The Mitochondrial Genome of Spotted Green Pufferfish Tetraodon Nigroviridis

Genes Genet. Syst. (2006) 81, p. 29–39 The mitochondrial genome of spotted green pufferfish Tetraodon nigroviridis (Teleostei: Tetraodontiformes) and divergence time estimation among model organisms in fishes Yusuke Yamanoue1*, Masaki Miya2, Jun G. Inoue1†, Keiichi Matsuura3, and Mutsumi Nishida1 1Ocean Research Institute, University of Tokyo, 1-15-1 Minamidai, Nakano-ku, Tokyo 164-8639, Japan 2Department of Zoology, Natural History Museum & Institute, Chiba, 955-2 Aoba-cho, Chuo-ku, Chiba 260-8682, Japan 3Department of Zoology, National Science Museum, 3-23-1 Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan (Received 12 November 2005, accepted 19 December 2005) We determined the whole mitochondrial genome sequence for spotted green pufferfish, Tetraodon nigroviridis (Teleostei: Tetraodontiformes). The genome (16,488 bp) contained 37 genes (two ribosomal RNA genes, 22 transfer RNA genes, and 13 protein-coding genes) plus control region as found in other vertebrates, with the gene order identical to that of typical vertebrates. The sequence was used to estimate phylogenetic relationships and divergence times among major lineages of fishes, including representative model organisms in fishes. We employed partitioned Bayesian approaches for these two analyses using two datasets that comprised concatenated amino acid sequences from 12 protein-coding genes (excluding the ND6 gene) and concatenated nucleotide sequences from the 12 protein-coding genes (without 3rd codon positions), 22 transfer RNA genes, and two ribosomal RNA genes. The resultant trees from the two datasets were well resolved and largely congruent with those from previous studies, with spotted green pufferfish being placed in a reasonable phylogenetic position. The approx- imate divergence times between spotted green pufferfish and model organisms in fishes were 85 million years ago (MYA) vs. torafugu, 183 MYA vs. three-spined stickleback, 191 MYA vs. medaka, and 324 MYA vs. zebrafish, all of which were about twice as old as the divergence times estimated by their earliest occurrences in fossil records. Key words: mitochondrial genome, Takifugu rubripes, fugu, partitioned Baye- sian analysis fishes have been sequenced and compared with the INTRODUCTION human and other vertebrates’ genomes. Torafugu (Tak- The human genome sequence was generated by the ifugu rubripes) became the second vertebrate organism to Human Genome Project (Venter et al., 2001), and using be sequenced to draft quality prior to tetrapods (Aparicio it we can identify and characterize human genes. Most et al., 2002). Other genome projects for several species genetic information that governs how humans develop of ray-finned fishes, such as spotted green pufferfish (Tet- and function is encoded in their genome sequence. Com- raodon nigroviridis), zebrafish (Danio rerio), medaka paring the genomes of different organisms will guide (Oryzias latipes), and three-spined stickleback (Gasteros- future approaches to understanding gene function, regu- teus aculeatus), are also ongoing. lation, and evolution. Recently, the genomes of several Although ray-finned fishes and humans diverged from their common ancestor as long as 450 MYA (Kumazawa Edited by Norihiro Okada * Corresponding author. E-mail: [email protected] et al., 1999; Hedges and Kumar, 2003), their genomes † Present address: School of Computational Science, Florida have essentially the same genes and regulatory State University, Tallahassee, FL32306 4120, USA sequences. Furthermore, some ray-finned fishes have 30 Y. YAMANOUE et al. great advantages over mammals for research purposes: many are small and easy to maintain and have short MATERIALS AND METHODS reproductive cycles. Among ray-finned fishes, puffer- fishes have a genome of approximately 400 Mb, and the Sample and DNA extraction. A portion of the epax- effort needed for sequencing to obtain a comparable ial musculature (about 0.25g) was excised from a fresh amount of information is modest by comparison with the specimen and immediately preserved in 99.5% ethanol. effort needed for mammalian genomes (2.0–5.0 Gb). The Total genomic DNA was extracted using a Qiagen spotted green pufferfish, Tetraodon nigroviridis, was pro- DNeasy tissue kit (Qiagen) following the manufacturer’s posed as a model organism for genomic studies (Crnogo- protocol. The voucher specimen was deposited at the rac-Jurcevic et al., 1997), and recently its draft sequence Department of Zoology, Natural History Museum & Insti- was published (Jaillon et al., 2004). tute, Chiba (CBM-ZF 10554). The correct interpretation of any kind of comparative biological data requires an evolutionary framework, PCR and sequencing. The mitochondrial genome of namely, well-supported phylogeny with precise diver- spotted green pufferfish was amplified in its entirety gence times. Compared with the relationships among using a long polymerase chain reaction (PCR) technique other vertebrate groups, relationships among major lin- (Cheng et al., 1994). Long PCR primers (Table 1) were eages of ray-finned fishes, comprising over half of all ver- used so as to amplify the entire mitochondrial genome in tebrate species, remain ill defined, and thus the proposed two reactions. The long-PCR products were diluted with relationships among model organisms in fishes have tra- TE buffer (1:19) for subsequent use as PCR templates. ditionally relied on morphology-based hypotheses. Only A total of 67 fish-versatile PCR primers and a species- recently, several efforts are underway that rely on exten- specific primer (Table 1) were used in various combina- sive taxonomic coverage of the diversity of ray-finned tions to amplify contiguous, overlapping segments of the fishes to estimate higher-level relationships (e.g., Miya et entire mitochondrial genome for spotted green pufferfish. al., 2003). There are a few studies on divergence time Long PCR and subsequent short PCR were carried out as estimation for ray-finned fishes, and the estimates based previously described (e.g., Miya and Nishida, 1999; Inoue on the earliest fossil records and molecular clock were et al., 2003). very different. Molecular clock estimates were much Double-stranded PCR products, purified using ExoSAP- older than fossil estimates; for example, fossil records IT (USB), were subsequently used for direct cycle have shown that the radiation of Perciformes dates back sequencing with dye-labeled terminators (Applied to the early Cenozoic (Benton, 1993, 2005), but a molecu- Biosystems). The primers used were the same as those lar clock estimate has placed it from the Jurassic to the for PCR. All sequencing reactions were performed early Cretaceous (Kumazawa et al., 1999). In studies on according to the manufacturer’s instructions. Labeled model organisms in fishes, the proposed divergence times fragments were analyzed using a Model 377 DNA have exclusively referred to the estimates based on the sequencer (Applied Biosystems). earliest fossil records (e.g., Volff et al., 2003; Chen et al., 2004). Alignments. We chose five model species for genome In this study, we determined the whole mitochondrial science (spotted green pufferfish, torafugu, three-spined genome sequence for the spotted green pufferfish, Tetra- stickleback, medaka, and zebrafish) and representatives odon nigroviridis, and analyzed its genome contents. from major lineages of fishes (total 21 species, Table Recent studies have used many genes to estimate diver- 2). Final rooting was done using the small-spotted cat- gence times in the hope of reducing the effect of rate vari- shark. ation (Nei and Glazko, 2002). We used the whole The DNA sequences were edited and analyzed with mitochondrial genome sequence to estimate the diver- EditView ver. 1.0.1, AutoAssembler ver. 2.1 (Applied Bio- gence times between the model organisms and major fish systems), and DNASIS ver. 3.2 (Hitachi Software Engi- lineages because they have been demonstrated in recent neering Co. Ltd.). The individual gene sequences for the studies to be useful for estimating the divergence times 21 species were aligned manually using DNASIS based on among basal lineages within tetrapods (Kumazawa et al., the previously aligned sequences of 48 teleosts (Miya et 2004), primates (Schrago and Russo, 2003), and actinop- al., 2001). Amino acids were used for alignments of the terygians (Inoue et al., 2005). The partitioned Bayesian protein-coding genes and secondary structure models approach, which does not assume constancy of evolution- (Kumazawa and Nishida, 1993) were used for the align- ary rates (Thorne and Kishino, 2002), was employed for ment of tRNA genes. Since strictly secondary-structure- estimation of divergence times because evolutionary rate based alignment for the two rRNA genes was impractical heterogeneity was observed in the mitochondrial genes for the large dataset, we instead employed machine align- among the species used in this study. ment, which would minimize erroneous assessment of the positional homology of the rRNA molecules. The two Mitochondrial genome of green pufferfish 31 Table 1 PCR and sequencing primers used in the analysis of spotted green pufferfish mitochondrial genome L primers Sequence (5´–3´) H primers Sequence (5´–3´) Long PCR primers L2508-16S CTC GGC AAA CAT AAG CCT CGC CTG TTT ACC S-LA-16S-H TGC ACC ATT RGG ATG TCC TGA TCC AAC AAA AAC ATC L12321-Leu GGT CTT AGG AAC CAA AAA CTC TTG GTG CAA H15149-CYB GGT GGC KCC TCA GAA GGA CAT TTG KCC TCA PCR and sequencing primers L708-12S TTA YAC ATG CAA GTN TCC GC H690-12S GCG

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