Metazoan deep phylogenies – can the Cambrian explosion be resolved with molecular markers? Martin Schlegel1, Guido Fritzsch1,2 and Peter F. Stadler2 1Institut für Zoologie Universität Leipzig Talstraße 33 04103 Leipzig 2Interdisziplinäres Zentrum für Bioinformatik und Institut für Informatik Universität Leipzig Kreuzstraße 7b 04103 Leipzig Fossils are our primary window to the history of life. Already Darwin was aware that the abundance of the fossil record dramatically increased in the Cambrian some 530 million years ago. Arthropods, annelids, molluscs, brachiopods, echinoderms, chordates, i.e. representatives of most of the recent animal phyla appeared (and a considerable number of life forms that did not survive and cannot be assigned to recent taxa). The appearance and radiation within ten million years is referred to the Cambrian explosion. To date it remains a problem to resolve the order of ramification within the extant major lineages and thus to understand the evolution of the various metazoan body plans, developmental processes, genome and proteome organisation as well as the phylogenetic position of important model organisms, such as Caenorhabditis and Drosophila. Besides comparative morphology and anatomy molecular data are increasingly applied in reconstruction of phylogenetic relationships. However, a consistent view of Cambrian phylogeny did not yet emerge from molecular investigations, which are mainly based on small ribosomal subunit RNA sequence comparisons. It has been hypothesised that the phylogenetic ramification of the extant animal phyla was so rapid, that no apomorphies evolved between the short branching intervals. Alternatively, the phylogenetic separation of major extant phyla may have already began long before the Cambrian, but fossilisable material was not evolved before the Cambrian. Hints for such an evolution come from Precambrian fossils of sponges, cnidarians and from traces of bilaterians. Consequently, if ramification occurred considerably before the Cambrian, a more intensive and extensive analysis of gene sequences should provide consistent information on deep metazoan phylogeny and an alternative to the Cambrian ”big bang” evolution. In order to test this alternative hypothesis we (and other working groups) started a comprehensive multi-gene sequence analysis in order to contribute to the solution of this outstanding evolutionary phenomenon in metazoan evolution. Here, we report on our first results of the analyses of mitochondrial genome sequence comparisons and the Histone genes H2a, H2b, H3 and H4. 100 Mammalia Crocodylia 74 Aves 99 100 Squamata 61 Amphibia Eutelostomia 100 Eutelostomia 100 100 90 Teleostei 82 Chondrichthyes 59 100 Hyperoatia 100 77 Hyperotreti Cephalochordata Hemichordata 97 Echinodermata 100 Urochordata 100 73 Insecta (Pterygota) Crustacea 88 Insecta (Collembola) 87 Myriapoda 57 Chelicerata 64 Crustacea 100 83 Plathelminthes 100 Nematoda 88 Nematoda 62 Mollusca (Bivalvia) Mollusca (Gastropoda) 100 Annelida Mollusca (Polyplacophora) Brachiopoda 100 Anthozoa 100 First results of the analysis of mitochondrial amino acid sequences with the maximum likelihood method. This tree shows a number of implausible features: (1) the placement of the urochordates outside the deuterostomes, (2) the disruption of the mollusca, (3) the placement of the plathelmintes inside the nematoda, (4) the interspersing of insecta and crustacea. A comprehensive taxon sampling is available from mitochondrial genome sequences. Phylogenetic analyses are congruent with traditional concepts of deuterostome monophyly and support classical subgroup division, with the exception of the position of the Urochordata, which are branching off before the deuterostomes. Within the protostomes mitochondrial analyses did not yield the desired improvement in resolution of branching patterns. Even monophyletic taxa such as the molluscs where not recovered. Thus, this character does not support the alternative hypothesis of phylogenetic ramification significantly before the Cambrian. At present we are analysing whether this is due to the lack of evolution of character differences (which would support the “big bang” hypothesis) or due to saturation effects by multiple substitutions. Caenorhabditis elegans Nematoda P 81 Platynereis dumerilii r 31 o 99 Urechis caupo t o Annelida Chaetopterus variopedatus s t o 100 Chironomus thummi 85 m Chironomus thummi thummi i a 98 Rhynchosciara americana Arthropoda 100 90 Drosophila hydei 69 Drosophila melanogaster 92 Psammechinus miliaris Strongylocentrotus purpuratus Echinodermata D 46 Homo sapiens e u 98 Bos taurus t e 38 Rattus norvegicus r 52 o 76 Mus musculus s Chordata t Cairina moschata o m 99 Gallus gallus i 99 a Xenopus laevis 67 Oncorhyncha mykiss Schizosaccharomyces pombe O u Saccharomyces cerevisiae t g 57 r Emericella nidulans . 0.02 First results of the analysis of Histone genes (H2A, H2B, H3, H4). It shows the analyse of amino acid sequences with neighbour-joining method (gamma model) A more promising result was obtained by analyses of concatenated histone genes. Although data are still lacking from some important lineages, such as the molluscs and the urochordates, clear character differences were detected in the sequences analysed so far which yield a consistent phylogenetic tree. Further sequences from repesentative samples are therefore urgently requested from histone genes, which where hitherto regarded to be evolutionarily too conserved to contain phylogenetic information. .