Proc. Natl. Acad. Sci. USA Vol. 95, pp. 6860–6865, June 1998 Evolution Secondary absence of mitochondria in Giardia lamblia and Trichomonas vaginalis revealed by valyl-tRNA synthetase phylogeny (amitochondriate protistsydiplomonadsyparabasalia) TETSUO HASHIMOTO*†‡,LIDYA B. SA´NCHEZ†,TETSUROU SHIRAKURA*, MIKLO´S MULLER¨ †, AND MASAMI HASEGAWA* *The Institute of Statistical Mathematics, 4–6-7 Minami-Azabu, Minato-ku, Tokyo 106, Japan; and †The Rockefeller University, New York, NY 10021 Communicated by William Trager, The Rockefeller University, New York, NY, March 27, 1998 (received for review December 29, 1997) ABSTRACT Nuclear-coded valyl-tRNA synthetase evolutionary origins of the amitochondriate condition. Before (ValRS) of eukaryotes is regarded of mitochondrial origin. any molecular phylogenetic data became available, cytological Complete ValRS sequences obtained by us from two amito- considerations led to the proposal by Cavalier-Smith that chondriate protists, the diplomonad, Giardia lamblia and the diplomonads, parabasalids, and microsporidia could be prim- parabasalid, Trichomonas vaginalis were of the eukaryotic itively amitochondriate (15, 16), and to the erection of the type, strongly suggesting an identical history of ValRS in all taxon Archezoa for these organisms. These three amitochond- eukaryotes studied so far. The findings indicate that riate lineages represented the first branches on phylogenetic diplomonads are secondarily amitochondriate and give fur- trees based on rRNA (17, 18) and some protein sequences (19). ther evidence for such conclusion reached recently concerning This observation was regarded as compelling evidence for an parabasalids. Together with similar findings on other amito- early separation of these groups from the main eukaryotic chondriate groups (microsporidia and entamoebids), this lineage, preceding the acquisition of mitochondria (20). Other work provides critical support for the emerging notion that no amitochondriate organisms, notably the type II ciliates and representatives of the premitochondrial stage of eukaryotic chytrid fungi, however, belong to lineages that consist domi- phylogenesis exist among the species living today. nantly of mitochondriate species, strongly suggesting their secondary amitochondriate nature (21). One of the goals of According to the generally accepted serial endosymbiotic current research on such organisms is to obtain information, theory, the complex eukaryotic cell is the result of the merger which could settle the question whether the amitochondriate of an eubacterium, probably an a-proteobacterium, into a condition, at least in some organisms, represents an ancestral, poorly understood ‘‘host,’’ assumed to be related to the ‘‘premitochondrial’’ feature or that all these organisms de- archaebacteria. According to this theory the eubacterium scended from ancestors that once harbored mitochondria and became the mitochondrion and the ‘‘host’’ provided the nu- lost secondarily the characteristic bioenergetic functions of the cleocytoplasmic compartment of the eukaryotic cell (1–3). mitochondrion and also the organelle itself. A widely exploited This integration was accomplished by endosymbiotic transfer approach is the search in amitochondriate species for genes of a considerable portion of the genes of the eubacterium to that could have been part of the genome of the ancestral the genome of the ‘‘host’’ (1, 3, 4). However, certain unicellular mitochondrion but were subsequently transferred to the nu- organisms belonging to diverse taxonomic groups, while they clear genome. display a number of characteristics of typical eukaryotic cells, Such genes have been recently detected in several amito- lack mitochondria, as demonstrated by biochemical and mor- chondriate organisms (discussed in refs. 22–25). The most phological findings (5–7). Whenever studied, no evidence of a compelling data concern heat shock proteins, molecular chap- second, organellar genome was found in such organisms (8, 9). erones, known to play a key role in mitochondrial biogenesis. Amitochondrial eukaryotes can be assigned to at least two Genes for such proteins are present in the nuclear genomes of major categories, based on the subcellular organization of their amitochondriate organisms belonging to microsporidia (26, metabolism (5, 6, 10, 11). Both groups comprise of several 27), parabasalids (28–31), and entamoebids (32). These results independent taxa. Type I organisms lack any compartmenta- argued strongly against an ancestral amitochondriate nature of tion of their core (energy) metabolism. Diplomonads (includ- these three groups. Of the better studied ‘‘early-branching’’ ing Giardia lamblia) and entamoebids belong to this type. organisms only G. lamblia and other diplomonads remained as Although the core metabolism of microsporidia remains un- possible candidates for being primitively amitochondrial, known, their morphology was taken as evidence suggesting seemingly in accord with widespread views that these organ- that they also are of type I (7, 11). Type II organisms, such as isms represent one of the most ancestral eukaryotic group (17, parabasalids (including Trichomonas vaginalis), certain heter- 20, 33). olobosea, some ciliates and chytrid fungi, contain a distinct, Aminoacyl-tRNA synthetases are essential enzymes for double-membrane bounded organelle, the hydrogenosome protein synthesis, and are useful for tracing the early evolution (12, 13). Although hydrogenosomal metabolism markedly of life (34). To explore the status of G. lamblia, we selected differs from that of mitochondria, the two organelles share a valyl-tRNA synthetase (ValRS) as a potential marker for a number of functional properties (12, 13). mitochondrial endosymbiotic event in the past. Only a single The existence of amitochondriate organisms challenged the gene for ValRS is present in fungi, and the product of this gene notion that the mitochondrion is a sine qua non attribute of eukaryotic cells (7, 14). It also raised the question of the Abbreviations: IleRS, isoleucyl-tRNA synthetase; JTT, Jones-Taylor- Thornton; ML, maximum likelihood; QP, quartet puzzling; ValRS, The publication costs of this article were defrayed in part by page charge valyl-tRNA synthetase. Data deposition: The sequences reported in this paper have been payment. This article must therefore be hereby marked ‘‘advertisement’’ in deposited in the GenBank database (accession nos. AB008525– accordance with 18 U.S.C. §1734 solely to indicate this fact. AB008530). © 1998 by The National Academy of Sciences 0027-8424y98y956860-6$2.00y0 ‡To whom reprint requests should be addressed. e-mail: hasimoto@ PNAS is available online at http:yywww.pnas.org. ism.ac.jp. 6860 Downloaded by guest on October 2, 2021 Evolution: Hashimoto et al. Proc. Natl. Acad. Sci. USA 95 (1998) 6861 has been shown to be functional not only in cytosol but also in vaginalis (this work), G. lamblia (this work), Escherichia coli mitochondria (35, 36). A single ValRS gene has been detected (X05891), Thermus thermophilus (Y10900), Bacillus subtilis also in Homo sapiens with its product similarly assumed to have (X77239), Bacillus stearothermophilus (M16318), Lactobacillus cytosolic and mitochondrial functions (37). A ValRS phylog- casei (L08854); [IleRS]: H. sapiens (D28473), Caenorhabditis eny rooted by isoleucyl-tRNA synthetases (IleRS) revealed elegans (Z81038), S. cerevisiae (X07886), Schizosaccharomyces that eukaryotic ValRS sequences form a monophyletic clade pombe (AB004538), Tetrahymena thermophila (M30942), that shows the closest affinity with Gram-negative bacteria Nosema locustae (L37097), Mycobacterium tuberculosis (38). On the basis of these findings Brown and Doolittle (38) (Z74020), Staphylococcus aureus (plasmid) (X75439), Sulfolo- concluded that the gene coding for this protein in mitochon- bus acidocaldarius (L37106), Methanobacterium thermoau- drion-containing eukaryotes has been transferred to the nu- totrophicum (M59245), Pyrococcus furiosus (L37105), E. coli cleus from the ancestral mitochondrion (38, 39). Thus the (X00776), Campylobacter jejuni (U15295), Pseudomonas fluo- presence or absence of typical eukaryotic ValRS in amito- rescens (X80132), Aquifex pyrophilus (L37096), S. aureus chondriate protists could provide an important clue to deter- (X74259), Thermotoga maritima (L37104),and S. cerevisiae mine whether any given organism did harbor mitochondria in (mitochondrion) (L38957). its evolutionary past. For the following bacterial species, the ValRS andyor IleRS We sequenced the complete gene encoding ValRS from G. sequences were obtained from the Institute for Genomic lamblia and also from T. vaginalis and aligned the deduced Research Microbial Databases (http:yywww.tigr.orgytdby): amino acid sequences with all ValRS and IleRS sequences Haemophilus influenzae, Helicobacter pylori, Synechocystis sp., available in the databases.The evidence obtained shows that G. Chlamydia trachomatis, Borrelia burgdorferi, B. subtilis (IleRS), lamblia represents no exception and also carries the relics of a Mycoplasma genitalium, Mycoplasma pneumoniae, Archaeoglo- past mitochondrial endosymbiotic event. This conclusion is in bus fulgidus, Methanobacterium thermoautotrophicum accord with earlier data pointing in the same direction (40– (ValRS), and Methanococcus jannaschii. 42). The very recent demonstration that this species contains Phylogenetic Analysis. The maximum likelihood (ML) and expresses a chaperonin 60 gene closely related to the method of