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Mitochondrial DNA, Early Online: 1–2 ! 2013 Informa UK Ltd. DOI: 10.3109/19401736.2013.809451

MITOGENOME ANNOUNCEMENTS Characterization of the mitochondrial genome of Rousettus leschenaulti

Michał Szczes´niak1, Misako Yoneda2, Hiroki Sato2, Izabela Makałowska1, Shigeru Kyuwa3, Sumio Sugano4, Yutaka Suzuki4, Wojciech Makałowski4,5, and Chieko Kai2

1Laboratory of Bioinformatics, Adam Mickiewicz University, Poznan, Poland, 2Institute of Medical Science, University of Tokyo, Tokyo, Japan, 3Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan, 4Department of Medical Genome Sciences, University of Tokyo, Chiba, Japan, and 5Institute of Bioinformatics, University of Muenster, Muenster, Germany

Abstract Keywords We present a complete sequence of mitochondrial genome of echolocating megabat Fruit bats phylogeny, next generation Rousettus leschenaulti (16,704 nt, GenBank record KC702803) and provide its annotation. sequencing, Rousettus leschenaulti We also show that phylogenetic analysis based on mitochondrial genomes of eighteen mitochondrion bat species, including R. leschenaulti, supports division of Chiroptera into Yinpterochiroptera and Yangochiroptera. History Received 23 May 2013 Accepted 24 May 2013 Published online 2 July 2013

Megabats or fruit bats constitute a monophyletic family We also show that phylogenetic analysis based on mitochondrial Pteropodidae whose members, unlike other chiropterans, do not genomes of eighteen bat species, including R. leschenaulti, use laryngeal echolocation. Instead, they rely on sight and supports division of Chiroptera into Yinpterochiroptera and olfaction to locate food with the exception of Rousettus genus Yangochiroptera (Figure 1). Next generation sequencing with that acquired an ability to use vocal echolocation. Traditionally, Illumina technology was performed on genomic DNA extracted

For personal use only. bats are divided into two suborders: Megachiroptera and from a spleen sample of a single R. leschenaulti specimen. In Microchiroptera. However, in recent years this morphology- total, 531,016 reads were obtained corresponding to 3200 based division has been re-examined and, based on growing times coverage. The extracted reads were assembled using molecular data, classification into Yinpterochiroptera and Platanus 1.0.0 software (Yokohama, Japan) resulting in two Yangochiroptera groups becomes widely accepted (Springer contigs. Sequence alignment with R. aegyptiacus mitochondrial et al., 2001). Yinpterochiroptera comprises Pteropodidae and genome showed that the missing fragment was located in a D-loop their close relatives – echolocating bats of the superfamily and almost entirely overlapped with a perfect repeat Rhinolophoidea, while Yangochiroptera represent the remaining (CATACACGTACG)23. Coordinates of protein coding genes, echolocating bats. The fact that the echolocating bats from transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), origin of Rhinolophoidea are closer related to megabats than to other replication and D-loop region were identified by sequence chiropterans seems to contradict morphological data and is similarity with R. aegyptiacus mitogenome. The genome encodes constantly a subject of scientific debate. thirty-seven genes, including thirteen protein coding genes, Mitochondrial DNA Downloaded from informahealthcare.com by University of Tokyo on 07/03/13 Here we present a complete sequence of mitochondrial twenty-two tRNAs and two rRNAs. Twelve protein-coding genome of echolocating megabat Rousettus leschenaulti (16 genes are encoded on heavy strand (H-strand) and only ND6 on 704 nt, GenBank record KC702803) and provide its annotation. light strand (L-strand). Also, a majority of tRNAs (fourteen)2013 and both rRNAs are coded on H-strand. The mitochondrial genome of animals contains non-coding regions such as origin of replication of light strand (OriL), D-loop and intergenic spacers, which are important for maintenance and replication of the genome (Fernandez-Silva et al., 2003, Hassanin et al., 2005). The OriL region is located between tRNAAsx and tRNACys genes and is thirty-three bases long. However, we were unable to determine the exact copy number of CATACACGTACG sequence within D-loop. Nevertheless, comparison with R. aegyptiacus suggests that there are twenty-three or twenty-four copies of the repeat. The genome exhibits a high level of similarity to mitogenome of R. aegyptiacus (99.7% sequence Correspondence: Chieko Kai, Laboratory Animal Research Center and identity), which is reflected in the cladogram (Figure 1). Although International Research Center for Infectious Diseases, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo morphological data seem to support microbat monophyly, 108-8639, Japan. Tel: +81-3-5449-5497. Fax: +81-3-5449-5379. E-mail: molecular studies provide a growing evidence for microbats [email protected] being paraphyletic and show a closer relationship of 2 M. Szczes´niak et al. Mitochondrial DNA, Early Online: 1–2

Figure 1. Maximum likelihood cladogram of eighteen chiropteran mitochondrial genomes.

Rhinolophoidea to megabats (Teeling et al., 2005, Van den References Bussche & Hoofer, 2004), which are referred to as Fernandez-Silva P, Enriquez JA, Montoya J. (2003). Replication and Yinpterochiroptera, while the rest of the bats belong to the transcription of mammalian mitochondrial DNA. Exp Physiol 88: suborder Yangochiroptera. Our analysis supports these findings 41–56. and clearly demarcates Yinpterochiroptera and Yangochiroptera Hassanin A, Leger N, Deutsch J. (2005). Evidence for multiple reversals within Chiroptera. of asymmetric mutational constraints during the evolution of the mitochondrial genome of Metazoa, and consequences for phylogenetic Acknowledgements inferences. Syst Biol 54:277–98. Springer MS, Teeling EC, Madsen O, Stanhope MJ, de Jong WW. (2001). For personal use only. We are grateful to Dr Noboru Manabe, Animal Resource Science Center, Integrated fossil and molecular data reconstruct bat echolocation. The University of Tokyo, for breeding animals and providing us the Proc Natl Acad Sci U S A 98:6241–6. tissues. Teeling EC, Springer MS, Madsen O, Bates P, O’brien SJ, Murphy WJ. (2005). A molecular phylogeny for bats illuminates biogeography and Declaration of interest the fossil record. Science 307:580–4. This study was supported by Grant-in-Aid from the Ministry of Van den Bussche RA, Hoofer SR. (2004). Phylogenetic relationships Education, Science, Culture, and Sports, Japan and partially by the among recent chiropteran families and the importance of choosing FP7-People-2009-IRSES Project ‘‘EVOLGEN’’ No. 247633. The authors appropriate out-group taxa. J Mammal 85:321–30. declare no conflict of interest. Mitochondrial DNA Downloaded from informahealthcare.com by University of Tokyo on 07/03/13