|ELECTORONIC SUPPLEMENTARY MATERIALS
1. MATERIALS & METHODS
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.
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).
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 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.
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.
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.
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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.
Carcharhiniformes Scyliorhinidae Scyliorhinus canicula Y16067
Triakidae Mustelus manazo AB015962
Ingroups (lobe-finned fishes)
Coelacanthiformes Latimeriidae Latimeria menadoensis AP006858
Ceratodontiformes Ceratodontidae Neoceratodus forsteri AJ584642
Ingroups (ray-finned fishes)
Polypteriformes Polypteridae Polypterus ornatipinnis AP004351
Polypterus senegalus senegalus AP004352
Erpetoichthys calabaricus AP004350
Acipenseriformes Acipenseridae Acipenser transmontanus AB042837
Scaphirhynchus cf. albus AP004354
Polyodontidae Polyodon spathula AP004353
Lepisosteiformes Lepisosteidae Lepisosteus oculatus AB042861
Atractosteus spatula AP004355
Amiiformes Amiidae Amia calva AB042952
Hiodontiformes Hiodontidae Hiodon alosoides AP004356
Osteoglossiformes Osteoglossidae Osteoglossum bicirrhosum AB043025
Pantodon buchholzi AB043068
Albuliformes Notacanthidae Notacanthus chemnitzi AP002975
Anguilliformes Anguillidae Anguilla japonica AB038556
Muraenidae Gymnothorax kidako AP002976
Congridae Conger myriaster AB038381
Clupeiformes Engraulidae Engraulis japonicus AB040676
Clupeidae Sardinops melanostictus AB032554
Cypriniformes Cyprinidae Cyprinus carpio X61010
Danio rerio AC024175
Balitoridae Crossostoma lacustre M91245
Salmoniformes Salmonidae Coregonus lavaretus AB034824
Salmo salar U12143
Oncorhynchus mykiss L29771
Esociformes Esocidae Esox lucius AP004103
Aulopiformes Chlorophthalmidae Chlorophthalmus agassizi AP002918
Polymixiiformes Polymixiidae Polymixia japonica AB034826
Gadiformes Gadidae Gadus morhua X99772
Atheriniformes Atherinopsidae Menidia menidia AB370893
Melanotaenidae Melanotaenia lacustris AP004419
Notocheilidae Iso hawaiiensis AB373006
Cyprinodontiformes Aplocheilidae Aplocheilus panchax AB373005
Goodeidae Xenotoca eiseni AP006777
Cyprinodontidae Jordanella floridae AP006778
Beloniformes Scomberesocidae Cololabis saira AP002932
Exocoetidae Exocoetus volitans AP002933
Hemiramphidae Hyporhamphus sajori AB370892
Oryzias latipes group
Oryzias luzonensis AB498064
Southern Japanese populations
Oryzias latipes (Hd-rR) AB498065
Oryzias latipes (Nago) AP008946
Oryzias latipes AP004421
Northern Japanese populations
Oryzias latipes (HNI) AB498066
Oryzias latipes (Hirosaki) AP008941
China and West Korean populations
Oryzias latipes (SOK) AP008947
Oryzias latipes (Shanghai) AP008948
Oryzias javanicus group
Oryzias javanicus AB498067
Oryzias minutillus AB498068
Oryzias dancena AB498069
Oryzias celebensis group
Oryzias celebensis AB498070
Oryzias marmoratus AP005981
Oryzias sarasinorum AB370891
Beryciformes Berycidae Beryx splendens AP002939
Holocentridae Sargocentron rubrum AP004432
Gasterosteiformes Gasterosteidae Gasterosteus aculeatus AP002944
Scorpaeniformes Scorpaenidae Helicolenus hilgendorfi AP002948
Perciformes Cichlidae Oreochromis sp. AP009126
Neolamprologus brichardi AP006014
Tropheus duboisi AP006015
Astronotus ocellatus AP009127
Paretroplus maculatus AP009504
Etroplus maculatus AP009505
Hypselecara temporalis AP009506
Ptychochromoides katria AP009507
Paratilapia polleni AP009508
Tylochromis polylepis AP009509
Pomacentridae Abudefduf vaigiensis AP006016
Amphiprion ocellaris AP006017
Labridae Pseudolabrus sieboldi AP006019
Halichoeres melanurus AP006018
Pleuronectiformes Paralichthyidae Paralichthys olivaceus AB028664
Tetraodontiformes Tetraodontidae Takifugu rubripes AJ421455
Tetraodon nigroviridis AP006046
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)
B 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)
C 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 (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 The upper and lower bounds of separation between Madagascar and
L 85 Indian (Smith et al. 1994; Storey 1995)
T U 145 The upper and lower bounds of separation between
L 112 Indo-Madagascar landmass and Gondwanaland (Smith et al. 1994; Storey 1995; Masters et al. 2006)
U U 120 The upper and lower bounds of separation between African and L
L 100 South American landmasses (Smith et al. 1994; Storey 1995)
Supplementary Table S3. Comparisons of divergence time estimates between the present study and previous studies
Node This study Azuma et al. Yamanoue et al.
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
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.