ELECTORONIC SUPPLEMENTARY MATERIALS
1. MATERIALS & METHODS
TAXONOMIC SAMPLING
In addition to 54 fish species used in Azuma et al. (2008), 22 atherinomorphs (including 14
medakas) were newly added in this study, making a total number of species analyzed 76 (table
S1). The 14 medakas included all of the three known species groups (celebensis, javanicus
and latipes species groups), with the latter comprising two species (Oryzias luzonensis and O.
latipes) and four regional populations of O. latipes (Shanghai, South Korea, southern and
northern Japanese populations).
SPECIMENS AND DNA EXTRACTION
A portion of epaxial musculature (ca. 0.25 g) from fresh specimens of each species was
excised and the tissue immediately preserved in 99.5% ethanol. Total genomic DNA from the
ethanol-preserved tissue was extracted using DNeasy (Qiagen) or Aquapure genomic DNA
isolation kit (Bio-Rad Laboratories, Inc.) following manufacturer’s protocols.
PCR AND SEQUENCINGS
Whole mitogenomes of the eight medaka species were amplified in their entirety using a long
PCR technique (Cheng et al. 1994). Seven fish-versatile PCR primers for the long PCR were
used in the following four combinations: L2508-16S + H12293-Leu; L2508-16S +
H15149-CYB; L8343-Lys + H1065-12S; and L12321-Leu + S-LA-16S-H (for locations and
sequences of these primers, see Inoue et al. 2000, 2001; Ishiguro et al. 2001, Kawaguchi et al.
2001; Miya & Nishida 2000) to amplify the entire mitogenome in two reactions. Long PCR
reaction conditions followed Miya and Nishida (1999). Long PCR products diluted with TE
buffer (1:19) were subsequently used as templates for short PCR reactions employing
fish-versatile PCR primers in various combinations to amplify contiguous, overlapping
segments of the entire mitogenome. The short PCR reactions were carried out following
protocols previously described (Miya and Nishida 1999), then purified using Exosap-IT
enzyme (GE Healthcare Bio-Sciences Corp.), and subsequently sequenced with dye-labeled
terminators (BigDye terminator ver. 1.1/3.1, Applied Biosystems) and the primers used in the
short PCRs. Sequencing reactions were conducted following the manufacturer’s instructions,
followed by electrophoresis on ABI Prism 3100 or 3130 DNA sequencers (Applied
Biosystems). A list of PCR primers used in this study is available from MM upon request.
SEQUENCE EDITING AND ALIGNMENT
Each sequence electropherogram was edited with EditView (ver. 1.01; Applied Biosystems)
and the multiple sequences were concatenated using AutoAssembler (ver. 2.1; Applied
Biosystems). The concatenated sequences were carefully checked and annotated using
DNASIS (ver. 3.2; Hitachi Software Engineering) and a sequence file was created for each
gene.
1
Mitogenome sequences from the 22 atherinomorphs were concatenated with the
pre-aligned sequences used in Azuma et al. (2008) in a FASTA format, which was subjected to
multiple alignment using MAFFT ver. 6 (Katoh & Toh 2008). The aligned sequences were
imported into MacClade ver. 4.08 (Maddison & Maddison 2000) and the resulting gaps in the
pre-aligned sequences were manually removed to reproduce the alignment used in Azuma et
al. (2008). The dataset comprises 6966 positions from first and second codon positions of the
12 protein-coding genes (excluding ND6 gene), 1673 positions from the two rRNA genes and
1407 positions from the 22 tRNA genes (total 10,046 positions). The third codon positions of
the protein-coding genes were entirely excluded because of their extremely accelerated rates
of changes that may cause high level of homoplasy (Miya & Nishida 2000) and
overestimation of divergence time (Benton & Ayala 2003).
PHYLOGENETIC ANALYSIS
Unambiguously aligned sequences were divided into four partitions (first, second codon
positions, rRNA and tRNA genes) and subjected to the partitioned maximum-likelihood (ML)
analysis using RAxML ver. 7.0.4 (Stamatakis 2006). General time reversible model with sites
following a discrete gamma distribution (GTR + ; the model recommended by the author)
was used and a rapid bootstrap (BS) analysis was conducted with 1000 replications (–f a
option). This option performs BS analysis using GTRCAT, which is GTR approximation with
optimization of individual per-site substitution rates, and classification of those individual
rates into certain number of rate categories. After implementing the BS analysis, the program
uses every fifth BS tree as a starting point to search for ML tree using GTR +  model of
sequence evolution to obtain more stable likelihood values.
DIVERGENCE TIME ESTIMATION
A relaxed molecular-clock method for dating analysis developed by Thorne and Kishino
(2002) was used to estimate divergence times. This method accommodates unlinked rate
variation across different loci (“partitions” in this study), allows the use of time constraints on
multiple divergences, and uses a Bayesian MCMC approach to approximate the posterior
distribution of divergence times and rates based on a single tree topology estimated from the
other method (ML tree in this study). A series of software in a program package
multidistribute (v9/25/2003) was used for these analyses.
Baseml in PAML ver. 3.14 was used to estimate model parameters for each partition
separately under the F84 +  model of sequence evolution (the most parameter-rich model
implemented in multidistribute). Based on the outputs from baseml, branch lengths and the
variance-covariance matrix were estimated using estbranches in multidistribute for each
partition. Finally multidivtime in multidistribute was used to perform Bayesian MCMC
analyses to approximate the posterior distribution of substitution rates, divergence times, and
95% credible intervals. In this step, multidivtime uses estimated branch lengths and the
variance-covariance matrices from all partitions without information from the aligned
sequences.
MCMC approximation with a burnin period of 100,000 cycles was obtained and every
100 cycles taken until a total of samples reaching 10,000. To diagnose possible failure of the
2
Markov chains to converge to their stationary distribution, at least two replicate MCMC runs
were performed with two different random seeds for each analysis.
Application of multidivtime requires values for the mean of the prior distribution for the
time separating the ingroup root from the present (rttm) and its standard deviation (rttmsd) and
we set conservative estimates of 4.45 (= 445 Mya) and 4.45 SD, respectively. The tip-root
branch lengths were calculated using TreeStat v. 1.1 for all terminals and their average was
divided by rttm (4.45) to estimate rate of the root node (rtrate) and its standard deviation
(rtratesd), which were set to 0.074 and 0.074, respectively. The priors for the mean of the
Brownian motion constant, brownmean and brownsd, were both set to 0.5, specifying a
relatively flexible prior.
The multidivtime program allows for both minimum (lower) and maximum (upper) time
constraints and it has been argued that multiple calibration points would provide overall more
realistic divergence time estimates. We therefore sought to obtain an optimal phylogenetic
coverage of calibration points across our tree, although we could set maximum constraints
based on fossil records only for the three basal splits between Sarcopterygii and
Actinopterygii, Polypteriformes and Actinopteri, Acipenseriformes and Neopterygii (A–C in
figure 1; table S2). We also set lower and upper time constraints for three nodes in cichlids
divergence, which show excellent congruities with Gondwanan continental fragmentations,
assuming that they have never dispersed across oceans. Accordingly we set a total of 27 time
constrains based on both fossil record and biogeographic events as shown in figure 1 and table
S2.
2. RESULTS
GENOME ORGANIZATION
The whole mitogenome sequences from the eight medaka species reported here for the first
time were registered in DDBJ/EMBL/GenBank (table S1 in ESM). The genome contents
(including 13 protein-coding, two rRNA and 22 tRNA genes and the control region) and gene
orders were identical to those of typical vertebrates.
3. ACKNOWLEDGMENTS
We thank Y. Azuma, Y. Yamanoue and other members of Marine Molecular Biology
Laboratory, Ocean Research Institute, The University of Tokyo, for their invaluable advice
and discussions. Sincere thanks are also go to J.L. Thorne for his advice in performing
multidivtime analysis.
4. REFRENCES
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Inoue, J.G.., Miya, M., Tsukamoto, K. & Nishida, M. 2000 Complete mitochondrial DNA
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Ishiguro, N.B., Miya, M. & Nishida, M. 2001 Complete mitochondrial DNA sequence of ayu,
Plecoglossus altivelis. Fish. Sci. 67, 474–481. (doi: 10.1046/j.1444-2906.2001.00283.x)
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Katoh, K. & Toh, H. 2008 Recent developments in the MAFFT multiple sequence alignment
program. Briefings Bioinformat. 9, 286–298. (doi:10.1093/bib/bbn013)
Kawaguchi, A., Miya, M. & Nishida, M. 2001 Complete mitochondrial DNA sequence of
Aulopus japonicus (Teleostei: Aulopiformes), a basal Eurypterygii: longer DNA sequences
and higher-level relationships. Ichthyol. Res. 48, 213–223. (doi:
10.1007/s10228-001-8139-0)
Maddison, W.P. & Maddison, D.R. 2000 MacClade version 4. Sunderland, Massachusetts:
Sinauer Associates.
Masters, J.C., de Wit, M.J. & Asher, R.J. 2006 Reconciling the origins of Africa, India and
Madagascar with vertebrate dispersal scenarios. Folia Primatol. 77, 399–418. (doi:
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early radiation of the pikes in North American fresh waters. J. Paleontol. 66, 839–846.
Yamanoue, Y., Miya, M., Inoue, J.G., Matsuura, K. & Nishida, M. 2006 The mitochondrial
genome of spotted green pufferfish Tetraodon nigroviridis (Teleostei: Tetraodontiformes)
and divergence time estimation among model organisms in fishes. Gene Genet. Syst. 81,
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5
Supplementary Table S1. List of species used in this study with DDBJ/GenBank/EMBL
accession numbers. Taxonomic treatment of species of the family Adrianichthyidae follows
Parenti (2008)
—————————————————————————————————————
Order
Family
Species
Accession No.
—————————————————————————————————————
Outgroups (sharks)
Carcharhiniformes
Scyliorhinidae
Scyliorhinus canicula
Y16067
Triakidae
Mustelus manazo
AB015962
Ingroups (lobe-finned fishes)
Coelacanthiformes
Latimeriidae
Ceratodontiformes
Ceratodontidae
Ingroups (ray-finned fishes)
Polypteriformes
Polypteridae
Acipenseriformes
Acipenseridae
Lepisosteiformes
Polyodontidae
Lepisosteidae
Amiiformes
Hiodontiformes
Osteoglossiformes
Amiidae
Hiodontidae
Osteoglossidae
Albuliformes
Anguilliformes
Cypriniformes
Notacanthidae
Anguillidae
Muraenidae
Congridae
Engraulidae
Clupeidae
Cyprinidae
Salmoniformes
Balitoridae
Salmonidae
Clupeiformes
Esociformes
Aulopiformes
Polymixiiformes
Gadiformes
Atheriniformes
Esocidae
Chlorophthalmidae
Polymixiidae
Gadidae
Atherinopsidae
Melanotaenidae
Latimeria menadoensis
Neoceratodus forsteri
AP006858
AJ584642
Polypterus ornatipinnis
AP004351
Polypterus senegalus senegalus AP004352
Erpetoichthys calabaricus
AP004350
Acipenser transmontanus
AB042837
Scaphirhynchus cf. albus
AP004354
Polyodon spathula
AP004353
Lepisosteus oculatus
AB042861
Atractosteus spatula
AP004355
Amia calva
AB042952
Hiodon alosoides
AP004356
Osteoglossum bicirrhosum
AB043025
Pantodon buchholzi
AB043068
Notacanthus chemnitzi
AP002975
Anguilla japonica
AB038556
Gymnothorax kidako
AP002976
Conger myriaster
AB038381
Engraulis japonicus
AB040676
Sardinops melanostictus
AB032554
Cyprinus carpio
X61010
Danio rerio
AC024175
Crossostoma lacustre
M91245
Coregonus lavaretus
AB034824
Salmo salar
U12143
Oncorhynchus mykiss
L29771
Esox lucius
AP004103
Chlorophthalmus agassizi
AP002918
Polymixia japonica
AB034826
Gadus morhua
X99772
Menidia menidia
AB370893
Melanotaenia lacustris
AP004419
6
Cyprinodontiformes
Beloniformes
Beryciformes
Gasterosteiformes
Scorpaeniformes
Perciformes
Notocheilidae
Iso hawaiiensis
Aplocheilidae
Aplocheilus panchax
Goodeidae
Xenotoca eiseni
Cyprinodontidae
Jordanella floridae
Scomberesocidae
Cololabis saira
Exocoetidae
Exocoetus volitans
Hemiramphidae
Hyporhamphus sajori
Adrianichthyidae
Oryzias latipes group
Oryzias luzonensis
Southern Japanese populations
Oryzias latipes (Hd-rR)
Oryzias latipes (Nago)
Oryzias latipes
Northern Japanese populations
Oryzias latipes (HNI)
Oryzias latipes (Hirosaki)
China and West Korean populations
Oryzias latipes (SOK)
Oryzias latipes (Shanghai)
Oryzias javanicus group
Oryzias javanicus
Oryzias minutillus
Oryzias dancena
Oryzias celebensis group
Oryzias celebensis
Oryzias marmoratus
Oryzias sarasinorum
Berycidae
Beryx splendens
Holocentridae
Sargocentron rubrum
Gasterosteidae
Gasterosteus aculeatus
Scorpaenidae
Helicolenus hilgendorfi
Cichlidae
Oreochromis sp.
Neolamprologus brichardi
Tropheus duboisi
Astronotus ocellatus
Paretroplus maculatus
Etroplus maculatus
Hypselecara temporalis
Ptychochromoides katria
Paratilapia polleni
Tylochromis polylepis
Pomacentridae
Abudefduf vaigiensis
7
AB373006
AB373005
AP006777
AP006778
AP002932
AP002933
AB370892
AB498064
AB498065
AP008946
AP004421
AB498066
AP008941
AP008947
AP008948
AB498067
AB498068
AB498069
AB498070
AP005981
AB370891
AP002939
AP004432
AP002944
AP002948
AP009126
AP006014
AP006015
AP009127
AP009504
AP009505
AP009506
AP009507
AP009508
AP009509
AP006016
Amphiprion ocellaris
AP006017
Labridae
Pseudolabrus sieboldi
AP006019
Halichoeres melanurus
AP006018
Pleuronectiformes
Paralichthyidae
Paralichthys olivaceus
AB028664
Tetraodontiformes
Tetraodontidae
Takifugu rubripes
AJ421455
Tetraodon nigroviridis
AP006046
—————————————————————————————————————
8
Supplementary Table S2. Maximum (U) and minimum (L) time constrains (Ma) used for
dating at nodes in figure S2
—————————————————————————————————————
Node Constraints
Calibration information
—————————————————————————————————————
A
U 472
The minimum age for the basal split of bony fish based on the earliest
known acanthodian remains from Late Ordovician (Janvier 1996)
L 419
The †Psarolepis fossil (sarcopterygian; Zhu et al. 2006) from
Ludlow (Silurian) (Hurley et al. 2007)
U 419
The minimum age for the Sarcopterygii/Actinopterygii split
L 392
The †Moythomasia fossil (actinopteran) from the Givetian/Eifelian
boundary (Hurley et al. 2007)
U 392
The minimum age for the Polypteriformes/Actinopteri split
L 345
The †Cosmoptychius fossil (neopterygian or actinopteran) from
Tournasian (Hurley et al. 2007)
D
L 130
The †Protopsephurus fossil (Polyodontidae) from Hauterivian
(Cretaceous) (Hurley et al. 2007)
E
L 284
The †Brachydegma fossil (stem amiids) from Artinskian (Permian)
(Hurley et al. 2007)
F
L 136
The †Yanbiania fossil (Hiodontidae) from the Lower Cretaceous
(Hurley et al. 2007)
G
L 112
The †Laeliichthys fossil (Osteoglossidae) from the Aptian
(Cretaceous) (Patterson 1993)
H
L 151
The †Anaethalion, †Elopsomolos, and †Eoprotelops fossil
(Elopomorpha) from Kimmeridgian (Jurassic) (Hurley et al. 2007)
I
L 94
The †Lebonichthys (Albulidae) fossil from the Cenomanian
(Cretaceous) (Patterson 1993)
J
L 49
The Conger (Congridae) and Anguilla (Anguillidae) fossils from the
Ypresian (Tertiary) (Patterson 1993)
K
L 146
The †Tischlingerichthys fossil (Ostariophysi) from Tithonian
(Jurassic) (Hurley et al. 2007)
L
L 56
The †Knightia fossil (Clupeidae) from the Thanetian (Tertiary)
(Patterson 1993)
M
L 49
The †Parabarbus fossil (Cyprinidae) from the Ypresian (Tertiary)
(Patterson 1993)
N
L 74
The †Esteseox foxi fossil (Esociformes) from the Campanian
(Cretaceous) (Wilson et al. 1992)
O
L 94
The †Berycopsis fossil (Polymixiidae) from the Cenomanian
B
C
9
(Cretaceous) (Patterson 1993)
P
L 50
The pleuronectiform fossil from the Ypresian (Tertiary) (Patterson
1993)
Q
L 98
The tetraodontiform fossil from the Cenomanian (Tyler & Sorbini
1996)
R
L 32
The estimated divergence time between Takifugu and Tetraodon
(Benton and Donoghue 2007)
S
U 95
L 85
The upper and lower bounds of separation between Madagascar and
Indian (Smith et al. 1994; Storey 1995)
T
U 145
L 112
The upper and lower bounds of separation between
Indo-Madagascar landmass and Gondwanaland (Smith et al. 1994;
Storey 1995; Masters et al. 2006)
U
U 120
L 100
The upper and lower bounds of separation between African and L
South American landmasses (Smith et al. 1994; Storey 1995)
—————————————————————————————————————
10
Supplementary Table S3. Comparisons of divergence time estimates between the present
study and previous studies
—————————————————————————————————————
Node
This study
Azuma et al. Yamanoue et al.
(2008)*
(2006)
—————————————————————————————————————
Sarcopterygii vs. Actinopterygii
428 (419–442) 429 (417–449) 470 (415–524)
Teleostei vs. Neopterygii
364 (346–378) 365 (348–378) 390 (340–442)
Euteleostei vs. Otocephala
289 (269–310) 288 (268–307) 315 (270–363)
Cyprinus vs. Danio
153 (125–183) 147 (120–174) 167 (131–208)
Acanthopterygii vs. Paracanthopterygii 209 (191–225) 207 (190–224) 223 (191–264)
Percomorpha vs. Berycomorpha
200 (185–217) 198 (183–215) 206 (174–245)
Oryzias vs. Tetraodontiformes
180 (166–195) 176 (163–191) 184 (154–221)
Oryzias vs. Cichlidae
150 (139–161) 152 (141–165)
——
Gasterosteus vs. Tetraodontidae
173 (159–189) 170 (156–185) 192 (153–235)
Takifugu vs. Tetraodon
78 (63–93)
78 (65–93)
73 (57–94)
—————————————————————————————————————
* Estimated with biogeography-based time constraints on cichlid divergence
11
FIGURE LEGEND
Figure S1. Maximum likelihood tree from analysis of whole mitogenome sequences (10,046
positions excluding third codon positions) from 76 fish species using RAxML ver. 7.0.4.
Numerals beside internal branches indicate bootstrap probabilities based on 1000 replicates.
Scale indicates expected number of substitutions per site.
12