Sex in Penicillium: Combined phylogenetic and experimental approaches M. López-Villavicencio a, * ,1 , G. Aguileta b,1 , T. Giraud b , D.M. de Vienne a,b , S. Lacoste a , A. Couloux c , J. Dupont a a Origine, Structure, Evolution de la Diversité, UMR 7205 CNRS-MNHN, Muséum national d’histoire naturelle, CP39, 57 rue Cuvier, 75231 Paris Cedex 05, France b Ecologie, Systématique et Evolution, UMR 8079, Bâtiment 360, Université Paris-Sud, F-91405 Orsay cedex, France; UMR 8079, Bâtiment 360, CNRS, F-91405 Orsay cedex; France c Genoscope – Centre National de Séquençage: BP 191, 91006 EVRY cedex, France article info Article history: Received 25 January 2010 Accepted 6 May 2010 Available online 9 May 2010 Keywords: Positive selection Relaxed selection dN dS Talaromyces Experimental crosses abstract We studied the mode of reproduction and its evolution in the fungal subgenus Penicillium Biverticillium using phylogenetic and experimental approaches. We sequenced mating type (MAT) genes and nuclear DNA fragments in sexual and putatively asexual species. Examination of the concordance between indi- vidual trees supported the recognition of the morphological species. MAT genes were detected in two putatively asexual species and were found to evolve mostly under purifying selection, although high sub- stitution rates were detected at some sites in some clades. The first steps of sexual reproduction could be induced under controlled conditions in one of the two species, although no mature cleistothecia were produced. Altogether, these findings suggest that the asexual Penicillium species may have lost sex only very recently and/or that the MAT genes are involved in other functions. An ancestral state reconstruction analysis indicated several events of putative sex loss in the genus. Alternatively, it is possible that the supposedly asexual Penicillium species may have retained a cryptic sexual stage. Ó 2010 Published by Elsevier Inc. 1. Introduction Despite the costs of sex (Otto and Lenormand, 2002), most eukaryotes engage in sexual recombination at least at some point in their life cycle. The predominance of sexual reproduction sug- gests that sex must provide some advantages. Although asexual reproduction is common in nature, exclusively asexual taxa are rare and they are considered to be short-lived (Judson and Normark, 1996). Many models have been built to explore short- and long-term advantages of sex to explain its maintenance, but evidence from natural cases is still scarce (but see De Visser and Elena, 2007, for recent experimental evidence for direct benefits of sex). More information is needed on suitable biological models that could be used to tackle these issues. The empirical study of sex and recombination has been based on vertebrates, insects and plants models, while other groups of eukaryotes, including fungi, have been neglected (Birky, 1999). Biological groups that exhibit a diversity of reproductive strategies provide unique opportunities to study the evolution of sex. Groups such as fungi are thus excellent models as they present a great range of reproductive strategies, including obligatory sexual spe- cies, those that alternate sexual and asexual reproduction, and oth- ers that appear to be strictly asexual (Taylor et al., 1999). Fungi also appear to have had multiple transitions from sexuality to asexual- ity (Lobuglio et al., 1993). In Fungi, asexual reproduction by pro- duction of asexual propagules (e.g. conidia) has been considered to be particularly common, with a quarter of fungal species thought to reproduce only by asexual means (Taylor et al., 1999). However, recent studies have shown that a great number of fungal ‘‘asexual” species are in fact capable of sexual reproduction. Sex in these species is only difficult to observe in nature and challenging to induce in the laboratory. Some species for which sex has not been observed, like Coccidioides immitis, present population struc- tures consistent with recombination, suggesting the existence of cryptic sex in nature (Burt et al., 1996). Sex has been successfully induced under controlled conditions in species such as Candida albicans (Hull et al., 2000), Aspergillus fumigatus (O’Gorman et al., 2009), A. flavus and A. parasiticus, which were long thought to be asexual (Horn et al., 2009a,b). Finally, apparently functional mating type genes (i.e. genes that define mating compatibility in fungi) have recently been identified and characterized in several species with no sexual cycle described, such as A. oryzae (Galagan et al., 2005), and recently in Penicillium chrysogenum and Acremonium chrysogenum (Hoff et al., 2008; Pöggeler et al., 2008). Other fungal species shown to be truly clonal from a population genetic stand- point, such as Penicillium marneffei, also present mating type genes, suggesting that sex has been lost recently (Woo et al., 2006; Fisher, 2007). Alternatively, sexual reproduction that would always occur between identical clones, as allowed under homothallism, would not be distinguishable from strictly asexual reproduction using population genetics. 1087-1845/$ - see front matter Ó 2010 Published by Elsevier Inc. doi:10.1016/j.fgb.2010.05.002 * Corresponding author. Fax: +33 1 69 15 73 53. E-mail address: mlopez@mnhn.fr (M. López-Villavicencio). 1 Both authors contributed equally to this paper. Fungal Genetics and Biology 47 (2010) 693–706 Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi