Fungi Evolution Revisited: Application of the Penalized Likelihood Method to a Bayesian Fungal Phylogeny Provides a New Perspective on Phylogenetic Relationships and Divergence Dates of Ascomycota Groups Ana Carolina B. Padovan, Gerdine F.O. Sanson, Adriana Brunstein, Marcelo R.S. Briones Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil Received: 26 May 2004 / Accepted: 15 December 2004 [Reviewing Editor: Dr. Nicolas Galtier] Abstract. The depiction of evolutionary relation- ships within phylum Ascomycota is still controversial because of unresolved branching orders in the radi- ation of major taxa. Here we generated a dataset of 166 small subunit (18S) rDNA sequences, represen- tative of all groups of Fungi and used as input in a Bayesian phylogenetic analysis. This phylogeny sug- gests that Discomycetes are a basal group of fila- mentous Ascomycetes and probably maintain ancestor characters since their representatives are intermingled among other filamentous fungi. Also, we show that the evolutionary rate heterogeneity within Ascomycota precludes the assumption of a global molecular clock. Accordingly, we used the penalized likelihood method, and for calibration we included a 400 million-year-old Pyrenomycete fossil considering two distinct scenarios found in the liter- ature, one with an estimated date of 1576 Myr for the plant–animal–fungus split and the other with an estimated date of 965 Myr for the animal–fungus split. Our data show that the current classification of the fossil as a Pyrenomycete is not compatible with the second scenario. Estimates under the first sce- nario are older than dates proposed in previous studies based on small subunit rDNA sequences but support estimates based on multiprotein analysis, suggesting that the radiation of the major Ascomy- cota groups occurred into the Proterozoic era. Key words: Ascomycota evolution — Bayesian inference — Penalized likelihood — Divergence date- estimation — Fossil constraint Introduction Molecular sequences have been used to infer evolu- tionary relationships among the main groups of Asc- omycota phyla, namely, filamentous Ascomycetes or Euascomycetes, Saccharomycetales, and Archiasco- mycetes (Bruns et al. 1992; Berbee and Taylor 1993; Gargas and Taylor 1995; Kurtzman 2000; Lumbsch 2000;Redeckeretal.2000;Berbee2001).Thesestudies propose that morphological characters are often insufficient to infer the evolutionary history of this phylum because morphological similarity is in most casescausedbyconvergence(BerbeeandTaylor1993). Phylogenies based on molecular characters have been useful in providing clues to the location of taxa with uncertain taxonomic position (e.g., mitosporic fungi). The most used marker is the nuclear small subunit (SSU)rDNA(or18SrDNA)(Brunsetal.1992;Berbee and Taylor 1993; Kurtzman 2000), although a few studieshaveexploredothergenomicregionsorprotein sequences (Liu et al. 1999; Heckman et al. 2001; Hedges et al. 2004). The concerted evolution of SSU rDNA copies within genomes precludes paralogy for this gene and makes it a useful tool for phylogenetic inference and subsequent molecular dating. These points cannot be apriori guaranteed for other genes. Correspondence to: Adriana Brunstein; email: adriana@ecb.epm.br J Mol Evol (2005) 60:726–735 DOI: 10.1007/s00239-004-0164-y