DNA sequence data from neglected animal groups support a controversial hypothesis of deep evolutionary history. Inferring that history using only whole-genome sequences can evidently be misleading.
Despite the comforting certainty of textbooks and 150 years of argument, the true relationships of the major groups (phyla) of animals remain contentious. In the late 1990s, a series of controversial papers used molecular evidence to propose a radical rearrangement of animal phyla1,2,3. Subsequently, analyses of whole-genome sequences from a few species showed strong, apparently conclusive, support for an older view4,5,6. Philippe et al., writing in Molecular Biology and Evolution7, now provide evidence from expanded data sets that supports the newer evolutionary tree, and also show why whole-genome data sets can lead phylogeneticists seriously astray.
Traditional trees group together phyla of bilaterally symmetrical animals that possess a body cavity lined with mesodermal tissue, the coelom (for example, the human pleural cavity), as Coelomata. Those without a true coelom are classified as Acoelomata (no coelom) and Pseudocoelomata (a body cavity not lined by mesoderm). We call this tree the A–P–C hypothesis. Under A–P–C, humans are more closely related to the fruitfly Drosophila melanogaster than either is to the nematode roundworm Caenorhabditis elegans5,6 (Fig. 1).
In contrast, the new trees1,2,3,7 suggest that the basic division in animals is between the Protostomia and Deuterostomia (a distinction based on the origin of the mouth during embryo formation). Humans are deuterostomes, but because flies and nematodes are both protostomes they are more closely related to each other than either is to humans. The Protostomia can be divided into two ‘superphyla’: Ecdysozoa (animals that undergo ecdysis or moulting, including flies and nematodes) and Lophotrochozoa (animals with a feeding structure called the lophophore, including snails and earthworms). We call this tree the L–E–D hypothesis (Fig. 1). Importantly, in this new tree, the coelom must have arisen more than once, or have been lost from some phyla.
Molecular analyses have been divided in their support for these competing hypotheses. Trees built using single genes from many species tend to support L–E–D8, but analyses using many genes from a few complete genomes support A–P–C5,6. The number of species represented in a phylogenetic study can have two effects on tree reconstruction. First, without genomes to represent most animal phyla, genome-based trees provide no information on the placement of the missing taxonomic groups. Current genome studies do not include any members of the Lophotrochozoa. More notably, if a species' genome is evolving rapidly, tree reconstruction programs can be misled by a phenomenon known as long-branch attraction9.
In long-branch attraction, independent but convergent changes (homoplasies) on long branches are misconstrued as ‘shared derived’ changes, causing artefactual clustering of species with long branches. Because these artefacts are systematic, confidence in them grows as more data are included, and thus genome-scale analyses are especially sensitive to long-branch attraction. Long branches can arise in two ways. One is when a distantly related organism is used as an ‘outgroup’ to root the tree of the organisms of interest. The other is when one organism of interest has a very different, accelerated pattern of evolution compared with the rest. Unfortunately for whole-genome studies, the usual outgroup, yeast, is very distantly related to animals, and C. elegans is a long-branch species5. Long-branch attraction will therefore tend to result in nematodes moving to the base of the tree, generating erroneous support for A–P–C. Not all whole-genome studies are tainted: analysis of rare insertions and deletions of genomic features (introns) in some animal genomes, characters that may be immune to the insidious charms of long-branch attraction, does not support A–P–C10.
Philippe et al.7 have overcome these problems by using data from ‘expressed sequence tags’ (ESTs) in addition to complete genome sequences. Sequencing ESTs efficiently samples just the genes in any genome, avoiding the non-coding parts. The vastly lower cost of an EST project compared with sequencing a complete genome means that large numbers of ESTs have been generated for a much wider range of organisms, and we and others have been decorating the animal tree with EST data, including data from the neglected Lophotrochozoa11.
Using this expanded data set, Philippe et al.7 find convincingly in favour of L–E–D (Fig. 1). They include many more data than previous non-genomic studies (35,371 amino acids from 146 genes) and more species than genome studies (35 species representing 12 animal phyla and 14 outgroups including choanoflagellates, thought to be the protozoan phylum most closely related to animals).
When only a distant outgroup (yeast) was used7, nematodes emerge at the base of the tree. But with closer outgroups (protozoans related to animals, and jellyfish), nematodes cluster with arthropods, as predicted by the L–E–D hypothesis. In the complete data set, however, lophotrochozoan flatworms cluster with the ecdysozoan nematodes, and not with their supposed lophotrochozoan relatives (the molluscs and annelid worms). Suspecting that this was another long-branch artefact, Philippe et al. selectively eliminated genes expected to contribute most to long-branch attraction — those with a greater evolutionary rate in some species (such as nematodes) compared with others (such as deuterostomes). Indeed, as the most biased genes were removed, support for Ecdysozoa and Lophotrochozoa increased.
Will this be the last, defining statement in the controversy? There remain some unresolved problems with Philippe and colleagues' analysis, such as the position of the phylum Tardigrada (water bears). Tardigrades are unquestionably close to arthropods (they have eight stumpy legs), but appear as the sister phylum to the Nematoda. Have the nematodes lost the legs they once had, or are tardigrades misplaced?
Additionally, only 12 of the 35 animal phyla are currently represented: will addition of more phyla — particularly lophotrochozoan phyla — change the tree significantly? Have coeloms in protostome and deuterostome animals very different developmental origins: have they arisen independently? Some really obscure bilaterally symmetrical animal phyla, such as acoel flatworms, are thought to have separated from the main animal lineage before the divergence of protostomes and deuterostomes. Will these illuminate the evolution of our own complex bodies?
Genome sequences from many other animals are now being gathered, and EST projects are under way or planned for many more. No doubt the tree will sprout new shoots — and new controversies — very soon.
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