Tunicates or urochordates (appendicularians, salps and sea squirts), cephalochordates (lancelets) and vertebrates (including lamprey and hagfish) constitute the three extant groups of chordate animals. Traditionally, cephalochordates are considered as the closest living relatives of vertebrates, with tunicates representing the earliest chordate lineage. This view is mainly justified by overall morphological similarities and an apparently increased complexity in cephalochordates and vertebrates relative to tunicates. Despite their critical importance for understanding the origins of vertebrates, phylogenetic studies of chordate relationships have provided equivocal results. Taking advantage of the genome sequencing of the appendicularian Oikopleura dioica, we assembled a phylogenomic data set of 146 nuclear genes (33,800 unambiguously aligned amino acids) from 14 deuterostomes and 24 other slowly evolving species as an outgroup. Here we show that phylogenetic analyses of this data set provide compelling evidence that tunicates, and not cephalochordates, represent the closest living relatives of vertebrates. Chordate monophyly remains uncertain because cephalochordates, albeit with a non-significant statistical support, surprisingly grouped with echinoderms, a hypothesis that needs to be tested with additional data. This new phylogenetic scheme prompts a reappraisal of both morphological and palaeontological data and has important implications for the interpretation of developmental and genomic studies in which tunicates and cephalochordates are used as model animals.

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Correspondence to H.P. email: herve.philippe@umontreal.ca -2 - PrefaceAs more complete genomes are sequenced, phylogenetic analysis is entering a new era -that of phylogenomics. One branch of this expanding field aims to reconstruct the evolutionary history of organisms based on the analysis of their genomes. Recent studies have demonstrated the power of this approach, which has the potential to provide answers to a number of fundamental evolutionary questions. However, challenges for the future have also been revealed. The very nature of the evolutionary history of organisms and the limitations of current phylogenetic reconstruction methods mean that part of the tree of life may prove difficult, if not impossible, to resolve with confidence. Introductory paragraphUnderstanding phylogenetic relationships between organisms is a prerequisite of almost any evolutionary study, as contemporary species all share a common history through their ancestry. The notion of phylogeny follows directly from the theory of evolution presented by Charles Darwin in "The Origin of Species" 1 : the only illustration in his famous book is the first representation of evolutionary relationships among species, in the form of a phylogenetic tree. The subsequent enthusiasm of biologists for the phylogenetic concept is illustrated by the publication of Ernst Haeckel's famous "trees" as early as 1866 2 .Today, phylogenetics -the reconstruction of evolutionary history -relies on using mathematical methods to infer the past from features of contemporary species, with only the fossil record to provide a window on the evolutionary past of life on our planet. This reconstruction involves the identification of HOMOLOGOUS CHARACTERS that are shared among different organisms, and the inference of phylogenetic trees from the comparison of these characters using reconstruction methods (BOX 1). The accuracy of -3 -the inference is therefore heavily dependent upon the quality of models for the evolution of such characters. Because the underlying mechanisms are not yet well understood, reconstructing the evolutionary history of life on Earth based solely on the information provided by living organisms has turned out to be difficult.Until the 1970s, which brought the dawn of molecular techniques for sequencing proteins and DNA, phylogenetic reconstruction was essentially based on the analysis of morphological or ultrastructural characters. The comparative anatomy of fossils and extant species has proved powerful in some respects; for example, the main groups of animals and plants have been delineated fairly easily using these methods. However, this approach is hampered by the limited number of reliable homologous characters available; these are almost non-existent in micro-organisms 3 and are rare even in complex organisms.The introduction of the use of molecular data in phylogenetics 4 led to a revolution.In the late 1980s, access to DNA sequences increased the number of homologous characters that could be compared from less than 100 to more than 1,000, ...

Background: Thanks to the large amount of signal contained in genome-wide sequence alignments, phylogenomic analyses are converging towards highly supported trees. However, high statistical support does not imply that the tree is accurate. Systematic errors, such as the Long Branch Attraction (LBA) artefact, can be misleading, in particular when the taxon sampling is poor, or the outgroup is distant. In an otherwise consistent probabilistic framework, systematic errors in genome-wide analyses can be traced back to model mis-specification problems, which suggests that better models of sequence evolution should be devised, that would be more robust to tree reconstruction artefacts, even under the most challenging conditions.