Département de Zoologie et Biologie Animale, Université de Genève, Switzerland
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Abstract |
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Introduction |
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The 18S rDNA gene is the most commonly used gene in phylogenetic analyses, and its database is the most complete for all living organisms (Hillis and Dixon 1991
; Maidak et al. 1999
). Like other ribosomal genes, the 18S gene is very easy to amplify because of its large number of copies per genome. Furthermore, the alternation in a single molecule of very conserved and much more variable regions makes 18S rDNA a powerful tool for molecular phylogeny at different taxonomic levels. However, it has been shown that in many phyla, such as Rhombozoa, Dicyemida, Nematoda, Gnathostomulida, and Chaetognatha, 18S rDNA genes do not evolve at a constant rate (Katayama et al. 1995
; Winnepenninckx et al. 1995
; Pawlowski et al. 1996
; Littlewood et al. 1998
). As a result, phylogenetic analyses lead to an artifactual basal clustering of the fast-evolving phyla, known as the long-branch attraction (LBA) phenomenon. The Acoela, most of which have been shown to be fast-clock organisms for the 18S rDNA gene (Carranza, Baguña, and Riutort 1997
), represent a particularly good example of the LBA effect.
There are two main ways to overcome the LBA problem. The first one is to broadly sample problematic groups in order to find non-fast-clock species among each of them. This method was successfully used with the phylum Nematoda, shown to be closely related to the Arthropoda (Aguinaldo et al. 1997
), and led to a better comprehension of general metazoan phylogeny, with the separation of the triploblastic animals into three main clades, the Lophotrochozoa, the Ecdysozoa, and the Deuterostomia (Halanych et al. 1995
; Aguinaldo et al. 1997
). Ruiz-Trillo et al. (1999)
applied this method to the Acoela and retained the unique non-fast-clock sequence of Paratomella rubra out of 18 acoel species. When only taxa with uniform rates of change were included in their analyses, the sequence of P. rubra appeared in a very basal position, as the first offshoot of the triploblastic animals. Therefore, the authors concluded that the acoel flatworms do not belong to the phylum Platyhelminthes and are probably the closest living relatives of the first Bilateria.
A second way to avoid the LBA effect is to find another gene for which rates of substitution are more constant between all metazoan phyla. The problem in finding such a gene is that no other database of metazoan sequences is as complete as the one for 18S rDNA. This is the main reason why so few studies of animal phylogeny are based on other genes, despite the general agreement that molecular phylogenetic inference requires congruent results from multiple gene sequences to be really conclusive (Baldauf and Palmer 1993
; Regier and Schultz 1997
; Abouheif, Zardoya, and Meyer 1998
).
Elongation factor 1-alpha (EF1a) is a protein found in all eukaryotic lineages. EF1a is very abundant in the cytosol, where it is involved in the GTP-dependent binding of aminoacyl-tRNAs to the A site of the ribosomes in the second step of translation from mRNAs to proteins. Its universal occurrence and good amino acid sequence conservation makes it an ideal phylogenetic tool with which to determine very ancient relationships, such as the relative branching order of the most primitive Eukaryotes (see, e.g., Hashimoto et al. 1994
; Nordnes, Krauss, and Johansen 1994
; Baldauf, Palmer, and Doolittle 1996
). Furthermore, the nucleotide sequences of EF1a genes were used at a much lower level to study phylogenies of families and genera, using the degenerated third position of codons (see, e.g., Cho et al. 1995
; Mitchell et al. 1997
). EF1a has also been used for resolving the phylogenetic relationships between animal phyla and classes as a good means to confirm or cast doubt on results based on other genes (Kojima et al. 1993
; Regier and Shultz 1997
; Kojima 1998
).
In this study, we use EF1a in the first attempt to infer the phylogenetic position of the Acoela using a protein-coding gene. A fragment of about 950 nt of EF1a was amplified and sequenced for the acoel flatworm Convoluta roscoffensis, as well as for three species of Tricladida (Crenobia alpina, Schmidtea polychroa, and Girardia tigrina) and two species of Polycladida (Prostheceraeus vittatus and Leptoplana tremellaris). We compared it to other metazoan sequences in order to establish the phylogenetic position of the Acoela and test the validity of the results obtained with 18S rDNA sequences.
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Materials and Methods |
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DNA from one individual of each species was extracted in guanidine lysis buffer, precipitated with isopropanol, and dissolved in distilled water (Tkach and Pawlowski 1999
).
PCR Amplification, Cloning, and Sequencing
PCR amplifications were all performed in a total volume of 50 µl with an amplification profile consisting of 40 cycles of 30 s at 94 °C, 30 s at 52 °C, and 120 s at 72 °C, followed by 5 min at 72 °C for final extension. To avoid contamination with the endosymbiotic algae of C. roscoffensis, a set of four metazoan-specific primers was designed for the PCR-amplification of a fragment of about 950 nt of EF1a (see table 1
for the sequences and positions of these primers). These metazoan-specific primers were used to amplify the same fragment of EF1a for the two polyclads and the three triclads. All PCR products were purified using the High Pure PCR purification kit (Roche) and then ligated in the p-GEM-T vector (Promega) and cloned in XL-2 Ultracompetent Cells (Stratagene), all according to the manufacturers' instructions. The clones were then sequenced on an ABI 377 Prism sequencer. The sequences reported in this paper have been deposited in the GenBank database under accession numbers AJ250908AJ250914.
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Results |
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Thirty-eight sequences of diverse metazoan and fungal species were retained for the phylogenetic analyses, with the latter being used as a designated outgroup in all analyses. The choice of Fungi as an outgroup is congruent with previous studies based on several proteins (Baldauf and Palmer 1993
; Nikoh et al. 1994
) which confirmed that they are the closest eukaryotic relatives of the Metazoa. Sequences of some groups (nematodes, molluscs, echinoderms) were not selected because of their artifactual branching at the base of the Metazoa. Three hundred two amino acid residues, corresponding to the amplified nucleotide fragments excluding the primers and the short insert in C. roscoffensis and the triclads, were used in the analyses (the complete alignment is available at our web site, http://www.unige.ch/sciences/biologie/biani/msg). Of these 302 amino acids, 149 are constant (48.2%). Twenty-three variable positions are parsimony-uninformative, and 130 are parsimony-informative.
Prior to phylogenetic analyses, we performed two tests. First, we ran a relative-rate test (Robinson-Rechavi and Huchon 2000) on both nucleotide and amino acid sequences of EF1a to determine if this gene shows a relatively constant rate of substitution among lineages. The test confirmed at a 5% level that the differences in substitution rates among all lineagesincluding the two acoel sequenceswere nonsignificant; in all lineages, the EF1a's evolve at similar rates. Then, we performed a likelihood-mapping analysis (Strimmer and von Haeseler 1997
) to evaluate the phylogenetic content of the data. Likelihoods of all 148,995 possible quartets of sequences were mapped on a triangle, which was partitioned in seven regions. Of these quartets, 84.3% had completely resolved phylogenies, and only 7.8% were in the star-tree region (fig. 2 ), indicating that the 302 amino acid residues of EF1a used in our analyses contain a satisfying degree of phylogenetic information. NJ, MP, and ML analyses were then performed.
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Next, an ML tree was constructed with the quartet puzzling (QP) algorithm (Strimmer and von Haeseler 1996
), which automatically assigns estimations of support to each internal branch. Dayhoff's PAM substitution model was used. The frequency of each amino acid was estimated from the data set, and a 5% level chi-square test was performed to confirm that the amino acid composition of each sequence was identical to the average amino acid composition of the whole alignment. The chosen model of rate heterogeneity was a discrete gamma distribution with eight categories, with all necessary parameters being estimated automatically from the data set. The QP search was conducted with 10,000 puzzling steps. As shown by the likelihood-mapping analysis, 11,592 of the 148,995 quartets analyzed (7.8%) were unresolved, leading to a multifurcating tree with a basal polytomy in the metazoan clade. The tree has a log likelihood value of -6,507.07. Six groups of metazoans are defined: the diploblastid clade, the vertebrate clade, the arthropod clade, the clade formed by annelids plus pogonophorans, and two platyhelminth clades. As in the NJ tree, the two sequences of C. roscoffensis appear as the sister group to the two polyclads, with a QP reliability of 65%. However, the monophyly of the Platyhelminthes was recovered in only 28% of all intermediate trees generated in the 10,000 puzzling steps, and the three triclad species cluster in a distinct group (data not shown).
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Discussion |
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However, the monophyly of the Platyhelminthes was never well supported in our analyses, as shown by very low bootstrap values (8% in the NJ analysis and 21% in the MP analysis). Interestingly, this low support for the platyhelminth clade is apparently mainly due to the important differences between polyclad and triclad sequences. Indeed, if the acoel sequences are not included in the analysis, the five Turbellaria no longer appear monophyletic with the NJ method (data not shown), with the triclads being the sister group to the annelid plus pogonophoran clade. On the other hand, when the three triclad sequences are not included in the analysis, the grouping of the acoel sequences with the two polyclads is strengthened, with a bootstrap support value of 74% (data not shown). Inversely, when the two polyclad sequences are removed from the analysis, the two acoel sequences group with the triclads with a bootstrap support value of 61% (data not shown). Similarly, with the MP method, removing the two polyclad sequences from the analysis strengthens the support value for the acoel plus triclad clade (71%, data not shown). In addition, if the triclad sequences are removed from the analysis, the two sequences of C. roscoffensis cluster with the polyclad sequences with a bootstrap support value of 62%. In all cases, the Acoela branch with one of the lineages of Turbellaria, and the platyhelminth clade always remains the sister group to the other lophotrochozoan species. In fact, the two sequences of C. roscoffensis share several common amino acid substitutions with the polyclads on the one hand and with the triclads on the other hand, with very few of them being common to the five Turbellaria. This may explain why the monophyly of the platyhelminth clade is never well supported, whereas the monophyly of the acoel plus triclad clade alone and that of the acoel plus polyclad clade alone are supported by quite confident bootstrap values.
Given the weak support of EF1a data for the monophyly of the Platyhelminthes, we were interested in testing their support for the hypothesis of an early branching of the Acoela, suggested by 18S rDNA data. With this aim, we used the exhaustive search mode of the protml program in MOLPHY, version 2.3 (Adachi and Hasegawa 1996
), which allows constrained tree topologies, to determine the likelihood values of the totally resolved trees compatible with the six metazoan groups present in the QP tree. We retained only the 20 best tree topologies out of the 945 that were compatible with the QP analysis. Nine of them show the lophotrochozoan clade to be monophyletic, and in four, the Platyhelminthes are monophyletic and a sister group to the annelid plus pogonophoran clade. Moreover, the three diploblastid sequences are the first offshoot of the Metazoa in 18 of the 20 best trees. The Kishino-Hasegawa test (Kishino and Hasegawa 1989
) showed at a 5% level that none of the 20 best topologies could be rejected as significantly worse than the other. Next, we performed the same test on two constrained topologies, defined as follows: we used the general topology obtained by Ruiz-Trillo et al. (1999)
as a constrained topology, first with the Platyhelminthes monophyletic, then with the two acoel sequences at the base of all other triploblastic species. The first constrained topology was included in the 20 best tree topologies; it has a log likelihood value of -6,373.49. However the topology which shows the Acoela as the first offshoot after the diploblasts, which has a log likelihood value of -6,392.86, was rejected as significantly worse than the best tree at a 5% level. This test shows that even if EF1a sequences cannot resolve the problem of the monophyly of the phylum Platyhelminthes, they give good evidence in favor of rejecting the hypothesis that acoel flatworms represent the first offshoot of the Bilateria.
A problematic feature of our analyses is the fact that the two sequences of C. roscoffensis grouped with the polyclad sequences in both NJ and ML analyses, whereas they grouped with the triclad sequences in the MP analysis. This can be explained by the fact that in both NJ and ML analyses, amino acid substitutions were weighted using Dayhoff's PAM substitution model, whereas in the MP analysis, all amino acid substitutions were equally weighted. As stated above, the two acoel sequences share a few more amino acid substitutions with the three triclads than with the two polyclads, leading to the grouping of C. roscoffensis with the triclads in the MP analysis. However, when Dayhoff's PAM substitution model is taken into account, these substitutions are weighted differently, and C. roscoffensis groups with the polyclads. Nevertheless, the sequence signature shared by C. roscoffensis and the three triclads suggests that the Acoela might be more closely related to the Tricladida than to the Polycladida. Interestingly, Campos et al. (1998)
also found a close relationship between Tricladida and Acoela in their molecular phylogeny of the phylum Platyhelminthes, based on partial 18S rDNA sequences. In the analyzed fragment of EF1a, about 15 specific amino acid substitutions define the triclad clade, whereas only about 10 define the polyclad clade. NJ and ML analyses might thus be misleading because of the numerous amino acid substitutions that define the triclad clade, which tend to group the acoel and polyclad sequences.
More sequences from representatives of all turbellarian orders and of members of the Neodermata would perhaps help to clarify this situation. It has been shown (Lecointre et al. 1993
; Giribet and Ribera 1998
) that increasing the number of taxa in a clade can help to strengthen its monophyly, although it also very rapidly increases the duration of the analyses. However, even more EF1a sequences may not definitively resolve the relationships within the Platyhelminthes. As shown by our analyses, the phylogeny of the phyla for which more EF1a sequences are available is not always well resolved. Whereas the internal topology of the arthropod clade is always well defined in all trees, the one of the annelid plus pogonophoran clade is much vaguer. This shows that the phylogenetic information contained in EF1a sequences is not the same for all metazoan groups. EF1a is thus probably not the perfect gene for inferring good metazoan phylogenies, but neither is 18S rDNA, as shown by Abouheif, Zardoya, and Meyer (1998)
. Both genes give conflicting results, which prompts us to search for another molecular marker to conclusively resolve the phylogenetic position of Acoela. Our study stresses the need for the use of several different genes to properly determine the phylogenetic position of any group of organisms.
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Acknowledgements |
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Footnotes |
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1 Abbreviations: CI, consistency index; EF1a, elongation factor 1-alpha; LBA, long-branch attraction; NJ, neighbor joining; ML, maximum likelihood; MP, maximum parsimony; QP, quartet puzzling; rDNA, ribosomal DNA; RI, retention index.
2 Keywords: elongation factor 1-alpha
molecular phylogeny
Convoluta roscoffensis,
Acoela
Platyhelminthes
3 Address for correspondence and reprints: Jan Pawlowski, Université de Genève, Station de Zoologie, 154, route de Malagnou, 1224 Chêne-Bougeries, Genève, Switzerland. E-mail: jan.pawlowski{at}zoo.unige.ch
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