Introduction

Acoelomorph flatworms (Acoela+Nemertodermatida) constitute a small group of bilaterian marine worms that recently came to the limelight. Several morphological similarities suggest that acoelomorphs belong to the Platyhelminthes [1]–[3]; however, these characters are often ill-defined or mere symplesiomorphies [4], leaving the status of the phylum Platyhelminthes unsettled. Molecular phylogenies based on SSU rRNA [5], [6], combined SSU and LSU rRNA [7], myosin II [8] and mitochondrial genomes [9], provide strong statistical support for excluding acoelomorphs from Platyhelminthes. Rather, these markers locate them as a sister-group to all remaining Bilateria.

However, because acoelomorphs evolve at a very high rate (except perhaps for myosin II), their basal emergence can be easily explained by a long branch attraction (LBA) [10] artefact triggered by the distantly related outgroup (Cnidaria). Although careful approaches (e.g. selection of the slowest evolving rRNA sequence among 18 acoels [5]) have been used in an attempt to avoid LBA artefacts, the position of acoelomorphs remains unsettled. In particular, acoelomorphs are often considered as secondarily simplified organisms [11], [12], potentially explaining their fast evolutionary rate. However, finding acoelomorphs at or close to the base of Bilateria, instead of belonging to Platyhelminthes, would allow to polarize several key bilaterian characters including the brain, the coelom, the nephridium, and the possession of a primary larval stage.

We therefore decided to reanalyse this important question by applying the powerful simultaneous analysis of multiple orthologous nuclear protein-coding genes [13] and using up-to-date tree reconstruction methods in order to enhance the phylogenetic signal [14].

Results and Discussion

We sequenced 2304 ESTs from the acoel Convoluta pulchra, a number that has been suggested to provide a sufficient amount of homologous positions [15]. A total of 68 different protein-coding genes was unequivocally assigned to a dataset of conserved single copy genes previously cloned from other multicellular animals and related taxa [14], [16]. A rich taxon sampling of 51 species containing the main bilaterian lineages (Deuterostomia, Ecdysozoa and Lophotrochozoa) and several close and slow-evolving outgroup species (four Cnidaria, three Porifera, three Choanoflagellata and three Ichthyosporea) was selected to reduce as much as possible potential LBA artefacts. After the removal of ambiguously aligned positions and portions that have not been sequenced in Convoluta, an alignment of 11,959 positions was obtained.

The sequences from Convoluta are extremely divergent, which make its placement likely to be subject to the LBA artefact. Indeed, when parsimony, a method highly sensitive to this artefact [10], is used, Convoluta is invariantly attracted by the longest unbroken branch: the tunicate Oikopleura with a Bootstrap Support (BS) of 96% (Figure S1), or the Platyhelminthes (BS = 72%) when Oikopleura is excluded (Figure S2). We therefore turned to probabilistic methods that are less sensitive to LBA [17], [18]. Moreover, we used the CAT model [19] because it has been shown to overcome the LBA artefact when other models fail [14], [20]. The resulting tree (Figure 1) is in excellent agreement with current knowledge [12] and with recent large scale analyses [14], [16], [21]–[24]: the monophyly of all animal phyla and of most super-phyla (Bilateria, Protostomia, Ecdysozoa, and Lophotrochozoa) is recovered with strong support (BS>99%). Importantly, Convoluta does neither cluster with Platyhelminthes nor with Oikopleura, albeit these two groups display the longest branches. Although the precise position of Convoluta is not robustly resolved, two highly supported nodes (Protostomia and Lophotrochozoa) separate acoels from Platyhelminthes. The rejection of the traditional morphological hypothesis is therefore very strong.

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Figure 1. Phylogenetic analysis of genomic data strongly rejects the grouping of Convoluta and Platyhelminthes.

Bayesian tree obtained from the analysis of 11,959 aligned amino-acid positions with the CAT model. Bootstrap values obtained are indicated when <100%, otherwise a bullet is present on the node. The scale bar indicates the number of changes per site.

https://doi.org/10.1371/journal.pone.0000717.g001

However, the use of the standard WAG model provides strong support for the grouping of the two long branches platyhelminths and acoels (Figure S3). To further confirm that this grouping is an artefact of both parsimony and the WAG model, we looked for genes that are present in Convoluta and in deuterostomes, but absent from protostomes (see supplementary for details). Despite having analysed only 1,664 contigs from Convoluta (mainly encoding universal proteins such as ribosomal proteins), we found one gene, the guanidinoacetate N-methyltransferase, that is present in sponges, cnidarians, deuterostomes and Convoluta, but absent from all protostomes, except the basal chaetognaths [24]. The tree of the WAG model (Figure S3), which locates acoels as a sister-group to platyhelminths, implies at least three independent losses, whereas the tree of Figure 1 implies a single loss in the protostomes, after the emergence of chaetognaths. The congruence of the tree inferred by the CAT model and of the distribution of guanidinoacetate N-methyltransferase strongly argues that the tree inferred using the WAG model is biased by an LBA artefact, pointing towards a reduced sensitivity of the CAT model to this artefact [14], [20].

Although our phylogenomic tree of Bilateria is generally well resolved, three nodes within protostomes, the positions of chaetognaths, tardigrades and platyhelminths, are poorly supported. More strikingly, most of the relationships among deuterostomes are unsupported. Except for the monophyly of Olfactores [16] and of Ambulacraria [21], the remaining nodes, including deuterostomes and chordates, receive BS below 55%. The very fast evolving Convoluta emerges in this part of the tree, as a sister-group to deuterostomes (Figure 1, BS = 14%), to chordates (BS = 33%), or to Xenoturbella (BS = 20%). Convoluta shows affinity with deuterostomes in 90% of the bootstrap replicates, being basal to Bilateria in only 7% of the replicates and to Protostomia in only 3%. It should be mentioned that the lack of support within deuterostomes is not due to the presence of the long branch of Convoluta, since the BS remains low (between 42% and 73%) when Convoluta is removed (Figure S4). In consequence, obtaining a reliable placement of Convoluta is hampered by two difficulties: (i) its very fast evolutionary rate, and (ii) a location in, or close to, deuterostomes, a region for which the powerful phylogenomic approach only yields poorly supported results [16], [21], [23].

To enhance the phylogenetic signal [25], we removed the outgroup (i.e. non-bilaterian species). Surprisingly, a single topological change occurs (Figure S5): Xenoturbella becomes the sister-group to Convoluta, albeit with low support (41%). Furthermore, the weak support for the recently proposed Xenoturbella+Ambulacraria [21] is not due to the presence of Convoluta (Figure S6). In summary, our large alignment of 11,959 positions strongly rejects the grouping of acoels with platyhelminths, and more generally protostomes. It favours deuterostome affinity for acoels, potentially as a sister-group to xenoturbellids. However, more data, especially from slowly evolving acoels, are needed to solve this challenging phylogenetic problem.

The rejection of the grouping of acoels with platyhelminths sensu stricto (see [4] for a thorough taxonomical discussion) have several morphological implications. First, the lack of protonephridia in acoels, traditionally regarded as derived by the loss from a platyhelminth ancestor bearing them, may now be considered the retention of a primitive condition, a state shared with diploblasts. A similar argument could be applied to the sack-like gut of acoels (and of its sister group, the Nemertodermatida; [26], [27]) which may now be a symplesiomorphy shared with the similar state in diploblasts and independent from the similar sack-like condition of the Platyhelminthes sensu stricto within the Lophotrochozoa. In addition, the peculiar duet-spiral cleavage of acoels, also traditionally considered to be derived from the quartet spiral cleavage of other platyhelminths, could now be considered as having originated either from a form of radial or biradial cleavage characteristic of more primitive metazoans. Interestingly, the grouping of Acoelomorpha and Xenoturbellida (Figure S5) has been proposed on the basis of the ultrastructures of epidermal locomotory ciliary rootlets [28] and deserves future studies.

Our results contradict not only the morphological view but also previous molecular phylogenies that strongly support an emergence of acoels at the base of Bilateria [5]–[9], since this position receives here a bootstrap support of only 7%. But, such a basal emergence is expected to be reinforced by a LBA artefact between the long branch of Convoluta and the one of the distantly related outgroup. The use of the CAT model, which is less sensitive to LBA [14], [20], partly explains the observed lack of support for a basal emergence, since the latter support increases when the WAG model is used (Figure S7). However, the main reason for the discrepancy with previous molecular studies [5]–[9] is likely that the outgroup is less distantly related in our dataset, thereby reducing the LBA artefact: the distance from cnidarians to the last common ancestor of Bilateria represents 55.5% of the cnidarians/vertebrates distance for our nuclear proteins (Figure 1), but 63.9% for mitochondrial proteins [9] and 73.7% for 18S rRNA [5].

In more conceptual terms, the position of acoels out of the Platyhelminthes should warn us against the naive view that considers some features as ‘lost’, ‘absent’, or ‘reduced’ in clades (e.g. acoels) than might never have had them in the first place. Indeed, the possible position of acoels as a sister-group to Xenoturbellida, another group composed of simple organisms, at the base of deuterostomes leaves open the question of the evolutionary origin of their morphological simplicity.

Materials and Methods

Supporting Information

Acknowledgments

We would like to thank Peter Ladurner (University of Innsbruck, Austria) for providing specimens of Convoluta pulchra and Ketil Malde (University of Bergen, Norway) for helping in the EST data analysis. The Réseau Québecois de Calcul de Haute Performance, the Barcelona Supercomputing Centre (Mare nostrum) and the Centre de supercomputació de Catalunya are acknowledged for computational resources.