The origins of neural systems remain unresolved. In contrast to other basal metazoans, ctenophores, or comb jellies, have both complex nervous and mesoderm-derived muscular systems. These holoplanktonic predators also have sophisticated ciliated locomotion, behaviour and distinct development. Here, we present the draft genome of Pleurobrachia bachei, Pacific sea gooseberry, together with ten other ctenophore transcriptomes and show that they are remarkably distinct from other animal genomes in their content of neurogenic, immune and developmental genes. Our integrative analyses place Ctenophora as the earliest lineage within Metazoa. This hypothesis is supported by comparative analysis of multiple gene families, including the apparent absence of HOX genes, canonical microRNA machinery, and reduced immune complement in ctenophores. Although two distinct nervous systems are well-recognized in ctenophores, many bilaterian neuron-specific genes and genes of “classical” neurotransmitter pathways either are absent or, if present, are not expressed in neurons. Our metabolomic and physiological data are consistent with the hypothesis that ctenophore neural systems, and possibly muscle specification, evolved independently from those in other animals.

The origin of many of the defining features of animal body plans, such as symmetry, nervous system, and the mesoderm, remains shrouded in mystery because of major uncertainty regarding the emergence order of the early branching taxa: the sponge groups, ctenophores, placozoans, cnidarians, and bilaterians. The "phylogenomic" approach [1] has recently provided a robust picture for intrabilaterian relationships [2, 3] but not yet for more early branching metazoan clades. We have assembled a comprehensive 128 gene data set including newly generated sequence data from ctenophores, cnidarians, and all four main sponge groups. The resulting phylogeny yields two significant conclusions reviving old views that have been challenged in the molecular era: (1) that the sponges (Porifera) are monophyletic and not paraphyletic as repeatedly proposed [4-9], thus undermining the idea that ancestral metazoans had a sponge-like body plan; (2) that the most likely position for the ctenophores is together with the cnidarians in a "coelenterate" clade. The Porifera and the Placozoa branch basally with respect to a moderately supported "eumetazoan" clade containing the three taxa with nervous system and muscle cells (Cnidaria, Ctenophora, and Bilateria). This new phylogeny provides a stimulating framework for exploring the important changes that shaped the body plans of the early diverging phyla.

To reconstruct the evolutionary origin of multicellular animals from their unicellular ancestors, the genome sequences of diverse unicellular relatives are essential. However, only the genome of the choanoflagellate Monosiga brevicollis has been reported to date. Here we completely sequence the genome of the filasterean Capsaspora owczarzaki, the closest known unicellular relative of metazoans besides choanoflagellates. Analyses of this genome alter our understanding of the molecular complexity of metazoans’ unicellular ancestors showing that they had a richer repertoire of proteins involved in cell adhesion and transcriptional regulation than previously inferred only with the choanoflagellate genome. Some of these proteins were secondarily lost in choanoflagellates. In contrast, most intercellular signalling systems controlling development evolved later concomitant with the emergence of the first metazoans. We propose that the acquisition of these metazoan-specific developmental systems and the co-option of pre-existing genes drove the evolutionary transition from unicellular protists to metazoans.

The large phylogenetic distance separating eukaryotic genes and their archaeal orthologs has prevented identification of the position of the eukaryotic root in phylogenomic studies. Recently, an innovative approach has been proposed to circumvent this issue: the use as phylogenetic markers of proteins that have been transferred from bacterial donor sources to eukaryotes, after their emergence from Archaea. Using this approach, two recent independent studies have built phylogenomic datasets based on bacterial sequences, leading to different predictions of the eukaryotic root. Taking advantage of additional genome sequences from the jakobid Andalucia godoyi and the two known malawimonad species (Malawimonas jakobiformis and Malawimonas californiana), we reanalyzed these two phylogenomic datasets. We show that both datasets pinpoint the same phylogenetic position of the eukaryotic root that is between "Unikonta" and "Bikonta," with malawimonad and collodictyonid lineages on the Unikonta side of the root. Our results firmly indicate that (i) the supergroup Excavata is not monophyletic and (ii) the last common ancestor of eukaryotes was a biflagellate organism. Based on our results, we propose to rename the two major eukaryotic groups Unikonta and Bikonta as Opimoda and Diphoda, respectively. eukaryote phylogeny | phylogenomics | Opimoda | Diphoda | LECA

Regionalised activation of canonical Wnt signalling via β-catenin stabilisation is a key early step in embryonic patterning in many metazoans, including the basally diverging cnidarians, but the upstream maternal cues appear surprisingly variable. In Clytia, regionalised β-catenin stabilisation defining a presumptive 'oral' territory is determined by two maternally coded Frizzled family Wnt receptors of opposite localisation and function. We have identified a maternally coded ligand, CheWnt3, the RNA of which is localised to the animal cortex (future oral side) of the egg. Antisense morpholino oligonucleotide experiments showed that CheWnt3 is required maternally for regionalised oral β-catenin stabilisation in the early embryo, being only the second clear example of a maternally required Wnt ligand after Xenopus Xwnt11. In line with the determinant role of the maternally localised Frizzleds, CheWnt3 overexpression by RNA injection initially had little effect on establishing the oral domain. Subsequently, however, overexpression had dramatic consequences for axis development, causing progressive expansion of β-catenin stabilisation to yield spherical 'oralised' larvae. Upregulation of both CheFz1 and CheFz3 RNAs in CheWnt3 morpholino embryos indicated that CheWnt3 participates in an active axial patterning system involving reciprocal downregulation of the receptors to maintain oral and aboral territories. Localised introduction of CheWnt3 RNA induced ectopic oral poles in CheWnt3 morpholino embryos, demonstrating its importance in directing oral fate. These findings suggest that the complete ligand-dependent Wnt signalling cascade is involved in axial patterning in ancestral eumetazoans. In Clytia, two variant Frizzled receptors and one Wnt ligand produced from localised RNAs cooperate to initiate regionalised Wnt pathway activation.

Many of the eukaryotic phylogenomic analyses published to date were based on alignments of hundreds to thousands of genes. Frequently, in such analyses, the most realistic evolutionary models currently available are often used to minimize the impact of systematic error. However, controversy remains over whether or not idiosyncratic gene family dynamics (i.e., gene duplications and losses) and incorrect orthology assignments are always appropriately taken into account. In this paper, we present an innovative strategy for overcoming orthology assignment problems. Rather than identifying and eliminating genes with paralogy problems, we have constructed a data set comprised exclusively of conserved single-copy protein domains that, unlike most of the commonly used phylogenomic data sets, should be less confounded by orthology miss-assignments. To evaluate the power of this approach, we performed maximum likelihood and Bayesian analyses to infer the evolutionary relationships within the opisthokonts (which includes Metazoa, Fungi, and related unicellular lineages). We used this approach to test 1) whether Filasterea and Ichthyosporea form a clade, 2) the interrelationships of early-branching metazoans, and 3) the relationships among early-branching fungi. We also assessed the impact of some methods that are known to minimize systematic error, including reducing the distance between the outgroup and ingroup taxa or using the CAT evolutionary model. Overall, our analyses support the Filozoa hypothesis in which Ichthyosporea are the first holozoan lineage to emerge followed by Filasterea, Choanoflagellata, and Metazoa. Blastocladiomycota appears as a lineage separate from Chytridiomycota, although this result is not strongly supported. These results represent independent tests of previous phylogenetic hypotheses, highlighting the importance of sophisticated approaches for orthology assignment in phylogenomic analyses.

By exploiting the large body of genome data and the considerable progress in phylogenetic methodology, recent phylogenomic studies have provided new insights into the relationships among major eukaryotic groups. However, confident placement of the eukaryotic root remains a major challenge. This is due to the large evolutionary distance separating eukaryotes from their closest relatives, the Archaea, implying a weak phylogenetic signal and strong long-branch attraction artifacts. Here, we apply a new approach to the rooting of the eukaryotic tree by using a subset of genomic information with more recent evolutionary origin-mitochondrial sequences, whose closest relatives are α-Proteobacteria. For this, we identified and assembled a data set of 42 mitochondrial proteins (mainly encoded by the nuclear genome) and performed Bayesian and maximum likelihood analyses. Taxon sampling includes the recently sequenced Thecamonas trahens, a member of the phylogenetically elusive Apusozoa. This data set confirms the relationships of several eukaryotic supergroups seen before and places the eukaryotic root between the monophyletic "unikonts" and "bikonts." We further show that T. trahens branches sister to Opisthokonta with significant statistical support and question the bikont/excavate affiliation of Malawimonas species. The mitochondrial data set developed here (to be expanded in the future) constitutes a unique alternative means in resolving deep eukaryotic relationships.

Stramenopiles or heterokonts constitute one of the most speciose and diverse clades of protists. It includes ecologically important algae (such as diatoms or large multicellular brown seaweeds), as well as heterotrophic (e.g. bicosoecids, MAST groups) and parasitic (e.g. Blastocystis, oomycetes) species. Despite their evolutionary and ecological relevance, deep phylogenetic relationships among stramenopile groups, inferred mostly from small-subunit (SSU) rDNA phylogenies, remain unresolved, especially for the heterotrophic taxa. Taking advantage of recently released stramenopile transcriptome and genome sequences, as well as data from the genomic assembly of the MAST-3 species Incisomonas marina generated in our laboratory, we have carried out the first extensive phylogenomic analysis of stramenopiles, including representatives of most major lineages. Our analyses, based on a large dataset of 339 widely distributed proteins, strongly support a root of stramenopiles lying between two clades, Bigyra and Gyrista (Pseudofungi plus Ochrophyta). Additionally, our analyses challenge the Phaeista-Khakista dichotomy of photosynthetic stramenopiles (ochrophytes) as two groups previously considered to be part of the Phaeista (Pelagophyceae and Dictyochophyceae), branch with strong support with the Khakista (Bolidophyceae and Diatomeae). We propose a new classification of ochrophytes within the two groups Chrysista and Diatomista to reflect the new phylogenomic results. Our stramenopile phylogeny provides a robust phylogenetic framework to investigate the evolution and diversification of this group of ecologically relevant protists.