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

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

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

4. 4289 genera 453 families 44 orders extant ray-finned fish 27,580 spp. The knowns and unknowns PaleozoicMesozoicCenozoic 1.When 2.Where 3.How ray-fiinned fishes have diversified during these 420 Myr ??? common ancestor The late Silurian 420 Mya

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

6. Four separate analyses: An overview Otocephala 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 6864 spp. Basal Actinopterygii Inoue et al. (2003) Mol. Phyl. Evol. 26: 110–120 Inoue et al. (2004) Mol. Phyl. Evol. 32: 274–286 10 spp. 34 spp. 217 spp. 801 spp. Basal Euteleostei Ishiguro et al. (2003) Mol. Phyl. Evol. 27: 476–481 322 spp. Higher Teleostei 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) 321 spp. 219 spp. 272 spp. 535 spp. 209 spp. 13,866 spp.

7. common ancestor The late Silurian 420 Mya PaleozoicMesozoicCenozoic 4289 genera 453 families 44 orders extant ray-finned fish 27,580 spp. Evolutionary Mitogenomics of Fishes (EMFish) 1.16,500 bp x 327 spp. 2.Phylogenetic analysis 3.Divergence time estimation 4.Macroevolutionary analysis

8. EMFish: Some statistics 906Number of species*** 1045Total number 88Other groups/individuals** 957Our research group 1. Number of whole mitogenome sequences* * As of 9 August 2007 ** From NCBI database *** Excluding duplicated species (3.7%)90624,618Species (79.3%)383482Family (18.3%)7784,257Genus 2. Taxonomic coverage of the data ProportionSeq**Numbers*Rank * Nelson (1994) ** Including unpubl. data

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

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

11. Fish-BOLNumber of “leaves”

12. EMFish Topology of “branches”

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

14. EMFishFish-BOL

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

17. Schematic figure of a cell Size (human)3.0 × 10 8 bp16,500 bp (redundant)(compact) Number of genes 30,00013 Mode of inheritanceMendelianmaternal (recombination)(no recombination) Mode of variations DNA substitutions/indels substitutions/indels Gene duplications/indels/translocations Chromosome same as above + inversion –– Genome polyploidy –– Homology assessment difficulteasy Evolutionary rateslow–fastmedium–fast Nuclear vs. mitochondrial genomes Features Nuc Mito Configuration linearcircular

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

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

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

21. A project summary—1. Published studies 1–4. Foundation of mitochondrial (phylo)genomics of fishes (10 published papers) 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

22. A project summary—1. (continued) 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.

23. 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 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 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 continued Continued — total, 54 papers cited >1000 times 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.

24. A project summary—2. Ongoing studies 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.)

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

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