Copyright 0 1996 by the Genetics Society of America The Complete Nucleotide Sequence of the Mitochondrial Genome of the Lungfish (A-otqPterus dolloi) Supports Its Phylogenetic Position as a Close Relative of Land Vertebrates Rafael Zardoya and Axel Meyer Department of Ecology and Evolution and Program in Genetics, State University of New York, Stony Brook, New York 11 794-5245 Manuscript received June 10, 1995 Accepted for publication January 13, 1996 ABSTRACT The complete DNA sequence (16,646 bp) of the mitochondrial genome of the African lungfish, Protopterusdolloi, was determined. The evolutionary position of lungfish as possibly the closest living relative among fish of land vertebrates made its mitochondrial DNA sequence particularly interesting. Its mitochondrial gene order conforms to the consensus vertebrate gene order. Several sequence motifs and secondary structures likely involved inthe regulation of the initiation of replication and transcription of the mitochondrial genome are conserved in the lungfish and are more similar to those of land vertebrates than those of ray-finned fish. A novel feature discovered is that the putative origin of L- strand replication partially overlaps the adjacent tRNAcys.The phylogenetic analyses of genes coding for tRNAs and proteins confirm the intermediate phylogenetic position of lungfish between ray-finned fishes and tetrapods. The complete nucleotide sequence of the African lungfish mitochondrial genome was used to estimate which mitochondrial genes are most appropriate to elucidate deep branch phyloge- nies. Only a combined set of either protein or tRNA mitochondrial genes (but not each gene alone) is able to confidently recover the expected phylogeny among vertebrates that have diverged up to but not over -400 mya. HE transition from life in water to life on land, tologists, comparative morphologists and recently de- T -360 mya (BENTON1990), was one of the most velopmental biologists. consequential events in the history of vertebrates. It was The other extant groupof lobe-finned fish, the coe- accompanied by a variety of refined morphological and lacanths were believed to have gone extinct -80 mya, physiological modifications, e.g., reductions and rear- but in 1938, the only surviving species of this lineage rangements of the skull bones and modifications of of fishes was discovered off the coast of East Africa. swimming fins into load-bearing limbs (e.g., PANCHEN Since its sensational rediscovery, the coelacanth is of- and SMITHSON1987). Two groups of lobe-finned fish ten depicted in textbooks as the “missing link” be- (Table l),the lungfish and the coelacanth (Latimm’u tween fish and all land vertebrates i.e., amphibians, chalumnae) have both been implicated as the closest reptiles, birds and mammals (ROMER 1966).However, living relative of tetrapods (reviewed in MEYER 1995). many morphological, paleontological (e.g., reviewed Lungfish were discovered > 150 years ago (BISCHOFF in PATTERSON1980; ROSENet al. 1981), andmost mo- 1840) and for several reasons, e.g., they are obligate lecular (MEYERand WILSON1990; MEYERand DOLVEN airbreathers, were initially believed to be amphibians, 1992; HEDGESet al. 1993; reviewed in MEYER1995; but not fish. In the lower Devonian (-400 mya) lungfish see YOKOBORI et al. 1994) data suggest that lungfish were a speciesrich group that inhabited both marine and not the coelacanth are more closely related to and freshwater environments (e.g., reviewed in tetrapods.Hence, the nucleotide sequence of the CLOUTIER1991). However, onlya very small number of lungfish mitochondrial genome is of interest, both in “relict” species survive today. These are the Australian terms of the evolution of the mitochondrial gene or- lungfish, Neoceratodus forsteri, the South American lung- der in vertebrates and in terms of the phylogeny of fish, Lqbidosiren paradoxa, and four species in the genus land vertebrates. Protqterus from Africa. These living fossils are of inter- Until now, the complete mitochondrial DNA se- est to evolutionary biology since their morphology, quences of 19 vertebrate species have been reported. physiology, and biochemistry might be representative Thirteen of them are from mammals, four from fishes, of that of the common ancestor of all land vertebrates. but only one of an amphibian and one of a bird have Therefore, lungfish have been widely studied by paleon- been determined. Remarkably, the structure and orga- nization of vertebrate mitochondrial genomes is quite Corresponding authm: Rafael Zardoya, Department of Ecology and Evolution, State University of New York, Stony Brook, NY 117945245. conserved and only minor rearrangements have been E-mail: [email protected] described for the chicken (DESIARDINSand Mows Genetics 142 1249-1263 (April, 1996) 1250 R. Zardoya and A. Meyer TABLE 1 HindIlI Systematic position of lungfish Class: Osteichthyes (bony fish) Subclass: Actinopterygii (ray-finned fish) HindllI Chondrostei (sturgeon, Acipenser; bichir, Polypterus) Neopterygii (gar, Lepisosteus; bowfish, Amia; modern ray-finned fish, Teleostei) Protopterus dolloi \:\ Subclass: Sarcopterygii (lobe-finned fish) Actinistia (coelacanth, Latimeria) Rhipidistia Dipnoi (lungfish) Porolepiformes" Osteolepiformes" Tetrapoda (land vertebrates) Lissamphibia (modern amphibians) Amniota (reptiles, birds, mammals) EcoRI /} 2 \ Modified from CARROIL(1988) and AHIBERG (1991). Posi- Hind111 EroRl tion of lungfish in bold. " Extinct. FIGURE1 .-Restriction map and gene organization of the Protopterus dolloi mitochondrial genome. All protein coding 1990) and the opossum ( JANKE et al. 1994). Changes genes are encodedby the H-strand with the exception of iW6, which is coded by the Lstrand. Each tRNA gene is identified in gene order seem to be associated with the potential by the single letter amino acid code and depicted according capability of tRNAs to translocate, although sometimes to the coding strand. Only the EcoRI and Hind111 restriction other genes are involved in transpositions as well. It is sites used for cloning are shown. not yet clear when the establishment of the vertebrate consensus gene order occurred during their evolution. MATERIALSAND METHODS The lamprey, one of the earliest vertebrates, has a pecu- Mitochondrial DNA was purified from fresh eggs of a single liar gene order (LEEand KOCHER 1995). Although it individual of the African lungfish (Protopterus dolloi) as pre- is similar to that of other vertebrates, it has enough viously described (ZARDOYAet al. 1995a). After homogeniza- tion, intact nuclei and cellular debris were removed by a low- differences (i.e., rather than one major noncoding two speed centrifugation (1000 X g). Mitochondria were pelleted regions, the missing 01,in the WANCY region, and by spinning at 10,000 X g for 20 min and subjected to a translocations of cytochrome b, and tRNApr", tRNA?"", standard alkaline lysis procedure followed by a phenol/chlo- and tRNA"'" are found)to be considered to bea unique roformextraction. The isolated mtDNA was cleaved with and possibly a derived condition from the vertebrate EcoRI and HindIII restriction enzymes (see Figure 1 for posi- tions of restriction sites). Three EcoRI fragments of 7.4, 3.1 consensus mitochondrial gene order. Despite the slow and 0.7 kb and five HindIII fragments of 3.3, 2.9, 2.9, 2.2 and rate of evolution of gene order, nucleotide sequence 1.1 kb were cloned into pUC18 covering the entire lungfish evolution of animal mitochondrial DNA is rapid. The mtDNA molecule. In addition, the 7.4-kb EcoRI fragment was dynamic evolution of mitochondrial DNA sequences subcloned with Suu3A to facilitate sequencing. occurs through the accumulation of point mutations Plasmid DNA was extracted from each clone using a Magic and make them particularly valuable for estimating phy- miniprep kit (Promega). After ethanol precipitation, cloned DNA was used as template for Taq Dye Deoxy Terminator logenetic relationships among closely related species cycle-sequencing reactions (Applied Biosystems Inc.) follow- (BROWNet al. 1979). However, not all mitochondrial ing manufacturer's instructions. Sequencing was performed genes evolve at the same rate (e.g., reviewed in MEYER with an automated DNA sequencer (Applied Biosystems 373A 1993), and some genes are more appropriate than oth- Stretch). Sequences were obtained using both M13 universal ers for inferring phylogenetic relationships among dis- sequencing primers and 40 specific oligonucleotide primers. The sequences obtained from each clone were -350 bp in tantly related species. length and eachsequence overlapped the nextcontig by We determined the complete nucleotide sequence -100 bp. In no case were differences in sequence observed and the gene order of the African lungfish mitochon- between the overlapping regions. The location and sequence drial genome. The aims of this study were to reconstruct of these primers will be provided by the authors uponrequest. mitochondrial genome evolution, estimate which mito- Sequence data were analyzed by use of the GCG program package (DEVEREUXet al. 1984) and alignments and phyloge- chondrial genes (tRNA, rRNA, or proteincoding) are netic analyses were performed using CLUSTAL V (HIGGINS most appropriate to elucidate deep branch phyloge- and SHARP1989), PAUP Version3.1.1 (SWOFFORD 199S), nies, and clarify lungfish relationships to tetrapods and PHYLIP Version 3.5 (FELSENSTEIN1989), and MOLPHY Ver- to ray-finned fish (Actinopterygii, Table 1).We are pres- sion 2.2 (ADACHI and HASEWAGA,1992). ently