Unusually High Evolutionary Rate of the Elongation Factor 1␣ 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 1␣ (EF-1␣) 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-1␣ 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-1␣ 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-1␣ 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-1␣ 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, ␣- and ␤-tubulin, glyceraldehyde-3- derived from EF-1␣ analyses has been regarded as ar- phosphate dehydrogenase, and the protein elongation tifactual. Several arguments have been put forward to factor 1␣ (EF-1␣) 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-1␣ 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 1␣, 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- ᭧ 1999 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038 tain groups that emerge arti®cially as early branches,

234 Eukaryotic EF-1␣ 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 1␣ coding sequences (around 1,270 bp) were ampli®ed the trees. with different combinations of the forward primer 1F The EF-1␣-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-1␣-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-1␣ 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-1␣-coding regions, the smaller, intronless sequences, together with a selection of published eu- loci might have been preferentially ampli®ed. PCR am- karyotic EF-1␣ 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-1␣ genes, was of the ciliate EF-1␣ 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 interactions with the cytoskeleton, which has quencing (around 1,000 nt) in order to detect possible undergone important modi®cations in the Ciliophora. different clones not distinguishable by the restriction analysis. Sequences were obtained automatically with a Materials and Methods Vistra DNA Sequencer 725 (Amersham). Both DNA Strains and Growth Conditions strands of one representative of each distinct clone, as identi®ed by the restriction analysis or by the partial Species from seven different classes of ciliates sequencing, were completely sequenced, as was one were used in this study. Cells of Spathidium sp. (class strand of another clone to check for possible Taq poly- Litostomatea) and of Kentrophoros sp. (class Karyore- merase errors. When discrepancies were observed, they lictea) were kindly provided by Dr. A. Baroin-Touran- were resolved by sequencing of additional clones. Se- cheau. The rest of the species were grown on liquid quences have been submitted to GenBank under the ac- media containing the following nutrients: 1.5% bacto- cession numbers AF056096±AF056109. proteose peptone (Difco) for T. pyriformis (class Oli- gohymenophorea); infusion of the marine green alga Phylogenetic Analysis Lactuca sp. and living cells of the bacterium Aerobacter Assembly and translation of the ciliate sequences aerogenes for Paranophrys carnivora, Telotrochidium were carried out using the programs of the GCG Wis- henneguyi (class Oligohymenophorea), and Colpoda in- consin Package, version 9.1 (Genetics Computer Group, ¯ata (class Colpodea); living cells of the cyanobacteri- Madison, Wis.). All the eukaryotic EF-1␣ sequences um Phormidium autumnale for Naxella sp. (class Nas- available in data banks were identi®ed using a BLAST sophorea); living cells of the ciliate T. pyriformis for search mail facility ([email protected]). The Euplotes aediculatus and Stylonychia mytilus (class Hy- blast2retp and retp2ali programs (P. Lopez, personal potrichea); wheat and rice infusion for Blepharisma ja- communication) allowed the automatic retrieval of these ponicum (class Heterotrichea); and living cells of the sequences and their writing into a MUST-compatible cryptophyte alga Cryptomonas sp. for Stentor coeruleus ®le. Partial sequences or those containing likely se- (class Heterotrichea). All the strains except Naxella sp. quencing errors were discarded, leading to a ®nal num- (found in a pond in Tivoli, Italy) were isolated from ber of 197 eukaryotic sequences. Thirteen archaeal EF- different ponds and soil samples from the campus of the 1␣ sequences were included as the outgroup. All of Paris XI University (Orsay, France). these sequences were manually aligned using the ED 236 Moreira et al. program of the MUST package, version 1.0 (Philippe Mutational saturation of the EF-1␣ sequences from 1993), and an unambiguous alignment of 387 positions a given taxonomic group was measured by comparison was obtained. It contained the regions 22±121, 129±172, of the number of substitutions inferred by MP or ML 174±185, 187±206, 208±214, 216±275, 277±325, 327± methods with the number of differences observed for 336, 338±343, 345±379, 381±389, and 391±419 of the each pair of sequences (Philippe et al. 1994). molecule (following the numbering of the T. pyriformis EF-1␣ sequence). Analysis of Sequence Variability Different sequence subsets (see Results) were To analyze the number of substitutions of the ciliate aligned in order to study the impact of the addition of EF-1␣ sequences and compare it with those of other new sequences on the EF-1␣ phylogeny. Well-known taxonomic groups, we distributed 162 eukaryotic and 13 monophyletic groups (fungi, metazoans, and angio- archaeal aligned sequences into 8 different groups. Cil- sperms) were represented by small but representative iates were analyzed as a single group (``all Ciliophora'') numbers of sequences to have data sets of limited size as well as being decomposed into the sequences from to facilitate the time-consuming analyses. Additionally, the genus Euplotes (``Euplotes spp.'') and those from EF-1␣ sequences known to be under developmental reg- the rest of the ciliates (``other Ciliophora''). Thus, ulation or low expression control were removed (e.g., groups were de®ned as follows: Metazoa (118 sequenc- Xenopus laevis thesaurin, Strongylocentrotus purpura- es), Fungi (20 sequences), Magnoliophyta (angio- tus, Porphyra purpurea tef-s). On the other hand, almost sperms) (14 sequences), all Ciliophora (19 sequences), all available protist sequences were used. The most im- Euplotes spp. (4 sequences), other Ciliophora (15 se- portant exception was the sequence from the hypotrich quences), Diplomonadida (5 sequences), and Archaea ciliate S. lemnae, which was eliminated to reduce the (13 sequences). The most parsimonious tree was com- number of taxa, considering that this genus was well puted for each group using PAUP, version 3.1, with 10 represented by the species S. mytilus (97.5% identity random additions of taxa and the TBR branch-swapping with S. lemnae). The sequence from the microsporidian option. The number of variable positions as well as the Glugea plecoglossi (Kamaishi et al. 1996) was also ex- number of substitutions for each sequence position with- cluded from the analysis, since it is extremely divergent in each group was thus inferred. The numbers of vari- and likely to be misplaced in phylogenetic trees, taking able positions for the same taxonomic groups were also into account the compelling evidence to consider micro- calculated using SSU RNA and ␣-tubulin sequence data sporidians very derived fungi (MuÈller 1997). for comparison with the EF-1␣ data. Similarly, the number of substitutions for each po- Phylogenetic trees were constructed using distance sition of the EF-1 alignment was inferred and displayed (neighbor-joining [NJ]) (Saitou and Nei 1987), maxi- on an alignment of the consensus sequences of the dif- mum-parsimony (MP), and maximum-likelihood (ML) ferent taxonomic groups. Constant positions were rep- methods with the MUST version 1.0 package (program resented by their amino acid letters, those undergoing NJ) (Philippe 1993), PAUP version 3.1 (Swofford one or two substitutions were replaced by light gray 1993), and PROTML version 2.3 (Adachi and Hasegawa boxes, while those that have undergone three or more 1996), respectively. Phylogenetic distances were com- substitutions were replaced by dark gray boxes. puted with the method of Kimura (1983). MP trees were obtained from 100 random addition heuristic search rep- licates and the tree bisection-reconnection (TBR) Results branch-swapping option. ML trees were constructed by Characterization of Ciliate EF-1␣ Sequences the quick-add operational taxonomic units search em- PCR products of about 1,270 bp ampli®ed from ploying the JTT model of amino acid substitution and ciliate genomic DNAs using the primer pairs 1F-10R1, retaining the 5,000 top-ranking trees. An additional ML 1F-10R2, 1A-10R1, and 1A-10R2 were cloned and analysis taking into account a gamma distribution for screened by restriction analysis (30 clones for each am- among-site variation was carried out using the quartet pli®cation) and partial sequencing. The EcoRI restriction puzzling method of the program PUZZLE (Strimmer analysis proved to be especially suitable for differenti- and von Haeseler 1996). Bootstrap proportions (BPs) ation of multiple tef genes within a given species. Thus, were estimated by analysis of 1,000 replicates with sim- two different tef loci (showing different EcoRI restric- ple addition of taxa for the MP analyses and by analysis tion patterns) were detected in the species B. japonicum, of 1,000 replicates for the NJ analysis. For the ML anal- E. aediculatus, Spathidium sp., and S. coeruleus, while ysis, bootstrap proportions were computed using the only single tef loci were identi®ed in the rest of the RELL method (Kishino, Miyata, and Hasegawa 1990) species (C. in¯ata, Kentrophoros sp., Naxella sp., P. on the 5,000 top-ranking trees. carnivora, P. tetraurelia, S. mytilus, and T. henneguyi). Indices of tree symmetry (Is) were estimated using Fifteen new EF-1␣ genes were cloned and sequenced. the method of Heard (1992). In the case of a subset of None of these contained introns in the region covered sequences within a tree, Is was estimated by manual re- by the primers used. Failure to detect introns could have construction of a partial tree for these sequences with been a result of PCR bias, since the reactions designed respect to the topology they showed within the complete to amplify large DNA fragments could have led to the tree. Afterward, the Is of the partial tree was calculated recovery of only the smaller, intronless loci. To elimi- applying the method of Heard (1992). nate this possibility, we additionally ampli®ed several Eukaryotic EF-1␣ Evolution 237

FIG. 1.ÐML tree for the ciliate EF-1␣ sequences. Numbers at each node are MP (above) and ML (below) bootstrap values. Sequences determined in this study are indicated by asterisks. The scale bar represents 5 substitutions per 100 positions.

speci®c small fragments covering almost the complete hood values, were analyzed. Only the monophyly of het- EF-1␣-coding genes for all the tested species (see Ma- erotrichs (B. japonicum and S. coeruleus) was retrieved, terials and Methods). Sequencing of these fragments supported by a high BP value (100) (®g. 1). Conse- con®rmed a lack of introns. The relatively high number quently, the topology of our EF-1␣-based tree was in- of ampli®cations and clones screened supported the the- congruent with results of rRNA sequence analysis, ory that, indeed, ciliate tef loci, at least in these species, which support the monophyly of all of these classes are intronless. (Baroin-Tourancheau et al. 1995; Wright and Lynn EF-1␣-Based Ciliate Phylogeny 1997). Furthermore, depending on the reconstruction method applied (NJ, MP, or ML), ciliates showed very A phylogenetic analysis of the ciliate EF-1␣ se- different relationships, except for the monophyly of het- quences rooted with apicomplexan sequences (®g. 1), erotrichs, the two sequences from Spathidium sp., and using NJ, MP, and ML methods, was carried out. The those from the genus Euplotes (data not shown). The phylogenetic distribution of the duplicated tef sequences bootstrap support for the deep nodes sustaining these clearly indicates that several independent duplications diverse topologies was very low (BP Յ 45). Altogether, took place in ciliate tef gene evolution, instead of one these results showed that EF-1␣ was not a reliable mark- ancestral event followed by some losses. Thus, each pair er for inferring ciliate phylogeny. of duplicated sequences from the heterotrichs B. japon- The aberrant ciliate phylogeny may be due to the icum and S. coeruleus and from the litostomate Spathi- accumulation of multiple substitutions, which erases dium sp. appears as a monophyletic group. This strongly suggests that the divergence of these species preceded part of the phylogenetic signal. In fact, ciliate sequences the duplication of their tef loci. In contrast, the dupli- became saturated above a value of approximately 90 ob- cated sequences from the two Euplotes species revealed served differences, reaching a plateau around 130 ob- a more complex situation. Their observed phylogeny served differences, whereas the inferred substitutions could be explained by an ancestral duplication of the tef (calculated by MP) reach a value as high as 336 (®g. locus before the divergence of the two species, followed 2A). This saturation level has an order of magnitude by the loss of one of the E. aediculatus alleles and fur- comparable to that of the complete eukaryotic and ar- ther duplication of its remaining copy. An alternative chaeal data set, which displays a maximum number of explanation might be two independent duplications hid- observed differences of nearly 200, almost three times den by the high divergence degree of the second E. cras- as small as the maximum number of inferred substitu- sus sequence, which leads to its emergence at the base tions (560) (®g. 2B). The mutational saturation becomes of the Euplotes clade by a long-branch attraction artifact. especially critical when sequences show unequal evo- At present, the available information does not favor ei- lutionary rates, as in the case of ciliates. In fact, some ther hypothesis. ciliate sequences, especially those from Spathidium and The ML tree did not recover the monophyly of two Euplotes species, exhibit branches longer than the rest, ciliate classes, oligohymenophoreans and hypotrichs, which indicates important differences in evolutionary even when the ®rst 5,000 trees, ordered by their likeli- rates. The combination of both factors, mutational sat- 238 Moreira et al.

the original situation prior to our studies. The phyloge- netic analysis led to a polyphyly of ciliates (®g. 3A), which displayed four independent offshoots as previ- ously reported (Hashimoto and Hasegawa 1996). Three of the ciliate sequences branched out of the crown, and the second E. crassus EF-1␣ sequence was even the ®rst offshoot in the NJ tree (earlier than the Giardia lamblia sequence). The addition of our new ciliate sequences (®g. 3B) or of several new protist sequences recently determined by other groups (®g. 3C) again failed to recover the monophyly of ciliates. Tree B again separated ciliates into four independent groups, while tree C (with a small- er ciliate sampling) produced two ciliate assemblages. Interestingly, the addition of several nonciliate sequenc- es to the initial data set led to the recovery of the mono- phyly of three ciliate sequences (T. pyriformis and both E. crassus sequences), although with very low support (BP ϭ 22). Finally, all the sequences combined into a fourth data set (D) generated a tree topology in which ciliates did not form a monophyletic clade but were distributed as three discrete groups (®g. 3D), regardless of the re- construction method employed. A large ciliate group comprised 14 of the 18 ciliate sequences, although their monophyly was very weakly supported (BP ϭ 3). In addition, the apicomplexans P. falciparum and C. par- vum and the amoeba E. histolytica branch artifactually within this group. Moreover, in the NJ and MP trees, all the Euplotes sequences emerged out of this ciliate group, with the most divergent E. crassus sequence be- ing the earliest offshoot of the eukaryotic tree (data not shown). The two Spathidium sp. sequences were also located outside of the main ciliate clade with a very unstable position, varying from a basal emergence to- gether with the diplomonads and the trichomonads in the MP tree (with a very low BP of 8) to a more central region after the emergence of these phyla in the NJ and FIG. 2.ÐSaturation curves for the ciliate (A) and eukaryote plus ML trees. All these observations re¯ect the extreme in- archaea (B) data sets. On the y axis, the numbers of observed differ- stability of the phylogenetic positions of the Euplotes ences between pairs of sequences are shown; on the x axis, the num- bers of substitutions between the same two sequences inferred by the and Spathidium sequences, a result possibly related to MP method are shown. Each dot thus represents one pair of sequences, long-branch attraction artifacts stemming from their considering the observed over the inferred number of substitutions. high evolutionary rates. Finally, the ML tree also dis- The diagonal represents the ideal case of no more than one substitution played the hypotrich S. mytilus and the karyorelictean per sequence position. Open circles (B) correspond to eukaryotic ver- Kentrophoros sp. outside of the main ciliate group, as sus archaeal sequence comparisons. the ®rst offshoot next to a Euglenozoa-Magnoliophyta clade. uration and unequal evolutionary rates, can explain the A positive effect of sequence addition was detected abnormal phylogeny of ciliates. through the evolution of the tree symmetry. This param- eter is expressed with an index (Is) that ranges from 0, Phylogenetic Analysis of the Expanded Eukaryotic for a completely symmetric tree, to 1, for a completely EF-1␣ Data Set asymmetric tree (Heard 1992). All the trees obtained To test the model explaining the asymmetric base with the initial data set were highly asymmetric (Is of of the eukaryotic EF-1␣ tree, four different data sets (A, 0.73) (table 2), especially in their basal regions. Inter- B, C, and D) were analyzed by all three methods. Since estingly, the increase in sequence number within the two ciliate sequences exhibit notable unequal evolutionary intermediate data sets with ciliate (data set B) and non- rates, only the ML analysis, which is less affected by ciliate species (data set C) strongly affected the sym- this factor (Hasegawa and Fujiwara 1993), will be de- metry of the new trees (Is values of 0.33 and 0.39, re- scribed. The initial data set studied (A) contained a lim- spectively). Finally, the most complete data set (D) ited sampling of sequences (20 eukaryotic sequences showed very low tree asymmetry (Is ϭ 0.18). We esti- plus 8 archaeal sequences as the outgroup) to represent mated the net gain of symmetry by calculating the Is of Eukaryotic EF-1␣ Evolution 239

FIG. 3.ÐThe impact of sequence addition. ML trees were constructed using data sets A (initial data set), B (addition of ciliate sequences), C (addition of diverse protist sequences), and D (combination of data sets B and C). Ciliate species are shown in bold. Numbers at each node are bootstrap values. Scale bars represent 5 substitutions per 100 positions. 240 Moreira et al.

Table 1 Differences in Numbers of Steps and Likelihoods for the Trees Imposing Ciliate Monophyly and Those Imposing Alveolate Monophyly

DATA SET A B C D TOPOLOGY (28 species) (42 species) (37 species) (51 species) Ciliate monophyly ...... D ϭ 15 (0.79%) D ϭ 14 (0.55%) D ϭ 16 (0.67%) D ϭ 10 (0.33%) ⌬LnL ϭ 0.76 SE ⌬LnL ϭ 2.52 SE ⌬LnL ϭ 0.91 SE ⌬LnL ϭ 0.59 SE Alveolate monophyly . . . D ϭ 17 (0.89%) D ϭ 15 (0.59%) D ϭ 16 (0.67%) D ϭ 10 (0.33%) ⌬LnL ϭ 0.88 SE ⌬LnL ϭ 1.38 SE ⌬LnL ϭ 0.91 SE ⌬LnL ϭ 0.19 SE

NOTE.ÐD is the difference in the numbers of steps, and ⌬LnL is the difference in likelihoods between the best unconstrained tree and trees constraining ciliate or alveolate monophyly. Numbers in parentheses are the percentages of the numbers of steps with regard to the shortest tree. the 20 species from the initial data set within the new quences (4 in data set A) is more dif®cult to retrieve tree topologies obtained with the other three data sets than that of a larger number (18 in data sets B and D), (subset A in table 2). The Is for subset A, with an initial which demonstrates the positive effect of sequence ad- value of 0.73 (i.e., that of tree A), decreased to 0.56 and dition. 0.55 in trees B and C, respectively, and reached 0.45 in These results are related to the fact that statistical the tree D. Comparison of the different reconstruction support for the different nodes of the tree varies de- methods showed that the ML retrieved the most sym- pending on the addition of new sequences. Actually, the metric trees from almost all data sets, while the NJ and tree derived from the smallest data set (A) showed three MP methods did not show important differences be- different regions. A ®rst region comprising the earliest tween them (table 2). The observed increase of Is is thus branches supported by BPs above 50, a central region in good agreement with the predictions of the model. with BPs ranging from 39 to 48 (except for the BP of The improvement of the inferred phylogeny is also 21 for the branching of the Magnoliophyta), and a third well manifested by the fact that in the ®nal tree, the region containing some taxa of the crown supported by monophyly of ciliates requires a difference of likelihood very high BPs (above 98). The addition of new taxa to of only Ϫ25.5 (representing an SE of 0.59) against the the analysis (trees B and C) did not alter this regional- most likely tree (with a likelihood of Ϫ17,757), which ization of BP values, although these values strongly de- is statistically insigni®cant (Kishino and Hasegawa creased in the central region (approximately from 39± 1989). A tree with monophyletic ciliates corresponds to 48 to 10±40). Using data set D, this decrease was ac- 3,041 steps, compared with 3,031 for the most parsi- centuated (central-region BP values were between 3 and monious tree. Moreover, the monophyly of alveolates 23) (®g. 3). Interestingly, BP values in the other two (i.e., the ciliates plus the two apicomplexans P. falci- regions of the tree, especially those of the crown, did parum and C. parvum) was recovered with the same not experience pronounced changes. small increase of 10 steps (table 1). The taxonomic sam- The monophyly of Diplomonadida, Euglenozoa, pling had an interesting effect on these values, since in Fungi, Metazoa, and Mycetozoa (including dictyostel- data set A, the monophyly of the four ciliate sequences ids, myxogastrids, and protostelids) (BPs close to 100), requires an increase of 15 steps (1,925 instead of 1,910 the early emergence of diplomonads (Giardia and rela- for the most parsimonious tree), similar to the values tives) (BPs of 57±91) and of the parabasalian Tricho- obtained using data sets B and C (14 and 16 steps, re- monas (BPs of 71±88), and the sisterhood of Fungi and spectively) (table 1). However, these values, expressed Metazoa (BPs of 96±100) were well supported by all of as percentages of the respective total numbers of steps, the data sets and methods employed. However, the sister are much smaller for data sets B and C (0.55 and 0.67, group of this last clade could not be reliably determined respectively) than for data set A (0.79). As expected, the with the available data. Depending on the taxonomic percentage is still smaller for data set D (0.33). Alveo- sampling and on the method used, the Mycetozoa or the late monophyly requires similar values (table 1). Nota- rhodophyte P. purpurea is the closest group to the an- bly, the monophyly of a smaller number of ciliate se- imal-fungal clade (data not shown). Interestingly, a monophyletic group containing the Euglenozoa and the Table 2 Magnoliophyta (as ®rst noticed by Roger et al. 1999) Symmetry Indices for the Trees Built Using All Sequences was observed in all the trees after the addition of new (all taxa) or Only Those from Data Set A (subset A) species (ciliates or other protists), with BPs ranging from 40 to 68. RECONSTRUCTION METHOD ML MP NJ Analysis of Variation Rates DATA SET All Taxa Subset A All Taxa Subset A All Taxa Subset A We determined the numbers of variable positions A ..... 0.73 0.73 0.77 0.77 0.74 0.74 for eight taxonomic groups (Metazoa, Fungi, Magno- B ..... 0.33 0.56 0.39 0.69 0.39 0.65 liophyta, all Ciliophora, genus Euplotes, the rest of Cil- C ..... 0.39 0.55 0.26 0.50 0.41 0.53 iophora, Diplomonadida, and Archaea) and three differ- D ..... 0.18 0.45 0.25 0.65 0.32 0.60 ent genes (EF-1␣, SSU rRNA, and ␣-tubulin) (table 3). Eukaryotic EF-1␣ Evolution 241

Table 3 Numbers of Variable and Signature Positions in the EF-1␣, ␣-Tubulin, and SSU rRNA Sequences from Eight Different Taxonomic Groups

MARKER EF-1␣ ␣-Tubulin SSU RNA (387 positions) (375 positions) (875 positions)

GROUP N Variable Signature N Variable Signature N Variable Signature Metazoa ...... 118 221 (57.1) 1 26 145 (38.7) 1 26 272 (31.1) 0 Fungi ...... 20 125 (32.3) 1 14 224 (59.7) 0 20 204 (23.3) 0 Magnoliophyta . . . 14 59 (15.2) 11 12 75 (20) 3 5 25 (2.8) 0 Ciliophora ...... 19 251 (64.8) 0 14 65 (17.3) 1 16 237 (27.1) 1 Euplotes spp. .... 4 157 (40.6) 0 NA NA NA NA NA NA Other ciliates .... 15 217 (56) 0 NA NA NA NA NA NA Metamonada ..... 5 148 (38.2) 18 4 36 (9.6) 31 5 385 (44) 14 Archaea ...... 13 276 (71.3) 7 Ða Ð Ð 11 352 (40.2) 64

NOTE.ÐN ϭ number of sequences analyzed. NA ϭ not analyzed. Numbers in parentheses are the numbers of variable positions as percentages of aligned positions. a No eukaryotic-type tubulin has been found in Archaea.

Among the eukaryotic EF-1␣ sequences, ciliates exhib- centrated in other regions. Among these motifs, some ited the highest number of variable positions (251 out putative motifs of interaction with actin are most inter- of 387 aligned positions, twice as many as the taxonom- esting. ically diverse fungi). This large proportion of variable positions indicates that, during the evolutionary history Discussion of this group, a large fraction of amino acid positions Ciliate tef Gene Evolution were free to vary, i.e., a large number of covarions ex- isted (Fitch and Markowitz 1970). This number was The analysis of the 15 new ciliate tef genes ana- comparable to that of the Archaea (276), which repre- lyzed here showed three interesting characteristics: their sents a taxonomic domain as broad as the whole eu- lack of introns, the fact that multiple tef copies in dif- karyotic one. Interestingly, the Diplomonadida, a protist ferent ciliate species appear to be the result of several group not branching within the terminal crown, with independent duplications, and strong unequal evolution- only ®ve EF-1␣ sequences, showed a high number of ary rates. Intron absence might have been the result of variable positions (148). a bias in the PCR ampli®cations, but we tried to dimin- In contrast, ciliates did not show high variability ish this possibility as much as possible and consistently for the other two markers, which were analyzed using found no introns. The fact that tef genes directly cloned similar taxonomic samplings (table 3). They possess 237 from genomic DNA of E. crassus (Bergemann et al. variable positions for SSU RNA, similar to Metazoa 1996) and S. lemnae (Bierbaum, Donhoff, and Klein (272) and Fungi (204). On the other hand, their ␣-tu- 1991) also lacked introns strongly supports actual intron bulin is less variable (65 variable positions) than are absence. On the other hand, the existence of more than those of Metazoa (145) and Fungi (224). An important one tef locus per genome is very frequent among eu- consequence of the great ciliate EF-1␣ variability is the karyotes, and evidence exists for multiple independent absence of speci®c signatures for this group. In fact, duplication events within different taxonomic groups. signatures are the main reason that the monophyly of At ®rst sight, the high variability of the ciliate EF- groups is recovered even when the sequences show a 1␣ sequences with regard to their strong differences of high degree of saturation (Lopez, Forterre, and Philippe evolutionary rate seemed not to affect the functional do- 1998). A good example is provided by the diplomonads, mains of the molecule. Indeed, even the most divergent which exhibit a high number of variable positions (148) of these sequences show a general high degree of con- but also a remarkable number of signature positions (18) servation of all of the characterized motifs involved in that are different or variable in the other groups. This the translational activity, such as the GDP-, GTP-, and fact leads to very high support (a BP of 100 for all of tRNA-binding regions (®g. 4). Other ciliate molecules the methods) for the monophyly of this group. playing a major role in the translation process, such as Additionally, we studied the distribution of the var- rRNAs (Van de Peer, Van der Auwera, and de Wachter iable positions along the EF-1␣ primary structures to 1996), do not display an increased evolutionary rate verify whether some known protein motifs are affected equivalent to that exhibited by the EF-1␣ sequences (see by the unusual variability of the ciliate sequences. For table 3). Both observations suggest that the translational this purpose, the inferred number of substitutions for activity of ciliates has not undergone a very divergent each position was displayed on an alignment of the con- evolution. However, it has recently been demonstrated sensus sequences of these groups (®g. 4). Notably, some that the EF-1␣ plays additional roles in eukaryotic cells. EF-1␣ motifs involved in the translational activity, such Compelling evidence exists of the EF-1␣ interaction as the GDP-, GTP-, and tRNA-binding regions, are well with cytoskeletal proteins, particularly actin (Demma et conserved in Ciliophora, while high variability is con- al. 1990; Itano and Hatano 1991), but also tubulin (Shi- 242 Moreira et al.

FIG. 4.ÐPosition-by-position analysis of sequence variability. Consensus sequences were obtained for eight taxonomic groups, and the number of substitutions for each position within each group, inferred by the MP method, is represented. Amino-acid letters correspond to constant positions, light gray boxes correspond to positions that have undergone one or two substitutions, and dark gray boxes correspond to positions that have undergone three or more substitutions. GTP- (bold letters), tRNA-, and actin- (A-b) binding domains are indicated. ina et al. 1994), microtubule-organizing centers (Numata ciliate actin and EF-1␣, or even to a loss of their inter- et al. 1991), and calmodulin (Kurasawa et al. 1996). action. However, more data, especially from biochemi- Moreover, EF-1␣ has been found to promote the bun- cal approaches, will be needed to test this hypothesis. dling of actin ®laments and the severing of microtubules (Demma et al. 1990; Shiina et al. 1994). Such activities The Evolutionary Position of Ciliates and Their have also been described for protists, including ciliates Impact on the Eukaryotic EF-1␣-Based Phylogeny (Kurasawa et al. 1996). The phylogenetic analysis of the enhanced EF-1␣ Interestingly, the overall actin-based phylogeny of data set including 15 new ciliate sequences still dis- eukaryotes is congruent in some respects with that based played an artifactual polyphyletic distribution of this on EF-1␣ sequences. It shows a similar polyphyly of phylum in the eukaryotic tree. At ®rst sight, this seems ciliates and a very early emergence of some sequences, to contradict the idea that the increase of the number of especially those from the genus Euplotes (Bhattacharya sequences should improve phylogenies (Lecointre et al. and Weber 1997; Philippe and Adoutte 1998). Actin is 1993; Graybeal 1998). In fact, the tree topologies ob- a quantitatively minor protein in ciliates, and its function tained from our different data sets suggest that the appears to be limited to food vacuole formation (Cohen, branching of several ciliate sequences outside of the Garreau de Loubresse, and Beisson 1984). This limita- main ciliate group is due to a long-branch attraction phe- tion implies a relaxation on its selective constraints that nomenon. This artifact particularly affects the Euplotes could explain its accelerated evolutionary rate (Philippe sequences, since the NJ and MP methods do not place and Adoutte 1998). Hence, the acceleration of the actin them within the main ciliate group, displaying these se- mutational rate might have been accompanied by a co- quences as very early branches. Thus, the order of emer- evolutionary acceleration of the mutational rate of the gence of the ciliate species correlates roughly with the involved EF-1␣ interacting regions. These regions are branch lengths inferred from the analysis of the internal not well characterized, though they have been tentative- ciliate phylogeny (®g. 1) and contributes to the in- ly circumscribed to some short motifs within the amino- creased asymmetry of the basal region of the EF-1␣ tree. terminal and central domains of the protein (Edmons, The simple increase in the number of sequences Murray, and Condeelis 1995). For almost all these of may not overcome the long-branch attraction problems; putative motifs, the highest variability among the groups the addition of slow-evolving sequences is the most ef- analyzed is seen in ciliates (®g. 4). Altogether, these fective approach to reduce problems of phylogenetic in- results could point to a coevolutionary acceleration of consistency (Kim 1996). For instance, the addition of Eukaryotic EF-1␣ Evolution 243

fast-evolving sequences, such as those of the Euplotes However, several problems make the reliability of this and Spathidium species, did not solve the long-branch marker questionable for the intermediate and basal re- attraction problem for ciliates in the NJ and MP analys- gions of the eukaryotic tree, where most protist species es. Thus, although the attraction of the E. crassus are located. branches toward the tree base was notably attenuated As shown, this problem arose from two main fac- after addition of the E. aediculatus sequences, they join tors: high mutational saturation and the long-branch at- the rest of ciliates only in the ML analysis. Therefore, traction artifact. In fact, mutational saturation erases it seems that the choice of adequate sequences could be most of the phylogenetic signal, thereby allowing the an even more decisive factor than the simple increase long-branch attraction artifact to be the main factor in in the amount of sequences (Aguinaldo et al. 1997). Ob- determining the af®nities of the sequences during phy- viously, the lack of a priori criteria to foresee the quality logenetic analysis. In addition, the outgroup used to of any given sequence constitutes a problem. Hence, in build EF-1␣-based eukaryotic phylogenies presents an practice, the sole strategy with which to approach prob- interesting problem due to its sequence variability. Ar- lematic phylogenies is to increase the number of se- chaeal EF-1␣ sequences exhibit an extreme degree of quences, with the hope that this will also provide se- variability, with 276 variable positions out of 387 quences of good phylogenetic quality, i.e., slow-evolv- aligned ones, the highest number noted among the ing ones. In addition, the careful selection of sequences, groups analyzed (table 3). The sole well-conserved po- taking into account only the slow-evolving sequences, sitions of the archaeal EF-1␣ correspond mainly to con- can be of great help. stant positions in eukaryotic EF-1␣ (®g. 4). These po- Although the addition of new sequences has not sitions are scarcely informative for phylogenetic infer- resolved the problem of long-branch attraction for the ence, since they do not allow differentiation among EF-1␣ data set, the behavior of the tree symmetry, an groups or they represent nonpolarizable characters. This important parameter in describing the tree topologies, may also increase the long-branch attraction phenome- appears to con®rm the assumption of our model of eu- non, since those highly variable eukaryotic sequences karyotic EF-1␣ evolution. In fact, the addition of new will be strongly attracted by the highly variable out- sequences led to a strong increase in the global tree sym- group, as proven for other markers (Lockhart et al. metry, from an initial Is value of 0.73 (data set A) to a 1996). This seems to especially affect the branches in ®nal one of 0.18 (data set D) (table 2). A part of this the basal and middle regions of the tree, which appear gain of symmetry comes from the simple increase of more sensitive to sampling differences in the outgroup species within several clades. The most important gain (Roger et al. 1999). In addition, the position-by-position of symmetry with the model is due to the joining of variability analysis has shown that the large numbers of some basal branches emerging asymmetrically in the EF-1␣ positions free to vary, or covarions, are dissimilar ®rst tree (data set A) to monophyletic groups in the ®nal among the different ciliate groups, as seen for the di- tree (data set D). The increase of the sequence number vergent Euplotes sequences, in which 32 of the 157 var- leads from an initial Is value of 0.73 to a ®nal one of iable positions are constant within the rest of ciliates 0.45 for species subset A. This net gain of symmetry (®g. 4). This suggests the existence of a high degree of due to the breaking of some long branches supports the heterogeneity in the ciliate covarion composition, not notion that the asymmetric base of the EF-1␣ tree is only as compared with other eukaryotic groups, but also highly artifactual and probably derived from long- within this phylum. A complex set of factors may be branch attraction artifacts. The comparison of the to- related to this heterogeneity, such as the unequal rates pologies derived from the different reconstruction meth- of other elements of the ciliate translation machinery, ods lends additional evidence for this conclusion. In especially the rRNAs, imposing different coevolutionary fact, the ML trees displayed the highest symmetry val- constraints on the EF-1␣ genes. This heterogeneous cov- ues in almost all cases (table 2). Since the ML method arion composition has important consequences, since is the least sensitive to long-branch attraction (Kishino correction models such as the gamma distribution (Yang and Hasegawa 1989), the fact that the ML tree topolo- 1996), valid for sequences with homogeneous among- gies are more symmetric also allows establishment of a site variation, cannot be successfully applied to sequenc- likely correlation between asymmetry and artifactual re- es with such heterogeneous covarion composition. In construction from a methodological point of view, as fact, our analyses of the EF-1␣ data set using this cor- con®rmed by simulation approaches (unpublished data). rection method did not overcome the problems dis- These observations may be true even for some groups cussed here, yielding results almost identical to those of placed as early branches with high BPs, such as the the classical ML analysis (data not shown). microsporidians, diplomonads, and trichomonads. In contrast, the most apical region of the tree ex- hibits more consistency, probably owing to the fact that EF-1␣ as a Phylogenetic Marker the level of mutational saturation is much lower there The eukaryotic EF-1␣-based phylogeny exhibits than in the rest of the tree. Therefore, the usefulness of important divergences from that based on rRNA se- EF-1␣ for the inference of the eukaryotic phylogeny quences, some of which have also been shown by other cannot be fully rejected, since it seems to be a suitable phylogenetic markers and biological data. A clear ex- marker for analyzing some phyla in the crown, as dem- ample is the monophyly of mycetozoans and their in- onstrated by the phylogenetic analysis of the Mycetozoa, clusion within the crown (Baldauf and Doolittle 1997). misplaced by some other markers (Baldauf and Doolittle 244 Moreira et al.

1997). Another example may be the interesting grouping ciliate Euplotes crassus are very dissimilar in their sequenc- of Euglenozoa and angiosperms into a well-supported es, copy numbers and transcriptional activities. Gene 168: terminal crown assemblage (®g. 3), in contradiction to 109±112. the rRNA-based phylogeny. Several biological data BHATTACHARYA, D., and K. WEBER. 1997. The actin gene of agree with this observation, although some others clear- the glaucocystophyte Cyanophora paradoxa: analysis of the coding region and introns, and an actin phylogeny of eu- ly differentiate the two groups (Cavalier-Smith 1987). karyotes. Curr. Genet. 31:439±446. Therefore, this putative assemblage should be cautiously BIERBAUM, P., T. DONHOFF, and A. KLEIN. 1991. Macronuclear regarded until the evaluation of additional phylogenetic and micronuclear con®gurations of a gene encoding the pro- markers leads to a de®nitive answer. tein synthesis elongation factor EF-1␣ in Stylonychia lem- The comparison of different phylogenies suggests nae. Mol. Microbiol. 5:1567±1575. that no presently known marker avoids all phylogenetic CAVALIER-SMITH, T. 1987. Glaucophyceae and the origin of reconstruction artifacts. All of them yield more-or-less plants. Evol. Trends Plants 2:75±78. contradictory results and display trees with presumably COHEN, J., N. GARREAU DE LOUBRESSE, and J. BEISSON. 1984. problematic regions (e.g., asymmetric bases). Most like- Actin micro®laments in Paramecium: localization and role ly, a reasonable picture of eukaryotic evolution will be in intracellular movements. Cell Motil. 4:443±468. obtained only in the form of a composite that integrates DEMMA, M., V. WARREN,R.HOCK,S.DHARMAWARDHANE, and J. CONDEELIS. 1990. 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