<<

Copyright 0 1996 by the Society of America

The Complete Mitochondrial DNA Sequence of the ( ornutipinnis), a Ray-Finned : Ancient Establishment of the Consensus Gene

Katharina Noack, Rafael Zardoya and Axel Meyer

Department of and , and Program in Genetics, State University of New York, Stony Brook, New York 11794-5245 Manuscript received May 27, 1996 Accepted for publication July 20, 1996

ABSTRACT The evolutionary position of bichirsis disputed, andthey have been variously alignedwith ray-finned fish () or lobe-finnedfish (),which also include . Alternatively, they have been placed into their own group, the Brachiopterygii. The phylogenetic position of as possibly the most primitive living bony fish ()made knowledge about their mitochondrial of considerable evolutionary interest.We determined the complete nucleotide sequence(16,624 bp) of the mitochondrial genome of a bichir, Polypterus ornutipinnis. Its genome contains 13 - coding genes, 22 WAS,two rRNAs and one major noncoding region. The genome’s structure and organization show that this is the most basal vertebrate that conformsto the consensus vertebratemtDNA gene order. Bichir mitochondrial protein-coding and ribosomal RNA genes have greater sequence similarity to ray-finned fish than to either or . Phylogenetic analyses suggest the bichir’s placement as the most basal living member of the ray-finned fish and rule outits classification as a lobe- finned fish. Hence, its lobe- are probably not a shared-derived trait with those of lobe-finned fish (Sarcopterygii).

OW fish (Osteichthyes) are typically divided into Figure 1 summarizes some of the various phyloge- B two major groups,the actinopterygians (ray- netic hypotheses involving bichirs. One of the earliest finned fish) and the sarcopterygians (lobe-finned fish). hypotheses of polrpterid ancestry held that they were With >25,000 known , the ray-finned fish com- sarcopterygians, and bichirs were placed into thesubor- prise by far the largest of . Ray-finned der Crossopterygii along with rhipidistians, coela- fish date back to the early (CARROLL 1988), canths and Iungfish (Figure la) (HUXLEY1861; COPE and their diversity has since thenincreased steadily both 1871; reviewed in PATTERSON1980, 1982). Goodrich in terms of morphologcal diversity and in terms of (1928), for the first time, considered placing polypter- numbers of species. By contrast, the once highly suc- ids within the actinopterygians as a taxonomically dis- cessful and diverse lobe-finned fish, now only include tinct lower .Since then, thealternative two main two extant fish groups, thelungfish and thecoelacanth. hypotheses of polypterine relationships have been that During the last 150 yr, both of these latter groups have polypterids are either classified as a distinct subclass of been variously thought to be the closest living relatives Osteichthyes, the Brachiopterygii (Figure lb),or as of land vertebrates (e.g., ZARDOYA and MEYER 1996a; basal actinopterygians (Figure IC). Themain problems reviewed in MEYER 1995). of assessingpolypterine relationships are that the fossil While the classification of bony fish into either the record for these fish only extends back to the ray-finned or the lobe-finned fish is without problems, (GREENWOOD1974; CARROLL1988) and that they show the phylogenetic placement of species of the order a large number of primitive ancestral as well asuniquely Polypteriformes into one of these two categories (e.g., derived characteristics that make the determination of PATTERSON1982; LAUDER and LIEM1983, also referred their phylogenetic position, and hence their classifica- to as ) has been historically difficult, and it tion, difficult. Bichirs have a row of dorsal finlets that still remains somewhat uncertain (Table 1). Unlike any extend down their back, primitive rhombicganoid other groupof living fish (reviewed in PATTERSON 1982; scales, numerous spiracular plates, and lobed pectoral LAUDER and LIEM 1983), polypteriform fish (bichirs, fins. Their pectoral fins have a fleshy external appear- Polypterus, and , Calamoichthys) have been ance similar to those of lungfish and the . a number ofwidely differing taxonomic placed into Polypterine fish also have a well-vascularized that groups during the last century. arises from the ventral foregut and a pair of external in juveniles, a rare feature, not seen in any other Correspondingauthor Axel Meyer, Department ofEcoIogy and Evolu- tion, State University of New York, Stony Brook, NY 117945245, fish, except in some species of lungfish. E-mail: [email protected] Many authors still argue that Polypteriformes are

Genetics 144: 1165-1180 (November, 1996) 1166 K. Noack, R. Zardoya and A. Meyer

TABLE 1 morphologically too distinct from actinopterygians to Systematic position of bichirs be classified as such (JESSEN 1973; NELSON1973; JARVIK 1980; BJERRING 1985).This view, however, does not clar- Superclass: ify what other group of fish polypterids are most closely Class related to. Most recent authorshave, basedon morpho- Subclass: () logical characters such asscale structure and neuro- Subclass: (, rays) Class Sarcopterygii cranial , placed polypterids with actinoptery- Subclass: Coelacanthimorpha () gians (Figure IC) (GARDINER1973; SCHAEFFER1973; Subclass: Dipnoi () PATTERSON1980,1982; LAUDER and LIEM1983; GARDI- Subclass: Tetrapoda (, , , NER and SCHAEFFER 1989). However, the placement of ) Polypteriformes within the Actinopterygii is also uncer- Class Actinopterygii Subclass: tain. Often polypterids are placed within the Chondros- Order: Polypteriformes (bichirs, Polypterus, tei, a group to which most authors also add the stur- , Calamoichthys) geons and their relatives (Table 1, e.g., GARDINER1967; Order: (, ; ANDREWS 1967; CARROLL1988; NELSON1994). Whereas , Polyodont) Subclass: other researchers classify polypterids as a of equal Order: (, ) rank and basal to the Chondrostei (reviewed in PAT- Order: (, ) TERSON 1982; LAUDER and LIEM1983). Division: Teleostei Among other reasons, knowledge ofthe phylogenetic Modified from NELSON(1994). position of the bichir is of importance for the recon- struction of the development and evolution of verte-

A Bichirs

Lobe-finned fish HUXLEY 1861 COPE 1871 Tetrapods

Ray-finned fish

B

Bichirs I FIGURE1.-Alternative hwotheses of rela- ,I JESSEN 1973 tionships between bichir, ray-finned fish and Ray-finned fish NELSON1973 lobe-finned fish. (A) Polypteriformes are lobe- JARVIK 1980 finned fish. (B) Polypteriformes are a third sub Lobe-finned fish BJEMNG 1985 class of Osteichthyes with an unresolved posi- tion in respect with ray-finned and lobe-finned fish. (C) Polypteriformes are primitive ray- Tetrapods finned fish.

C Bichirs GOODRICH 1928 GARDINER 1973 -Ray-finned fish SCHAEFFER19n PAlTERSON 1982 I I Lobe-finned fish L*EM 1982 GARDINER & SCHAEFFER 1989 Tetrapods NELSON 1994 Polypterus Mitochondrial DNA 1167 brate traits such as paired limbs from fins (e.g., SHUBIN 3.1-kb fragment was amplified using specific oligonucleotide primers and then cloned into pGEM-T (Promega). and ALBERCH 1986; SORDINOand DUBOULE1996). How- Plasmid DNA was extracted from each cloneusing a Wizard ever, so far, only two molecular phylogenetic studies miniprep kit (Promega). After ethanol precipitation, purified have incorporated nucleotide sequence datafrom bich- DNAwas used as template for Taq Dye Deoxy Terminator irs. NORMARKet al. (1991) used partial DNA sequences cyclesequencing reactions (Applied Biosystems Inc.) follow- of the mitochondrial qtb, COI and COII genes to infer ing manufacturer’s instructions. Sequencing was performed with an automated DNA sequencer (373A Stretch, Applied phylogenetic relationships of Neopterygians, using the Biosystems Inc.). Sequences were obtained using both M13 bichir as outgroup taxon. LE et al. (1993) used portions universal sequencing primers and 37 specificallydesigned oli- of the large nuclear ribosomal gene in an attempt to gonucleotide primers. The sequencesobtained from each infer the phylogeny among major fish lineages. Their clone were -350 bp in length and each sequence overlapped study supported theview that polypterids are aprimitive the next by -100 bp. In no case were differences in sequence ray-finned fish lineage, albeit with low confidence. observed among the overlapping regions. Sequence data were analyzed by use of the GCG program Mitochondrial genes are increasingly used to infer package (DEVEREUXet al. 1984) and alignments were per- phylogenetic relationships both among closely related formed using CLUSTAL W (THOMPSONet al. 1994). The data species as well as distantly related ones (KUMAZAWAand were subjected to maximum parsimony (MP) analyses using NISHIDA1993; CAO et al. 1994; RUSSO et al. 1996; ZAR- PAUP Version 3.1.1 (SWOFFORD1993). MP analyses were per- formed using heuristic searches (TBR branch swapping, DOYA and MEYER1996~). ZARDOYA and MEYER(1996a) MULPARS option in effect, simple stepwise addition of taxa). show that combined sets of all mitochondrial protein Maximum likelihood (ML) analyses and neighbor-joining coding and tRNA genes can with strong supportresolve (NJ) analyses were performed using PHYLIP Version 3.5 the relationships among major groups of jawed verte- (FELSENSTEIN1989). For NJ analyses, distance matrices were brates. Until now the complete mitochondrial genome calculated based on Kimura corrected distances (KIMLJRA sequences of 22 vertebrate species have been reported; 1980). TheKishino-Hasegawa test to assess the statistical con- fidence of maximum likelihood trees was performed using 15 arefrom mammals, five from fish, only one from an MOLPWYVersion 2.2 (ADACHI and HASEGAWA 1992). Confi- and one from a . The structure and dence levels for MP and NJ analyses were assessedby bootstrap organization of vertebrate mitochondrial is analyses based on 1000 replications (FELSENSTEIN1985) using remarkably conserved. Minor rearrangements, appar- PHYLIP Version 3.5 (FELSENSTEIN1989). ently caused by the translocation of tRNA genes, have The complete mtDNA sequence of the bichir has been deposited in GenBank under the accession No. U62532. been described for chicken (DESJARDINSand MORAIS 1990) and for the opposum (JANKE et al. 1994). It is RESULTSAND DISCUSSION not yet clear when the establishment of the vertebrate consensus gene order occurred during their evolution. Genome organization: The total length of the bichir Lampreys, one of the earliest vertebrates, have a pecu- mitochondrial genome is 16,624 bp, it is similar to that liar gene order (LEEand KOCHER 1995), but without of the mitochondrial genomes of other bony fish(Table the study of more , it is impossible to deter- 2). The overall base composition of the L-strand is A mine if this gene order represents an ancestral or a = 32.2%, T = 28.1%, C = 25.6% and G = 14.1%. As uniquely derived condition. We determined the com- found in other vertebrates, the bichir’s mitochondrial plete nucleotide sequenceand gene orderof the bichir genome contains 13 protein coding genes, 22 transfer mitochondrial genome with the aim to investigate the RNA genes, and two ribosomal RNA genes. Interest- evolution of the vertebrate mitochondrial genome and ingly, the orientation and relative position of all genes to clarify the bichir’s relationships to lobe-finned and and the major noncoding region is identical to the ver- ray-finned fish. Furthermore, we assessed the perfor- tebrate consensus mitochondrial gene order (Figures 2 mance of the bichir as an outgroup taxon for inferring and 3, Table 3). The presence of this gene order in relationships among major groups ofjawedvertebrates. the bichir documents the early establishment of the particular vertebrate organization of the mitochondrial genome. All genes except the gene encoding for ND6 MATERIALSAND METHODS and eight transfer RNAs are encoded on the H-strand, Mitochondrial DNA was purified from fresh and kid- and the genes encoded on both strands of the bichir ney tissue of commercially obtained bichir (Polypterus mati- mtDNA are of similar length as those in other verte- pinnis) as previously described (ZARDOYA et al. 1995). After brates (Table 3). Peptide encoding genes were identi- homogenization, intact nuclei and cellular were re- moved by low-speed centrifugation (1000 X g). Mitochondria fied by comparison with the rainbow mtDNA were pelleted by centrifugation at 10,000 X gfor 20 min and (ZARDOYA et al. 1995) and by the presence of initiation subjected to a standard alkaline lysis procedure. The isolated and stop codons. Transfer RNA genes were identified mtDNA was cleaved with EcoRI and Hind111 restriction en- by sequence similarity to homologous vertebrate tRNAs, zymes. Three EcoRI fragments of 4.0, 3.9 and 3.0 kb and four their specific anticodons, and their ability to fold into Hind111 fragments of3.1, 1.0, 0.6, and 0.3 kb (See Figure 2 for positions of restriction sites) were cloned into pUC18 putative cloverleaf structures. covering theentire bichir mtDNA molecule except for a As seen in other vertebrates there is an overlap be- stretch of 3.1 kb spanning the GO1 and GO11 region. This tween the genes encoding for ND4 and ND4L, ATPase 1168 K. Noack, R.and Zardoya A. Meyer

TABLE 2 Lengths of fish mitochondrial genes

Species

~ ~~ ~ ~~~~ G ene LungfishLamprey GeneBichir Trout Control region 1184 1003 927 896 1068 49 1 1.2s rRNA 937 944 951 937 950 900 16s rRNA 1591 1680 1681 1680 1655 1621 Cytb 1144 1141 1141 1144 1141 1191 m1 966 972 975 975 958 966 m.2 1028 1050 1047 1047 1036 1044 m3 346 349 35 1 351 346 35 1 ND4 1384 1381 1383 1383 1378 1377 m4L 297 297 297 297 297 29 1 m5 1836 1839 1824 1837 1842 1797 m6 513 522 519 522 504 519 COI 1548 1551 1551 1551 1557 1554 COII 69 1 69 1 691 69 1 688 690 COIII 784 784 786 768 784 786 APT6 682 670 684 684 682 714 A TP8 168 168 165 168 168 168 Total 16646 16642 16575 16558 16624 16201 Values expressed as base pairs.

6 andATPase 8, and ND5 and ND6. The bichir mtDNA chondrialgene order. Once a complete chondrich- also has an overlap of nine nucleotides involving COI thyan mitochondrial genome has been sequenced, the and tR.htA''~ucNI, which are encoded on opposite DNA exact phylogenetic timing of the evolution of this gene strands. This overlap is also found in the lamprey (LEE order in vertebrates will have been established. and KOCHER1995) but not in any other fish (TZENGet Protein coding genes: The bichir mitochondrial ge- al. 1992; CHANC et al. 1994; ZARDOYA et al. 1995; ZAR- nome encodes for 13 . Painvise comparisons DOYA and MEYER 1996a) where these two genes either of the protein coding genes of the bichir with their directly abutt or are separated by a few nucleotides. homologues of , loach, carp, lungfish and These datashow that the birchir is the most basal verte- lamprey showed that bichir proteins consistently have brate known so far with the consensus vertebrate mito- greater sequence similarity to those from ray-finned fish

HindIII

FIGURE2,"Restriction map and gene organi- zation of the mitochon- Eco R drial genome. All protein coding genes are en- coded by the H-strand with the exception of ND6, which is coded by the Lstrand.Each tRNA gene is identified by the single letter amino acid codeand depicted according to thecoding strand. Only the EcoRI and Hind111 restriction sites used for cloning are shown.

EcoRI A/ 2 Hind111 Polypterus Mitochondrial DNA 1169

tRNA-Phe+ - 12s rRNA+ 100 1 >CAAAGATTTGGTCCTAGTCTTACTATCAT 200 101 TTCTCGACTAAACTTATACATGCAAGTATCCGCGCTCCGGTGAAAATGCCCTCAATCTTCCTAGTAGAGGATAAGGAGCTGGCATCAGGCTCGTGTCCAC 201 GGCCCCAAAACGCCTTGCTTTGCCACACCCCCACGGGATTTCAGCAGTAGTAAACATTAGGCAATCAGCGAAAGCTAGACCTAGTTATAGTTAATTAGAG 300 301 CCGGTAAAACTCGTGCCAGCCACCGCGGTTATACGAGGGGCTCCAAATGATGGTTATACGGCGTATGGCGTGGTTMCAATTACAAT~CTGAGAAC 400 401 AAAATACCTTTAAGCTGTCATACGCTAATAAGTCCATGAACTGG 500 501 GATTAGATACCCCACTATGCTTGGTCTTAAACTAAGGCGGCACTTATACTAAGCCGCTCGCCAGGTTACTACGAGCGCAAGCTTAAAACCCAAAGGACTT 600 601 GGCGGTGCTTCAGAACCACCTAGAGGAGCCTGTTCTGTAATCGATAATCCTCGTTCAACCTCACCGCATCTTGCATTCCAGCCTATATACCGCCGTCGCC 700 701 AGTCTACCTTTTGAGAGACCATAAGTAGGCCCAATAAGT~ACTTAAGACTAAAACACGTCAGGTCAAGGTGTAGCTTATGATGCGGGAAG~TGGGCTACATTTT e00 801 CTAGATTAGAATACACGAAAGACACTATGAAACCTGTGTCTGAAGGAGGATTTAGCAGTAAATGGGGAGTAGAGTGCCCACTGAAGCTTGGCGCTGAAGC 900 90 GCGCACACACCGCCCGTCACTCTCCTCCAAACATTTTATAATTTTTAAGACTAAAATA~CACTTCCGACAATAAGAGGAGGCAAGTCGTAACATGGTAAGTGTACC1 1000 tRNA-Val+ - 16s rRNA+ 1001 GGAAGGTGCACTTGGAATAACCAAAATGTAGCTTAATAGAGCATTATA 1100

1101 GCTAGCCTGACCACACACTACCAAACACTATT~TAAATATACTTATAAT~C~ACATTTGTTAACTTCAGTATAGGCGATAGAAAGAGAA 1200

1201 CAAAAGAGCTATAGCAACAGTACCGCAAGGG~GCTGAAAGAGAAATGAAACAAATCGTTAAAGCACGACATAGCAGAGATTAAATCTCGTACCTTTTG 1300 1301 CATCATGATCTAGTAAGTAAGCCCAAGC~ATGATTTATAGTTTGACCCCCCGAAACTAGACGAGCTACTTCGAGGCAGTTGAAAGGACCACCCCGTCT 1400 1401 CTGTGGAAAAAGAGTGGGAAGACTTCCAAGTAGAGGTGACAAGCCTAACGAGCCTAGTGAGCCTAGTGATAGCTGGTTACTTGAGAAATGGATAAAAGTCCAGCCTCAA 1500 1501 AATTTCTAAAAAATATACATTCCGTTTAARAAATTTTTAAGAACATTTGAGAGTTATTCACAGGAGGTA~GCTCCTATGAACTGGGAAACAACCCAAT 1600 1601 AAGGAGGAAAAAGRTCATAATTTACAAGGACAAAATCCAAGTGGGCCTGAAAGCAGCCACCTTTAAAGAAAGCGTTATAGCTTAAATAATATATTATTCC 1700 1701 GTATATCCGGATAAAATCTCTGAATCCCCTACAAATATCAAGTTATTCTATTTGAGTAGAAGAAATTATGCTAGAACTAGTAATAAGAAAAATGATTTTC 1800 1801 TCCTAGCACAAGTGTAAGTTAGAACGGACAAACCACTAACAATTAGACGAACCCAACAGAGGGCCAAATAACGCCTATAATAAAAACAAGAAAACCCTAT 1900

1901 TAATCTTATCGTAAATCTTACACAAGAGTGCCTAAAGGAAAGACTAAAAGACT~AGAGAAAAAAGGAACTCGGCAAATCCGAGCCCCGCCTGTTTACCAAAAACAT 2000 2001 CGCCTTCAGCTTTTCATGTATTGAAGGTCCTGCCTGCCCAGTGACATGAGTTTAACGGCCGCGGTATCCTGACCGTGCAAAGGTAGCGTAATCACTTGTT 2100

2101 CTTTAAATGAGGACTGGTATGAATGGCCCCACGAGGGCTCAACTGTCTCTTTTTCTCCGGTCAATTAAACTGATCTCCCCTGTGCAG~GCGGGCAT~A 2200 2201 GACATAAGACGAGAAGACCCTGTGGAGCTTTAGACTAAATCCAAACACTCCTCACTATATTTTACCGTATAGACAAACACAGCGTTATGGCCAT~GTC 2300 2301 TTAGGTTGGGGCGACCACTGAGAACAAATAATCCTCAGCGATGATTGAAGCACAGCTTTATAAACTAAGAATGACAATTCAAAGCATCAGGACACCTGAC 2400 2401 ATTAGGATCCAGACTAATCTGATCAACGRACCAAGTTACCCCAGGGATAACAGCGCAATCTTTTCCAAGAGCCCAAATCGACGAAAAGGTTTACGACCTC 2500 2501 GATGTTGGATCAGGACATCCTAATGGTGCATCCGCTATTAAGGGTTCGTTTGTTCAGCGATTAARGTCfTACGTGATCTGAGTTCAGACCGGAGCAATCC 2 600 2601 AGGTCAGTTTCTATCTATGTAGTCATTTATCCTAGTACGAAAGGATTGGA~AAATGGGGCCTATATT~~ACACGCCCCCTTTTAACCTACTGAAGCCA2700 tRNA-Leu (UUR) -3 - 2701 AATCAAGTAGATAATAAAAAACATACCCACGCCCTAGAACAGGGTTM 2800 NADH1-3 MTLITLIINPLMYIIPILLAMAFLTL 2801 GGTTCBBJ;CCCCCTCTTTAACTATGACCTTAATCACATTAATTATTAACCCACTCATATATATTATCCCCATTCTGCTAGCAATAGCCTTCCTCACTCTA 2900 VERKMLGYMQLRKGPNIVGPYGLLQPIADGVKLF 2901 GTAGAACGAAAAATATTAGGCTACATGCAACTCCG~AGGACCTAACATCGTAGGCCCATACGGTCTCCTCCAACCCATCGCAGACGGTGTAAAACTAT 3000 IKEPVKPSTSSPTLFLLTPTLALTLALILWIPL 3001 TTATTAAAGAACCAGTAAAACCATCAACCTCTTCCCCAACACTTTTTCTACTCACCCCGACACTTGCCCTCACCTTAGCCCTCATTTTATGAATTCCTCT 3100 PMPLALTDLNLTILFILAVSSLSVYSILGSGWA 3101 TCCTATACCACTAGCCCTCACAGACCTAAATTTAACTATTCTTTTCATTTTAGCCGTATCAAGCCTATCAGTCTATTCAATCTTAGGTTCAGGTTGAGCT 3200 SNSKYALIGALRAVAQTISYEVTLGLIIISLIMF 3201 TCAAATTCCAAATATGCCCTAATTGGAGCATTACGAGCAGTAGCTCAGACAATTTCATATGAAGTCACCTTAGGCCTCATCATCATCTCCTTAATTATAT 3300 TGGFTLTTFNTAQEAVWLILPAWPLAAMWFIST 3301 TTACAGGGGGATTCACATTAACCACCTTCAATACCGCACAAGAAGCCGTATGACTTATCCTACCAGCCTGACCACTAGCCGCAATATGATTCATTTCAAC 3400 LAETNRAPFDLTEGESELVSGFNVEYAGGPFAL 3401 CCTAGCCGAAACAAACCGAGCACCATTTGATCTTACAGAAGGAGAGTCCGAACTAGTATCTGGATTTAATGTTGAATACGCAGGCGGACCATTCGCCCTC 3500 FFLAEYTNILLMNALSTILFLGPSFNTLTINLNW 3501 TTCTTCCTGGCGGAATACACTAACATCTTACTAATAAATGCTCTCTCTACAATTCTATTTCTCGGCCCATCATTCAACACATTAACTATTAACCTTAACT 3600

FIGURE3.-Complete nucleotide sequence of the Lstrand of the bichir mitochondrial DNA molecule. Position 1 1refers to the first nucleotide of tRNAp"".Direction of transcription for each gene is denoted by arrows. The deduced aminoacid sequence for each gene product is shown above the nucleotide sequence (one-letter amino acid abbreviation is placed above the first nucleotide of each codon). Termination codons areindicated by an asterisk. tRNA genes are underlined and the corresponding anticodons are overlined. In the control region,CSB (Conserved Sequence Block) and TAS (Termination Associated Sequence) are underlined. 1170 K. Noack, R. Zardoya and A. Meyer

AIKTMILASMFLWVRASYPRFRYDQLMHLVWKN 3601 O~~~A~~~TATPCCI3700 PLPLTLALITWHISLPISMAGSPPQL tRNA-Ile+ 3701 ~C~~TA~A~ACATA?T3800 - - 3801 3900 +tRNA-Gln NADH 24 "Met4 - MNPY 3901 4000 ILSIMLISLGLGTTLTFASSNWLLAWMGLEINT 4001 C~~~~~~~~AA~4 100 LAIIPLMANNHHPRAVEAATKYFITQAAAAALLL 4 101 CTACICCATCATTCCACTPATAaCCAACAATCATCACCCA 4200 FSSLINAWQSGQWMIQDMSMPMSALMTIAIAIK 4201 TA~~M~~~TC~~TM~~TA~T~TA~ATAA~~~~M4300 LGVAPVHFWLPEVMQGIKLNTGLILATWQKLAP 4301 A~A~~~~~ATA~~~~~~T~~~AA~~A~~4400 LALLYQISNNLMPELMIALGLUSTIVGGWGGLNQ 4401 C~~AAT~~~TAA~A~~TAC4500 TQIRKIMAYSSIAHLGWIISIMHFUPSLAIINL 4501 AA~~A~~~A~~A~~~T~~TA~~~~A~M~4600 IMYIIMTTTMFHIFNTLNSTTINALAINWSKFP 4601 AA~ATATATAPATTATGACAACAACAATATPCATAATPT4700 ALSAITMLALLSLGGLPPLSGFLPKWLILQELTN 4701 ~~~CATA~~~~A~A~~A~A~~~C~~A~~~~~A4800 QNLALTATVMALSALLSLYFYLRLSYSLTTTIM 4801 ATCAAAATCTCaCAerAACCGCCAC~~T~C~A~~~A~A~ATA~A~~~TA~~A~~~~AT4900 PNTYQHMLNWNIKTKITFILPTMMIMTIAMLPI 4901 ACCCAACACATACCAACATAT~~~~~~~~T~C~A~~A~~T~TAA~ATAA~A~~T~AA~5000 S P S I I S M F tRNA-Trp - 5001 TCACa;naAITA~TATPCT mmcz 5100 - +-tRNA-Ala 5101 3CmA 5200 - +tRNA-Am 5201 GGCPTPCCTCCCGCCGGGGAG 5300 - +tRNA-Cp - 5301 ePGCATGCAACTACACPACAAGGCI"n;ATAAGAGAAGOG 5400 coI4 ttIU?A-TyrMTITRWLFSTNHKDIGTLDLIFGAWAGM 5401 ~~~~~C~~~A~~~~C~~AA~T5500 VGTALSLLIRAELGQPGALMGDDQIYNVVGTAH 5501 AGTAGGAACCGCACTAAGCCTCCPAATPCGCGCAGAACPA 5600 AFVMIFFMVMPIMIGGFGNWLVPLMIGAPD~AFP 5601 G~T~~~~~~~AC~~~T~~~C~A~TA~C5700 RMNNMSFWLLPPSLLLLLTSSAVEAGVGTGWTV 5701 CACGAATAAATAACATAACI'I"ITCPGATPACTTCCACC 5800 YPPLGGNLAHAGASVDLAIFSLHLVGVSSILGA 5801 ATATCCCCCATTAA~T~A~T~~~~~~~~~~A~~~~~~A5900 INFITTIINMKPPSTSQYQTPLFVWSVLVTAVLL 5901 A~M~~~~AA~A~~TA~CC~ATC~C~A~T~C~~C~~A~~AT~TCA~A~A~A~~6000 LLSLPVLAAGITMLLTDRNLNTTFFDPAGGGDP 6001 TA~A~CC~~~~~TAGCCGCCGGAATTACC6100 ILYQHLFWFFGHPEVYILILPGFGMVSHIVAYY 6101 AA~~CAACATPTATPCM~A~~OGCCATCCAGAA6200 SGKNEPFGYMGMVWAMMAIGLLGFIVWAHHMPTV 6201 T~A~~A~A~~~~~TATAGGAATAGTATG6300 GMDVDTRAYFTSATMIIAIPTGVKVFSWLATLH 6301 ~ATAGACGC~C~T~~A~A~~A~~~A~A~~~~ACA~~6400 GGAIKWETPMLWALGFIFLLTVGGLTGIILANS 6401 ~~~A~~TGc~~TA~A~~A~T~A'IT~~A~~~~~A~A~~~A6500 SLDIMLHDTYYVVAHFHYVLSMGAVLAIUGGLVH 6501 ~~TATPATA'ITACACGACACATACTACGTGGT6600 FIGLIRE 3.- ConLinupd P olypteru s Mitochondrial Polypterus DNA 1171

WFPLFPGYTLHPTWTKIHFGVMFIGVNLTFSPQ 6601 A~~A~~ATA~~~~~MTA~A~MCA~6700 HFLGLAGMPRRYSDYPDAYTLWUSLSSIGSMIS 6701 A~~~~A~~A~~~ATM~6800 LTAVIMFLFILWEAFAAKREVQTVELTHTNVEWL 6801 ~~lTATA~A~A~~~~~~A~~~~lTM~~M~~C6900 HGCPPPYHTYEEPAFVQSPQARE' TIcA"""cAAm 6901 TIcA"""cAAm 7000 - ttRNA-Ser( IEN) tRUA-) - 7001 7100 COI I+ MAHPTQLGLQDASSPIMBELLLFHDHALMT 7101 ~A~~~~A~AcAA~~~AlTATA~~~A~~~~~MT~7200 VFLISTLVLYIIMTAVSTKLTNKHLLDAQEIEIV 7201 6A~MlT~~A~ATA~A~ATM~~~M~~~~~~~~7300 WTVMPALVLIAIALPSLRILYLMDEINDPHLTI 7301 T~M~AT7400 KATGHQWYWSYEYTDYDTLNFDSYMIPTQDLLP 7401 T~~~~T~A~TAT~lT~lT~~~T~TM~C~~A~A~7500 GQFRLLDTDURMVVPTGSPVRMLITAEDVLHSWA 7501 GXAATCGMT~~TACKAlT~GACQl'A~CTCAWG7600 VPSLGLKMDAVPGRLNQTTFIATRPGVFFGQCS 7601 CAdACCATCATTAGaCePAAAAATAL;ATGCGGTOCCAOG 7700 EICGANHSFMPITIESAPVKYFESWSSSMLAES 7701 A~~~~A~~A~~~~~~~TA~~~~TcAAT~~~~7800 tRNA-Lys-1 ATPase 8-1 - MPQLNPNPW 7801 T TGCCAcAAClTAAFXAAM3XOCCrO 7900 FTILIFTWAVFLTILPUKVTSHKMPNELLTKDP 7901 A~A~~M~A~~~~A~~~A~~C~~~T~TA~M~~AlTM~~~8000 ATPase 6-1 MTLSFLDQFASQSFLGIPLIAIA SNLLTEIWYWPWH* 8001 ~~~~~T~~~A~~~~~~A~~CA~~~A~8100 ILIPWHLPPSPYKRWMSNRLITFQSWFIARTT?? 8101 ~~M~~TAlTA~A~~~~~~~T~~~MlT~~~~A~~~MT8200 QLMLPLNTGAHKWAMILTALLLFLMTLNLLGLLP 8201 CAACTPATAlTA~~~CAT~~ATM~~A~A~MTM~~~~AlTAC8300 YTFTPTTQLSHNMALAVPLWLATVLIGMRUQPT 8301 CGTACCACTATGACTAGCTACCCAACTATCAATAAATAC~C8400 ASLAHLLPEGTPTPLIPILIIIETISLFIRPLA 8401 A~~A~~~~~~A~~~M~~~lTAlTATC~~lTA~~A~~~8500 LGVRLTANLTAGHLLIQLISTATFVMLSIMPTIA 8501 CTGGGAC~TACGACPAACAC~CAAACPTAA~~GCAOGCCAG8600 TLTFIVLALLTILEIAVAMIQAYVLVLLLSLYL 8601 CCACACPCACAmATPGTACTA~~MCTA~A~~~~~~AC~A~~AlTM~A~8700 QE N V COIII"1 MAHQAHAYHMVDPSPWPLTGAVAALLLTS 8701 AcAA~~M~TcAA~~TA~ATA~C~C~~~MC~A~M~8800 GLAVWFHFKSLTLLAMGLLLMILTMIQWWRDII 8801 CCATM~T~~~~~~T8900 REGTFQGHHTPPVQKGLRYGMILFITSEVFFFL 8901 T~~A~~~A~TM~A~A~AcATc~A~~9000 GFFWAFYHSSLAPTPELGGIWPPTGITPLDPFEV 9001 GG~~TCIY;AC~~~TA~A~~~C~A~ACA~A~~A~G9100 PLLNTAVLLASGVTVTWTHHSLMEGKRTEATQA 9101 TTCCPCTTCTTAATA~~~A~~~~~CA~~~A~ACA~MTA~~~~~~AC~9200 LTLTILLGLYFTALQAMEYYEAPFTIADGVYGT 9201 AerAACCTTAACCAmTATTAGGCTPATAerPCACCGCCCI 9300 TFFVATGFHGLHVIIGSTFLAGCLLRQIL~HFTS 9301 A~A~~~A~~A~~~~A~~~A~TA~~~AT~~A~9400 SHHFGFEAAAWYWHFVDVVWLFLYVSIYWWGS 9401 ~A~~~~A~~A~A~A~M~A~w~~~9500 FIGURE3.- Continued 1172 K. Noack, R. Zardoya and A. Meyer

NADH 3-3 tRNA-Gly-3 - ~NLIL~~ILI 9501 n;AAmAATcpTAATATAA"TAATP 9600 SSLISTILAIVAFWLPQMNPDMEKLSPYECOFDP 9601 T~TATA~~TA~~9700 LGSARLPFS~RFFLVAILFLLFDLEIALLLPLP 9701 CCl'AGIVSCCAlTCTA~~CCI'AGAAA~~9800 WSTHLDPTLMLMWAFTIIILLTIGLIYEWLQGG 9801 A~~~ATACl'MTA~~~A~TTA~A~M~A~~~9900 LEWAE t.mDH 4- tRNA--9-3 - ~T~IM 9901 lTAG?iAlGA" TGACCCATATCAT 10000 FTFSTAFMLGLSGLTFNRTHLLSALLCLEG~~L 10001 A~~~ATA~A~lTA~~AA~~~~A~A~~~ATAATAlTA10100 SLFIALAMWCTQNETMHFSSAPLLLLALSACEAG 10101 T~~~G10200 NADH 4-1 ~LKLLIPT LGLSLLVATARAHGSDHLQNLNLLQC* 10201 GGCCA~C~~~AA~~~~mAA~10300 IMLFPMIWTLNPKWLWSATTTHSLIIASLSLTLF 10301 ATTATmA~CA~~~~~CATA~~~A~A~A~~AT10400 KCYSTTQWSNLNYMLATDMISTPLIILTCWLLP 10401 TTAAATOCTACIY3CACAACCCAATGATCAT~~~~AA~A~~~~~10500 LMIIASQNHHSTEPINRQRSYITLLVSLQALLI 10501 ACT~TAAlTA~A~~TATATC~~C~TC~~~ACA~~~A~A~A~~~A~10600 ~AFSATEIILFYI~FESTLIPTLIIITRWGNQTE 10601 AT~~CATA~ATA~~~~~A~A~A~A~A~~~~~G10700 RLNAGIYFLFYTLAGSLPLLVALLYLYNTAGSL 10701 AAT~~A~~~~~~~ATA~ATAT~~~~10800 SFISMNLISIPPNTWTNTFLWVACVTAFLVK~P 10801 OTCATCPAmCAATAAACTTAA~~AA~C~~~A~~A~~~~~~TA~10900 LYGVHLWLPKAHVEAPVAGS~ILAAILLKLGGYG 10901 T~~~~~~CAAAGCCCATGT11000 ~IRMTI~LEPATKSLAYPFIILALWGII~TGSI 11001 OAATAATTCGAATAACCATA~~~~~~A~~C~~~A~~A~~~lTA~~~T11100 CMRQSDMKSLIAYSSVS~MGLVASGILIQTTWG 11101 T~~~~~~ATATA~Cl'A~~~~~~~~~11200 FTGAIILMIAHGLTSSALFCLANTAYERTHSRTL 11201 mA~~ATCTPAATAATPGCCCACGGdTAC11300 LLARGMQIILPLMATWWFIMSLAN~ALPPLPNL 11301 ~~Cl'A~ACCCAATGAT~TAC~~~~CA~MTA~~~~A~AT~~~AWL~TA~AC~~~~11400 MGELHILVSMFNWSNWTILLTGTGTLITASYSL 11401 AATAGGAGAAePAATAATCCPAGTA'PePATAmAACTGA 11500 YLYMSSQRGPTPNNLTFMELSUTREHLLLTL~II 11501 TA~ATATA~~~~C~~CCAACCCCCAAC11600 PIILLMIKPELIWGWCW tRNA-His+ - 11601 TPCCAATTATCCPCC~C~~~A~~~~T~~T 11700 tRNA-Ser ( AGY ) -3 - "Leu 11701 GCl'CQ2GCACaATm11800 NADH-5+ (CW--$ - M~ISQLSQMFMTC 11801 T3ETAl"XCAATTATCACAWLTA~ATAACAT 11900 LSLTMIILILPITFSFITKPSNKWPFQVKNAVK 11901 G~Tc~~A~~T~~~~~~~12000 LSFFVSLIPSITCLNLNLQSFTIYYQWFSISST 12001 ATTA~~~AA~A~~A~~~~~~~~~cAA~~~A~~12100 KINISLQFDQYSMIFMTIALYVTWSILEFAIYY~ 12101 AAAATPAACATPAGCCPACAA~GATCAATACPCCATRAT~12200 HTDILINRFFKYLLTFLIAM~ILVTANN~FQLF 12201 TA~~~~AA~AC~~~TACCPAT12300 IGWEGVGIMSFLLIGWWYGRADANMAALQAVIY 12301 TATCGCATA~~~A~~~T~TATCATPCCPACP12400 FIGURE3.- Continued Polypterus Mitochondrial DNA 1173

NRVGDIGLMMTMSWLLINTNSWDIQQLFGLT~~M 12401 M~~~~TMT~TA~~~~~G~TA~A~~A12500 DTTLPATGLLLAATGKSAQFGLHPWLPAAMEGP 12501 T~UXGNXWTICTAGCAGCAACA~T~XUXAA~TGA~AC~"~'A~12600 TPVSALLHSSTNVVAGIFLLIRL~PLIENNNNI M""TA"AA-m-=TAA=Tm 12601 M""TA"AA-m-=TAA=Tm 12700 LTAALCLGAITTLFTATCALTQNDIKKIVGFSTS 12701 ClTA~A"ll'ACX"C~~~~~~A~T12600 SQLGLMHVAIGLNQPQLAFLHICTHAFFKAMLF 12601 ~~MTMT~~~AA~~A~~~~~~T~12900 LCSGSIIHSLNDEQDIRKMGGINKTLPLTSSCL 12901 C~~T~~MT~~~~A13000 TIGSLALMGTPFLAGFFSKDAIIEAINTSHLNAW 13001 ~A~~~A~~~~~~~~~AA~~~~~13100 ALVLTLIATSFTAVYSLRIIYFVLMNHPRTLPL 13101 Gh~A~A~~~AA~~~A~~A~A~~A~~~~~A~~13200 SPVNENNPLIANPIKRLAWGSIIAGLILCQYIL 13201 CAT~~~~~AA~TAT~13300 PNKTQTLTMTPMLKLTALIVSLLGLLTALELASM 13301 ~AAT~~~TAA~T~~AA~A~A~~A~A~~A~GCA~~~13400 ANKQIKINPTKFTHNFSNMLGFYPHIMHRLMSK 13401 T~TAA~T~A~TA~ATAC~AATA~AA13500 LPLMLGQISATQMSDQLWMEKLGPKGIAHTQLL 13501 A~A~MT~~~~~TA~~A~TA~~~A~~~~~~C~13600 VTQKITHVHRGLIKTYLSIMMLSIIIITIIIMIT 13601 ~A~~~a~A~T~~AA~~CATA~A~~~ATAATA~A~~~A~A~~A~A~AT~~A13700 t ~VARLCGRSYGRTLELVVLLA~LLVWGAIFLMY 13701 CCTAACCOCACOTAAACAAC~~M~A~C~~AAT~~A~AATAATA~T~~~TATM13800 GGANYVEAVGVYRRMAGFYEEVDDFLEMDGVFA 13601 C~~TATIY3ePCKX3TACCCCAACATATI13900 VWAVIVLFMYVLVYSFVEWSGWANPYPEAALAPS 13901 O~CM~~~A~A~TAA~~~14000 YAFVVLMGGLYILFLIMSLFT~GCQMLMGCGVG 14001 AT~~TA~A~AA~~~~T~~~~~AT~T~~ATA~ACA~~A~AA~~ACA~C~14100 +NADH 6 AGVMLGFAAFYPSPNSAVAILSILFMVSFVVML 14101 G~~~~~~~~~~TA~A~~~TAA~A14200 CYtb VM - ttRNA-Glu M A I I R K T H 14201 CCATTPC"KGCAA~A'IC~OXX 14300 PLAKIINSAFIDLPAPSNISSWWN~GSLLGLCLI 14301 ~~~A~AAC~~A~~~CA~~~~~~~~T~A~~A~AA14400 AQIITGLFLAMHYVSDINSAFSSVAHICRDVNY 14401 TCOCACAORTPATCACAOGA~~~A~A~~A~~~~A14500 GWLIRNFBANGASLFFICIYLRIARGLYYGSYL 14501 ~~~AA~~~~C~~~A~~~A~A~TA~ATA~A~~A~14600 YTETWNMGVILLLLTMMTAFVGYVLPWGQMSFWG 14601 T~~~~TA~~~A~CCATAAT~~A~A~A~A~~TA~G14700 ATVITNLLSAIPYIGDTLVQWIWGGFSVDKPTL 14701 C~TA~~~TA~A~~~~~~~T~~~14600 TRFFAFHFILPFAIAAASLVHIVFLHETGSNNP 14801 TA~~~~A~~~~C~C~TA~A~~C~~~14900 VGINSDADQIPFHPYFTFKDLLGFIILLLIIIML 14901 OTAOOAATPAATPCCG~~CC~~~A~~A~AC~~~A~A~~A~A~A~~~A~A~ATAT15000 ALLSPNLLNDPGNFTPANPLITPPHIKPEWYFL 15001 TA~~~~~A~~~A~~~~~AA~A~cc~TA~~c~~TA~15100 FAYAILRSIPNKLGGVLALLFSILILMLV~LLH 15101 A~~M~~A~=~~A~~~~A~A~cA~~A~~~~~~~A~c15200 TSKIRSATFRPLFKITLWILAADVLILTWfOOQP 15201 A~~~~~A~~A~~~~~~~~~~~~~15300 VEDPYIIIGQAASILYFLIFLVLMPLSGWLENK 15301 CA~AOAAGACCCGTACATPA~~~ATA~A~A~~~A~=15400 FIGURE3. -Continued 1174 K. Noack, R. Zardoya and A. Meyer

M L N R D tRNA-Thr+ - 15401 AAT-m 15500 - +tRNA-ProControl Region-, msd 15501 A"TA'L"ATATAXACACAATATTTAAA"lTACAT 15600

15601 TCATA~~A~~A~A~A~T15700 TBs-3 TAS-4 15101 A~A~~A~A~~~~A~~~~A~ATATA~~~~~~AT~~ATATA~TAA~~T~~15800

15801 ~~A~~~~~~TA~A~~C~~~~TA~~~A~~~A~TATATA15900 15901 ~~~~~~T~~~~A~~~~~~~~~~GK~~T~A~~TA~16000

16001 O~TA~16100 ce-r, 16101 A~A~A~TA~A~AT~~CA~16200 16201 ~~CA~AAACA~~~A~~"PI?AGC~16300 mB3 16301 CA~~A~~~A~~~~A~~~~~~~~~TA~~16400 - 16401 T~TA~~16500 CSB-11 CSB-I11 16501 CCACXXEFTAAAAWGKC~~CCG'l'W~~~AW~~~16600

16601 "CAWCTACGA 16624 FIGURE3. - Continued than to those of either the lungfish or the lamprey in the TYC stem, 5 bp in the anticodon stem and 4 bp (Table 4). in the DHU stem. In most vertebrates tRNA"fA''Y' has All protein encoding genes in the bichir mitochon- a reduced DHU arm. The bichir tRNAsm(A"Y),similar to drial genome use the initiation codon ATG except for that of the lamprey tRNAs"'A"y', has no recognizable COI, which uses GTG. The use of GTG for initiation of DHU stem and loop. Rainbow trout, carp and on translation in COI is shared with other fish mitochon- the other hand posses a tRNAS"'A"Y'which is much less drial genomes that have been sequenced completely truncated (ZARDOYA et al. 1995). and with the chicken mitochondrial genome. Three Ribosomal RNA genes: The 12s and 16s ribosomal genes (ATPase 8, ND4L, and AD51 use TAA as stop RNA genes in bichir are 950 and 1655 nucleotides long, codon, eight genes (AD1,ADZ, COII, ATPase 6, COIII, respectively. They are situated as in other vertebrates ND3, AD4, and cyt 6) use the incomplete stop codonT, between tRNApheand tRNAL"'UUR'and are separated by and the remaining two genes (CUI and ND6) use an the tRNAvaf.The ribosomal RNA genes follow the same AGR stop codon (Table 3). Of the complete vertebrate trend as the protein encoding genes and show greater genomes sequenced, no ray-finned fish uses AGR as a sequence similarity to ray-finned fish than to lamprey stop codon, whereas lamprey, lungfish, , chicken or lungfish (Table 4). and mammals do use AGR stop codons. Noncoding regions:The control region,which is the The overall codon usage of the 13 protein genes of largest noncoding region found in the bichir mtDNA, the bichir mtDNA genome shows a strong bias against is 1068 nucleotides long and is situated between the guanine at third positions. The anti-G bias in bichir is tRNAp" and tRNAph"genes. The control region of verte- the second strongest (4.4%) after lamprey (3.8%), brates contains the site of initiation for heavy strand while other fish and frog areless strongly biased(MEYER replication as well as promoters for the transcription of 1993) (Table 5). As seen in other mitochondrial ge- both the light strand and the heavy strands. The bichir nomes (NAYLORet al. 1995) pyrimidines are overrepre- control region has an overall base composition that is sented compared with purines in the second codon rich in A and T (percent A+T = 64.3). Between posi- position (percent C+T = 68.3) due to the hydrophobic tions 16,100 and 16,300 (Figure 3), a Grich region character of proteins encoded by mitochondrial genes. is found within which the central conserved block D Transfer RNA genes: The bichir mitochondrial ge- (SOUTHERNet al. 1988) was identified. This sequence nome contains 22 transfer RNA genes that are inter- block is conserved between bonyfish and mammals spersed between ribosomal RNA and protein coding (LEEet al. 1995), buthas not been foundin the lamprey genes. The transfer RNA genes range in size from 67 control repon (LEE andKOCHER 1995). The consemed to 75 nucleotides. All bichir tRNA genes can be folded sequence blocks CSB-I1 and CSB-111, which are thought into conventional cloverleaf secondary structures. As to be involved in the generation of the RNA primer seen in other vertebrate tRNAs formation of G-U, and needed for initiation of heavy strand replication (WAL other atypical pairings are found in the stem regions. BERG and CLA~ON1981), are in the right do- All proposed cloverleaf structures, exceptthat of main of the bichir control region. Similar to lungfish tmkSer(AGY) contain 7 bp in the amino acid stem, 5 bp (ZARDOYAand MEYER1996a) and frog ( et al. 1985), Polypterus Mitochondrial DNA 1175

TABLE 3 also been found at the 3' end of other vertebrate con- Localization of features in the mitochondrial genomeof trol regions and are thought to act as a recognition Polypterus mtipinnk site for the termination of heavy strand elongation by forming stable hairpin-loop structures (SACCONEet al. Codon 1991). In the left domain, we could also identify four FeatureFrom To Size, bp StopStart putative termination associated sequences (TASs) based on the consensus mouse TAS sequence proposed tRNA-Phe 1 71 71 by DODAet al. (1981) (Figure 3). 950 12s rRNA 72 1021 The origin of light strand replication (0,)is located tRNA-Val 1022 1092 71 16s rRNA 1093 2747 1655 between the tRNAA""and tRNAcy,' genes and is 46 nucleo- tRNA-bu(UUR) 2748 2822 75 tides in length (Figure 3). The sequencehas the poten- NADH 1 2823 3780 958 ATG T- tial to form a stable stem-loop structure with 11 bp in tRNA& 3781 3853 73 the stem and 12 nucleotides in the loop.The conserved tRNA-GEn 3854 3920 67 (L) motif 5'-GCCGG3' at the base of the stem within the tRNA-Met 3989 69 3921 tRNAcys gene, whichhas been shown to be involved NADH 2 3990 5025 1036 ATG T- tRNA-T? 5026 5094 69 in the transition from RNA synthesis to DNA synthesis tRNA-Ala 5100 5168 69 (L) (HIXSONet al. 1986), is also conserved in the bichir. tRNA-Asn 5170 5242 73 (L) The most interesting feature of the bichir OLis a stretch tRNA-Cys 5278 5346 69 (L) of T-rich sequence in the loop. This stretch of T-rich / tRNA-Tyr 5347 5416 70 (L) sequence in humans has been shown to be involved in I 5418 6974 1557 GTG AGG co the synthesis of a RNA primer for initiation of light tRNA-Sm (UCN) 6966 7036 71 (L) tRNA-Asp 7039 7107 69 strand replication (WONGand CLAYTON1985). All co II 7111 7801 688 ATG T- mammalian mtDNAs sequenced so far have this T-rich ~RNA-LJLs 7802 7874 73 sequence, whereas all other fishmtDNAs sequenced ATPase 8 7875 8042 168 ATG TAA have a stretch of cytosines or, as is the case for lungfish, ATPase 6 8033 8714 682 ATG T- a stretch ofcytosines and thymines (ZARDOYA and Co III 8716 9499 784 ATG T- MEYER1996a). tRNA-Gly 9500 9570 71 NADH 3 9571 9916 346 ATG T- Phylogeneticanalyses of bichirmitochondrial ge- tRNA-Arg 9917 9986 70 nome: To phylogenetically correctly place the bichir NADH 4L 9987 10283 297 ATG TAA among vertebrates, and especially to assess its relation- NADH 4 10277 11654 1378 ATG T- ship to ray-finned and lobe-finned fish, we used the tRNA-His 11655 11723 69 mitochondrial genes of the following species for phylo- tRNA-Sm (AGY) 11724 11789 66 genetic analyses: human (ANDERSON et al. 1981), tRNA-Leu (CW) 11792 11863 72 NADH 5 11864 13705 1842 ATG TAA (hASON and GULLBERG1993), (JANKEet NADH 6 13701 14204 504 (L) ATG AGG al. 1994), chicken (DESJARDINSand Mows 1990), frog tRNA-Gh 14205 14273 69 tL) (ROEet al. 1985),lungfish (Z~OYAand MEYER1996a), CYT b 14277 15417 1141 ATG T- rainbow trout (ZARDOYA et al. 1995), carp(CHANG et al. tRNA-Thr 15418 15488 71 1994), loach (TZENGet al. 1992) and lamprey (LEE and tRNA-Pro 15489 15556 68 (L) KOCHER 1995). Protein-encoding genes were aligned Control region 15557 16624 1068 and gaps were introduced according to the deduced amino acid sequences. Variation among the 13protein a putative CSB-I with a reduced motif of GACAT can coding genes was mainly found in the carboxyl end of be identified at position 16,398 (Figure 3). the polypeptides and in few cases in the amino end. At the 3' end of the control region, the palindromic However, the central core of the mitochondrial pro- sequence motives TACAT and ATGTA are repeated teins was found to be highly conserved. Therefore, am- four and three times, respectively. These motives have biguous alignments at 5' and 3' ends of proteincoding

TABLE 4 Percentage nucleotide similarity of bichir mitochondrial genes against their homologues of other available fish

12s 16s12s hDl ND2 hD3 ND4 ND4L hD5 hD6 ATP6 ATP8 COI COII COIII CYT b bichir-lamprey 68.3 66.866.8 63.956.9 6157.7 57.4 61.9 68.4 50.6 77.6 71 65.674.4 bichir-ray- finned69.470.5 fish"63.7 71 71.2 73.9 67.7 67.3 61 70.9 61.772.277.1 75.3 78.5 bichir-lungfish 67.468.9 71.2 61.9 66.2 65 58.6 62.9 75.859.373.8 76.2 57.1 66.7 69.3

a Ray-finned fish refers to the average sequence similarity between bichir against trout, carp and loach. 1176 K. Noack. R. Zardoya and A. Meyer TABLE 5 Base composition for the 13 proteincoding genes of fish and an amphibian

Codon position A G C T Lamprey 1 30.4 22.6 22.9 24.1 2 19 12.9 26.5 41.6 3 3.8 41.3 21.5 33.4 Polypterus 1 29.9 23 23.7 23.4 2 18.9 12.8 18.9 2 27 41.3 3 42.7 4.4 27.8 25.1 Trout 1 26.4 25.4 26.8 21.4 2 18.2 2 13.8 27.7 40.3 3 33.4 8.9 33.9 23.8 Carp 1 20.6 26.427.1 25.9 2 18.5 2 14 28.2 39.3 3 44.2 5.918.6 31.3 Loach 1 27.2 26.4 25.6 20.8 2 40.118.5 27.7 13.7 3 35.8 9.6 34.6 20 Bichir Lungfish 1 23.6 27.6 23.4 25.4 FIGURE 4.-Fifty percent majority rule bootstrap (FELSENSTEIN 2 18.4 2 13.341.1 27.2 1985) consensus tree ofjawed vertebrates based on 1000 replica- 3 34.7 3 28.4 28.5 8.4 tions. Two data sets were subjected to MP (bootstrap values above Frog 1 29.9 21 23.3 25.8 branches) and NJ (bootstrap values below branches) analyses. 2 20.5 11.6 27.2 40.7 The first data set comprises a combination of all mitochondrial 3 41.2 6.5 41.2 3 22.3 30 genes (bootstrap values upper of each pair of numbers). The second data set includes all mitochondrial protein coding genes Values are expressed as percentage. combined (bootstrap values lower of each pair of numbers). ML analyses with the same two data sets yielded the same topology. genes were excluded from the phylogenetic analyses. Bichir, rainbow trout, carp, and loach were used as outgroup for The analyseswere performed using nucleotide se- all analyses. quences and third codon positions were excluded since they are largely saturated with back mutations at this investigate the performance of the bichir as outgroup levelof phylogenetic inquiry (e.g., CAO et al. 1994; in resolving vertebrate phylogenetic relationships. The RUSSO et al. 1996; ZARDOYA and MEYER 1996a). Simi- phylogenetic analyses were performed using a data set larly, all tRNA genes were aligned taking their second- of all mitochondrial genes combined and individual ary structures into account. DHU and TQC arms were data sets of combined protein coding genes, tRNAs or omitted due to ambiguity in alignments, and all posi- rRNAs only. All three commonly used phylogenetic tions were weighted equally. Moreover, both rRNA methods (MP, NJ, ML) arrived at identical and strongly genes were aligned also taking their secondary struc- supported topologies when bichir, rainbow trout, carp tures into account and questionably aligned sequences and loach were used as outgroup taxa and the com- were omitted from the analyses. For rRNA genes, all bined set of all mitochondrial genes was analyzed (Fig- positions were weighted with a transition/transversion ure 4). ratio of 2:l to account for thefaster rate for transitions With the separate protein data set, the expected phy- overtransversions in these genes (e.g., MEYER 1993; logeny was also obtained with all three phylogenetic ORT~and MEYER 1996). In all analyses, gaps in align- methods used (Figure 4). Strikingly, the separate data ments were treated as missing data. sets for tRNAs or rRNAs were unable to resolve the In theirinvestigation of lungfish relationships among expected vertebrate relationships. In the case of the vertebrates, ZARDOYA and MEYER (1996a) found that if rRNA data set, the MP and ML analyses placed frog the lamprey is used as outgroup, vertebrate trees with and lungfish as the sistergroup of and this unexpected topologies and low bootstrap values are grouping was supported by a bootstrap value of 56% found regardless of which phylogenetic method is used. (MP). NJ analysis of this data set arrived at a tree that The same results are obtained when bichir is added to placed frog and lungfish into a monophyletic group the analysis and lamprey is used as outgroup. It seems basal to tetrapods, this was supported by a boot- that too many multiple substitutions might have accu- strap value of 52%. For the tRNA data set, all three mulated along the fast evolving lamprey lineage, hin- phylogenetic analyses (MP, NJ, and ML) arrived at un- dering the recovery of correct phylogenetic relation- expected trees in which the lungfish was basal to teleosts ships among vertebrates (RUSSO et al. 1996; ZARDOYA and tetrapods. This topology was supported with boot- and MEYER1996a,c). We used the same taxa that were strap values of74 and 57% for MP and NJ analyses, included in the ZARDOYA and MEYER (1996a) study to respectively. In theparsimony analysesof the tRNA and Polypterus Mitochondrial DNA 1177

TABLE 6 Statistical confidence of maximum likelihood trees

tRNAs rRNAs

Tree topology log 1 Ala SE log 1 Ali SE

1. (bichir,(trout,(carp,loach)),(lungfish,(frog,(chicken,(marsupial,(whale,human)))))) -8746 -6.7 6.3 -11,004 -11.2 16.6 2. (bichir,((frog,lungfish),(uout,(carp,loach))),(chicken,(marsupial,(whale,human)))) -8762-22.6 11 -10,992 - - 3. (bichir,lungfish,((trout,(carp,loach)),(frog,(chicken,(marsupial,(whale,human)))))) -8739 - - -11,01217.1-19.4 Ali represents the difference in log-likelihood between the best tree (i) obtained for each data set and the expected tree

(tree #l).SE is the standard error. Trees are declaredSignificantly v different when the difference in log-likelihood is larger than 1.96 standard error. rRNA data sets, only a few more steps are needed to (Figure 4) rule out the hypothesis that the bichir is a recover the expected topology (Figure 4), suggesting sarcopterygian (Figure la). Without the addition of an that the shortest trees obtained for those two data sets appropriate, and yet unavailable, outgroup, it remains, are poorly supported and notstatistically different from however, difficult to distinguish between the two the expected tree. This finding from the MP analysis hypotheses of bichir as a third major bony fish lineage was confirmed with ML when standard errors of the (Figure lb) or the birchir as a basal actinopterygian difference in log-likelihood between the ML tree given (Figure IC).One problem in assessing polypterine rela- by each data set and that of the correct treewere calcu- tionships is that the bichir is one of the mostbasal lated by the formulaof KISHINO and HASEGAWA (1989). vertebrate taxa for which the complete mitochondrial Both the rRNA data set and the tRNA data set exhibited genome is currently available. Lamprey hasbeen shown log-likelihood ratios for the expected tree thatwere not to be unable to resolve vertebrate relationships and is significantly lower than those of the best tree obtained too distant to be used as outgroup (RUSSO et al. 1996; in each case (Table 6). This suggests that the expected ZARDOYA and MEYER1996a,c). Therefore, the phyloge- tree (Figure 4) cannot be statistically ruled out for the netic position of the bichir can only be determined in tRNA and rRNA separate data sets. This conclusion is the future if members of the Chondrichthyes (sharks, also confirmed by thehigher bootstrap values(with rays and chimaeras) are used as outgroup and a stur- MP and NJ) obtained for the combined data set of geon mitochondrial genome has been sequenced. all mitochondrial genes compared with the bootstrap The Cytochrome b gene has been sequenced for both the values (MP and NJ) obtained for the separate data set white (Carcharodon carcharias) and the shark of all protein-coding genes (Figure 4). This stronger (Gukocerh cuvier) (MARTIN and PALUMBI1993). Addition- support of the expected topology is due to theaddition ally, the cyt b sequence is also available (BROWN of the phylogenetic information contained in the rRNA et al. 1989). To further test polypterine relationships,the and tRNA data sets to that contained in the protein- same analyses as described above were performed with coding data set. this gene using the same 10 taxa as above plus sturgeon Phylogeneticposition of bichirs: Analyses of the and the two sharks. Thisdata set was analyzed with MP,FJJ combined proteincoding genes byMP, NJ, and ML and ML using both nucleotide sequences (third positions

human A human whale fiwhale marsupial 82 Imarsupid chicken 62

62 frog FIGURE 5.”Majority rule bootstrap (FEUEN- lungfish STEIN 1985) consensus trees of the ~tochromb I lungfish data set. (A) Tree was obtained from Mp analy- bichir sis. (B) Tree was obtained from NJ analysis. loach 100 Numbers above branches indicate bootstrap values (1000 replicates). ML analysis arrived at trout the same topology shown for tree A. 96 I sturgeon I bichir sturgeon r white shark 1178 K. Noack, R. Zardoya and A. Meyer

/ f"

/ \ rhipidistian

/ FIGURE6.-Phylogenetic hypothesis and pectoral evolution. The basal (b) and radial (r) are indicated in each of the /limb schematics. Only up to three basals and radials are indicated,often their number is larger. Thehomology of some basal bones, e.g., in thebirchir and the (Amia calua) is disputed, The , andnum- bichir bers of some of the axial elements between ray-finned and lobe-finnedfish is ambiguous (from ROMERand PARSONS1977).

/ -vteleost

\

shark were excluded) and amino acid sequences and the sharks theory that thebichir might be a distinct piscine lineage were declared outgroups. MP and ML analyses place the (Brachiopterygii, sensu BJERRING1985) that is more bichir as sistergroup to all other ray-finned fish included closely related to sarcopterygians than to actinopterygi- in the analysis; ie., the bichir is placed basal to sturgeon, ans (Figure 5b). This tentative topology is only weakly supporting the view that bichir should not be placed to- supported by a low bootstrap value of 62%. gether with the Acipenseriformes (Table 1) intothe For all three phylogenetic methods (MP, NJ and ML), Chondrostei (contra NELSON1994). However, the boot- the relationships between lungfish and frog arenot strap value for the node combining bichir with the other convincingly resolved withthe cytochrome b data set. The ray-finned fishin MP analysis is low (36%),leading to an discordant results and thelow bootstrap values for gen- unresolved trichotomy between bichir, ray-finned and fish erallywell-established tetrapod relationships support lobe-finned fish in a 50% majority rule consensus tree the notion that single genes often are unable to obtain (Figure 5a). thecorrect phylogeny among such lineages that di- Interestingly, the NJ analysis places the bichir as the verged >400 mya (CAO et al. 1994; RUSSO et al. 1996; sistergroup to lungfish and tetrapods supporting the ZARDOYA and MEYER 1996a,c). Polypterus Mitochondrial DNA 1179

Our results show that thebichir is a reliable outgroup complete mtDNA sequences of the blue and the fin whale, two species that can hybridize in . J. Mol. Evol. 37: 312-322. to assess relationships amongjawed vertebrates, leading BJERRING,H. C., 1985 Facts and thoughts onpiscine phylogeny, pp. to well-supported topologies when large data sets (all 31-57 in Evolutionaty Biology of Primitive , edited by R. E. Foreman, A. GORBMAN, 1. M. DODD and R. Or.ssoN. Plenum mitochondrial genes combined or at least all protein ,_I Press, New York. coding genes combined) areassayed. It can be expected BROWN,J. R., T. L. GILBERT,D. J. KOWELL, P. J. O’,N. E. that this newly obtained mitochondrial sequence will BUROKERel al., 1989 Nucleotide sequence of the apocytoch- rome b gene in mitochondrial DNA. Nucleic be especially useful to polarize mitochondrial data sets Y Acids Re: 17: 4389. with whichone can hope toinvestigate the relationships CAO,Y., J. ADACHI, A. JANKE,S. PAABOand M. HASEGAWA,1994 Phylo- among the most species-rich class of vertebrates. The genetic relationships among eutherian orders estimated from firm establishment of the phylogenetic position of the inferred sequences of mitochondrial proteins:instability of a tree based on a single gene. J. Mol. Evol. 39: 519-527. bichir awaits the collection of additional data sets, e.g., CARROLL, R. L., 1988 Vettebrate andEvolution. Freeman, the complete mitochondrial genomes of an acipenseri- New York. form fish (e.g., sturgeon) and a cartilaginous fish. CHANG,Y. S., F. L. HUANG andT. B. Lo, 1994 The complete nucleo- tide sequence and gene organization of carp ( carpio) The resolution of the bichir’s phylogenetic position mitochondrial genome. J. Mol. Evol. 38: 138-155. has bearing on the understanding of the evolutionary COATES,M. I., 1994 The origin of vertebrate limbs. Development history, establishment of homology, and the evolution (SUPPI.) 169-180. COPE, E. D., 1871 Contribution to the of the Lesser of developmental mechanisms that led from fish fins to Antilles. Trans. Am. Phil. SOC.14: 445-483. vertebrate limbs. Pentadactyl tetrapod limbshave DESJARDINS,P., and R. MORAIS, 1990 Sequence and gene organiza- evolved from sarcopterygian lobed fins (e.g., AHLBERC tion of the chicken mitochondrial genome. J. Mol. Biol. 212 599-634. and MILLNER1994; COATES1994). The bichir’s phyloge- DEVEREUX,J., P. HAEBERLIand 0. SMITHIES,1984 A comprehensive netic position would suggest that its lobed fins might set of sequence analysis programs for the VAX. Nucleic Acids not be a sharedderived trait that unites them with sar- Res. 12: 387-395. DODA,J. N., C. T. WRIGHTand D. A. CIAWON,1981 Elongation of copterygians but rather that thebirchir’s lobed fins had displacement loop strands in human and mouse mitochondrial evolved independently (Figure 6). Fossil evidence (e.g., DNAis arrested near specific template sequences. Proc. Natl. reviewed in CARROLL1988) already indicated that fossil Acad. Sci. USA 78: 6116-6120. FEUENSTEIN,J., 1985 Confidence limits on phylogenies: an ap actinopterygian fish hadendochondrial radial bones proach using the bootstrap. Evolution 39: 783-791. that did not extend far out into the fin. The birchir’s FELSENSTEIN,J., 1989 PHYLJP-phylogeny inference package (Ver- probable phylogenetic positions as an actinopterygian sion 3.4.). 5: 164-166. GARDINER,B. G., 1967 Further notes on palaeoniscoid fishes with would therefore argue for independent evolution of a classification of the Chondrostei. Bull. Br.Mus. (Nat. Hist.) its lobed-fins from the plesiomorphic actinopterygian Geol. 14: 143-206. condition. The disputed homologies (reviewed in e.g., GARDINER,B. G., 1973 Interrelationships of teleostomes, pp. 105- 135 in Interrelationships of Fisha, edited byP. H. GREENWOOD, PATTERSON1980, 1982; AHLBERG and MILLNER1994; R. S. MILES and C. PATTERSON. Academic Press, London. COATES1994) for the axial elements of the pectoral GARDINER,B. G., and A.B. SCHAEFFER,1989 Interrelationships of of fish and land vertebrates and the evolu- lower actinopterygian fishes. Zool. J. Linn. SOC.97: 135-187. GOODRICH,E. S., 1928 Polypterus apalaeoniscid? Palaeobiologica tion of their development (Figure 6) will be facilitated 1: 87-92. by the firm establishment of the phylogenetic position GREENWOOD,P. H., 1974 Review of of the bichir. in . Ann. Geol. Surv. Egypt 4: 211-232. HIXSON,J. E., T. W.WONC and D. A. CIAWON, 1986 Both the We thank GREGERICKSON for insightful comments on the manu- conserved stem-loop and divergent 5’-flanking sequences are re- script. Partial financial support came through grants from the Na- quired for initiation at the humanmitochondrial origin of light- strand replication. J. Biol. Chem. 261: 2384-2390. tional Science Foundation (BSR-9107838 and BSR-9119867), and the HUXLEY,T. H., 1861 Preliminary essay upon the systematic arrange- Max-Planck-Society,Germany to A.M. R.Z. is a postdoctoral fellow of ment of the fishes of the Devonian . Mem. Geol. Surv. the Ministerio de Educacion y Ciencia of Spain. This publicationwas U.K. Decade 10: 1-40, prepared during A.M.’S tenure as John Simon Guggenheim Fellow JANKE,A., G. FELDMAIER-FUCHS,K. THOMAS,A. VON HAESELERand and a Miller Visiting Research Professor at theUniversity of California S. PAABO,1994 The marsupial mitochondrial genome and the at Berkeley. The kind hospitality of the Departments of Integrative evolution of placental mammals. Genetics 137: 243-256. Biology and Molecular and and the Miller Institute at JARWK, E., 1980 Basic Structure andEvolution of Vertebrates., Vol. 1. Berkeley is gratefully acknowledged. Academic Press, London. JESSEN, H. L., 1973 Inten-elationships of actinopterygians and bra- chiopterygians: evidence from pectoral , pp. 227-232 LITERATURE CITED in Interrelationships of Fishes, edited by P. H. GREENWOOD,R. S. MILESand C. PAITERSON.Academic Press, London. ADACHI, J., and M. HASEGAWA,1992 M0LPHY:Program.s forMolecular KIMURA, M., 1980 A simple method for estimating evolutionary rate I-PROTML: Maximum Likelihood Infeence of Protein of base substitutions through comparative studies of nucleotide Phylogeny. Institute of Statistical Mathematics, Tokyo. sequences. J. Mol. Evol. 16: 11 1 - 120. AHLBERG, P. E., and A. R. MILNER,1994 The origin and early diversi- KISHINO,H., and M. HASEGAWA, 1989 Evaluation of the maximum fication of tetrapods. Nature 368: 507-514. likelihood estimateof the evolutionary tree topologies from DNA ANDERSON,S., A. T. BANNER,B. G. BARRELL, M.H. DE BRUIJN,A. R. sequence data, andthe branching order in Hominoidea. J. Mol. COULSON et al., 1981 Sequence and organization of the human Evol. 29: 170-179. mitochondrial genome. Nature 290 457-464. KUMAZAWA,Y., and M. NISHIDA,1993 Sequence evolution of mito- ANDREWS, S. M., 1967 Class Crossopterygii, pp. 641-642 in TheFossil chondrial tRNA genes and deep-branchanimal phylogenetics. J. Record, edited by W. B. HAIU.AND. Geological Society, London. Mol. EvoI. 37: 380-398. ~NASON,U., and A. GUI.I.BERG,1993 Comparison between the LAUDER,G. V., and K. F. LIEM,1983 The evolution and interrelation- 1180 K. Noack, R. Zardoya and A. Meyer

ships of the actinopterygian fishes. Bull. Mus. Comp. Zool. 150: region of mammalianmitochondrial DNA structure-function 95-197. model and evolutionary pattern. J. Mol. Evol. 33 83-91. LE, H. L. V., G. LECOINTREand R. PERASSO,1993 A 28s based SMTOU, N., and M. NEI, 1987 The neighbor-joining method: a new phylogeny of theGnathostomes: first steps in the analysis of method for reconstructing phylogenetic trees. Mol. Biol. Evol. conflict andcongruence with morphologically based cladc- 4: 406-425. grams. Mol. Phyl. Evol. 2: 31-51. SCHAEFFER,B., 1973 Interrelationships of chondrosteans, pp. 207- LEE,W.-J., W. CONROY,H. HOWELLand T. D. KOCHER,1995 Struc- 226 in Interrelationships of Fishes, edited by P. H. GREENWOOD, ture and evolution of mitochondrial control regions. J. R. S. MILESand C. PATTERSON.Academic Press, London. Mol. Evol. 41: 54-66. SHUBIN,N. H., and P. ALBERCH,1986 A morphogenetic approach LEE, W. J., and T. D. KOCHER,1995 Complete sequence of a to the origin and basic organization of the tetrapod limb, pp. lamprey (Petromyzonmarinus) mitochondrial genome: early estab- 319-387 in , edited by M. K. HECHT,B. WM.. lishment of the vertebrate genome organization. Genetics 139: IACE, andG. I. PRANCE.Plenum Press, New York. 873-887. SORDINO, P.,and D. DUBOULE,1996 A molecular approach to the MARTIN, A. P., and S. R. PALUMBI,1993 Protein evolution in differ- evolution of vertebrate paired appendages. Trends Ecol. Evol. ent cellular environments: cytochromebin sharks and mammals. 11: 114-119. Mol. Biol. Evol. 10: 873-891. SOUTHERN,S. O., P. J. SOUTHERNand A. E. DIZON,1988 Molecular MEYER, A.,1993 Evolution of mitochondrial DNA in fishes, pp. 1- characterization of a cloned dolphin mitochondrial genome. J. 38 in and of Fishes, edited by H. A. Mol. Evol. 28: 32-42. MOMMSEN.Elsevier Science, New York. SWOFFORD,D. L., 1993 PAW: Phylogenetic Analysis Using Parsimony. MEYER,A,, 1995 Molecular evidence on the origin of tetrapods and Illinois Natural History Survey, Champaign, IL. the relationships of the coelacanth. Trends Ecol. Evol. 10 11 1 - THOMPSON,J. D., D. G. HIGGINS and T.J. GIBSON,1994 CLUSTAL 116. W. improving the sensitivityof progressive multiple sequence NAYLOR,G. J., T. M. COLLINSand W.M. BROWN,1995 Hydropho- alignmentthrough sequence weighting, position specific gap bicity and phylogeny. Nature 373 555-556. penalties and weight matrix choice. Nucleic Acids Res. 22 4673- NELSON,G. J., 1973 Relationships of clupeomorphs, with remarks 4680. on the structureof the lowerjaw in fishes, pp. 333-349 in Interre- TZENG,C. S., C. F. HUI, S. C. SHENand P. C. HUANG,1992 The lationships ofFishes, edited by P. H. GREENWOOD,R. S. MILESand complete nucleotide sequence of the Crossostomalacustre mito- C. PATTERSON.Academic Press, London. chondrialgenome: conservation and variations among verte- NELSON,G. J., 1994 .John Wiley & Sons, New York. brates. Nucleic Acids Res. 20: 4853-4858. NORMARK,B. B., A. R. MCCUNEand R. G. HARRISON,1991 Phyloge- WALBERG,M. W., and D. A. CLAYTON,1981 Sequence and properties netic relationships of neopterygian fishes, inferred from mito- of the human KB cell and mouse L cell D-Loop regions of mito- chondrial DNA sequences. Mol. Biol. Evol. 8: 819-834. chondrial DNA. Nucleic Acids Res. 9: 5411-5421. ORTI, G., and A. MEYER,1996 Molecular evolution of Ependymin WONG,T. W., and D. A. CLAYTON,1985 In vitro replication of hu- and the phylogenetic resolution of early divergences among eu- manmitochondrial DNA accurateinitiation at the origin of light-strand synthesis. Cell 42 951-958. teleost fishes. Mol. Biol. Evol. 13: 556-573. ZARDOYA,R., and A. MEYER,1996a Thecomplete nucleotide se- PATTERSON,C., 1980 Origin of tetrapods: historical introduction to quence of the mitochondrial genome of the lungfish ( the problem, pp. 159-175 in The Terrestrial Environment and the doZloi), supports its phylogenetic position as a close relative of origzn of Land Vertebrates, edited by A.L. PANCHEN.Academic land vertebrates. Genetics 142: 1249-1263. Press, London. ZARDOYA,R., and A. MEYER,1996b The origin of tetrapods based PATTERSON,C., 1982 Morphology and interrrelationships of primi- on 28s ribosomal RNA sequences. Proc. Natl. Acad. Sci. USA 93: tive actinopterygian fishes. Am. Zool. 22: 241-259. 5449-5454. ROE, B. A,, M. DIN-POW,R. K. WILSONand J. F. WONG,1985 The ZARDOYA, R., and A. MEYER,1996c Phylogenetic performance of complete nucleotide sequence of the Xenopus Zaevis mitochon- mitochondrial protein coding genes in resolving relationships drial genome. J. Biol. Chem. 260: 9759-9774. among vertebrates. Mol. Biol. Evol. 13: 933-942. ROMER,A. S., and T. S. PARSONS,1977 The VertebrateBody, Ed. 5, ZARDOYA,R., A. GARRIDO-PERTIERRAand J. M. BAUTISTA,1995 The Saunders Company, Philadelphia. complete nucleotide sequence of the mitochondrial DNA ge- Russo, C. A. M., N. T~ZAKIand M. NEI, 1996 Efficiencies of differ- nome of the Rainbow trout, mykiss. J. Mol. Evol. ent genes and different tree-building methods in recovering a 41: 942-951. known vertebrate phylogeny. Mol. Biol. Evol. 13: 525-536. SACCONE,C., G. PESOI.Eand E.SBISA, 1991 The main regulatory Communicating editor: N. TAKAHATA