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.     

Back in time: a new systematic proposal for the Bilateria

Conventional wisdom suggests that bilateral organisms arose from ancestors that were radially, rather than bilaterally, symmetrical and, therefore, had a single body axis and no mesoderm. The two main hypotheses on how this transformation took place consider either a simple organism akin to the planula larva of extant cnidarians or the acoel Platyhelminthes (planuloid-acoeloid theory), or a rather complex organism bearing several or most features of advanced coelomate bilaterians (archicoelomate theory). We report phylogenetic analyses of bilaterian metazoans using quantitative (ribosomal, nuclear and expressed sequence tag sequences) and qualitative (HOX cluster genes and microRNA sets) markers. The phylogenetic trees obtained corroborate the position of acoel and nemertodermatid flatworms as the earliest branching extant members of the Bilateria. Moreover, some acoelomate and pseudocoelomate clades appear as early branching lophotrochozoans and deuterostomes. These results strengthen the view that stem bilaterians were small, acoelomate/pseudocoelomate, benthic organisms derived from planuloid-like organisms. Because morphological and recent gene expression data suggest that cnidarians are actually bilateral, the origin of the last common bilaterian ancestor has to be put back in time earlier than the cnidarian-bilaterian split in the form of a planuloid animal. A new systematic scheme for the Bilateria that includes the Cnidaria is suggested and its main implications discussed.     

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.     

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.     

Phylogenetic analysis of the Wnt gene family. Insights from lophotrochozoan members

The Wnt gene family encodes secreted signaling molecules that control cell fate specification, proliferation, polarity, and movements during animal development. We investigate here the evolutionary history of this large multigenic family. Wnt genes have been almost exclusively isolated from two of the three main subdivisions of bilaterian animals, the deuterostomes (which include chordates and echinoderms) and the ecdysozoans (e.g., arthropods and nematodes). However, orthology relationships between deuterostome and ecdysozoan Wnt genes, and, more generally, the phylogeny of the Wnt family, are not yet clear. We report here the isolation of several Wnt genes from two species, the annelid Platynereis dumerilii and the mollusc Patella vulgata, which both belong to the third large bilaterian clade, the lophotrochozoans (which constitute, together with ecdysozoans, the protostomes). Multiple phylogenetic analyses of these sequences with a large set of other Wnt gene sequences, in particular, the complete set of Wnt genes of human, nematode, and fly, allow us to subdivide the Wnt family into 12 subfamilies. At least nine of them were already present in the last common ancestor of all bilaterian animals, and this further highlights the genetic complexity of this ancestor. The orthology relationships we present here open new perspectives for future developmental comparisons.     

The Origin of Patterning Systems in Bilateria——Insights from the Hox and ParaHox Genes in Acoelomorpha

Hox and ParaHox genes constitute two families of developmental regulators that pattern the Anterior-Posterior body axis in all bilaterians.The members of these two groups of genes are usually arranged in genomic clusters and work in a coordinated fashion,both in space and in time. While the mechanistic aspects of their action are relatively well known,it is still unclear how these systems evolved. For instance,we still need a proper model of how the Hox and ParaHox clusters were assembled over time.This problem is due to the shortage of information on gene complements for many taxa (mainly basal metazoans) and the lack of a consensus phylogenetic model of animal relationships to which we can relate our new findings.Recently, several studies have shown that the Acoelomorpha most probably represent the first offshoot of the Bilateria. This finding has prompted us,and others, to study the Hox and ParaHox complements in these animals,as well as their activity during development.In this review,we analyze how the current knowledge of Hox and ParaHox genes in the Acoelomorpha is shaping our view of bilaterian evolution.     

Revaluation of deuterostome phylogeny and evolutionary relationships among chordate subphyla using mitogenome data

The traditional knowledge in textbooks indicated that cephalochordates were the closest relatives to vertebrates among all extant organisms. However, this opinion was challenged by several recent phylogenetic studies using hundreds of nuclear genes. The researchers suggested that urochordates, but not cephalochordates, should be the closest living relatives to vertebrates. In the present study, by using data generated from hundreds of mtDNA sequences, we revalue the deuterostome phylogeny in terms of whole mitochondrial genomes （mitogenomes）. Our results firmly demonstrate that each of extant deuterostome phyla and chordate subphyla is monophyletic. But the results present several alternative phylogenetic trees depending on different sequence datasets used in the analysis. Although no clear phylogenetic relationships are obtained, those trees indicate that the ancient common ancestor diversified rapidly soon after their appearance in the early Cambrian and generated all major deuterostome lineages during a short historical period, which is consistent with ＂Cambrian explosion＂ revealed by paleontologists. It was the 520-million-year＇s evolution that obscured the phylogenetic relationships of extant deuterostomes. Thus, we conclude that an integrative analysis approach rather than simply using more DNA sequences should be employed to address the distant evolutionary relationship.     

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.     

Expression of the 22 nucleotide let‐7 heterochronic RNA throughout the Metazoa: a role in life history evolution?

The 22 nucleotide let-7 small temporal RNA has been found consistently in samples from diverse bilateria but not from sponge or cnidarians. Here we further examine the phylogenetic distribution of this regulatory RNA by sampling representatives of diverse metazoan lineages. The 22 nucleotide let-7 RNA is detectable in triclad and polyclad platyhelminths, nemertean, and chaetognath but not ctenophore or acoel metazoans. These results support recent arguments that acoels are distinct from other acoelomate platyhelminths. We argue that let-7 is not a bilaterian or triploblast synapomorphy but instead evolved later in metazoan evolution, perhaps in association with complex life history traits.     

The Position of Arthropods in the Animal Kingdom: A Search for a Reliable Outgroup for Internal Arthropod Phylogeny

Morphological evidence for the phylogeny of the animal kingdom has been discussed by numerous authors. DNA sequencing and phylogenetic methods for analyzing these data are alternative approaches to animal phylogeny, but the phenomenon of long branch attraction and poor taxonomic sampling have caused misinterpretations of metazoan relationships. Here we report a cladistic approach to metazoan evolution including 133 18S rDNA sequences of 31 animal phyla. Despite the difficulties associated with analyzing large data sets, our data suggest that the Bilateria and Protostomia are monophyletic. The internal phylogeny of the protostomes is divided into two main clades. One clade includes the classical protostome worms (annelids, sipunculans, echiurans, pogonophorans, and vestimentiferans), mollusks, nemerteans, “lophophorates,” platyhelminths, rotiferans, and acanthocephalans, although the internal resolution of the clade is very low. The second clade includes arthropods and other molting animals: tardigrades, onychophorans, nematodes, nematomorphans, kinorhynchs, and priapulans. The arthropods and related phyla lack a ciliated larvae, lack a multiciliate (locomotory) epithelium, and share many features, notably, a reduced coelomic cavity and the presence of a cuticle which molts. The use of these outgroups within the molting clade to root arthropod phylogenies is recommended instead of using annelids or other spiralians. The data are quite conclusive in those phyla with a good taxonomic sampling (i.e., platyhelminths and arthropods).     

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.     

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.      

Hidden diversity of Acoelomorpha revealed through metabarcoding

Animals with bilateral symmetry comprise the majority of the described species within Metazoa. However, the nature of the first bilaterian animal remains unknown. As most recent molecular phylogenies point to Xenacoelomorpha as the sister group to the rest of Bilateria, understanding their biology, ecology and diversity is key to reconstructing the nature of the last common bilaterian ancestor (Urbilateria). To date, sampling efforts have focused mainly on coastal areas, leaving potential gaps in our understanding of the full diversity of xenacoelomorphs. We therefore analysed 18S rDNA metabarcoding data from three marine projects covering benthic and pelagic habitats worldwide. Our results show that acoels have a greater richness in planktonic environments than previously described. Interestingly, we also identified a putative novel clade of acoels in the deep benthos that branches as sister group to the rest of Acoela, thus representing the earliest-branching acoel clade. Our data highlight deep-sea environments as an ideal habitat to sample acoels with key phylogenetic positions, which might be useful for reconstructing the early evolution of Bilateria.     

Evolution of Pentameric Ligand-Gated Ion Channels: Pro-Loop Receptors

Pentameric ligand-gated ion channels (pLGICs) are ubiquitous neurotransmitter receptors in Bilateria, with a small number of known prokaryotic homologues. Here we describe a new inventory and phylogenetic analysis of pLGIC genes across all kingdoms of life. Our main finding is a set of pLGIC genes in unicellular eukaryotes, some of which are metazoan-like Cys-loop receptors, and others devoid of Cys-loop cysteines, like their prokaryotic relatives. A number of such “Cys-less” receptors also appears in invertebrate metazoans. Together, those findings draw a new distribution of pLGICs in eukaryotes. A broader distribution of prokaryotic channels also emerges, including a major new archaeal taxon, Thaumarchaeota. More generally, pLGICs now appear nearly ubiquitous in major taxonomic groups except multicellular plants and fungi. However, pLGICs are sparsely present in unicellular taxa, suggesting a high rate of gene loss and a non-essential character, contrasting with their essential role as synaptic receptors of the bilaterian nervous system. Multiple alignments of these highly divergent sequences reveal a small number of conserved residues clustered at the interface between the extracellular and transmembrane domains. Only the “Cys-loop” proline is absolutely conserved, suggesting the more fitting name “Pro loop” for that motif, and “Pro-loop receptors” for the superfamily. The infered molecular phylogeny shows a Cys-loop and a Cys-less clade in eukaryotes, both containing metazoans and unicellular members. This suggests new hypotheses on the evolutionary history of the superfamily, such as a possible origin of the Cys-loop cysteines in an ancient unicellular eukaryote. Deeper phylogenetic relationships remain uncertain, particularly around the split between bacteria, archaea, and eukaryotes.     

The Molecular Symplesiomorphies Shared by the Stem Groups of Metazoan Evolution: Can Sites as Few as 1 % Have a Significant Impact on Recognizing the Phylogenetic Position of Myzostomida?

Although it is clear that taxon sampling, alignments, gene sampling, tree reconstruction methods and the total length of the sequences used are critical to the reconstruction of evolutionary history, weakly supported or misleading nodes exist in phylogenetic studies with no obvious flaw in those aspects. The phylogenetic studies focusing on the basal part of bilaterian evolution are such a case. During the past decade, Myzostomida has appeared in the basal part of Bilateria in several phylogenetic studies of Metazoa. However, most researchers have entertained only two competing hypotheses about the position of Myzostomida—an affinity with Annelida and an affinity with Platyhelminthes. In this study, dozens of symplesiomorphies were discovered by means of ancestral state reconstruction in the complete 18S and 28S rDNAs shared by the stem groups of Metazoa. By contrastive analysis on the datasets with or without such symplesiomorphic sites, we discovered that Myzostomida and other basal groups are basal lineages of Bilateria due to the corresponding symplesiomorphies shared with earlier lineages. As such, symplesiomorphies account for approximately 1–2 % of the whole dataset have an essential impact on phylogenetic inference, and this study reminds molecular systematists of the importance of carrying out ancestral state reconstruction at each site in sequence-based phylogenetic studies. In addition, reasons should be explored for the low support of the hypothesis that Myzostomida belongs to Annelida in the results of phylogenomic studies. Future phylogenetic studies concerning Myzostomida should include all of the basal lineages of Bilateria to avoid directly neglecting the stand-alone basal position of Myzostomida as a potential hypothesis.     

Hox genes from the Polystomatidae (Platyhelminthes, Monogenea)

Hox genes form a multigenic family that play a fundamental role during the early stages of development. They are organised in a single cluster and share a 60 amino acid conserved sequence that corresponds to the DNA binding domain, i.e. the homeodomain. Sequence conservation in this region has allowed investigators to explore Hox diversity in the metazoan lineages. Within parasitic flatworms only homeobox sequences of parasite species from the Cestoda and Digenea have been reported. In the present study we surveyed species of the Polyopisthocotylea (Monogenea) in order to clarify Hox identification and diversification processes in the neodermatan lineage. From cloning of degenerative PCR products of the central region of the homeobox, we report one ParaHox and 25 new Hox sequences from 10 species of the Polystomatidae and one species of the Diclidophoridae, which extend Hox gene diversity from 46 to 72 within Neodermata. Hox sequences from the Polyopisthocotylea were annotated and classified from sequence alignments and Bayesian inferences of 178 Hox, ParaHox and related gene families recovered from all available parasitic platyhelminths and other bilaterian taxa. Our results are discussed in the light of the recent Hox evolutionary schemes. They may provide new perspectives to study the transition from turbellarians to parasitic flatworms with complex life-cycles and outline the first steps for evolutionary developmental biological approaches within platyhelminth parasites.