Complete mitochondrial genome sequence of the Indian pipistrelle Pipistrellus coromandra (Vespertilioninae)

ABSTRACT

We characterized the complete mitogenome of Pipistrellus coromandra (Indian pipistrelle) for comparative analysis of mitogenomes and for resolving the phylogenetic relationship of four tribes in the subfamily Vespertilioninae. The mitogenome size of P. coromandra was 17,153 bp, with a control region and a typical set of 37 mitochondrial genes. The nucleotide composition of the P. coromandra mitogenome showed an AT bias with a nucleotide composition of 33.5% A, 30.7% T, 13.3% G, and 22.5% C. The mitochondrial protein-coding genes in P. coromandra use the standard start codon (ATN), two stop codons (TAA and AGA), and two incomplete stop codons (TA- and T--). The intertribal relationship of four tribes was highly resolved from the phylogenetic analysis of mitogenome sequences.

KEYWORDS:

• Pipistrellus coromandra
• Vespertilioninae
• mitogenome
• Pipistrellini

1. Introduction

Animal mitogenomes are generally 16–18-kb double-stranded circular molecules (Boore 1999; Jeon & Park 2015) that consist of 13 protein-coding genes (PCGs), two ribosomal RNA genes (12S rRNA and 16S rRNA), 22 transfer RNA genes (tRNAs), and a non-coding control region (CR) that contains signals required for replication and transcription (Wolstenholme 1992; Ruokonen & Kvist 2002; Gissi et al. 2008). Mitogenomes have been used as reliable tools in various phylogenetic and molecular evolution studies because of their special characteristics, such as uniparental inheritance, a unique genetic code, high mutation rates, and low number of recombination events. A few rapidly evolving regions (e.g. the CR) within the mitogenome have been used in phylogenetic analyses to investigate intraspecific relationships and relationships between closely related species (Kim et al. 2010), while genome-level analyses, which include base compositions, codon usages, gene order arrangements, and secondary structures of tRNA and rRNA genes, have been used to identify higher level phylogeny among taxa (Boore 1999; Lei et al. 2010). Thus, the generation and identification of characteristics of complete mitogenome sequences are useful when investigating phylogenetic relationships among taxa at higher levels.

The Chiroptera is the second largest order in mammals and includes about 1240 bat species (Schnitzler & Kalko 2001). Among bats, the family Vespertilionidae has over 300 species distributed worldwide and is the largest and most well-known family. Approximately, 240 species of this family have been placed in the subfamily Vespertilioninae (Simmons 2005). Although various studies have assessed the phylogenetic relationships within Vespertilioninae (Roehrs et al. 2010; Koubínová et al. 2013), intertribal relationships based on complete mitogenome sequences have not yet been determined.

In the present study, the complete mitogenome of the Indian pipistrelle, Pipistrellus coromandra (Vespertilioninae), was characterized, and compared with previously published Vespertilioninae mitogenomes. Based on the mitogenome DNA sequences, we investigated the intertribal relationships in the subfamily Vespertilioninae, including the tribes Pipistrellini, Vespertilionini, Plecotini, and Lasiurini.

2. Materials and methods

2.1. Sample collection and DNA extraction

P. coromandra is distributed throughout most of South Asia, parts of southern China, and much of mainland Southeast Asia. A wing membrane tissue sample was collected from a P. coromandra individual after it was caught in the academic research forests of the University Putra Malaysia (Malaysia) using a mist-net (Avinet, Dryden, NY, USA). The tissue sample was stored at −40°C until total genomic DNA extraction. Total genomic DNA was extracted from the wing membrane tissue sample using the DNeasy® Blood & Tissue Kit (Qiagen, Valencia, CA, USA), according to the manufacturer's protocols.

2.2. Primer design, PCR amplification, and DNA sequencing

For PCR amplification of the complete mitogenome of P. coromandra, we used 10 newly designed primer sets (Supplementary Table 1) from sequence-conserved regions on the multiple alignments of the complete mitogenomes of Vespertilio sinensis (NC024558) and Pipistrellus abramus (NC005436), which are available from GenBank (Nikaido et al. 2001; Yoon et al. 2014).

PCR amplification was performed in a final reaction volume of 25 μL, which contained 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 4 mM MgCl₂, 200 mM of each dNTP, 50 pmol of each primer, 2 U ExTaq polymerase, and 1 μL of DNA sample, under the following conditions: 94°C for 5 min (initial denaturation); then 94°C for 1 min (denaturation), 48–56°C for 30 s (annealing), and 72°C for 1 min (extension) for 35 cycles; with a final extension at 72°C for 10 min. The PCR products were resolved by electrophoresis in 1.0% agarose gel, and were extracted using a DNA Gel Extraction Kit (Qiagen, Valencia, CA, USA). The extracts were then sent to Biomedic Co. Ltd. (Bucheon, South Korea) for sequencing from both directions using a primer-walking strategy.

2.3. Genome annotation

The complete mitogenome of P. coromandra was annotated and characterized based on the V. sinensis and P. abramus mitogenomes, and then aligned with the other six Vespertilioninae species using the Clustal W program found in Geneious Pro 5.5.9 (Biomatters, Auckland, New Zealand). The 13 mitochondrial PCG sequences were translated into amino acid sequences using the vertebrate mitogenome genetic code. The tRNA scan-SE search server and the ARWEN web server with default parameters were used to identify the tRNA genes and potential stem-loop secondary structures within these tRNA genes (Lowe & Eddy 1997; Bernt et al. 2013). Nucleotide skewness was calculated according to the formulae: AT skew = [A − T]/[A + T] and GC skew = [G − C]/[G + C] (Lobry 1996). The tandem repeats were searched in the CR using the Tandem Repeats Finder program (Benson 1999).

2.4. Phylogenetic analysis

Phylogenetic relationships among the four tribes (Pipistrellini, Vespertilionini, Lasiurini, and Plecotini) were inferred using a concatenated nucleotide dataset (11,785 bp) of the mitochondrial 13 PCGs, two RNA genes (12S rRNA and 16S rRNA), and 22 tRNA genes except for the CR and the 3rd codon positions and stop codons of 13 PCGs, which are highly variable. Among the complete mitogenome sequences available from GenBank, we selected two mitogenome sequences from closely (Phyllostomidae: Artibeus lituratus) (NC016871) and more distantly (Rhinolophidae: Rhinolophus pumilus) (NC005434) related families to the family Vespertilionidae as outgroup (Agnarsson et al. 2011).

The phylogenetic relationships were inferred using Bayesian inference (BI) procedures in MrBayes version 3.2.2 program (Ronquist et al. 2012) and the maximum likelihood (ML) method in MEGA v.6.06 (Adachi & Hasegawa 1996), respectively. For the BI analysis, general time reversible model (GTR) + I + G was selected as the best substitution model under Akaike information criterion by Modetest 3.7 (Posada & Crandall 1998). The concatenated multi-gene dataset was partitioned as three parts of 13 PCGs, two rRNA genes (12S rRNA and 16S rRNA), and 22 tRNA genes. The BI analysis was performed with two simultaneous runs of two million generations and one out of every 500 generations was sampled. The first 25% of trees were discarded and the resulting trees were used to generate a majority consensus tree based on posterior probabilities. For the ML analysis, the GTR with gamma distributed invariant sites (G + I) was selected as the best model of evolution by the MEGA v 6.06. The number of discrete gamma categories was specified by the default. The ML heuristic method for tree search was the Nearest Neighbor Interchange starting with the initial tree selected automatically under the default NJ/BioNJ. The reliability of the phylogenetic tree was supported with 1000 bootstrap replications.

3. Results and discussion

3.1. Genome organization

The complete mitogenome (KP688404) of P. coromandra is a circular molecule that consists of a CR and a typical set of 37 vertebrate mitochondrial genes containing 13 PCGs, 22 tRNA genes, and 2 rRNA genes (12s rRNA and 16s rRNA) (Table 1). One Nd6 and eight tRNAs were transcribed from the light strand, while the other 12 PCGs, 14 tRNAs, and 2 rRNAs were located on the heavy strand. The gene order and orientation of the P. coromandra mitogenome were identical to those of all the other Vespertilioninae species (Figure 1).

Figure 1. Map of the P. coromandra mitogenome. Gray color indicates the PCG regions. Genes for tRNAs are designated by a single letter code for the corresponding amino acid. The heavy strand in the outer circle encodes 28 genes, whereas 9 genes are encoded in the light strand in the inner circle.

Table 1. Gene organization and characterization of P. coromandra mitogenome.

	Start position	Stop position	Length (bp)	Anticodon	Start codon	Stop codon	Strand
tRNA^Phe	1	73	73	GAA			+
12S rRNA	74	1029	956				+
tRNA^Val	1030	1097	68	TAC			+
16S rRNA	1097	2673	1577				+
tRNA^Leu ^(CUN)	2674	2749	76	TAA			+
Nd1	2755	3710	956		ATG	TA-	+
tRNA^Ile	3711	3779	69	GAT			+
tRNA^Gln	3777	3851	75	TTG			−
tRNA^Met	3852	3920	69	CAT			+
Nd2	3921	4962	1042		ATA	T--	+
tRNA^Trp	4963	5033	71	TCA			+
tRNA^Ala	5041	5109	69	TGC			−
tRNA^Asn	5110	5182	73	GTT			−
OR	5183	5217	35				+
tRNA^Cys	5215	5280	66	GCA			−
tRNA^Tyr	5281	5347	67	GTA			−
Cox1	5349	6893	1545		ATG	TAA	+
tRNA^Ser ^(UCN)	6897	6967	71	TGA			−
tRNA^Asp	6973	7039	67	GTC			+
Cox2	7040	7723	684		ATG	TAA	+
tRNA^Lys	7727	7793	67	TTT			+
ATP8	7795	7998	204		ATG	TAA	+
ATP6	7956	8636	681		ATG	TAA	+
Cox3	8636	9420	785		ATG	TA-	+
tRNA^Gly	9420	9488	69	TCC			+
Nd3	9489	9835	347		ATT	TA-	+
tRNA^Arg	9837	9906	70	TCG			+
Nd4L	9908	10204	297		ATG	TAA	+
Nd4	10198	11575	1378		ATG	T--	+
tRNA^His	11576	11644	69	GTG			+
tRNA^Ser ^(AGY)	11645	11704	60	GCT			+
tRNA^Leu ^(UUR)	11705	11773	69	TAG			+
Nd5	11774	13594	1821		ATA	TAA	+
Nd6	13578	14105	528		ATG	TAA	−
tRNA^Glu	14106	14174	69	TTC			−
Cytb	14180	15319	1140		ATG	AGA	+
tRNA^Thr	15320	15389	70	TGT			+
tRNA^Pro	15389	15455	67	TGG			−
D-loop	15456	17153	1698				+

The total P. coromandra mitogenome was 17,153 bp, which is similar in size to the other completely sequenced mitogenomes of the subfamily Vespertilioninae (Table 2). A greater size variation was observed in the CRs when compared with variations in the other mitochondrial gene regions. The variations in the CRs are mainly due to differences in the number of repeated motifs at the 3′-end of the CR.

Table 2. Size and nucleotide composition of the subfamily Vespertilioninae mitogenomes.

Species	Total length (bp)	Gene region				Non-gene region			Nucleotide composition (%) and skewness						Accession no.	References
		13 PCGs		two rRNAs	22 tRNAs	CR	OR	Intergenic spacers	A	T	G	C	AT skew	GC skew
		Size (bp)	No. of codons	Size (bp)	Size (bp)	Size (bp)	Size (bp)	Size (bp)
P. coromandra	17,153	11,408	3793	2533	1524	1698	35	32	33.5	30.7	13.3	22.5	0.04	−0.26	KP_688404	This study
P. abramus	16,976	11,409	3794	2518	1520	1536	35	35	33.6	30.1	13.2	23.0	0.06	−0.27	NC_005436	Nikaido et al. (2001)
V. sinensis	16,971	11,406	3794	2525	1521	1531	35	30	32.6	29.6	14.0	23.7	0.05	−0.26	NC_024558	Yoon et al. (2014)
C. tuberculatus	16,818	11,420	3796	2513	1501	1392	35	43	33.2	29.2	13.6	24.1	0.06	−0.28	NC_002626	Lin and Penny (2001)
P. auritus	16,900	11,406	3794	2521	1513	1464	35	37	32.8	28.7	14.3	24.2	0.07	−0.26	NC_015484	Unpublished
P. rafinesquii	16,438	11,404	3794	2509	1512	1013	31	41	32.1	26.2	15	26.8	0.09	−0.28	NC_016872	Meganathan et al. (2012)
L. borealis	17,048	11,407	3794	2535	1515	1591	32	42	32.7	31.3	14.2	21.8	0.02	−0.21	NC_016873	Meganathan et al. (2012)

3.2. Nucleotide composition

The mitogenome of P. coromandra showed AT bias with a nucleotide composition of 33.5% A, 30.7% T, 13.3% G, and 22.5% C, which is similar to that in the other Vespertilioninae mitogenomes (Table 2). As observed in the mitogenomes of other mammals (Reyes et al. 1998; Gibson et al. 2005), the Vespertilioninae mitogenomes, including that of P. coromandra, have a nucleotide composition order of A > T > C > G, which indicates that the AT content is higher than the GC content (Table 2). The AT skew of all the Vespertilioninae mitogenomes was positive and their GC skew was negative, respectively. The GC skew value was −0.26 for P. coromandra, Plecotus auritus, and V. sinensis mitogenomes, and their mitochondrial GC skew value was less negative than that (−0.28) for Chalinolobus tuberculatus and Plecotus rafinesquii. The asymmetrical base composition seen in the Vespertilioninae mitogenomes was probably due to the selection pressures such as mutational pressure (Sueoka 1962, 1994; Reyes et al. 1998; Hassanin et al. 2005; Castellana et al. 2011), purifying (Ruiz-Pesini et al. 2004; Castellana et al. 2011; Bi & Sokouri 2015), and adaptive selection (Ruiz-Pesini et al. 2004; da Fonseca et al. 2008; Castellana et al. 2011).

3.3. Codon usage and the sequence features of PCGs

The total length of the 13 mitochondrial PCGs in P. coromandra was 11,408 bp, which could be translated into 3793 amino acids (Table 2). It has been reported that mammalian mitogenomes contain some compact and overlapping regions between PCGs (Fernandez-Silva et al. 2003). We observed a 43 bp overlap between Atp8 and Atp6, a 17 bp overlap between Nd5 and Nd6, a 7 bp overlap between Nd4L and Nd4, and a single bp overlap between Atp6 and Cox3 (Table 1). Similar gene overlapping has been observed in other bat mitogenomes (Meganathan et al. 2012).

The 13 mitochondrial PCGs in P. coromandra use the standard start codon (ATN), two stop codons (TAA and AGA), and two incomplete stop codons (TA- and T--) for translation initiation and termination (Table 1). Ten PCGs used ATG as a start codon; the exceptions were Nd2 and Nd5 (ATA), and Nd3 (ATT). TAA was used as a stop codon in seven PCGs (Cox1, Cox2, Atp8, Atp6, Nd4L, Nd5, and Nd6). The stop codon AGA was used in Cytb and Nd2, and the incomplete stop codon (T--) was used in Nd4, which may be completed by poly-adenylation of the 3′-end of the mRNA after transcription (Anderson et al. 1981; Boore 1999; Yoon et al. 2014).

Loss of unnecessary genes, gene transfer to the nucleus, and reduction of genome size required for rapid organelle replication could account for the compactness of mitochondrial genomes (Selosse et al. 2001). Thus, incomplete stop codons or overlaps between genes may be a product of the selective pressure to reduce genome size in mitochondria (Rand 1993; Selosse et al. 2001).

Codon usage bias for the start and stop codons in the seven Vespertilioninae mitogenomes is shown in Figure 2 and Supplementary Table 2. The four different start codons (ATG, ATA, ATT, and ATC) were detected in all 13 Vespertilioninae mitogenome PCGs. ATG was the most common start codon, accounting for 77.78% of the overall 91 start codons and appearing in 10 PCGs (excluding Nd2, Nd3, and Nd5), whereas the rare start codon ATC was only used in the Nd2 of P. abramus and P. auritus. The three stop codons (TAA, AGA, and TAG) and the two incomplete stop codons (TA and T--) were detected in 13 Vespertilioninae mitogenome PCGs. The most common stop codon was TAA, which accounted for 53.85% of the 91 stop codons and appeared in Cox1, Cox2, Atp8, Atp6, Nd4L, Nd5, and Nd6. The rare stop codon AGA was only detected in Cytb. The codon usage pattern of the P. coromandra mitogenome, apart from the absence of the start codon ATC, was similar to the other Vespertilioninae mitogenomes.

Figure 2. Usage bias of the start and stop codons in the 13 mitochondrial PCGs of the seven Vespertilioninae species. The number in the parenthesis on each codon indicates the number of times the codon appears in the 13 mitochondrial PCGs of the seven Vespertilioninae species.

3.4. Ribosomal RNA and transfer RNA genes

The rRNA genes in the P. coromandra mitogenome were located between tRNA^Phe and tRNA^{Leu (CUN)} and separated by tRNA^Val (Figure 1). The combined size of the two P. coromandra rRNA genes was 2533 bp, which was longer than the other Vespertilioninae mitogenomes, with the exception of the Lasiurus borealis mitogenome (Table 2).

The 22 tRNA genes of the P. coromandra mitogenome were interspersed in the mitogenome, and the combined size of the 22 tRNA genes was 1524 bp, ranging from 60 bp in tRNA^{Ser (AGY)} to 76 bp in tRNA^{Leu (CUR)} (Table 1). Its size is a little longer than that of the other Vespertilioninae mitogenomes (Table 2). Most of the tRNA genes of P. coromandra mitogenome could be folded into the canonical cloverleaf secondary structure. The DHU arm had been deleted in the secondary structure of the tRNA^{Ser (AGY)}, which is thought to be common in the metazoan mitogenome (Lavrov et al. 2000; Kilpert & Podsiadlowski 2006).

3.5. Non-coding regions

The metazoan mitogenome contains some non-coding regions, such as the origin of replication (O_R), the CR, and some intergenic spacers. These non-coding regions are important during the replication and maintenance of the mitogenome (Annex & Williams 1990; Fernandez-Silva et al. 2003).

The mitochondrial O_R of P. coromandra is located between tRNA^Asn and tRNA^Cys in the WANCY region, which consists of a cluster of five tRNA genes (tRNA^Trp, tRNA^Ala, tRNA^Asn, tRNA^Cys, and tRNA^Tyr) (Figure 1), as in other vertebrates (Seutin et al. 1994). The size mitochondrial O_R in the Vespertilioninae, including P. coromandra, is generally 35 bp except for two species P. rafinesquii (31 bp) and L. borealis (32 bp), respectively (Table 2).

The CR of P. coromandra is located between tRNA^Pro and tRNA^Phe, and 1698 bp long (Figure 1). It is longer than that of the other Vespertilioninae bats, ranging from 1013 bp long in P. rafinesquii to 1591 bp long in L. borealis. In comparison with the other genes, the CR showed the higher variation in size, which is caused of repeated sequences.

Previous studies show the presence of termination-associated sequences, many complex repeat regions, and some conserved regions within the CR of bat mitogenomes (Wilkinson et al. 1997; Pumo et al. 1998). These features have been observed in the mitogenomes of all Vespertilioninae species.

Intergenic spacers were present at 10 sites in the P. coromandra mitogenome, and ranged from a single bp spacer (between tRNA^Tyr and Cox1, tRNA^Lys and Atp8, tRNA^Arg and Nd3, and tRNA^Arg and Nd4L) to a 7-bp spacer (between tRNA^trp and tRNA^Ala) (Table 1). In Vespertilioninae, the combined sizes of the mitochondrial intergenic spacers ranged from 30 bp in V. sinensis to 43 bp in C. tuberculatus. Such intergenic spacers have generally been observed in various metazoan mitogenomes, including mammalian mitogenomes (Fernandez-Silva et al. 2003).

3.6. Phylogenetic relationship

The monophyly of the subfamily Vespertilioninae is supported by high bootstrap values in the BI analysis, but by relatively lower bootstrap values in the ML analyses (Figure 3). The two Pipistrellus species, P. coromandra and P. abramus, are well grouped. The two species of the tribe Vespertilionini, V. sinensis and C. tuberculatus, also form a sister group. As in a previous study (Koubínová et al. 2013), the tribes Pipistrellini and Vespertilionini are sisters, which in turn are sisters to the tribe Lasiurini. The tribe Plecotini is basal to the other tribes. In the ML analysis, however, phylogenetic position of C. rafinesquii (Plecotini) and the sister relationship between the two species of the Vespertilionini (V. sinensis and C. tuberculatus) are unclear. Therefore, it is required inclusion of mitogenomes from further taxa for resolving the exact relationship between its members.

Figure 3. Higher phylogeny of the subfamily Vespertilioninae. The phylogenetic relationships among the four tribes (Pipistrellini, Vespertilionini, Lasiurini, and Plecotini) were inferred from concatenated nucleotide dataset of 13 mitochondrial PCGs, 2 rRNA genes, and 22 tRNA genes using Bayesian (BI) analysis and maximum likelihood (ML) method. Numbers on the nodes represent BI posterior probability/ML bootstrap value, respectively.

4. Conclusions

We sequenced the completed mitogenome of P. coromandra and characterized a typical set of 37 mitochondrial genes and a CR on the mitogenome. In the mitogenome comparison across seven species of the subfamily Vespertilioninae, the general features of the mitogenomes, including gene region size, base composition, and codon usage for translation initiation and termination, were relatively well conserved within Vespertilioninae. In the phylogenetic analysis, P. coromandra was placed within the tribe Pipistrellini, forming a sister clade to P. abramus. The phylogenetic relationship of the four tribes was relatively well resolved by the BI analysis based on the mitogenome sequences. However, since the current results depend on only seven mitogenome sequences, more mitogenome sequences need to be included for understanding higher phylogeny of the subfamily Vespertilioninae.

Acknowledgements

The authors would like to thank the anonymous reviewers for their valuable comments and suggestions to improve the quality of the paper.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was supported by a grant from National Research Foundation of Korea (NRF) Joint Research Program [grant number 2013K2A1A2055207].