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Unusually High Evolutionary Rate of the Elongation Factor 1 Genes From Unusually High Evolutionary Rate of the Elongation Factor 1a Genes from the Ciliophora and Its Impact on the Phylogeny of Eukaryotes David Moreira, Herve Le Guyader, and Herve Philippe Laboratoire de Biologie Cellulaire, Unite de Recherche AssocieÂe 2227, Centre National de la Recherche Scienti®que, Universite Paris±Sud, Orsay, France The elongation factor 1a (EF-1a) has become widely employed as a phylogenetic marker for studying eukaryotic evolution. However, a disturbing problem, the artifactual polyphyly of ciliates, is always observed. It has been suggested that the addition of new sequences will help to circumvent this problem. Thus, we have determined 15 new ciliate EF-1a sequences, providing for a more comprehensive taxonomic sampling of this phylum. These sequences have been analyzed together with a representation of eukaryotic sequences using distance-, parsimony-, and likelihood-based phylogenetic methods. Such analyses again failed to recover the monophyly of Ciliophora. A study of the substitution rate showed that ciliate EF-1a genes exhibit a high evolutionary rate, produced in part by an increased number of variable positions. This acceleration could be related to alterations of the accessory functions acquired by this protein, likely to those involving interactions with the cytoskeleton, which is very modi®ed in the Ciliophora. The high evolutionary rate of these sequences leads to an arti®cial basal emergence of some ciliates in the eukaryotic tree by effecting a long-branch attraction artifact that produces an asymmetric topology for the basal region of the tree. The use of a maximum-likelihood phylogenetic method (which is less sensitive to long-branch attraction) and the addition of sequences to break long branches allow retrieval of more symmetric topologies, which suggests that the asymmetric part of the tree is most likely artifactual. Therefore, the sole reliable part of the tree appears to correspond to the apical symmetric region. These kinds of observations suggest that the general eukaryotic evolution might have consisted of a massive radiation followed by an increase in the evolutionary rates of certain groups that emerge arti®cially as early branches in the asymmetric base of the tree. Ciliates in the case of the EF-1a genes would offer clear evidence for this hypothesis. Introduction The advent of molecular phylogeny has forever phylogenetic questions have been successfully ad- changed our understanding of protist evolution by pro- dressed using this marker, including the phylogenies of viding new tools and a phylogenetic framework based some groups of metazoans (Kobayashi, Wada, and Satoh on criteria more objective than the analysis of structural 1996; McHugh 1997), the sisterhood of the fungal and characters. Initial work was carried out using the small- animal clades (Baldauf and Palmer 1993; Hasegawa et subunit ribosomal RNA (SSU rRNA) as a phylogenetic al. 1993), and the monophyly of the slime molds and marker. The eukaryotic phylogeny that emerged from their inclusion within the crown of the eukaryotic tree SSU rRNA sequence comparison consisted of a basal (Baldauf and Doolittle 1997). However, it was quite dis- region with several protist groups emerging in a step- turbing that ciliates included so far in several analyses wise fashion and a large apical unresolved radiation (Tetrahymena pyriformis, Euplotes crassus, and Stylon- (known as the ``crown'') grouping together plants, ani- ychia lemnae) did not form a monophyletic clade in any mals, fungi, and several protist phyla (Sogin 1991). EF-1a tree (Adoutte et al. 1996; Baldauf and Doolittle Nevertheless, the rRNA-based phylogenies were soon 1997; Yamamoto et al. 1997). This seriously contradicts revealed to be sensitive to problems derived from drastic a large amount of morphological, developmental, and differences in GC content among taxa (Loomis and molecular data that clearly support the monophyly of Smith 1990; Galtier and Gouy 1995). As a consequence, the phylum Ciliophora (for review, see Schlegel and Eis- since they are not affected by this problem, several pro- ler 1996). As a result, the hypothetical ciliate polyphyly teins such as actin, a- and b-tubulin, glyceraldehyde-3- derived from EF-1a analyses has been regarded as ar- phosphate dehydrogenase, and the protein elongation tifactual. Several arguments have been put forward to factor 1a (EF-1a) are currently used as alternative phy- explain this apparent contradiction. First, only a few cil- logenetic markers. iate sequences, ill representing the diversity of this phy- In particular, EF-1a has become widely employed. lum, were available (Baldauf and Doolittle 1997). Sec- This protein plays a central role in protein synthesis, ond, the ciliates have a faster rate of sequence diver- forming a ternary complex with aminoacyl-tRNA and gence, which would generate a long-branch attraction GTP (Yager and von Hippel 1987). In addition, it inter- phenomenon (Philippe and Adoutte 1996; Adoutte et al. acts with some cytoskeletal proteins, especially actin 1996; Baldauf and Doolittle 1997; Roger et al. 1999). (Durso and Cyr 1994; Shiina et al. 1994). Some relevant Indeed, long-branch attraction (Felsenstein 1978) is a common artifact affecting phylogenetic reconstruction. Key words: Ciliophora, elongation factor 1a, eukaryotic phylog- When a distant outgroup is used, fast-evolving taxa are eny, long-branch attraction, taxonomic sampling, tree symmetry. attracted by the long branch of the outgroup, and the Address for correspondence and reprints: Herve Philippe, Labor- resulting trees display artifactual asymmetric bases. atoire de Biologie Cellulaire, BaÃtiment 444, Universite Paris±Sud, Based on this idea, it has been proposed that eukaryotic 91405 Orsay Cedex, France. E-mail: [email protected]. evolution might have consisted of a massive radiation Mol. Biol. Evol. 16(2):234±245. 1999 followed by an increase in the evolutionary rates of cer- q 1999 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038 tain groups that emerge arti®cially as early branches, 234 Eukaryotic EF-1a Evolution 235 producing an asymmetric tree base (Philippe and Adout- Gene Ampli®cation, Cloning, and Sequencing te 1998). In addition, since the groups exhibiting higher Ciliate genomic DNA was isolated using a hexad- evolutionary rates can be different for different phylo- ecyltrimethylammoniumbromide (CTAB) puri®cation genetic markers, this model also helps to explain the method as previously described in Winnepenninckx, incongruences observed among different phylogenetic Backeljau, and de Wachter (1993). Almost complete EF- markers for the likely artifactual asymmetric regions of 1a coding sequences (around 1,270 bp) were ampli®ed the trees. with different combinations of the forward primer 1F The EF-1a-based phylogeny displays an asymmet- and the reverse primers 10R1 and 10R2, described by rical base, and the artifactual polyphyly of ciliates, lo- Baldauf and Doolittle (1997), and a new forward primer cated in this region, is an interesting case study. Addi- 1A (GTT ATT GGA CAY GTY GAY TCN GG), cor- tion of taxa to break long branches was suggested as a responding to the peptide VIGHVDSG. Partial regions means to alleviate the long-branch attraction artifact from the EF-1a-coding sequences (350±700 bp) were (Hendy and Penny 1989), and therefore, if the model ampli®ed with the above described primers, together explaining the asymmetric bases is correct, the addition with the forward primers 3F and 4F2 and the reverse of new ciliate sequences to the EF-1a phylogeny must primers 4R and 7R (Baldauf and Doolittle 1997). These lead to a more symmetric tree topology. partial amplicons were screened to study the possible We thus determined 15 new ciliate sequences, in- presence of introns, since during PCR ampli®cations of cluding representatives of their main classes. The new complete EF-1a-coding regions, the smaller, intronless sequences, together with a selection of published eu- loci might have been preferentially ampli®ed. PCR am- karyotic EF-1a sequences, were analyzed using dis- pli®cations were carried out as described by Baldauf and tance-, parsimony-, and likelihood-based phylogenetic Doolittle (1997). methods. These analyses still failed to recover the After each ampli®cation and cloning assay, plasmid monophyly of Ciliophora, with an artifactual basal DNAs from 30 clones containing inserts of the expected emergence of the fast-evolving species. Nevertheless, size were analyzed by endonuclease restriction with the the comparison of different data sets has shown that, as enzymes EcoRI, HindIII, SalI, and Sau3AI (New Eng- expected, the tree becomes increasingly symmetrical land Biolabs). In some cases, the coexistence of two with the addition of new sequences, supporting the mod- bands with different restriction patterns, which were el described above. The unusually high evolutionary rate subsequently shown to be duplicated EF-1a genes, was of the ciliate EF-1a genes is in part produced by an detected. Plasmid DNAs were sequenced using the increased number of variable positions. Their particular Thermo Sequenase premixed cycle sequencing kit mode of evolution is dif®cult to explain by modi®ca- (Amersham). For each ampli®cation product (including tions in the translational activity. Conversely, it could all those differentiated by their restriction pro®les), a be related to alterations of the accessory functions in- minimum of 10 clones were analyzed by partial se- volving
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