phylogenetic place of mitochondrion-lacking protozoan, giardia

Phylogenetic Place of Mitochondrion-Lacking Protozoan, Giardia Zamblia, Inferred from Amino Acid Sequences of Elongation Factor 2

Tetsuo Hashimoto, * Yoshihiro Nakamura,$ Takashi Kamaishi,$ll Fuminori Nakamura,§ Jun Adachi,? Ken-ichi Okamoto,§ and A4asami Hasegawa*? *Institute of Statistical Mathematics; -/=Department of Statistical Science, Graduate University for Advanced Studies; /Laboratory of Gene Manipulation, Showa University; $Department of Medical Biology, Showa University; )I Department of Fisheries, University of Tokyo

Partial regions of the mRNA encoding a major part of translation elongation factor 2 (EF-2) from a mitochondrion- lacking protozoan, Giardia lamblia, were amplified by polymerase chain reaction, and their primary structures were analyzed. The deduced amino acid sequence was aligned with other eukaryotic and archaebacterial EF-2’s, and the phylogenetic relationships among eukaryotes were inferred by the maximum likelihood (ML) and the maximum parsimony (MP) methods. The ML analyses using six different stochastic models of amino acid substitutions and the MP analysis consistently suggest that among eukaryotic species being analyzed, G. lamblia is likely to have diverged from other higher eukaryotes on the early phase of eukaryotic evolution.

Introduction

There are more than a thousand species of protozoa and a few fungi that have no mitochondrion, although they are eukaryotes. Phylogenetic placing of mitochondrion-lacking protozoa is crucial in clarifying the early evolution of eukaryotes. Some of these protozoa may be living relics of the earlist phase of eukaryotic evolution before the symbiotic origin of mitochondria, while the others may have secondarily lost mitochondria due to their parasitic lifestyle (Cavalier-Smith 1987, 1989, 199 1) . On the basis of the sequence comparisons of small subunit ribosomal RNA ( SrRNA ), Sogin ( 199 1) , Sogin et al. ( 1989a, 1989b), and Leipe et al. ( 1993) proposed a eukaryotic tree including mitochondrion-lacking protozoa. According to the up-to-date SrRNA tree by Leipe et al. ( 1993)) among mitochondrion-lacking protozoa, Vairimorpha necatrix, Tritrichomonas foetus, and Giar- dia Zamblia represent the earliest, the second earliest, and the third earliest offshoots, respectively, while the other mitochondrion-lacking protozoan, Entamoeba histolytica, separates from higher eukaryotes after mitochondrion-containing protozoa, such as Euglena gracilis and Trypanosoma brucei, have diverged.

However, the G+C content of SrRNA is sometimes biased drastically among species, as well as genome G+C content. Because it is difficult in general to take account

Key words: Giardia lamblia, mitochondrion-lacking protozoa, eukaryotes, elongation factor 2, protein phylogeny, bias of G+C content.

Address for correspondence and reprints: Tetsuo Hashimoto, In- stitute of Statistical Mathematics, 4-6-7 Minami-Azabu, Minato-ku, Tokyo 106, Japan. E-mail: [email protected].

Mol. Biol. Evol. 12(5):782-793. 1995. 0 1995 by The University of Chicago. All rights reserved. 0737-4038/95/1205-0007$02.00

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of the effect of the bias in inferring evolutionary tree, the SrRNA tree may sometimes be unreliable (Hasegawa and Hashimoto 1993). From a protein phylogeny of translation elongation factor 1 a ( EF- 1 a), we previously suggested that as well as G. lamblia, E. histolytica also represents an outgroup to E. gracilis and higher eukaryotes in disagreement with the widely accepted SrRNA tree (Hasegawa et al. 1993; Hashimoto et al. 1994). In addition, we clearly demonstrated that amino acid compositions of conservative proteins are free from the drastic bias of genome G+C content and that a protein phylogeny will give a more robust estimation of the early divergence of eukaryotes (Hasegawa et al. 1992a; Hashimoto et al. 1992, 1994; Shirakura et al. 1994). Hence, it seems better to infer an evolutionary tree from the amino acid sequences of conservative proteins, such as translation elongation factors, RNA polymerases, and ATPases, rather than from SrRNA sequences. Reex- amination of the place of mitochondrion-lacking protozoa by using these conservative proteins is highly desirable.

Recently we have reported a major part of the amino acid sequence of translation elongation factor 2 ( EF-2 ) from a mitochondrion-lacking protozoan, E. histolytica. According to the phylogenetic analyses on the basis of the ML method, the divergence of E. histolytica was likely to be earlier than those of the other higher eukaryotic species ( Shirakura et al. 1994). Here we also report a major part of EF-2 from the other mitochondrion-lacking protozoan, G. Zamblia, and further analyze the phylogenetic relationships among eukaryotes. Our ML analyses using six different stochastic models of amino acid substitutions and an MP analysis

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Phylogenetic Place of Giardia 783

consistently suggest that G. lamblia is the earliest offshoot of the EF-2 tree among eukaryotic species being analyzed.

Giardia lamblia, classified to the order Diplomon- adida (see Cavalier-Smith 1993)) is a single-celled par- asite which infects the intestine of humans and other mammals and may cause diarrhea. It has a number of unique metabolic features, including a predominantly anaerobic metabolism, complete dependence on salvage of exogenous nucleotides, a limited ability to synthesize and degrade carbohydrates and lipids (see Adam 199 1) . Furthermore, it has two nuclei that are haploid by all criteria having been tested (Kabnick and Peattie 1990, 199 1) . EF-2, the homologue of eubacterial elongation factor G (EF-G), catalyzes the GTP-hydrolysis dependent translocation of peptidyl-tRNA from the aminoacyl site to the peptidyl site on the ribosome and is essential for protein synthesis in all organisms. EF-2 /EF-G is a useful protein for tracing the early evolution of life, because of its universal occurrence in all living organisms on earth and because of its relatively slow evolutionary rate (Iwabe et al. 1989; Miyata et al. 199 1).

Material and Methods Nucleotide Sequence Analysis

Giardia lamblia Polish strain was maintained in culture at 37°C in Diamond’s medium TYI-S-33 (Keis- ter 1983) supplemented with 10% newborn calf serum, bovine bile (0.5 mg ml -’ ) , and L-cystein ( 2 mg ml -’ ) . Trophozoites adherent to the inner surface of the culture bottles ( 1 X 10 6 ml -I ) were harvested at 3,000 X g for 10 min after cooling the culture on ice for 10 min. Ex- traction of total and poly (A)+ RNAs was performed by standard techniques (Sambrook et al. 1989). Poly (A)+ RNA was treated with Murine reverse transcriptase ( Pharmacia LKB ) , and the resultant RNA:cDNA duplex was used as a template for polymerase chain reaction (PCR) using Taq polymerase ( Perkin-Elmer Cetus) . Cycling conditions were 95°C (1 min), 55°C (2 min), 72°C (5 min) for 30 cycles. Primers for PCR amplifications described in the legend of figure 1 were synthesized on the Applied Biosystems model 38 1 A DNA Synthesizer. Amplified fragments were purified and li- gated with the plasmid, pUC 18, digested with Sma I.

Escherichia coli strain JM 109 was transformed by the plasmid using the method of Hanahan ( 1983). Plas- mid DNA was prepared by the alkaline method (Sam- brook et al. 1989). For sequencing the PCR amplified and then the cloned fragments, more than three inde- pendent clones were sequenced with both strands using double-stranded dideoxy sequencing method (Sambrook et al. 1989, Sequenase ver. 2, USB).

Southern Blot Analysis

Chromosomal DNA of G. lamblia was isolated by standard techniques ( Sambrook et al. 1989). Five ug of the DNA was digested with restriction endonucleases. The resultant fragments were separated by electropho- resis on a 1 .O% agarose gel, transferred onto nylon mem- brane (Hybond-N, Amersham), and fixed by UV stra- talinker (Funakoshi Co.). The plasmid containing the PCR amplified 1.3 kb product (fragment II in fig. 1) was digested with restriction endonucleases Eco RI and Pst I, and the fragment was 32P-labeled by the Ramdom Primer Extension Labeling System ( DuPont / NEN) . The labeled product was purified by NENSORBTM 20 (DuPont /NEN), and used as a probe. Hybridization with the 32P-labeled probe was carried out at 60°C over- night in a buffer containing 6 X SSC, 5 X Denhardt’s solution, 0.5% SDS, and 50 ug/ml denatured salmon sperm DNA (Sambrook et al. 1989).

Phylogenetic Analyses

EF-2 sequences from seven eukaryotes and three archaebacteria in addition to that from G. lamblia were used for phylogenetic analyses. Species were at first classified into following seven lineages: ( 1) Animalia- Homo sapiens (Rapp et al. 1989)) Drosophila melanogaster (Grinblat et al. 1989)) Caenorhabditis elegans (Ofulue and Candid0 199 1) ; ( 2) Fungi- Saccharo- myces cerevisiae (Perentesis et al. 1992); (3) green algae-Chlorella kessleri ( Schnelbogl and Tanner 199 1) ; (4) Dictyostelium discoideum (Toda et al. 1989); (5) Entamoeba histolytica (Shirakura et al. 1994); (6) Giardia lamblia (this work); and ( 7) archaebacteria- Sulfolobus acidocaldarius ( Schroder and Klink 199 1) , Methanococcus vannielii ( Lechner et al. 1988), Halo- bacterium halobium (Itoh 1989 ) .

Pairwise alignments of amino acid sequences were inferred by a maximum likelihood alignment method (Thorne et al. 199 1, 1992). The method assumes an evolutionary model by allowing multiple-residue insertion and deletion events as well as regional heterogeneity in the substitution process. By an expectation-maximi- zation (EM) algorithm introduced by Thorne and Churchill ( 1995 ), we can obtain a maximum likelihood alignment with parameter estimates of insertion /deletion and substitution events. All possible pairwise alignments among the above 11 sequences were inferred by the program Statprot (kindly provided by Dr. J. L. Thorne) . On the basis of these results, a multiple alignment was constructed by manually adjusting inconsis- tencies found among the pair-wise alignments. A total of 945 unrooted tree topologies, corresponding to the above seven lineages, were examined by the ML and the MP methods. We assumed a relationship among the three


784 Hashimoto et al.

archaebacterial species as (Sulfolobus, (Halobacterium, Methanococcus)), according to Miyata et al. ( 199 1). However, a trifurcating tree topology was assumed for the three animals because we could not resolve relationships among the Animalia with statistical significance.

To evaluate the robustness of the ML analyses against the violation of assumed Markov models, six different models of the amino acid substitutions were used: ( 1) the JTT model using recently compiled amino acid substitution tables by Jones et al. ( 1992) (Cao et al. 1994a) ; (2) the JTT-F model ( “F” option of the JTT model) (Cao et al. 19946); (3) the Dayhoff model (Kishino et al. 1990); (4) the Dayhoff-F model (“F” option of the Dayhoff model) (Cao et al. 1994b); (5 ) the proportional model (Hasegawa et al. 19923); and (6) the Poisson model (Hasegawa et al. 19923). When the “F” option is introduced, models are reconstructed by using the actual amino acid compositions of the protein under analysis as the equilibrium compositions. The proportional model corresponds to the “F” option of the Poisson model. Standard errors (SEs) of log-likelihood differences were estimated by equation ( 12) in Kishino and Hasegawa ( 1989 ) . Bootstrap probability for tree i, Pi, being the ML tree among the 945 alternatives, was estimated by the RELL method (Kishino et al. 1990; Hasegawa and Kishino 1994). The goodness of approximation for different models was compared by the Akaike Information Criterion ( AIC), defined as AIC = -2 X (log-likelihood) + 2 X (no. of free parameters). The model that minimizes AIC is considered to be the most appropriate (see Sakamoto et al. 1986). All com- putations for the ML analyses were performed by the program PROTML in MOLPHY ver. 2.2 (Adachi and Hasegawa 1994).

The maximum parsimony analysis based on Fel- senstein’s program, PROTPARS in PHYLIP version 3.2 was also applied for the same data set to compare the results presented by the ML analyses. Standard errors of the differences of number of substitutions were estimated by equation ( 26) in Kishino and Hasegawa (1989).

Results Nucleotide Sequence Analysis

Sets of PCR primers used, locations of amplified fragments, and a restriction map deduced from the sequencing results are shown schematically in figure 1. First, a pair of degenerate oligonucleotide primers, Al and A2, corresponding to the highly conserved amino acid sequences, GAGELHLE and FPQCVFDHW, respectively (see fig. 2), was used, and a 0.8 kb amplified product (fragment I) was cloned into pUC 18. After sequence analysis of the fragment I, another pair that included a degenerate primer, B 1, and a unique primer,

61 82

Cl - c2 Al is

I Sal1

/I\ \ Sal1 Sac I Hind111 Sac I

I I‘\ HIndUI BarnHI MI PstI

FIG. 1 .-Locations of PCR primers and sequencing strategies. Regions of the primers used for PCR amplifications are shown over the bold line, which represents a major region of Giardia lumblia EF- 2 gene. The primers synthesized were: Al: 5’-GGNGCNGGNGAR- YTNCAYYTNGA-3’, A2: 5’-CCARTGRTCRAANACRCAYTGN- GGRAA-3’, B 1: 5’-GGNGTNTGYGTNCARACNGARACNGT-3’, B2: 5’-GATCCGAGACGCGGATGTCCAT-3’, C 1: 5’-AAYATGWS- NGTNATHGCNCAYGTNGA-3’, and C2: 5’-ACTTTGTT- GAGCATGAGGCACG-3’. Restriction sites deduced from sequencing results are shown under the bold line. Amplified fragments corresponding to the three sets of primers are shown by thin lines (see text).

B2, was used for further amplification of the upstream region. The degenerate primer, Bl, corresponded to the conserved amino acid sequence, GVCVQTETV (see fig. 2), and the unique primer, B2, was synthesized com- plementarily to the 3’ portion of the primer, Al, the nucleotide sequence of which had already been deter- mined. An amplified product (fragment II) showed 1.3 kb, and was cloned into pUC 18 for sequence analysis. Similarly, the final amplification was performed by using a degenerate primer, Cl, corresponding tc NMSVIAHVD (see fig. 2), and a unique primer, C2: complementary to the 3 ’ portion of the primer, B 1. This yielded a 0.5 kb product ( fragment III), and it was cloned and sequenced.

By sequence analyses of the fragments, I, II, and III, 775, 1,279, and 475 bases, respectively, were deter- mined. A total of 2,457 bases, excluding overlapped regions between the fragments I and II and between the fragments II and III revealed an uninterrupted reading frame, corresponding to the 8 19 amino acid sequence that was highly similar to a major part of the eukaryotic EF-2. Thus, the frame was identified as a part of the Giardia lamblia EF-2 mRNA. The G+C content of all the 2,457 bases and of the third codon positions showed 56.9% and 74.6%, respectively. The high G+C conteni in the third codon positions resulted in biased codon usage preferring G and C (data not shown), but the bias was not so great in comparison with the codon usage pattern of the previously characterized EF- 1 a coding region of G. Zamblia. The G+C content of the third codon positions of G. Zamblia EF-la was 99.5% (Hashimotc et al. 1994).

To make sure that the EF-2 sequence obtained by the PCR method really originated from G. Zamblia and



to estimate a copy number of EF-2 gene, Southern blot analysis of genomic DNA was performed. The analysis using the 1.3 kb probe containing the fragment II shown in figure 1 clearly suggested that there is only one copy of the gene for EF-2 in the G. Zamblia genome (data not shown ) .

Protein Sequence Comparisons

The predicted amino acid sequence excluding both the N- and C-terminal regions of G. lamblia EF-2 was aligned with the other seven eukaryotic and three archaebacterial EF-2’s. Alignment is shown in figure 2. The seven eukaryotic sequences were closely related, and an unambiguous alignment was obtained among them. As remarkable gap regions, insertions of 12 and 35 residues, respectively, in Caenorhabditis elegans and G. lamblia (positions 96- 130)) a 10 residue insertion in G. lamblia (positions 234-244)) a 13 residue insertion in Homo sapiens (positions 288-300), and a 2 1 residue deletion in Dictyostelium discoideum (positions 658- 678) were introduced to align seven sequences. Among the three archaebacterial sequences, an unambiguous alignment was also constructed with minor insertions and deletions. However, when eukaryotic and archaebacterial sequences were aligned with each other, very wide deletions were necessary for the archaebacterial sequences between positions 274 and 393. The 35 residue insertion in G. Zamblia (positions 96-l 30) and the 2 1 residue deletion in D. discoideum (positions 658-678)) both of which occurred in the alignment among eukaryotic EF-2s, were also necessary to align these sequences with the archaebacterial ones. With the exceptions of the above wide gap regions, eukaryotic and archaebacterial EF-2s demonstrated a high degree of conservation. On the basis of the alignment, 530 sites unambiguously aligned were selected and used for an- alyzing phylogenetic relationships among eukaryotes. Regions selected are indicated by aligned positions as follows: 32-83, 135-229, 255-273, 35 l-364, 394- 451,472-485, 507-536, 574-607,611-630,641-657, 679-694, 696-700, 702-709, 7 19-728, 749-858, and 864-89 1.

Phylogenetic Analyses

Phylogenetic relationships among six eukaryotic lineages, G. lamblia, Entamoeba histolytica, D. discoideum, Chlorella kessleri, Saccharomyces cerevisiae, and Animalia, were analyzed by using archaebacteria as an outgroup. The ML methods based on the JTT, the JTT- F, the Dayhoff, the Dayhoff-F, the proportional, and the Poisson models and the MP method were examined for a total of 945 possible tree topologies. On the basis of the results of an ML analysis using the JTT-F model,

we selected 13 alternative trees as candidates for a real tree, because the JTT-F model best approximated the data. These are an ML tree and 12 alternatives which could not be significantly discriminated from the ML tree by the criterion of 1 SE of log-likelihood differences. Table 1 only summarizes the results of the JTT-F model and of the MP analysis for these 13 trees. The ML analyses, assuming six different models of amino acid substitutions, consistently gave an ML tree (tree- 1)) in which G. Zamblia represents the earliest offshoot among eukaryotes and in which C. kessleri and D. discoideum, respectively, represent the second and the third earliest offshoots next to G. lamblia. The AICs of the ML trees showed 16450.9, 164 14.5, 16642.6, 16507.6, 17494.4, and 17992.9 for the JTT, the JTT-F, the Dayhoff, the Dayhoff-F, the proportional, and the Poisson models, respectively. Comparison of these values clearly indicates that the JTT-F model is by far the best among the alternative models used in the ML analyses. On the other hand, being inconsistent with the ML analyses, the MP analysis showed tree-5 as the MP tree, in which G. Zam- blia represents the earliest offshoot, and E. histolytica and D. discoideum, respectively, represent the second and the third earliest offshoots next to G. Zamblia, although the difference of substitution numbers of the tree- 1 (ML tree) from the MP tree (tree-5 ) was only 5 f 9.1. Each tree shown in table 1 places G. Zamblia as the earliest offshoot of eukaryotes, but the species that branches off next to G. lamblia is not always consistent among the 13 alternative trees.

Totals of bootstrap probabilities for the trees that support various topological elements were summarized in table 2 for the JTT-F model and the MP analysis. Totals of those for the 105 trees in which G. Zamblia represents the earliest offshoot of eukaryotes amounted to more than 0.85 both in the JTT-F model and the MP analysis. Totals of those in which E. histolytica or C. kessleri represents the earliest offshoot showed far lower values, but we cannot entirely exclude this possibility. However, the placing of D. discoideum, S. cerevisiae, or Animalia as the earliest offshoot was quite unlikely (data not shown). With respect to the second earliest offshoot next to G. Zamblia, totals of bootstrap probabilities for the 15 trees were shown in table 2 for three different possibilities. We cannot deny any possibility that E. histolytica, D. discoideum, or C. kessleri represent the second earliest offshoot next to G. lamblia. As well as the differences of log-likelihoods and of substitution numbers shown in table 1, the totals of bootstrap probabilities also cannot confirm the species that would have diverged next to G. lamblia.

On the other hand, totals of bootstrap probabilities for the 105 trees linking Animalia with S. cerevisiae


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110 120 130 J&j__ 150 160 170 180 190_______200 G3 G4

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210 220 230 240 250 260 270 280 290 300

VEDMMKKLWGDRYFDPANGKFSKSATSPEGKKLPRTFCQLILDPIFKVFDAIMNFKKEETAKLIEKLDI--KLDSEDKDKEGKPLLKAVMRBWLPAGDAL VVKLMNRLWGENFFNAKTKKWQKQKEADN----KRSFCMYILDPIYKVFDAIMNYKKEEIGTLLEKIGV--TLKHEDKDKDGKALLKTVMRTWL AGEAL VDKLMKNLWGDRFFDLKTKKWSSTQTDES----KRGFCQFVLDPIFMVFDAVMNIKKDKTAALVEKLGI--KLANDEKDLEGKPLMKVFMRKWL AGDTM KAKMMDRLWGDSFFNPKTKKWTNKDTDAEGKPLERAFNMFILDPIFRLFTAIMNFKKDEIPVLLEKLEI--VLKGDEKDLEGKALLKVVMRKFL AADAL TKRh(MEKLWGDNFFDATTRKWTKKHTGADTC--KRGFCQFIYEPIKTVIEAAMNDNKDKLFDLLKKLNVYSKLKPEDRELMGKPLMKRVMQTWL AHEAL EDKLMGRLWGDSYFDATAKKWTSNPQSADGKALPRAFCQFVLEPIYQLTRAIVDEDAVKLEKMMKTLQI--TLAPEDAEIKGKQLVKAVMRKFL AADAI RKRMLEKLWGDNYWDAKAKKm(KNGKGDHGEVLQRGFVQFCFDPITKLFNAIMEGRKADYEKMLTNLQI--KLSADDKEKEGKELLKTVMKLWL AGVTL LSTWMKNLWGNRFLNEKTGKWTGKSQCDNGEKNQRGFAIYVMDPILQLFDAVMTEQKKKYTKMLKQLNV--TLTPDEEDMTGKRLLKAVMQKFL AADAL _----__----___---____--____--____________~-~___~--P~GKRGVKFSDVVNAYTS__GDKAKIEELASK_-_____-__V IHEAL ____________________---___-_-______~_____~~____~~_ PYMQKSG]SFKD[]DYC_____EQEKQSELADK_-________A LHEVl

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310 320 330 340 350 360 370 380 390 400

LQMITIHLPSPVTAQKYRCELLYEGPPDDEAAMGlKSCDPKGPLMMYISKM--------VPTSDKGRFYAFGRVFSGLVSTGLKVRIMCPNYTPG--KKE QMIAIHL S VVA KY MEMLYE PHDDEAAIAVKSC PDGPLMMYISKM--------VPTSDKGRFYAFG V A KVATGQKCRIMGPNYTPG--KKE QMIAFHL S VTA KY MEMLYE PHDDEAAVAIKTC PNGPLMMYISKM--------VPTSDKGRFYAFG V S KVATGMKARIQGPNYVPG--KKE EMIVLHL S VTA AY AEQLYE PADDANCIAIKNC PKADLMLYVSKM--------VPTSDKGRFYAFG V A TVKSGQKVRIQGPNYVPG--KKD EMMIWHL S AKA KY VDVLYE PLDDTYATAVRNC ADGPLMMYVSKM-------- IPAADKGRFYAFG V S RIATGRKVRIMGPNYVPG--QKK SMIVTHL S LVA KY CANLYE PMDDECAVAIQKC PNGPLMMYVSKM-------- VPTSDKGRFYAFG V S I IVPVKRSELWVSTYVPG--KKD EMIVLHL S VVA KY TSNLYT PMDDEAAKAMANC EKGPLMMYVSKM-------- IPTNDKGRFYAFG V S TIRTGGKARICGPNYVPG--KKD EMIIVHL S KKA QY VDTLYT PLDDPAAEAIRNC PNGPLMLYVSKM---- ----VPTVDKSRFFAFG V S VVQTGQKVHIMCPEYHPGTSKKD DAVIKFV N RDS KY IPKIWK DLDSEIAKAMINA PNGPIVMMINDMKVDPHAGLVAT---------G V S TLRAGEEVWLVNAKRQQR----- DMAIKHL N LQA KY IPNIWK DAESEVGKSMAMC PNGPLAGVVTKIIVDKHAGSISA- --------C L S RIKQGDELYLVGSKQKAR----- DMVAEHF N IDA PR IPTVWR DADSEIAASMRLV EDGEVVLMVTDIGVDPHAGEIAA---------G V S TLEKGQELYVSGTAGKNR-----

410 420 430 440 450 460 470 480 490 500

FIG. 2.-Alignment of the amino acid sequences of eukaryotic and archaebacterial EF-2’s. Abbreviations of species: H.s., Homo sapiens; D.m., Drosophila melanogaster; C.e., Caenorhabditis elegans; S.C., Saccharomyces cerevisiae; C.k., Chlorella kessleri; D.d., Dictyostelium discoideum; E.h., Entamoeba histolytica; G.I., Giardia lamblia; S.a., SuIfolobus acidocaldarius; M.v., Methanococcus vannielii; H.h., Halobacterium halobium. Deletions needed for the alignment are indicated by -. In the G. lamblia and E. histolytica (our previous work) sequences, unidentified regions which we could not determine are indicated by =. In positions occupied by an identical amino acid throughout all species, the amino acid is shown only for the top sequence. Regions for synthesizing degenerate primers, A 1, A2, Bl , and C 1, are indicated over the alignment. Regions designated by Gl through G4 under the alignment are sequence motifs highly conserved among proteins from GTPase superfamily. Posttranslationaly modified residues in mammalian EF-2 are indicated by - over the alignment.


II. s. DLYLKPIQRT1LMMGRYVEPIEDVPCCNIVGLVGVD---------QFLVKTGTIT--TFEHAHNMRVMKFSVSPVVRVAVEAKNPADLPKLVEGLKRLAK D.m. DLYEKAIQRTILM RYVEAIEDVPS ICGLV VD---------QFLVKTGTIT--TFKDAHNMKVMKFSVS VRVAV PKNPA P LVEGLKRLAK C.e. DLYEKTIQRTILM RFIEPIEDIPS IAGLV VD---------QYLVKGGTIT--TYKDAHNMRVMKFSVS VRVAV AKNPA P LVEGLKRLAK S.C. DLFIKAIQRVVLM RFVEPIDDCPA IIGLV ID---------QFLLKTGTLT--TSETAHNMKVMKFSVS VQVAV VKNAN P LVEGLKRLSK C. k. DLTVKTVQRTVLC RRQEAVEDVPC TVALV LD---------QFITKNATLTDEKCEDAHTIKAMKFSVS VRVAV PKVAS P LVEGLKRLAK D.d. DLFLKSIQRTVLM RKTEQIEDCPC IVGLV VD- --------QFLVKSGTIT--TSEVAHNIRVMKFSVS VRVAV PKNPS P LVEGLKRLAK E.h. DCVIKNIQRTMLM RYTDPIDECPC VIGLV VD--------- QYLLKSGTIT--DSDTAHIIKDMKFSVS VRVAV TKNPS P LVEGMKRLSR c. 1. ELFIKNIQRTILM SRIEQIDDVPC TVGLV ID--------- QYLVKSGTIS--TYEQAHSIKPMKFSVS VRVAV PANPK P LLEGMKRLDK S.a. ------ILQVSLY AIRELAEEIPV IAAAL MDAARSGETGVDIRFKDSVLG--SFEKLHYISE------ VTISV PRNPK T MI DALRKLSI M.V. ------AQQVAIF AERVQVPSISA ICALT LREATAGETVCSPS--KILEP--GFESLTHTSE------ ITVAI AKNTK P LIEILRQIGR H.h. ------VQSVGIY GEREEVDEVPA IAAVT LKDAIAGSTVSNEE-----MT--PFESIDHISE------ ITKSI AQNMD P LIETLRQVSK

H. s. SDPMVQCII-EESGEHIIAGAGELHLEICLK-DLEED-HACIPIKKSDPVVSYRETVSEESNVLCLSKSPNKHNRLYMKARPFPDGLAEDIDKG-EVSAR D.m. S PMVQCII-EESGEHIIA A E L ICLK-DLEED-HACIPLKKSD VVSY TVSEESDQMCLSKSPNKHNRLLMKALPMPDGLPEDIDN -EVSAK C.e. S PMVQCIF-EESGEHIIA A E L ICLK-DLEED-HACIPLKKSD VVSY TVQSESNQICLSKSPNKHNRLHCTAQPMPDGLADDIEC -TVSAR S.C. S PCVLTYM-SESCEHIVA T E L ICLQ-DLEHD-HAGVPLKISP VVAY TVESESSQTALSKSPNKHNRIYLKAEPIDEEVSLAIEN -1INPR C. k. S PMVQCTI-EETGEHIIA A E L ICLK-DLQDDFMGGAEIRVSE VVSF TVIGTSDHVVMSKSPNKHNRLYMQARPMEDGLAEAIDE -KIGPR D.d. S PCVLCYS-EESGEHIVA A E L ICLK-DLAED-HAGIEIKTTD VVSF SV---------------------KASPISMELQDLIEA SDISSK E.h. S PLCLCYT-EESGEHIVA A E L VCLK-DLQEDYCSGVPLIVTE VVSF TITEPSRIQCLSKSANNQNRLFMRAFPFAEGLAEDIEA -EIKPD c.1. S PCVMCICDKDENQNIIA A E L ICLK-DLREDFCGGMDIRVSD VVSY TVTEKSTKVVMAKSANKHNRLYFEAEPISEEVIEAIKD -EITSE S.a. E SNLVVKINEETGEYLLS M F L VSLQ-LLKENY--GLDVVTTP IVVY SIRNKS-QVFEGKSPNKHNKLYISVEPLNNQTIDLIAN -TIKED M. v. E NTVRIEINEETGEHLIS M E 1 VITDTKIGRDG--GIEVDVGE IIVY TITGTS-PEIEGKSPNKHNKLYMIAEPMEESVYAAYVE -KIHDE H.h. E PTISIEINEDTGEHLIS Q E L VQTQ-RIERNQ--GIPVTTGE IVVY TPTSDS-QEVEGVSPNRHNKFYITVEQLSDDVLEEIRL -EVSMD

H. s. D.m. C. e. S.C. C.k. D.d. E.h. G. 1. S.a. M.V. H.h.

H. s. GGQIIPTARRCLYASVLTAQPRLMEPIYLVEIQCPEQVVGGIYGVLNRKRGHVFEESQVAGTPMFVVKAYLF D.m. CC II TT RCLYAAAITAKPRLM VYLCE QC EVAV GIYGVLNRR HVFEENQVV TPMFVVKAYL C.e. GG II TA RVFYASVLTAEPRLL VYLVE QC EAAV GIYGVLNRR HVFEESQVT TPMFVVKAYL S.C. GG II TM RATYAGFLLADPKIQ VFLVE QC EQAV GIYSVLNKK QVVSEEQRP TPLFTVKAYL C. k. GG II TA RSMYAAQLTAQPRLL VYLVE QC EQAM GVYSVLNQK MVFEELQRP TPIFNLKAYL D.d. GG II TA RVLYAAELTASPTLL IYLVE TA ENAI GIYSVLNRR IVIGEERRI SPLFSVKAHL E.h. GA MI CA RCCFACVLTGAPSLL MYLAE QC ESAI GlYtVMSRR KIISEEQRP TPLFNVRAYL G. 1. AC LT AT RGLYAACLYASPMLM FYLVD LA EGCM GIYSTMSKR VVISEEPRE QPLTEVKAHL %a. PA LY AV NAIFAGILTSKPTLL LQKLD RI MEYL NVTAVITRK KVINVVQ-T N-VARVYAEI M.V. PS II AI FGVRDAVSSAKPILL MQKIY NT QDYM DAIREINNR QIVDMEQ-E D-MAIIKGSV H.h. PA VI AT DAVHRALIDADIRLL IQDVR DV SEHM AASGEVQGR RVDDMYQ-E D-LMVVEGIA

H. s. D.m. C. e. S.C. C.k. D.d. E.h. G. 1. S.a. M.V. H.h.

510 520 530 540 550 560 570 580 590 600

Al

610 620 630 640 650 660 670 680 690 700

QELKQRARYLAE----KYEWDVAEARKIWCFGPDCTGPNILTDI----TKGVQYLNElKDSVVAGFQWATKEGALCEENMRGVRFDVHDVTLHADAliiRG DEFKARARYLSE---- KYDYDVTEARKIWCFGPDGTGPNFILDC---- KSVQYLN IKDSVVAGFQWASKE ILAD NLRGVRFNIY VTL A Al DEFKARAKYPGE----KYEYAVTEARKIWCFGPDGTGPNLLMDV---- KGVQYLN IKDSVVAGFQWATRE VLSD NMRGVRFNVH VTL A AI DDFKARARIMAD----DYGWDVTDARKIWCFGPDGNGPNLVIDQ---- KAVQYLH IKDSVVAAFQWATKE PIFG EMRSVRVNIL VTL A AI DDPKVRSKILSE----EFGWDKELAKKILAFGPDTTGPNMVTDI---- KGVQYLN IKDSVVAAFQWASKE VLAE NMRGIVFEVC VVL A AI DDPKARANYLAD----NHEWDKNDAMNIWSFGPEGNGANLLVNV---- KGVQYLN IKDSFVGAFQWATKE VVCD NMRGIRFNLY VTL T Al TDFKERAKFLSE----KYGWDVDEARKIWCFGPDNCGPNLFVDV---- KGIQYLN VKDSIVNGFNNAMHD VVCN QIRGVRINLE VKL A Al QDSKVRARILTD----KYGWDSDEAKQIWSFGPVGASSGHMTNLILEA KGVQYVK SKEHIVSGFQIVCRN VLAG ELVGTCFKLR ATF A AI MDNKEMAKILRD----QAEWDYDEAKKI-------VAIDENINVFIDA SGVQHLR IMDTLLQGFRLAMKE PLAF PVRGVKVVLH AVV E PA -DFKKKTNVDAETRLIEAGLEREQAKKV-------MSIYNG-NMIVNM KGIVQLD ARELIIEGFKEGVKG PLAS RAQGVKIKLI ATF E AI MPEQERREVLQE-----AGMDKETSQDV-------ENIIGR-NIFIDD KGIQHLN TMELVVDGLTDSLED PLAA PVEGALIRLH ARL E AI

710 720 730 740 750 760 770 780 790 800

810 820 830 840 850 860 870

QILPGDPFDNSSRPSQVVAETRKRKGLKEGiPALDNFLDKL------------ QVLPGDPSEPSSKPYAIVQDTRKRKGLKEGLPDLSQYLDKL------------ QVLPGDPLEAGTKPNQIVLDTRKRKGLKEGVPALDNYLDK~----------- STLGSDPLDPTSKAGEIVLAARKRHGMKEEVPGWQEYYDKL------------ EAMGSDPTQVGSQANTLVMDIRKRKGLKPEPAALSEYEDKL------------ --ASIGVVNKDKKATEVALATRKRKGLAPElPALDKFHRKTtNNLSHTLSFQI _____--____--_-~~----~~~~~~-_~~~~~~~----__-____--_-__ -_---____--_________~~~~~-~~~~~~~~~~--___~~~~~-_-_~~~ _____--___-_-____-_-_-~~~~~__-_~_~~~________-__-_____ ------~~_--~~~~~~-~~~~~~~~~~~~~~~~~~-_____~~~~_-_-__~ APVPDSIL------VDLIMKIRERKGKPKQLPKVEDFIS-------------- ERVPNEIQv ____ TKVVAQIRDRKGLKSE --s-s_ _ ________________

RVMADNLB_-----REIIMEIRERKGMKTELPESITHF--------------- 910 920 930 940 950

A2 'VNESFGFTADLRSNTGGQAFPgCVFDHW N SFGFTADL SNTG QAFPQCVFDHW N SFGFTADL SNTG QAFPQCVFDHW N SFGFTGEL QATG QAFPQMVFDHW I SFGFTSTL AATA QAFPQCVFDHW L SLRFTADL SHTA QAFPQCVFDHW C SFGFTADL SHTS QA=========

A SFGFDADL AATS QA=========

G SFELASEL ASSA RAFWGTEFSRW A MFGFAGAI GATQ RCLWSVEFSGF D MlGFSSDl SATE RASWNTENAGF

880 890 900

787 Downloaded from https://academic.oup.com/mbe/article-abstract/12/5/782/974540by gueston 25 March 2018


Table 1 Phylogenetic Relationships among Eukaryotes by ML and MP Analyses Using Archaebacteria as an Outgroup

ML METHOD (JTT-F Model) MP METHOD

TREE TOPOLOGY* AL&: P, A, P,

1. (G,(Ck,(Dd,(E,(S,A))))) . (-8170.3) 0.2828 +5 _+ 9.1 0.0405 2. (G,(Dd,(Ck,(E,(S,A))))) . . . . . -3.8 + 8.1 0.1120 +9 + 8.9 0.0190 3. (G,(E,((Dd,Ck),(S,A)))) . . -6.4 + 14.9 0.1057 +2 + 4.9 0.0707 4. (G,((Dd,Ck),(E,(S,A)))) . . . . -7.0 -t 7.2 0.0159 +12 + 7.9 0.0005 5. (G,(E,(Dd,(Ck,(S,A))))) . . -9.2 + 17.5 0.0676 ( 1673)f 0.2546 6. (G,(Ck,((E,Dd),(S,A)))) . . . . -11.1 + 11.4 0.0366 +1 +- 8.7 0.1990 7. (G,((E,Dd),(Ck,(S,A)))) . -14.7 * 14.7 0.0113 +5 f 7.0 0.0140 8. (G,(E,(Ck,(Dd,(S,A))))) . . -15.9 + 16.5 0.0002 +8 + 4.2 0.0016 9. (G,(E,(Dd,(S,(Ck,A))))) . . - 18.0 + 24.4 0.0538 +12 + 6.6 0.0034

10. (G,(Dd,(E,(S,(Ck,A))))) . . . -18.7 _+ 20.8 0.0295 +ll + 8.7 0.0126 11. (G,(E,(S,(A,(Dd,Ck))))) . . -19.2 + 19.9 0.0214 +9 + 8.1 0.0294 12. (G,((E,Dd),(S,(Ck,A)))) . . . . -22.0 + 23.1 0.0180 +11 + 8.4 0.005 1 13. (G,(E,(S,(Dd,(Ck,A))))) . . . -25.3 + 25.6 0.0108 +16 + 9.0 0.0034

a G, Giardia lamblia; E, Entamoeba histolytica; RI, Dictyostelium discoideum; Ck, Chlorella kessleri; S, Saccharomyces cerevisiae; A, Animalia.

b Ali is the difference of log-likelihood of tree-i from that of the ML tree (tree-l), and f is 1 SE. c Bootstrap probability estimated by lo4 replications. d Ai is the difference of number of substitutions of tree-i from that of the MP tree (tree-5) and + is 1 SE. ’ Log-likelihood of the ML tree. ‘Number of substitutions of the MP tree.

showed 0.7336 and 0.7306 in the JTT-F model and the MP analysis, respectively, whereas those linking Ani- malia with C. kessleri showed lower values, 0.1429 and 0.0450, respectively. Among Animalia, S. cerevisiae (Fungi), and C. kessleri (green algae), the linking of Animalia with S. cerevisiae is more likely than the other links.

The ML analyses of the other five models also gave consistent results, although the values of total bootstrap

Table 2 Totals of Bootstrap Probabilities for the Trees That Support Various Topological Elements

Topological Element” No. of ML Method MP

Topologies (JTT-F Model) Method

(Outgroup,(G, . . . . . . . . . 105 0.88 19 0.8684 (Outgroup,(G,(E, . . . . . 15 0.2628 0.3653 (Outgroup,(G,(Dd, . . . . 15 0.1939 0.1260 (Outgroup,(G,(Ck, . . . . 15 0.35 11 0.2916

(Outgroup,(E, . . . . . . . , 105 0.0393 0.0660 (Outgroup,(Ck, . . . . . . 105 0.0709 0.0287 (S, A) . . . . . . . . . . . . . 105 0.7336 0.7306 (Ck, A) . . . . . . . . . . . . 105 0.1429 0.0450 (S, Ck) . . . . . . . . . . . . . 105 0.0003 0.0028

’ G, Giardia lamblia; E, Entamoeba histolytica; IX, Dictyostelium discoideum; Ck, Chlorella kessleri; S, Saccharomyces cerevisiae; A, Animaha.

probabilities are slightly different among alternative models. On the basis of the phylogenetic analyses as described above, a eukaryotic tree of EF-2 was inferred by the ML method using the JTT-F model and is shown in figure 3. Ambiguous branching orders were represented by a multifurcation. Table 3 represents the estimated number of amino acid substitutions along each branch in the ML tree with its 1 SE.

Discussion Features of Eukaryotic EF-2

The N-terminal quarters of the eukaryotic and archaebacterial EF-2’s and eubacterial EF-G’s can be aligned with the corresponding N-terminal regions of EF- 1 a’s and their eubacterial homologues, elongation factor Tu’s (EF-Tu’s), because these two types of proteins might have evolved by a gene duplication event that occurred prior to the divergence of the three primary kingdoms of life (Iwabe et al., 1989; Miyata et al., 199 1). About 200 amino acid residues of the N-terminal portion of these elongation factors contain highly conservative motifs commonly found in the GTPase superfamily (Kohno et al. 1986; Bourne et al. 199 1). Regions, Gl through G4, defined by Bourne et al. ( 199 1) are shown in figure 2. Three-dimensional structures of Escherichia coli EF-Tu and mammalian p2 1 ras have shown that these segments are critical in GDP/GTP exchange, GTP-in-



the SrRNA tree may sometimes be unreliable because of the extreme bias of G+C contents. For example, the

..I - Saccharomyces cerevisiae G+C content of Entamoeba histolytica SrRNA (Que Chlorella kessleri

1 Dictyostelium discoideum and Reed 199 1) is very low ( 38.3%), whereas that of G.

Entamoeba histolytica lamblia (Sogin et al. 19893) is extremely high (74.7%). Obviously the base compositions of SrRNA seem to be affected by the genome base compositions of the species.

Figure 4A shows the base compositions of the 530

0.1 substitutions I site sites of EF-2 used in the present phylogenetic analyses.

FIG. 3.-EF-2 tree of eukaryotes with archaebacteria as an out- The base composition of the third codon position is sig-

group inferred by the ML method based on the JTT-F model. The nificantly different among species. The G+C content is

horizontal length of each branch is proportional to the estimated num- very low in E. histolytica ( l&2%), whereas in Chlorella ber of substitutions. The root of this tree is located either between kessleri it is extremely high ( 9 1.7%). Probably the base nodes 0 and 1 or between nodes 0 and 5 (Miyata et al. 199 1; Rivera compositions of the third codon position are directly and Lake 1992). affected by the mutational GC (AT) pressure operating

duced conformational change, and GTP hydrolysis (see Bourne et al. 199 1) . Putative GTP binding domain of Giardia lamblia EF-2 including these four regions shows high similarity to both the other eukaryotic and the archaebacterial EF-2’s, although the 35 residue insertion that contains many lysine residues is specific only to G. lamblia EF-2 (positions 96- 130 in fig. 2 ) .

Ca *+ / calmodulin-dependent protein kinase III ( Price et al. 199 1). All of these modification sites are entirely conserved in the G. lamblia EF-2, indicating that the G. lamblia EF-2 has a common eukaryotic feature. In the Dictvostelium EF-2, however, both of the two phos-

Mammalian EF-2 has been shown so far to undergo three types of posttranslational modification, all of which result in inhibition of translational elongation. These are ADP-ribosylation of histidine-7 15 by diphtheria toxin (Van Ness et al. 1980; Nilsson and Nygard 1985; Kohno et al. 1986), oxidation of tryptophan-22 1 by N- bromosuccinimide (NBS) (Guillot et al. 1993a, 19933), and phosphorylation of threonine residues 57 and 59 by

in genome DNA. However, at the first and the second codon positions in figure 4A, the biases of base com-

Table 3

positions are not so great. Especially in the second position, the compositions of each base of all four species are within error bars. Because a base substitution at the

Estimated Number of Amino Acid Substitutions per Site

second position tends to result in an amino acid substitution to a physicochemically different amino acid, the

along Each Branch of the Tree in Figure 3

position may be very conservative among species. It is consistent with amino acid composition data shown in figure 4B. The amino acid compositions of the 530 sites of EF-2 from four species do not differ significantly from each other. These results clearly indicate that the amino acid composition of EF-2 is free from the drastic bias of genome base composition, and that the EF-2 phylogeny would give a robust estimation for the early divergence of eukaryotes. It has been suggested also for EF-1 a

phorylation sites are replaced by methionine at position 59 and by cysteine at position 6 1 (fig. 2). If there is a

Branch Leading to Estimated Number

of Substitutions

common eukaryotic regulatory mechanism of the translation elongation step by phosphorylation of these two sites, this feature of Dictyostelium EF-2 is quite unusual. Furthermore, a wide deletion of Dictyostelium EF-2 corresponding to positions 658 through 678 is also a very unusual feature specific only to Dictyostelium. It would be interesting to know whether or not a drastic evolutionary event has happened on the EF-2 and other molecules involved in translation elongation in the branch leading to Dictyostelium.

Robustness of EF-2 Phylogeny

Homo . . . . Drosophila . . Caenorhabditis . . . . Saccharomyces Chlorella . . . . . Dictyostelium . . . . . . Entamoeba . . . . . Giardia . . . . . . Sulfolobus . . . . . . . Methanococcus . . . Halobacterium . . . . . 1 . . . . . . . . . . . . . . . . 2 . . . . . . . . . . . . . . . . 3 * . . . . . . . . . . . . . . .

0.074 z!I 0.013 0.077 + 0.0 13 0.079 f 0.0 13 0.208 + 0.024 0.256 + 0.027 0.236 f 0.025 0.272 f 0.027 0.238 -t 0.029 0.389 + 0.040 0.298 f 0.034 0.352 -t- 0.037 0.448 f 0.045 0.136 f 0.025 0.086 f 0.0 17

The SrRNA sequences have been widely used in clarifying deep branchings of eukaryotes (Sogin et al. 1989a, 19893; Sogin 1991; Leipe et al. 1993). However,

4 . . . . . . . . . . . . . . . . 5 . . . . . . . . . . . . . . . .

NOTE.--+ refers to 1 SE.

0.108 f 0.018 0.211 f 0.034



[“a

50.0

40.0

30.0

20.0

10.0

0.0

4

. ..A.

P.

A o q

ATGC ATGC ATGC Iat 2nd 3rd

0.0 WCHMYFQNPRDIICTEVSGAL

amino acid

[%I

50.0

40.0

30.0

20.0

10.0

0.0

.

A A

A . . _.....,

ADGC

- AVERAGE

0 Chhvella

x Oktpstellum

0 Entmnmbn

A Gkdh

FIG. 4.-Compositions of bases (A) and amino acids (B) for 530 sites of EF-2, and compositions of bases for 9 18 sites of SrRNA (C) from four eukaryotic species, Giardia lamblia, Entamoeba histolytica, Dictyostelium discoideum, and Chlorella kessleri. In panel A, the composition for each codon position, first, second, and third, was calculated from 530 aligned amino acid sites used in the phylogenetic analyses. On the basis of the 44-nomial distribution, standard error (SE) of the averaged composition among four species was calculated, and 2 SE was shown by an error bar for each base. For details, see the work of Hasegawa and Kishino (1989). In panel B, the composition for each amino acid was calculated from 530 sites. Amino acids represented by one letter code are shown in the abscissa in the order of normalized compositions by Jones et al. ( 1992). The SE of the averaged composition was calculated on the basis of the 204-nomial distribution, and 2 SE was shown by an error bar. In panel C, an alignment of the SrRNA was constructed for three archaebacteria and more than 20 species of eukaryotes, including the four species represented in this figure. Un- ambiguously aligned 9 18 sites were selected and used to calculate the base composition and the SE of averaged composition.

(Hasegawa et al. 1992a; Hashimoto et al. 1994)) for the largest subunit of RNA polymerase III (Hashimoto et al. 1992), and for mitochondrially encoded proteins (Adachi and Hasegawa 1992; Adachi et al. 1993) that amino acid compositions are relatively free of the biased base compositions of genome DNA.

In contrast, figure 4C represents the base composition of 9 18 SrRNA sites. Almost all the 918 selected sites would have been used in the widely accepted universal tree of SrRNA. The base compositions of SrRNA are significantly different among four species. The bias is very great, especially in G. lamblia.

Phylogenetic Relationships among Eukaryotes

The ML methods on the basis of six different stochastic models of amino acid substitutions and the MP method consistently suggested that G. lamblia shows the earliest offshoot of EF-2 tree among the eukaryotic lineages being analyzed. It is consistent with the results of the EF- 1 a phylogenies recently performed by our group (Hashimoto et al. 1994) and by Baldauf and Palmer ( 1993 ) . In our previous EF-2 analyses based on the data set without G. lamblia EF-2, E. histolytica have shown to diverge earlier than other eukaryotes with about 80% bootstrap support (Shirakura et al. 1994). However, in our present analyses based on a slightly different alignment including G. lamblia EF-2, the divergence of E. histolytica next to G. Zamblia was not entirely conclusive. We could not deny the possibility that C. kessleri or D. discoideum branches off next to G. lamblia (tables 1 and 2). Since C. kessleri belongs to unicellular green algae which possess chloroplasts and mitochondria, it should be regarded as a lower plant. Unfortunately, there are no EF-2 data from higher plants. If the data from higher plants were added to the present data set, the placings of E. histolytica, C. kessleri, and D. discoideum might become more clear. We have previously shown by ML analyses of EF- la that Animalia are highly likely to be closer to Fungi than to Plantae (Hasegawa et al. 1993 ) . Total of the bootstrap probabilities for the 15 EF- la trees linking Fungi with Animalia amounted to greater than 0.9, when E. histolytica and archaebacteria were used as outgroups. A consistent conclusion favoring the linking of Saccharomyces cerevisiae with Animalia has also been shown in the present EF-2 analyses, although the total values of bootstrap probabilities were slightly lower than those of the EF-la analyses. Recently, Bal- dauf and Palmer ( 1993) concluded from MP analyses of actin, a-tubulin, P-tubulin, and EF- 1 a that Animalia and Fungi are each other’s closest relatives. Furthermore, Nikoh et al. ( 1994) recently analyzed 23 different proteins by ML, MP, and neighbor-joining methods and reached the same conclusion by totally evaluating the sum of log-likelihoods in the ML analyses, In spite of


Phylogenetic Place of Giardia 79 1

some exceptions, such as a phylogeny of the largest subunit of DNA dependent RNA polymerase II in which Animalia has been shown to be closer to Plantae (Sidow and Thomas 1994), an overall evaluation on the basis of many proteins confirms us that a close relationship between Animalia and Fungi is very robust. Although there are several uncertainties in the tree shown in figure 3 and in the results of analyses shown in tables 1 and 2, the placing of G. Zamblia as the earliest offshoot and the linking of Animalia with 5’. cerevisiae are likely by the present analyses.

Since the sequence data of EF-2 from other protozoa, such as Trypanosoma, Euglena, Plasmodium, and Tetrahymena, have not yet been reported, the results derived from the present EF-2 analyses may not always be comparable to those on the basis of SrRNA and EF-la. There still remain many problems on the phylogenetic placing of protozoa including mitochondrion- lacking species. Further sequence data of the genes encoding conservative proteins, such as elongation factors, RNA polymerases, and ATPases, from several protozoa would be highly desirable to understand the early evolution of eukaryotic cells.

Sequence Availability

The nucleotide sequence data reported here appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases with the accession number D29835.

Acknowledgments

We thank Dr. T. Nakamura for providing trophozoites of Giardia lamblia; Dr. W. Zillig for providing a sample of G. lamblia genomic DNA; Dr. J. L. Thorne for providing computer programs of maximum likelihood alignment; Drs. N. Okada, E. Otaka, K. Mizuta, and K. Suzuki for technical support and discussion; and Y. Cao, A. Tokui, and C. Oda for technical assis- tance. This work was carried out under the ISM Co- operative Research Program (931SM l CRP-A64 and 941SM l CRP-A60), and was supported by grants from the Ministry of Education, Science, and Culture of Japan.

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MITIKO GO, reviewing editor

Received September 14, 1995

Accepted May 11, 1995


phylogenetic place of mitochondrion-lacking protozoan, giardia

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