Downloaded from www.microbiologyresearch.org by IP: 54.237.57.119 On: Fri, 20 May 2016 14:30:59 Microbiology (2001), 147, 891–907 Printed in Great Britain Hybrid genotypes in the pathogenic yeast Cryptococcus neoformans Teun Boekhout, 1 Bart Theelen, 1 Mara Diaz, 2 Jack W. Fell, 2 Wim C. J. Hop, 3 Edwin C. A. Abeln, 1 Franc  oise Dromer 4 and Wieland Meyer 5 Author for correspondence : Teun Boekhout. Tel : 31 30 2122671. Fax : 31 30 2512097. e-mail : boekhoutcbs.knaw.nl 1 Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands 2 Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, 4600 Rickenbacker Causeway, Key Biscayne, FL 33149, USA 3 Department of Epidemiology & Biostatistics, Erasmus University Medical Centre, PO Box 1738, 3000 DR Rotterdam, The Netherlands 4 Institut Pasteur, Unite  de Mycologie, Rue du Dr Roux, 75724 Paris Cedex 15, France 5 Molecular Mycology Laboratory, University of Sydney at Westmead Hospital, Westmead, NSW, Australia Amplified fragment length polymorphism (AFLP) genotyping of isolates of the pathogenic fungus Cryptococcus neoformans suggested a considerable genetic divergence between the varieties C. neoformans var. neoformans and C. neoformans var. grubii on the one hand versus C. neoformans var. gattii on the other. This divergence is supported by additional phenotypic, biochemical, clinical and molecular differences. Therefore, the authors propose the existence of two species, C. neoformans (Sanfelice) Vuillemin and C. bacillisporus Kwon-Chung, which differ in geographical distribution, serotypes and ecological origin. Within each species three AFLP genotypes occur, which differ in geographical distribution and serotypes. Differences in ecological origin (AIDS patients, non-AIDS patients, animals or the environment) were found to be statistically not significant. In C. neoformans as well as in C. bacillisporus one of the genotypes represented a hybrid. The occurrence of hybridization has consequences for the reproductive biology of the species, as new genotypes with altered virulence or susceptibility to antifungal drugs may arise through the exchange of genetic material. Keywords : Cryptococcus neoformans, systematics, hybrids, geography, serotypes, AFLP INTRODUCTION Cryptococcus neoformans is a clinically important basidiomycetous yeast (Howard & Kwon-Chung, 1995). The fungus belongs to the order Tremellales (jelly fungi) of the Hymenomycetes (Fell et al., 2000), a group of fungi commonly occurring on woody substrates. The species is known in both the asexual (anamorph) and sexual (teleomorph) state, for which the respective names Cryptococcus neoformans (Sanfelice) Vuillemin and Filobasidiella neoformans Kwon-Chung are used. In nature the fungus occurs, to our present knowledge, only in the asexual state. C. neoformans can cause life- threatening infections in humans, especially in immuno- compromised patients. Estimates of the incidence rate in ................................................................................................................................................. Abbreviations : AFLP, amplified fragment length polymorphism ; CCV, cophenetic correlation value ; CSF, cerebrospinal fluid ; IGS, intergenic spacer ; RAPD, random amplified polymorphic DNA ; UPGMA, unweighted pair group method using arithmetic means. AIDS patients range from 5 to 30%, with the highest numbers occurring in sub-Saharan Africa (Mitchell & Perfect, 1998), while other estimates set the upper limit at 6–12 % (Casadevall & Perfect, 1995). The main sites of infection are the lungs and the central nervous system, including cerebrospinal fluid (CSF), while most skin infections are probably due to disseminated systemic infections (Casadevall & Perfect, 1998 ; Schupbach et al., 1976). According to the current classification, the species consists of three varieties : C. neoformans var. neo- formans (serotype D), C. neoformans var. grubii Franzot et al. (serotype A) (Franzot et al., 1999), both comprising the teleomorph F. neoformans var. neoformans Kwon- Chung, and C. neoformans variety gattii Vanbreu- seghem & Takashio (serotypes B and C) with the teleomorph F. neoformans var. bacillispora Kwon- Chung (Franzot et al., 1999 ; Kwon-Chung, 1975, 1976 ; Kwon-Chung & Bennett, 1984 ; Kwon-Chung et al., 1978, 1982a, b ; Pfeiffer & Ellis, 1993). Variety grubii 0002-4387  2001 SGM 891