Complete Mitochondrial Genome of Three Species of the Genus Microtus (Arvicolinae, Rodentia)

Simple Summary

The knowledge and analysis of mitochondrial genomes can be useful to answer some interesting questions about the biology of some species. This is the case of vole species that are characterized by unusual genetic features related to sex chromosomes and variation of their karyotypes. In this work, we describe the mitochondrial genomes of three emblematic vole species, demonstrating that they are highly conserved in the organization of their protein coding, transfer RNA and ribosomal RNA genes. In addition, we performed a detailed analysis of their control regions identifying several domains and conserved boxes related to mitochondrial DNA regulation. Finally, a phylogenetic analysis with the mitochondrial DNA confirmed the established phylogeny of the analyzed voles.

Abstract

The 65 species of the genus Microtus have unusual sex-related genetic features and a high rate of karyotype variation. However, only nine complete mitogenomes for these species are currently available. We describe the complete mitogenome sequences of three Microtus, which vary in length from 16,295 bp to 16,331 bp, contain 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes and a control region. The length of the 13 PCGs and the coded proteins is the same in all three species, and the start and stop codons are conserved. The non-coding regions include the L-strand origin of replication, with the same sequence of 35 bp, and the control region, which varies between 896 bp and 930 bp in length. The control region includes three domains (Domains I, II and III) with extended termination-associated sequences (ETAS-1 and ETAS-2) in Domain I. Domain II and Domain III include five (CSB-B, C, D, E and F) and three (CSB-1, CSB-2, and CSB-3) conserved sequence blocks, respectively. Phylogenetic reconstructions using the mitochondrial genomes of all the available Microtus species and one representative species from another genus of the Arvicolinae subfamily reproduced the established phylogenetic relationships for all the Arvicolinae genera that were analyzed.

1. Introduction

The rodent subfamily Arvicolinae (lemmings, muskrats and voles) includes numerous species that have been classified into 21 genera [1], of which the genus Microtus is the most species-rich (65 extant species). This genus underwent fast recent radiation about 1.2–2 million years ago (MYA) [2] in what was one of the most explosive speciation events ever recorded in vertebrates [1].

Some species from the genus Microtus are characterized by a series of unusual sex-related genetic features [3,4,5,6,7,8,9,10]. Moreover, the genus Microtus shows one of the highest rates of karyotype variation of all mammals [11], with highly variable chromosome numbers ranging from 2n = 18 in M. oregoni [12] to 2n = 62 in M. duodecimcostatus and M. lusitanicus [13]. Despite this variability in their karyotypes, the majority of Microtus species have similar external morphologies and therefore are hard to distinguish using traditional taxonomic characteristics [14,15,16,17]. In addition, the genus Microtus underwent a recent rapid radiation resulting in the emergence of numerous cryptic species with unclear phylogenetic relationships [18,19,20,21]. Although some of these cryptic species can be distinguished using mitochondrial and/or nuclear markers [22,23], Microtus phylogeny is not clearly established and many systematic relationships within this genus are still unresolved [20,24].

Phylogenetic reconstructions have been carried out using karyotype banding, chromosome painting and repeated DNA sequence analysis [3,8,25,26]. Despite lacking the reliability to solve intra-specific relationships [27], mitochondrial markers COI and NADH are nevertheless regarded as very precise tools for deciphering interspecific relationships between Arvicolinae species [16,17,24,28]. Several nuclear genetic markers such as the growth hormone receptor (GRH) including exon 10 [27] have been employed to solve intra-specific relationships in the genus Microtus. An interesting phylogenomic approach employing partial mitogenomes and genotyping-by-sequencing single nucleotide polymorphism (SNP) data from seven Microtus species has also been recently described [29].

Traditionally, complete mitogenomes have been sequenced by PCR amplification of overlapping fragments [30,31]. However, next-generation sequencing (NGS) now allows us to assemble much more easily the whole mitochondrial genome due to its high-copy portion in relation to the nuclear DNA [32,33,34,35,36,37]. Nevertheless, although complete mitochondrial genomes are very useful for phylogenetic reconstructions, the number of species with fully described mitogenomes is still low [38,39]. Only eight complete mitogenomes are currently available for Microtus species, and therefore, in light of the fast radiation processes taking place in this genus, the description of mitogenomes from other Microtus species is potentially of great interest. In this paper, we report the sequencing and characterization by next-generation sequencing (NGS) data of the complete mitogenome of three further Microtus species, M. cabrerae, M. chrotorrhinus and M. thomasi.

2. Materials and Methods

2.1. DNA Extraction, Sequencing and Mitogenome Assembly

A single male specimen of M. cabrerae (Siles, Spain (TOPC-0902)) and one of M. thomasi (Edessa, Greece, (R231)) were used for DNA extraction, while for M. chrotorrhinus (North America), an established cell line from a male individual was employed [4,40]. All capture and sacrifice protocols were approved by the Junta de Andalucía Ethics Committee for Animal Experimentation (code: 04/09/2018/130). Tissue samples from the specimens were stored in 100% ethanol at −20 °C. The total DNA was extracted from tissues and from cultured cells using the Gentra Puregene Tissue Kit (Qiagen, Hilden, Germany). The extracted genomic DNA of M. cabrerae was sequenced using the Illumina technology in Macrogen (Europe) and M. thomasi and M. chrotorrhinus DNAs in Novogene (Hong Kong). Briefly, for genome sequencing, we used approximately 3 µg of genomic DNA for the construction of a library of 750-bp-long fragments for M. cabrerae and 350-bp-long fragments for the other two species. These libraries were sequenced using the Illumina^® Hiseq™ 2000 (San Diego, CA, USA) platform with the 2 × 100 nt paired-end option. Two Gbp of sequences were obtained for each species. Graph-based clustering analysis [41,42] was performed using RepeatExplorer implemented within the Galaxy environment (http://repeatexplorer.org/). Sequence clusters corresponding to mtDNA were selected and assembled. We also used the Geneious v.R7.1 software [43] to assemble and annotate the mitogenomes. Both assembled versions of each species’ mitogenome were aligned and revised manually using the complete M. arvalis mitogenome as reference [38] (accession number: MG948434). In addition, the sequence of the control region (D-loop) of the mitochondrial genomes was confirmed by PCR amplification and sequencing using the primer pair Pro+ (5′-ACCATCAGCACCCAAAGCTG-3′) and Phe- (5′-AAGCATTTTCAGTGCTTTGCTT-3′) [35,44].

2.2. Annotation and Sequence Analysis

The annotation of these three mitogenomes was performed using web-based services MITOS (http://mitos.bioinf.uni-leipzig.de/help.py) [45] and tRNA scan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/) [46]. The annotations of the protein-coding genes (PCGs), transfer RNAs (tRNAs) and rRNA genes were refined by manual comparison, according to Markova et al. [47], with the M. arvalis mitogenome [38]. The base composition was estimated using the Bioedit program (v7.0.9.0) (http://www.mbio.ncsu.edu/BioEdit/bioedit.html), and codon usage was analyzed using MEGA version 10 [48]. The circularized drawing of the mitogenome was performed with the OrganellarGenomeDRAW tools (http://ogdraw.mpimp-golm.mpg.de/) [49].

2.3. Phylogenetic Analysis

For the phylogenetic analyses, in addition to the sequenced mitogenomes described above, we also included nine complete mitochondrial genomes from Microtus species available in GenBank and from other representative species of the Arvicolinae subfamily [1,38,50,51,52,53,54,55,56,57,58,59,60,61,62]. As outgroup, we used a species of the genus Akodon (A. montensis) [63] from the subfamily Sigmodontinae.

Complete mitogenomes were aligned using ClustalW, and the phylogenetic relationships were reconstructed using the Bayesian inference (BI) implemented in MrBayes v. 3.1 [64]. Runs of two million generations were conducted. Trees were sampled every 1000 generations with a burn-in of 25%. The best-fit nucleotide substitution model with the lowest BIC (Bayesian Information Criterion) value was chosen (GTR + G + I) using MEGA version X [48].

3. Results and discussion

3.1. Gene Organization

The complete mitogenomes of M. cabrerae (MN058077), M. chrotorrhinus (MN058078) and M. thomasi (MN058079) analyzed were 16,331 bp, 16,297 bp and 16,295 bp in length, respectively. These values are similar to the mitogenomes of other species from this genus, which range between 16,283 bp (M. rossiaemeridionalis) and 16,312 bp (M. kikuchii) [1,60]. They were also comparable in size to those from other species of the Arvicolinae subfamily such as Proedromys liangshanensis (16,296 bp) [57] and Neodon forresti (16,397 bp; GenBank accession number: KU891252.1). All our results confirm that the mitochondrial genomes in the Arvicolinae subfamily are very similar in size.

The mitogenomes from M. cabrerae, M. chrotorrhinus and M. thomasi include a control region (D-loop) and a conserved set of 37 vertebrate mitochondrial genes, with 13 protein-coding genes (PCGs), 22 tRNA genes and two rRNA genes (12S rRNA and 16S rRNA) (

Table 1. Gene organization of the three Microtus mitogenomes (M.ca: M. cabrerae; M.ch: M. chrotorrhinus; M.th: M. thomasi).

Gene	Nucleotide Positions			Size (bp) 1			Strand 2	Anticodon	Intergenic Nucleotide 1
	M.ca	M.ch	M.th	M.ca	M.ch	M.th			M.ca M.ch M.th
tRNAPhe	1–66			66			H	TTC
12S rRNA	69–1018	69–1015	69–1016	950	947	948	H		2
tRNAVal	1019–1089	1018–1085	1016–1086	71	70	70	H	GTA
16S rRNA	1088–2655	1084–2645	1085–2651	1568	1562	1567	H		−2
tRNALeu (uur)	2656–2730	2647–2721	2653–2727	75			H	TTA		1	1
Nd1	2731–3685	2722–3676	2728–3682	955			H
tRNAIle	3686–3753	3677–3745	3683–3751	68	69	69	H	ATC
tRNAGln	3751–3822	3743–3814	3749–3820	72			L	CAA	−3
tRNAMet	3824–3892	3817–3885	3822–3890	69			H	ATG	1	2	1
Nd2	3893–4927	3886–4920	3891–4925	1035			H
tRNATrp	4929–4995	4922–4988	4927–4993	67			H	TGA	1
tRNAAla	4997–5065	4990–5058	4995–5063	69			L	GCA	1
tRNAAsn	5067–5136	5061–5130	5066–5135	70			L	AAC	1	2	2
Rep_origin	5137–5171	5131–5165	5136–5170		35		H
tRNACys	5169–5236	5163–5230	5168–5235	68	69	68	L	TGC	−3
tRNATyr	5237–5303	5231–5297	5236–5302	67			L	TAC
Cox1	5305–6849	5299–6843	5304–6848	1545			H		1
tRNASer (ucn)	6847–6915	6841–6909	6846–6914	69			L	TCA	−3
tRNAAsp	6919–6986	6913–6980	6918–6985	68			H	GAC	3
Cox2	6988–7671	6982–7665	6987–7670	684			H		1
tRNALys	7675–7737	7669–7732	7674–7737	63	64	64	H	AAA	3
Atp8	7738–7941	7733–7936	7738–7941	204			H
Atp6	7899–8578	7894–8573	7899–8578		680		H		−43
Cox3	8579–9362	8574–9357	8579–9362	784			H
tRNAGly	9363–9430	9358–9425	9363–9430	68			H	GGA
Nd3	9431–9778	9426–9773	9431–9778	348			H
tRNAArg	9780–9845	9775–9841	9780–9846	66	67	67	H	CGA	1
Nd4L	9849–10145	9844–10140	9850–10146	297			H		3	2
Nd4	10139–11516	10134–11511	10140–11517	1378			H		−7
tRNAHis	11517–11572	11512–11579	11527–11585	56	68	59	H	CAC			9
tRNASer	11584–11642	11580–11638	11586–11644	59			H	AGC	11
tRNALeu	11642–11711	11638–11707	11644–11713	70			H	CTA	−1
Nd5	11712–13523	11708–13519	11714–13525	1812			H
Nd6	13523–14044	13516–14040	13522–14046	525			L		−4
tRNAGlu	14045–14113	14041–14109	14047–14115	69			L	GAA
CytB	14119–15261	14115–15257	14121–15263	1143			H		5
tRNAThr	15264–15333	15260–15326	15266–15331	70	67	66	H	ACA	2
tRNAPro	15334–15401	15327–15394	15332–15399	68			L	CCA
D-loop	15402–16331	15395–16297	15400–16295	930	903	896	H

¹ The corresponding three values are only indicated when they are different. ² Strand: H: heavy and L: light.

). As expected, the organization and structures of these three mitogenomes were identical to those described for other Microtus and mammal species (Figure 1). Hence, twelve PCGs, 14 tRNAs and two rRNAs are located on the heavy strand, while Nd6 and eight tRNAs are found on the light strand. The D-loop is emplaced between the tRNA-Pro and tRNA-Phe genes [37,38,57]. The percentage of identity observed in pairwise comparing of these three complete mitogenomes varied between 86.34% (M. thomasi–M. cabrerae pair-wise comparison) and 87.55% (M. thomasi–M. chrotorrhinus pair-wise comparison). These results fall into the range of the identity values that we calculated for comparisons between the available Microtus mitogenomes (85.50–98.75%).

3.2. Nucleotide Composition

We identified a bias towards A and T nucleotides, which is commonly reported in mitogenome sequences in mammals [35,37]. Hence, the A+T compositions of the H-strands are 58.19%, 59.27% and 59.77% in the M. cabrerae, M. chrotorrhinus and M. thomasi mitogenomes, respectively. The 13 mitochondrial PCGs are AT-biased, with an A+T content ranging from 54.34–56.63% for Cox3 to 64.71–66.67% for the Atp8 gene. The control region, the two rRNA genes and the 22 tRNAs are also AT-biased in all three species (

Table 2. Data about the 13 PCG of the three Microtus mitogenomes (M.ca: M. cabrerae; M.ch: M. chrotorrhinus; M.th: M. thomasi).

Gene	Gene Length (bp)	% Identity			A + T Content (%)			Start/Stopcodons	Protein Length (aa)
		M.ca/M.ch	M.ca/M.th	M.ch/M.th	M.ca	M.ch	M.th	M.ca/M.ch/M.th
Nd1	955	84.50	85.86	86.39	56.96	57.49	57.70	GTG/T--	318
Nd2	1035	82.22	82.03	83.96	59.32	61.84	61.93	ATT/TAA	344
Cox1	1545	86.21	86.15	86.28	55.34	57.86	56.50	ATG/TAA	514
Cox2	684	86.11	86.11	85.38	56.29	57.46	58.33	ATG/TAA	227
Atp8	204	85.78	87.75	89.71	64.71	66.67	65.69	ATG/TAA	67
Atp6	680	86.93	84.58	86.78	56.39	56.98	59.77	ATG/TA-	226
Cox3	784	85.33	84.44	85.84	54.34	56.63	56.12	ATG/T--	261
Nd3	348	83.33	83.33	84.48	58.62	57.76	59.48	ATT/TAA	115
Nd4L	297	82.83	82.83	82.49	57.91	55.89	59.60	ATG/TAA	98
Nd4	1378	82.15	80.48	83.74	57.84	58.35	60.16	ATG/T--	459
Nd5	1812	83.17	82.34	85.10	56.29	57.73	58.44	ATA/TAA ATT/TAG ATA/TAA	603
Nd6	525	85.14	85.52	87.62	60.76	63.05	60.76	ATG/TAG	174
CytB	1143	87.66	87.75	88.10	56.52	56.26	58.53	ATG/TAA	380
Total	11,390								3786

).

3.3. Protein-Coding Genes and Codon Usage

The 13 mitochondrial PCGs from the three analyzed species are 11,390 bp in length (11,358 bp in codons and 32 bp in stop codons) and encode 3786 amino acids (Table 2). The three described mitogenomes also contain some overlapping nucleotides and gaps between PCGs or between PCGs and tRNAs (Table 1). The longest overlap of 43 bp is located between the Atp8 and Atp6 genes (Table 1).

Twelve mitochondrial PCGs use exactly the same start codon for translation initiation in all the three species: GTG for Nd1, ATT for Nd2 and Nd3, ATG for the other nine PCGs. Only the Nd5 gene has variation in the start codon: ATA in M. cabrerae and M. thomasi, and ATT in M. chrotorrhinus. Similarly, 12 PCGs genes use exactly the same five stop codons for translation termination in all three species, two incomplete (T-- for Nd1, Cox3 and Nd4; TA- for Atp6) and the three other complete (TAG for Nd6, TAA for the rest of PCGs). However, Nd5 uses TAA in M. cabrerae and M. thomasi, and TAG in M. chrotorrhinus (Table 2).

The most abundant start and stop codons were ATG and TAA, respectively, a finding that agrees with previous work on other mammal mitogenomes [30,31,35,37,65,66]. Incomplete stop codons (T-- or TA-), like those used in three PCGs (Nd1, Cox3, and Nd4), are commonly observed in metazoan mitogenomes. They might be further completed by poly-adenylation of the 3′-end of the mRNA occurring after transcription, giving rise to the complete functional TAA stop codon [67,68].

The length of the 13 PCGs is the same in all three species and, consequently, in the coded proteins as well. The percentages of nucleotide identity range between 82.03%, observed when comparing the M. cabrereae and M. thomasi Nd2 gene sequences, and 89.71%, obtained when comparing the M. chrotorrhinus and M. thomasi Atp8 gene sequences (Table 2).

Due to the A+T richness of the Microtus mitogenomes, a strong bias toward A+T-rich codons in the codon usage of the PCGs was observed (

Table 3. Codon usage of mitochondrial genomes protein-coding genes (PCG) of the three Microtus species.

Codon	n	%	RSCU	Codon	n	%	RSCU
UUU(F)	69–92	1.8–2.4	0.58–0.79	UAU(Y)	53–56	1.4–1.5	0.87–0.91
UUC(F)	142–169	3.7–4.5	1.21–1.42	UAC(Y)	65–69	1.7–1.8	1.09–1.13
UUA(L)	84–105	2.2–2.8	0.86–1.10	UAA(*)	11–12	0.3	3.38–3.69
UUG(L)	15–20	0.4–0.5	0.15–0.20	UAG(*)	1–2	0–0.1	0.31–0.62
CUU(L)	82–90	2.2–2.4	0.85–0.92	CAU(H)	13–30	0.3–0.8	0.26–0.61
CUC(L)	79–100	2.1–2.6	0.82–1.02	CAC(H)	68–87	1.8–2.3	1.39–1.74
CUA(L)	251–254	6.6–6.7	2.58–2.62	CAA(Q)	66–67	1.7–1.8	1.72–1.74
CUG(L)	35–46	0.9–1.2	0.36–0.47	CAG(Q)	10–11	0.3	0.26–0.28
AUU(I)	142–178	3.7–4.7	0.77–1.00	AAU(N)	35–43	0.9–1.1	0.43–0.52
AUC(I)	178–229	4.7–6.0	1.00–1.24	AAC(N)	122–130	3.2–3.4	1.48–1.57
AUA(M)	164–173	4.3–4.6	1.48–1.59	AAA(K)	83–94	2.2–2.5	1.66–1.86
AUG(M)	45–60	1.2–1.6	0.41–0.53	AAG(K)	7–17	0.2–0.4	0.14–34
GUU(V)	22–33	0.6–0.9	0.54–0.73	GAU(D)	20–26	0.5–0.7	0.53–0.70
GUC(V)	46–50	1.2–1.3	1.01–1.22	GAC(D)	48–55	1.3–1.4	1.30–1.47
GUA(V)	73–86	1.9–2.3	1.74–1.82	GAA(E)	65–83	1.7–2.2	1.38–1.75
GUG(V)	19–24	0.5–0.6	0.46–0.53	GAG(E)	12–29	0.3–0.8	0.25–0.62
UCU(S)	37–38	1	0.76–0.80	UGU(C)	6–8	0.2	0.38–0.50
UCC(S)	62–76	1.6–2.0	1.30–1.56	UGC(C)	24–26	0.6–0.7	1.50–1.63
UCA(S)	117–122	3.1–3.2	2.40–2.55	UGA(W)	75–96	2–2.5	1.47–1.88
UCG(S)	6–12	0.2–0.3	0.13–0.25	UGG(W)	6–27	0.2–0.7	0.12–0.53
CCU(P)	33–50	0.9–1.3	0.64–1.01	CGU(R)	4–9	0.1–0.2	0.25–0.56
CCC(P)	34–56	0.9–1.5	0.68–1.09	CGC(R)	10–19	0.3–0.5	0.63–1.19
CCA(P)	99–114	2.6–3.0	1.95–2.22	CGA(R)	38–40	1–1.1	2.38–2.50
CCG(P)	2–17	0.1–0.4	0.04–0.33	CGG(R)	3–5	0.1	0.19–31
ACU(T)	55–58	1.4–1.5	0.69–0.75	AGU(S)	12–17	0.3–0.4	0.25–0.36
ACC(T)	87–94	2.3–2.5	1.12–1.19	AGC(S)	36–43	0.9–1.1	0.75–0.88
ACA(T)	153–159	4.0–4.2	1.97–2.01	AGA(*)	0	0	0
ACG(T)	7–12	0.2–0.3	0.09–0.15	AGG(*)	0	0	0
GCU(A)	39–52	1.0–1.4	0.60–0.80	GGU(G)	37–41	1–1.1	0.69–0.75
GCC(A)	116–134	3.1–3.5	1.81–2.05	GGC(G)	50–65	1.3–1.7	0.93–1.19
GCA(A)	78–82	2.1–2.2	1.19–1.28	GGA(G)	58–86	1.5–2.3	1.06–1.61
GCG(A)	8–11	0.2–0.3	0.12–0.17	GGG(G)	41–54	1.1–1.4	0.75–0.99

RSCU: relative synonymous codon usage. * Termination codon.

). The most frequently used codons are CTA (Leu), ATC (Ile), ATA (Met), ATT (Ile), TTC (Phe), ACA (Thr), AAC (Asn), TCA (Ser), CCA (Pro) and GCC (Ala) (Table 3). Accordingly, leucine (15.1–15.5%), isoleucine (8.9–9.7%), threonine (8.2–8.3%), serine (7.5–7.7%), alanine (6.8–6.9%), phenylalanine (6.2–6.3%), methionine (5.7–6.0%) and glycine (5.6–5.7%) are the most common amino acids found in the mitochondria protein sequences, comprising approximately 65% of the coded amino acids. The high proportion of A+T in PCGs appears to be a shared feature in mammal species [35,66]. For any amino acid, the relative synonymous codon usage value (RSCU) is equivalent to the number of times that a codon appears in a gene in relation to the number of expected occurrences under an assumption of equal codon usage. The six codons with the highest RSCU values described in the PCGs from Microtus are as follows: CTA(L) (2.58–2.62), TCA(S) (2.40–2.55), CGA(R) (2.38–2.50), ACA(T) (1.97–2.01), CCA(P) (1.95–2.22) and GCC(A) (1.81–2.05), three of which are A+T rich (Table 3).

3.4. rRNAs, tRNA Genes and Non-Coding Regions

The tRNA-Val is located between the rRNA genes 12S and 16S. rRNAs genes appeared flanked by tRNA-Phe and tRNA-Leu(UUR) (Table 1; Figure 1). The combined sizes of the two rRNA genes in M. cabrerae, M. chrotorrhinus and M. thomasi are 2518 bp, 2509 bp and 2515 bp, respectively. The combined sizes of the 22 tRNA genes in M. cabrerae, M. chrotorrhinus and M. thomasi are 1488 bp, 1500 bp and 1489 bp, respectively. The length of the tRNA genes ranged between 56 bp for tRNA-Hys in M. cabrerae to 75 bp for tRNA-Leu(UUR) in the three mitogenomes (Table 1).

Non-coding regions are important during replication and for the maintenance of the mitogenomes. These included the L-strand origin of replication (OL), intergenic spacers and the control region [69]. The three mitogenomes from Microtus species analyzed here have identical ORs of 35 bp. This region is located between tRNA-Asn and tRNA-Cys in the WANCY region, which refers to a cluster of five tRNA genes (tRNA-Trp, tRNA-Ala, tRNA-Asn, tRNA-Cys and tRNA-Tyr). The same organization is present in other Microtus and Arvicolinae species, as well as in most mammal species [31,37,38,57]. Hence, the OR from the mitogenomes of other Microtus species varies in length from 34 to 40 bp, while in most Arvicolinae species it is 34 bp in length and is highly conserved (Figure 2). Intergenic spacers were also found in the mitogenomes, with sizes in the range 1–11 bp (Table 1).

The D-loops in M. cabrerae, M. chrotorrhinus and M. thomasi are 930 bp, 903 bp and 896 bp in length, respectively, and are located between tRNA-Pro and tRNA-Phe (Table 1; Figure 1). In other Arvicolinae species, the length of this region ranges between 657 bp (M. ochrogaster) and 1089 bp (Myodes rufocanus). Control regions are the most clearly differentiated regions in the mitogenomes of the three Microtus species analyzed, with a nucleotide similarity of 80.67–82.09%. Similar values are observed when comparing the D-loop regions of different Microtus species (77.1–97.3%) and those from other Arvicolinae species (64.8–97.8%).

The control region includes three domains (Domains I, II and III). In Domain I, the extended termination-associated sequences (ETAS-1 and ETAS-2) were identified (Figure 3). The ETAS-1 sequence is better conserved than the ETAS-2 sequences in these species. Thus, pair-wise comparisons of ETAS-1 sequences showed a similarity of 89.83–96.6%, while the ETAS-2 sequence similarity was 67.3–76.9%.

The conserved sequence blocks CSB-1, CSB-2 and CSB-3 [70,71] were identified within Domain III. CSB-1 is the best-conserved block, having similarity values of 66.7–84% when comparing the three Microtus species with each other. CSB-2 and CSB-3 are less well conserved, with similarities of 52.6–68.4% and 54.1–68.0%, respectively (Figure 3). No repetitive DNA sequences were found to be present between CSB1 and CSB2 on the Microtus D-loop, as occurs in other mammal species [35]. Five other conserved sequence blocks (CSB-B, C, D, E and F) were identified in central Domain II [72], all of which are well conserved with nucleotide similarities of 78.95–100% (Figure 3).

3.5. Phylogenetic Analysis

The phylogenetic positions of the three analyzed species were assessed using Bayesian inference (Figure 4). All Microtus species are clustered in a well-supported clade that also includes Neodon and Lasiopodomys species. The Alexandromys species are grouped in the same branch with genera Neodon and Lasiopodomys, which have been considered as subgenera of Microtus genus, although phylogenetic relationships are not clearly established [20,24]. Proedromys is also close to this Microtus clade since it appears grouped as part of a well-supported node (posterior probability values = 1). The Microtus-Proedromys group is also associated with the clade of Myodes and Eothenomys (posterior probability values = 0.93). Finally, Dicrostonyx and Ondatra show a basal position. These results agree with the previously established phylogenetic relationships for these genera [73,74,75,76].

A number of conclusions can be drawn for the Microtus species analyzed. The two species from the Terricola group included here, M. (Terricola) thomasi and M. (Terricola) subterraneus, are closely associated with M. arvalis and M. rossiaemeridionalis. The close phylogenetic relationship between the Terricola and Microtus subgenera has been previously reported [20,29]. Although the two subgenera Aulacomys and Pedomys are not resolved the three North American species, M. chrotorrhinus, M. ochrogaster and M. richardsoni are grouped together. These Nearctic species fall within the phylogeny of Microtus, in line with previous studies [20], rather than being basal, as recently reported [29]. M. cabrerae and M. agrestis are grouped, which could support their inclusion in the subgenus Agricola as has been previously proposed [20]. Ostensibly, M. cabrerae and M. agrestis share certain unusual genetic features, including the presence of giant sex chromosomes, which could be regarded as additional proof of their phylogenetic proximity. However, several studies have clearly demonstrated that these enlarged sex chromosomes arose and evolved independently in the genus Microtus [4], and hence, their presence is not a robust criterion for the inclusion of M. cabrerae and M. agrestis in the same subgenus. A previous mitochondrial phylogenetic reconstruction obtained similar results, with M. cabrerae and M. agrestis clustered in the same clade, but the level of genetic divergence indicated that both species could be considered as members of two different subgenera (Agricola and Iberomys) [29]. The genus Iberomys, which is based on the description of archaic morphological characters, has been proposed, with only the species Microtus cabrerae [77,78]; however, no support is obtained for this genus [20].

The phylogenetic results obtained here with mitochondrial data need to be validated by the use of other nuclear markers because of the limitation inherent to mitogenomes, maternal inheritance, accelerated rates of substitution, introgression, effective population size and neutrality [79]. However, sequencing and characterization of mitogenomes from other species of the genus Microtus and closely related taxa from the subfamily Arvicolinae will help our understanding of the phylogenic relationships of this rodent species group and hence resolve some of the issues that remain open.

4. Conclusions

The complete mitogenomes of three Microtus species are described. Our results demonstrated that these mitogenomes have the organization and characteristics of the described mitogenomes of voles and mammalian species and contain 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes and a control region. We identified the conserved domain and the sequence-conserved blocks of the region control. Phylogenetic reconstructions reproduced the established phylogenetic relationships for all the Arvicolinae genera that were analyzed. Our results could be useful in future studies about the identifications and phylogeny of Arvicolidae species, especially of the genus Microtus.