Final Activity Report Summary - AFTOL (The Uncertain Fungal Tree of Life: the Phylogenomics Solution (FTOL)) This is the final work review of the achievements made by Carlos Echavarri-Erasun (CEE) in his research project financed with EU Marie Curie International Reintegration funds. Carlos started his work on a Marie Curie grant to work on phylogenomics and Tree of life (TOL) in May 2006, submitted his first annual evaluation in May 2007, and now presents his final report in 2008 after 2 years working for the Fungal Diversity Center (CBS, KNAW) in Utrecht, the Netherlands. This project mainly focused on using a wide spectrum of bioinformatics analyses to obtain a reliable picture on the evolution of the fungi to extract a representation of the fungal TOL (AFTOL). The approach was based on theoretical in silico analyses using genomic data available of 132 genomes (updated to March, 208), plus testing this theoretical approach in real life experiments to solve complex phylogenetic issues. Therefore, in this two years, CEE has performed an impressive amount of bioinformatics and computational work in which a large array of tools was used to manage very large amounts of genomic-data. As a result of this phylogenomic information, a very revealing picture was extracted that gives new perspectives about the evolution of the eukaryotes in general, and the fungi particular, in a very high detail. This work has been developed in a team-work effort in our lab with great contributions from Dr Eiko Kuramae and bio-informatician Dr Vincent Robert. As expected, there were some project difficulties early on, however, new ideas emerged to shortcut them including focusing on secondary metabolism as a source of valuable phylogenetic markers. CEE studies in yeast metabolism during his United States graduate school experience had a positive impact on this approach because his familiarity with the metabolic pathways involved. The secondary metabolism in eukaryotes produces a great diversity of chemicals to fulfill many different cellular tasks. Although, there is a rich metabolic diversity, the metabolic backbone from where these pathways emerged is quite conserved. Indeed, the enzymes present in the mevalonic, isoprenoid and terpenoid pathways are quite conserved in bacteria, archaea and eukaryotes. These pathways appeared in a primitive anoxic world, however, the emergence of oxygen as a main atmospheric component due to the proliferation of oxygenic photosynthetic organisms, speed up the development of new pathways in which oxygen played a vital role. In our studies, we have identified the sterol and steroid pathways as key elements in the modelling of the eukaryote lineage and their phylogeny. Indeed, the ergosterol pathway seems to act as a relevant marker that produces accurate phylogeny signal for the elucidation of the TOL. Furthermore, relevant implications on how a pathway evolves and develops seem to reflect precisely what occurred in the evolution of the eukarya, with emphasis on the animal-fungal split. There are, therefore, very significant findings that are currently being subject to testing for near future publication.