Short Report Mitochondrial genomes and phylogeny of the ocean sunfishes (Tetraodontiformes: Molidae) Yusuke Yamanoue 1,2* , Masaki Miya 3 , Keiichi Matsuura 2 , Masaya Katoh 4 , Harumi Sakai 5 , and Mutsumi Nishida 6 1 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan 2 Department of Zoology, National Science Museum, 3-23-1 Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan (e-mail: yamanoue@kahaku.go.jp) 3 Department of Zoology, Natural History Museum & Institute, Chiba, Chiba, Japan 4 Ishigaki Tropical Station, Seikai National Fisheries Research Institute, Fisheries Research Agency, Ishigaki, Japan 5 National Fisheries University, Department of Applied Aquabiology, Shimonoseki, Japan 6 Ocean Research Institute, University of Tokyo, Tokyo, Japan Received: November 6, 2003 / Revised: February 19, 2004 / Accepted: March 3, 2004 Abstract We determined the complete nucleotide sequences of the mitochondrial genomes for the three currently recognized species of ocean sunfish: Mola mola, Masturus lanceolatus, and Ranzania laevis (Tetraodontiformes: Molidae). Each genome contained the 37 genes as found in teleosts, with the typical gene order in teleosts. Bayesian, maximum-likelihood, and maximum-parsimony analyses were conducted with the data set comprising concatenated nucleotide sequences from 36 genes (excluding the ND6 gene) of three molids and four outgroups (three tetraodontiforms plus a caproid). The resultant trees supported monophyly of the Molidae and its intrarelationships ((Mola, Masturus), Ranzania), which were congruent with previous morphology-based hypotheses. Key words Molidae · Tetraodontiformes · Complete mtDNA sequence · Phylogeny · Ocean sunfishes The purpose of the present study was to corroborate the hypotheses of molid phylogeny using complete nucleotide sequences of mitochondrial (mt) genomes. Materials and Methods DNA extraction.—A portion of the epaxial musculature (ca. 0.25 g) was excised from fresh specimens of each species and immediately preserved in 99.5% ethanol. Total genomic DNA was extracted using the Qiagen DNeasy tissue kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The voucher specimens and/or tissues/extracted DNA samples were deposited at the Department of Zool- ogy, National Science Museum (NSMT; specimens), and Ocean Research Institute, University of Tokyo (ORIUT; tissue/DNA): Mola mola (ORIUT 910), Masturus lanceolatus (NSMT-P 63168, ORIUT 911), and Ranzania laevis (ORIUT 912). PCR and sequencing.—The mitochondrial genomes of the three molids were amplified in their entirety using a long polymerase cham reaction (PCR) technique (Cheng et al., 1994). Six fish-versatile long-PCR primers (L2508– 16S, L12321-Leu, H12293-Leu, H15149-CYB, H1065–12S, and S-LA-16S-H; for locations and sequences of these prim- ers, see Miya and Nishida, 1999) were used in various com- Ichthyological Research ©The Ichthyological Society of Japan 2004 Ichthyol Res (2004) 51: 269–273 DOI 10.1007/s10228-004-0218-6 T he members of the family Molidae, commonly called ocean sunfishes, are greatly modified puffers (Tetrao- dontiformes). The family comprises three monotypic genera, Mola, Masturus, and Ranzania (Tyler, 1980; Nelson, 1994), but Fraser-Brunner (1951) had a different view, recognizing five species in the three genera. Ocean sunfishes are pelagic and distributed in temperate and tropical waters all over the world. They share several unique anatomical features, of which the most striking is the lack of almost all osteological elements of the caudal fin structures, which are replaced by a pseudocaudal fin (Tyler, 1970, 1980). Various morphological aspects of ocean sunfishes have been investigated (osteology: Fraser-Brunner, 1951; Tyler, 1980; Santini and Tyler, 2002; myology: Winterbottom, 1974; development of early stages: Leis, 1977; fossils: Tyler and Bannikov, 1992; Uyeno and Sakamoto, 1994). As for the intrarelationships of the family, several authors (Bonaparte, 1841a,b; Gill, 1897; Fraser-Brunner, 1951; Tyler, 1980) hy- pothesized that Mola and Masturus are more closely related to each other than to Ranzania on the basis of similarity in morphology. Santini and Tyler (2002) provided the first cla- distic analysis of the intrarelationships of the Molidae using 48 osteological characters, confirming the previous hypoth- esis. Although morphological studies have reached a con- sensus regarding the molid intrarelationships, no molecular study has been conducted for their phylogeny.