Masaki Miya 1 and Mutsumi Nishida 2

1. Second International Barcode of Life Conference 18–20 September 2007 in Taipei Evolutionary History of Fishes: An Overview Based on >1000 Whole Mitochondrial Genome Sequences Masaki Miya1 and Mutsumi Nishida2 1 Natural History Museum and Institute, Chiba 2 Ocean Research Institute, University of Tokyo

2. Agnatha (108 spp.) Chondrichthyes (980 spp.) Mammal (4554 spp.) Actinopterygii (27,580 spp.) Aves (9924 spp.) 8.0% 17.5% 48.7% Sarcopterygii (27,971 spp.) 13.2% 10.6% Reptilia (7483 spp.) Amphibia (6002 spp.) “Basal Sarcopterygii” (8 spp.) Ray-finned fish in vertebrates

3. Agnatha Chondrichthyes Actinopterygii Sarcopterygii 420 Mya Origin of ray-finned fish

4. common ancestor extant ray-finned fish 27,580 spp. The late Silurian 420 Mya ??? • When • Where • How ray-fiinned fishes have diversified during these 420 Myr 4289 genera 453 families 44 orders Paleozoic Mesozoic Cenozoic The knowns and unknowns

5. 1. Development of the new method • Miya & Nishida (1999) Mar. Biotechnol. 1: 416–426 2. Use of mitogenomic data in phylogenetic analysis • Miya & Nishida (2000) Mol. Phylogenet. Evol. 17: 437–455 3. Ability of mitogenomic data for controversial issues • Inoue et al. (2001) Mol. Phylogenet. Evol. 20: 275–285 4. Gene rearrangement as a new phylogenetic marker • Inoue et al. (2000) J. Mol. Evol. 52: 311–320 Resolution of the higher-level relationships of ray-finned fish (Actinopterygii) A summary of the initial stage

6. 10 spp. Basal Actinopterygii 34 spp. • Inoue et al. (2003) Mol. Phyl. Evol. 26: 110–120 • Inoue et al. (2004) Mol. Phyl. Evol. 32: 274–286 217 spp. 801 spp. Otocephala 6864 spp. • Saitoh et al. (2003) J. Mol. Evol. 56: 464–472 • Ishiguro et al. (2005) J. Fish Biol. 67: 561–569 • Lavoué et al. (2005). Mol. Phyl. Evol. 37: 165–177 • Saitoh et al. (2006) J. Mol. Evol. 63: 826–841 • Lavoué et al. (2007). Mol. Phyl. Evol. 43:1096–1105 322 spp. 321 spp. Basal Euteleostei 219 spp. • Ishiguro et al. (2003) Mol. Phyl. Evol. 27: 476–481 272 spp. Higher Teleostei 535 spp. • Miya et al. (2001) Mol. Biol. Evol. 18: 1993–2009 • Miya et al. (2003) Mol. Phyl. Evol. 26: 121–138 • Miya et al. (2005) Biol. J. Linn. Soc. 85: 289–306 • Mabuchi et al. (2007) BMC Evol. Biol. 7: 10 • Kawahara et al. (2007) Mol. Phyl. Evol. (in press) 209 spp. 13,866 spp. Four separate analyses: An overview

7. common ancestor extant ray-finned fish 27,580 spp. The late Silurian 420 Mya 4289 genera 453 families 44 orders Paleozoic Mesozoic Cenozoic Evolutionary Mitogenomics of Fishes (EMFish) 16,500 bp x 327 spp. Phylogenetic analysis Divergence time estimation Macroevolutionary analysis

8. 1. Number of whole mitogenome sequences* Our research group 957 Other groups/individuals** 88 Total number 1045 * As of 9 August 2007 ** From NCBI database *** Excluding duplicated species Number of species*** 906 2. Taxonomic coverage of the data Rank Numbers* Seq** Proportion Family 482 383 (79.3%) Genus 4,257 778 (18.3%) * Nelson (1994) ** Including unpubl. data Species 24,618 906 (3.7%) EMFish: Some statistics

9. Fish-BOL extant ray-finned fish Correct estimation of genetic diversity (≈ species diversity) 27,580 spp. 655 bp x 4123 spp. = 2.7 Mb 4289 genera 453 families 44 orders Fish-BOL: character and taxon samplings

10. EMFish extant ray-finned fish Correct estimation of gene tree (≈ species phylogeny) 27,580 spp. 16,500 bp x 327 spp. = 5.4 Mb 4289 genera 453 families 44 orders EMFish: character and taxon samplings

11. Number of “leaves” Fish-BOL

12. EMFish Topology of “branches”

13. common ancestor extant ray-finned fish 27,580 spp. The late Silurian 420 Mya Divergence Time Rates of Diversification Paleozoic Mesozoic Cenozoic 4289 genera 453 families 44 orders New Insights into Evolutionary History New insights into evolutionary history Molecular Phylogeny

14. Fish-BOL EMFish

16. Second International Barcode of Life Conference 18–20 September 2007 in Taipei Evolutionary Genomics of Fishes: A New Perspective Based on Whole Mitochondrial Genome Sequences from >1000 species Masaki Miya1 & Mutsumi Nishida2 1Natural History Museum & Institute, Chiba 2Ocean Research Institute, University of Tokyo

17. Nuclear vs. mitochondrial genomes Features Nuc Mito Configuration linear circular Evolutionary rate slow–fast medium–fast Schematic figure of a cell Size (human) 3.0 × 108 bp 16,500 bp (redundant) (compact) Number of genes 30,000 13 Mode of inheritance Mendelian maternal (recombination) (no recombination) Mode of variations DNA substitutions/indels substitutions/indels Gene duplications/indels/translocations Chromosome same as above + inversion –– Genome polyploidy –– Homology assessment difficult easy

18. Long PCR product #1 Long PCR product #2 Development of the new method 1. Long PCRs 2. Full-nested short PCRs 3. Direct cycle sequencing Miya & Nishida (1999) Mar. Biotechnol. 1: 416–426

19. Sister of the Salmonidae Sister-group of the Salmonidae

20. Sister of the Salmonidae Sister-group of the Salmonidae

21. 5. Higher-level relationships of Actinopterygii • Miya et al. (2001) Mol. Biol. Evol. 18: 1993–2009. First attempt at resolving higher teleostean relationships • Inoue et al. (2003) Mol. Phylogenet. Evol. 26: 110–120 . Basal actinopterygian relationships • Miya et al. (2003) Mol. Phylogenet. Evol. 26: 121–138. Analysis of 100 mitogenomes of higher teleosts • Saitoh et al. (2003) J. Mol. Evol. 56: 464–472. Ostariophysan phylogeny and evolution • Ishiguro et al. (2003) Mol. Phylogenet. Evol. 27: 476–488. Basal euteleostean relationships • Inoue et al. (2004) Mol. Phylogenet. Evol. 32: 274–286. Mitogenomic evidence for the elopomorph monophyly • Lavoué et al. (2005) Mol. Phylogenet. Evol.37: 165–177. Gonorynchiform phylogeny within the Otocelphala • Ishiguro et al. (2005) J. Fish Biol. 67: 561–569. Phylogenetic position of Sundasalangidae • Miya et al. (2005) Biol. J. Linn. Soc. 85: 289–306. Phylogenetic position of toadfish in higher actinopterygians A project summary—1. Published studies 1–4. Foundation of mitochondrial (phylo)genomics of fishes (10 published papers)

22. 5. Higher-level relationships of Actinopterygii (continued) • Saitoh et al. (2006) J. Mol. Evol. 63: 826–841. Higher-level relationships of the Cypriniformes. • Mabuchi et al. (2007) BMC Evol. Biol. 7: 10. Polyphyly of the Labroidei. • Lavoué et al. (2007) Mol. Phylogenet. Evol. 43: 1096–1105. Higher-level relationships of the Clupeiformes. • Yamanoue et al. (2007) Mol. Phylogenet. Evol. (In press.) Phylogenetic position of Tetraodontiformes. • Miya et al. (2007) Ichthyol. Res. 54 (In press.) Phylogenetic position of Stylephoridae. • Kawahara et al. (2007) Mol. Phylogenet. Evol. (In press.) Polyphyly of the Gasterosteiformes. A project summary—1. (continued)

23. continued 6. Species level phylogeny • Yamanoue et al. (2004) Ichthyol. Res. 51: 269–273. Relationships of ocean sunfishes • Minegishi et al. (2004) Mol. Phylogenet. Evol. 34: 134–146. Phylogeny and evolution of freshwater eel genus Anguilla 7. Molecular biology and evolution • Inoue et al. (2003) Mol. Biol. Evol. 20: 1911–1924. Evolution of the gulper eel mitogenomes and analysis of lower teleostean phylogeny • Mabuchi et al. (2004) J. Mol. Evol. 59: 287–297. Evolution of pseudogenes in the scarid mitogenomes 8. Theoretical issues in molecular systematics • Simmon et al. (2004) Mol. Biol. Evol. 21: 188–199. How meaningful are posterior probabilities in Bayesian analysis? • Simmon & Miya (2004) Mol. Phylogenet. Evol. 31: 351–362. Efficient resolution of higher-level relationships; taxonomic sampling vs. character sampling 9. Divergence time estimation • Inoue et al. (2005) Gene 349: 227–235. Divergence time of the two coelacanths • Yamanoue et al. (2006) Gene Genetic Syst. 81:29–39. Continued — total, 54 papers cited >1000 times

24. 1. Global analysis of phylogeny and evolution • Elasmobranch relationships based on 31 whole mitochondrial genome sequences (Shirai et al.) • Actinopterygian phylogeny and evolution based on 327 whole mitochondrial genome sequences (Miya et al.) • Evolution of mitochondrial genomes of fishes based on the data from 250 species (Satoh et al.) 2. Local analysis of phylogeny and evolution Osteoglossomorpha (Lavoué et al.); Anguilliformes (Inoue et al.); Clupeiformes (Lavoué et al); Cypriniformes (Miya et al.); Characiphysi (Nakatani et al.); Aulopiformes (Kawaguchi et al.); Lophiiformes (Miya et al.); Gadiformes (Satoh et al.); Pleuronectiformes (Suzuki et al.); 3. Species/population level phylogeny and evolution • Natural selection along temperature gradient in two populations of Japanese medaka (Mukai et al.) • Patterns of variation in Japanese flounder mitogenomes (Shigenobu et al.) A project summary—2. Ongoing studies

25. Top page BLAST homology search Taxonomic search A project summary—3. Database

26. AToL Cypriniformes Tree of Life (CToL) Earth’s Most Diverse Clade of Freshwater Fishes • Grant proposal accepted for “Assembling the Tree of Life” project from NSF in August 2004 • Five years project beginning from September 2004 • PIs: R. L. Mayden and six US members with 16 scientists participated (including M. Miya and K. Saitoh) from 12 countries Phylogeny of the Cypriniformes Mitogenomic Resolution of a Big Phylogeny Cypriniformes 350 genera, 3285 spp. • Grant proposal accepted for Grants-in-Aid (Scien-tific Research A) from JSPS on 18 April 2005 Family Tribe • Four years project beginning from April 2005 Genus Species • PI: M. Miya; Co-PIs: M. Nishida and K. Saitoh International collaboration