The phylogenetic position of Acoela as revealed by the complete mitochondrial genome of <Emphasis Type="Italic">Symsagittifera roscoffensis</Emphasis>

Background  

Acoels are simply organized unsegmented worms, lacking hindgut and anus. Several publications over recent years challenge the long-held view that acoels are early offshoots of the flatworms. Instead a basal position as sister group to all other bilaterian animals was suggested, mainly based on molecular evidence. This led to the view that features of acoels might reflect those of the last common ancestor of Bilateria, and resulted in several evo-devo studies trying to interpret bilaterian evolution using acoels as a proxy model for the "Urbilateria".     

Are the Platyhelminthes a monophyletic primitive group? An assessment using 18S rDNA sequences

In most zoological textbooks, Platyhelminthes are depicted as an early- emerging clade forming the likely sister group of all the other Bilateria. Other phylogenetic proposals see them either as the sister group of most of the Protostomia or as a group derived from protostome coelomate ancestors by progenesis. The main difficulty in their correct phylogenetic placing is the lack of convincing synapomorphies for all Platyhelminthes, which may indicate that they are polyphyletic. Moreover, their internal phylogenetic relationships are still uncertain. To test these hypotheses, new complete 18S rDNA sequences from 13 species of "Turbellaria" have been obtained and compared to published sequences of 2 other "Turbellaria," 3 species of parasitic Platyhelminthes, and several diploblastic and deuterostome and protostome triploblastics. Maximum-parsimony, maximum-likelihood, and neighbor-joining methods were used to infer their phylogeny. The results show the order Catenulida to form an independent early- branching clade and emerge as a potential sister group of the rest of the Bilateria, while the rest of Platyhelminthes (Rhabditophora), which includes the parasites, form a clear monophyletic group closely related to the protostomes. The order Acoela, morphologically considered as candidates to be ancestral, are shown to be fast-clock organisms for the 18S rDNA gene. Hence, long-branching of acoels and insufficient sampling of catenulids and acoels leave their position still unresolved and call for further studies. Within the Rhabditophora, our analyses suggest (1) a close relationship between orders Macrostomida and Polycladida, forming a clear sister group to the rest of orders; (2) that parasitic platyhelminthes appeared early in the evolution of the group and form a sister group to a still-unresolved clade made by Nemertodermatida, Lecithoepitheliata, Prolecithophora, Proseriata, Tricladida, and Rhabdocoela; and (3) that Seriata is paraphyletic.      

The Nemertodermatida are basal bilaterians and not members of the Platyhelminthes

Recent hypotheses on metazoan phylogeny have recognized three main clades of bilaterian animals: Deuterostomia, Ecdysozoa and Lophotrochozoa. The acoelomate and 'pseudocoelomate' metazoans, including the Platyhelminthes, long considered basal bilaterians, have been referred to positions within these clades by many authors. However, a recent study based on ribosomal DNA placed the flatworm group Acoela as the sister group of all other extant bilaterian lineages. Unexpectedly, the nemertodermatid flatworms, usually considered the sister group of the Acoela together forming the Acoelomorpha, were grouped separately from the Acoela with the rest of the Platyhelminthes (the Rhabditophora) within the Lophotrochozoa. To re-evaluate and clarify the phylogenetic position of the Nemertodermatida, new sequence data from 18S ribosomal DNA and mitochondrial genes of nemertodermatid and other bilaterian species were analysed with parsimony and maximum likelihood methods. The analyses strongly support a basal position within the Bilateria for the Nemertodermatida as a sister group to all other bilaterian taxa except the Acoela. Despite the basal position of both Nemertodermatida and Acoela, the clade Acoelomorpha was not retrieved. These results imply that the last common ancestor of bilaterian metazoans was a small, benthic, direct developer without segments, coelomic cavities, nephrida or a true brain. The name Nephrozoa is proposed for the ancestor of all bilaterians excluding the Nemertodermatida and the Acoela, and its descendants.     

Coordinated spatial and temporal expression ofHox genes during embryogenesis in the acoelConvolutriloba longifissura

Background  

Hox genes are critical for patterning the bilaterian anterior-posterior axis. The evolution of their clustered genomic arrangement and ancestral function has been debated since their discovery. As acoels appear to represent the sister group to the remaining Bilateria (Nephrozoa), investigatingHox gene expression will provide an insight into the ancestral features of theHox genes in metazoan evolution.     

The Hox gene complement of acoel flatworms, a basal bilaterian clade

Several molecular data sets suggest that acoelomorph flatworms are not members of the phylum Platyhelminthes but form a separate branch of the Metazoa that diverged from all other bilaterian animals before the separation of protostomes and deuterostomes. Here we examine the Hox gene complement of the acoel flatworms. In two distantly related acoel taxa, we identify only three distinct classes of Hox gene: an anterior gene, a posterior gene, and a central class gene most similar to genes of Hox classes 4 and 5 in other Bilateria. Phylogenetic analysis of these genes, together with the acoel caudal homologue, supports the basal position of the acoels. The similar gene sets found in two distantly related acoels suggest that this reduced gene complement may be ancestral in the acoels and that the acoels may have diverged from other bilaterians before elaboration of the 8- to 10-gene Hox cluster that characterizes most bilaterians.     

To Be or Not to Be a Flatworm: The Acoel Controversy

Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear synapomorphies among the three large flatworm taxa - the Catenulida, the Acoelomorpha and the Rhabditophora - have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the acoel flatworm Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives.     

Acoel flatworms are not platyhelminthes: evidence from phylogenomics

Acoel flatworms are small marine worms traditionally considered to belong to the phylum Platyhelminthes. However, molecular phylogenetic analyses suggest that acoels are not members of Platyhelminthes, but are rather extant members of the earliest diverging Bilateria. This result has been called into question, under suspicions of a long branch attraction (LBA) artefact. Here we re-examine this problem through a phylogenomic approach using 68 different protein-coding genes from the acoel Convoluta pulchra and 51 metazoan species belonging to 15 different phyla. We employ a mixture model, named CAT, previously found to overcome LBA artefacts where classical models fail. Our results unequivocally show that acoels are not part of the classically defined Platyhelminthes, making the latter polyphyletic. Moreover, they indicate a deuterostome affinity for acoels, potentially as a sister group to all deuterostomes, to Xenoturbellida, to Ambulacraria, or even to chordates. However, the weak support found for most deuterostome nodes, together with the very fast evolutionary rate of the acoel Convoluta pulchra, call for more data from slowly evolving acoels (or from its sister-group, the Nemertodermatida) to solve this challenging phylogenetic problem.     

The phylogenetic position of the comb jellies (Ctenophora) and the importance of taxonomic sampling

The transition to a vermiform body shape is one of the most important events in animal evolution, having led to the impressive radiation of Bilateria. However, the sister group of Bilateria has remained obscure. Cladistic analyses of morphology indicate that Ctenophora is the sister group of Bilateria. Previous analyses of SSU rRNA sequences have yielded conflicting results; in many studies Ctenophora forms the sister group of Cnidaria + Bilateria, but in others the ctenophores group with poriferans. Here we re‐examine the SSU sequence by analyzing a dataset with 528 metazoan + outgroup sequences, including almost 120 poriferan and diploblast sequences. We use parsimony ratchet and jackknife methods, as well as Bayesian methods, to analyze the data. The results indicate strong phylogenetic signals for a cnidarian + bilaterian group and for the comb jellies to have branched off early within a group uniting all epithelial animals [(Ct,(Cn,Bi))]. We demonstrate the importance of inclusive taxonomic coverage of ribosomal sequences for resolving this problematic part of the metazoan tree: topological stability increases dramatically with the addition of taxa, and the jackknife frequencies of the internal nodes uniting the lineages [(Cn,Bi) and ((Ct,(Cn,Bi))] also increase. We consider the reconstructed topology to represent the current best hypothesis of the interrelationships of these old lineages. Some morphological features supporting alternative hypotheses are discussed in the light of this result. © The Willi Hennig Society 2004.     

Dismissal of Acoelomorpha: Acoela and Nemertodermatida are separate early bilaterian clades

We used new 18S and 28S rRNA sequences analysed with parsimony, maximum likelihood and Bayesian methods of phylogenetic reconstruction to show that Nemertodermatida, generally classified as the sister group of Acoela within the recently proposed Phylum Acoelomorpha, are a separate basal bilaterian lineage. We used several analytical approaches to control for possible long branch attraction (LBA) artefacts in our results. Parsimony and the model based phylogenetic reconstruction methods that incorporate 'corrections' for substitution rate heterogenities yielded concordant results. When putative long branch taxa were experimentally removed the resulting topologies were consistent with our total evidence analysis. Deletion of fast-evolving nucleotide sites decreased resolution and clade support, but did not support a topology conflicting with the total evidence analysis. Establishment of Acoela and Nemertodermatida as two early lineages facilitates reconstruction of ancestral bilaterian features. The ancestor of extant Bilateria was a small, benthic direct developer without coelom or a planktonic larval stage. The previously proposed Phylum Acoelomorpha is dismissed as paraphyletic.     

Early evolution of the bilateria

The phylogeny of the Bilateria and especially the early steps in the evolution of the bilaterian bauplan are still a controversial topic. In this context the relationships of the platyhelminths and the nematodes play a crucial role. Previous molecular studies of the relationships of these groups, which were based on 18S ribosomal DNA sequences, yielded conflicting results. In the present study a new framework is developed for the phylogenetic analysis of bilaterian relationships, using concatenated amino acid sequences of several nuclear genes. In this analysis, the rhabditophoran platyhelminths are probably the sister group of all other analyzed Bilateria, the Eubilateria, which are characterized by a one-way intestine with an anus. The Eubilateria are split into the nematode lineage and the coelomates. The phylogenetic results of the present study indicate that genetic features found in the model organisms Caenorhabditis and Drosophila might be found in all Eubilateria. Estimations of the divergence times show that the major bilaterian phyla did not originate in an explosive radiation during the Cambrian but rather that the Bilateria have a several hundred million years long Precambrian history.     

Hox and ParaHox Genes in Flatworms: Characterization and Expression

Flatworms (phylum Platyhelminthes) are favourite organisms inDevelopmental Biology and Zoology because of their extraordinarypowers of regeneration and because they may hold a pivotal placein the origin and evolution of the Bilateria. Hox genes playkey roles in both processes: setting up the new anteroposteriorpattern in the former, and as qualitative markers of phylogeneticaffinities among bilaterian phyla in the latter. We have searchedfor Hox and ParaHox genes in several flatworm groups spanningfrom freshwater triclads to marine polyclads and, more recently,in the acoels, the likely earliest extant bilaterian. We haveisolated and sequenced eight Hox genes from the freshwater tricladGirardia tigrina and three Hox and two ParaHox genes from thepolyclad Discocelis tigrina. Data from the acoels Paratomellarubra and Convoluta roscoffensis is also reported. FlatwormHox sequences and 18S rDNA sequence data support clear affinitiesof Platyhelminthes to spiralian lophotrochozoans. The basalposition of acoel flatworms supported from recent 18S rDNA data,remains still uncertain. Expression of Hox genes in intact andregenerating adult organisms show nested patterns with gradedanterior expression boundaries, or ubiquitous expression. Newapproaches to study the function of Hox genes in flatworms,such as RNA interference are briefly discussed.     

Molecular phylogeny of gastrotricha based on 18S rRNA genes comparison: rejection of hypothesis of relatedness with nematodes

Gastrotrichs are meiobenthic free-living aquatic worms whose phylogenetic and intra-group relationships remain unclear despite some attempts to resolve them on the base of morphology or molecules. In this study we analysed complete sequences of the 18S rRNA gene of 15 taxa (8 new and 7 published) to test numerous hypotheses on gastrotrich phylogeny and to verify whether controversial interrelationships from previous molecular data could be due to the short region available for analysis and the poor taxa sampling. Data were analysed using both maximum likelihood and Bayesian inference. Results obtained suggest that gastrotrichs, together with Gnathostomulida, Plathelminthes, Syndermata (Rotifera + Acanthocephala), Nemertea and Lophotrochozoa, comprise a clade Spiralia. Statistical tests reject phylogenetic hypotheses regarding Gastrotricha as close relatives of Nematoda and other Ecdysozoa or placing them at the base of bilaterian tree close to acoels and nemertodermatides. Within Gastrotricha, Chaetonotida and Macrodasyida comprise two well supported clades. Our analysis confirmed the monophyly of the Chaetonotidae and Xenotrichulidae within Chaetonida as well as Turbanellidae and Thaumastodermatidae within Macrodasyida. Mesodasys is a sister group of the Turbanellidae, and Lepidodasyidae appears to be a polyphyletic group as Cephalodasys forms a separate lineage at the base of macrodasyids, whereas Lepidodasys groups with Neodasys between Thaumastodermatidae and Turbanellidae. To infer a more reliable Gastrotricha phylogeny many species and additional genes should be involved in future analyses.     

Gastrotricha and metazoan phylogeny

The phylogenetic position of the Gastrotricha within Bilateria and relationships among gastrotrich subgroups are reanalysed using morphological, developmental, nonsequence molecular, and ecological characters, together with the conserved regions of small-subunit ribosomal RNA genes (SSU rDNA). The analysis shows that traditional 'Macrodasyida' is a paraphyletic stemline of Chaetonotida, with Dactylopodolida, Redudasys , and Turbanellida as the basalmost gastrotrich groups. The 'Cycloneuralia hypothesis', which assumes sister group relationships between Gastrotricha and Ecdysozoa is supported. The sensitivity analysis of the combined dataset yields the following scheme of relationships of the main bilaterian clades: (1) Acoelomorpha is a basalmost bilaterian clade; (2) both Deuterostomia and Protostomia (less Acoelomorpha) are monophyletic; (3) the phylogenetic position of Ectoprocta, Brachiopoda + Phoronida, and Cycloneuralia within Protostomia is unstable; (4) Trochozoa (incl. Entoprocta, Nemertea, Lobatocerebrum , and possibly Jennaria ), Platyhelminthes s.s ., and Gnathifera-Myzostomida form a clade ('Spiralia'); (5) Cycliophora and possibly also Chaetognatha may be close to the gnathiferans. Evolution of metazoan ciliation and cycloneuralian cuticle is discussed. It is concluded that cycloneuralian and gastrotrich ancestors were multiciliate and had epidermal cilia covered by cuticular sheaths.     

The Acoela: on their kind and kinships, especially with nemertodermatids and xenoturbellids (Bilateria incertae sedis)

Acoels are among the simplest worms and therefore have often been pivotal in discussions of the origin of the Bilateria. Initially thought primitive because of their “planula-like” morphology, including their lumenless digestive system, they were subsequently dismissed by many morphologists as a specialized clade of the Platyhelminthes. However, since molecular phylogenies placed them outside the Platyhelminthes and outside all other phyla at the base of the Bilateria, they became the focus of renewed debate and research. We review what is currently known of acoels, including information regarding their morphology, development, systematics, and phylogenetic relationships, and put some of these topics in a historical perspective to show how the application of new methods contributed to the progress in understanding these animals. Taking all available data into consideration, clear-cut conclusions cannot be made; however, in our view it becomes successively clearer that acoelomorphs are a “basal” but “divergent” branch of the Bilateria.     

Mesodermal Gene Expression in the Acoel Isodiametra pulchra Indicates a Low Number of Mesodermal Cell Types and the Endomesodermal Origin of the Gonads

Acoelomorphs are bilaterally symmetric small marine worms that lack a coelom and possess a digestive system with a single opening. Two alternative phylogenetic positions of this group within the animal tree are currently debated. In one view, Acoelomorpha is the sister group to all remaining Bilateria and as such, is a morphologically simple stepping stone in bilaterian evolution. In the other, the group is a lineage within the Deuterostomia, and therefore, has derived a simple morphology from a more complex ancestor. Acoels and the closely related Nemertodermatida and Xenoturbellida, which together form the Acoelomorpha, possess a very limited number of cell types. To further investigate the diversity and origin of mesodermal cell types we describe the expression pattern of 12 orthologs of bilaterian mesodermal markers including Six1/2, Twist, FoxC, GATA4/5/6, in the acoel Isodiametra pulchra. All the genes are expressed in stem cells (neoblasts), gonads, and at least subsets of the acoel musculature. Most are expressed in endomesodermal compartments of I. pulchra developing embryos similar to what has been described in cnidarians. Our molecular evidence indicates a very limited number of mesodermal cell types and suggests an endomesodermal origin of the gonads and the stem cell system. We discuss our results in light of the two prevailing phylogenetic positions of Acoelomorpha.     

How the worm got its pharynx: phylogeny, classification and Bayesian assessment of character evolution in Acoela

Acoela are marine microscopic worms currently thought to be the sister taxon of all other bilaterians. Acoels have long been used as models in evolutionary scenarios, and generalized conclusions about acoel and bilaterian ancestral features are frequently drawn from studies of single acoel species. There is no extensive phylogenetic study of Acoela and the taxonomy of the 380 species is chaotic. Here we use two nuclear ribosomal genes and one mitochondrial gene in combination with 37 morphological characters in an analysis of 126 acoel terminals (about one-third of the described species) to estimate the phylogeny and character evolution of Acoela. We present an estimate of posterior probabilities for ancestral character states at 31 control nodes in the phylogeny. The overall reconstruction signal based on the shape of the posterior distribution of character states was computed for all morphological characters and control nodes to assess how well these were reconstructed. The body-wall musculature appears more clearly reconstructed than the reproductive organs. Posterior similarity to the root was calculated by averaging the divergence between the posterior distributions at the nodes and the root over all morphological characters. Diopisthoporidae is the sister group to all other acoels and has the highest posterior similarity to the root. Convolutidae, including several "model" acoels, is most divergent. Finally, we present a phylogenetic classification of Acoela down to the family level where six previous family level taxa are synonymized.     

Ancient origins of axial patterning genes: Hox genes and ParaHox genes in the Cnidaria

Among the bilaterally symmetrical, triploblastic animals (the Bilateria), a conserved set of developmental regulatory genes are known to function in patterning the anterior–posterior (AP) axis. This set includes the well-studied Hox cluster genes, and the recently described genes of the ParaHox cluster, which is believed to be the evolutionary sister of the Hox cluster ( Brooke et al. 1998 ). The conserved role of these axial patterning genes in animals as diverse as frogs and flies is believed to reflect an underlying homology (i.e., all bilaterians derive from a common ancestor which possessed an AP axis and the developmental mechanisms responsible for patterning the axis). However, the origin and early evolution of Hox genes and ParaHox genes remain obscure. Repeated attempts have been made to reconstruct the early evolution of Hox genes by analyzing data from the triphoblastic animals, the Bilateria ( Schubert et al. 1993 ; Zhang and Nei 1996 ). A more precise dating of Hox origins has been elusive due to a lack of sufficient information from outgroup taxa such as the phylum Cnidaria (corals, hydras, jellyfishes, and sea anemones). In combination with outgroup taxa, another potential source of information about Hox origins is outgroup genes (e.g., the genes of the ParaHox cluster). In this article, we present cDNA sequences of two Hox-like genes ( anthox2 and anthox6 ) from the sea anemone, Nematostella vectensis. Phylogenetic analysis indicates that anthox2 (=Cnox2) is homologous to the GSX class of ParaHox genes, and anthox6 is homologous to the anterior class of Hox genes. Therefore, the origin of Hox genes and ParaHox genes occurred prior to the evolutionary split between the Cnidaria and the Bilateria and predated the evolution of the anterior–posterior axis of bilaterian animals. Our analysis also suggests that the central Hox class was invented in the bilaterian lineage, subsequent to their split from the Cnidaria.