58 13.23 25.57 
Ochotonidae (Ochotona curzoniae17131 31.02 25.87 13.46 29.65 28.70 27.63 13.35 30.32 
Family or Subfamily (Species) Size (bp) Nucleotide compositions of complete mt sequence Nucleotide compositions of 13 protein coding genes 
  %A %T %G %C %A %T %G %C 
Arvicolinae (Microtus fortis fortis16310 32.66 26.22 13.42 27.70 30.79 27.14 13.12 28.94 
Arvicolinae (Microtus kikuchii16312 32.71 25.99 13.59 27.71 30.72 27.02 13.42 28.84 
Arvicolinae (Microtus levis16283 32.95 27.29 13.59 26.17 30.93 28.50 13.41 27.15 
Arvicolinae (Neodon fuscus16328 31.91 26.54 14.36 27.20 30.63 26.47 13.41 29.48 
Arvicolinae (Neodon irene16367 32.33 26.78 13.89 26.99 30.32 27.84 13.59 28.25 
Arvicolinae (Proedromys liangshanesis16296 33.01 25.99 13.64 27.37 32.17 26.22 12.31 29.30 
Cricetinae (Mesocricetus auratus16264 32.63 31.07 13.07 23.22 31.26 32.85 12.51 23.38 
Murinae (Rattus rattus16305 34.01 27.89 12.57 25.53 31.72 29.32 12.37 26.59 
Murinae (Mus musculus musculus16300 34.61 28.55 12.31 24.53 32.66 29.83 12.19 25.33 
Sciurinae (Scinrus vulgaris16507 32.11 30.86 12.55 24.48 30.16 32.55 12.44 24.85 
Leporidae (Lepus capensis17722 31.64 29.53 13.19 25.63 29.62 31.58 13.23 25.57 
Ochotonidae (Ochotona curzoniae17131 31.02 25.87 13.46 29.65 28.70 27.63 13.35 30.32 

Protein-coding genes

Twelve of the 13 protein-coding genes were identified on the H-strand, while the remaining gene (nad6) was on the L-strand. The total length of protein-coding genes of N. fuscus was 11396 bp, accounting for 69.79% of the complete mt genome. As shown in Table 3, the genes encoding COX1-3, ATP6, ATP8, ND4L, ND4 and Cytb started with the common start codon ATG, whereas nad3 and the three remaining genes (nad1-2 and nad5) used GTG and ATA/ATT as start codons, a situation occasionally seen in other mammals. Seven genes (cox1, cox2, atp6, atp8, nad3, nad4L and cytb) were terminated by the general stop codon TAA, whereas nad2, nad5 and nad6 used TAG as the stop codon. The remaining genes (nad1, cox3 and nad4) used incomplete stop codons T(–), presumably transformed into complete stop codons through post-transcriptional polyadenylation [25]. This phenomenon is widespread in other animals [26–28].

Table 3
Location, size, and other information of genes in the mt genome of Neodon fuscus
Genes Begins Ends Size (bp) Strand Start/stop condon Intergenic nucleotides 
tRNAPhe 66 66   
12S rRNA 69 1015 947  
tRNAVal 1016 1086 71  
16S rRNA 1087 2648 1562  
tRNALeu 2650 2724 75  
nad2722 3681 960 ATA/TAG -2 
tRNAIle 3680 3747 68  
tRNAGln 3745 3816 72  -3 
tRNAMet 3818 3886 69  
nad3887 4921 1035 ATT/TAG 
tRNATrp 4923 4989 67  
tRNAAla 4991 5059 69  
tRNAAsn 5062 5131 70  
OL 5132 5164 33  
tRNACys 5163 5230 68  -2 
tRNATyr 5231 5297 67  
cox5299 6843 1545 ATG/TAA 
tRNASer 6841 6909 69  -3 
tRNAAsp 6913 6980 68  
cox6982 7665 684 ATG/TAA 
tRNALys 7669 7732 64  
atp7733 7936 204 ATG/TAA 
atp7894 8574 681 ATG/TAA 57 
cox8574 9357 784 ATG/T -1 
tRNAGly 9358 9426 69  
nad9427 9774 348 GTG/TAA 
tRNAArg 9776 9842 67  
nad4L 9844 10140 297 ATG/TAA 
nad10134 11511 1378 ATG/T -7 
tRNAHis 11512 11579 68  
tRNASer 11580 11638 59  
tRNALeu 11638 11707 70  -1 
nad11708 13519 1812 ATA/TAG 
nad13516 14040 525 ATG/TAG -4 
tRNAGlu 14041 14109 69  
Cyt14115 15257 1143 ATG/TAA 
tRNAThr 15260 15327 68  
tRNAPro 15328 15395 68  
D-loop 15396 16328 933  
Genes Begins Ends Size (bp) Strand Start/stop condon Intergenic nucleotides 
tRNAPhe 66 66   
12S rRNA 69 1015 947  
tRNAVal 1016 1086 71  
16S rRNA 1087 2648 1562  
tRNALeu 2650 2724 75  
nad2722 3681 960 ATA/TAG -2 
tRNAIle 3680 3747 68  
tRNAGln 3745 3816 72  -3 
tRNAMet 3818 3886 69  
nad3887 4921 1035 ATT/TAG 
tRNATrp 4923 4989 67  
tRNAAla 4991 5059 69  
tRNAAsn 5062 5131 70  
OL 5132 5164 33  
tRNACys 5163 5230 68  -2 
tRNATyr 5231 5297 67  
cox5299 6843 1545 ATG/TAA 
tRNASer 6841 6909 69  -3 
tRNAAsp 6913 6980 68  
cox6982 7665 684 ATG/TAA 
tRNALys 7669 7732 64  
atp7733 7936 204 ATG/TAA 
atp7894 8574 681 ATG/TAA 57 
cox8574 9357 784 ATG/T -1 
tRNAGly 9358 9426 69  
nad9427 9774 348 GTG/TAA 
tRNAArg 9776 9842 67  
nad4L 9844 10140 297 ATG/TAA 
nad10134 11511 1378 ATG/T -7 
tRNAHis 11512 11579 68  
tRNASer 11580 11638 59  
tRNALeu 11638 11707 70  -1 
nad11708 13519 1812 ATA/TAG 
nad13516 14040 525 ATG/TAG -4 
tRNAGlu 14041 14109 69  
Cyt14115 15257 1143 ATG/TAA 
tRNAThr 15260 15327 68  
tRNAPro 15328 15395 68  
D-loop 15396 16328 933  

In terms of codon usage patterns, N. fuscus prefers to use U+A codons, as observed in most rodent mt genomes. The codon CUA was the most commonly used, accounting for 4.05% of the total codons (3832) while CGG was the least used (0.47%).

tRNA and rRNA genes

The mt genome of N. fuscus had two genes for rRNAs (12S rRNA and 16S rRNA) and 22 tRNA-coding genes (specifying 20 amino acids). The 12S rRNA and 16S rRNA genes of N. fuscus mtDNA were 947 and 1562 bp in length, respectively, and were separated by tRNAVal. The 22 tRNA genes (14 on the H-strand and 8 on the L-strand) ranged from 59 to 75 bp in length (Table 3). The tRNASer(GCU) gene lacked the dihydrouridine stem and loop (DHU), a common phenomenon among vertebrates [29,30], whereas the 21 remaining tRNA genes had typical clover-leaf structures (not shown).

Noncoding regions

The two noncoding regions included a control region (CR) and L-strand replication origin (OL). The length of the CR was 933 bp, capable of forming a stable D-loop structure and was located between tRNAPro and tRNAPhe. The D-loop region was composed of an extended termination-associated sequence (ETAS), the central domain (CD), conserved sequence blocks (CSBs), L-strand promoter and H-strand promoter. Based on the situation for other species, we could identify in the ETAS a likely hypervariable region, 251 bp in length and two conserved blocks, ETAS1 (59 bp) and ETAS2. The CD was 308 bp in length, rich in G (19.48%). Three CSBs were identifiable: CSB-1, CSB-2 and CSB3 [31,32]. CSB-1 (here, 24 bp) is the most conserved block present in most mammals, while CSB-2 only partially exists in some species and CSB-3 is sometimes absent [32,33]. The OL region was 34 bp in length, extending from the 3′ end of tRNALys to the 5′ end of tRNAAsn within the WANCY cluster of tRNAs. It could fold into a stable stem-loop secondary structure with an 11 bp stem and a 12 bp loop (Figure 1). A comparison of the OL region of four members of the Arvicolinae shows the stem region to be very well conserved while the loop region was slightly less conserved.

The predicted structure of the OL region of Neodon fuscus, Microtus levis, Proedromys liangshanensis and Neodon irene

Diversity of mt DNAs among vole individuals

The alignment of the cytb gene and D-loop for 37 individuals of N. fuscus, including specimen 1, revealed that there were no differences in the cytb gene, and that only two variable sites (384 and 691) existed in the D-loop sequences (Supplementary Figure S2).

Phylogenetic analyses

Phylogenetic trees (Figure 2) demonstrate the monophyly of each subfamily or family of rodents. In both ML (not shown) and Bayesian analyses, N. fuscus had a closer relationship with Neodon species than with any member of the genus Microtus or Lasiopodomys mandarinus. Trees inferred from cytb genes or an alignment of 13 concatenated protein-coding genes also grouped N. fuscus and the other Neodon species to the exclusion of other species (Figure 3; Supplementary Figure S1). In these trees, the genus Microtus is shown to be paraphyletic, rendered so by the position of Microtus levis.

The phylogenetic relationship of Neodon fuscus with 32 other rodent species inferred from a Bayesian method based on an alignment of complete mt genomes

Figure 2
The phylogenetic relationship of Neodon fuscus with 32 other rodent species inferred from a Bayesian method based on an alignment of complete mt genomes

The numbers at a node represent bootstrap values. ★ Indicates N. fuscus examined in our study. O. collaris and O. curzoniae are used as outgroups.

Figure 2
The phylogenetic relationship of Neodon fuscus with 32 other rodent species inferred from a Bayesian method based on an alignment of complete mt genomes

The numbers at a node represent bootstrap values. ★ Indicates N. fuscus examined in our study. O. collaris and O. curzoniae are used as outgroups.

Phylogenetic trees inferred from cytb gene sequences (from 34 species) using Bayesian method

Figure 3
Phylogenetic trees inferred from cytb gene sequences (from 34 species) using Bayesian method

The number at each node represents the bootstrap value. ★ Indicates N. fuscus examined in our study. Mus musculus is used as outgroup.

Figure 3
Phylogenetic trees inferred from cytb gene sequences (from 34 species) using Bayesian method

The number at each node represents the bootstrap value. ★ Indicates N. fuscus examined in our study. Mus musculus is used as outgroup.

Discussion

In the present study, we determined the complete nucleotide sequence of the N. fuscus mt genome, which is similar to that of other rodents in gene number, arrangement, composition and other features. For instance, the content of A+T in the mt DNAs for Microtus fortis fortis, M. kikuchii, Neodon irene, Proedromys liangshanesis and O. curzoniae was under 60%, similar to that of N. fuscus.

In all phylogenetic analysis reported here, N. fuscus, N. irene and Neodon sikimensis were sister taxa. Similarly, the phylogenetic trees constructed by Chen et al (2012) [11] demonstrated a close relationship between N. fuscus, N. irene and N. sikimensis.

N. irene [34] (Irene’s mountain vole, Chinese scrub vole) occurs in alpine meadows and shrubby slopes in West China (Qinghai, Gansu, Sichuan, Xizang and Yunnan) and Myanmar, at elevations ranging from 2800 to over 4000 meters [8,35]. Its complete mt genome was sequenced recently [34,36].

Despite slight differences between IQ-TREE (ML) and Bayesian analyses, N. fuscus is clearly not close to Lasiopodomys mandarinus (Microtus mandarinus), which is clustered with species of Microtus. In other words, N. fuscus does not belong to genus Microtus and it is also inappropriate for it to be named L. fuscus. Liu et al [10] have suggested that Microtus fuscus should be transferred to Neodon fuscus according to morphological and molecular analysis. Our work using whole mt genomes further confirms this conclusion. Another species regularly appearing close to N. fuscus in phylogenetic trees was N. leucurus, which had previously been assigned commonly to genus Phaiomys. Much of their sequence data for N. leucurus were derived from the study by Galewski et al (2006) [37], who also found a close relationship between

N. irene and N. leucurus, but N. fuscus was not included in their study. Synonymy of N. fuscus with N. leucurus appears never to have been suggested. A possible explanation might be the misidentification of some of the tissues used in previous studies.

The speciose genus Microtus includes more than 60 species, accounting for ∼50% of Arvicoline rodents [34,38]. The apparently rapid evolution rate of Microtus is problematic for classification. In the past, the taxonomy of Microtus was mainly based on morphological differentiation, distinct karyotypes and different chromosome numbers [39,40]. However, some species have similar chromosome number and karyotype but appear morphologically different, while some are opposite [41,42]. Therefore, this genus clearly requires revision to fit phylogenetic relationships indicated by molecular data [10,11,37,43,44]. Both Neodon and Proedromys have been included within Microtus at various times [42,45–48] but have eventually become recognized as independent genera. Molecular systematic approaches to revision of the paraphyletic genus Microtus, and the Arvicolinae more broadly, are urgently needed. The molecular systematic analysis of Microtus based on molecular data including both mitochondrial and nuclear markers [49,50]. In the past decades, most phylogenetic studies focused on mt genomes and some species received a good distinction [41,51]. Recently, some researchers are beginning to use both mtDNA and nuclear DNA to analyze the phylogeny of genus Microtus and obtain good results [52–54]. In the future, the mtDNA and nuclear loci, especially the combined application of both, may play an important role in taxonomy of intricate genera.

Briefly, phylogenetic trees based on complete mtDNAs and mt cytb genes for the subfamily Arvicolinae suggested that N. fuscus, and the other Neodon species are sister taxa. It is therefore appropriate that the vole, typically in the book ‘Wilson & Reeder’s Mammal Species of the World’ used as L. fuscus, should be called or renamed N. fuscus.

Acknowledgments

We thank Rui-Lin Ma, Jin-Shan Cai, Yan-Li Shen and Guang-Wei Hu for their support in the collection of vole samples at the Center for Animal Disease Prevention and Control, Qinghai Province.

Ethics Statement

All animals were handled in strict accordance with good animal practice according to the Animal Ethics Procedures and Guidelines of the People’s Republic of China, and the study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (No. LVRIAEC2012-007).

Funding

This study was financially supported by Project support was provided by National Key Basic Research Program (973 Program) of China [grant number 2015CB150300], the National Natural Science Foundation of China [grant numbers 31401148, 31402191], the National Key Research and Development Program of China [grant numbers 2017YFD0501301, 2018YFC1602], Central Public-interect Scientific Institution Basal Research Fund [grant numbers 1610312017001; 1610312016012].

Author Contribution

J. L., L. L., H. Y. and W. J. conceived and designed the experiments. J. L. and L. L. performed the experiments and the data analyses. J. L. prepared the figures and wrote the manuscript, W. J. and H. Y. provided improving paragraph and B. F. provided very constructive suggestions for revisions.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Abbreviations

     
  • CD

    central domain

  •  
  • CR

    control region

  •  
  • CSB

    conserved sequence block

  •  
  • cytb

    cytochrome b

  •  
  • ETAS

    extended termination-associated sequence

  •  
  • ML

    maximum likelihood

  •  
  • mt

    mitochondrial

  •  
  • PCR

    polymerase chain reaction

  •  
  • rRNA

    ribosomal RNA

  •  
  • tRNA

    transfer RNA

References

References
1.
Boore
J.L.
,
Macey
J.R.
and
Medina
M.
(
2005
)
Sequencing and comparing whole mitochondrial genomes of animals
.
Methods Enzymol.
395
,
311
348
[PubMed]
2.
Brown
W.M.
and
Wilson
G.A.C.
(
1979
)
Rapid evolution of animal mitochondrial DNA
.
Proc. Natl. Acad. Sci. U.S.A.
76
,
1967
1971
3.
Curole
J.P.
and
Kocher
T.D.
(
1999
)
Mitogenomics: digging deeper with complete mitochondrial genomes
.
Trends Ecol. Evol.
14
,
394
398
[PubMed]
4.
Wu
X.B.
,
Wang
Y.Q.
,
Zhou
K.Y.
,
Zhu
W.Q.
,
Nie
J.S.
and
Wang
C.L.
(
2003
)
Complete mitochondrial DNA sequence of Chinese alligator, alligator sinensis, and phylogeny of crocodiles
.
Chin. Sci. Bull.
48
,
2050
2054
5.
Knudsen
B.
,
Kohn
A.B.
,
Nahir
B.
,
McFadden
C.S.
and
Moroz
L.L.
(
2006
)
Complete DNA sequence of the mitochondrial genome of the sea-slug, Aplysia californica: Conservation of the gene order in euthyneura
.
Mol. Phylogenet. Evol.
38
,
459
469
[PubMed]
6.
Lavoue
S.
,
Miya
M.
,
Saitoh
K.
,
Ishiguro
N.B.
and
Nishida
M.
(
2007
)
Phylogeneticrelationships among anchovies, sardines, herrings and their relatives (Clupeiformes), inferred from whole mitogenome sequences
.
Mol. Phylogenet. Evol.
43
,
1096
1105
[PubMed]
7.
Morin
P.A.
,
Archer
F.I.
,
Foote
A.D.
,
Vilstrup
J.
,
Allen
E.E.
,
Wade
P.
et al. .
(
2010
)
Complete mitochondrial genome phylogeographic analysis of killer whales (Orcinus orca) indicates multiple species
.
Genome. Res.
20
,
908
916
[PubMed]
8.
Büchner
E.
(
1894
)
Wissenschaftliche resultate der von n. M. Przewalski nach central-asien unternommenen reisen auf kosten einer von seiner kaiserlichen hoheit dem grossfürsten thronfolger nikolai alexan-drowitsh gespendeten summe herausgegeben von der kaiserlichen akademie der wissenschaften st. Petersburg
.
Zoologischer, Theil Band I Säugethiere
3
,
232
9.
Smith
A.
and
Xie
Y.
(
2008
)
Guide to the mammals of China
,
Princeton University Press
,
Priceton
10.
Liu
S.Y.
,
Sun
Z.Y.
,
Liu
Y.
,
Wang
H.
,
Guo
P.
and
Robert
W.M.
(
2012
)
A new vole from Xizang, china and the molecular phylogeny of the genus Neodon (cricetidae: Arvicolinae)
.
Zootaxa
3235
,
1
22
11.
Chen
W.C.
,
Hao
H.B.
,
Sun
Z.Y.
,
Liu
Y.
,
Liu
S.Y.
and
Yue
B.S.
(
2012
)
Phylogenetic position of the genus Proedromys (arvicolinae, rodentia): evidence from nuclear and mitochondrial DNA
.
Biochem. Syst. Ecol.
42
,
59
68
12.
Zhang
X.X.
,
Lou
Z.Z.
,
Huang
S.Y.
,
Zhou
D.H.
,
Jia
W.Z.
,
Su
C.
et al. .
(
2013
)
Genetic characterization of Toxoplasma gondii from qinghai vole, plateau pika and tibetan ground-tit on the qinghai-tibet plateau, China
.
Parasit. Vectors
6
,
291
[PubMed]
13.
Ma
J.
,
Wang
H.
,
Lin
G.
,
Zhao
F.
,
Li
C.
,
Zhang
T.
et al. .
(
2015
)
Surveillance of echinococcus isolates from qinghai, china
.
Vet. Parasitol.
207
,
44
48
[PubMed]
14.
Li
J.Q.
,
Li
L.
,
Fan
Y.L.
,
Fu
B.Q.
,
Zhu
X.Q.
,
Yan
H.B.
et al. .
(
2018
)
Genetic diversity in Echinococcus multilocularis from the plateau vole and plateau pika in Jiuzhi County, Qinghai Province, China
.
Front. Microbiol.
9
,
2632
[PubMed]
15.
Xu
X.
,
Guan
Y.Y.
,
Tian
T.
et al. .
(
2011
)
Distribution of the intermediate hosts of Echinococcus multilocularis in Shiqu County, Sichuan, China
.
Chi. Med. J. (Engl).
124
,
2834
2837
16.
Wang
L.M.
,
Song
X.Y.
,
Zhu
X.P.
,
Xie
F.
,
Li
G.Q.
,
Luo
Z.D.
et al. .
(
2009
)
Monitoring the microtus fuscus plague epidemic in sichuan province during 2000-2008
.
Zhonghua Liu Xing Bing Xue Za Zhi
30
,
1175
1178
,
(In Chinese)
[PubMed]
17.
Yang
Y.
,
Wang
M.
,
Zhao
X.
,
Zhao
Z.
,
Zhang
A.
,
Wei
R.
et al. .
(
2014
)
Analysis on the results of etiology and serology of plague in qinghai province from 2001 to 2010
.
Chin. J. Epidemiol.
48
,
124
127
,
(In Chinese)
18.
Fan
Y.L.
,
Lou
Z.Z.
,
Li
L.
,
Yan
H.B.
,
Han
X.M.
,
Li
J.Q.
et al. .
(
2014
)
Preliminary study on taxonomic status of cysticerci from Microtus fuscus in Qinghai-Tibet Plateau of China
.
Chin. Veterinary Sci.
44
,
789
793
,
(In Chinese)
19.
Swindell
S.R.
and
Plasterer
T.N.
(
1997
)
Seqman, Sequence Data Analysis Guidebook
, pp.
75
89
,
Springer
,
Totowa, Germany
20.
Thompson
J.D.
,
Higgins
D.G.
and
Gibson
T.J.
(
1994
)
Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice
.
Nucleic Acids Res.
22
,
4673
4680
[PubMed]
21.
Hall
T.A.
(
1999
)
A user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt
.
Nucleic Acids Symp. Ser.
95
98
[PubMed]
22.
Lowe
T.M.
and
Eddy
S.R.
(
1997
)
Trnascan-se: a program for improved detection of transfer rna genes in genomic sequence
.
Nucleic Acids Res.
25
,
955
964
[PubMed]
23.
Kalyaanamoorthy
S
et al. .
(
2017
)
ModelFinder: fast model selection for accurate phylogenetic estimates
.
Nat. Methods
14
,
587
589
[PubMed]
24.
Ronquist
F.
and
Huelsenbeck
J.P.
(
2003
)
Mrbayes 3: Bayesian phylogenetic inference under mixed models
.
Bioinformatics
19
,
1572
1574
[PubMed]
25.
Ojala
D.
,
Montoya
J.
and
Attardi
G.
(
1981
)
Trna punctuation model of rna processing in human mitochondria
.
Nature
290
,
470
474
[PubMed]
26.
Gissi
C.
,
Gullberg
A.
and
Arnason
U.
(
1998
)
The complete mitochondrial DNA sequence of the rabbit, Oryctolagus cuniculus
.
Genomics
50
,
161
169
[PubMed]
27.
Peng
R.
,
Zeng
B.
,
Meng
X.
,
Yue
B.
,
Zhang
Z.
and
Zou
F.
(
2007
)
The complete mitochondrial genome and phylogenetic analysis of the giant panda (Ailuropoda melanoleuca)
.
Gene
397
,
76
83
[PubMed]
28.
Zhong
H.M.
,
Zhang
H.H.
,
Sha
W.L.
,
Zhang
C.D.
and
Chen
Y.C.
(
2010
)
Complete mitochondrial genome of the red fox (Vuples vuples) and phylogenetic analysis with other canid species
.
Zoological Res.
31
,
122
130
,
(In Chinese)
29.
Garey
J.R.
and
Wolstenholme
D.R.
(
1989
)
Platyhelminth mitochondrial DNA: evidence for early evolutionary origin of a trnaseragn that contains a dihydrouridine arm replacement loop, and of serine-specifying aga and agg codons
.
J. Mol. Evol.
28
,
374
387
[PubMed]
30.
Pereira
S.L.
(
2000
)
Mitochondrial genome organization and vertebrate phylogenetics
.
Genet. Mol. Biol.
23
,
745
752
31.
Larizza
A.
,
Makalowski
W.
,
Pesole
G.
and
Saccone
C.
(
2002
)
Evolutionary dynamics of mammalian mrna untranslated regions by comparative analysis of orthologous human, artiodactyl and rodent gene pairs
.
Comput. Chem.
26
,
479
490
[PubMed]
32.
Sbisa
E.
,
Tanzariello
F.
,
Reyes
A.
,
Pesole
G.
and
Saccone
C.
(
1997
)
Mammalian mitochondrial d-loop region structural analysis: identification of new conserved sequences and their functional and evolutionary implications
.
Gene
205
,
125
140
[PubMed]
33.
Saccone
C.
,
Pesole
G.
and
Sbisa
E.
(
1991
)
The main regulatory region of mammalian mitochondrial DNA: structure-function model and evolutionary pattern
.
J. Mol. Evol.
33
,
83
91
[PubMed]
34.
Thomas
O.
(
1912
)
The duke of bedford’s zoological exploration of Eastern Asia. On mammals from the provinces of Szechwan and Yunnan, Western China
.
Proc. Zool. Soc. Lond.
127
141
35.
Musser
G.
and
Carleton
M.
(
2005
)
Superfamily muroidea
. In
Mammals species of the world. A taxonomic and geographic reference
(
Wilson
D.E.
and
Reeder
D.M.
, eds),
Johns Hopkins University Press
,
Baltimore 2142, Maryland
36.
Fan
L.
,
Fan
Z.
,
Yue
H.
,
Zhang
X.
,
Liu
Y.
,
Sun
Z.
et al. .
(
2011
)
Complete mitochondrial genome sequence of the Chinese scrub vole (Neodon irene)
.
Mitochondr. DNA
22
,
50
52
37.
Galewski
T.
,
Tilak
M.K.
,
Sanchez
S.
,
Chevret
P.
,
Paradis
E.
and
Douzery
E.J.
(
2006
)
The evolutionary radiation of Arvicolinae rodents (voles and lemmings): relative contribution of nuclear and mitochondrial DNA phylogenies
.
BMC Evol. Biol.
6
,
80
[PubMed]
38.
Chaline
J.
,
Brunet-Lecomte
P.
,
Montuire
S.
,
Viriot
L.
and
Courant
F.
(
1999
)
Anatomy of the arvicoline radiation (Rodentia): palaeogeographical, palaeoecological history and evolutionary data
.
Ann. Zool. Fenn.
36
,
239
267
39.
Maruyama
T.
and
Imai
H.T.
(
1981
)
Evolutionary rate of the mammalian karyotype
.
J. Theor. Biol.
90
,
111
121
[PubMed]
40.
Gu
W.
,
Wang
T.Z.
and
Zhu
B.C.
(
1999
)
Study on the morphology of sex chromosomes pairing of the synaptonemal complexes in mandarin vole (Microtus mandarinus)
.
Acta Theriol. Sin.
19
,
150
154
41.
Golenishchev
F.N.
,
Malikov
V.G.
,
Yu
B.S.
et al. .
(
2018
)
Toward assembling a taxonomic puzzle: case study of Iranian gray voles of the subgenus, Microtus, (Rodentia, Cricetidae)
.
Mamm. Biol.
94
,
98
105
42.
Triant
D.A.
and
Dewoody
J.A.
(
2006
)
Accelerated molecular evolution in microtus (rodentia) as assessed via complete mitochondrial genome sequences
.
Genetica
128
,
95
108
[PubMed]
43.
Bannikova
A.A.
,
Lebedev
V.S.
,
Lissovsky
A.A.
,
Matrosova
V.
,
Abramson
N.I.
,
Obolenskaya
E.V.
et al. .
(
2010
)
Molecular phylogeny and evolution of the asian lineage of vole genus Microtus (rodentia: Arvicolinae) inferred from mitochondrial cytochrome b sequence
.
Biol. J. Linn. Soc. Lond.
99
,
595
613
44.
Robovský
J.
,
ŘIčánková
V.
and
Zrzavý
J.
(
2008
)
Phylogeny of Arvicolinae (mammalia, cricetidae): utility of morphological and molecular data sets in a recently radiating clade
.
Zool. Scr.
37
,
571
590
45.
Ellerman
J.R.
(
1941
)
The families and genera of living rodents trustees of the british museum
,
Trustees of the British Museum
,
London
46.
Ellerman
J.R.
and
Morrison-Scott
T. C.S.
(
1951
)
Checklist of palaearctic and indian mammals 1758 to 1946
.
Trustees of the British Museum, London
47.
Luo
Z.
,
Chen
W.
and
Gao
W.
(
2000
)
Fauna sinica (mammalia vol. 6 rodentia II cricetidae)
,
Science Press
,
Beijing, China
,
(In Chinese)
48.
Nowak
R.M.
(
1999
)
Walker’s mammals of the world
,
The Johns Hopkins University Press
,
Baltimore, U.S.A.
49.
Buzan
E.V.
,
Krystufek
B.
,
Hänfling
B.
and
Hutchinson
W.F.
(
2008
)
Mitochondrialphylogeny of Arvicolinae using comprehensive taxonomic sampling yieldsnew insights
.
Biol. J. Linn. Soc. Lond.
94
,
825
835
50.
Martinkova
N.
and
Moravec
J.
(
2012
)
Multilocus phylogeny of arvicoline voles (Arvicolini, Rodentia) shows small tree terrace size
.
Folia. Zool.
61
,
254
267
51.
Haring
E.
,
Sheremetyeva
I.N.
and
Kryukov
A.P.
(
2011
)
Phylogeny of palearctic vole species (genus Microtus, Rodentia) based on mitochondrial sequences
.
Mamm. Biol.
76
,
258
267
52.
Barbosa
S.
,
Paupério
J.
,
Pavlova
S.V.
et al. .
(
2018
)
The Microtus voles: resolving the phylogeny of one of the most speciose mammalian genera using genomics
.
Mol. Phylogenet. Evol.
125
,
85
92
[PubMed]
53.
Laura
R.M.
,
Hernández-Canchola
G.
and
León-Paniagua
L.
(
2018
)
Molecular systematics and phylogeography of the endemic osgood’s deermouse, osgoodomys banderanus, (rodentia: cricetidae) in the lowlands of western mexico
.
Mol. Phylogenet. Evol.
127
,
867
877
[PubMed]
54.
Li
K.
,
Kohn
M.H.
,
Zhang
S.
,
Wan
X.
et al. .
(
2017
)
The colonization and divergence patterns of brandt’s vole (lasiopodomys brandtii) populations reveal evidence of genetic surfing
.
BMC Evol. Biol.
17
,
145

Author notes

*

These authors contributed equally to this work.

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