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SUPPLEMENTARY MATERIALS
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SPECIMENS AND DNA EXTRACTION
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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 the manufacturer’s protocols.
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PCR AND SEQUENCINGS
1. MATERIALS & METHODS
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Whole mitochondrial genomes of the 31 anguilliform species were amplified in their entirety
using a long PCR technique (Cheng et al. 1994). Seven fish-versatile PCR primers for 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, 2001a; Ishiguro et al. 2001, Kawaguchi et al.
2001; Miya & Nishida 2000) to amplify the entire mitochondrial genome in two reactions.
Species-specific primers were designed in cases where no appropriate fish-versatile primers
for the long PCR were available. Long PCR reaction conditions followed Miya & 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 mitochondrial
genome. When these primers did not work effectively, taxon-specific primers were designed
from the obtained sequences for the primer walking strategy. The short PCR reactions were
carried out following protocols previously described (Miya & 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 an ABI Prism 377 or 3100 DNA
sequencer (Applied Biosystems). A list of PCR primers used in this study is available from
JGI upon request.
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SEQUENCE EDITING AND ALIGNMENT
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Whole mitochondrial genome sequences from 58 species were arranged into the typical gene
order of vertebrates (beginning from tRNA-Phe) and the dataset was subjected to multiple
alignment using MAFFT ver. 6 (Katoh & Toh 2008). We imported the aligned sequences into
MacClade 4.08 (Maddison & Maddison 2005) and adjusted the alignment by eye. Amino
acids were used to guide the alignment of the protein-coding genes and secondary structure
models guided the alignment of tRNA genes. Regions that could not be unambiguously
aligned, such as the 5´ and 3´ ends of several protein-coding genes and loop regions of rRNA
and several tRNA genes, were excluded. The dataset comprises 10,821 positions from the 12
protein-coding genes (excluding ND6 gene), 1728 positions from the two rRNA genes and
1152 positions from the 22 tRNA genes (total 13,701 base pairs).
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PHYLOGENETIC ANALYSIS
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Unambiguously aligned sequences were divided into five partitions (first, second and third
codon positions, rRNA and tRNA genes) and subjected to the partitioned ML analysis using
RAxML ver. 7.2.4 (Stamatakis 2006). A 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 a GTR approximation with
optimization of individual per-site substitution rates and classification of those individual
rates into a certain number of rate categories. After implementing the BS analysis, the
program uses every fifth BS tree as a starting point for another ML search using the GTR + 
model of sequence evolution. The top 10 best-scoring ML trees are saved (fast ML searches),
and BS probabilities are shown on the best-scoring ML tree.
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TRACING CHARACTER EVOLUTION
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The ancestral growth habitat was reconstructed on the best-scoring ML tree under the MK1
model (Lewis 2001) in Mesquite ver. 2.6 (Maddison & Maddison 2009). Four character states
were assigned to the growth habitats (Miller & Tsukamoto 2004): shallow water (character state
0), outer shelf and slope (state 1), oceanic midwater (state 2) and freshwater (state 3).
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To confirm the results from the ML reconstruction, a Bayesian approach implemented in
SIMMAP ver. 1.0 Beta 2.4 (Bollback 2006) was performed for two ancestral nodes (A and B in
figure 2). For the latter approach, partitioned Bayesian phylogenetic analysis was conducted
with MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003) using the same evolutionary models as in
the RAxML analysis. We assumed that all model parameters were unlinked and that rate
multipliers were variable across partitions. The default settings for the priors on the proportion
of invariable site (0–1) and the gamma shape parameter (0.1–50.0) were used. A Dirichlet
distribution was assumed for the rate matrix and base frequencies and every tree topology was
assumed to be equally probable. The Markov chain Monte Carlo (MCMC) process was set so
that four chains (three heated and one cold) ran simultaneously. We continued the runs for
1,000,000 cycles, with 1 in every 1000 trees being sampled. “Stationarity” (lack of
improvement in the likelihood score) was checked graphically and all trees and parameters
before reaching the stationarity were discarded (“burn-in”). All trees after the “burn-in” period
(200,000 cycles) were pooled and these trees (800 trees) were used for the reconstruction of the
ancestral character states.
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This study was supported in part by Research Fellowships of the Japan Society for the
Promotion of Science for Young Scientists (07304), JSPS Postdoctoral Fellowships for
Research Abroad, and Grants-in-Aid from the Ministry of Education, Culture, Sports, Science
and Technology, Japan (12NP0201, 15380131, 17207007 and 19207007). We thank the
captain, officers, crew, scientists and students on board of the KT-96-1, KT-96-19, KT-99-6
and KT-01-6 cruises of the R/V Tansei Maru and the KH-95-2, KH-00-1, KH-00-5 and
KH-01-1 cruises of the R/V Hakuho Maru for their assistance in collecting samples.
2. ACKNOWLEDGEMENTS
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3. REFRENCES
Bollback, J.P. 2006 SIMMAP: Stochastic character mapping of discrete traits on phylogenies.
BMC Bioinformatics 7, 88.
Cheng, S., Higuchi, R. & Stoneking, M. 1994 Complete mitochondrial genome amplification.
Nat. Genet. 7, 350–351. (doi:10.1038/369684a0)
Inoue, J. G., Miya, M., Aoyama, J., Ishikawa, S., Tsukamoto, K. & Nishida, M. 2001b
Complete mitochondrial DNA sequence of the Japanese eel Anguilla japonica. Fish. Sci.
67, 118–125. (10.1111/j.1444-2906.2001.00207.x)
Inoue, J.G., Miya, M., Tsukamoto, K. & Nishida, M. 2000 Complete mitochondrial DNA
sequence of the Japanese sardine Sardinops melanostictus. Fish. Sci. 66, 924–932. (doi:
10.1111/j.1444-2906.2000.00148.x)
Inoue, J. G., Miya, M., Tsukamoto, K. & Nishida, M. 2003 Evolution of the oceanic gulper eel
mitochondrial genomes: large-scale gene rearrangements originated within the eels. Mol.
Biol. Evol. 20, 1917–1927. (doi: 10.1093/molbev/msg206)
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)
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)
Kawahara, R., Miya, M., Mabuchi, K., Lavoue, S., Inoue, J. G., Satoh, T. P., Kawaguchi, A. &
Nishida, M. 2007 Interrelationships of the 11 gasterosteiform families (sticklebacks,
pipefishes, and their relatives): a new perspective based on whole mitogenome sequences
from 75 higher teleosts. Mol. Phylogenet. Evol. 46, 224–236. (doi:
10.1016/j.ympev.2007.07.009)
Lewis, P. O. 2001. A likelihood approach to estimating phylogeny from discrete morphological
character data. Syst. Biol. 50, 913–925. (doi: 10.1080/106351501753462876)
Maddison, W.P. & Maddison, D.R. 2005 MacClade version 4.08. Sunderland, Massachusetts:
Sinauer Associates.
Miller, M. J. & Tsukamoto, K. 2004 An introduction to leptocephali: Biology and
identification. Tokyo: Ocean Research Institute, The University of Tokyo.
Minegishi, Y., Aoyama, J., Inoue, J. G., Miya, M., Nishida, M. & Tsukamoto, K. 2005
Molecular phylogeny and evolution of the freshwater eels genus Anguilla based on the
whole mitochondrial genome sequences. Mol. Phylogenet. Evol. 34, 134–146. (doi:
doi:10.1016/j.ympev.2004.09.003)
Miya, M. & Nishida, M. 2000 Use of mitogenomic information in teleostean molecular
phylogenetics: a tree-based exploration under the maximum-parsimony optimality
criterion. Mol. Phylogenet. Evol. 17, 437–455. (doi:10.1006/mpev.2000.0839)
Robins, C.R. 1989 The phylogenetic relationships of the anguilliform fishes. In Orders
Anguilliformes and Saccopharyngiformes, Fishes of the western North Atlantic (ed. E.B.
Böhlke) Part 9, Vol. 1, pp. 9–23. New Haven: Sears Foundation for Marine Research.
Ronquist, F. & Huelsenbeck, J.P. 2003 MrBayes 3: Bayesian phylogenetic inference under
mixed models. Bioinformatics 19, 1572–1574.
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Table S1. List of the species used in this study.
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Order/Suborder a
Family
Species
Accession No.
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Outgroup
Elopiformes
Elopidae
Elops hawaiensis
AB051070
Albuliformes
Notacanthidae
Notacanthus chemnitzi
AP002975
Ingroup
Anguilliformes
Anguilloidei Anguillidae
Anguilla japonica
AB038556
A. reinhardtii
AP007248
A. megastoma
AP007243
A. celebesensis
AP007239
A. marmorata
AP007242
A. nebulosa nebulosa
AP007246
A. nebulosa labiata
AP007245
A. interioris
AP007241
A. obscura
AP007247
A. bicolor bicolor
AP007236
A. bicolor pacifica
AP007237
A. mossambica
AP007244
A. borneensis
AP007238
A. dieffenbachii
AP007240
A. australis australis
AP007234
A. australis schmidtii
AP007235
A. rostrata
AP007249
A. anguilla
AP007233
Heterenchelyidae
Pythonichthys microphthalmus AP010842
Moringuidae
Moringua edwardsi
AP010840
M. microchir
AP010841
Muraenoidei
Chlopsidae
Kaupichthys hyoproroides
AP010845
Robinsia catherinae
AP010846
Myrocongridae
Myroconger compressus
AP010847
Muraenidae
Anarchias sp.
AP010843
Gymnothorax kidako
AP002976
Rhinomuraena quaesita
AP010844
Congroidei
Synaphobranchidae Ilyophis brunneus
AP010848
Synaphobranchus kaupii
AP002977
Simenchelys parasitica
AP010849
Ophichthidae
Ophisurus macrorhynchus
AP002978
Myrichthys aki
AP010862
Colocongridae
Coloconger cadenati
AP010863
Derichthyidae
Nessorhamphus ingolfianus
AP010850
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176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
Muraenesocidae
Nemichthyidae
Congridae
Nettastomatidae
Serrivomeridae
Derichthys serpentinus
Muraenesox bagio
Cynoponticus ferox
Nemichthys scolopaceus
Avocettina infans
Labichthys carinatus
Heteroconger hassi
Paraconger notialis
Ariosoma shiroanago
Conger myriaster
Nettastoma parviceps
Hoplunnis punctata
Facciolella oxyrhyncha
Serrivomer sector
S. beanii
Stemonidium hypomelas
Thalassenchelys sp.
Type II larva
AP010851
AP010852
AP010853
AP010854
AP010855
AP010856
AP010859
AP010860
AP010861
AB03838
AP010864
AP010865
AP010866
AP007250
AP010857
AP010858
AP010867
AP010868
Unidentified
Unidentified
Saccopharyngiformes
Cyematidae
Cyema atrum
AP010870
Saccopharyngidae
Saccopharynx lavenbergi
AB047825
Eurypharyngidae
Eurypharynx pelecanoides
AB046473
Monognathidae
Monognathus jesperseni
AP010869
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a
Classifications follow Robins (1989) and Miller & Tsukamoto (2004)
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