Table S1. Whole mitochondrial genome sequencing primers. List of primers used for amplification and sequencing of the complete mitochondrial genome of E. maclovinus and C. rastrospinosus. 20-60 ng of genomic DNA were amplified in 25 µl under the following conditions: Buffer 1X (Promega, 50mM KCl, 10mM Tris-HCl pH=9 at 25°C, 0.1% TritonX-100), 0.4 mM of each primer, 0.4mM dNTPs, 1 unit of Taq (Promega). Amplification profiles were: a) pre-denaturing at 94°C 2 min, b) 35-40 cycles of denaturing 94 °C 1 min, annealing at 42–55 °C (according to primer Tm) 1 min, extension 72 °C 3 min, 4) additional extension for 5 min at 72 °C.

Nos / Primer name / Primer sequence / Ref.
1 / 12S-H / 5'TGACTGCAGAGGGTGACGGGCGGTGTGT3' / 1
2 / 12S-L / 5'AAAAAGCTTCAAACTGGGATTAGATACCCCACTAT3' / 1
3 / gob-12SF1 / 5'TGAAGGAGGATTTAGCAGTAAG3' / 2
4 / gob-16SR1 / 5'AAGTGATTGCGCTACCTTCGCAC3' / 2
5 / 16S-BR-H / 5'CCGGTCTGAACTCAGATCACGT3' / 3
6 / 16S-AL-L / 5'CGCCTGTTTATCAAAAACAT3' / 3
7 / FISH16SF / 5'GGTTTACGACCTCGATGTTGGATC3' / *
8 / Lati Met R / 5'TCGGGGTATGGGCCCGAAAGCTT3' / 4
9 / FISHGLNF / 5'TCAAAACTCTTGGTGCTTCCACTACAC3' / *
10 / FISHCOIR / 5'ATTACRTTATAAATTTGGTCATC3' / *
11 / FISHTRPF / 5'ACCAARAGCCTTCAAAGC3' / *
12 / FISHCOIIR / 5'TAYTCATAGCTTCARTATCA3' / *
13 / COIII.2R / 5'GTTAGTGGTCAKGGGCTTGGRTC3' / *
14 / FISHCOIIF / 5'ATAGAAGAACTTCTTACYTTCCAYGA3' / *
15 / ATP8.2F / 5'AAAGCRTYRGCCTTTTAAGC3' / 5
16 / FISHCOIIIR / 5'ACGTCTACRAARTGTCAGTATCA3' / *
17 / FISHCOIIIF / 5'TACCACTTYACATCTGAACACCA3' / *
18 / FISHND4R / 5'ATTAGKGATTTTAGGTCTGTTTG3' / *
19 / FISHND4F / 5'CACCTTTGACTYCCAAAAGCCCAYGTAGAA3' / *
20 / Lati Leu R / 5'TTTGCACCAAGAGTTTTTGGTTCCTA3' / 4
21 / FISHHISF / 5'AAAACATTAGATTGTGATTCTAA3' / *
22 / Lati ND5 R1 / 5'CCYATYTTTCKGATRTCYTGYTC3' / 4
23 / FISHND5F / 5'TGCACCCACGCATTCTTTAAAGC3' / *
24 / FISHCYTBR / 5'AGKGAGCCAAAGTTTCATCA3' / *
25 / FISHGLUF / 5'GAAAAACCACCGTTGTTATTCAACTACA3' / *
26 / FISHPROR / 5'AGTTTAATTTAGAATYTTRGCTTTGG3' / *
27 / FISHTHRF / 5'ARAGCRYCGGTCTTGTAATCCG3' / *
28 / FISH12SR / 5'TTATCGGTTCTAGAACAGGCTCCTCTA3' / *

* this study


Table S2. Dot blotting primers. List of specific primers used for mtDNA amplification of, H. antarcticus, B. variegatus, C. rastrospinosus, E. maclovinus and D. rerio. The whole genome amplification for the latter was performed in four overlapping fragments, whereas for H. antarcticus, C. rastrospinosus and E. maclovinus mtDNA amplification was obtained by the amplification of six overlapping fragments. For B. variegatus only the ND6 region was amplified.

Species / Primer name / Primer sequence / PCR profile / Ref.
D. rerio mtDNA / 16S-BLf / 5'TGGTGCAGCCGCTATTAAGG3' / Tdn§ 68-60°C / 6
CoIAHR / 5'TAGTCAGAGTACCGTCGTGG3' / Ta§ 60°C / *
CoIBLF / 5'TGATTCCCACTATTTACAGGCT3' / Tdn 68-60°C / *
NapAHRbis / 5'AGGTTAGTCCGTGGGCGATT3' / Ta 60°C / *
ARG-BLf / 5'CAAGACCCTTGATTTCGGCTCA3' / see ref. / 6
ND5-AHr / 5'AATGCAATTCCGCATGGTCC3' / see ref. / 6
ND5-BLf / 5'TTATTCTTATGCTCAGGGGC3' / see ref. / 6
16S-AHr / 5'ATGTTTTTGGTAAACAGGCG3' / see ref. / 6
Notothenioids mtDNA primers
12Sfornot / 5'YGGGAACAAGGAGCWGGTATCA3' / Tdn 68-60°C / *
ND1revnot / 5'GGSAATAACAGGGGCASRATRA3' / Ta 62°C / *
16Sfornot / 5'TGGYAYGTTAGGGTGGCAGAGC3' / Tdn 68-60°C / *
COIrevnot / 5'ARKAGGAGGAARGAGGGAGGKA3' / Ta 62°C / *
COIfornot / 5'ATTGTTACCGCYCAYGCCTTYG3' / Tdn 68-60°C / *
ND3revnot / 5'AAGCCGCAYTCRTAGGGGGAKA3' / Ta 62°C / *
ND3fornot / 5'TCTYGCCSTCGTATCRTTYTGK3' / Tdn 68-60°C / *
ND5revnot / 5'TGGTGTTCGTTGYRSGTYGTYC3' / Ta 60°C / *
ND5fornot / 5'AYTMAYYKCCCYYTCWSKCSCM3' / Tdn 68-60°C / *
Cytbrevnot / 5'RCAWYGTCCGCGKTGAKAYCAR3' / Ta 60°C / *
cytbfornot / 5'CTGAAACRTCGGRGTMGTCCTW3' / Tdn 68-60°C / *
d-looprevnot / 5'GGCAGTGAGAGGGGTTTKTKWW3' / Ta 62°C / *
Species specific primers for / *
H. antarcticus / ND3Harpagiferfor / 5'GGTCTTGGTTAAACCCCAAGGAAA3' / Tdn 55-50°C / *
ND5Harpagiferrev / 5'ATGACCCTGCTGGATATTGCT3' / Ta 50°C / *
ND6extHarpagiferfor / 5'AGCAATATCCAGCAGGGTCAT3' / Tdn 56-50°C / *
ND6intHarpagiferrev / 5'AGTTTAGGGATGGCTTGGTGT3' / Ta 50°C / *
CytbHarpagiferfor** / 5'ACACCAAGCCATCCCTAAAST3' / Tdn 55-50°C / *
Ta 50°C / *
B. variegatus / ND6extBovictusfor / 5'GTTTACCTCATGCTCTTTGTCTT3' / Tdn 55-50°C / *
ND6extBovictusrev / 5'ACSATSGTTTTTCAAGCCATTAG3' / Ta 50°C / *
C. rastrospinosus / Nd3Chionodracofor / 5'TTCTCGCCGTCGTATCATTTTG3' / Tdn 68-60°C / *
Nd5Chionodracorev / 5'GGTGGATGAGGGTGGGATAGAA3' / Ta 60°C / *
Nd5Chionodracofor / 5'CGCCCTCATCGTCACAATCCTT3' / Tdn 68-60°C / *
CytbChionodracorev / 5'ATAGTGCGATTGTGGCGAGTGC3' / Ta 60°C / *
E. maclovinus / Nd5Eleginopsfor / 5'TGATTCACCTCCCTTTCTCTCCCC3' / Tdn 68-60°C / *
CytbEleginopsrev / 5'ACCATAGTTGCGCCTGCAACA3' / Ta 60°C / *

** with d-looprevnot

* this study

§ Tdn (touch down cycles) and Ta (annealing temperature), for details see the section “Materials and Methods”


Table S3. List of species. Species list and genes retrieved from Genbank and used in the phylogenetic reconstruction (Fig. 4).

Species / Abbreviations / Gene sequence / Genebank Acc. Nos / Ref.
Scyliorhinus canicula / S.can / ATPase 6, ATPase 8,
Cytochrome b / Y16067** / 7
Polypterus ornatipinnis / P.orn / ATPase 6, ATPase 8, NADH6, Cytochrome b / AP004351 / 8
Amia calva / A.cal / ATPase 6, ATPase 8, NADH6, Cytochrome b / AB042952 / 8
Polyodon spathula / P.spa / ATPase 6, ATPase 8, NADH6, Cytochrome b / AP004353 / 8
Osteoglossum bicirrhosum / O.bic / ATPase 6, ATPase 8, NADH6, Cytochrome b / AB043025 / 8
Anguilla japonica / A.jap / ATPase 6, ATPase 8, NADH6, Cytochrome b / AB038556 / 8
Conger myriaster / C.myr / ATPase 6, ATPase 8,
NADH6 / AB038381 / 8
Sardinops melanostictus / S.mel / ATPase 6, ATPase 8, NADH6, Cytochrome b / AB032554 / 8
Cyprinus carpio / C.car / ATPase 6, ATPase 8, NADH6, Cytochrome b / X61010 / 9
Danio rerio / D.rer / ATPase 6, ATPase 8, NADH6, Cytochrome b / NC002333 / 6
Salmo salar / S.sal / ATPase 6, ATPase 8, NADH6, Cytochrome b / U12143 / 10
Lophius americanus / L.ame / ATPase 6, ATPase 8, NADH6, Cytochrome b / AP004413 / 11
Pagrus major / P.maj / ATPase 6, ATPase 8,
NADH6, Cytochrome b / AP002949 / 11
Enedrias crassispina / E.cra / ATPase 6, ATPase 8, NADH6, Cytochrome b / AP004449 / 11
Lycodes toyamensis / L.toy / ATPase 6, ATPase 8,
NADH6 / AP004448 / 11
Emmelichthys struhsakeri / E.str / ATPase 6, ATPase 8, NADH6, Cytochrome b / AP004446 / 11
Pterocaesio tile / P.til / ATPase 6, ATPase 8, NADH6, Cytochrome b / AP004447 / 11
Gerlachea australis / G.aus / Cytochrome b / AF490631 / 12
Harpagifer antarcticus / H.ant / Cytochrome b / AF490647 / 12
Chaenocephalus aceratus / C.ace / Cytochrome b / AF037121 / 13
Pagetopsis macropterus / P.mac / Cytochrome b / AF037119 / 13

**whole mtDNA genome sequence


References to the supplementary material

1.  Kocher TD, Thomas WK, Meyer A, Edwards SV, Pääbo S, Villablanca FX, Wilson, AC (1989) Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. USA 86:6196–6200

2.  Ruber L, Van Tassell JL, Zardoya R (2003) Rapid speciation and ecological divergence in the American seven-spined gobies (Gobiidae, Gobiosomatini) inferred from a molecular phylogeny. Evolution 57:1584–1598

3.  Palumbi S, Martin AP, Romano S, McMillan WO, Stice L, Grabowski G (1991) The Simple Fool's Guide to PCR, Version 20 Honolulu, HI, University of Hawaii, pp 46

4.  Zardoya R, Meyer A (1997) The complete DNA sequence of the mitochondrial genome of a “living fossil,” the coelacanth (Latimeria chalumnae). Genetics 146:995–1010

5.  San Mauro D, Gower DJ, Oommen OV, Wilkinson M, Zardoya R (2004) Phylogeny of Caecilian amphibians (Gymnophiona) based on complete mitochondrial genomes and nuclear RAG1. Mol. Phylogenet. Evol. 33:413–427

6.  Broughton RE, Milam JE, Roe BA (2001) The complete sequence of the zebrafish (Danio rerio) mitochondrial genome and evolutionary patterns in vertebrate mitochondrial DNA. Gen. Res. 11:1958–1967

7.  Delarbre C, Spruyt N, Delmarre C, Gallut C, Barriel V, Janvier P, Laudet V, Gachelin G (1998) The complete nucleotide sequence of the mitochondrial DNA of the dogfish, Scyliorhinus canicula. Genetics 15:331-44

8.  Inoue JG, Miya M, Tsukamoto K, Nishida M (2003) Basal actinopterygian relationships: a mitogenomic perspective on the phylogeny of the "ancient fish". Mol. Phylogenet. Evol. 26:110-20

9.  Chang YS, Huang FL, Lo TB (1994) The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome. J. Mol. Evol. 38:138-55

10.  Hurst CD, Bartlett SE, Davidson WS, Bruce IJ (1999) The complete mitochondrial DNA sequence of the Atlantic salmon, Salmo salar. Gene 239:237-42

11.  Miya M, Takeshima H, Endo H, Ishiguro NB, Inoue JG, Mukai T, Satoh TP, Yamaguchi M, Kawaguchi A, Mabuchi K, et al (2003) Major patterns of higher teleostean phylogenies: a new perspective based on 100 complete mitochondrial DNA sequences. Mol. Phylogenet. Evol. 26:121–138

12.  Derome N, Chen W-J, Dettai A, Bonillo C, Lecointre G (2002) Phylogeny of Antarctic dragon fishes (Bathydraconidae, Notothenioidei, Teleostei) and related families based on their anatomy and two mitochondrial genes. Mol. Phylogenet. Evol. 24:139-152

13.  Chen W-J, Bonillo C, Lecointre G (1998) Phylogeny of the Channichthyidae (Notothenioidei, Teleostei) based on two mitochondrial genes. In di Prisco, G, Pisano, E, Clarke, A (eds) Fishes of Antarctica: a Biological Overview Milan: Springer Verlag, pp 287-298

2