Long-held ideas regarding the evolutionary relationships among animals have recently been upended by sometimes controversial hypotheses based largely on insights from molecular data. These new hypotheses include a clade of moulting animals (Ecdysozoa) and the close relationship of the lophophorates to molluscs and annelids (Lophotrochozoa). Many relationships remain disputed, including those that are required to polarize key features of character evolution, and support for deep nodes is often low. Phylogenomic approaches, which use data from many genes, have shown promise for resolving deep animal relationships, but are hindered by a lack of data from many important groups. Here we report a total of 39.9 Mb of expressed sequence tags from 29 animals belonging to 21 phyla, including 11 phyla previously lacking genomic or expressed-sequence-tag data. Analysed in combination with existing sequences, our data reinforce several previously identified clades that split deeply in the animal tree (including Protostomia, Ecdysozoa and Lophotrochozoa), unambiguously resolve multiple long-standing issues for which there was strong conflicting support in earlier studies with less data (such as velvet worms rather than tardigrades as the sister group of arthropods), and provide molecular support for the monophyly of molluscs, a group long recognized by morphologists. In addition, we find strong support for several new hypotheses. These include a clade that unites annelids (including sipunculans and echiurans) with nemerteans, phoronids and brachiopods, molluscs as sister to that assemblage, and the placement of ctenophores as the earliest diverging extant multicellular animals. A single origin of spiral cleavage (with subsequent losses) is inferred from well-supported nodes. Many relationships between a stable subset of taxa find strong support, and a diminishing number of lineages remain recalcitrant to placement on the tree.

The Wnt gene family encodes secreted signalling molecules that control cell fate in animal development and human diseases. Despite its significance, the evolution of this metazoan-specific protein family is unclear. In vertebrates, twelve Wnt subfamilies were defined, of which only six have counterparts in Ecdysozoa (for example, Drosophila and Caenorhabditis). Here, we report the isolation of twelve Wnt genes from the sea anemone Nematostella vectensis, a species representing the basal group within cnidarians. Cnidarians are diploblastic animals and the sister-group to bilaterian metazoans. Phylogenetic analyses of N. vectensis Wnt genes reveal a thus far unpredicted ancestral diversity within the Wnt family. Cnidarians and bilaterians have at least eleven of the twelve known Wnt gene subfamilies in common; five subfamilies appear to be lost in the protostome lineage. Expression patterns of Wnt genes during N. vectensis embryogenesis indicate distinct roles of Wnts in gastrulation, resulting in serial overlapping expression domains along the primary axis of the planula larva. This unexpectedly complex inventory of Wnt family signalling factors evolved in early multi-cellular animals about 650 million years (Myr) ago, predating the Cambrian explosion by at least 100 Myr (refs 5, 8). It emphasizes the crucial function of Wnt genes in the diversification of eumetazoan body plans.

factor). Except for mef2, the expression of these genes is largely restricted to the endodermal layer, the gastrodermis. mef2 is restricted to the ectoderm. The temporal and spatial expression of these 'mesoderm' genes suggests that they may play a role in germ layer specification. Furthermore, the predominantly endodermal expression of these genes reinforces the hypothesis that the mesoderm and endoderm of triploblastic animals could be derived from the endoderm of a diploblastic ancestor. Alternatively, we consider the possibility that the diploblastic condition of cnidarians is a secondary simplification, derived from an ancestral condition of triploblasty.

The human oncogene beta-catenin is a bifunctional protein with critical roles in both cell adhesion and transcriptional regulation in the Wnt pathway. Wnt/beta-catenin signalling has been implicated in developmental processes as diverse as elaboration of embryonic polarity, formation of germ layers, neural patterning, spindle orientation and gap junction communication, but the ancestral function of beta-catenin remains unclear. In many animal embryos, activation of beta-catenin signalling occurs in blastomeres that mark the site of gastrulation and endomesoderm formation, raising the possibility that asymmetric activation of beta-catenin signalling specified embryonic polarity and segregated germ layers in the common ancestor of bilaterally symmetrical animals. To test whether nuclear translocation of beta-catenin is involved in axial identity and/or germ layer formation in 'pre-bilaterians', we examined the in vivo distribution, stability and function of beta-catenin protein in embryos of the sea anemone Nematostella vectensis (Cnidaria, Anthozoa). Here we show that N. vectensis beta-catenin is differentially stabilized along the oral-aboral axis, translocated into nuclei in cells at the site of gastrulation and used to specify entoderm, indicating an evolutionarily ancient role for this protein in early pattern formation.

Over 99% of modern animals are members of the evolutionary lineage Bilateria. The evolutionary success of Bilateria is credited partly to the origin of bilateral symmetry. Although animals of the phylum Cnidaria are not within the Bilateria, some representatives, such as the sea anemone Nematostella vectensis, exhibit bilateral symmetry. We show that Nematostella uses homologous genes to achieve bilateral symmetry: Multiple Hox genes are expressed in a staggered fashion along its primary body axis, and the transforming growth factor-beta gene decapentaplegic (dpp) is expressed in an asymmetric fashion about its secondary body axis. These data suggest that bilateral symmetry arose before the evolutionary split of Cnidaria and Bilateria.

BackgroundHox genes were critical to many morphological innovations of bilaterian animals. However, early Hox evolution remains obscure. Phylogenetic, developmental, and genomic analyses on the cnidarian sea anemone Nematostella vectensis challenge recent claims that the Hox code is a bilaterian invention and that no “true” Hox genes exist in the phylum Cnidaria.Methodology/Principal FindingsPhylogenetic analyses of 18 Hox-related genes from Nematostella identify putative Hox1, Hox2, and Hox9+ genes. Statistical comparisons among competing hypotheses bolster these findings, including an explicit consideration of the gene losses implied by alternate topologies. In situ hybridization studies of 20 Hox-related genes reveal that multiple Hox genes are expressed in distinct regions along the primary body axis, supporting the existence of a pre-bilaterian Hox code. Additionally, several Hox genes are expressed in nested domains along the secondary body axis, suggesting a role in “dorsoventral” patterning.Conclusions/SignificanceA cluster of anterior and posterior Hox genes, as well as ParaHox cluster of genes evolved prior to the cnidarian-bilaterian split. There is evidence to suggest that these clusters were formed from a series of tandem gene duplication events and played a role in patterning both the primary and secondary body axes in a bilaterally symmetrical common ancestor. Cnidarians and bilaterians shared a common ancestor some 570 to 700 million years ago, and as such, are derived from a common body plan. Our work reveals several conserved genetic components that are found in both of these diverse lineages. This finding is consistent with the hypothesis that a set of developmental rules established in the common ancestor of cnidarians and bilaterians is still at work today.

The Sox and Forkhead (Fox) gene families are comprised of transcription factors that play important roles in a variety of developmental processes, including germ layer specification, gastrulation, cell fate determination, and morphogenesis. Both the Sox and Fox gene families are divided into subgroups based on the amino acid sequence of their respective DNA-binding domains, the high-mobility group (HMG) box (Sox genes) or Forkhead domain (Fox genes). Utilizing the draft genome sequence of the cnidarian Nematostella vectensis, we examined the genomic complement of Sox and Fox genes in this organism to gain insight into the nature of these gene families in a basal metazoan. We identified 14 Sox genes and 15 Fox genes in Nematostella and conducted a Bayesian phylogenetic analysis comparing HMG box and Forkhead domain sequences from Nematostella with diverse taxa. We found that the majority of bilaterian Sox groups have clear Nematostella orthologs, while only a minority of Fox groups are represented, suggesting that the evolutionary pressures driving the diversification of these gene families may be distinct from one another. In addition, we examined the expression of a subset of these genes during development in Nematostella and found that some of these genes are expressed in patterns consistent with roles in germ layer specification and the regulation of cellular behaviors important for gastrulation. The diversity of expression patterns among members of these gene families in Nematostella reinforces the notion that despite their relatively simple morphology, cnidarians possess much of the molecular complexity observed in bilaterian taxa.

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